(Claudio Nicolini) Nanobiotechnology Nanobioscie

Nanobiotechnology
&
Nanobiosciences
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Volume
Pan Stanford Series on NANOBIOTECHNOLOGY

Nanobiotechnology
&
Nanobiosciences
CLAUDIO NICOLINI
University of Genoa, Italy
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Published by
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A catalogue record for this book is available from the British Library.
Pan Stanford Series on Nanobiotechnology — Vol. 1

NANOBIOTECHNOLOGY AND NANOBIOSCIENCES
Copyright © 2009 by Pan Stanford Publishing Pte. Ltd.
All rights reserved. This book, or parts thereof, may not be reproduced in any form or by any means,
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ISBN-13 978-981-4241-38-0
ISBN-10 981-4241-38-5
Printed in Singapore.
Rhaimie - Nanobiotechnology.pmd 1 6/19/2008, 3:00 PM

To My Mother Camilla
Preface
This volume introduces in a coherent and comprehensive fashion the

Stanford Series on Nanobiobiotechnology by defining and reviewing the
major sectors of Nanobiotechnology and Nanobiosciences with respect to
the most recent developments. Nanobiotechnology indeed appears
capable of yielding a scientific and industrial revolution along the routes
correctly foreseen by the numerous programs on Nanotechnology
launched over the last decade by numerous Councils and Governments
worldwide, beginning in the late 1995 by the Science and Technology
Council in Italy and by the President Clinton in USA and ending this
year with President Putin in Russian Federation.
The aims and scope of the Series and of this Volume are to cover the
basic principles and main applications of Nanobiotechnology as an
emerging field at the frontiers of Biotechnology and Nanotechnology.
The publishing policy of the Series consists of at least one volume per
year up to two per year with the title and authors chosen among leading
scientists active in the field and will have the form of full manuscript
single or multiauthored, and of a coherent collection of chapters-reviews
edited by one or more leading scientists to cover a given topics. A
Scientific Committee formed by Wolfgang Knoll (Max Planck Institute
Mainz), Joshua LaBaer (Harvard University, Boston), Michael
Kirpichnikov (Moscow University) and Christian Riekel (ESRF,
Grenoble) has been established by the Publisher to act as an Editorial
Board to discuss with myself as Series Editor for the purpose of sourcing
and peer-reviewing manuscripts and providing advice on the series of
future high quality Volumes.
vii
viii Nanobiotechnology and Nanobiosciences
I am particularly grateful to all numerous members of the Nanoworld

Institute and Fondazione EL.B.A. active in this field cited in the list of
publications contained in the Bibliography. Our research activities here
reported were supported by several multinational companies, by the
Italian Ministry of University and Scientific - Technological Research
through an annual allocation granted to the Fondazione EL.B.A. and
through numerous FIRB and FISS research contracts on Organic and
Biological Nanosciences and Nanotechnology to both the Fondazione
Elba and the CIRSDNNOB-Nanoworld Institute of the University of
Genoa, including a very recent one on Functional Proteomics jointly with
Harvard University.
Claudio Nicolini
(www.claudionicolini.it)
Genoa, 30 March 2008

Contents
Preface vii
1. Nanoscale Materials 1
1.1 Produced Via LB Technology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
1.1.1 Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
1.1.2 Proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
1.1.2.1 Light sensitives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6
1.1.2.2 Metal-containings . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8
1.1.2.3 Others . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12
1.1.3 Genes and Oligonucleotides . . . . . . . . . . . . . . . . . . . . . . . . . . . 14
1.1.4 Lipids and Archaea . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16
1.2 Produced Via Organic Chemistry . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17
1.2.1. Conductive polymers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18
1.2.1.1 Amphipilic conjugated polymers . . . . . . . . . . . . . . . . . 22
1.2.2 Carbon nanotubes and their nanocomposites . . . . . . . . . . . . . . 25
1.2.2.1 Interactions between conjugated polymers and
single-WN . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32
1.2.2.2 SWNT for hydrogen storage . . . . . . . . . . . . . . . . . . . . 33
1.2.3 Nanoparticles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 34
1.3 Produced Via LB Nanostructuring . . . . . . . . . . . . . . . . . . . . . . . . . . . 37
1.3.1 Langmuir-Blodgett . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 39
1.3.1.1 Protective plate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 41
1.3.1.2 Heat-proof and long range stability . . . . . . . . . . . . . . . 43
1.4 Produced Via APA Nanostructuring . . . . . . . . . . . . . . . . . . . . . . . . . . 48
1.4.1 Focus ion beam . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 49
2. Nanoscale Probes 54
2.1 Surface Potential . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 54
2.2 Atomic Force Microscopy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 58
2.2.1 AFM spectroscopy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 64
2.2.2 Scanning tunneling microscopy . . . . . . . . . . . . . . . . . . . . . . . . 65
ix
x Nanobiotechnology and Nanobiosciences
2.3 Nuclear Magnetic Resonance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 67

2.3.1 Circular dichroism . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 69
2.4 Brewster-Angle Microscopy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 70
2.4.1 Ellipsometry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 71
2.5 Electrochemistry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 72
2.6 Infrared Spectroscopy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 74
2.7 Nanogravimetry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 77
2.7.1 Quality factor . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 79
2.8 Biomolecular Microarrays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 81
2.8.1 Gene expression via DNASER . . . . . . . . . . . . . . . . . . . . . . . . . 82
2.8.2 Protein expression via Nucleic Acid Programmable Array . . . 86
2.9 Biophysical Informatics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 90
2.9.1 Bioinformatics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 91
2.9.2 Biophysical molecular modelling . . . . . . . . . . . . . . . . . . . . . . . 93
2.9.2.1 Three-dimensional structure of octopus rhodpsin . . . . 94
2.9.2.2 Three-dimensional structure of cytochrome P450scc . 96
2.9.2.3 Protein crystallization . . . . . . . . . . . . . . . . . . . . . . . . . 97
2.9.2.4 Nanobiodevice implementation . . . . . . . . . . . . . . . . . 100
2.10 Mass Spectrometry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 101
2.10.1 Mass spectrometry of label-free NAPPA . . . . . . . . . . . . . . 105
2.11 Synchrotron Radiation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 106
2.11.1 Diffraction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 109
2.11.2 Grazing Incidence Small Angle X-ray Scattering . . . . . . . . 111
3. Nanoscale Applications in Health and Science 118

3.1 Nanobiocrystallography . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 118
3.1.1 Radiation resistance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 127
3.1.2 New protein structures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 128
3.1.3 Three-dimensional engineering . . . . . . . . . . . . . . . . . . . . . . . . 132
3.1.4 Basics of crystal formation . . . . . . . . . . . . . . . . . . . . . . . . . . . 135
3.1.4.1 Cytochrome P450scc . . . . . . . . . . . . . . . . . . . . . . . . . 136
3.1.4.2 Lysozyme . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 145
3.2 Nanomedicine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 148
3.2.1 Carbon nanotubes biocompatibility and drug delivery . . . . . . 148
3.2.2 Photosensitization of titanium dental implants . . . . . . . . . . . . 152
3.2.3 Biopolymer sequencing and drug screening chip . . . . . . . . . . 155
3.3 Nanogenomics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 158
3.3.1 Human T lymphocytes cell cycle . . . . . . . . . . . . . . . . . . . . . . 158
3.3.2 Organ transplants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 167
3.3.3 Osteogenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 173
3.4 Nanoproteomics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 174
3.4.1 Cell cycle . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 175
Contents xi
3.4.2 Cell transformation and differentiation . . . . . . . . . . . . . . . . . . 180

3.5 Nanomechanics and Nanooptics . . . . . . . . . . . . . . . . . . . . . . . . . . . . 183
3.5.1 Nanocontacts for addressing single-molecules . . . . . . . . . . . . 183
3.5.2 Nanofocussing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 191
3.5.3. Optical tweezers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 193
3.5.4 Magnetism . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 195
3.6 Cell Nanobioscience . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 197
3.6.1 Nucleosome core . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 197
3.6.1.1 DNA deformation . . . . . . . . . . . . . . . . . . . . . . . . . . . . 201
3.6.1.2 Water and ions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 201
3.6.2 Protein stability to heat and radiation . . . . . . . . . . . . . . . . . . . 202
3.6.2.1 Bioinformatic analysis . . . . . . . . . . . . . . . . . . . . . . . . 204
3.6.2.2 Structural comparisons . . . . . . . . . . . . . . . . . . . . . . . . 207
3.6.2.3 Structural comparisons of homologous
thermophilic/mesophilic pairs . . . . . . . . . . . . . . . . . . 207
3.6.2.4 Water comparisons of homologous
thermophilic/mesophilic pairs . . . . . . . . . . . . . . . . . . 209
3.6.2.5 Detailed comparison of mesophilic versus
thermophilic thioredoxin . . . . . . . . . . . . . . . . . . . . . . 212
4. Nanoscale Applications in Industry and Energy Compatible with

Environment 219
4.1 Nanobioelectronics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 220
4.1.1 Nanosensors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 220
4.1.1.1 Protein-based nanosensors . . . . . . . . . . . . . . . . . . . . . 220
4.1.1.2 Organic nanosensors . . . . . . . . . . . . . . . . . . . . . . . . . . 237
4.1.2 Passive elements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 246
4.1.2.1 Resistors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 247
4.1.2.2 Capacitors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 250
4.1.2.3 Wires . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 254
4.1.3 Active elements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 255
4.1.3.1 Schottky diode . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 256
4.1.3.2 Led . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 257
4.1.3.3 Optical filtering and holography 261
4.1.3.4 Displays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 265
4.1.3.5 Monoelectronic transistors . . . . . . . . . . . . . . . . . . . . . 267
4.1.4 Quantum dots and quantum computing . . . . . . . . . . . . . . . . . . 271
4.2 Nanoenergetics Compatible with Environment . . . . . . . . . . . . . . . . . 275
4.2.1 Photovoltaic cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 275
4.2.1.1 Reaction centers-based . . . . . . . . . . . . . . . . . . . . . . . . 278
4.2.1.2 Purple-membrane based . . . . . . . . . . . . . . . . . . . . . . . 279
4.2.2 Batteries . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 282
xii Nanobiotechnology and Nanobiosciences
4.2.2.1 Lithium ion batteries elements . . . . . . . . . . . . . . . . . . . 285

4.2.2.2 The cathode . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 285
4.2.2.3 The anode . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 288
4.2.2.4 The electrolyte . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 291
4.2.3 Hydrogen storage and fuel cells . . . . . . . . . . . . . . . . . . . . . . . 296
4.3 Nanobiocatalysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 301
4.3.1 Bioreactors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 306
4.3.1.1 From lab scale to industrial scale . . . . . . . . . . . . . . . . 309
4.3.2 Bioactuators . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 312
Bibliography 313
Index 363
Chapter 1
Nanoscale Materials
This chapter overviews the present status of new materials by organic

and biological nanotechnology and their applications with respect to the
development of organic and biological nanotechnology defining
Nanobiotechnology and Nanobiosciences capable to yield a significant
scientific and technological progress.
Particular emphasis is placed on what has been accomplished in our
laboratory in the last eight years, whereby the details on the
supramolecular layer engineering and its application to industrial
nanotechnology can be found in recent complete reviews (Nicolini et al.,
2001; 2005; Nicolini and Pechkova, 2006). Material technology has
changed our lives within only a few decades. Sand, the starting material,
has been turned into a versatile “high-tech” product. Techniques have
been developed for the production of silicon wafers and for the
modification of this raw material into the final “high-tech” product -
miniature electronic logic functions. Research now focuses on the
molecular manufacturing (Nicolini, 1996c), from the microstructures to
the nanostructures for a new generation of nanomaterials. The rapid
increase in our knowledge of the function of both biological and organic
materials has also directed interest into this area. The functionality,
efficiency, and flexibility of biomaterials (see paragraph 1.1) are
impressive; they are beyond the capabilities of synthetic chemistry
already quite powerful (see paragraph 1.2).
The development of gene-technological methods has opened the way
to the controlled modifications of proteins, which appear already in
position to allow the production of newly designed biomaterials. Several
molecules of native organic and biological origin being investigated in
1
2 Nanobiotechnology and Nanobiosciences
the last years are here summarized to exemplify potentially useful

nanomaterials. They can be divided into two major classes, namely those
produced via biotechnology and those produced via chemical synthesis.
1.1 Produced Via LB Technology
1.1.1 Cells
Several well-established cell culture systems, like mammalian CHO-K1,

H9c2 or HeLa cells or microorganisms, are utilized in different context
for various applications (Adami et al., 1992, 1995a; Hafemann et al.,
1988; Garibaldi et al., 2006, Spera and Nicolini, 2007).
Chinese hamster ovary fibroblasts (CHO clone K1) for
Nanoproteomics, as supplied by American Type Culture Collection,
Rockville, MD, is a stable hypodiploid cell line derived by spontaneous
transformation from a fibroblast culture (Misteli, 2001). Cells were
cultured in F12 medium supplemented with 10% fetal calf serum and
0.2% gentamycin at 37 °C in 5% CO2 atmosphere. To reverse transform
the cells they were treated with a solution 10-3 M dibutyryl cyclic 3’,5’
monophosphate adenosine sodium salt (Bt2cAMP, Sigma Chemical Co.,
St. Louis, MO) that was added to the normal culture medium for six
hours before the analysis. It has been shown that single cells of CHO-K1
in the native state grow equally well on plastic surfaces or in suspension
(Figure 1.1). In the presence of reverse transformation conditions,
however, excellent growth is still achieved on the plastic surface but no
growth whatever occurs in suspension (Hsie and Puck, 1971) as
monitored by a phase contrast microscope (Wilovert, Wesco).
Primary cultures of rat hepatocytes for Nanosensors were also used
for experiments with biosensing potentiometric systems because they are
easy to obtain and, like all hepatocytes, they maintain, in the first hours
in vitro, their metabolic skills practically unchanged with respect to the
in vivo situation (Nicolini et al., 1995a). Hepatocytes were isolated from
liver of Sprague-Dawley albino rats (200–250 g) by in situ collagenase
perfusion according to Williams (Williams, 1977).
Nanoscale Materials 3
Figure 1.1 Chinese hamster ovary fibroblasts (CHO clone K1). Electronic micrographs of
CHO-K1 cells at t equal 0 hour (mitosis), 2 hours (G1 phase), 3 hours (S phase) and 6
(G2 phase) hours after mitosis. (Nicolini and Rigo, Biofisiche e Tecnologie Biomediche,
Zanichelli Editore, 1992).
Cardiac muscle cells for Drug Delivery via Carbon Nanotubes are a
rat heart cell line H9c2 (2-1), obtained from American Type Culture
Collection (Rockville, MD) at passage 16. Cells at passages from 22 to
24 were used for the experiments. H9c2 cells were cultured in Dulbecco's
modified Eagle's medium with 4 mM L-glutamine adjusted to contain 1.5
g/l sodium bicarbonate, 4.5 g/l glucose, 1.0 mM sodium pyruvate, 10%
FBS, with 50 units penicillin/ml, 100 mg streptomycin/ml at 37°C in 5%
CO2, and the medium was changed every 2–3 days. Subconfluent cells
were detached with trypsin and seeded 24 hours before treatment at a
density of 25000–30000 cells/cm2 in 100 mm petri dishes. Cells were
seeded in Petri dishes and let to grow for 24 hours before treatments.
While untreated control cells were administered the complete medium
without nanotubes, sonicated in parallel with Single Wall NanoTube
(SWNT) suspension, treated cells were administered complete medium
with suspended 0.2 mg/ml SWNT for serial time steps. A positive
control was set to induce cell damage and death by treating H9c2 with
200 µM and 1 mM hydrogen peroxide. The experiments were performed
in triplicate. Cardiomyocytes treated or not with SWNT were evaluated
at relevant time points by light microscopy to assess cell proliferation
and viability. Digital photomicrographs were recorded and the number of

viable cells computed. Myocytes were incubated for 24, 48 and 72 hours
at standard culture conditions to determine the viability following
treatments. The number of viable cells was determined with trypan blue
exclusion. In brief, cell monolayers were rinsed twice with PBS and
resuspended with trypsin and EDTA. The cells were immediately stained
with 0.4% trypan blue, and the number of viable cells was determined
using a hemocytometer under a light microscope. In order to assessment
apoptosis the cells were labeled with annexin V-FITC and propidium
iodide, and fluorescent positive cells were detected by flow cytometry.
Cellular fluorescence was determined by a flow cytometry apparatus
(FACS-SCAN, Becton-Dickinson, Franklin Lakes, NJ, USA). Flow
cytometric analysis was performed on a minimum of 1 × 104 unfixed
cells per sample. Cardiomyocytes were trypsinized with trypsin-EDTA,
resuspended in PBS, and loaded with 10 µM propidium iodide (PI;
Sigma-Aldrich) and 1 µM annexin V-FITC (Sigma Aldrich) at 4°C for
10 min. Apoptosis was identified as cells with low forward scatter (FSC)
on side scatter (SSC)/FSC dot plots, PI dim staining on FSC/PI dot plots,
and annexin V-positive and PI-negative staining on annexin V/PI dot
plots. Fluorescence probes were excited with an 488 nm argon ion laser.
The emission fluorescence was monitored at 525 nm for annexin V-FITC
and 620 nm for PI. Data were analyzed with the Cell Quest software
package (Becton-Dickinson).
Human T lymphocytes for Nanogenomics, as previously shown
(Nicolini et al., 2006a) are obtained by a sample of heparinized
peripheral blood specimens diluted 1:1 with sterile phosohate buffer
saline (PBS, pH 7.2). The sample was centrifuged on a Ficoll-Hypaque
gradient (specific gravity 1.080) at 1500 rpm for 40 min at room
temperature without brake (Abraham et al., 1980). The lymphocytes rich
interface was collected and washed twice with PBS, and the number of
cells counted in presence of trypan blue. The 2 × 106 cells were seeded
into each well containing 2ml of RPMI 1640 culture medium
supplemented with 10% heat-inactivated fetal bovine serum, penicillin
100 unit/mL and streptomicin 50 µg/mL. The cells has then been
incubated at 37 °C in a humid chamber with 5% of CO2 for 24, 48, 72
hours. The cells have been then collected and counted and then it has
been proceeded to RNA extraction.
1.1.2 Proteins
Recombinant protein expression and purification is the single most

important prerequisite for the effective engineering of nanostructured
protein-based materials.
Typically the cDNA encoding the mature form of the protein being
immobilized is subcloned into the proper expression vector, highly
expressed in Escherichia coli and the expressed protein is typically
purified by affinity chromatography (Pernecky and Coon, 1996;
Ghisellini et al., 2004; Wada et al., 1991; Amann et al., 1988) and
evaluated spectrophotometrically using the appropriate extinction
coefficient at the appropriate wavelength.
In the case of overall cell proteins extraction, as required in Mass
Spectrometry at the nanoscale, a protein extraction kit (Subcellular
Proteome Extraction Kit, Calbiochem) is typically chosen for the total
protein extraction from cells (Spera and Nicolini, 2007). It is designed
for extraction of cellular proteins from adherent and suspension-grown
cells according to their subcellular localization. For the sequential
extraction of the cell content, the kit takes advantage of the differential
solubility of certain subcellular compartments in special reagent
mixtures. Upon extraction of culture cells, four partial proteomes of the
cells are obtained:
cytosolic proteins,
membrane and membrane organelle proteins,
nuclear proteins,
cytoskeleton proteins
The kit contains four extraction buffers, a protease inhibitor cocktail
to prevent protein degradation and a nuclease (Merck) to achieve an
efficient removal of contaminating nucleic acids. The sub-cellular
extraction was performed according to the protocol for freshly prepared
adherent culture cells. We performed extraction from 106 CHO-K1 cells
in logarithmic phase at 80% confluence (Spera and Nicolini, 2007). To
monitor the extraction procedure, morphological changes of the cells
were examined by a phase contrast microscope (Wilovert, Wesco). The

protein fractions were stored at -80°C until the Mass Spectrometry
analysis. The amount of total proteins recovered from each extraction
step has been evaluated by Bradford assay (Tjio and Puck, 1958), using
bovine serum albumin as standard.
1.1.2.1 Light sensitives
Photosynthetic reaction centers from Rhodobacter sphaeroides and

bacteriorhodopsin (bR) from purple membrane (PM) have been used for
their unique optoelectronic properties and for their capability of
providing light-induced proton and electron pumping.
Once assembled they display extremely high thermal and temporal
stability (Nicolini 1997a, 1998a,b, Nicolini et al., 1999). These features
make them two of the most promising biological molecules for
developing devices (Nicolini 1996a, Nicolini et al., 2005). RC proteins
can conduct electrons in only one direction in presence of a suitable light
source (photoconduction) or of an external electric field (conduction).
Both these conductive processes are due to the tunnelling of a single
electron into the structure of the protein.
In the case of bR the situation is different (Figure 1.2). BR is the main
part of purple membranes (about 80%) and is already close packed in it.
It is difficult to extract bR in the form of individual molecules, for they
are very unstable (Shen et al., 1993). The modification of the retinal
protein bacteriorhodopsin gave birth to the generation of new versatile
media for optical processing (Zeisel and Hampp, 1992). Gene technology
as the key technique in this new direction of nanomaterials research may
be the counterpart to photolithography in semiconductor technology. The
design of complex molecular functions is a goal extremely difficult to
achieve with the classical approach of chemistry and supramolecular
chemistry, whereas nature developed a large variety of such
“nanomaterials”. It is indeed now possible to begin the construction of
such complex nanomaterials ab initio, by starting out from the molecules
that we discover in nature and try to modify their properties and adapt
them towards the demands of technical applications, for both
metalloproteins and light-sensitive proteins. Modification of the genetic
code facilitates manipulations of organisms that can produce these new

“high-tech” nanomaterials with conventional biotechnological methods.
Figure 1.2. PDB image of (left) Bacteriorhodopsin (PDB id 2BRD, Grigorieff, et al.,
1996) and (right) Octopus Rhodopsin (PDB id 2AUL, Sivozhelezov and Nicolini 2006),
top) front view, middle) top view, bottom) side view.
Therefore, the price of such products will be affordable. An intensive

screening for functional biopolymers with technically relevant functions
and their variation and identification will create a new class of
“biomimetic” materials. The experimental studies done with
bacteriorhodopsin have shown that this new approach leads to
competitive materials in selected areas of opto-electronics and optical
filtering; a similar route is presently being pursued with metallo-proteins

also here summarized, as it will be shown see later.
1.1.2.2 Metal-containings
Expression and purification of a typical metal-protein, i.e. recombinant

native P450scc, occur in E. coli JM109cells, transformed with pTrc99A-
P450scc expression plasmid, being grown and induced as described in
Wada et al. (1991) with few modifications.
The δ-aminolevulinic acid (1 mM), a precursor of heme biosynthesis,
was added at the same time as isopropyl-1-thio-β-D-galactopyranoside (1
mM) and the cells were grown for 72 h at 28 °C by shaking at 150 rpm.
Typically expressed cytochrome P450scc is purified by three different
chromatographic steps: DEAE cellulose, hydroxyapatite and Adx-
sepharose 4B columns. The sample is solubilized in 10 mM K phosphate
buffer (pH 7.4) containing 0.1 mM EDTA, 0.2 sodium cholate and 20%
glycerol and stored at -80°C. The properties of two P450scc
cytochromes, namely wild-type versus recombinant, were systematically
characterized in structural and functional terms (Nicolini et al., 2001), in
order to probe if conformational variations are associated with the two
different processing pathways used for protein maturation (co-
translational and post-translational). Initially sequencing was carried out
using Pulsed-liquid Phase Sequencer Mod. 477A (Applied Biosystems)
in order to determine the primary structure of both proteins. As
recombinant type with two signal sequences (secretory and
mitochondrial ones) and wild-type (“wt”) with mitochondrial sequence
were examined according to the same routine, the obtained results show
identity of the primary structure of the N-terminus of both proteins
(Ghisellini et al., 2002, 2004). The first aminoacid was determined to be
Ile, which agrees with theoretical suggestion about the position of the
digestion site of the signal protease. These data give evidence that the
recombinant protein is the mature one and goes trough the entire
processing pathway. The fact that the recombinant construction has two
functioning digestion sites for signal proteases seems to be very
promising in view of biotechnological applications. The identity of their
primary structure, in the presence of significant structural (Figure 1.3)
and functional alterations, is consistent with the hypothesis that the

processing pathways have a role in determining the function and the
structure of proteins. Proteins are either wild-type, namely isolated from
the corresponding cell tissues, or recombinant, namely obtained from
clones properly genetic engineered with quite high yield and extreme
purity and homogeneity.
Figure 1.3. PDB image of cytochrome P450scc, top) front view, middle) top view,
bottom) side view (Sivozhelezov et al., 2006).
In the latter, the cDNA of the cytochrome P450scc obtained from

genomic library of the bovine surrenal cortex (precursor gene) was
cloned “down-stream” the secretory signal of Kluviromyces lactis in the
ORF of GAL UAS promoter in the vector derived from pYeDP 1/10.
The yield of the product detected in the culture broth after induction of
GAL UAS promoter was quite satisfactory.
The signal-appended cytochrome P450scc was translocated across the
cells compartments and processed to yield authentic, haem-assembled
cytochrome P450scc and was exported from the yeast cells. The eventual
processing of the P450scc precursor was somehow unstable but unusual
with respect to the presence of mitochondrial signal and the known
reaction specificity of signal peptidase. The secreted protein was tested
spectrophotometrically, electrophoretically, immunologically and by
visual transformation of the culture broth into red color. Thus, the
mitochondrial membrane protein (that carries the signal of post-
translational transfer) was efficiently processed to mature haemo-protein
and secreted from S. cereviseae by way of co-translational transfer. On
the basis of the HPLC data it was concluded that the purity level of this
protein as secreted was extremely high (approximately 98%). The
production rate of correctly matured exported cytochrome P450scc was
10.2 mg per liter of culture. The obtained mature haem-bound
cytochrome P450scc was indistinguishable in many of its characteristics
from the native counterpart or was even better, with the only exception
that its production was quite unstable. Because of this instability we have
then utilized homologous expression within the E. coli bacteria and
isolated the same cytochrome P450scc with similarly high degree of
purity and yield, but with the needed reproducibility and stability
(Ghisellini et al., 2002, 2004). The problem of obtaining metalloproteins
from microorganisms is crucial for development of applications in
nanobiotechnology. As shown above, in comparison with a method of
extraction from natural source this approach is cheaper and more
advantageous for obtaining high quality protein of interest in large
amounts. Cytochrome P450scc in nature is localized in inner membrane
of mitochondria of the bovine adrenal cortex and plays a crucial role in
the steroid metabolism. This protein is globular, contains the heme group
and two active sites: “oxygen pocket” and “substrate pocket”. The
NADPH, adrenodoxine and adrenodoxine reductase are necessary for the
functioning of the protein as a “mini chain of electron transfer”. The
hem-assembled P450scc in some of the characteristics is superior to its
natural analogue. It can have as substrates cholesterol (5-cholesten-3-
βol), styrene (99% pure grade), clozapine (8-chloro-11-(4’-

methyl)piperazine-5-dibenzo[b,e]-1,4-diazepine) and several other
chemicals. Cytochrome P450scc native recombinant (product of
CYPA11A gene) was cloned in E. coli system expression: cDNA gene of
P450scc mature form was sub-cloned in the pTrc99A vector to obtain
bacterial expression. The cDNA gene of the mature protein was obtained
by deleting the N-terminal mitochondrial targeting sequence coding the
first 39 aminoacid residues (Wada et al., 1991; Amann, et al., 1988).
Figure 1.4. PDB image of crystal structure of the catalytic subunit of human protein
kinase CK2 (PDB ID 1NA7), top) front view, middle) top view, bottom) side view
(Pechkova et al., 2003).
The cDNA encoding for another mature form of cytochrome called

P4502B4 was also subcloned for applications in Nanobiotechnology into
the pGEX-KN expression vector and highly expressed in Escherichia
coli by isopropylβ-d-thiogalactoside (IPTG) induction. The expressed
protein, fused to glutathione-S-transferase (Figure 1.4) was purified by
glutathione sepharose 4B affinity chromatography (Pernecky and Coon
1996). The purified cytochrome P4502B4 recombinant was in a 10 mM

K-phosphate buffer (pH 7.4) containing 0.1 mM EDTA, 0.005% Tween
21 and 20% glycerol.
Cytochrome P4501A2 cDNA clone was inserted into the expression
vector and used to transform Escherichia coli. Extraction and
purification of cytochrome P4501A2 recombinant was performed
according to Fischer et al. (1992). The purified cytochrome P4501A2
was solubilized in 100 mM K phosphate buffer (pH 7.25) containing 1
mM EDTA, 1 mM DTT, and 20% glycerol. Protein concentration was
determined using the BCA assay (Pierce) or by the Bradford method,
using bovine serum albumin as a standard. 10% SDS-polyacrylamide gel
electrophoresis was performed as described by Laemmli (1970). Spectra
were recorded using a JASCO 7800 Spectrophotometer (Japan) at room
temperature. The purified cytochromes (P4501A2, P4502B4, P450scc)
were found to be almost completely in the low spin iron configuration
(Guryev et al., 1996, 1997). The heme content for all iso-forms was
measured according to Omura and Sato (1964) using an extinction
coefficient of 91 mM-1 cm-1 for the absorbance difference between 450
and 490 nm. The concentration of the recombinant cytochrome is 1.2
mg/ml, and the pure grade of each sample was determined taking into
consideration the absorbance values at A280 and the absorbance
maximum at A417 (Soret peak). The absorbance A417/A280 ratios for
cytochromes P4501A2, P4502B4, and P450scc were 0.5, 0.8, and 0.9
respectively.
1.1.2.3 Others
Other different proteins, both water-soluble and membrane-bound, have

been utilized and extensively characterized by a wide variety of
biophysical probes in our efforts to develop new protein-based
nanotechnology (Nicolini, 1995; Nicolini and Pechkova, 2006).
In the case of most proteins, the best way to examine the 3D structure
is to make X-ray analysis. However, some proteins are not suitable for
this technique, as the size of the crystal is too small, or the protein is very
difficult to produce and cost too much to make large (submillimeter) 3D
crystals. Instead, for high-resolution nuclear magnetic resonance (NMR)
only minute amount of protein is necessary to make 3D atomic structure

in solution. Thus NMR is very suitable for this task. As model systems
for soluble proteins we have explored several proteins, namely
glutathione-S-transferase, three metalloproteins, namely cytochromes
P450scc (Figure 1.3), P4502B4 and P4501A2, lysozymes, human kinase
CK2 (Pechkova et al., 2003), thermophilic and mesophilic thioredoxin,
an ubiquitous protein with many functions, such as in thiol-dependent
redox reactions.
Table 1.1 Key proteins of interest to Nanobiotechnology present in RCSB PDB Data
Bank.
Protein Source Method Reference

P450scc Bovine Ab-initio Sivozhelezov et al., 2006b
Lysozyme LB Egg X-ray Pechkova and Nicolini, 2006
Thioredoxin Bacillus acidocaldaricus NMR Bartolucci et al., 1997
Thioredoxin Escherichia coli NMR Jeng et al., 1994
Kinase CK2α Human X-ray Pechkova et al., 2003
IF2β Sulfolobus NMR Vasile et al., 2008
Bacteriorhodopsin Halobacterium halobium X-ray Chou et al., 1992
Rhodopsin Octopus Ab-initio Sivozhelezov and Nicolini
2006
As model system for membrane-bound proteins we have chosen

photosynthetic reaction centers from Rhodobacter sphaeroides and from
Rhodopseudomonas viridis, bovine rhodopsin, octopus rhodopsin
(Sivozhelezov and Nicolini 2006) and bacteriorhodopsin (bR). In the
system of Halobacterium halobium, bacteriorhodopsin is found as a 2D
crystalline lattice in purple membranes. This purple membrane protein is
usually 1000 nm in diameter and 50 Å in thickness and makes part of the
overall cell membrane of Halobacteria (Chou et al., 1992).
Bacteriorhodopsin is the main protein in the biological function of
these bacteria. BR converts light energy into chemical energy by
transferring protons from the inside to the outside of the cell membrane,
thus functioning as a light-driven proton pump. As a result, ATP is
regenerated from ADP by an enzyme called ATP-synthase. Another class
of proteins of wide interest is the ribosomal proteins such as Initiation
Factor 2 from Sulfolobus solfataricus (Vasile et al., 2008), and Bone
Morphogenetic Proteins representing a family of 13 proteins capable to

stimulate bone formation. Nearly all the chosen proteins have been well
characterized in solution by X-ray crystallography or solution NMR and
their three-dimensional structure is now known at the atomic resolution
for all proteins of our interests in Nanobiotechnology and
Nanobiosciences as discussed later (Table 1.1).
Figure 1.5. PDB image of Thioredoxin from Escherichia coli (PDB ID 1XOB, Jeng et
al., 1994) and from Bacillus acidocaldaricus (PDB ID 1QUW Nicastro et al., 2000), top)
front view, middle) top view, bottom) side view.
1.1.3 Genes and Oligonucleotides
To analyze either a fluorescently labelled DNA sample of unknown

sequence (Jacobs and Fodor, 1994) or gene expression (Butte, 2002) in
human tissues or cells, chip comprising an array of short
oligonucleotides or of genes as hybridization probe constitutes the core
technology of nanogenomics (Nicolini, 2006).
DNA microarray (see chapter 3) allows the study at the nanoscale of

an immense amount of genes (over 10,000) with only one experiment
and therefore can draw a picture of a whole genome.
The expression of each gene is analyzed by the hybridization of each
gene spot with cDNA prepared by reverse transcription of total RNA
isolated from cell or tissue being characterized. The CyScriptRT enzyme,
together to RNA and to dCTP Cy3 and dCTP Cy5 nucleotides, is
employed in a synthesis reaction lead to 42 °C for 1 hour and ½ in order
to obtain the selective fluorescence labelling by retrotranscription (Figure
1.6).
Figure 1.6. The cDNA coming from control cell and from cell to be tested are labeled
with two different fluorescent probes, respectively with green (Cy3) and red (Cy5)
emission, allowing thereby to monitor differentially in the same experiment the test genes
and the control genes.
The synthesis product of this reaction, marked cDNA, has been then
subordinate to a purification step. Total RNA was extracted and
amplified using T-7 in vitro transcription. The cDNA marked samples
have been purified employing a purification kit and chromatographic
columns supplied by Amersham Biosciences. The cDNA obtained has
been precipitated and resuspended in bi-distilled water to quantify, by the
employing of the spectrophotometer, the samples and to verify the
labelling. The cDNA marked samples have been subsequently
lyophilized and resuspended in an opportune volume (120 µl) of

hybridization buffer (Salt-Based ibridation). For the array hybridization 1
µg of cDNA marked with Cy3 and 1 µg of cDNA marked with Cy5 they
have been mixed in a tube and resuspended in the hybridization buffer
together to the control sample (Arabidopsis control). The sample thus
obtained has been denatured and spotted on the array. The matrices have
been then put in a hybridization chamber at 42 °C for 20 hours. To
eliminate the aspecific binding the array has been washed with SSC
buffers of decreasing concentration. On the other side in genome
sequencing (Jacobs and Fodor, 1994) conventional solid-phase
oligonucleotide synthesis was time ago involved to step-wise assembly
5'-dimethoxytrityl (DMT)-protected nucleoside monomers in the 3' to 5'
direction. In a typical coupling procedure, the 5'-hydroxyl of an
immobilized nucleoside is deprotected with mild acid, the liberated
hydroxyl group is phosphatylated with a DMT-protected
deoxynucleoside 3'-phosphoramidite, and the resulting phosphate is
oxidized to a phosphotriester (Caruthers, 1985). The process is repeated
until the desired oligonucleotide has been prepared. This technique was
adapted to include light-directed parallel chemical synthesis by replacing
the 5'-protecting group DMT with a substituted nitroveratryl derivative,
and incorporating a nitroveratryloxycarbonyl (Nvoc), protected hydroxyl
linker into the synthesis substrate (Fodor et al., 1993). Hydroxyl groups
are selectively photo-deprotected as described previously, and arrays of
oligonucleotides assembled using standard peptide chemistry. It is worth
to notice that using the orthogonal-stripe method (Jacobs and Fodor,
1994), it was possible time ago to assemble all 65566 possible
octanucleotides (48) in only 32 chemical steps (4 × n; where n = 8)
(Pease et al., 1994).
1.1.4 Lipids and Archaea
Archaea, a philogenetically coherent separate group of microorganisms,

which differs from Eubacteria and Eukaria, comprises a variety of
extremophilic bacteria living under extreme conditions such as high
temperature, acidic or alkaline pH, and saturated solutions (Kandler,
1992).
Later we will see how nanotechnology may allow to mimic and even
enhance their thermophilic properties in mesophilic proteins
(Sivozhelezov et al., 2007; Nicolini and Pechkova, 2006a). In this
context we will discuss only the archaea membrane lipids useful to
construct nanodevices for their unusual properties and structure
(Gambacorta et al., 1994), based on isoprenoid chains of different
lengths with ether linkage to glycerol or to more complex polyols. In
particular, membrane lipids of thermophilic archaea, such as Sulfolobus
solfataricus (optimal growth conditions are 87 °C and pH 3), possess
bipolar architecture (De Rosa et al., 1983, 1986; Luzzati et al., 1987)
characterized by the presence of two polar heads and hydrophobic
isoprenoid moiety of practically double average length with respect to
that of classical ester lipids. The lipids play a key role in stabilization of
the membrane under extreme conditions. Intense X-Ray scattering
studies of the lipids from S. solfataricus carried out time ago by Gulik et
al. (1985) pointed to a variety of phases observed when the temperature
or water content in the sample was changed. In this respect, it was
interesting to compare the structure of artificial multilayer systems
created from these bipolar lipids with that of conventional amphiphilic
molecules (Nicolini, 1996a).
1.2 Produced Via Organic Chemistry
Among the numerous organic compounds produced via chemical

synthesis one of the first ones used in Nanobiotechnology was fullerene,
with a structure consisting of C60 molecules investigated by X-ray
diffraction (Meiney et al. 1991), electron diffraction (Krätschmer et al.,
1990) and scanning tunnelling microscopy (STM) (Wilson et al., 1991)
techniques. STM images of C60 samples show close packing of spherical
molecules with lattice spacing of about 1.1 nm, in accordance with X-ray
diffraction studies yielding a face-centred cubic (FCC) lattice spacing of
1.404–1.411 nm.
In the last decade are conductive polymers, carbon nanotubes,
nanoparticles and their nanocomposites in combination with biopolymers
to yield the most interesting properties in a wide range of applications.
The major drawback of the polymeric materials is the multi-step

synthesis required for the functionalization and the stringent process
requirements of the condensation polymerization. It was therefore our
efforts in the last few years (Nicolini et al., 2005) to find shorter
synthetic routes to process the polymers with predictable absorption
wavelengths of light. Several types of polymer derivatives have been
synthesized in our laboratory:
poly(p-phenylenevinylene) (PPV), namely poly(2-methoxy-5-(2’-
ethyl)hexyloxy-p-phenylenevinylene) (MEHPPV), whereby the
Gilch route has been modified in order to increase the
processability for specific device application;
poly(phenylene vinylene) (PPVs) are main-chain conjugated
polymers, which have very interesting electrical and
photoconjugated properties, and that make them suitable for
applications in opto-electronics and microelectronics devices, such
the PV cells;
inorganic nanoparticles, such as CdS, PbS and TiO2, and organic
systems, namely conducting polymer, dyes, fullerene (C60)
nanocrystals, were prepared and organized in thin films to fabricate
Donor (D) - Acceptor (A) supramolecular assemblies.
While the full details on the research efforts in nanotechnology-based
organic materials can be found for the past in Nicolini et al. (2001a) and
for the present in Nicolini et al. (2005), I will present in this subchapter
only few key examples.
1.2.1. Conductive polymers
The structures of polymeric nanoscale materials are summarized in

Figure 1.7. The conventional polymers, plastics, are traditionally used for
their interesting chemicals, mechanicals and insulating properties, but not
for theirs electronics properties.
The idea of using polymers also for their electronic semi-conductive
properties in molecular electronic devices is relatively new (Su et al.,
1979; Nicolini, 1996a,b). Conjugated polymers behaving as insulators or
semiconductors in their neutral form, can reach metallic conductivity as a
result of the doping process (Salaneck and Brédas, 1994; Nicolini,
1996a). From 1977, the dream of combining the mechanical and

processing properties of the polymers with the electronic and optical
properties of the metals has been the driving force of the science and
technology of the conjugated conducting polymers (Nicolini et al.,
2001a, 2005; Nicolini, 1996a). Conjugated polymers play a fundamental
role in transistors, integrated circuits and photovoltaic devices, Light
Emitting Devices and solid-state laser (Nicolini, 1996a).
Figure 1.7. Structural formulae of some common polymers. The bonds with hydrogen
atoms are not shown. (I) Polyethylene (PE); (II) trans-polyacetylene (PA); (III)
poly(para-phenylenevinylene) (PPV); (IV) poly(para-phenylene) (PPP); (V)
polythiophene (PT) (Reprinted with the permission from Nicolini et al., New materials by
organic nanotechnology and their applications, in Recent Research Development in
Materials Chemistry 6, pp. 17–40, © 2005, Transworld Publishing).
A central topic in the physic of the π-conjugated polymers (and their

parent oligomers) is the strong connections between electronic structure,
geometrical structure and chemical structure. The latter can be termed
lattice in parallel with the nomenclature of the condensed matter physics.
In the ’80 in the contest of transport and optical properties, was
developed the polarons, bipolarons and solitons concepts (Nicolini,
1996a). The essential idea about the unusual nature of the species bearing
charges and of the excited states of the conjugated systems has been
intensively discussed in the last twenty years (see also Nicolini, 1996a).
Recently the refinement of these ideas and the perceptions on the
physical nature of the unique electronic properties of these conjugated
polymers, as isolated molecules and as molecular solids leaded to the
development of an higher sophisticated treatment level and consequently
to a better understanding of some essential characteristics of the
electronic structure of these polymers. In this contest the understanding

of the nature of the π-conjugated systems is complicated by the
electronic interactions, and by the strong interconnection and mutual
influence of the electronic and geometrical structures (phonons).
The “electron-lattice interaction” is the strong influence that an extra
electron or a hole or an excitation plays on the local geometry of the
molecule (lattice, in the solid state physics terminology). Another
fundamental point is that, despite the strong coupling between electrons
or holes and the underlying lattice, the bearing charged species, or the
neutral species in the excited states are surprisingly mobile (André et al.,
1991). The geometrical structure of some discussed polymers is reported
in Figure 1.7, where as a convention is reported only the monomeric
repetition unit, or “unit cell”.
The high band gap value in exclusively σ bonded polymer, Eg(σ),
makes this materials electrically insulating and generally not able to
absorb the visible light. In the polyethylene, for instance, with
monomeric repetitive unit defined by -(CH2-CH2)-, the optical band gap
is about 8 eV. In the conjugated polymers exist a continuous network,
often a simple chain, of carbon atoms in the hybridized state sp2 or sp.
This chain of atoms with π-overposition of atomic orbital and with
the periodical conditions imposed by the unit cell, leads to π-delocalized
states along the polymeric backbone. As a result the π-bands forms the
electronic structure border. In a monodimentional systems this π-states
gives a π-band gap, Eg(π) < Eg(σ), allowing to optical absorbance at
lower photonic energies.
The essential properties of systems with delocalized π-electrons are
the following:
The electronic band gap Eg is relatively small (1–4 eV). This
allows electronic excitation at low energies, and semiconducting
behavior.
The polymeric chain can be oxidized or reduced in a relatively
simple way, generally by a charge transfer with the doping
molecules.
The mobility of the carriers is large enough to have high electrical
conductivity in the doped state (oxidized or reduced).
The charge bearing species are not free electron or holes, but
quasi-particles, that can move freely through the materials, or
along the uninterrupted polymeric chains at least.
Due to the prevalently amorphous state of these polymers, the
macroscopic electrical conductivity in the samples needs the
hopping phenomena between the chains.
The geometrical structure is strongly dependent on the ionic states of
the molecule and leads to unusual charge bearing species. These bearing
species are self-localized (Table 1.2), in sense that the presence of an
extra electronic charge leads to local variations in the atomic geometry
(lattice), and at the same time, leading to localized variations in the
electronic structure. The charge bearing species can be generated by
optical absorbing of the neutral system, or by charge transfer doping.
Table 1.2. Self-localized excitations in the conjugated polymers. (Reprinted with the
permission from Nicolini et al., New materials by organic nanotechnology and their
applications, in Recent Research Development in Materials Chemistry 6, pp. 17–40, ©
2005, Transworld Publishing).
State Chemical term Charge Spin

Positive Soliton Cation +e 0
Negative Soliton Anion -e 0
Neutral Soliton Neutral Radical 0 1/2
Positive Polaron Radical Cation +e 1/2
Negative Polaron Radical Anion -e 1/2
Positive Bipolaron Di cation +2e 0
Negative Bipolaron Di anion -2e 0
Exciton singlet S1 0 0
Exciton triplet T1 0 1
Reassuming, the band gap value (Eg) in the conjugated polymers is

determined by the contribution of five terms, as shown in equation 1.1:
Eg = Ebla + Eθ + Eres + Esub + Eint (1.1)
where:
Ebla: bond length alternation - conjugation length.
Eθ: “rotational disorder” – mean deviation from co-planarity.
Eres: aromatic resonance – aromatic ring energy.
Esub: effects of substituents – inductive and mesomeric electronic

effects.
Eint: intermolecular coupling – interchain interactions in solid state.
This equation shows immediately, which are the structural variables
that need to be dominated in the band gap control of the conjugated
systems. As a consequence, the synthesis and assembling strategy
devoted to design conjugated polymer with controlled band gap, must
take into account the possibility of the control of energetic contribution
of one or more parameters of Eq. 1.1 (Nicolini et al., 2005).
The realization of electronic devices is composed by fundamental
steps, such as: synthesis and project of supramolecular devices,
development of architecture and chemical-physical assembling
techniques. This now day represents one of the bigger scientific and
technologic challenges of the new century. The researchers are called to
radically change their own approach to the problems of the chemistry
and physics of solid state. In research field of molecular control of
materials, are being new emphasis the molecular self-assembling
processes. This processes concerns the ability of single atoms or
molecules to organize themselves in a rational way to give controlled
macro and supramolecular structures. This new approach to the
atomic/molecular world can, in first instance, appear non conventional,
but in reality is typical of the biological world. In nature, in fact, every
process follow a bottom up self-assembling mechanism, starting from
single constituent atoms that self-assemble in more complexes structures
with extraordinaire regularity.
1.2.1.1 Amphipilic conjugated polymers
Polythiophenes (PTs) usually do not form stable monolayers at the air-

water interface because they are not sufficiently amphiphilic.
To obtain stable Langmuir films on the air-water interface, it is
essential that the active species exhibit the necessary degree of
amphiphilic character. It is well known that the low hydrophilic nature if
thiophene ring makes very difficult the stabilization of the Langmuir
films of the alkyl-substituted thiophenes on the interface and, as a
consequence, the achievement of good transfer ratios to a solid substrate.
An alternative approach to synthesize an amphiphilic PT with high

structural order was developed by Nicolini et al. (2005).
Figure 1.8. Polymerization of amphiphilic macromolecule starting from monomers with

different affinity with the reaction solvent (chloroform), (Reprinted with the permission
from Nicolini et al., New materials by organic nanotechnology and their applications,
Recent Research Development in Materials Chemistry 6, pp. 17–40, © 2005, Transworld
Publishing).
This method use the amphiphilic characters in the final polymer, and
allows the formations of biological membrane like structure. This is
possible using a simple chemical oxidative polymerization of 3-acetic
acid thiophene and 3-hexylthiophene monomers as reported in the
following scheme (Figure 1.8).
Table 1.3. – Comparison between the values of the peak absorption in the UV-vis spectra
of polymers obtained with different synthesis methods (Reprinted with the permission
from Nicolini et al., New materials by organic nanotechnology and their applications, in
Recent Research Development in Materials Chemistry 6, pp. 17–40, © 2005, Transworld
Publishing).
Chloroform solutions Uv-Vis (λmax)

PAHT (FeCl3 method) (Nicolini method) 442 nm
Polyhexylthiophene-PHAT (FeCl3 method) 436 nm
Polyhexylthiophene-PHAT (McCullough method) 442 nm
Its represents maybe, the simplest, easy and cheap method to obtain
this materials, in comparison with more expensive and sophisticated
methods, as shown in Table 1.3 reporting the values of the wavelength
absorbance maximum related to the band gap value of the polymer
electronic structure.
The high structural order is due to the amphiphilic character of the
polymer, which allows the coexistence of a fully conjugated and π-
stacked polymer structural motif and a membrane, forming motif. In the
Figure 1.9, it is shown how the PHAT have a different morphology

respect the polyalkylthiophenes and how it self assemble forming
ordered homogeneous films on solid surface. The amphiphilic nature of
the polymer allows it to assembly in solution and at the air-water
interface forming a densely packed monolayer with highly ordered
domains.
Using this self-assembling characteristic, it is possible use a bottom
up approach in order to deposit on solid support with different
dimensions and geometry polymeric films with variable thickness from
monolayers to microns.
Figure 1.9. (a) Polyhexylthiophene on hydrophobic substrate; (b) polyhexylthiophene on

hydrophilic substrate; (c) PHAT on hydrophilic substrate. The films are casted from
chloroform solutions and the images are taken by fluorescence microscopy. (Reprinted
with the permission from Nicolini et al., New materials by organic nanotechnology and
their applications, in Recent Research Development in Materials Chemistry 6, pp. 17–40,
© 2005, Transworld Publishing).
A schematic representation, relative to the self-assembling process

with Langmuir-Schaefer techniques using a polymer with amphiphilic
characteristics, is reported on the following Figure 1.10. Fluorescence
microcopy studies proves that at the air-water interface the hydrophilic
side chains go into the water subphase while the alkyl chains stick up in
the air, as depicted in Figure 1.10, forming dense and ordered films on
hydrophobic substrate, while the LS deposition leads to non
homogeneous monolayers on hydrophilic substrates with local domains.
The constant trends of increasing the density and complexity of
semiconductor chip circuitry have frequently stressed the need for
developing new revolutionary organic semiconductor technologies
“bottom-up” (Reed et al., 1997; Collier et al., 1999; Reed, 1999;

Nicolini, 1996a).
Figure 1.10. Images of PHAT monolayers deposited by LS technique on (left)

hydrophobic substrate; (right) is hydrophilic substrate. The images are taken by
fluorescence microscopy. (Reprinted with the permission from Nicolini et al., New
materials by organic nanotechnology and their applications, in Recent Research
Development in Materials Chemistry 6, pp. 17–40, © 2005, Transworld Publishing).
The several notable advances in the field of conducting polymer

nanocomposite materials are indeed used for the photovoltaic and LED
devices (see chapter 4). The defects such as solitons, polarons and
bipolarons are the means to obtain the desired electrical conductivity in
the conducting polymers. It was indeed proposed that non-linear
excitations such as solitons and polarons play an important role in the
transport of electrical charge in conducting polymers. Conducting
polymers contain a variety of other defects such as cross-links, branch-
point and conformational defects. Such defects may arise due to
chemical linking of monomer units yielding an undesired linkage and/or
breakage of chemical regularity. The cross-linking results in the rubbery
nature of a conducting polymer. Some of the problems that have retarded
practical applications of conjugated polymers are related to
environmental stability, and loss of desirable mechanical properties on
doping.
1.2.2 Carbon nanotubes and their nanocomposites
Carbon Nanotubes (NT) are new carbon allotropes (Figure 1.11), sharing
similarities with graphite.
Their structure is a molecular scale wire shaped, called single-walled

nanotubes (SWNT) or in concentric wires, called multi-walled nanotubes
(MWNT), with dimensions of 1–1.5 nm in diameter and about up to
many µm in length.
Single-wall carbon nanotubes (SWNT) are produced using catalytic
metal particles in carbon arc vaporization, catalytic decomposition of
organic vapors, plasma-enhanced chemical vapor deposition, and laser
vaporization techniques (Iijima, 1991; Dillon et al., 1997) SWNTs can
be self-organized into ropes that consist of hundreds of aligned SWNTs
on a two-dimensional triangular lattice, with an intertube spacing of van
der Waals gap of approximately 3.2 Å (Odom et al., 2002; Jost et al.,
2004). NT possess properties exploited in various fields as electronics or
aerospace, and have recently received a great interest related to their use
in biological systems (Baughman et al., 2002, Penn et al., 2003).
Figure 1.11. Structure of carbon nanotube.
The unique electrical and optical properties render nanotubes very

sensitive to chemical or physical modifications of the surrounding
environment. It follows that in vivo implants of bioelectronic sensors or
delivery of molecules to cells could be improved with a targeted delivery
with NT.
In recent years, two classes of organic materials like conducting

polymers and carbon nanotubes have gained enormous interest for their
attractive chemical-physical properties (Iijima, 1991). These
characteristics have led to an interesting application of both the materials
by the embedding of little quantity of carbon nanotubes, either single
walled carbon nanotubes or multi walled carbon nanotubes, inside the
polymer matrix for the synthesis of nanocomposites (Steuerman et al.,
2002). The fabrication of such nanocomposites materials can be obtained
by means of either simple reaction carried out by easy steps of synthesis
in the presence of a dispersion of carbon nanotubes or by assembling
carbon nanotubes and just synthesized conducting polymers (Valentini et
al., 2004a; Bavastrello et al., 2004; Erokhina et al., 2002).
It is natural that the possibility of synthesizing materials by
employing economic methods, can pave the path for the industrial
applications of nanocomposite materials.
Figure 1.12. Schematic of reaction for the synthesis of nanocomposite materials by means
of oxidative polymerisation. (Reprinted with the permission from Nicolini et al., New
materials by organic nanotechnology and their applications, in Recent Research
Among the conducting polymers utilizable for the synthesis of

nanocomposite materials, the polyaniline and its derivatives can be
chosen as good candidate since they have been deeply studied and have
shown good electric properties, easy methods of synthesis and high

environmental stability (Paul et al., 1985; Ram et al., 1999; Genies et al.,
1990; Nicolini et al., 2001a).
The first method consists in the following steps. The monomers are
dissolved into the medium of reaction, constituted by a dispersion of
carbon nanotubes in an aqueous solution of hydrochloric acid. The
dispersion is always obtained by means of ultrasonic equipment. Thus,
by adding the oxidizing agent constituted by ammonium persulfate the
reaction of polymerization is started. The growing polymeric chains
embed the carbon nanotubes and form a matrix that contains them. The
reaction is always continued for 12 hours at a temperature of 0–4°C, and
before obtaining the final nanocomposite, the crude material undergoes
an undoping process and several steps of purification carried out with
different solvents. A schematic representing the reaction of synthesis of
the nanocomposite materials is shown in Figure 1.12.
This method of synthesis implies the formation of non covalent bonds
between the conducting polymer chains and the carbon nanotubes
themselves. Anyway, in spite of the weak bonds obtained from the
synthesis, this method represents a good way to dissolve non-
functionalised carbon nanotubes into organic solvents. A specific
example can be shown by the embedding of carbon nanotubes inside a
polymeric matrix soluble in chloroform. The resulting synthesized
nanocomposite will be then soluble inside the same solvent allowing
carbon nanotubes in solution.
The second method consists in assembling carbon nanotubes
previously deposited on a substrate and a consequent deposition of thin
layer of the conducting polymer (Bavastrello et al., 2004). In this method
of fabrication carbon nanotubes are not embedded in the polymeric
matrix but they are previously deposited on a silica substrate and then a
layer of conducting polymers is spread on them. A good method for the
preparation of a substrate of aligned carbon nanotubes is that known as
Plasma Enhanced Chemical Vapour Deposition. For the deposition of
the conducting polymers upon the mat of carbon nanotubes different
techniques can be employed, according to the final task to be obtained.
This depends on the thickness and the macromolecule order required.
Among them we can use in addition to the Langmuir-Schaefer, spin

coating and solution casting techniques.
The first method of deposition, as shown in a previous paragraph, can
be useful for the fabrication of thin films with a high grade of order, but
the final thickness is maintained in nanometers scale since thicker layers
would require long period of time.
The spin coating technique can be used to obtain thicker layers
sacrificing something in the final order of the molecules constituting the
film itself.
The solution casting technique is surely the less appropriate to obtain
highly ordered films but is appreciate to get good thickness in only few
depositions.
The nanocomposite materials usually show different properties with
respect to the polyaniline derivative used in the synthesis. The
nanocomposite materials based on carbon nanotubes and polyaniline
derivatives can be successfully employed for the fabrication of different
devices. An interesting application is for example the fabrication of
sensors for acidity (Bavastrello et al., 2004).
This is due to the fact that this class of conducting polymers is doped
by means of protic acids and during the doping process it is able to
change its conductivity of several orders of magnitude. This
phenomenon, fundamental for the fabrication of sensor devices for acids,
is deeply affected by the conducting polymers themselves, since the
chemistry of polyanilines is generally more complex with respect to
other CP.
This fact is due to their dependence on both the pH value and the
oxidation states, described by three different forms known as
leucoemeraldine base, the fully reduced form, emeraldine base, 50%
oxidised form, and pernigraniline base, fully oxidised form.
The most important is the emeraldine base form and its protonation
by means of H+ ions generated from protic acids gives the emeraldine
salt form, responsible of the strong increment of conducting properties
(Epstein and MacDiarmid, 1991), and the mechanism of reaction is
represented in Figure 1.13.
This process is reversible and it is possible for the presence of imine
group basic sites located along the conducting polymer backbone. The
remarkable fact that the chemical-physical properties of polyaniline and

its derivatives are pH sensitive has led to the study of these materials as
sensors (Lindino and Bulhoes, 1996).
Figure 1.13. Mechanism of reaction for the doping process of polyaniline by means of
protic acids of the emeraldine base form. (Reprinted with the permission from Nicolini et
al., New materials by organic nanotechnology and their applications, in Recent Research
One of most important properties of nanocomposite materials is

surely individuated in the possibility of enhancing the electrical
properties with respect to the pure conducting polymers.
Recent studies carried out on a nanocomposite material based on
poly(o-methoxyaniline) with multi walled carbon nanotubes,
demonstrated that the insertion of carbon nanotubes embedded in the
polymeric matrix provide better conducting properties with respect to the
parent pure polymer.
For films of 30 monolayers, the nanocomposite material showed a
specific conductivity of 2 S/cm, while for the pure conducting polymer a
specific conductivity of 1.1 × 10-3 S/cm was found (Bavastrello et al.,
2004a). Interestingly the conducting polymer in the doped form can be
maintained for long periods of time till the material reacts with basic
reagents and strongly changes its chemical-physical properties.
In other words, the reversibility of the process is not spontaneous.
Anyway, studies carried out on the nanocomposite material obtained by
embedding multi walled carbon nanotubes in the polymeric matrix,

showed an anomalous behavior.
In fact, this nanocomposite tends to spontaneously release the doping
agent.
Figure 1.14: Representation of the conducting polymer chains wrapped around bundles of
carbon nanotubes (Reprinted with the permission from Nicolini et al., New materials by
organic nanotechnology and their applications, in Recent Research Development in
Materials Chemistry 6, pp. 17–40, © 2005, Transworld Publishing).
It means that the system is maintained in the doped only under a

constant support of doping agent (Bavastrello et al., 2004b). The possible
explanation of this singular phenomenon is related to the chemical
structure of the conducting polymer, where the aromatic rings
constituting the polymeric chains bear two methyl groups. In Figure 1.14
is shown the representation of the conducting polymer chains wrapped
around bundles of carbon nanotubes.
The methyl groups are then responsible of an increased sterical
hindrance that impedes the conformational rearrangement that takes
place during the doping process. It is also possible that the presence of
carbon nanotubes inserted in the polymeric matrix accentuates this
phenomenon (Bavastrello et al., 2004).
Indeed the electrical behavior of the doped nanocomposite material
along the time is such that the conductivity of the material, deposited as a
thin film increased of about two orders of magnitude in 30 hours
(Bavastrello et al., 2004b).
1.2.2.1 Interactions between conjugated polymers and single-WN
The remarkable physical and chemical properties of single-walled carbon

nanotubes (SWNTs) have made these materials attractive candidates for
a host of structural and electronic tasks. However, a number of
challenges must still be met before nanotubes can be exploited for most
of these envisioned applications.
Those challenges include separating the tubes by conductivity types,
scaling synthetic approaches to large-scale production, and developing
chemical techniques for manipulating nanotubes as rational molecular
materials. Progress on all, of these fronts has been proceeding rapidly
over the past few years as shown by Gimzewski and co-workers
(Schlittler et al., 2001). In cooperation with California Nanosystem
Institute (Steuerman et al., 2002) we have recently carried out the
chemical interactions between single walled carbon nanotubes (SWNTs)
and two structurally similar polymers, poly{(m-phenylenevinylene)-co-
[(2,5-dioctyloxy-p-phenylene)vinylene]}, or PmPV, and poly{(2,6-
pyridinylenevinylene)-co-[(2,5-dioctyloxy-p-phenylene)vinylene]}, or
PPyPV, are investigated. The fundamental difference between these two
polymers is that PPyPV is a base and is readily protonated via the
addition of HCl. Both polymers promote chloroform solubilization of
SWNTs. We find that the SWNT/PPyPV interaction lowers the pKa of
PPyPV. Optoelectronic devices, fabricated from single polymer-wrapped
SWNT structures, reveal a photogating effect on charge transport, which
can rectify or amplify current flow through the tubes. For PmPV
wrapped tubes, the wavelength dependence of this effect correlates to the
absorption spectrum of PmPV. For PPyPV, the wavelength dependence
correlates with the absorption spectrum of protonated PPyPV, indicating
that SWNTs assist in charge stabilization.
To further explore the interaction of conjugated polymers
organization on SWNTs surface we have recently (Narizzano and
Nicolini 2005) studied the interactions between poly{(2,6-
pyridinylenevinylene)-co-[(2,5-dioctyloxy-p-phenylene)vinylene]}, or
PPyPV, and SWNTs by UV-Vis absorption spectroscopy, suggesting a
semi-quantitative mechanism with ropes formation of SW carbon
nanotubes (Figure 1.15). The SWNTs appear to promote the polymer
organization. The PPyPV is a Lewis base and can be doped by strong and
weak Lewis acids. The basicity strength of the PPyPV depends on the
polymer interchain interactions enhanced by the SWNTs presence, as the
SWNT concentration is increased, and a Kb increment of PPyPV is
observed.
Figure 1.15. Semi-quantitative mechanism of the interaction process and ropes formation
of SWNTs by a conjugate polymer (PPyPV), taking into account all the species in
solution is proposed. Particular attention is paid to study the interactions as a function of
nanotube and polymer concentrations ((Reprinted with the permission from Narizzano
and Nicolini, Mechanism of conjugated polymer organization on SWNT,
Macromolecular Rapid Communications 26, pp. 381–385 © 2005, Wiley-VCH Verlag
GmbH & Vo. KGaA).
1.2.2.2 SWNT for hydrogen storage
Finally SWNTs appear to have many potential advantages for hydrogen

storage (De Heer et al., 1995) over currently available adsorbents. They
have large theoretical surface areas that are on the order of those for
high-surface-area activated carbons, but many open problems still
remain. The key to this potential lies in the nanotube's unique structure,
which in turn depends on the unique properties of its building material
and the defects that can form in the network of carbon bonds.
Crystallized arrays of SWNTs have a very narrow pore-size
distribution that has virtually all their surface area in the micropore
region. In contrast, surface area in activated carbons is broadly
distributed between macropores, mesopores, and micropores. The pore
sizes in an array of tubes could be controlled by tuning the diameter of
the SWNTs making up the array. Theoretical calculations by Ye et al.

(1999) predicted that carbon nanotubes have very strong capillary forces
for encapsulating both polar and nonpolar fluids. Dillon and coworkers
(1997) used hydrogen adsorption on carbon soots containing small
amounts of SWNTs with high hydrogen uptake.
With SWNT materials, hydrogen apparently adsorbs and desorbs over
a narrower range of pressure, as shown by Ye et al. (1999), so storage
systems could be designed to operate without wide pressure excursions.
Similarly Darkrim and Levesque (1998) computed hydrogen adsorption
by grand canonical Monte Carlo simulations and stressed that the results
depended on the choice of intermolecular potentials between the
hydrogen molecules and the carbon atoms. These studies prove the
potential usefulness of carbon nanotube technology in this field and must
be continued to search for new energy solutions compatible with the
environment, a big challenge in front to the entire humanity in the near
future.
1.2.3 Nanoparticles
Nanometer size semiconductor particles of cadmium sulphide inside

Langmuir-Blodgett (LB) films of cadmium arachidate can be obtained by
exposing films to an atmosphere of hydrogen sulphide according to the
following reaction:
(CH3 (CH 2 )18 COO)2 Cd + H 2S → 2(CH3 (CH 2 )18 COOH ) + CdS
where protons from hydrogen sulphide protonate the head groups of
the arachidic acid, while sulphur binds to cadmium. Works on mercury,
lead and cadmium sulphide (Zylberajch et al., 1989, Erokhin et al.,
1995a, 1998; Facci et al., 1994; Nicolini, 1996c) point out this approach
as a general one for the formation of particles of sulphur salts of bivalent
metals. Optical and electron diffraction investigations on the particles
formed by this technique allowed to estimate that their sizes range
between 5 nm and 20 nm.
Assembly of nanoparticles in organic conjugated heterostructures or
superlattices is of great importance in current material science (Figure
1.16), as well as in molecular electronics (Gao et al., 1997).
Figure 1.16. Atomic force microscopy of gold nanoparticles (Larosa et al., in

preparation).
Controlled thickness and order of these ultrathin structures by LB

techniques play a crucial role in obtaining high quality molecular films
(Nicolini, 1996b). CdS nanoparticles were formed (Smotkin et al., 1988)
by exposing cadmium arachidate to an atmosphere of H2S gas, whereby
carboxylic groups of arachidic acid were protonated, and nanometer size
CdS was produced (Figure 1.17a).
H2S gas was prepared by the reaction of FeS with diluted H2SO4 and
samples are exposed into the chamber to the H2S atmosphere for 12 h,
that is, a time interval to complete the reaction.
Figure 1.17. a). Images taken by fluorescence microscopy of 20 layers films of

copolymer. (a) before exposure to H2S; (b) after exposure to H2S. (Reprinted with the
permission from Narizzano et al., A heterostructure composed of conjugated polymer and
copper sulfide nanoparticles, The Journal of Physical Chemistry B 109, pp. 15798–
15802, © 2005, American Chemical Society).
Distribution of nanoparticles observed by CCD camera, shown in

Figure 1.17a, reveals their random growth resulting in the formation of
isolated islands. The technique allowed us to obtain very good resolution
of inorganic layer formation, 0.6 nm for each precursor bilayer.
The methodology was employed to prepare CdA (Facci et al., 1995),
PbS, CuS, HgS, etc. layers (Erokhin et al., 2002). It was found that the
technique does not yield continuous semiconductor monolayers and for
films less than 25 nm thick low conductivity is observed (Figure 1.17b).
The use of a conjugated matrix results in conductive connection of the
semiconductor islands and, therefore, in the formation of a continuous
conductive layer at film thickness less than 25 nm (Figure 1.17b). A
conjugated polymer with amphiphilic properties that allowed the
formation of a stable monolayer at the air-water interface was indeed
needed in order to achieve structures with controlled thickness, by means
of the LS technique (Narizzano et al., 2005). This was obtained by
chemical copolymerization of two different monomers: the 3-
thiopheneacetic acid (3TAA) and 3-hexylthiophene (3HT). The former
allows for reaction with copper ions, and consequently the formation of
nanoparticles, and it has a hydrophilic character. The latter has a
hydrophobic character and makes the final polymer soluble in common
organic solvents. CuS nanoparticles were grown directly in the polymeric
matrix using the carboxylic groups as nucleation centers. The reactions
were monitored by quartz crystal microbalance (Figure 2.16 in chapter
2), Brewster angle (see Figure 2.11 in chapter 2), and fluorescence
microscopy (Figure 1.17a). This new conjugated amphiphilic polymer
acts as a growth matrix for semiconducting CuS nanoparticles in which
they form islands/domains three-dimensional randomly distributed.
Interestingly (Narizzano et al., 2005) the electrical properties and the
conductivity of the heterostructure strongly depend on the formation of
nanoparticle islands in the conjugated polymer (Figure 1.17b). In thicker
film large nanocrystal domains grow at the expense of smaller ones,
reflecting a three-dimensional diffusion mechanism of individual
particles from smaller to larger domains. This is driven by a decrease in
the surface free energy as larger domains grow, since only few
nanoparticles remain in energetically unfavorable sites.
In summary it is conservative to conclude that the nanoscale

methodology via chemical synthesis represents a promising general-
purpose tool for the design of new materials, products and processes for
a wide variety of applications.
1.3 Produced Via LB Nanostructuring
The design, the engineering and the properties of nanostructured protein

biofilms represent the nanomaterials at the higher level of
supermolecular organization of both polymers and biopolymers. Each
nanostructured biofilm exhibits numerous and interesting properties in
vivo and in vitro, which deserve attention.
A large number of potential applications for organized protein
monolayers had motivated considerable research activity in this field
(Nicolini, 1997; Boussaad et al., 1998; Kiselyova et al., 1999), as shown
also by numerous reviews (Nicolini et al., 1995b, 2001a; Nicolini
1996a,b, 1998b; Vianello et al., 2004; Hampp, 2000; Hampp and
Brauchle, 2003).
Construction of specific interaction-directed, self-assembled protein
films has been performed at the air-water interface. The Langmuir-
Blodgett (LB) technique has been extensively used to order and
immobilize natural proteins on solid surfaces (Nicolini, 1997; Tronin et
al., 1994, 1995; Facci et al., 1993).
A surface pressure ranging between 15 and 25 mN/m was used to
transfer the monolayers of the various proteins being immobilized from
air-water interface to solid supports, and the subphase is buffered and
contains high ionic strength.
The layer-by-layer (LBL) assembly processes based on electrostatic
or other molecular forces represent instead a unique technique that
presents an alternative approach to the formation of nanostructured
architectures by adsorption of consecutively alternating polyelectrolytes.
An application of the LBL technique is the fabrication of homogenous
ultrathin film of conjugated polymers.
Molecular-level processing of conjugated polymers (i.e., polypyrrole,
polyaniline, poly(phenylene vinylene), poly(o-anisidine) by the LBL
technique (Ferreira and Rubner, 1995; Ram et al., 1999a; Decher 1996,
1997; Decher et al., 1992) was indeed shown in the literature (Ram and
Nicolini, 2000) (Figure 1.18).
Figure 1.18. Layer-by-Layer deposition. (a) Schematic of in situ self-assembled layer-by-

layer films of PPY with PSS. (b) Schematic of in-situ self-assembly of PPY on PSS
surface as function of time (Reprinted with the permission from Ram and Nicolini, Thin
conducting polymeric films and molecular electronics, in Recent Research Development
in Physical Chemistry 4, pp. 219–258, © 2000, Transworld Publishing).
The self-assembly of charged polyelectrolytes (i.e., proteins, nucleic

acids, conducting polymers, zirconium phosphate, optical dyes, metal
nanoparticles, aluminosilcates, and clay) by LBL can be considered an
alternative approach to spin-coating and chemical vapor deposition
techniques (Nicolini et al., 2005).
None however appears for proteins as efficient as the Langmuir-
Blodgett technique namely if utilizing our “protective plate”modification
(Troitsky et al., 1996a).
1.3.1 Langmuir-Blodgett
The fact that oil forms thin layers over the water surface is known from
the ancient time. First statement that such films must be monomolecular
in thickness was done by Lord Rayleigh (1879). Nevertheless systematic
scientific study of such objects began from the works of Irving Langmuir
and can be divided into several phases. During the first phase main
attention was paid to the behavior of monolayers of amphiphilic
molecules at the air/water interface.
The phase is connected with early works of Langmuir in 1920. As a
result, monolayer formation process was characterized and several phase
transitions were determined in two-dimensional system at the air/water
interface. During the second phase it was shown that the layers could be
transferred onto surfaces of solid substrates. The works were performed
by Langmuir in collaboration with Blodgett, whose names began to be
used to term the method itself (Blodgett, 1934, 1935; Blodgett and
Langmuir, 1937). Their technique consisted in deposition of monolayers
when the substrate moved vertically through the monolayer. A little bit
later another deposition technique was suggested by Langmuir and
Schaefer, where the substrate touched the monolayer horizontally
(Langmuir and Schaefer, 1939). The method now is called in a literature
as “horizontal lift” or Langmuir-Schaefer technique. It is interesting to
note that the method was developed for deposition of protein layers (first
attempt to work with protein layers was done in 1938 by Langmuir and
Schaefer on pepsin and urease (Langmuir and Schaefer, 1938). After the
initial interest in the subject at the beginning of the century resulting in a
Nobel prize received by Langmuir in 1932, the activities in the field were
not numerous involving only some academic interest for such two-
dimensional systems. Third phase in the LB films investigations began
with the works done in the group of Kuhn (Kuhn 1965; Drexhage and
Kuhn, 1966). The works have demonstrated that it was possible to form
complex structures with desired mutual orientation of functional groups
of molecules by the method (Bücher et al., 1967; Inacker et al., 1976).
The works on energy transfer in the films (Kuhn, 1981) created big
resonance in the scientific world attracting a huge number of researchers
to be involved in investigation of films. It is possible to consider that the
fourth stage of the LB investigations began when the first international

LB conference was organized (Nicolini, 1996a). It demonstrated that
scientific forces of differing background began to be included into LB
films investigations. During this stage the films became to be
characterized by practically all experimental techniques available
nowadays (Nicolini, 1996; Nicolini and Pechkova, 2006a). Application
aspect of the films began also be taken into account (Nicolini et al.,
2001a; Nicolini and Pechkova, 2006a), with their development in an
alternative technology for manifacturing.
Figure 1.19. Schematic of the protein monolayer formation by the LB Trough (A-B), at
the optimal surface pressure automatically controlled and monitored (C), and its manual
horizontal transfer to a siliconized glass circle cover slide accordingly to the Langmuir
Schaefer deposition (D-E). (Reprinted with the permission from Nicolini and Pechkova,
Nanostructured biofilms and biocrystals, Journal of Nanoscience and Nanotechnology 6,
pp. 2209–2236, © 2006, American Scientific Publishers, http://www.aspbs.com).
The floating monolayer can be transferred onto the surface of solid

supports (Figure 1.19). Two main techniques are usually considered for
the monolayer deposition, namely, Langmuir-Blodgett (or vertical lift)
and Langmuir-Schaefer (or horizontal lift). In LB technique (Sánchez-
González et al., 2003), a specially prepared substrate is passed vertically
through the monolayer. The monolayer is transferred onto the substrate
surface during this passage. In those cases where it is important to have

the monolayer electrically neutral, the deposition will not be performed
when some charges in the monolayer molecule head groups are
uncompensated and the electrostatic interaction of this charge with water
molecules will be higher than the hydrophobic interactions of chains with
the hydrophobized substrate surface. The Langmuir-Schaefer method of
monolayer transfer from the air/water interface onto solid substrates is
most frequently utilized for protein immobilization (Owaku et al., 1989)
and it is illustrated in Figure 2.2. It was developed in 1938 by Langmuir
and Schaefer for deposition of protein layers. Prepared substrate
horizontally touches the monolayer, and the layer transfers itself onto the
substrate surface.
1.3.1.1 Protective plate
A modification of the original LB/LS technique, called “protective plate”

method (Troitsky et al., 1996a) and shown in Figure 1.20, appeared
however mandatory to enhance the physical and functional properties of
all class of proteins, mainly of enzymes.
The “protective plate” apparatus, described in Figure 1.20 was used
to produce the multilayered nanostructures. The “protective plate”
method allows us to combine the LB technique with adsorption of
soluble compounds in such a way that during LB assembly formation the
surface of the protein layer never crosses the air-water interface.
This feature eliminates the main reason for protein denaturation.
Utilizing this apparatus, the deposition of LB monolayers and/or
adsorption of dissolved compounds are carried out in different
compartments in the required sequence.
To transfer the sample from one compartment to another, the
deposited film is protected by a thin layer of water, which is held by
capillary forces between the solid support and the protective plate.
In accordance with the required deposition sequence, the sample
moves automatically between the compartments with different
monolayers or dissolved compounds and the protective plate shifts up
and down to either open or close the film surface.
Figure 1.20. Schematic of “protective plate” method deposition. Principle of the proposed
method. Monolayer is deposited (a), substrate is closed by plate (b), system substrate-
plate is pulled out from aqueous subphase (c). (Reprinted with the permission from
Troitsky et al., Deposition of alternating LB monolayers with a new technique, Thin
Solid Films 285, pp. 122–126, © 1996a, Elsevier).
The “protective method” technique (Troitsky et al., 1996a, 2003) has

several advantages not available with LB method:
a) protein monolayer formed at an air-water interface can be
functionally protected by the thin water layer and then transferred
to many different surfaces, including those prepared for electronic
studies; protein multi-layers are obtained by repeating the
monolayer transfer process as many times as desired;
b) the transferred film can be studied in different environments, such

as air or solution (Guryev et al., 1997).
The success of the technique draws on the property of amphiphilic
molecules when spread and compressed at an air-water interface to form
a compact monolayer, with hydrophobic and hydrophilic parts directed to
air and water, respectively. When this technique is applied to non-
amphiphilic water-soluble proteins, difficulties can be encountered which
can be overcame by the “protective plate” method.
Simple water-soluble proteins at the air-water interface would tend
indeed to expose their hydrophobic interior to the air, causing dramatic
changes of their native structure, which may be detrimental to the
function of the proteins or even cause the liganded cofactors to dissociate
from the protein. In-plane order can be greatly enhanced by exploiting
the combination of planar orientation and mobility, which this interface
provides.
1.3.1.2 Heat-proof and long range stability
The physical properties of Langmuir-Blodgett (LB) films of proteins

(Nicolini and Pechkova, 2006a; Tiede 1985; Hwang et al., 1977) and
lipid-protein complexes (Lvov et al., 1991; Fromherz 1971; Phillips et
al., 1975; Heckl et al., 1987; Kozarac et al., 1988) were intensively
studied and characterized by different techniques.
Numerous observations on the thermal stability of proteins organized
in dense solid films, deposited by LB (Nicolini et al., 1993; Facci et al.,
1994; Erokhin 1995a) or by self-assembling (Shen et al., 1993), point to
the role of decreased water content and molecular close packing
(Nicolini et al., 1993).
Bacteriorhodopsin (bR) and photosynthetic reaction centers from
Rhodopseudomonas Viridis (RC) were studied in solution, LB film and
self-assembled film. RC was extracted from the membranes by detergent
(lauryldimethylamineoxide-LDAO); the solution contains the individual
protein molecules surrounded by a detergent “belt” shielding the
hydrophobic areas of the protein surface.
The initial solution of bR was instead the solution of sonicated
membrane fragments. Described differences in the initial protein solution
conditions, of course, differentiates the processes of film formation, both

in the case of the LB technique and in the case of self-assembling.
Circular dichroism spectra of different samples of RC and bR after
heating at different temperatures are presented in Figure 1.21 (Erokhin,
et al., 1996a).
Comparison of the CD spectra of RC and BR in solution allows one
to conclude that bR in solution is much more heat resistant with respect
to RC. The other interesting point is that the temperature behaviors of the
bR in LB and self-assembled films are absolutely identical.
Both of them demonstrate high thermal stability, and significant
differences in the CD spectra appear only after heating up to 200 °C. The
situation is absolutely different in the case of RC.
In fact, LB films show high thermal stability, and significant
differences appear, as in case of bR, only after heating to 200 °C. Self-
assembled films of RC, in contrast, are much more affected by thermal
treatment.
Such differences in the secondary structure behavior with respect to
temperature can be explained by suggesting that molecular close packing
of proteins in the film is the main parameter responsible for the thermal
stability.
In fact, as in the case of bR, we have close packing of molecules even
in the solution (membrane fragments); there are practically no
differences in the CD spectra of bR solution at least till 75 °C
(denaturation takes place only for the sample heated to 90 °C).
RC in solution begins to be affected even at 50 °C and is completely
denatured at 75 °C, for the solution contains separated molecules.
Figure 1.21. CD spectrum of RC (A) and bR (B) in solution, LB and spread films.
(Reprinted with the permission from Erokhin et al., On the role of molecular close
packing on the protein thermal stability, Thin Solid Films 285, pp. 805–808, © 1996a,
Elsevier).
Langmuir-Blodgett monolayers of photosynthetic reaction centers

from Rhodobacter sphaeroides have been studied by scanning tunnelling
microscopy (Facci et al., 1994a) (Figure 1.22).
Figure 1.22. Heat and cooling of Photosynthetic Reaction Center films. STM image of an
RC monolaycr after heating at 150 °C for 10 min. (a) Image size 57.6 x 57.6 nm2 (b)
zoomed area (21.3 x 21.3 nm2 from the outlined area. Images in the light and in the dark
were identical. (Reprinted with the permission from Facci et al., Scanning tunnelling
microscopy of a monolayer of reaction centers, Thin Solid Films 243, pp. 403–406, ©
1994a, Elsevier).
Freshly deposited films were studied both in the dark and in the light.
In the dark, images revealed molecular structure with 64Å and 30 Å
periodicities, which correspond to protein and sub-unit sizes known from
X-ray crystallography, while no periodic structure appeared in the light
due to the tip action on the excited proteins.
STM voltage-current measurements showed the charge separation in
single protein molecules in the film and their different behavior in the
dark and light. Together with surface potential measurements at the
macroscopic level, they indicated the preservation of reaction center
activity in the monolayer. By fixing the protein layer with
glutaraldehyde, it was possible to prevent the perturbing tip action and
obtain a periodic molecular structure with 30 Å spacing even in the light.
After heating at 150° C, the unfixed film reorganized itself into a long-
range ordered state with a hexagonal structure of 27 Å spacing but with
no activity. It is important to notice that the heat-proof in LS protein film
is systematically associated with long range stability at room temperature
(Paddeu et al., 1996; Facci et al., 1998) (Figure 1.23).
Figure 1.23. Long range stability of protein films. Enzymatic activity of fresh and of long
term stored (one year) GST LB monolayer immobilised on silanized silicon surface. Each
point represents the average value with the confidential interval. (Reprinted with the
permission from Paddeu et al., Kinetics study of glutathione s-transferase Langmuir-
Blodgett films, Thin Solid Films 284-285, pp. 854–858, © 1996, Elsevier).
In summary, LB organization of protein molecules in film not only

preserved the structure and functionality of the molecules, but also
resulted in the appearance of new useful properties, such as enhanced
thermal stability coupled to long-range stability (Nicolini et al., 1993;
Erokhin et al., 1995).
These are the properties that open the areas of application of protein
films to many fields of potential industrial implications, namely
nanobiocatalysis, nanosensors, nanoactuators and nanoelectronics
(chapter 4).
1.4 Produced Via APA Nanostructuring
The process of ordered porous alumina fabrication and microstructuring

(Nicolini and Pechkova, 2006a; Grasso et al., 2006) is shown
schematically in Figure 1.24.
Figure 1.24. APA method. (Reprinted with the permission from Grasso et al.,
Nanostructuring of a porous alumina matrix for a biomolecular microarray,
Nanotechnology 17, pp. 795–798, © 2006, IOP Publishing Limited).
Briefly, ordered nanopore arrays are prepared by using a

photolitographic technique that microstructures the nanopore arrays. A
negative resist (SU-8, Micro-Chem) was used, that is a high contrast,
epoxy based photoresist designed for micromachining and other
microelectronic applications. SU-8 shows very high optical transparency
above 360 nm, which makes it ideally suited for imaging near vertical
sidewalls in very thick films. SU-8 is best suited for permanent
applications since; when imaged, cured and left in place it gives
hydrophobic properties to final nanostructured surfaces. An aluminium
sheet (250 µm thick) was cleaned to obtain maximum process reliability
followed by isopropyl alcohol cleaning and deionized water rinse. After
the resist has been applied to the substrate, it was soft baked in order to
evaporate the solvent and thicken the film. The resist used is optimized
for near UV (350–400 nm) exposure and is virtually transparent and
insensitive above 400nm. Hexagonally ordered pore domains were
prepared by a self-organization process under specific anodization

conditions. This nano-patterning technique leads to a sharp edge. The
anisotropy of the process can be seen with FIB system. The sidewalls of
the structures are very steep, and their roughness is determined by the
quality of the mask (Figure 1.25). This second resist having hydrophobic
properties increases specificity to biological sample linking.
Figure 1.25. FIB system images of cross-sectional morphologies of the microarray spot,
resulting at the end of photolithographic microstructuring technique and 2 step
anodization process. FIB - FEI - measurement were done with gallium ions - 37 pA -
both for cutting and imaging (Reprinted with the permission from Grasso et al.,
Nanostructuring of a porous alumina matrix for a biomolecular microarray,
Nanotechnology 17, pp. 795–798, © 2006, IOP Publishing Limited).
1.4.1 Focus ion beam
APA is a partially transmissive substrate, thus a luminous signal emitted

into APA can be seen either at the top (direct vision) or at the bottom
side (through the bottom layer). These advantages can be exploited in
order to differentiate the instruments for sample analysis (laser scanner,
fluorescence microscopy, DNA Array). APA can be obtained by
removing the remaining aluminum substrate in a saturated HgCl2
solution.
In alternative to a substrate in aluminum, a glass surface can be used,
being possible to evaporate an aluminum layer with desired thickness
directly on glass. The DNA solution is placed onto the hydrophilic spots
of alumina of the arrays, while surface covered by the resist that
surrounds the alumina spot shows hydrophobic properties. The DNA
binding can be improved by means of a surface functionalization with

Poly-L-Lysine.
The Poly-L-Lysine linkage exploits the phosphate groups onto
alumina (Figure 1.26) and the final biomolecular nanostructured APA
gave excellent results (Figure 1.27) with far reaching potential
application in microarray technology for both DNA and protein.
Figure 1.26. Gene-APA linkage via Poly-L-Lysine (Reprinted with the permission from
Nicolini and Pechkova, Nanostructured biofilms and biocrystals, Journal of Nanoscience
and Nanotechnology 6, pp. 2209–2236, © 2006, American Scientific Publishers,
http://www.aspbs.com).
This photolithographic microstructuring technique for the ordered

nanopore arrays fabrication is reported consists of a negative resist with
hydrophobic properties increasing specificity to biomolecules linking.
Figure 1.27. Nanostructured APA for gene microarrays probed by fluorescent labeling.
(fluorescence microscope image, magnification 2X, filter set n° 15. (Reprinted with the
permission from Grasso et al., Nanostructuring of a porous alumina matrix for a
biomolecular microarray, Nanotechnology 17, pp. 795–798, © 2006, IOP Publishing
Limited).
Nanoporous alumina is formed by anodic process and yields straight

holes with high aspect ratio: its use as substrates for DNA-microarray or
protein-chip application offers several advantages over conventional
supports, making them very attractive to use as supports for biological
sample microarrays application. Oligonucleotide and antibody
microarrays are currently intensively investigated for a broad range of
applications in biomedical diagnostics, to simultaneously determine
several parameters in individual samples from a limited amount of
material (Templin et al., 2002). On the other hand, the fabrication of
nanochannel-array materials has attracted considerable scientific and
commercial attention. Moreover, the application of ordered nanochannel
arrays as two-dimensional photonic crystals has generated increasing
interest in recent years.
Among the applications for these materials are the inhibition or
enhancement of spontaneous emission and the fabrication of tunable
optical filters and microcavities (Foresi et al., 1997), waveguides
(Yablonovitch 1987) and catalytic combustion (Suzuki et al., 2003).
Figure 1.28. Schematic view illustrating the direct electron transfer between the
cytochrome P450scc catalytic “core” and the APA modified working electrode. In the
box is shown the specific interaction between the cytochrome P450sccnegative surface
(blue) and the positive charges of Poly-L-Lysine. (Reprinted with the permission from
Stura et al., Anodic porous alumina as mechanical stability enhancer for Ldl-cholesterol
sensitive electrode, Biosensors and Bioelectronics 23, pp. 655–660, © 2007, Elsevier).
Anodic porous alumina, which has been studied extensively over the
last five decades (See et al., 1980; Thompson and Wood, 1983) has
recently been reported to be a typical self-ordered nanochannel material

(Masuda and Fukuda, 1995; Masuda et al., 1997). Self-organization
during pore growth, leading to a densely packed hexagonal pore
structure, has been reported in oxalic, sulfuric, and phosphoric acid
solution (Jessensky et al., 1998).
The interpore distances of the regularly ordered pore arrangement
have been extended to the large range of 50÷420 nm (Li et al., 1998).
Masuda and Satoh (1996) first reported a two-step fabrication method for
straight nanoholes in a thin membrane of alumina. The structural
characteristics of the ordered porous alumina make it not only a perfect
template material for the fabrication of nanoscale structures, but also an
outstanding candidate material for two-dimensional (2D) photonic
crystals, which may show photonic bandgaps that are adjustable in the
visible to ultra-violet spectral region.
This material offers several advantages over conventional supports:
high surface area enlargement, improved microfluidic properties, easy
and cheap manufacturability, flexibility in porous dimension and
confinement effect as a mean for selectivity and maximization of light
emission.
Figure 1.29. Topographic AFM image of rhodium-graphite s.p.e working electrode

before, after the APA deposition and after functionalization. (a) Top view of rhodium-
graphite s.p.e working electrode before the increase of its surface by APA membrane; (b)
Top view of the same rhodium-graphite s.p.e after the APA deposition on its working
electrode surface. Scale; (c) Top view of APA nanopores after the working electrode
surface functionalization (physical adsorption) with PLL and cytochrome P450scc. Scale
bars correspond to 1 µm. (Reprinted with the permission from Stura et al., Anodic porous
alumina as mechanical stability enhancer for Ldl-cholesterol sensitive electrode,
Biosensors and Bioelectronics 23, pp. 655–660, © 2007, Elsevier).
For protein array applications APA was also used (Stura et al., 2007)
as Mechanical Stability Enhancer of Cytochromes Electrodes to improve
the performances of the electrodes based on P450scc for LDL-
cholesterol detection and measure, APA (Anodic Porous Alumina) was
used. To optimize the adhesion of P450scc to APA, a layer of poly-L-
lysine, a poly-cathion, was successfully implemented as intermediate
organic structure (Figure 1.28).
This inorganic APA matrix has been used to functionalise the
rhodium-graphite working electrode (Figure 1.28), and the corresponding
pores can be tuned in diameter modifying the synthesis parameters in
order to obtain cavities 275 nm wide and 160 µm deep (as demonstrated
with AFM and SEM measurement (Stura et al., 2007).
This allows the immobilization of P450scc macromolecules (Figure
1.29) preserving their electronic sensitivity to its native substrate, i.e. the
cholesterol. Even if the sensitivity of the APA+P450scc system was
slightly reduced with respect to the pure P450scc system, the readout was
stable for a much longer period of time, and the measures remained
reproducible inside a proper confidentiality band, as demonstrated with
several cyclic voltammetry measures (Stura et al., 2007).
Chapter 2
Nanoscale Probes
Over the last few decades numerous probes have been emerging at the
nanoscale level which are being used in developing new basic scientific
knowledge (see chapter 3), novel nanostructured materials (see chapter
1) and revolutionary applications to health (see chapter 3) and industry
(see chapter 4). Several biophysical probes employed to characterize
films and crystals, as well as single polymer or biopolymer earlier
introduced, are here summarized stressing their unique contributions and
features ranging from surface potential and AFM (Atomic Force
Microscopy) to µGISAXS (micro Grazing Incidence Small Angle X-ray
Scattering).
2.1 Surface Potential
As already mentioned in this book (see paragraph 1.3), LB technique

allows to operate with biological objects, such as proteins, and to
organize them in regular layers, which can be probed with surface
potential technology. It is worth mentioning that LB films are of
fundamental interest as they represent 2D systems, which can give origin
to new types of phenomena not found in 3D objects.
As mentioned before, classic materials for the LB method are
amphiphilic molecules, one side of which is a polar head-group and the
other, a long hydrocarbon chain (Hann, 1990). In order to spread the
molecules, they must be dissolved in a “strong” solvent at concentration
that does not permit the formation of aggregates. Drops of the solution
are placed on the water surface. The amount of the molecules in the drop
must be rather small in order to have after spreading a monolayer where
54
Nanoscale Probes 55
molecules are far from each other and do not interact. When such
molecule is placed on the water surface, its polar head group interacts
with water, while the hydrocarbon chain faces towards air, as it cannot be
surrounded by water for entropy reasons. Floating molecules can be
compressed by the barrier until condensed state is achieved and
monitored with surface potential measurements (Figure 2.1). Usually the
parameter under control during the compression is the surface pressure,
which characterizes the decrease in the surface tension of the water
surface due to the presence of the monolayer:
π = σ H O − σ ml
2
where σH2O is the surface tension of water without monolayer and σml
is the surface tension of the water surface covered by monolayer. One of
important characteristics of the monolayers on the water surface is the
dependence of surface pressure on the area occupied by single molecule
in the monolayer. The dependence is usually called “compression
isotherm” or “π-A isotherm” (the curve is measured at fixed
temperature). This characteristic (Figure 2.1) is rather important since it
allows to calculate the area per molecule in the monolayer and to reveal
phase transitions in the monolayer structure.
The Langmuir-Blodgett (LB) technique was in recent time extended
to quite more interesting biopolymers as proteins (Nicolini et al., 1993;
Nicolini 1997; Sanchez-González et al., 2003; Owaku et al., 1989).
These molecules, being placed at the air/water interface, arrange
themselves in such a way that their hydrophilic part penetrates water due
to its electrostatic interactions with water molecules, which can be
considered electric dipoles. The hydrophobic part (aliphatic chain)
orients itself to air, because it cannot penetrate water for entropy reasons.
Therefore, a few molecules placed at the water surface form a two-
dimensional system at the air/water interface. Figure 2.1 shows the
dependence of surface pressure upon area per molecule, obtained at
constant temperature (Nicolini, 1997; Paternolli et al., 2004; Nicolini and
Pechkova, 2006a).
The compression of the interface yields a surface potential (Figure
2.2) for the cytochromes at the air-water interface (Nicolini et al., 2001).
Initially, the compression does not result in significant variations in
surface pressure. Molecules at the air/water interface are rather far from
each other and do not interact.
Figure 2.1 Typical π-A isotherm of cytochrome P450 monolayer obtained by the shown
spreading and Wilhelmy plate on 10 mM K-phosphate, pH 7.4, buffer subphase.
This state is referred to as a two-dimensional gas. Further

compression results in an increase in surface pressure. Molecules begin
to interact. This state of the monolayer is referred as two-dimensional
liquid, which can be separated in liquid-expanded and liquid-condensed
phases. Continuation of the compression results in the appearance of a
two-dimensional solid-state phase, characterized by a sharp increase in
surface pressure, even with small decreases in area per molecule. Dense
packing of molecules in the monolayer is reached. Further compression
results in the collapse of the monolayer.
Surface potential measurements are typically made on the solid
substrate using a home-made device (Erokhin et al., 1995). This
technique, which has been widely used for investigations of monolayers
both at air/water interfaces (Tredgold and Smith, 1983) and, more
recently, on solid substrates (Facci et al., 1994a, Erokhin et al., 1995),
can measure the surface potential (with a sensitivity of 1 mV) that arises
Nanoscale Probes 57
from dipole and charge distributions in one or more deposited

monolayers (Figure 2.2); the resulting current is monitored in a circuit,
one end of which is equipped with a vibrating electrode placed near
(around 1 mm) the monolayer surface and which oscillates at its
resonance frequency (282 Hz). Early reference (Erokhin et al., 1995)
illustrates the experimental setup and the measuring principle as
implemented originally on the LB oriented RC layer. In the dark, LB
provides a specific surface potential due to the preferential orientation of
its molecules, which have a static dipole moment. On exposing to light,
RC molecules begin to show an additional dipole behavior due to the
charge separation inside them (electrons are displaced by about 3 nm)
(Facci et al., 1994a).
Figure 2.2. Typical surface potential versus barrier position of a monolayer of

cytochrome P450 at the air-water interface. The subphase was a 10 mM K-phosphate, pH
7.4 (Reprinted with the permission from Paternolli et al., Recombinant cytochrome P450
immobilization for biosensor applications, Langmuir 20, pp. 11706–11712 © 2004,
American Chemical Society).
In that early paper by measuring the difference between the surface

potential values of the RC layer in the dark and in the light, direct
information is provided about the activity of the proteins in an LB film.
Such an approach, moreover, seems to be very useful-especially for
multiple investigations on the same monolayer as it couples the protein
monolayer with a solid state electrode in a "soft" fashion (Facci et al.,
1994a). Non-oriented films do not show any preferential molecular
orientation, and so it is impossible to monitor the protein activity using
surface potential measurement. Only when the given protein, such as any
cytochrome P450 displays a self-assembly significant surface potential
appears evident (Figure 2.2) even when the surface pressure is zero
(Figure 2.1).
2.2 Atomic Force Microscopy
AFM utilizes a sharp probe to scan across the surface of a sample with
the laser being focused on the tip, and the beam being reflected to the
split photodiode detector (Figure 2.3).
Figure 2.3. Atomic force microscopy configuration as described in the text.
As the cantilever is deflected over the sample, the photodiode

monitors the changes of the laser beam. The changes are stored in the
computer to produce a topographic image of the sample surface. There
are three different modes in atomic force microscopy on protein film:
contact mode, tapping mode, and non-contact mode.
In contact mode, the tip physically comes in contact with the sample.
Atomic resolution can be reached in contact mode; however, there is a
risk of damage to samples being soft.
During tapping mode, the cantilever is oscillated at or near its
resonance frequency. The scanner, in a pendulum-type motion, enables
the tip to “tap” the sample surface as the scanner comes to the bottom of
Nanoscale Probes 59
its swing. There is less damage in tapping mode and higher lateral
resolution; however, there is a slightly slower scan speed than in contact
mode.
The cantilever is oscillated at a frequency slightly above the
cantilever's resonance frequency during non-contact AFM. The tip
oscillates above the adsorbed fluid layer on the surface. It does not come
in contact with the sample surface. The sample is not damaged during
non-contact mode; however, the scan speed is much slower, there is
lower lateral resolution, and it may be used only with very hydrophobic
samples. Our original homemade AFM instruments (Sartore et al., 2000)
operated in air or in water, at constant deflection with triangular-shaped,
gold-coated Si3N4 microlevers. Originally the tips of the microlevers had
a standard aspect ratio (about 1:1), and the levers had a nominal force
constant of 0.03 N/m. The constant-force set point was about 0.1 nN,
while the images acquired were 256 × 256 pixel maps. All images are
standard top-view topographic maps, where the brightness is
proportional to the quota of the features over the sample surface i.e., light
means mountain, dark means valley. Figure 2.4 shows an AFM of
lysozyme LB film (Pechkova et al., 2005a).
Figure 2.4. AFM image of LB lysozyme film. Images of the lysozyme template, obtained
in tapping mode in a dry atmosphere (AFM: cantilever I type NSC14/Cr-Au MikroMash)
(Reprinted with the permission from Pechkova et al., µGISAXS and protein
nanotemplate crystallization methods and instrumentation, Journal of Synchrotron
Radiation 12, pp. 713–716, © 2005a, Blackwell Publishing).
A custom AFM instrument further optimised for protein crystal

imaging in solution has been recently introduced (Figure 2.5) and tested
on crystals and Langmuir-Blodgett films of two proteins having quite

different molecular weight (Pechkova et al., 2007b).
Figure 2.5. Piezo movers assembly in the AFM used for the experiments. Piezo tubes
were purchased by Physik Intruments and perform a maximum 10x10 µm travel in XY
and 2.5µm in Z direction (Reprinted with the permission from Pechkova et al., Atomic
force microscopy of protein films and crystals, Review of Scientific Instruments 78, pp.
093704_1–093704-7, © 2007b, American Institute of Physics).
AFM is a topography sensitive method, which in this last case is used

with protein crystals in wet environments (Pechkova et al., 2007b). In the
“noncontact-tapping mode”, the AFM derives topographic information
from measurements of attractive forces. Images of the lysozyme crystal
(Figure 2.6) have been grabbed in tapping mode with a cantilever I type
NSC14/Cr-Au MikroMash in dry atmosphere utilizing our instrument
based on in house SPMagic controller (Pechkova et al., 2007b). The
freeware WSxM© (http://www.nanotec.es) was utilized for the
processing of the acquired images. The typical resonance frequency of
the cantilever tip is between 110 [kHz] and 220 [kHz], and the proper
positioning of the cantilever on the tip holder of AFM has been found at
frequency of 92 [kHz] with intensity 0.6 Volt. The set point for loop
control was at 0.2 Volt. The integral gain value during image grabbing
has been set at 4.4 (I Gain) and the proportional gain value during image
acquisition has been set at 8.03 (P gain). This approach allows to study
the crystal periodicity and morphology in their mother liquid, preserving
Nanoscale Probes 61
the native periodic protein crystal structure, typically destroyed with

drying. Comfortingly it appears to distinguish the protein crystals from
the salt crystals (Figure 2.6a), which under the optical microscope is
frequently quite similar and often their difference is revealed only during
X-ray analysis. AFM estimates of the given single proteins packing,
order and morphology appear quite similar in the LB thin film and in the
crystals, thereby allowing routine crystal measurements at high
resolution. The AFM consists of a custom measuring head with a flexible
SPM controller in house produced which can drive the head for contact,
non-contact and spectroscopy modes, providing the user with an high
degree of customization for the crystal measurement (Figure 2.6b).
Figure 2.6. AFM images and profiles of (a) lysozyme crystal 4 µm × 4 µm and (b) salt
crystal 4 µm × 4 µm (Reprinted with the permission from Pechkova et al., Atomic force
microscopy of protein films and crystals, Review of Scientific Instruments 78, pp.
093704_1–093704-7, © 2007b, American Institute of Physics).
The described AFM system for crystal was tested with standard
samples (Figure 2.7a) in order to prevent any possible problem when
working with more protein samples. We have imaged several portions of
CD-ROMs, both burned and not, because they show regular geometries
at standardized distances. Figure 2.7a shows a typical result of an
unwritten sample. The image was acquired in tapping mode using a

NSC-11 cantilever from MikroMasch. Another test was preformed on
samples with a regular geometry and known dimensions. This test indeed
can be used to calibrate the instrument in all scan directions. We have
used several types of calibration gratings, namely TGX01, TGZ01,
TGZ02 and TGZ03 from MikroMasch.
Figure 2.7. (a) AFM image of the standard sample: a typical result obtained with
unwritten CD-ROM sample in tapping mode using a NSC-11 cantilever from
MikroMasch; (b) AFM image of the standard sample: typical result obtained with TGZ01
grid in tapping mode using a NSC-18 cantilever from MikroMasch. The picture also
shows a line profile (below on the left) taken along a step portion where a dust particle
was imaged (zoomed image on the right) (Reprinted with the permission from Pechkova
et al., Atomic force microscopy of protein films and crystals, Review of Scientific
Instruments 78, pp. 093704_1–093704-7, © 2007b, American Institute of Physics).
The former grid shows a square pattern with 3µm pitch and is mostly
used to calibrate X and Y directions. The latter ones show line step
profiles with horizontal pitch of 3 µm and distinct vertical (Z) steps.
Figure 2.7b shows a typical result obtained when imaging the TGZ01
grid in tapping mode. A NSC-18 cantilever from MikroMasch was used.
The picture also shows a line profile taken along a step portion where a
dust particle was imaged (zoomed in the same figure). The profile clearly
Nanoscale Probes 63
indicates that the vertical dimensions are correctly calibrated, that no

particular convolution or spherical effects are present and that small
details can be reasonably acquired along the vertical axis.In conclusion,
atomic force microscopy (AFM) has been frequently used to study
protein, nucleic acid, and crystals in situ, in their mother liquors and as
they grow.
From the sequential AFM images taken at brief intervals over many
hours, or even days, the mechanisms and kinetics of the growth process
was tentatively defined time ago (McPherson et al., 2000; Wiechmann et
al., 2001). In few case three-dimensional microcrystals of integral
membrane proteins as OmpC porin, air-dried slowly and imaged by
AFM (Kim et al., 2000), give some correspondence with X-ray
diffraction even if only recently new insights emerge from a detailed
unexpected study using laser irradiation on classical versus LB crystal
(Pechkova et al., in preparation). AFM studies allow also analyzing in
details the molecularity of growth steps of tetragonal lysozyme crystals
(Li et al., 1999).
The incorporation of a wide range of impurities, ranging in size from
molecules to microns or larger microcrystals, and even foreign particles
were visually recorded. Thanks to these observations and measurements,
a more complex understanding of the detailed character of
macromolecular crystals has been emerging, one that reveals levels of
complexity previously unsuspected. The new Atomic Force Microscopy
configuration (Pechkova et al., 2007b) allows acquiring “on real time”
images at the atomic resolution also of very small micro crystals
previously impossible.
For crystal characterization at subnanometric resolution precise
control of operating conditions is indeed a critical aspect, in which the
strong dependency of measurements on environmental factors, such as
acoustic and mechanical noises, temperature, and humidity play an
important role. This was shown initially using the tapping mode with the
AFM measurements of protein in water giving a resolution ten times
higher than in air (Pechkova and Nicolini, 2002a). Indeed, with the
measurements in water, the disturb created by dust or other particles
attached onto the surface by adhesion can be corrected, and the AFM
image of P450scc micro crystal yielded the same geometric parameters
of the P450scc protein obtained by homology modeling (Pechkova and

Nicolini, 2002a; Sivozhelezov et al., 2006). The present AFM system for
crystal (Pechkova et al., 2007b) has grown from an AFM for surface
investigation installed in a chamber with controlled atmosphere
developed time ago with hardware-software configuration based on
neural network (Sartore et al., 2000; Salerno et al., 1999).
2.2.1 AFM spectroscopy
With AFM we can typically obtain from the force in action apparent in
Figure 2.8 the intensity of the force versus the sample-tip distance.
Figure 2.8. Forces in action.
Moving the tip along the Z-axis (i.e., orthogonal to the sample) and
recording the cantilever deflection gives then rise to the force-distance
spectroscopy, which can be readily applied to the study of protein-
protein interaction without the utilization of fluorescence labelling.
Doing the AFM analysis shown in Figure 2.8 with the proteins yields
multi snap-off segments, where the two interacting proteins are fixed
respectively on the sample and on the tip, namely gives rise to the AFM
spectroscopy of proteins (Figure 2.9).
Nanoscale Probes 65
We have recently designed based on the above principles an AFM-

based Label Free analysis of NAPPA microarrays (see paragraph 2.8.2)
containing an opto-mechanical system for an AFM head capable to house
multiple cantilevers, for differential measurements and driven by an
appropriate electronics in order to detect the emerging signals with
significant improvement in protein detection (Sartore et al., in
preparation).
Figure 2.9. Spectroscopy of proteins, namely cantiliver deflection versus piezo Z position
(a) and AFM force versus Piezo movement (b).
2.2.2 Scanning tunneling microscopy
Time ago Scanning Tunneling Microscopy (STM) was also used (Facci
et al., 1994) to characterize nanomaterials in terms of voltage-current (V-
I) characteristic at single points.
All the images are typically obtained in air in constant current mode
within a tunneling current range of 0.1–0.5 nA and voltage from -1.5 V
to 1.5 V (MDT-AsseZ, Moscow-Padua).
STM analysis on different areas of several samples showed similar
features, some of which are presented in Figure 2.10.
In all these cases it is possible to distinguish features shaped like
wells in a corrugated matrix, sometimes isolated, sometimes
preferentially arranged along lines.
Figure 2.10. STM images of cadmium arachidate. (a) STM image of CdS film from one
bilayer of cadmium arachidate on top of highly oriented pyrolytic graphite: constant
current mode; image size 25.6 x 25.6 nm2; maximum corrugation 1 nm; tunnelling
parameters Vt = 0.1 V, It = 1 nA: scanning rate 12 Hz. (b) STM image of highly oriented
pyrolytic graphite plate: constant current mode; image size 25.6 x 25.6 nm2; maximum
corrugation 1 nm; tunnelling parameters Vt = 0.15 V, It = 1 nA; scanning rate 12 Hz.
(Reprinted with the permission from Facci et al., Formation of ultrathin semiconductor
films by Cds nanostructure aggregation, The Journal of Physical Chemistry 98, pp.
13323–13327, © 1994, American Chemical Society).
Their dimensions, ranging typically between 5–10 nm, match well

with the sizes of CdS particles, formed by the above described
procedure, estimated by analysis of optical absorption spectra and by X-
ray and electron diffraction. This comparison allowed suggesting that the
wells in the images could be connected with the CdS particles formed in
LB film of arachidic acid (i.e., particles could lay inside the wells). This
fact is in agreement with general concepts coming from ellipsometry
(Smotkin et al., 1988) and X-ray analysis (Erokhin et al., 1991) and with
Nanoscale Probes 67
the suggestion that the initial 2D arrangement of cadmium atoms in the

film can be preserved to a certain extent in the final film after the
reaction.
2.3 Nuclear Magnetic Resonance

1
H NMR 500 MHz spectrometer has been used in this context to monitor
the atomic structure of conductive polymers can be determined as well
(Ram and Nicolini, 2000) and of proteins, either in solution as in the case
of ribosomal proteins (Figure 2.11) (Vasile et al., 2008) or derived from
the dissolved corresponding crystals (Pulsinelli et al., 2003; Pechkova
and Nicolini, 2006).
Figure 2.11. A) Stereoview of the best ten structure obtained for the β−subunit of aIF2
from S. Solfataricus. (PDB ID code 2NXU). B) Electrostatic surface plot generated with
MOLMOL of S. Solfataricus aIF2β, acidic and basic residues are colored in red and blue
respectively. The structures are rotated of 180° (Reprinted with the permission from
Vasile et al., Solution structure of the β-subunit of the translation initiaton factor Aif2
from Archaebacteria Sulfolobus solfataricus, Proteins Structure, Function and
Bioinformatics 70, pp. 1112–1115, © 2008, John Wiley & Sons, Inc.).
Nuclear magnetic resonance (NMR) is a phenomenon that occurs

when nuclei of certain atoms are immersed in a static magnetic field and
exposed to a second oscillating magnetic field. Some nuclei experience

this phenomenon, and others do not, depending upon whether they
possess a spin. Nuclear magnetic resonance spectroscopy is the use of the
NMR phenomenon to study physical, chemical, and biological properties
of matter. Many of the dynamic NMR processes are exponential in
nature. The simplest NMR experiment is the continuous wave (CW)
experiment.
There are two ways of performing this experiment. In the first, a
constant frequency, which is continuously switched on, probes the
energy levels while the magnetic field is varied. The CW experiment can
also be performed with a constant magnetic field and a frequency that
varies. The signal of NMR spectroscopy results from the difference
between the energy absorbed by the spins, and that depicts a transition
from the lower energy state to the higher energy state and the energy
emitted by the spins, which simultaneously make a transition from the
higher to the lower energy state. The signal is thus proportional to the
population difference between the states. NMR is a rather sensitive
spectroscopic technique since it is capable of detecting these very small
population differences. NMR samples are prepared by dissolving an
analyte in a deuterium lock solvent. The concentration of the sample
should be great enough to give a good signal-to-noise ratio in spectrum,
yet minimize exchange effects found at high concentrations. The exact
concentration of the sample in the lock solvent depends on the sensitivity
of the spectrometer.
Recently also the atomic structure of a new conjugated amphiphilic
polymer (Narizzano et al., 2005) was determined by 1H-NMR
spectroscopy in CDCl3 solution using a Bruker AMX 500 instrument.
The 1H-NMR spectra revealed that the polymer contains 20% of 3HT.
The molar fraction of HT was calculated by integrating the peak areas of
methyl protons (∼ 0.9 ppm), which are only present in the 3HT units, and
of aromatic protons (∼ 7.0 ppm). The normalized areas were found to be
0.52 for the methyl group, and 1.00 for the aromatic protons,
respectively. The polymer exhibits good solubility in CHCl3, maintaining
the typical absorption peak of 3-alkyl-substituted polythiophenes, which
are obtained via the FeCl3 method at about 431 nm. This absorption,
which is detected in the UV-visible spectrum of the non-doped sample in
Nanoscale Probes 69
chloroform solution correspond to the typical π-π* transition of the

conjugated backbone.
2.3.1 Circular dichroism
The Circular Dichroism (CD) spectra at various temperatures is at times

a very useful information on the secondary structure of polymers which
can be to acquired quickly whenever needed, as in monitoring the
increased stability of the protein as a result of their immobilization in the
thin films in the range of 25–300 °C (Figure 2.12), a very useful
information indeed to assess the unexpected and useful heat-proof
property intervening in proteins whenever immobilized by LB
technology.
Figure 2.12. Circular Dichroism of protein monolayer. CD spectra as a function of the

temperature. (A) Cytochrome P450 native recombinant film. (B) Mutant K201E. The
data were collected in the far ultraviolet region (180-250 nm) with a wavelength step of 2
nm. Each spectrum was the result of an accumulation of three scans, and it was recorded
at a rate of 50 nm/min with a time constant of 4 s. (Reprinted with the permission from
Ghisellini et al., P450scc mutant nanostructuring for optimal assembly, IEEE
Transactions on Nanobioscience 3, pp. 121–128, © 2004, IEEE).
CD spectra are typically acquired using a spectropolarimeter to obtain

the percentage of protein secondary structures in the film and in the
solution, namely in terms of α-helix, β-pleated and random coil.
2.4 Brewster-Angle Microscopy
Brewster Angle Microscopy (BAM) allows to follow the Langmuir film

formation and to display its morphology, as well as to correlate the
2D/3D transformations with the shape of the π-A isotherms recorded
simultaneously.
The method is based on the fact that polarized light does not reflect
from the interface when it reaches it at the Brewster angle, determined by
the equation:
tgϕ = n2/n1
where n1 and n2 are refractive indexes of the two media at the
interface. The value of this angle for the air/water interface is 53.1°.
Therefore, it is possible to adjust the analyzer position in such a way that
it will bear a dark field when imaging the air/water interface. Spreading
of the monolayer varies the Brewster conditions for both air/monolayer
and monolayer/water interfaces, making visible the morphology of the
monolayer. Langmuir film formation was imaged and analyzed by BAM.
It was also possible to detect film dishomogeneities and breaks as well as
the 2D-3D transformations, which occurred at different surface
pressures. Such typical morphologies related to the investigation carried
out on Langmuir films of both proteins and polymers are conformed in
the images obtained in Paddeu et al. (1997) and in numerous papers
reviewed by Nicolini and Pechkova (2006).
When semiconductor particles were grown directly on LS films of
copolymer with copper ions by exposure to H2S atmosphere, the
morphology of the films, studied by Brewster angle microscopy,
undergoes evident changes upon exposure to the H2S atmosphere. In fact,
the formation of CuS nanoparticles causes increased layer corrugation.
As can be observed in Figure 2.13 (Narizzano et al., 2005), the rather
good homogeneity of the pristine film surface changes drastically after
the reaction. Morphology of films was studied by Brewster angle
Nanoscale Probes 71
microscopy (BAM-2, Nanofilm Technologie GmbH, Germany) at each

step, i.e., after deposition and after the particle formation process.
Brewster angle microscopy images were acquired with an Instrument
BAM-2 (Nanofilm Technologie GmbH, Göttingen, Germany)
(Narizzano et al., 2005). The standard laser of the BAM is a diode laser
with wavelength of 690 nm. The high-power laser diode has 30 mW
primary output.
Figure 2.13. Brewster Angle Microscopic images taken for 20 layers films of
semiconductor particles grown directly on LS films of copolymer with copper ions
copolymer. (a) before exposure to H2S; (b) after exposure to H2S. The image size is 0.3 ×
0.2 mm (Reprinted with the permission from Narizzano et al., A heterostructure
composed of conjugated polymer and copper sulfide nanoparticles, The Journal of
Physical Chemistry B 109, pp. 15798–15802, © 2005, American Chemical Society).
2.4.1 Ellipsometry
Ellipsometry is a technique based on the measure of two parameters,

namely the ratio of the vibration of the electric vector in the plane of
incidence and perpendicular to it, and the difference of phases of these
two vectors. The theory of the ellipsometry allows connecting these two
parameters with the thickness and refractive index of a nanostructured
layer (Drude, 1902). Usually, the layer is assumed to be not absorbing
and isotropic. In principle, the assumption is not valid in the most of
cases. Nevertheless, for the thickness estimation it seems to vary the
value inside the experimental error.
The technique allows to determine the thickness of the monolayer

during compression, revealing the reorganization of molecules at the
water surface. It carries out the measurements of film thickness using a
laser as a light source. Typically the mean values of the measurements at
different angles are given for the thickness and the index of refraction
(Tronin et al., 1994). According to the two-layer model the first lower
layer accounts for the imperfections which always exist on the surface of
most substrate in the form of traces of polishing, intrusions of the
polisher, a thin oxide layer, etc.
2.5 Electrochemistry
Cyclic voltammetry offers important and sometimes unique approaches

to the electroactive species. The cyclic voltammetry (CV) is the most
versatile electroanalytical technique to study the electroactive species.
CV consists of cycling the potential of an electrode, which is immersed
in an unstirred solution and measuring the resulting current.
The detailed understanding of cyclic voltammetry technique can be
gained by considering the Nernst’s equation and the changes in
concentration that occur in solution adjacent to the electrode during the
electrolysis. The proper equilibrium ratio in reversible system at a given
potential is determined by the Nernst equation. The electron transfer
reaction between proteins such as cytochrome P450 in presence of
cholesterol is an important system for investigating fundamentals
regarding long-range electron transfer in biological systems; it has
attracted considerable experimental and theoretical attention (Nicolini et
al. (2001a). Cyclic voltammetry, as an efficient method to investigate
electron transfer reactions between proteins in films, is being exploited
increasingly for obtaining electrochemical information on proteins
(Figure 2.14).
The electrochemical measurements were made by a
potentiostat/galvanostat (either homemade or EG & G PARC, model
263A), which was supplied with its own software (M270). The working
cell was homemade. A standard three-electrode configuration was used,
where LB films of cytochrome P450scc were deposited on an ITO coated
Nanoscale Probes 73
glass plate, which acted as a working electrode, having platinum as a

counter electrode and Ag/AgCl as a reference electrode. The working
electrode was cycled between initial and switch potentials of 500 and
500 mV, respectively, after holding the electrochemical system at the
initial potential for 10 s. The scan rate used was 20 mV/s because the
cathodic peak was most evident at this speed and, at the same time, the
low background current was minimized.
Figure 2.14. Current-voltage characteristics of cytochrome film in presence of increasing

concentration of cholesterol. Cyclic voltammetry of cytochrome P450scc LB film with
the addition of (1) 100, (2) 400, and (3) 750 µM cholesterol. (Reprinted with the
permission from Paternolli et al., Recombinant cytochrome P450 immobilization for
biosensor applications, Langmuir 20, pp. 11706–11712, © 2004, American Chemical
Society).
All the measurements were repeated three times to verify the

reproducibility. The electrical behavior of all films of either polymers,
biopolymers or nanocomposites, is typically studied by I-V
measurements. For istance, after the formation of nanoparticles in the
polymeric matrix, the conductivity of LS films was changed, increasing
by about 2 orders of magnitude. Conductivity shows a linear dependence
on the film thickness (Narizzano and Nicolini, 2005). The relatively low
value of the conductance depends on the limited number of polymeric
layers and, as a consequence, on the low number of CuS nanoparticles as
well as their random distribution. What is interesting is the dependence
of the specific conductance (σ) of the polymer, with and without
nanoparticles, as a function of the monolayer number (Narizzano and
Nicolini, 2005). As a consequence of the increase in the number of

monolayers, we observed a net and linear increment of σ in the
PAET/CuS heterostructure, while in the pristine polymer σ remains
almost unchanged, going from 5 to 10 monolayers, with a slight
increment for the 20 monolayer system. This could be due to the
structural reorganization of the polymer that usually occurs as the
number of monolayers increase, thus leading to a longer conjugation
length. Nanoparticle formation is responsible for a monotonous increase.
The noticeable change brought about by increasing the number of
monolayers suggests that particles are able to move and to rearrange
themselves within the films, leading to a structural reorganization of the
PAET/CuS system (Narizzano and Nicolini, 2005).
2.6 Infrared Spectroscopy
Fourier transform infrared (FTIR) spectroscopy is a powerful analytical

tool for characterizing and identifying organic molecules. Infrared
radiation is defined as the electromagnetic radiation the frequency of
which varies between 14300 and 20 cm-1 (0.7–500 µm). Within this
region of the electromagnetic spectrum, chemical compounds absorb IR
radiation provided there is a change in dipole moment during a normal
molecular vibration, molecular orientation, and molecular rotation or
from combination of difference in overtones of normal vibration.
The FTIR spectrum of an organic compound, such as cytochrome C
(Figure 2.15) serves as its fingerprint and provides specific information
about chemical bonds and molecular structure. Samples are run either as
pure substance or in KBr pellets. A beam of infrared radiation is passed
through the sample and the detector generates a plot of percent
transmission of radiation versus the wave number or wavelength of the
transmitted radiation. When the percent transmission is below 100, some
of the light is absorbed by the sample. Each peak in the spectrum
represents absorption of light energy, and is called an absorption band. It
is a powerful analytical tool for characterizing and identifying organic
molecules. The IR spectrum of an organic compound serves as its
fingerprint and provides specific information about chemical bonding
Nanoscale Probes 75
and molecular structure (Bramanti et al., 1997; Pechkova et al., 2007b).

The FTIR involves the vibrations of molecular bonds. Molecules can
bend or stretch at their bonds. They can also wiggle around in a wag, or
twist. Molecules vibrate in certain modes, depending on the symmetry
properties of the molecules' shape (Bramanti et al., 1997; Pepe et al.,
1998).
Figure 2.15. Fourier transform infrared (FTIR) spectra of a) cytochrome C film obtained
by spreading the solution at pH 7.4, dried at 25 °C, b) cytochrome C film obtained by
spreading the solution at pH 1, dried at 100 °C, c) LB film in the 4000-2800 cm-1 region.
(Reprinted with the permission from Bramanti et al., Qualitative and quantitative analysis
of the secondary structure of cytochrome C Langmuir-Blodgett films, Biopolymers 42,
pp. 227–237, © 1997, John Wiley & Sons, Inc.).
The energy it takes to excite a vibrational mode varies depending on

the strength of the bond and the weight of the molecule. FTIR involves
the conversion of energy to molecular vibrations. Infrared radiation
(wave numbers of 4800–400 cm-1) can be converted to vibrations in the
molecule, which causes the molecule to go from a ground vibrational
state to an excited vibrational state.
The amount of energy required to stretch a bond depends on many
things, one of which is the strength of the bond and the masses of the
bonded atoms. The higher wave numbers are achieved when the bonds
are stronger and the atoms are found to be smaller. Two-dimensional
Fourier transform infrared spectroscopy has been recently applied to

study the thermal stability of multilayer Langmuir-Schaefer (LS) films of
lysozyme deposited on silicon substrates (Pechkova et al., 2007a).
The study has confirmed previous structural findings that the LS
cytochrome films have a high thermal stability that is extended in a
lysozyme multilayer up to 200 °C. 2D infrared analysis has been used
here to identify the correlated molecular species during thermal
denaturation (Figure 2.16).
Figure 2.16. Synchronous 2D infrared correlation spectra in the 1800–1400 cm-1 interval
of LS films during thermal treatment from 25 to 250°C. The analysis was done in
transmission and in situ (Reprinted with the permission from Pechkova et al., Thermal
stability of lysozyme Langmuir-Schaefer films by FTIR spectroscopy, Langmuir 23, pp.
1147–1151, © 2007a, American Chemical Society).
Asynchronous 2D spectra have shown that the two components of

water, fully and not fully hydrogen bonded, in the high-wave number
range (2800–3600 cm-1) are negatively correlated with the amine
stretching band at 3300 cm-1. On the grounds of the 2D spectra the FTIR
spectra have been deconvoluted using three main components, two for
water and one for the amine.This analysis has shown that, at the first
drying stage, up to 100 °C, only the water that is not fully hydrogen
bonded is removed. Moreover, the amine intensity band does not change
up to 200 °C, the temperature at which the structural stability of the
multilayer lysozyme films ceases.
Nanoscale Probes 77
2.7 Nanogravimetry
By means of this technique it was possible to estimate the surface density

and the area per molecule values by depositing the Langmuir-Blodgett
film at different surface pressures, as well as the reliability of the
deposition itself.
Gravimetric measurements were carried out with a Quartz Crystal
Balance (QCM) utilising 10 MHz quartz resonators according to the
procedure shown in literature. A layer of conducting polymer was
deposited on both sides of the resonator and dried with nitrogen flux, and
then the frequency shift due to the film deposition was registered. This
frequency variation ∆f was correlated to the mass change ∆M by the
Sauerbray equation, written as:
∆M = K ∆f
where constant K depends upon physical parameters of the utilized
resonator, and defined as “the sensitivity of the instrument”.
From the nanogravimetry curve (Figure 2.17), plotted as surface
density as a function of the number of protein monolayers, the
information on the uniformity and reproducibility of deposition can be
obtained.
Figure 2.17. The dependence of the surface density of deposited protein (cytochrome
P450scc and human kinase CK2α) LB thin film upon the number of the transferred
monolayers. (Reprinted with the permission from Pechkova and Nicolini, Protein
nanocrystallography: a new approach to structural proteomics, Trends in Biotechnology
22, pp. 117–122, © 2004a, Elsevier).
Moreover, knowing molecular weight of the protein molecule, it is

possible to experimentally evaluate the area per one molecule in the
obtained film and to compare it with that theoretically estimated for the
closely packed system (Pechkova and Nicolini, 2002a).
The latter value can be easily calculated knowing the geometrical
parameters of the protein from RCSB PDB data bank (Berman et al.,
2002). In case the structure of the protein is not resolved yet,
homologous protein parameters or geometrical features from molecular
modelling can be used for calculation. It uses quartz resonators with a
resonance frequency of about10 MHz to measure mass.
A simple circuit allows the quartz resonators to oscillate at their
resonance frequency. The shift in the resonance frequency, induced on a
quartz resonator by subsequent LB, deposition, is monitored by a
frequency meter. The frequency shift ∆f owing to the amount of mass ∆m
attached to the resonator surface is expressed in mass units by applying
the Sauerbrey equation (Sauerbrey 1964):
∆f ∆m
=−
f0 Aρl
where f0 is the resonance frequency of the quartz, ρ its density, and l
and A the thickness and the area covered by the deposited monolayer
respectively.
The knowledge of the area of the resonator covered by the deposited
layers (measured by an optical microscope) allows us to convert these
values to surface density units (ng mm-2) (Erokhin et al., 1990) to
calculate the packing degree (the ratio between the film surface density
at the air-water interface and that after deposition onto the solid
substrate).
Calibration of the quartz balance was performed according to Facci et
al. (1993) and Lvov et al. (1990).
The protein film is deposited on both sides of the resonator, and
afterwards dried by nitrogen flux; the frequency shift is registered after
the covering (Facci et al., 1993; Antolini et al., 1995a; Paddeu et al.,
1995a).
Nanoscale Probes 79
Figure 2.18. Example of specific reaction: a) quartz has been covered with BSA (by
silanization) and the flowing solution contains antibodies specific to BSA (1 mg/ml); b)
the same quartz has been contacted by flowing solution containing antibodies non-
specific to BSA (GAM 1 mg/ml).
Results of the typical gravimetric study of deposited monolayers are

presented in Figure 2.18.
2.7.1 Quality factor
In most situations the adsorbed film is not rigid and the Sauerbrey
relation becomes invalid (Adami et al., in preparation). Namely, a film
being “soft” (viscoelastic) will not fully coupled to the oscillation of the
crystal, hence the Sauerbrey relation will underestimate the mass at the
surface.
A soft film thereby dampens the crystal's oscillation, where the
damping, or dissipation, D of the crystal's oscillation (Figure 2.19),
which reveals the film's softness (viscoelasticity) is defined as:
D= Elost/ Estored
where Elost is the energy lost (dissipated) during one oscillation cycle
and Estored is the total energy stored in the oscillator.
Figure 2.19. Quartz dumping.
The coupling of the crystal surface to a liquid drastically changes the

frequency. When a quartz crystal oscillates in contact with a liquid, a
shear motion on the surface generates motion in the liquid near the
interface.
Figure 2.20. Quartz quality factor.
The oscillation surface generates plane-laminar flow in the liquid,

which causes a decrease in the frequency, depending on the liquid
density and viscosity: For a 10 MHz crystal with one face exposed to
diluted solutions, near room temperature, ∆f is about 2 KHz. The
dissipation of the crystal is measured by recording the response of a
Nanoscale Probes 81
freely oscillating crystal that has been vibrated at its resonance

frequency. D is also equal to 1/Q, the quality factor of the quartz crystal
(Figure 2.20). A typical 10 MHz, AT-cut crystal working in a vacuum or
gaseous environment has a dissipation factor in the range of 10-6–10-4. If
a substance slips on the electrode, frictional energy is dissipated and D
can be used to interfer the coefficient of friction of the adsorbed film; if
the film is viscous, energy is also dissipated due to the oscillatory motion
induced in the film and D can be used to interfer the internal friction in
the film. An experimental set-up is presently being developed to measure
D (Adami et al., in preparation), with the method being based on the
principle that when the driving power to an oscillator is switched off at
t=0, the amplitude of oscillation A decays as an exponential damped
sinusoid:
−t
A(t ) = A0 e τ sin (ω t + ϕ ) + constant
where τ is the decay time constant, φ is the phase and the constant is
the dc offset.
The decay constant is related to D by:
D= 1
π fτ
2.8 Biomolecular Microarrays
Advance in genomics and proteomics have created a demand for

miniaturize robot platforms for the high throughput (HT) study of
proteins on a solid surface at high spatial density. Biomolecular arrays
with a number of genes are currently available, chosen without a precise
consideration of the particular target of the study, as described in the
following subparagraph.
Two new methods nanotechnology-based to produce DNA-
microarray and protein-chip (alternative to the one commercially
available) were recently introduced, the first based on a novel
biomolecular patterning on glass (Troitsky et al., 2002) and the second
based on the nanostructuring of anodic porous alumina matrix for
biomolecular microarray (Grasso et al., 2006; and chapter 1).
2.8.1 Gene expression via DNASER
A new matrix-integral part of the new DNA microarray

instrumentation DNA analyzer (DNASER) here described was
introduced based on a novel DNA patterning on the solid support surface
(Troitsky et al., 2002). Such patterning found the way to modify a glass
surface for a precise positioning of small droplets of aqueous DNA
solutions, without special robots (arrayers), within the boundaries of the
modified regions. The physically heterogeneous surface consists of
highly hydrophilic spots surrounded by a highly hydrophobic area
leading to the surface patterning needed for a DNA microarray: a matrix
of hydrophilic spots properly activated for immobilization of
oligonucleotides has been fabricated on absolutely passive hydrophobic
surface (Figure 2.21). The optimal efficiency of the above
functionalitation technology of a glass-substrate in obtaining DNA
microarray was confirmed by the Cy3-dCTP-labeled DNA sample, as
shown by charge coupled device images of the DNASER previously
described.
Figure 2.21. Samples with the arrays of hydrophilic spots prepared for the deposition of
DNA solution. (Reprinted with the permission from Troitsky et al., DNASER II. Novel
surface patterning for biomolecular microarray, IEEE Transactions on Nanobioscience 1,
pp. 73–77, © 2002, IEEE).
Nanoscale Probes 83
The novel bioinstrumentation named DNASER was introduced for

the evaluation of gene expression via the real-time acquisition and
elaboration of images from fluorescent DNA microarrays (Nicolini et al.,
2002). A white light beam illuminates the target sample to allow images
grabbing on a high sensibility and wide-band charge-coupled device
camera (ORCAII, Hamamatsu).
This high-performance device permits to acquire DNA microarrays
images and to process them in order to recognize the DNA chip spots, to
analyze their superficial distribution on the glass slide and to evaluate
their geometric and intensity properties. Differently from conventional
techniques, the spots analysis is fully automated and the DNASER does
not require any additional information about the DNA microarray
geometry.
Figure 2.22. Nanogenomic diagnostics via DNASER. (A) The apparatus containing the
optimally designed CCD camera (Nicolini et al., 2002); (B) Fluorescent images of DNA
solution spots with two color codes taken under the optimal drying procedures of the
sample.
Using our DNASER (Nicolini et al., 2002; Troitsky et al., 2002), we

confirmed the results by analyzing changes in gene expression after 24,
48 and 72 hours.
The validity of the DNASER measurements was confirmed by
standard fluorescence microscopy equipped with CCD.
This experimental analysis proved that DNASER is appropriate for
monitoring gene expression during the human lymphocytes cell cycle
(Nicolini et al., 2006a).
We calculated a final map of interactions among these 8 high-ranking

genes in cell cycle of human T lymphocytes (Sivozhelevov et al., 2006a;
Chang et al., 2004).
Typically the experimental datasets are derived from pangenomic
microarrays as fully described elsewhere for the study of kidney
transplant in humans (Braud et al., 2008). Fifty-one individuals were
included in the study: 8 patients tolerating a kidney graft (TOL) without
any treatment and 18 patients with chronic rejection (CR) were evaluated
against 8 healthy volunteers (HV) using a subset of the pangenomic
(more than 35,000 genes) array displaying 6,865 genes (hence,
“individual fullchip”).
For every patient, 2 independent DNA amplifications were used. Data
were expressed as mean values (log2) of the relative intensities [Cy3
(grafted patient) /Cy5 (pool of 169 kidney grafted recipients with stable
graft function)].
This database emerges from our previous similar studies of original
datasets called west-genopole based on different microarrays utilizing
different gene nomenclature and obtained from 14 CR, 11 TOL and 6
HV patients (hence, “pool fullchip”).
The data validity of the microarray was in this case assayed in details
(Sivozhelezov et al., 2008) by counting the fraction (%) of valid data i.e.
data actually present in the microarray for each gene, separately for
tolerance and rejection samples.
In the pool fullchip, the total numbers of the tolerance and rejection
samples were 28 and 42 respectively. For example, for gene ABCA1-1A,
the fraction is 39/42=93% among the “rejection” samples, and
18/28=64% among the “tolerance” samples.
Clustering analysis according to that parameter showed that about
74% “pro-rejection” genes and 71% “pro-tolerance” genes were
classified in the top category, which we termed “reliable”. Three more
categories were revealed, termed “medium”, “unreliable”, and “very
unreliable”.
Nanoscale Probes 85
Figure 2.23. Top, percentages of classes of genes with belonging to the four categories
with respect to reliabilities in the old “fullchip” raw dataset. Bottom, the same values
(green) compared to fractions (%) of genes not adhering to the HGNC nomenclature
(“bad”) in each genes with respect to total “bad” genes. Left, CR data. Right, TOL data.
CR dataset consists of 42 samples in total and 6864 genes, while TOL dataset consists 28
samples in total and 6864 genes. Reliability is given by the percentage of proven
expression data by GENEPIX in such genes for 70 microarray samples. (Reprinted with
the permission from Sivozhelezov et al., Immunosuppressive drug-free operational
immune tolerance in human kidney transplants recipients: II Nonstatistical gene
microarray analysis, Journal of Cellular Biochemistry in press, © 2008, Wiley-Liss, Inc.,
a subsidiary of John Wiley & Sons, Inc.).
Figure 2.23 shows the presence of the genes in the four categories.
This limited validity does not appear however to introduce any bias in
the “pool fullchip” data for either pro-TOL or pro-CR genes
(Sivozhelezov et al., 2008), with the linear regression giving 93%
correlation coefficient with the slope of the regression line close to unity.
To further check if unreliability could be related to nomenclature
problem, we calculated (Sivozhelezov et al., 2008) the fraction of genes
not adhering to HGNC nomenclature in each of the four categories. If the
nomenclature problems did not affect the reliability, we could expect the
same fractions for the “bad” genes as for all genes. This is not the case.
Even though the fractions of “bad genes” are close to those for all genes,
occurrence of “bad” genes relative to all genes increases from category

to category (Figure 2.23 bottom). The fact that the observed differences
are small is readily explained by the fact that the disagreement with
HUGO nomenclature does not necessarily mean that the deposited
sample is unreliable. In fact, many of the genes obviously not adhering to
the HGNC nomenclature in the “fullchip” microarray analyzed, can be
assigned using parsing and database searches i.e., siUNC13Celegans is
resolved as the following gene, “Official Symbol: UNC13B and Name:
unc-13 homolog B (C. elegans) [Homo sapiens]”. Similarly,
“siRAB11amemberR” is resolved as “Official Symbol: RAB11A and
Name: RAB11A, member RAS oncogene family [Homo sapiens]”.
However, some gene specifications used in the “old fullchip” microarray
contain sequences that have been revoked from GenBank presumably by
their own authors, in which the GenBank record contains a note that it
has been discontinued, one example being siVoltLOC121358. Such
nucleotides do not necessarily contain gene sequences, and thus may
well be the cause of the entire absence of expression data, as well as in
poor reproducibility of the data when present. Our findings are in
agreement with the reported generally poor (32–33% correlation
coefficient) reproducibility of the microarray data across laboratories
[Members of the Toxicogenomics Research Consortium, 2005], which,
however, was increased to 56–59% after nomenclature and data handling
was standardized.
Further increase (in some cases up to 97%) was indeed achieved by
standardizing experimental procedures. This is indeed what appears in
our individual “fullchip”, which, in contrast to the pool “fullchip”, has as
much as 98% genes passing the 70% reliability criterium (Sivozhlezov et
al., 2008).
2.8.2 Protein expression via Nucleic Acid Programmable Array
Protein microarrays have found particular value in analyzing clustered

protein expression, revealing co-regulated protein networks; protein
expression analysis does readily predict protein abundance and does
provide information about protein function (LaBaer and Ramachandran,
2005) and protein-protein interactions (Figure 2.24).
Nanoscale Probes 87
Is really the combination of mass spectrometry (see later) and protein

microarrays that offer these features by allowing investigators to query
thousands of targets simultaneously (Spera and Nicolini, 2008).
Figure 2.24. Protein-protein interactions and protein small interactions.
Given the central role that proteins play in biology and physiology,
we need better methods to study protein abundance, structure and
activity in HT.
Protein microarrays and mass spectrometry offer indeed such
approaches that we intend to apply to deepen our study (Spera and
Nicolini, 2007, 2008; Spera et al., 2007) addressed to understand human
cell cycle progression and reverse transformation, a prerequisite for the
final goal of understanding and controlling human cancer at molecular
level.
However, the development of a MALDI MS-compatible protein
microarray is complex since existing methods for forming protein
microarrays do not transfer readily onto to a MALDI target. Actually we
are implementing a procedure to analyze by MALDI-TOF mass
specrotrometry (see later) Nucleic Acid Programmable Protein Array
(NAPPA) protein microarray (Ramachandran et al., 2004, and Figure
2.25).
Figure 2.25. (A) General scheme of a typical protein microarray experiment. A set of
capture ligands (proteins, antibodies, peptides) is arrayed onto an appropriate solid
support. After blocking surface unreacted sites the array is probed by incubation with a
sample containing the target molecules. If a molecular recognition event occurs, a signal
is revealed either by direct detection (mass spectrometry, surface plasmon resonance,
atomic force microscopy, quartz crystal microbalance) or by a labelled probe (CCD
camera, DNASER). (B) Assembly methods used to produce function-based protein
microarrays. (i) Expressed and purified proteins can be affixed directly to the surface of a
chemically activated matrix. By this method, native protein can be used and the proteins
will tend to position in random orientations, such that on average, each surface is likely to
be exposed to the interacting sample. However, the close attachment to the surface may
limit the overall solvent exposure of the protein and the chemical linkage may affect
protein folding. Fusion peptide tags added at the N- or C-terminus affix the protein
through an affinity capture reagent. Proteins are produced either by (ii) separate
expression and purification or (iii) by simultaneous expression and capture of the protein
on the array surface. The use of fusion tags allows the protein to be held at a distance
from the matrix, exposing more overall surface area to solvent, but sterically blocking
either the N- or C-terminus and requiring the addition of fusion tags to all target proteins.
(C) NAPPA approach. (continue)
Nanoscale Probes 89
Biotinylation of DNA: Plasmid DNA is cross-linked to a psoralen-biotin conjugate with

the use of ultraviolet light. (i) Printing the array. Avidin, polyclonal GST antibody, and
Bis suberate are added to the biotinylated plasmid DNA. Samples are arrayed onto glass
slides treated with 3-aminopropyltriethoxysilane and dimethyl suberimidate. (ii) In situ
expression and immobilization. Microarrays were incubated with rabbit reticulocyte
lysate with T7 polymerase. (iii) Detection. Target proteins are expressed with a C-
terminal GST tag and immobilized by the polyclonal GST antibody. All target proteins
are detected using a monoclonal antibody to GST against the C-terminal. (Reprinted with
the permission from Spera and Nicolini, Nappa microarray and mass spectrometry: new
trends and challenges Essential in Nanoscience Booklet Series, © 2008, Taylor & Francis
Group/CRC Press, http://nanoscienceworks.org).
NAPPA protein microarray together matrix-assisted laser desorption-

ionization time-of-flight (MALDI-TOF) mass spectrometry provides two
powerful and independent tools for the study of protein function and
structure in the human cell lines in vitro and in vivo. To study protein
abundance and function and to obviate the need to express, purify and
store the proteins we employ the self-assembling protein microarray
technology called NAPPA (nucleic acid programmable protein array). As
the proteins are freshly synthesized just in time for assaying, there is less
concern about protein stability. This approach produces a sizable amount
of protein per feature, averaging about 10 fmols. The microarrays are
stable dry at room temperature until they are activated to make protein.
This approach has been optimized for the detection of protein-protein
interactions and for the co-expression of both the target and query
proteins, eliminating the need for any purified proteins. In a protein
interaction mapping experiment recently reported by us among 30 human
DNA replication proteins, 85% of the previously biochemically verified
interactions were recapitulated. In NAPPA technology, full-length cDNA
molecules are immobilized on a microarray surface and expressed in situ
using a mammalian cell-free expression system (rabbit reticulocyte
lysate).
A fusion tag present on the protein is recognized by a capture
molecule arrayed (along with the cDNA) on the chip surface. This
capture reaction then immobilizes the protein on the surface in a
microarrayed format as shown in Figure 2.26 for the human kinase arrays
introduced at the Harvard Institute of Proteomics (LaBaer and
Ramachandran, 2005).
Figure 2.26. Human kinase array on gold surface (Reprinted with the permission from
Prof. Joshua Labaer at Harvard Institute of Proteomics).
To identify genes and proteins and their interactions key to the

specific cellular process, we integrate the above experimental
observations with molecular modelling and bioinformatics (see next
paragraph) utilizing existing database such as Gene, HomoloGene,
MeSh, Nucleotide and Protein Sequence, along with various advanced
software as String and MIM (Sivozhelezov et al., 2006a).
This opinion article, without being exhaustive, will focus on the
combined utilization of NAPPA arrays and mass spectrometry
highlighting some of open key technical challenges and the new trends
by means of a set of selected recent In house applications.
2.9 Biophysical Informatics
A new discipline has been emerging in the last few years to understand
the complex processes becoming apparent with the introduction of
nanobiotechnology in the study of proteins and genes, which we call here
Biophysical Informatics being at the merging of Bioinformatics and
Molecular Modelling based on Theoretical Biophysics.
Nanoscale Probes 91
2.9.1 Bioinformatics
Bioinformatics allows to identify the key genes or proteins involved in a

given biological process by the iterative searches of gene-related or
protein-related databases derived mainly from genes or proteins
microarray experimentation, revealing and predicting interactions
between those genes or those proteins, assigning scores to each of the
genes according to numbers of interaction for each gene weighted by
significance of each interaction, and finally applying several types of
clustering algorithms to genes (or proteins) basing on the assigned scores
(Sivozhelevov et al., 2006a).
All clustering algorithms applied, both hierarchical and K-means,
invariably selected the same six “leader” genes involved in controlling
the cell cycle of human T lymphocytes. Six genes were identified in the
cell cycle of human T lymphocytes, which appear to be uniquely capable
to switching between stages of cell cycle of human T lymphocytes
(Nicolini et al., 2006a; Giacomelli and Nicolini, 2006). In the recent
years mostly high-throughput, approaches has been used to identify key
genes for particular cellular processes, usage of the already-existing
knowledge bases on gene and protein interactions, combined from
heterogeneous data sources, is rare. The “leader gene” search/statistics
algorithm consists in:
(1) iteratively searching GenBank and PubMed databases to identify the
genes with proven involvement in the given cellular process,
(2) query of the STRING (von Mering et al., 2005) database to establish
links between the genes,
(3) assigning STRING association-based scores to each gene, and
(4) clustering of gene list according to those scores to yield the final
leader gene list.
The leader genes algorithm is being also applied to predicting genes
involved in cell cycle progression of human T lymphocytes (Nicolini et
al., 2006a; Giacomelli and Nicolini 2006), in osteogenesis (Marconcini
et al., Leader genes in osteogenesis, in preparation), in inflammatory
processes and in kidney graft rejection (Braud et al., 2008; Sivozhelezov
et al., 2008).
Figure 2.27. Relations between the genes induced during lymphocyte activation
according to gene-gene interaction (induction or suppression) (Reprinted with the
permission from Sivozhelezov et al., gene expression in the cell cycle of human T
lymphocytes, Journal of Cellular Biochemistry 97, pp. 1137–1150, © 2006a, Wiley-Liss
Inc., a subsidiary of John Wiley & Sons, Inc.).
Leader genes approach, with text-mining scoring option off, appear to

provide a list of the few most important genes relevant for the given
cellular processes according to the already-available experimental data,
and should be useful in interpreting the microarray expression data and
to guide clinical trials. A caution appear needed in the identification of
leader genes obtained using either text*mining or no text-mining scoring
and clustering which frequently do not have a single gene in common
(Braud et al., 2008; Sivozhelevov et al., A new algorithm for Leader
Gene identification”, in preparation).
Indeed, no text-mining approach produces more valid results
(Sivozhelevov et al., A new algorithm for Leader Gene identification, in
preparation). To perform the above analysis, two softwares are required
and need to be installed on a dedicated computer: MATLAB and
GenePix software. The above bioinformatics algorithm, based on the
scoring of importance of genes and a subsequent cluster analysis,
allowed indeed us to determine the most important genes, that we call
Nanoscale Probes 93
“leader genes” (Sivozhelezov et al., 2006a) in human T lymphocytes cell

cycle.
This particular cellular system is very well known and was
quantitatively characterized time ago (Cantrell, 2002; Abraham et al.,
1980); therefore, it can be a good starting point to verify our algorithm.
In particular, we identified 238 genes involved in the control of cell
cycle. Most important, only 6 of them were previously identified to be
the leader genes (Nicolini et al., 2006a); interestingly, they actually are
involved in the cell cycle control at important progression points, namely
the most important four at the transition from G0 to G1 phase (MYC;
(Oster et al., 2002), at the progression in G1 phase (CDK4; Modiano et
al., 2000), and at the transitions from G1 to S (CDK2; Kawabe et al.,
2002), and from G2 to M phases (CDC2; Baluchamy et al., 2003;
Torgler et al., 2004).
The two remaining “leader genes” (CDKN1A and CDKN1B) are
inhibitors of cyclin-CDK2 or -CDK4 complexes and thereby contribute
to the control of G1/S transition and of G1 progression (Jerry et al.,
2002; Chang et al., 2004).
Bioinformatics can be used also in protein microarrays for the study
of protein-protein and protein-gene interactions (Ramachandran et al.,
2004). Like for the DNA microarrays, the leader gene approach can
simplify their analysis, by reducing the protein displayed to the most
important ones to be subsequently tested by mass-spectrometry or by ad
hoc experimentation.
2.9.2 Biophysical molecular modelling
Homology modelling (Sivozhelezov and Nicolini, 2005, 2006, 2007;

Sivozhelezov et al., 2006b) has been recently used to provide useful
insights for the successful optimisation of numerous applications in
Nanobiotechnology, namely the crystallization of octR rhodopsin
(Sivozhelezov and Nicolini, 2006) and of cytochrome P450scc
(Sivozhelezov and Nicolini, 2005; Sivozhelezov et al., 2006b) and in the
optical computation (Sivozhelezov and Nicolini, 2007).
Octopus (octR), bovine (bovR), and bacterio (bR) rhodopsins all
belong to the structural super family of rhodopsin-like proteins sharing
the overall 7 transmembrane helix topology of bR except for some

details in distances and relative orientations of the helices (Faulon et al.,
2003).
This allows to use bR as the primary template in the homology
modeling for all structure/function studies with eventual applications,
basing on the huge body of such structure/function data (Hirai and
Subramaniam, 2003) with respect to bR, as well as biotechnology
applications (Fischer et al., 2003; Fischer and Hampp, 2004; Hampp
2000; Hampp and Juchem, 2004; Hillebrecht et al., 2004; Wise et al.,
2002). The structure of bR is typically compared with the homology
model of octopus rhodopsin (octR), which is similar in topology to bR
and as highly ordered in its native membranes as bR in purple
membranes (Sivozhelezov and Nicolini, 2006).
2.9.2.1 Three-dimensional structure of octopus rhodpsin
While classical homology modeling is still successfully used (Miedlich

et al., 2004), two other approaches were implemented for selecting a tool
for modeling the 3D structure of octR, one based on intraprotein
hydrogen-bond optimization (Pogozheva et al., 1997) and the other
based on first principles of transmembrane protein assembly (Trabanino
et al., 2004).
The first principles approach appeared more attractive considering
that was already tested on bovine rhodopsin. We started by predicting
positions of the helices, and then manually adjusting the resulting
alignment of octR versus bR using sequence identity and similarity. The
resulting approach (Sivozhelezov and Nicolini, 2006) is therefore a
hybrid between homology modeling and first-principles modeling.
The sequence identity level in the eventual model resulted 24.4%,
which is above the average 20% quoted for comparisons of vertebrate
versus invertebrate visual pigments and therefore supports the reliability
of our homology model (Figure 2.28), having as template the PDB entry
1U19 (Okada et al., 2002).
Model quality estimated by comparison of the model with its
template appears quite good (Sivozhelezov and Nicolini, 2006). Indeed
the model reproduces the positions of the residues surrounding the
Nanoscale Probes 95
chromophore (retinal) correctly, particularly the Lys306 residue

providing the Schiff base connection of the protein to the retinal.
Figure 2.28. Stereo view of the homology model of octopus rhodopsin as a Cα trace, with
the retinal shown as sticks. Invariant residues are numbered. Alpha helices are shown in
green. (Reprinted with the permission from Sivozhelezov and Nicolini, Theoretical
framework for octopus rhodopsin crystallization, Journal of Theoretical Biology 240, pp.
260–269, © 2006, Elsevier).
Notably, the model shows higher alpha helical content than its
template (203 versus 190 alpha helical residues), a rare occasion in
comparative modeling. This could be the basis for higher organization of
octR into 2D arrays compared to the bovR, as observe in vivo
experimentally (Davies et al., 2001). The good quality of the model was
also assessed by the model’s ability to reproduce the distinctive features
of all visual pigments described to date (Nathans, 1992), as the presence
of the sequence (Glu/Asp)-Arg-Tyr, the lysine in the middle of the
seventh putative transmembrane segment and the pair of cysteines
forming a disulfide bond connecting the first and second extra-cellular
loops. All are indeed present in our model.
Identity of the counterion is another important issue both with respect
to both the structure/function relationships and biotechnology
applications. The only other protein in addition to bR that we found to be
structurally similar to our model of octR was the transmembrane
cytochrome oxidase. This similarity is possibly related to similarity of

function between bR and the oxidase in that both of them involve
transfer of charged species, whether electron or proton, across the
membrane. The mismatch of the position of Helix4 provides the source
of most differences among the octR and bR structures, interestingly since
most of the conformational mobility of octR is mediated by Helix 4.
2.9.2.2 Three-dimensional structure of cytochrome P450scc
Similarly for the crystallization (Sivozhelezov et al., 2006b) and sensor

(Sivozhelezov and Nicolini, 2005) optimisation a new homology model
of bovine cytochrome P450scc has been obtained starting from the
recently determined crystal structure of mammalian cytochrome
P4502B4 (Sivozhelezov and Nicolini, 2005).
Figure 2.29. Hydrophobic patches around the molecule of cytochrome P450scc,

according to the homology model based on cytochrome P4502B4. (Reprinted with the
permission from Sivozhelezov et al., Mapping of electrostatic potential of a protein on its
hydrophobic surface: implications for crystallization of cytochrome P450scc, Journal of
Theoretical Biology 241, pp. 73–80, © 2006b, Elsevier).
The new emerging structure (Figure 2.29) appears compatible with

recent diffraction patterns of bovine P450scc microcrystals as obtained at
the Microfocus Beamline of the European Synchrotron Radiation Facility
Nanoscale Probes 97
in Grenoble (Nicolini and Pechkova, 2006). The same atomic structure is

utilized thereby to correctly predict the mutations needed for obtaining
both the modifying redox potential experimentally observed (Paternolli
et al., 2004) and the optimal LB assembly of the cytochrome obtained
experimentally (Ghisellini et al., 2004). Comfortingly these mutations
being predicted for the given functional modification are quite different
from what erroneously predicted by previous homology models present
in the RCSB Protein Data Bank (Berman et al., 2000).
2.9.2.3 Protein crystallization
Bacteriorhodopsin (bR) is presently a classical example of membrane

protein crystallization.
Comparing its structure with the homology model of octopus
rhodopsin (octR), the latter appears similar in topology and highly
ordered in its native membranes as bR in purple membranes. Such
comparison provides insights for optimization of octR crystallization
(Sivozhelezov and Nicolini, 2006). Our results suggest that for optimal
crystallization three new tryptophan residues should be introduced in the
resulting mutant octR and/or a new protein (RALBP) added to otherwise
non-crystallizable octR preparations. Experimentation is still under way.
The molecular manipulation techniques already used in the process of 2D
and 3D crystallization of both bovine rhodopsin (Caffrey, 2003; Okada et
al., 2002) and cytochromes (Hunte and Michel, 2003) are likely to
eventually provide a general paradigm for membrane protein
crystallization utilizing homology modeling. Indeed, they are promising
with octR because, in addition to the transmembrane domain, it has an
extensive soluble cytoplasmic domain that can potentially guide
crystallization in aqueous medium once hydrophobic surfaces are
shielded by detergent. Besides, techniques of molecular manipulation
using Langmuir-Blodgett technology are already described for OctR and
other photosensitive proteins (Pepe and Nicolini, 1996; Maxia et al.,
1995) allowing at least semi-qualitative testing of theoretical predictions.
As the first step, we build a 3D model of octR using bovR as a template
(Figure 2.28), and compare it with the structure of bR also in term of
biotechnology applications that have been reported for bR (Nicolini et

al., 1998, 1999) but can also be implemented in octR.
Similarly calculation and combined visualization of electrostatic and
hydrophobic properties of cytochrome P450scc based on two very
different homology models allowed to identify extensive hydrophobic
patches with neutral electrostatic potential and mutations removing such
patches and thus expecting to facilitate crystallization of cytochrome
P450scc (Sivozhelezov et al., 2006b), especially for the nanotemplate
crystallization method. These calculations allow to optimize
crystallization and other aspects of protein surface properties and protein
recognition. The most promising way for optimizing protein
crystallization turned to be nanotemplate crystallization method (as
shown later in chapter 3), as confirmed by both fluorescence labeling
studies (Pechkova et al., 2005) and by microGISAX spectra analysis
(Nicolini and Pechkova, 2006; Pechkova and Nicolini, 2006). The
resulting P450scc microcrystals diffraction rings were obtained by
synchroton microfocus diffraction (Nicolini and Pechkova, 2006).
However in order to further optimize P450scc crystallization for
obtaining the necessary entire diffraction data set, is necessary to take
into account that the plethora of the crystallized cytochrome P450 and
the corresponding number of reported 3D atomic structures suggest the
use of surface mutagenesis, specifically recommended for the case when
the obtained crystals do not have the sufficient quality, but crystallizable
homologs are available (Dale et al., 2003). The general recommendation
is to mutate residues predicted by homology models to be solvent-
exposed to those favorable for crystal contact formation. Statistically,
those desirable residues are arginine and glutamine (Baud and Karlin,
1999; Dasgupta et al., 1997). By homology modeling we have recently
proposed mutations that are expected to facilitate crystallization of
cytochrome P450scc according to the above indications, but with several
essential amendments (Sivozhelezov et al., 2006b) allowing to obtain the
more compact, stable and high ordered protein film, which can
significantly optimize the protein nanotemplate method, guaranteeing the
higher quality of nucleation for crystal formation. Here, we have
combined the above concept with the notion that it is the electrostatic
forces that primarily steer the molecules to each other in aqueous
Nanoscale Probes 99
solution, even though the eventual energetics of protein-protein contact

may be determined by the hydrophobic effect. The structure of
cytochrome P450scc predicted by homology with cytochrome P4502B4
(Figure 2.29) shows striking difference from the pattern exhibited by
cytochrome P4502B4 itself, with almost the entire surface of the protein
covered by the green “clouds” designating hydrophobic surfaces of the
proteins not screened by electrostatic potentials. Therefore, according to
the logics of Patro et al. (1996), no ordering should be observed in the
aggregates formed by the P450scc molecules, and thus very little chance
for P450scc molecules in their native form to yield well-diffracting
crystals. Our approach therefore suggests three options for altering the
structure of P450scc to assist crystallization. The first and foremost is
site directed mutagenesis. This approach has already a considerable
record of success reviewed in Dale et al. (2003). These successful
examples include, for instance, exhaustive mutagenesis of all 29 possibly
exposed hydrophobic residues of catalytic domain of HIV integrase,
resulting in just one mutant giving well-diffracting crystals (Dyda et al.,
1994). More rational approach (termed “crystal engineering”) involved
theoretical predictions and resulted in crystallizability introduced or
improved by inducing smaller number of mutations. For example 9
mutations resulted in improved crystallizability on the case of 24 kDa
fragment of DNA gyrase (D'Arcy et al., 1999) while only one mutation
was required for adding the propensity to crystallize in the case of human
leptin (Zhang et al., 1997a). This is the approach actually applied in this
study. The size and occurrence of electrostatically unscreened
hydrophobic patches (Figure 2.29) for the cytochrome P4502B4-based
cytochrome P450scc model is larger than for P4502B4 (not shown).
Some regions of the P450scc surface are completely covered by patches
and the corresponding residues must be considered for mutations
facilitating crystallization. They are marked on Figure 2.29: Tyr238,
Met240, Val245, Phe294, Leu311, Ile489, Phe507, and Phe513.
Following the guidelines of Baud and Karlin (1999) and Dasgupta et al.
(1997), each of the proposed residues should be replaced either by
arginine or glutamine, the latter two being most prone to crystal contact
formation. The mutations suggested herein have showed a considerable
effect on the hydrophobic patches decreased in size but, even more
importantly, with quite fewer contiguous patches in the mutant protein.

Therefore, the proposed mutation should further improve crystallization
and the work is in progress.
2.9.2.4 Nanobiodevice implementation
Recent modeling has provided insights also for optimization of present

octR experimentation for application in nanobiotechnology in a manner
similar to bR, and possibly even superior in optical computation
(Sivozhelezov and Nicolini, 2007).
Visual membranes of octopus, whose main component is the light-
sensitive signal transducer octopus rhodopsin (octR), are extremely
highly ordered, easily capture single photons, and are sensitive to light
polarization, which shows their high potential for use as a Quantum
Computing (QC) detector (Sivozhelezov and Nicolini, 2007). However,
artificial membranes made of octR are neither highly enough ordered nor
stable, while the bacterial homolog of octR, bacteriorhodopsin (bR),
having the same topology as octR, forms both stable and ordered
artificial membranes but lacks the optical properties important for optical
QC. In a recent study (Sivozhelezov and Nicolini, 2007), we investigate
the structural basis for ordering of the two proteins in membranes in
terms of crystallization behavior. We compare the atomic resolution 3D
structures of octR and bR and show the possibility for structural bR/octR
interconversion by mutagenesis. We also show that the use of
nanobiotechnology can allow (1) high-precision manipulation of the light
acceptor retinal, including converting its surrounding into that of
bacterial rhodopsin, the protein already used in optical-computation
devices and (2) development of multicomponent and highly regular 2D
structures with a high potential for being efficient optical QC detectors
(Sivozhelezov and Nicolini, 2007). The key property of bR allowing to
utilize it in optical memory devices (Fischer and Hampp, 2004), appears
to be its existence in two stable forms giving rise to novel types of
optical computation devices.
The implication of these studies has been successfully proved also
with P450scc for optimal sensor construction (Paternolli et al., 2004) and
for LB nanoassembly (Ghisellini et al., 2004). A previously reported
Nanoscale Probes 101
(Ghisellini et al., 2004) molecular modeling prediction was indeed

realized studying a hypothetical complex based on two P450scc
molecules. The aim of the mutation proved successful was to increase the
probability to form a pattern by improving electrostatic interactions and
simultaneously adding hydrophobic amino acid residues into the surface
regions included in the pattern. In particular, the mutation (K201E) was
identified by electrostatic calculations and caused the enhancement of the
stability/ordering during the protein immobilization.
2.10 Mass Spectrometry
The identification of different protein patterns in CHO-K1 cells grown

with and without cAMP (Spera et al., 2007) is performed with a RP-
HPLC-ESI MS apparatus, a Thermo Finnigan (San Jose, CA) Surveyor
HPLC connected by a T splitter to a PDA diode-array detector and to
Xcalibur LCQ Deca XP Plus Mass Spectrometer (Figure 2.30).
The mass spectrometer is equipped with an electrospray ion source
(ESI). The chromatographic column is a Vydac (Hesperia, CA) C8
column, with a 5 µm particle diameter (column dimensions 150 × 2.1
mm).
For HPLC-ESI MS analysis the protein solutions, stored at -80˚C,
were heated at room temperature and then lyophilized. The lyophilized
proteins were immediately dissolved in aqueous 0.2% TFA and
centrifuged at 12000 rpm for 5 minutes. After centrifugation, the
supernatant was analyzed by HPLC-mass spectrometry. The following
solutions were utilized for reversed-phase chromatography: eluent A,
0.056% aqueous TFA, and eluent B, 0.05% TFA in acetonitrile/water
80:20 (v/v).
The gradient applied was linear from 0 to 55% in 40 minutes, at a
flow rate of 0.30 ml/min. The T splitter gave a flow rate of about 0.20
ml/min toward the diode array detector and a flow rate of 0.10 ml/min
toward the ESI source. The diode array detector was set in the
wavelength range 214–276 nm.
Mass spectra were collected every 3 msec in the positive ion mode.
MS spray voltage was 4.50 kV, and the capillary temperature was 22 °C.
The deconvolution of the averaged ESI mass spectra was performed by

Mag-Tran1.0 software. Preliminary proteins identification from the mass
values of the intact protein was obtained from a search in Swiss-Prot
Data Bank (http://www.expasy.org).
The identification of CHO-K1 proteins via mass fingerprint is
performed coupling HPLC (Varian Inc) to separate the proteins, and
MALDI TOF MS, a Bruker Autoflex (Bruker Daltonics, Leipzig,
Germany) to analyze the tryptic digest of these samples (Spera et al.,
2007).
The HPLC measurements were carried out on a Varian Star HPLC
system which includes: 9012 Gradient Solvent Delivery System, 9050
UV-VIS Detector, 9300 Refrigerated AutoSampler (fitted with a 20 µl
loop), and a Star Chromatography Workstation (Varian Inc., USA).
Proteins will be separated on a C8 (250 × 4.6 mm; 5 µm particle size)
reverse phase column (Macherey-Nagel, Germany). For reversed-phase
chromatography we utilized the following solutions: eluent A, 0.056%
aqueous TFA, and eluent B, 0.05% TFA in acetonitrile/water 80:20 (v/v).
The gradient applied was linear from 0 to 50% in 30 minutes, at a
flow rate of 0.30 ml/min. For HPLC analysis the protein samples stored
at –80˚C, were heated at room temperature and then 100 µl of solutions
were injected. We used three samples of lysozyme from chicken egg
white (Sigma Chemical Co., St. Louis, MO) at different concentrations to
standardize the HPLC results and the protein amounts loaded to MS.
For protein fingerprint, the fractions collected from HPLC were
digested with trypsin overnight, according to the enzyme supplier
(Sigma-Aldrich).
For MALDI-TOF MS the tryptic digest samples were diluted to 4 nM
protein concentration in a 0.1% TFA solution. The matrix used for the
mass spectrometric analysis was a saturated solution of acid (α-Cyano-4-
hydroxycinnamic acid for peptides and light proteins and sinapinic acid
for heavy proteins, Bruker Daltonics) dissolved in 2/3 of 0.1% TFA and
1/3 of acetonitrile.
For the analysis 1.5 µl of matrix solution was mixed with 1.5 µl of
sample, then 1 µl of this mixture was spotted onto a suitable aluminum
plate and air-dried. MALDI-TOF MS was externally calibrated using
protein and peptide calibration standard solutions (Bruker Daltonics),
resulting in a mass accuracy < 100 ppm for intact proteins and < 10 ppm
for peptides.
The mass lists obtained were submitted to a data bank search. We use
a specific software for protein data interpretation, Biotools (Bruker
Daltonics), that allows an automated protein identification via library
search and that has a MASCOT Intranet search software (Matrix
Sciences, Ltd. www.matrixscience.com) fully integrated.
MS analysis started from the HPLC-ESI MS analysis of nuclear
proteins fraction from CHO-K1 cells and from cAMP treated cells
(Figure 2.30).
The total ion current (TIC) and the UV absorbance at 214 and 276 nm
(not reported) profiles obtained during chromatographic analysis of the
two protein solutions are reported in Figure 2.30. TIC and UV profiles
did not reveal significant signals between 0 and 10 min.
The highest variations are identifiable in the TIC plots (see
enlargement in the box) and show an inhibition of the nuclear protein
expression after treatment with cAMP.
In particular as shown in Spera et al. (2007):
- in the untreated CHO-K1 plot there is a peak at 42.45 min not
observable in the cAMP treated CHO-K1 plot;
- the intensity of the 44.90 min peak in the untreated CHO-K1 TIC
plot is double regarding the cAMP treated cells plot peak.
The total ion current of proteins of interest was used for their relative
quantification.
Since the total ion current depends not only from concentration but
also from the charge of the analyte, sample treatment was standardized
both in dilution and HPLC-ESI-MS procedures, to ensure similar bias,
namely same ion suppression, pH and organic solvent effects on the
analyte charge.
Under these conditions, the total ion current can be roughly
considered proportional to protein concentration, and it can be used to
evidence correlations existing among different proteins in different
samples (Messana et al., 2004).
Figure 2.30. HPLC-ESI-MS TIC and UV profile of nuclear protein fraction of CHO-K1
cells (grey line) and of CHO-K1 treated with cAMP (black line). Upper plot: HPLC-ESI-
MS TIC profile collected by ion-trap mass spectrometer (elution time 10-50 min).
Enlargement of the 41.7–45.5 min elution range showing the TIC profile is represented in
the box where the greater differences between the two plots are shown: the 42.45 min
peak (absent in the treated CHO-K1 plot) and the 44,90 min peak (its relative abundance
is double in the untreated CHO-K1 plot). Bottom plot: UV (214 nm) profile (elution time
10–50 min). (Reprinted with the permission from Spera and Nicolini, camp induced
alterations of Chinese hamster ovary cells monitored by mass spectrometry, Journal of
Cellular Biochemistry 102, pp. 473–482, © 2007, Wiley-Liss, Inc., a subsidiary of John
Wiley & Sons, Inc.).
2.10.1 Mass spectrometry of label-free NAPPA
In order to analyze NAPPA array it was necessary to modify the standard

MALDI target to host the NAPPA array, classically spotted on a standard
microscopy slide. For this reason an adequate lodging with the
dimension exactly the same of the array guarantees the immobility of the
glass once putted in the lodging.
Figure 2.31. MALDI-TOF-MS spectrum of human kinase NAPPA array after protein
synthesis (spot gene NA 7-A 12): high weights (20-35 kDa) for three different
spots/genes (from the top: gene NA7-A 12, gene NA 7-E 12, and gene NA 8-D 12)
(Reprinted with the permission from Spera and Nicolini, Nappa microarray and mass
spectrometry: new trends and challenges Essential in Nanoscience Booklet Series, ©
2008, Taylor & Francis Group/CRC Press, http://nanoscienceworks.org).
For an automated, reproducible and reliable MALDI identification of

each NAPPA spots we have created a new geometry file in FlexControl
(the Bruker software for MALDI-TOF control), since NAPPA geometry
is different from all the standard MALDI target geometry. Utilizing the
“Manual Fine Control” window of the software we have manually

identified each spots and then we have reordered its position in a new
file. We analyzed by MALDI TOF MS the kinase gold NAPPA array
(Figure 2.26). We followed expression of the NAPPA slides protocol by
Harvard Institute of Proteomics (joint manuscript NWI-HIP in
preparation) and once the proteins were synthesized we wash twice the
NAPPA with bi-distillated water and we proceeded according to the
previously described protocol (LaBaer and Ramachandran, 2005; Spera
and Nicolini, 2008).
Some of the identified peaks of our resulting MS in the low-medium
molecular weights are assimilable to that obtained by HIP MS analysis of
NAPPA before protein synthesis (private communication), due to the
components presents on the NAPPA (streptavidin, capture antibody,
plasmid DNA). To be sure of this assumption we have analyzed two
identical NAPPA, one before and the other after proteins synthesis (in
progress). The peaks shown in the high molecular weights (Figures 2.31)
are instead probably due to proteins synthesized on the spots. At the
moment we have some problems to perform protein fingerprint due to
the minimum drop dimension obtainable with the routine Pasteur
available and for this reason we are printing newly designed NAPPA
microarrays to overcome this problem.
The preliminary obtained results however encourage us to perform
new experiments on NAPPA of opportune geometry. Future activities
will be carried out on properly constructed NAPPA taking into
considerations all reported fluorescence, MS and bioinformatics for cell-
cycle study.
2.11 Synchrotron Radiation
The techniques of elastic scattering of X-rays are widely used as they

provide valuable tools to probe the order properties of protein crystals.
The diffraction range is reached when the incident radiation
wavelength is closed to the interatomic distances in a crystal and when
the scattering angles are wide. By a careful analysis of the integrated
diffracted intensities, one can access to the intimate atomic structure i.e.
the positions of nuclei and the spread of the electronic cloud around
atoms. Usually, the range of "diffuse scattering" involves techniques,
which allow to get statistical information at scales that are greater than
the interatomic distances. This domain is restricted in between the first
Bragg peak, which overlaps with the direct beam and the diffraction
peaks. With X-rays with a wavelength of a few angstroms, this domain
of small momentum transfer is reached in the small angle range in the
range 1–100 nm (Figure 2.32 below).
Figure 2.32. X-ray pattern of LB film containing 20 layers of wild-type (above) and
recombinant (middle) cytochrome P450scc. Below is the scattering and diffraction ranges
versus the wave vector transfer. (Part A,B: Reprinted with the permission from Nicolini
et al., Supramolecular layer engineering for industrial nanotechnology, in Nano-surface
chemistry, pp. 141–212 © 2001a, Marcel Dekker / Taylor & Francis Group LTD).
The scattering comes from strong variations of the mean electronic

density for X-rays and up to fifteen years ago, was limited to three
dimensional samples as the strong penetration depth of the radiations and
the low signal to noise ratio hampered the surface sensitivity. Quite
recently, thanks to synchrotron radiation, these techniques were extended
to surface geometry using the phenomenon of total reflection of X-rays
in the grazing incidence range. Several methods based on the utilization
of X-rays are used to study the structure of LB film structure.
Diffractometry and reflectometry are the most commonly used while
diffractometry is mainly used when well-ordered periodic structure is
under investigation and several Bragg reflections are present in the X-ray
pattern. The position of these reflections is determined by the Bragg
equation:
2Dsinθ = nλ
where D is the thickness of the periodic unit (period or spacing), λ is
the wavelength of the X-ray beam, θ is the incident angle, and n is the
number of the reflection. Therefore, the thickness of the periodic unit
(usually a bilayer) can be obtained directly from the angular position of
Bragg reflection. The other information, which can be directly obtained
from the X-ray pattern, is the correlation length (L). This parameter can
be considered as a thickness until which the film can be still considered
as an ordered one and it is determined by the following formula:
L = λ/2sin(∆θ)
where ∆θ is the half-width of the Bragg reflections. More information
can be obtained if several reflections were registered as in the case of LB
of only recombinant cytochrome P450scc but not of the wild-type (Figure
2.32). Each Bragg reflection can be considered as a component in the
Fourier row representing the electron density on the repeating unit of the
film. Kiessig fringes correspond to the interference of the X-ray beam
reflected from the air/film and film/substrate interfaces, and their angular
position gives the information about the total thickness of the LB film.
Similarly the Microfocus beamline of the European Synchrotron
Radiation in Grenoble is utilized to probe for the structure of several
protein systems in diffracting crystals.
2.11.1 Diffraction
Third generation synchrotron sources are now emitting synchrotron X-

ray beams that are trillion times more brilliant than those produced by X-
ray tubes, requiring quite smaller crystals for the 3D structure
determination. One key experimental component of nanocrystallography
is thereby an appropriately microfocussed Synchrotron Radiation
(Cusack et al., 1998), having microfocusing X-ray optics and a
microgoniometer (Figure 2.33.a) in addition to intense beams capable to
obtain X-rays diffraction patterns from microcrystals ranging in size
between 5 and 20 microns (Figure 2.33b).
Figure 2.33. The needle human protein CK2α crystal in the mounted loop (b). In the left
panel (a) is shown a photography of the crowded sample position on the microfocus
beamline with collimator (beam enters horizontally from the right), microscope (vertical),
cryo-cooling system and beam stop (not present) all very close to the crystal, which is
moved by means of a microgoniometer (Part A courtesy of Prof. Christian Riekel at
ESRF; Part B reprinted with the permission from Pechkova and Nicolini, Protein
22, pp. 117–122, © 2004a, Elsevier).
Protein microcrystallography requires indeed precision in alignment

and high mechanical stability. This calls for high power microscopes to
visualize both crystal and beam position and accurate motorized
adjustments to bring everything into alignment (Pechkova and Nicolini,
2004, 2004a, 2003a). However, one cannot indefinitely compensate for
small crystal size with increased beam intensity since at some stage so
much X-ray energy is being deposited in a small volume that the protein
structure and crystalline order will be destroyed by primary radiation
damage very quickly. Radiation damage to crystalline proteins using X-

rays is a problem which limits the structural information that can be
extracted from the sample (Garman and Nave, 2002) and only the
significant radiation stability induced in the crystal formed by our
nanofilm template method open new avenues in structural proteomics
(Pechkova et al., 2004). Recently, diffraction data could be collected on
a single extrasmall human CK2α (of about 20µm in diameter) and its
diffraction patterns were utilized to solve the structure of protein kinase
CK2α catalytic subunit 2.4 Å (Figure 2.22c). Interestingly, the collection
of data diffraction utilized for the subsequent 3D atomic structure
determination (Pechkova et al., 2003) lasted for long time. The needle
CK2α microcrystals appear quite stable to the incoming considerable
radiation preserving their shape and size without any apparent damage.
Radiation stability and high diffraction quality of nanotechnology-based
crystals were apparent on a recent systematic study at the Microfocus
Synchrotron Beamline (Pechkova et al., 2004), suggesting that protein
crystals grown by our method are less sensitive to radiation than those
obtained by classical method due to an intrinsic property of such
nanotechnology-induced microcrystals.
Earlier experiments with Microfocus were carried out with channel
receptors (Pebay-Peyroula et al., 1997) and with single
bacteriorhodopsin (bR) crystal of about 30 × 30 × 5 µm3 (one good out of
ten unutilizables), which produced a entire 2.4 Å data set and led to the
first high resolution model of bR obtained by X-ray crystallography
(Zhou et al., 2000). After that report, Ekström et al. (2003) were able to
perform an analysis of microcrystals on the actin-binding domain of
human actinin. Recently, microfocus has utilized with great success in
some other experiments leading to new atomic protein structures
determination (Luecke et al., 2001; Scheffzek et al., 2000; Berthet-
Colominas et al., 1999; Brige et al., 2002; Hanzal-Bayer et al., 2002;
Thom et al., 2002; Toeroe et al., 2001; Royant et al., 2000, 2001; Zouni
et al., 2001).
As mentioned above, the utilization of Microfocus is restricted by the
significant radiation damage to the crystal, which is one of the major
sources of error in data collection. This inherent problem in X-ray
microcrystallography has not so far been widely investigated. Radiation
damage can be partially avoided by cryo-techniques implementation, but

effect of ionizing radiation remains substantial even using moderate-
intense synchrotron facility, and for brighter sources it becomes indeed a
limiting factor (Burmeister, 2000). Beam defocusing can be used to
decrease the available flux density (Walsh et al., 1999), but the main
problem remains unsolved, especially for protein microcrystals, often
making the data collection impossible. Finally, although synchrotron
radiation damage is usually presumed to be nonspecific and manifested
as a gradual decay in the overall quality of data obtained, it was recently
reported that synchrotron radiation can cause highly specific damage
which can provide the information of structural and functional
significance (Weik et al., 2000; Ravelli et al., 2003; Matsui et al., 2002).
The curve obtained from the LB film of wild-type protein presents
neither Bragg reflections nor Kiessig fringes. Such a result means that
the film is not ordered and that there is not uniformity of the thickness
along the sample area. In the case of recombinant protein, we see Kiessig
fringes, whose angular position depends upon the number of deposited
layers. The average monolayer thickness calculated from these data is
about 6 nm, corresponding well to both the ellipsometric data and the
protein sizes from the RCSB Protein Data Bank (Berman et al., 2000).
2.11.2 Grazing Incidence Small Angle X-ray Scattering
µGISAXS is a technique of elastic scattering of X-rays widely used as

they provide valuable tools to probe the order properties of protein
crystals.
The diffraction range is reached when the incident radiation
wavelength is closed to the interatomic distances in a crystal and when
the scattering angles are wide. By a careful analysis of the integrated
diffracted intensities, one can access to the intimate atomic structure, i.e.,
the positions of nuclei and the spread of the electronic cloud around
atoms. Usually, the range of “diffuse scattering” involves techniques,
which allow getting statistical information at scales that are greater than
the interatomic distances. This domain is restricted in between the first
Bragg peak, which overlaps with the direct beam and the diffraction
peaks (Nicolini and Pechkova, 2004). With X-rays with a wavelength of
a few angstroms, this domain of small momentum transfer is reached in

the small angle range in the range 1–100 nm (Nicolini, 1996a). The
scattering comes from strong variations of the mean electronic density
for X-rays and up to fifteen years ago, was limited to three dimensional
samples as the strong penetration depth of the radiations and the low
signal to noise ratio hampered the surface sensitivity. Quite recently,
thanks to synchrotron radiation, these techniques were extended to
surface geometry using the phenomenon of total reflection of X-rays in
the grazing incidence range. The field of thin films growth brought a
need of knowledge about layer morphology and sizes of quantum dots,
supported islands or buried particles which, as shown in Nicolini and
Pechkova (2004) as pushed the development of Grazing Incidence Small
Angle X-Ray Scattering using the 5 micron beam size at ESRF in
Grenoble (µGISAXS), providing information about the dependence of
the electronic density perpendicular to the surface, like those due to the
roughness of a surface, the lateral correlations, sizes and shapes of
growing protein nanocrystals, of metallic islands or of the self organized
dots superlattices (Roth et al., 2003).
The protein templates were freshly plated in the hanging drop
container during the three-day experiment.
The glass substrate with the thin film template and the droplet was
removed from the container and the vacuum grease area was cleaned to
ensure no contamination of the signal. The droplet existed for > 60min in
air until complete evaporation. Hence an optimum timing had to be
found concerning the preparation, adjustment in the beam and data
acquisition time. The glass substrate was subsequently brought into the
beam and the incident angle αi adjusted to about 1°. This procedure was
restricted to less than 20 min in order to avoid precipitation of salt or
nanocrystals from the solution to the substrate, which would lead to a
contamination of the µGISAXS signal.
The droplet diameter was about 2 mm. Hence the footprint of the X-
ray beam will be fully within the droplet diameter. The small beam size
allows avoiding excessive liquid scattering and provides therefore a
reasonable signal-to-background ratio. The position of the drop relative
to the beam was determined by an absorption scan with a photodiode.
Experiments were performed with the beam at the center of the droplet at
the contact area droplet, protein template in order to optimize the signal
of the weakly scattering biopolymer samples. Data collection times for
individual droplets varied from 7 min to 20 min. This is well below
minimum droplet evaporation time of about 60 min. This “Stop”
procedure described above interrupts therefore crystal growth at specific
times and allows studying freshly grown thin films.
Figure 2.34 shows an enlargement of the Yoneda region of the
corresponding µGISAXS pattern. This region is most sensitive to
structural and morphological changes of the surfaces due to the
interference effect involved in the occurrence of the Yoneda peak. The
µGISAXS pattern is scaled to the same intensity. After 46 h clearly a
new Yoneda peak (N) emerges next to the Yoneda peak existing at
shorter times (G) with a critical angle below that of the substrate.
Figure 2.34. Development of Yoneda peak after plating (G) showing the development of
a new peak (N) due to scattering from a protein layer (see text). (Reprinted with the
permission from Pechkova et al., µGISAXS and protein nanotemplate crystallization
methods and instrumentation, Journal of Synchrotron Radiation 12, pp. 713–716, ©
2005a, Blackwell Publishing).
One could suppose that salt (NaAc) is precipitating on the substrate

thus leading to a new rough layer of small crystals. This is unlikely for
two reasons: (i) in view of the rapid data collection after removal of the
sample from the container. With the hanging drop method itself, salt
precipitation on the substrate should be reduced. In addition this second
peak does not appear at shorter times, (ii) one can calculate the nominal
critical angle of NaAc using the known molecular mass and composition
as αc(NaAc)=0.23°, which is larger than the critical angles of the glass
and the protein. Hence as a working hypothesis one can attribute the
peak at higher αf-values (G) to that of the glass substrate. The newly
developing peak (N) thus seems to be related to the protein itself.
The occurrence of a second Yoneda peak indicates the development
of a rough layer. Two models can be imagined both competing to
increase layer roughness (Figure 2.35). In model A the roughness of the
template layer is increased by holes.
Figure 2.35. Two possible models for the increasing layer roughness with time. Model A
favours increasing roughening via ablation of the nanostructured layer, while model B is
nucleation-based (Reprinted with the permission from Pechkova et al., µGISAXS and
protein nanotemplate crystallization methods and instrumentation, Journal of Synchrotron
Radiation 12, pp. 713–716, © 2005a, Blackwell Publishing).
That is to say this model assumes a decreasing density of the protein

template layer as the layer itself is removed by the protein solution thus
triggering or assisting the formation of new crystals in the solution.
Model B is motivated by a nucleation and growth process of possible
protein nanocrystals on the nanostructured template, whereby protein
molecules out of the solution might be adsorbed on the nanostructured
template. At the present state of experiments we cannot favour model A
or model B, i.e., a build-up of holes or a layer of small islands, acting as
nucleation centers for crystals. This question will be addressed in future
in situ µGISAXS experiments based on scaled intensity data. A
feasibility study on the template-assisted protein crystal growth using

µGISAXS has been reported in this paper. The small droplet size used in
hanging drop experiments necessitates the use of small X-ray beams and
hence the µGISAXS technique. The recording of diffuse scattering
implies measuring times up to several minutes due to the intensity
difference of several orders of magnitude between the specularly
reflected and the diffuse scattering signal. As compared to bulk protein
crystallography, the enlarged beam footprint results in less radiation
damage. We have collected a time series of µGISAXS patterns,
observing indeed a stable µGISAXS pattern at least up to the first few
images being acquired for the experiments here reported. It appears then
that we can exclude that the surface morphology changes are due to
beam damage since the comparison among the different crystallization
time intervals after plating is made among the corresponding pattern
being immediately acquired. In future experiments one could also
envisage to translate the beam laterally across the substrate surface in
order to further limit radiation damage.
As compared to topography-sensitive methods (i.e., AFM),
µGISAXS exploits the penetration depth of X-rays. This allows
investigating the interface solution-template where the significant signals
were observed in the present case. Working with a droplet permits to
avoid time-consuming cleaning of the sample, i.e., salt removing. Thus
the small remaining droplet is not hindering the measurement of the
interface solution-template, where nanocrystal growth is expected to take
place. The perspective to combine grazing incidence with wide-angle
scattering in order to really see crystallisation (Bragg peaks) appear quite
promising and we plan to do it in future in situ experiments.
Future experiments will be indeed extended to in situ investigations
of template assisted protein growth, which provides several advantages.
Thus crystal growth can be examined continuously. Furthermore,
background subtraction will be facilitated and any contamination of the
µGISAXS signal by cleaning or precipitating salt will be avoided. A
possible set-up, namely a hanging drop container with thin entry and exit
windows, is shown schematically in Pechkova and Nicolini (2003,
2004a). This set-up is inversed to the traditional µGISAXS set-up
(Müller-Buschbaum et al., 2003; Roth et al., 2003) in order to obtain an
upward-scattering geometry. This would allow for the first time

following protein crystal growth and nucleation from its very early stages
after plating. As compared to AFM, the hanging drop could be
investigated by µGISAXS in its natural growth position without
disturbing the growth process.
Figure 2.36. Schematic view of the microbeam grazing incidence small-angle X-ray
scattering setup (µGISAXS) at ID13/ESRF. The sample is mounted on a xyz-gantry and
a two-axis goniometer (φx, φy). The scan direction is y. αi denotes the angle between the
incident beam and the sample surface, αf the corresponding exit angle, and 2φ the out-of-
plane angle. The flight path (L=1.15 m) between sample and the 2-D detector (C) is
evacuated (10-2 mbar). The typical 2-D µGISAXS signal of the cytochrome P450scc drop
sitting on a LB layer is also shown. In the bottom right corner it is shown the
experimental layout of the protein solution typical droplet sitting on the protein layer
deposited over the glass. µGISAXS measured with 5 micron beam size points to the
present of P450scc nanoscrystal in the early stage of the crystallization with LB
((Reprinted with the permission from Nicolini and Pechkova, Structure and growth of
ultrasmall protein microcrystals by synchrotron radiation. I µGISAXS and
microdiffraction of P450scc, Journal of Cellular Biochemistry 97, pp. 544–552, © 2006,
Wiley-Liss, Inc., a subsidiary of John Wiley & Sons, Inc.).
The field of thin films growth brought a need of knowledge about

layer morphology and sizes of quantum dots, supported islands or buried
particles which, as shown in Figure 2.25 has pushed the development of

Grazing Incidence Small Angle X-Ray Scattering using the 5 micron
beam size at ESRF in Grenoble (µGISAXS), providing information
about the dependence of the electronic density perpendicular to the
surface, like those due to the roughness of a surface, the lateral
correlations, sizes and shapes of growing protein nanocrystals, of
metallic islands or of the self organized dots superlattices (Roth et al.,
2003). Such informations are of prime interest in understanding the link
between the mechanisms of growing crystal morphology and its physical
or chemical properties for both lysozymes (not shown) and cytochromes.
Chapter 3
Nanoscale Applications in Science and Health
This chapter overviews the present status of nanoscale applications of

organic and biological nanotechnology to science and health, namely of
those capable so far to yield a potential scientific and technological
progress both to protein crystallography, medicine, genomics, proteomics
and cell science, and to mechanics, optics and magnetism. Even if
particular emphasis is placed on what has been accomplished in our
laboratory in the last eight years, significant reference to the recent
activity of numerous other groups is also given.
3.1 Nanobiocrystallography
One of the newest approaches to structural proteomics is the emerging

field of protein nanocrystallography at the intersection between
nanotechnology and proteomics (Pechkova and Nicolini, 2003, 2004a).
This new field results from the combination of advanced
nanotechnologies – particularly atomic force microscopy (AFM), thin-
film nanotemplate technology, nanogravimetry – and of advances in
synchrotron radiation, namely micro-nanofocused diffraction and micro-
nanoGISAXS. It should be noted that nanocrystallography as described
here does not refer to the study of self-assembled nanocrystals (i.e.,
crystals of nanometer size) of silver, cobalt, gold and/or nanoparticles
created using reverse micelles or similar technologies. In addition, it does
not refer to the nanodrop or microdrop crystallization technology
resulting from the exciting advances made in microfluidic chips
(Pechkova and Nicolini, 2003), which are now commercially available
and show some accomplishement.
118
Nanoscale Applications in Science and Health 119
This progress is made now possible by the third generation

synchrotron sources emitting synchrotron X-ray beams that are trillion
times more brilliant than those produced by X-ray tubes, requiring quite
smaller crystals for the 3D structure determination. One key
experimental component of nanocrystallography is indeed an
appropriately microfocused synchrotron radiation (Cusack et al., 1998),
having micro-focusing X-ray optics and a microgoniometer (Figure 3.1)
in addition to intense beams capable to obtain X-rays diffraction patterns
from microcrystals ranging in size between 5 and 20 microns (Figure
3.2).
Figure 3.1. Synchrotron radiation with micro-focusing X-ray optics and

microgoniometer. Photograph of the crowded sample position on the microfocus
beamline with a collimator (beam enters horizontally from the right), microscope
(vertical), cryo-cooling system and beam stop (not present) all very close to the crystal,
which is moved using a microgoniometer. (Courtesy from Christian Riekel at ESRF).
Protein microcrystallography requires indeed precision in alignment

and high mechanical stability. This calls for high power microscopes to
visualize both crystal and beam position and accurate motorized
adjustments to bring microcrystals into alignment in order to obtain
proper diffraction pattern (Figure 3.2).
However, one cannot indefinitely compensate for small crystal size
with increased beam intensity since at some stage so much X-ray energy
is being deposited in a small volume that the protein structure and

crystalline order will be destroyed by primary radiation damage very
quickly.
Figure 3.2. Human kinase and cytochrome P450scc microcrystals. Human CK2α
microcrystal mounted on the nylon loop before (a) and after (b) exposure to the
microfocus Synchrotron Beamline for the collection of complete diffraction data set. (c)
Human CK2 microcrystals obtained by the protein nanofilm template. (d) Diffraction
pattern of bovine P450scc ultramicrocrystal powder (A,C: Reprinted with permission
from Pechkova and Nicolini, Atomic structure of a CK2α human kinase by microfocus
diffraction of extra-small microcrystals grown with nanobiofilms template, Journal of
Cellular Biochemistry 91, pp. 1010–1020, © 2004, Wiley-Liss, Inc., a subsidiary of John
Wiley & Sons, Inc.; B: Reprinted with permission from Pechkova and Nicolini, Protein
22, pp. 117–122, © 2004a, Elsevier; D: Reprinted with permission from Nicolini and
Pechkova, Structure and growth of ultrasmall protein microcrystals by synchrotron
radiation: I µGISAXS and microdiffraction of P450scc, Journal of Cellular Biochemistry
97, pp. 544–522, © 2006, Wiley-Liss, Inc., a subsidiary of John Wiley & Sons, Inc.).
Radiation damage to crystalline proteins using X-rays is a problem,

which limits the structural information that can be extracted from the
sample (Garman and Nave, 2002) and only the significant radiation
stability induced in the crystal formed by our nanofilm template method
open new avenues in structural proteomics (Pechkova et al., 2004).
A protein will stay in solution only up to a certain concentration.
Once this limiting concentration is reached, the solution will no longer
remain homogeneous, but a new state or phase will appear. This

phenomenon forms the basis of all protein crystallization experiments.
By changing the solution conditions, the crystallographer tries to exceed
the solubility limit of the protein so as to produce crystals (McPherson,
1999). This plan rarely runs smoothly. After changing the solution
conditions, one of several difficulties is usually encountered: (i) nothing
happens, i.e., the protein solution remains homogeneous; (ii) a new phase
appears, but it is not a crystal. Instead, it is an aggregate or a liquid; or
(iii) crystals do form, but they are unsuitable for structure determination
because they give a poor X-ray diffraction pattern. It is often possible to
overcome these difficulties by trial and error-repeated crystallization
attempts with many different conditions – but this strategy does not
always work. Even when it is successful, the lessons learned cannot be
easily generalized; the conditions, which work with one protein, are not
necessarily optimal for a different protein.
The problems associated with producing protein crystals have
stimulated fundamental research on protein crystallization. An important
tool in this work is the phase diagram. A complete phase diagram shows
the state of a material as a function of all of the relevant variables of the
system (Zemansky and Dittman, 1997).
For a protein solution, these variables are the concentration of the
protein, the temperature and the characteristics of the solvent (e.g., pH,
ionic strength and the concentration and identity of the buffer and any
additives). The most common form of the phase diagram for proteins is
two-dimensional and usually displays the concentration of protein as a
function of one parameter, with all other parameters held constant
(Saridakis et al., 1994).
Three-dimensional diagrams (two dependent parameters) have also
been reported (Sauter et al., 1999) and a few more complex ones have
been determined as well (Ewing et al., 1994).
When a protein crystal (Figure 3.3) is placed in a solvent, which is
free of protein, the crystals will begin to dissolve. If the volume of
solvent is small enough, the crystal will not dissolve completely; it will
stop dissolving when the concentration of protein in solution reaches a
specific value.
At this concentration, the crystal loses protein molecules at the same

rate at which protein molecules rejoin the crystal - the system is said to
be at equilibrium.
Figure 3.3. A schematic phase diagram of protein showing the solubility of a protein in
solution as a function of the concentration of the precipitant present (Reprinted with the
permission from Nicolini and Pechkova, Nanostructured biofilms and biocrystals, Journal
of Nanoscience and Nanotechnology 6, pp. 2209–2236, © 2006a, American Scientific
Publishers, http://www.aspbs.com).
The concentration of proteins in the solution at equilibrium is the

solubility. The solubility of a protein varies with the solution conditions.
A schematic diagram of a solubility curve, illustrating how the solubility
varies with the concentration of a precipitant (i.e., polyethylene glycol
(PEG) or a salt), is shown in Figure 3.4.
Crystals dissolve in the undersaturated region-where the
concentration is below the protein solubility – and grow in the
supersaturated region. The three subdivisions of the supersaturated
region are the metastable, labile, and precipitation zones) (Asherie,
2004).
In principle, crystals will form in any protein solution that is
supersaturated, i.e., when the protein concentration exceeds the
solubility. In practice, crystals hardly ever form unless the concentration
exceeds the solubility by a factor of at least three (Chernov, 1997). The
large supersaturation is required to overcome the activation energy

barrier, which exists when forming the crystal.
Figure 3.4 Crystallization process: free energy barrier overcoming (Reprinted with the
This barrier represents the free energy required to create the small
microscopic cluster of proteins – known as a nucleus – from which the
crystal will eventually grow (Figure 3.4). Since there is an energy barrier,
nucleation (the process of forming a nucleus) takes time. If the
supersaturation is too small, the nucleation rate will be so slow that no
crystals form in any reasonable amount of time (Kashchiev, 2000). The
corresponding area of the phase diagram is known as the “metastable
zone”. In the “labile” or “crystallization” zone, the supersaturation is
large enough that spontaneous nucleation is observable. If the
supersaturation is too large, then disordered structures, such as
aggregates or precipitates, may form. The “precipitation zone” is
unfavorable for crystal formation, because the aggregates and
precipitates form faster than the crystals.
The three zones are illustrated schematically in Figure 3.3. Since
these zones are related to kinetic phenomena, the boundaries between the
zones are not well defined (this in contrast to the solubility line which is
unambiguous description of the equilibrium between solution and
crystal).
Even though the division in zones is qualitative, the different

behaviors serve as guide when searching for the appropriate conditions to
produce crystals (Saridakis and Chayen, 2003).
When a precipitant is added to a protein solution to help produce
crystals, liquid drops sometimes form. These drops, also referred to as
“oils” or “coacervates”, can occasionally be observed by simply
changing the temperature, pH, or other solution condition. Such drops
generally contain a high concentration of protein. Under the influence of
gravity, the drops may separate from the rest of the solution. Eventually,
two liquid phases will form, and the concentrations of the various
components of the original solution will be different in the two phases.
This phenomenon is known as liquid-liquid phase separation (LLPS).
Although LLPS is analogous to water condensing from steam, there is a
significant difference. While liquid water is stable once it has formed, the
protein-rich and protein-poor liquid phases are not. The phases may exist
for days, even weeks, but LLPS is inherently metastable with respect to
crystallization (Asherie et al., 2001). In other words, the protein-rich
liquid phase can convert into crystals. LLPS can promote crystal
formation even without the formation of a macroscopic protein-rich
phase (Kuznestov et al., 2001). The precise mechanism by which LLPS
promotes crystal nucleation is still not known. One factor is the high
protein concentration, which exists in the droplets. This high
concentration corresponds to a large supersaturation and so increases the
crystal nucleation rate. Another factor may be the wetting of the surface
of the crystal by one of the liquid phases (Wolde and Frenkel, 1997).
Systematic studies have shown that in the case of lysozyme both factors
are important (Galkin and Vekilov, 2000). Crystal formation can be
initiated and its growth accelerated by a new method shown in Figure
3.5.
The protein thin-film nanotemplate is created using Langmuir-
Blodgett (LB) technology or modifications of it (Pechkova and Nicolini,
2004a), and is subsequently deposited on a solid glass support, to be
placed in the appropriate vapor-diffusion apparatus. This LB protein thin
film assumes the role of the template for protein nucleation and crystal
growth. During the screening procedure, the following parameters can be
varied: protein monolayer surface pressure, precipitant nature and
concentration, and number of protein thin-film monolayers. The protein

surface pressure chosen for the deposition should correspond to that of
the highly packed, ordered monolayer. In our experience, the surface
pressure corresponding to the closely packed system is 25 mN/m-1 for
chicken egg-white lysozyme, 20 mN/m-1 for human protein kinase CK2α
and 15 mN/m-1 for bovine cytochrome P450scc.
Figure 3.5. Vapour diffusion hanging drop nanotemplate method (Reprinted with the
permission from Pechkova et al., Three-dimensional atomic structure of a catalytic
subunit mutant of human protein kinase CK2, Acta Crystallographica D59, pp. 2133–
2139, © 2003, International Union of Crystallography).
It should be borne in mind that the surface pressure can also be

influenced by subphase composition. It should be noted that a possible
physical explanation for the increased crystal growth observed with our
method (Pechkova and Nicolini, 2002a,b) comes from the variation in
template surface pressure.
The increased anisotropy of the thin-film template associated with the
increased surface pressure and protein molecule orientation (Nicolini,
1997) causes a dipole moment in the LB monolayer. Indeed, in previous
studies (Facci et al., 1993), the surface potential has varied from 20.2
mV in the self-assembly (randomly oriented proteins) to 280 mV in the
LB film (ordered and oriented proteins).
Originally the nanotemplate induction of this dipole moment was
offered as the possible cause of the controllable and predictable
nucleation and growth of the protein crystal (Pechkova and Nicolini,
2002, 2002a).
Recently it was shown that the effect could also be due to the
migration of thin protein film fragments formed by highly packed and
ordered proteins into solution, in which they constitute the nucleation
centers of the protein microcrystals (Pechkova et al., 2005a).
Typically protein crystals are brittle and crush when touched with the
tip of a needle, while salt crystals that can sometimes develop in
macromolecule crystallization experiments will resist this treatment. This
fragility is consequence both of the weak interaction between
macromolecules within crystal lattices and of the high solvent content
(from 20% to more than 80%) in these crystals (Ducruix and Giege,
1992). For that reason macromolecular crystals have to be kept in a
solvent saturated environment, otherwise dehydration will lead to crystal
cracking and destruction.
The high solvent content, however, has useful consequences because
solvent channels permit diffusion of small molecules, as property used
for the preparation of isomorphous heavy atom derivatives needed to
solve the structure.
Further crystal structure can be considered as native structure as is
indeed directly verified in some cases by the occurrence of enzymatic
actions within crystals lattices upon diffusion of the appropriate ligands
(Mozzarelli and Rossi, 1996).
Other characteristic properties of macromolecular crystals are their
rather weak optical birefringence under polarized light: colors may be
intense for large crystals (isotropic cubic crystals or amorphous material
will not be birefringent).
Also, because the building blocks composing macromolecular are
enantiomers (L-amino acids in proteins-except in the case of some
natural peptides-and D-sugar in nucleic acids) macromolecules will not
crystallize in space group with inversion symmetries.
Accordingly, out of the 230 possible space groups, macromolecules
do only crystallize in the 65 space groups without such inversion. While
small organic molecules prefer to crystallize in space groups in which it
is easiest to fill space, proteins crystallize primarily in space groups,
which it is easiest to achieve connectivity.
Macromolecular crystals are also characterized by large unit cells

with dimension that can reach up to 1000 Å for virus crystals (Usha et
al., 1984).
In a crystalline form proteins are in a highly ordered three-
dimensional array where the protein molecules are bound to each other
with specific intermolecular interactions. Protein crystals contain not
only protein molecules but also uniform solvent-filled pores that
constitute 30–78% of the crystal volume depending on the protein and
the crystallization conditions.
3.1.1 Radiation resistance
The emerging technology presented in Figure 3.5 produces

nanostructured radiation-stable (Figure 3.6) biocrystals of any dimension
by nanobiofilm template and characterizes them by the nanotechnologies
earlier described in chapter 2, namely AFM, nanogravimetry and
microfocused synchrotron radiation in terms of scattering and diffraction.
Figure 3.6. Radiation resistant nanostructured lysozyme microcrystal as compared to

classical counterparts (Reprinted with the permission from Pechkova et al., Radiation
stability of protein crystals grown by nanostructured templates: synchrotron microfocus
analysis, Spectrochimica Acta 59, pp. 1687–1693, © 2004, Elsevier).
This new technology is generalizable to all classes of proteins which

can be immobilized in thin LB film at the air-water interface and is
thereby providing a new route to structural proteomics with far reaching
implications for the discovery of new drugs and for other interesting
applications in material science and electronics (see later).
Primary radiation damage is linearly dependent on the X-ray dose

even when the crystal is at cryogenic temperatures. Above a certain level
an excessive damage of the crystal develops which is interpreted as the
onset of secondary and/or tertiary radiation damage.
This upper limit of X-ray dose is compared with Henderson’s limit
(Henderson, 1990), and has profound implications for the amount of
useful X-ray diffraction data that can be obtained for crystals of a given
scattering power.
While the primary dose-dependent radiation damage is unavoidable,
secondary radiation damage can be partially avoided by cryo-techniques
implementation, but the effect of ionizing radiation remains substantial
even using moderately intense synchrotron facilities, and for brighter
sources it becomes indeed a limiting factor.
Radiation damage can cause specific changes in protein crystal
structure. Disulphide bonds break up and acidic side chains become
decarboxylated.
The unit cell volume increases, and the molecule might undergo small
rotations and translations. Non-isomorphism is introduced, which can
easily cause large differences in the structure factors.
The radiation stability induced in the biocrystal by the recently
introduced nanofilm template method is suggested by a recent systematic
study shown in Figure 3.6 (Pechkova et al., 2004).
For the first time, highly diffracting and uniquely radiation stable
protein microcrystals are consistently obtained by the nanotechnology-
based method.
3.1.2 New protein structures
Until now in the last century the atomic three-dimensional structures of

many proteins of different size up to the very large ribosomes have been
solved (Figure 3.7), but they represent only a small fraction of the
existing proteins in nature.
The significant radiation stability induced in the crystal formed by the
nanofilm template method has opened new avenues in structural
proteomics (Nicolini and Pechkova, 2004; Pechkova et al., 2003).
Figure 3.7. Protein crystal structures solved in the last century.
Indeed, diffraction data used for the subsequent determination of 3D

atomic structures were collected on a single, miniscule human CK2α
microcrystal (20 mm diameter) by the European Synchrotron Radiation
Facility (ESRF) microfocus beamline (beam size 20 × 20 µm2) and used
to solve the structure of the protein kinase CK2α catalytic subunit 2.4 Å
(Figure 3.8) (Pechkova et al., 2003; Pechkova and Nicolini, 2004).
For what concerns the application of protein biocrystal it must be
remembered that in early days the crystallization was an efficient protein
purification method. Now is very rare and is replaced by
chromatographic methods. However, protein purification by
crystallization has many advantages: high yield, high purity in one step,
unlimited scale up possibilities, and the product is highly concentrated
protein crystal slurry ready for further formulation. Some industrial
proteins have been purified by crystallization in large-scale. Furthermore
a majority of small molecular weight drugs are produced in crystalline
form because of the high storage stability, purity, and reproducibility of

the drug properties (Hancock and Zografi, 1997).
Figure 3.8. Atomic structure and diffraction patterns of human kinase microcrystal. Left:
cartoon representation of the three-dimensional atomic structure of human CK2α. The N-
terminal domain is in dark blue and the C-terminal domain is in light blue. Spheres mark
the positions of the three point mutations. Right: synchrotron diffraction pattern of human
CK2α microcrystals (Left: reprinted with the permission from Pechkova et al., Three-
dimensional atomic structure of a catalytic subunit mutant of human protein kinase CK2,
Acta Crystallographica D59, pp. 2133–2139, © 2003, International Union of
Crystallography; right: reprinted with the permission from Pechkova and Nicolini,
Atomic structure of a CK2α human kinase by microfocus diffraction of extra-small
microcrystals grown with nanobiofilm template, Journal of Cellular Biochemistry 91, pp.
1010–1020, © 2004, Wiley-Liss, Inc., a subsidiary of John Wiley & Sons, Inc.).
There are hundreds of macromolecular therapeutic agents used in

clinical trials or approved as drugs (Thayer, 2003). However, only
insulin is produced and administered in a crystalline form (Jen and
Merkle, 2001). According to Margolin and Navia (2001) the
crystallization of macromolecular pharmaceuticals can offer significant
advantages, such as:
a) protein purification by crystallization as presented above;
b) high-stability of the protein product compared with soluble or
amorphous forms;
c) crystals are the most concentrated form of proteins, which is a
benefit for storage, formulation, and for drugs that are needed in high
doses (e.g., antibiotics);
d) the rate of crystal dissolution depends on the morphology, size, and

additives; thus, crystalline proteins may be used as a carrier-free
dosage form.
The most important applications of biocrystal nanofilm-based are
presently the determination of protein structure at atomic resolution and
the construction of new innovative 3D materials and devices. The whole
process of structural determination by X-ray crystallography and the
state-of-the-art methods in the field have been reviewed recently in the
context of high-throughput crystallization, stressing the merits and
caveats of nanodrop setups for both crystal screening and growth.
Figure 3.9. Diffraction pattern of bovine P450scc ultramicrocrystal powder (Left)

apparently present in the microfocus loop (Right) containing the P450scc drop at 30
hours after the beginning of the crystallization process (Reprinted with the permission
from Nicolini and Pechkova, Structure and growth of ultrasmall protein microcrystals by
synchrotron radiation: I µGISAXS and µdiffraction of P450scc, Journal of Cellular
Biochemistry 97, pp. 544–552, © 2006, Wiley-Liss, Inc., a subsidiary of John Wiley &
Sons, Inc.).
The term “protein nanocrystallography” is indeed more appropriate

(Pechkova and Nicolini, 2003, 2004a; Pechkova et al., 2004) and gave
already the proof of principles with the three-dimensional atomic
structure of a catalytic subunit mutant of human protein kinase CK2
(Figure 3.8) (Pechkova et al., 2003) and of few others (Pechkova and
Nicolini, 2004a).
Microdiffraction was recently carried out at the Microfocus beamline
of the European Synchrotron Radiation in Grenoble also to probe for the
structure of protein system, such as P450scc cytochromes that has
remained unsolved until now. This characterization has been recently

done (Nicolini and Pechkova, 2004) in the form of “powder” P450scc
microscrystal obtained by the homologous nanotemplate method. The
periodic structure of the P450scc cytochrome appears evident from the
diffracting rings present in Figure 3.9; similarly Tripathi et al.,
(submitted) have determined the periodic structure of three different
ribosomal proteins so far unsolved utilizing powder diffraction
techniques.
3.1.3 Three-dimensional engineering
The construction of useful nanostructures via self-assembly of synthetic

organic molecules has been successfully demonstrated for various
applications, including molecular motors. The analogous use of
biological molecules such as DNA or proteins, as “building blocks” for
the in vitro construction of nanostructures is less developed and its
potential still far from being fully exploited.
Self-assembly of three dimensional nanostructured functional arrays
is a major challenge in nanotechnology, mainly in the fields of photonic
crystals, quantum dots arrays and metallic nanoparticles. Exploitation of
the inherent potential in the use of biological macromolecules, DNA and
proteins, as “guiding template” for the construction of one and two
dimensional nanoarrays of nanoparticles and quantum dots is just
beginning to emerge. The array of inner spacing of protein crystals
routinely prepared for X-ray crystallography attracted only little attention
until now: it was noted that different crystallization conditions applied to
same protein may result in different packing and attempts were made to
analyze protein-protein interactions for better understanding of packing
patterns. The use of protein crystal spacing as functional part for
practical purposes was limited to the use of chemically cross-linked
enzyme crystals as immobilized biocatalysts for a variety of synthetic
applications with effective diffusion taking place via small channels.
These studies have clearly demonstrated that protein crystals, routinely
obtained either via common crystallization procedures or by the
nanobiofilm templates here described, can be readily stabilized by
chemical cross-linking, e.g., with glutaraldehyde (Dotan et al., 2001)

(Figure 3.10).
Figure 3.10. Inner water distribution in 3D lysozyme crystal and their replacement after
glutaraldehyde staining with gold nanoparticles (Reprinted with the permission from
Dotan et al., Supramolecular assemblies made of biological macromolecules, in
Nanosurface chemistry, pp. 461–471, © 2001, CRC Press LLC, Taylor and Francis
Group LLC).
In this context it was also suggested that protein crystal spacing

“channels” could be used as “natural zeolites” for chromatographic
purposes. Particularly promising appears the use of directed self-
assembly of a binding protein via specific cross-linking into pre-designed
diamond-like protein crystal and the computer simulation of the array of
the enzyme crystal cavities. In this case, the construction of electronic
nanostructures has been successfully demonstrated (Dotan et al., 2001;
Pechkova and Nicolini, 2003), integrating in a novel “3D crystal
engineering” approach protein “building block” selection (a tetrahedral
lectin) and molecular modeling (for bi-ligand cross-linker design),
thereby potentially providing a versatile tool for the preparation of novel
composite nanostructured materials with their internal cavities as
“guiding template” with potential applications in the area of photonic
crystals and of semiconductors.
In summary, in many applications crystallized proteins are not

suitable for use as such as a result of their fragility and solubility. In
order to produce a crystalline protein matrix that is insoluble also in other
conditions than those used in crystallization, the crystals have to be
chemically cross-linked. In general, chemical cross-linking of protein
crystals (Figure 3.10) creates an active and microporous protein matrix
that can be used in catalytic and separation applications as molecular
electronics. Self-assembled crystals of nanometer size with potential
electronic applications can indeed be made of silver cobalt, gold and/or
nanoparticles and are fabricated by using reverse micelles or similar
technologies. Other examples of potentially interesting materials are
calcium carbonate crystals and nanohedra, which use symmetry to design
self-assembling protein cages, layers, crystals and filaments.
All the above materials based on organic and inorganic technologies
compete with the protein-based technologies earlier described in this
chapter, repeating the race to new electronic devices and systems which
is under way world-wide between inorganic, organic and biological
approaches.
A recent review by Nicolini and Pechkova (2006a) provides an
overview of the field of nanostructured biomaterials and biocrystals, with
particular emphasis on those being developed over the years by our
Institute. It intends mainly to exemplify the tremendous advancement of
this field and of their associated nanobiotechnologies (Gourley, 2005),
which in recent time have been accelerating at a very rapid pace and are
constantly redefining themselves worldwide (Zhao and Zhang, 2004;
Zhang et al., 2002; Wu and Payne, 2004; Fu et al., 2005). As a result
several areas of research, despite the numerous pending problems and
limitations previously outlined, appear to confirm their validity and the
various applications of the above technologies being here presented are
showing a significant increase in the potential market size to an
unprecedented rate.
3.1.4 Basics of crystal formation
µGISAXS information (see chapter 2) are of prime interest in

understanding the link between the basic mechanisms of growing crystal
morphology and its physical or chemical properties for both lysozymes
(not shown) and cytochromes (Nicolini and Pechkova, 2006) (Figure
3.11).
Figure 3.11. Full scattering and Yoneda regions of µGISAXS at the Microfocus beamline
of the European Synchrotron Radiation in Grenoble of cytochrome P450scc LB. Raw
µGISAXS patterns of a P450scc crystallizing drop sitting over one monolayer of
homologous P450 at 44 hours after plating using nanotemplate-assisted hanging vapor
diffusion method. Images were taken at 12 successive time intervals while salt and
protein crystals were sedimenting on the glass and the solution was evaporating. (Left)
total µGISAXS patterns of qy versus qz in A-1 unit, where, due to its high intensity, the
specular peak is covered by a beamstop. (Right) Yoneda region of αf dependency at
αi=0.92 (Reprinted with the permission from Nicolini and Pechkova, Structure and
growth of ultrasmall protein microcrystals by synchrotron radiation: I µGISAXS and
µdiffraction of P450scc, Journal of Cellular Biochemistry 97, pp. 544–552, © 2006,
The molecular mechanisms and the early steps of the growth of these
protein crystals have been characterized by µGISAXS at the Microfocus
beam line of the European Synchrotron Radiation Facility in Grenoble.
This new technology was indeed being utilized to investigate protein
crystal growth and nucleation mechanisms with and without

nanotemplate, with quite interesting results pointing to a more clear
understanding of the crystallization process (Nicolini and Pechkova,
2006; Pechkova and Nicolini, 2006). This new technology is indeed
being utilized to investigate protein crystal growth and nucleation
mechanisms with and without nanotemplate, with quite interesting
results pointing to a more clear understanding of the crystallization
process (manuscript in preparation).
It has been quite difficult so far to find the key to every existing
protein for developing the general procedure for protein crystallization.
Indeed, to be crystallized each protein requires its own specific
conditions, which are often difficult to determine and that require
empirical extensive searching (Thorsen et al., 2002; Rupp, 2003). That is
why protein crystallography is often called an art instead of a science.
For this reason the new approach based on nanotechnology had been
introduced with potential applications in life sciences and drug industry.
In addition to pharmaceutical industry, nanocrystallography can be also
useful to fabricate nanoscale arrays, which up to now are based on
lithographic techniques, utilizing protein crystals for the construction of
next-generation electronic and photonic devices (McMillan et al., 2002).
3.1.4.1 Cytochrome P450scc
Ultrasmall P450scc cytochromes microcrystals are grown by classical

hanging vapor diffusion and by its modification using homologous
protein thin-film template displaying a long-range order. The nucleation
and growth mechanisms of P450scc microcrystals are studied at the thin
cytochrome film surface by a new µGISAXS technique developed at the
microfocus beamline of the European Synchrotron Radiation Facility in
Grenoble.
The scattering comes from strong variations of the mean electronic
density for X-rays and up to fifteen years ago was limited to three
dimensional samples as the strong penetration depth of the radiations and
the low signal to noise ratio hampered the surface sensitivity. Quite
recently, thanks to highly focused synchrotron radiation (Riekel, 2000),
these techniques were extended to surface geometry using the
phenomenon of total reflection of X-rays in the grazing incidence range

(Müller-Buschbaum et al., 2003). The field of thin films growth brought
a need of knowledge about layer morphology and sizes of quantum dots,
supported islands or buried particles which has pushed down to the
micrometer size for incoming beam the development of Grazing
Incidence Small Angle X-ray Scattering (µGISAXS) (Müller-
Buschbaum et al., 2000, 2003; Roth et al., 2003), providing information
at the highest sensitivity down to the nanometer scale for the dependence
of the electronic density perpendicular to the surface, like those due to
the roughness of a surface, the lateral correlations, sizes and shapes of
gold nanoparticles (Roth et al., 2003). Such information are of prime
interest in understanding the link between growing ultrasmall crystal
morphology and its physical, chemical, structural properties, the latter
being also object of this work down to atomic resolution by X-ray
microfocus diffraction (Riekel, 2000).
For understanding the basic physical aspects of the template induced
P450scc cytochrome microcrystal nucleation and growth, the
synchrotron microfocus has being here used both for diffraction and
µGISAXS (Grazing Incidence Small-angle X-ray Scattering)
experiments (Roth et al., 2003; Lazzari, 2002).
As scanning force microscopy (SFM), sensitive only to surface
structures, grazing incidence small-angle x-ray scattering (GISAXS)
appears an excellently suited method for structural and morphological
studies of patterned thin films (Müller-Buschbaum et al., 1998, 2000)
and of cytochromes P450scc crystal growth and nucleation at its very
early stages, as induced by classical (Ducruix and Giege, 1999) and
nanotemplate-based (Pechkova and Nicolini, 2002; 2002a) vapour-
diffusion method. The innovative crystallization method is described in
Pechkova and Nicolini (2002; 2002a).
The protein thin-film nanotemplate is created using Langmuir-
Schaefer (LS) technology or modifications of it, and is subsequently
deposited on a solid glass support, to be placed in the appropriate vapor-
diffusion apparatus, namely the traditional hanging drop vapour-
diffusion method.
The optical elements used for the microbeam preparation and the
detector used is described in Roth et al. (2003) and in Pechkova et al.
(2005a).
The layout of the scattering measurements using the reference
cartesian frame which has its origin on the surface and is defined by its z-
axis pointing upwards, its x-axis perpendicular to the detector plane and
its y-axis along it. The light is scattered by any type of roughness on the
surface. Because of energy conservation, the scattering wave vector q is
the central quantity (A) to be monitored during the measurements. As
shown by Roth et al. (2003) the qy-dependence (out-of-plane scans)
reflects the structure and morphology parallel to the sample surface plane
(distances D, in-plane radius R) while the qz-dependence (detector scans)
reflects the height H of clusters, or the roughness parallel to sample
surface with:
qy=2π/λ sin(2θ)cos(αf)
qz=2π/λ sin (αi+αf)
As shown earlier the scattering intensity is recorded on a plane
ensuring that the angles are in the few degrees range and thus enabling
the study of lateral sizes of a few nanometers. The direct beam is here
suppressed by a beam stop to avoid the detector saturation as several
orders of magnitude in intensity separate the diffuse scattering from the
reflected beam. The protein solution droplets were placed in the hanging
drop container during the three-day experiment. The glass substrate with
the thin film template and the droplet was removed from the container
and the vacuum grease area was cleaned to ensure no contamination of
the signal. The 10 µliter droplet exists for about 60 min in air until
complete evaporation since the glass substrate and the atmosphere were
both cooled. Hence an optimum timing had to be found concerning the
preparation, adjustment in the beam and data acquisition time. The glass
substrate was subsequently brought into the beam and the incident angle
αi adjusted to about 1°. This procedure was restricted to less than 20 min
in order to avoid precipitation of salt or nanocrystals from the solution to
the substrate, which would lead to a contamination of the µGISAXS
signal.
A characteristic feature of a GISAXS pattern is the Yoneda peak (Y)

(Yoneda, 1963). This peak occurs at angles αi,αf=αc, where αc is the
critical angle of the sample. The critical angle depends on the material
via the real part of the refractive index and hence on the density and
roughness of the layer over the glass substrate. The relative intensities of
the Yoneda peaks can hence be interpreted in terms of build-up over the
glass substrate of protein crystal layers, islands of salts and protein
crystals and/or of holes in the protein films.
The 10 µliter droplet diameter was about 5 mm. Hence the footprint
of the X-ray beam will be fully within the droplet diameter. The small
beam size allows avoiding excessive liquid scattering and provides
therefore a reasonable signal-to-background ratio. The position of the
drop relative to the beam was determined by an absorption scan with a
photodiode. Experiments were performed with the beam at the center of
the droplet at the contact area droplet, protein template in order to
optimize the signal of the weakly scattering biopolymer samples. Data
collection times for individual droplets varied from 7 min to 20 min. This
is well below minimum droplet evaporation time of about 60 min. This
“Stop” procedure described above interrupts therefore crystal growth at
specific times and allows studying microcrystals freshly grown over thin
films. The full description of the µGISAXS method is contained in the
paper coauthored with the Grenoble beamline scientists (Pechkova et al.,
2005a). The quoted paper is one of a series of papers dealing with the
same set-up, and it is the first one describing the experimental details
sufficiently. Besides the technical details, the conclusions, results are not
derived here and there on a level of the available theory of surface
scattering. Several methods of interpreting the data are indeed only
mentioned, but not implemented because of the extreme difficulty in
utilizing ab initio considerations at the present stage of development. The
interpretations was stopped there simply in showing images of the
Yoneda peak, while here continue in more depth with in- and out-of-
planes data being presented and discussed in both cytochrome (this
paper) and lysozyme (Pechkova et al., 2005a).
The 2D scattering patterns (Figure 3.12) of qz, (detector scan) versus
qy (out-of-plane scan) allow to evaluate features like microcrystal cluster
diameters, heights and distances for the P450scc cytochromes grown

either by the nanotemplate-assisted method or the classical one.
Figure 3.12. The projections along the center column are shown with respect to the total
µGISAXS scattering (A) and the Yoneda regions (B) respectively as taken from Figure
3.11. (Reprinted with the permission from Nicolini and Pechkova, Structure and growth
of ultrasmall protein microcrystals by synchrotron radiation: I µGISAXS and µdiffraction
of P450scc, Journal of Cellular Biochemistry 97, pp. 544–552, © 2006, Wiley-Liss, Inc.,
a subsidiary of John Wiley & Sons, Inc.).
µGISAXS measurements of ten layers of P450scc cytochrome being

here used as substrate for the subsequent deposition and measurements of
crystallizing drop point to the existence of a long range order by plotting
the logarithmic signal of intensity versus the qy (not shown). Figure 3.12
shows the clear effect due to the P450scc nanocrystal being formed in the
droplet at 44 hours after plating and sedimenting over the above P450scc
multilayer. Interestingly while the lower peak in the Yoneda region
appears to increase in magnitude with increasing acquisition time, the

lower peak in the overall diffuse scattering region (likely corresponding
the solution scattering) is drastically decreasing during the same time
interval (Figure 3.12). The effect of sedimentation appears quite different
in the droplet containing both buffer and proteins with respect to the one
containing only buffer but without proteins: in the latter case the
scattering signal is lacking in the lower region even before sedimentation
(not shown).
Figure 3.13. Raw µGISAXS patterns of qy versus qz of three different drops deposited
over ten layers of P450scc. containing P450scc crystallizing solution at 17 hours after
plating either nanotemplate-based (A) or classical (B). The drop containing only the
buffer solution with precipitant as described in the text is shown in (C). (Reprinted with
the permission from Nicolini and Pechkova, Structure and growth of ultrasmall protein
microcrystals by synchrotron radiation: I µGISAXS and µdiffraction of P450scc, Journal
of Cellular Biochemistry 97, pp. 544–552, © 2006, Wiley-Liss, Inc., a subsidiary of John
Wiley & Sons, Inc.).
The raw µGISAX patterns of three different drops deposited over the
same ten Langmuir-Schaefer layers of P450scc exemplify in Figure 3.13
the kinetics of protein crystallization in the nanotemplate method versus
the classical one (not giving any signal), the role of buffer (not giving
any signal) and of thin solid film (giving a signal) in the observed
scattering profile. In summary, under the same conditions of buffer
solution and temperature, the µGISAXS images of the ten µliters drop
over 10 layers of P450scc acting as substrate display the following
features: (1) lacks any peak and appears quite less intense with diffuse
very low scattering signal at the critical angle of the substrate, when it
contains only buffer solution with salt and precipitant; in the Yoneda
region of the buffer drop three peaks do appear, with only the one with
highest αf being present before salt sedimentation but dominant and
slightly off-axis after salt sedimentation; (2) appears to change
significantly along the z-axis and the y-axis whenever successively
acquired at equally spaced time intervals over 7 minutes, at any time
after plating either with the “classical” method (see the lysozyme data
reported in Pechkova and Nicolini, 2006) or (mostly) with the protein
nanotemplate-based method, apparently due to sedimentation processes
of protein crystals from the solution; (3) along the z-axis changes from
the very low qz solution scattering of the total µGISAXS to the very low
αf protein substrate peak in the Yoneda region.
For this reason in order to compare the crystallization process taking
place with time in presence and absence of protein film as nanotemplate,
we have kept constant the measuring time, carrying out the
measurements quite rapidly immediately after removal from the
container (Figure 3.14). Under these conditions it appears that:
- the overall µGISAXS pattern of the drop containing P450scc
crystallizing solution based on “nanotemplate” at 44 hours after
plating is quite more pronounced with respect to the corresponding
“classical” one.
- in the Yoneda region of the corresponding µGISAXS pattern, being
the most sensitive to structural and morphological changes of the
surfaces due to the interference effect involved in the occurrence of
the Yoneda peak, contrary to “classical” drops “nanotemplate-based”
P450scc cytochrome drops taken at 17 and 44 hours after plating
(Figure 3.14) clearly show at least two pronounced Yoneda peaks
growing with time after plating.
A week Yoneda peak exists already at shorter times with a critical
angle corresponding to that of the protein substrate, unlikely that of the
salt precipitating on the substrate causing a new rough layer of small
crystals which yields a second peak not present at shorter times. Hence
all data are compatible with a working hypothesis, which attributes the
peak at higher αf-values to that of the glass substrate and/or to salt crystal
with the newly developing peak at lower αf-values being related to the
protein itself.
Figure 3.14. Yoneda regions of cytochrome P450scc “nanotemplate-based” drops taken

at 17 (A) and 44 hours (B) after plating versus P450scc cytochrome, where the αf
dependency is given at αi=0.96 and 0.92. Yoneda regions are indicated with arrows. The
patterns are shown on a logarithmic scale to enhance the features in the Yoneda regions.
With drops containing classical P450 crystallization solution or the buffer no significant
signal is apparent under the same conditions with the acquisition being carried out
immediately after drop deposition, After drop sedimentation with most liquid being
evaporated a µGISAXS signal ten times less pronounced is apparent (see panel C for the
corresponding buffer solution drop sitting on top of the same 10 layers of P450scc).
(Reprinted with the permission from Nicolini and Pechkova, Structure and growth of
ultrasmall protein microcrystals by synchrotron radiation: I µGISAXS and µdiffraction of
P450scc, Journal of Cellular Biochemistry 97, pp. 544–552, © 2006, Wiley-Liss, Inc., a
subsidiary of John Wiley & Sons, Inc.).
Finally, in the attempt to obtain structural information at the atomic

resolution we have then performed X-ray microdiffraction with the
largest microcrystals (about 5 micron in diameter) being obtained by the
homologous nanotemplate method after 44 hours plating time in the form
of “powder” P450scc microcrystals.
The kinetics and the structure of the ultrasmall P450scc cytochrome
microcrystals here investigated proves the superiority of the nanobiofilm
template method (Pechkova and Nicolini, 2003, 2004a) in inducing
nucleation and growth up to a 5 micron crystal size of a protein system
yet structurally unsolved. Our unprecedented approach with µGISAXS
combines the powerful thin film characterization method with the

micrometer-sized X-ray beam enhancing the spatial resolution used thus
far by two orders of magnitude (Roth et al., 2003). The acquired
scattering data along with the reproducible diffraction patterns of the
ultrasmall P450scc microcrystals allows for a nondestructive and
contact-free reconstruction of the crystallization process at the very early
stage and of the three-dimensional structure and morphology of the
nanocluster crystals. Such characterization and investigation on a
submicrometric scale of extra-small crystals cluster on top of the protein
film appears to allow a more clear understanding of the very early steps
of crystallization in cytochrome P450scc. It is worth to notice that even if
these data are quite illuminating, more conclusive data will be likely
obtained with the in situ experimentation already being planned. The old
paper of Yoneda (1963) has been frequently cited, and the underlying
models concerning surface roughness and the analytical interpretation of
the diffuse scattering has been utilized to reach a conclusion. The proper
models and conclusions were then decided by performing a zero-control
experiment using a template with a droplet containing the solvent alone
(no protein). Furthermore the influence of the surface coating on the
scattering has been measured separately, including a control diffraction
experiment with the surface coating alone. The available diffuse
scattering data have also been interpreted compatibly with state-of-the-
art knowledge. Considering that the Yoneda peak is one of the major
information extractable we have not made a simple generic comment, but
pointed to the role of the density and of the roughness of the protein
crystal and/or salt crystal layers. Detector scans are also shown in this
context to interpreter the data where micro GISAXS pattern were
provided and scaled to the same scattered intensity, taking into
considerations the critical angles of the glass and of the proteins.
Interestingly, from the scattering data here obtained the occurrence
and the time sequence of the various Yoneda peaks suggest the
development of a layer with significant increase in roughness due to the
significant increase in holes of the template layer as the P450scc crystal
grows in the hanging drop. That is to say this model assumes a
decreasing density of the protein template layer as the layer itself is
removed by the protein solution thus triggering or assisting the formation
of new crystals in the solution. In conclusion the data here presented

favor a build-up of holes in the nanotemplate acting as nucleation centers
for crystals formation in all protein systems including the ones so far
impossible to crystallize as the human kinase (Pechkova et al., 2004,
2003) and the P450scc here studied.
3.1.4.2 Lysozyme
To investigate the early steps of lysozyme crystallization and for this

reason we concentrate on the µGISAXS method, which appear
potentially able to utilize the out-of-plane cuts in the Yoneda regions of
its 2D scattering profiles in order to detect ultrasmall lysozyme crystals
quite before the light microscopy.
µGISAXS is indeed not restricted to the sample's surface, but is
sensitive to structures within the penetration depth of X-rays impinging
at small angles (grazing incidence) on a sample surface (Müller-
Buschbaum et al., 1998, 2000). In particular, its potential in
nondestructively determining the structure and morphology of patterned
thin protein films is exploited due to its order of magnitude increase in
the achievable resolution compared to conventional SAXS experiments
(Roth et al., 2003).
As shown before (Nicolini and Pechkova, 2006) the protein thin-film
nanotemplate is created using Langmuir-Blodgett (LB) technology or
modifications of it (Nicolini, 1997a) and is deposited on a solid glass
support, to be subsequently placed in the appropriate vapour-diffusion
apparatus as a modification of the “classical” hanging drop vapour-
diffusion method (Ducruix and Giege, 1999). This thin LB protein film
acts as nanostructured template for protein crystal nucleation and growth
up to 1000 micron size. This heterogeneous crystallization reduces the
level of supersaturation, allowing acceleration of lysozyme crystal
nucleation and growth (Pechkova and Nicolini, 2001). Moreover, this
method seems to produce radiation stable crystals (Pechkova et al.,
2004), quite more resistant than those obtained by classical techniques.
This aspect concerns also the crystals of miniscule thickness
dimensions (10–20 micron), such as those used for human kinase 3D
structure determination by synchrotron microfocus diffraction (Pechkova
et al., 2003). In the present work the data are obtained also with the
standard vapor diffusion hanging method called “classical” (Ducruix and
Giege, 1999). Every 10 µl drop contains equal proportion of lysozyme
containing solution of CH3COONa pH 4.5 and of CH3COONa 0.9 M
NaCl, pH 4.5. The experimental procedure and the schematic picture of
the µGISAXS setup are shown in Figure 3.15 of the accompanying paper
(Nicolini and Pechkova, 2006), where the sample is mounted on a xyz-
gantry and a two-axis goniometer.
Figure 3.15. The best case for lysozyme crystal size measured by light microscopy (in
microns) versus time (in hours) after plating utilizing the classical and the nanotemplate-
based vapour-diffusion method. (Reprinted with the permission from Pechkova and
Nicolini, Structure and growth of ultrasmall protein microcrystals by synchrotron
radiation: II. µGISAXS and microscopy of lysozyme, Journal of Cellular Biochemistry
97, pp. 553–560, © 2006, Wiley-Liss, Inc., a subsidiary of John Wiley & Sons, Inc.).
The incoming monochromatic X-ray beam (X=0.9775 Å) is 5µ wide,

and a two-dimensional (2D) high-resolution detector records the
scattered intensity from the sample surface. In the 2D pattern, structural
variations in z (depth of the sample), for example, a finite surface
roughness, lead to a specular and off-specular scattered intensity in qz-
direction. Structures in the x-y plane lead to out-of-plane (with respect to
the incoming beam and the sample surface normal) signals with finite qy
and 2θ ≠ 0.
Thin films investigated by GISAXS thus far (Holy and Baumbach,
1994) were of limited spatial resolution due to the large beam size
available (several hundred micrometers) (Gehrke, 1992). As shown
earlier (Roth et al., 2003) and in the accompanying paper (Nicolini and
Pechkova, 2006), to investigate our protein drops we utilized µGISAXS

combining a micrometer-sized synchrotron beam with a specifically
designed low-background GISAXS setup (Sennett and Scott, 1950;
Riekel, 2000; Lazzari, 2002). Typically the data were acquired at 0.88°
incident angle in a time ranging between 7 and 20 minutes to avoid
amorphous protein precipitation and salt crystals. We systematically
investigate the kinetics of the nanometer-sized crystals being formed
with and without nanobiofilm template of homologous proteins. We used
lysozyme as it can be easily crystallized and can thus be used as model
system to monitor the time-dependent crystals growth in size and number
as evaluated by light microscopy, in correlation with µGISAXS
measurements.
During a series of parallel experiments the lysozyme crystals appear
to grow in size with time, and significantly better for the nanotemplate-
assisted method, but not in number, which appears to remain constant
(Figure 3.16).
Figure 3.16. Out-of-plane scans of the Yoneda regions of the nanotemplate-assisted

samples at 8, 30 and 43 hours after plating. Included are the template and the glass
substrate. Clearly the enhancement of the diffuse scattering with increasing time is
visible. The detector dark noise has been subtracted and the intensity normalized to the
acquisition time. Curve 8h is shifted for clarity. The arrows indicate most-prominent in-
plane lengths ξ. The resolution is marked by a vertical line. (Reprinted with the
permission from Pechkova and Nicolini, Structure and growth of ultrasmall protein
microcrystals by synchrotron radiation: II. µGISAXS and microscopy of lysozyme,
Journal of Cellular Biochemistry 97, pp. 553–560, © 2006, Wiley-Liss, Inc., a subsidiary
of John Wiley & Sons, Inc.).
3.2 Nanomedicine
Nanomedicine represents a growing field of wide interest but still with

alternative results in clinical medicine and we take here only four
representative stimulating applications of nanoscale technologies organic
and biological to medicine.
3.2.1 Carbon nanotubes biocompatibility and drug delivery
Purified carbon nanotubes are new carbon allotropes, sharing similarities

with graphite, that have recently been proposed for their potential use
with biological systems, as probes for in vitro research and for diagnostic
and clinical purposes.
However the biocompatibility of carbon nanotubes with cells
represents an important problem that until recently remained largely
uninvestigated. It was only in 2006 that Garibaldi et al. have shown how
cardiovascular cells in vitro being exposed to purified carbon nanotube
material appear to possess a high level of cytocompatibility. Coating or
other functionalization procedures may render the single wall carbon
nanotube (SWNT) fully compatible with living cells. However, in the
perspective of an in vivo use of NT for clinical applications, it is
important to take into account that there is the possibility of a not fully
effective coating treatment, which could expose cells to a direct contact
with carbon nanotubes material. Moreover, while superposed materials
are often biodegradable, especially when using proteins or other biologic
molecules, carbon nanotubes are not degradable and overtime, by losing
functionalization, might come in direct contact with cells and tissues. In
relation to that, our observations (Garibaldi et al., 2006) point instead to
a lack of short-term toxicity induced by carbon nanotubes when added to
cardiomyocytes.
Figure 3.17 demonstrates that SWNT are biocompatible with
cardiomyocytes in culture, suggesting that long-term negative effects are
probably due to physical rather than chemical interactions.
Cardiac muscle cells (from rat heart cell line H9c2) viability in the
first three days of culture was not different between NT-treated cells and
untreated cells. However after 3 days of culture, cell death was slightly
higher in NT-treated cells.
Figure 3.17. Photomicrographs of H9c2 cells untreated (on the left) and after treatment
with carbon nanotubes (on the right): effect at day 1 (upper photomicrographs) and at day
3 (lower photomicrographs); magnification 40×; NT, nanotubes (Reprinted with the
permission from Garibaldi et al., Carbon nanotube biocompatibility with cardiac muscle
cells, Nanotechnology 17, pp. 391–397, © 2006, IOP Publishing Limited).
Trypan blue exclusion confirmed these observations (Figure 3.17):

compared to untreated after the corresponding time, trypan blue positive
cells increased by 5 and 9% after 1 and 3 days of treatment respectively
(p=ns). After reseeding non-viable cells coming from SWNT-treated
samples increased by 25%, when compared to reseeded cells not treated
with SWNT (p<0.05). Figure 3.18 shows the effect of SWNT treatment
on apoptotic death of cardiomyocytes as determined by annexin/PI
staining (Garibaldi et al., 2006). Exposure of cardiomyocytes to 0.2
mg/ml of SWNT induced little change in the apoptotic pattern at day 1
and 3 after SWNT treatment (mean differences did not reach
significance). After reseeding an increase in cell death could be observed
in SWNT-treated samples.
Figure 3.18. Flow cytometric analysis of annexin/PI staining; A) cells untreated or treated
with SWNT for the indicated time; B) positive control of cells treated with hydrogen
peroxide at the concentrations of 0.2 and 1 mM. FL1-H: relative Annexin V-FITC
fluorescence intensity; FL3-H: relative PI fluorescence intensity. (Reprinted with the
permission from Garibaldi et al., Carbon nanotube biocompatibility with cardiac muscle
cells, Nanotechnology 17, pp. 391–397, © 2006, IOP Publishing Limited).
This increase is accounted by annexin-positive/PI-negative cells

(9.3% vs 2.9% in reseeded untreated samples, p<0.05), that are apoptotic
cells (lower right quadrant), but at greater extent by annexin positive/PI
positive cells (18.7% vs 5.2% in the reseeded untreated samples,
p<0.05), that are late apoptotic or necrotic cells (upper right quadrant).
Figure 3.18b shows a positive control, with hydrogen peroxide at the
concentration of 0.2 with cells dying for apoptosis, while, at the
concentration of 1 mM hydrogen peroxide, most cells are necrotic.
To determine whether SWNT bound to cell membrane affect repeated

seeding of cardiomyocytes, subconfluent SWNT-treated cells were
detached and reseeded (1:4) in petri dishes (Figure 3.19) to eliminate
most of the unbound nanotubes material. At 24 and 72 hours after
reseeding, untreated cells showed the average rate of exponential growth,
while cells coming from trypsinized SWNT-treated sample showed a
limited ability to proliferate, with a definite difference in shape, with a
high degree of cell death. However, overtime cells from trypsinized
SWNT-treated sample continued to grow, and partially recovered the
original shape (Figure 3.19).
Figure 3.19. Photomicrographs of H9c2 cells untreated (on the left) and after treatment
with carbon nanotubes (on the right): long term effect at day 1 (upper photomicrographs)
and at day 3 (lower photomicrographs) after reseeding; magnification 40×; NT,
nanotubes (Reprinted with the permission from Garibaldi et al., Carbon nanotube
biocompatibility with cardiac muscle cells, Nanotechnology 17, pp. 391–397, © 2006,
IOP Publishing Limited).
In conclusion our results demonstrates that highly purified carbon

nanotubes possess no evident short term toxicity and can be considered
biocompatible with cardiomyocytes in culture, while the long term
negative effects, that are evidenced after reseeding, are probably due to
physical rather than chemical interactions. While more work is needed to
establish biological consequences associated to long term interactions of
SWNT with cells, our results encourages further research aimed to
establish the use of purified SWNT for in vivo applications in cell
systems, likewise drug delivery in vitro and in vivo systems (work in
progress).
3.2.2 Photosensitization of titanium dental implants
Dental implants are now day successfully used in dentistry for oral
rehabilitation supporting mobile or fixed prosthesis. Beside undisturbed
osseointegration and an adequate prosthetic design, clinical success of
dental implants can be jeopardized by bacterial infection inducing
mucositis or periimplantitis (Mombelli et al., 1987; Leonhardt et al.,
1992; Lindhe et al., 1992). Various methods have been proposed for the
treatment of periimplantitis including access flap procedures, the use of
locally or systemically administered antimicrobial agents as well as
decontamination of the exposed implant surfaces (Persson et al., 2001;
Kreisler et al., 2002).
The eradication of pathogenic microorganisms from implant surfaces
is a key step for successful treatment of failing implant. Several methods
for cleaning of implant surfaces have been described in order to treat
failing implants. Among others the laser treatment seemed to be effective
in terms of bacteria elimination (Kreisler et al., 2002, 2003; Dortbudak et
al., 2001; Shibli et al., 2003; Karacs et al., 2003; Swift et al., 1995;
Bereznai et al., 2003). Implants are generally supplied in sterile vessels
but infection may occur during the surgery. If bacterial infection of the
interface between the implant and the bone or tissue occurs remedial
surgery may be required. It has been demonstrated, in-vitro, that
illumination of an implant using high intensity visible radiation can clean
the surface. However, laser induced damage to the surface and undesired
heating of the implant have been reported. On the other side,
photosensitive dyes, for example toluidine blue, have also been
employed in-vitro to sterilise implants (Shibli et al., 2003). The use of
such dyes reduces the photon flux required to sterilise the implant. When
illuminated at the correct wavelength photosensitive dyes produce, with

high yield, singlet oxygen. It is this singlet oxygen, which reacts with
biological molecules and cleans the surface of the dental implants
(Matysik et al., 2002). Whilst the dye photosensitization strategy lowers
the photon flux required to sterilise an implant, and eliminates laser
induced structural damage, it necessitates the unwanted addition of an
organic dye to the specially engineered surface of the implant.
It is well known that commercially pure titanium is used to produce
implants owing to its excellent biocompatability. To promote bio-
efficacy the surface of metal implants are roughened. Grit balsting
followed by acid etching or titanium plasma spraying are employed to
produce the roughened nanoscale surfaces. Titanium is a very reactive
metal that is rendered corrosion resistant, and hence suitable for in-vivo
applications, by a passive TiO2 layer. Hence dental implants have
nanoscale TiO2 at their surface. TiO2 is a wide band gap semiconductor
that produces (hydr)oxyradicals when illuminated with UV light.
Figure 3.20. The rhodamine B concentration as a function of UV illumination time; ■ in

the presence of a TiO2 coated dental implant and □ with no implant present. The inset
shows the UV-absorption spectrum of rhodamine B (Reprinted with the permission from
Riley et al., An in-vitro study of the sterilization of titanium dental implants using low
intensity UV-radiation, Dental Materials 21, pp. 756–760, © 2005, Elsevier).
These free radicals, which are more reactive than singlet oxygen, may
be successfully employed in redox chemistry, killing bacteria (Riley et
al., 2005). Our group indeed recently reported the photosensitization of
titanium dental implants by TiO2. Namely the photosensitization of
titanium dental implants by TiO2 was successfully reported (Riley et al.,
2005), whereby the investigations of the decomposition of rhodamine B
indicate that the commercial implants are photoactive (Figure 3.20).
Experiments were performed on dental implants supplied by Premium
Implant System (Sweden & Martina, Padua, Italy). The implants were
3.75 mm in diameter and had an insertion depth of 8.5 mm. The implants
were of commercially pure titanium and had been sand blasted and acid
etched to promote osseointegration. All samples were unpacked from
their sterile containers immediately prior to use. Indeed our in vitro
studies of illuminated solutions containing Escherichia Coli and dental
implants indicate that photosterilisation may be achieved at low UV light
intensities (Figure 3.21) thereby suggesting a new possible utilization of
titanium in oral surgery.
Figure 3.21. The concentration of E. Coli in solution as a function of time; ● under UV

illumination in the presence of a TiO2 coated dental implant, □ in the presence of a dental
implant but no illumination and ■ under illumination but in the absence of a dental
implant. The lines are plotted as guides to the eye. (Reprinted with the permission from
Riley et al., An in-vitro study of the sterilization of titanium dental implants using low
intensity UV-radiation, Dental Materials 21, pp. 756–760, © 2005, Elsevier).
The photoreaction between TiO2 and rhodamine B (RH) under UV-

visible radiation is complex (Wu et al., 1998). Essentially two
mechanisms may operate in parallel. Absorption of UV radiation by the
TiO2 yields an electron and a hole at the particle surface. The electron
reacts with molecular oxygen to form an O2•- which, on protonation
yields HOO•. The HOO• may be further reduced, via hydrogen peroxide,
to a hydoxyl radical. Hydroxyl radicals are also formed by the reaction
between water or OH- and photogenerated holes. The photogenerated
oxyradicals may then react with RH to give mineralised products; either,
RH + {O2•-, HOO• or OH•} → intermediate →→ products
or
R + h+(TiO2) → R• + {O2•-, HOO• or OH•}→intermediate→→products
Alternatively the RH may adsorb visible light and then transfer an
electron to TiO2. As above, the electron on the TiO2 will react with
molecular oxygen to form the (hydr)oxyradicals that breakdown the dye.
This second process is termed sensitization.
3.2.3 Biopolymer sequencing and drug screening chip
Conventional DNA sequencing methodology is labour-intensive,

requiring significant sample preparation and electrophoretic size-
separation of radio-labelled DNA fragments. As an alternative, a chip
comprising an array of short oligonucleotides can be used as a
hybridization probe to analyse a fluorescently labelled DNA sample of
unknown sequence.
The resulting hybridization pattern could then be lysed to determine
the sequence of the target DNA. To test the feasibility of this approach,
an array containing all the 8-mer combinations of the nucleosides
guanine and cytosine (256 oligomers) was synthesized (Fodor et al.,
1993). After synthesis, the chip was incubated with 1 pmol of 5'-
fluorescein-CCCAAACCCAA-3' and scanned at 15 °C. Hybridization of
this target to the four complementary octanucleotides present on the
array was observed as brightly fluorescent spots, as were many of their
single-base mismatches. Methods and algorithms to reconstruct the target
sequence from such hybridization data were developed time ago (Fodor
et al., 1993).
More than 100 million bases in the DNA from different sources have
been already sequenced with various techniques. However, even this was
absolutely insufficient, and the Human Genome Project introduced
procedures able to sequence up to megabases. Four groups introduced
originally the “sequencing by hybridization” (SbH) technique almost
simultaneously: one in Russia (Lysov et al., 1988; Khrapko et al., 1989),
two in the UK (Southern, 1996; Bains and Smith, 1988), and one in
Yugoslavia (Drmanac et al., 1996). This technique was based on
sequence-specific DNA hybridization to a large set of oligonucleotides
of specified length. By identifying overlapping sets of oligonucleotides
that form perfect duplexes with the target DNA sequence, unknown
DNA sequences were determined with a model SbH experiment
introduced by Mirzabekov in 1994.
Incorporation of the SbH procedures into DNA sequencing
microchips were manufactured time ago for detecting hybridization of
the target DNA to immobilised oligonucleotides (Mirzabekov, 1994).
The application of technologies with resolution threshold of 10 µm (such
as printing or micromanipulation) permitted to reduce even further the
dimensions of the chip. To overcome another drawback of the approach,
the costly procedure of oligomer synthesis, methods of parallel synthesis
on various surfaces in 2D arrays were developed (Cantor et al., 1992). A
matrix incorporating 256 purine 8-mers has been manufactured by solid-
phase oligonucleotide synthesis (Cantor et al., 1992). Spectacular
opportunities became available through the use of addressable photo-
activated chemistry in the parallel synthesis of oligonucleotides directly
on a glass surface. This technology provided the means for effective
industrial manufacture of highly complex sequencing microchips. When
large numbers of small-dimension oligonucleotide matrices are to be
produced industrially, and quality control for every immobilized
oligomer was essential, then using robots to apply pre-synthesized
oligomers to the matrix (Khrapko et al., 1989) proved advantageous.
This method is also applicable to the immobilisation of a much wider
variety of oligonucleotide analogs, as well as other compounds, such as
proteins, antibodies, antigens and low-molecular weight ligands, on
matrices. Such microchips finded applications in many other areas of

nanoproteomics and nanogenomics besides large-scale sequencing; for
example, in diagnostics or molecular screening.
The cost of a microchip will incorporate the cost of manufacturing.
By analogy with electronic microchip production, the per-unit cost
should not be excessive for large-scale production. The challenge has
been to establish manufacturing procedures able to produce,
reproducibly, matrices incorporating the oligonucleotides synthesized to
the degree of accuracy required. Traditional methods for generating and
identifying biologically active compounds for pharmaceutical
development are typically tedious and time-consuming. Screening
natural products from animal and plant tissues, or the products of
fermentation broths, or the random screening of archived synthetic
molecules have been the most productive avenues for the identification
of new, biologically active lead compounds. “Rational” drug-design is a
more sophisticated approach, in which the tools of synthetic organic
chemistry, X-ray crystallography and molecular modelling are employed
to produce pharmaceutically active molecules (as shown in previous
party of this chapter. This is an extremely time-consuming, labour
intensive and expensive process, during which compounds are designed,
synthesized and tested in an iterative fashion. Although successful, this
approach has been slow to yield products with desired properties. Ideally,
one would like to have access to an infinite source of compounds that
could be screened rapidly for individual molecules with desired
biological properties. The new field of research early referred to as
combinatorial chemistry represented a first step towards this goal. For
example, peptides are built up from the 20 naturally occurring, gene-
encoded amino acids. The combinatorial assembly of these amino acids
can generate up to 8000 (203) unique tripeptides, 160000 tetra peptides,
64 × 106 hexapeptides, etc. Practical technologies for achieving such
random assembly of molecular subunits were first developed for the
synthesis of peptides and oligonucleotides (Ellington and Szostak, 1990;
Tuerk and Gold, 1990).
3.3 Nanogenomics
A new approach for medical diagnostics and therapy named

“Nanogenomics” is emerging from the interplay of bioinformatics and
biomolecular microarray to a previously unforeseeable level. This
editorial summarizes here its major features with few key examples of
molecular genomics application to medicine.
DNA microarrays have emerged as one of the most promising
methods for the analysis of gene expression (Butte, 2002; Nicolini et al.,
2006a). This technique allows the study of an immense amount of genes
(over 10000) with only one experiment and therefore can draw a picture
of a whole genome. Anyway, the huge number of data coming out from
microarray experiments may often raise experimental complications and
difficulties in the analysis. Moreover, the greatest part of genes displayed
on an array is often not directly involved in the cellular process being
studied.
3.3.1 Human T lymphocytes cell cycle
Human lymphocytes gene expression is monitored before and after PHA

stimulation over 72 hours, using DNA microarray technology. Results
are then compared with our previous bioinformatics predictions, which
identified 6 leader genes of highest importance in human T lymphocytes
cell cycle.
Experimental data are strikingly compatible with bioinformatic
predictions of the specific role and interaction of PCNA, CDC2 and
CCNA2 leader genes at all phases of the cell cycle and of CHEK1 leader
gene in regulating DNA repair and preservation. It does not escape our
notice that the conception and use of ad hoc arrays, based on a
bioinformatics prediction which identifies the most important genes
involved in a particular biological process, can really be an added value
in cell biology and cancer research alternative to massive frequently
misleading molecular genomics.
Recently, we proposed a bioinformatics algorithm, based on the
scoring of importance of genes and a subsequent cluster analysis, which
allowed us to determine the most important genes, that we call “leader
genes” (Sivozhelezov et al., 2006a) in human T lymphocytes cell cycle.

The basis of the scoring system relies upon the calculation of interactions
among genes, performed with software available in the web, such as
STRING (von Mering et al., 2005). The number of links for each gene is
then weighted and the final weighted numbers of links are clustered, in
order to make a hierarchical classification of genes (Sivozhelezov et al.,
2006a). In this way, it becomes possible to draw and to update maps of
the major biological control systems, and to integrate them in a concise
manner to discern common patterns of interactions between gene
expression and their correlated coding of proteins. We chose, as a model
system, human T cell lymphocytes stimulated to entry cell cycle with
PHA (Nicolini et al., 2006a). This particular cellular system is very well
known and was quantitatively characterized time ago (Cantrell, 2002;
Isakov and Altman, 2002; Oosterwegel et al., 1999a,b; Abraham et al.,
1980); therefore, it can be a good starting point to verify our algorithm.
In particular, we identified 238 genes involved in the control of cell
cycle. Most important, only 6 of them were previously identified to be
the leader genes (see table 1 from Nicolini et al., 2006a); interestingly,
they actually are involved in the cell cycle control at important
progression points, namely the most important four at the transition from
G0 to G1 phase (MYC; Oster et al., 2002), at the progression in G1
phase (CDK4; Modiano et al., 2000), and at the transitions from G1 to S
(CDK2; Kawabe et al., 2002), and from G2 to M phases (CDC2;
Baluchamy et al., 2003; Torgler et al., 2004). The two remaining “leader
genes” (CDKN1A and CDKN1B) are inhibitors of cyclin-CDK2 or -
CDK4 complexes and thereby contribute to the control of G1/S transition
and of G1 progression (Jerry et al., 2002; Chang et al., 2004).
We also confirmed our results by analyzing changes in gene
expression after 48 hours, using a newly developed and simple
technology called DNASER, which is a novel bioinstrumentation for
real-time acquisition and elaboration of images from fluorescent DNA
microarrays developed in our laboratories (Nicolini et al., 2002; Troitsky
et al., 2002). The validity of the DNASER measurements was confirmed
by standard fluorescence microscopy equipped with CCD. This
experimental analysis proved that DNASER is appropriate for
monitoring gene expression during the human lymphocytes cell cycle

(Nicolini et al., 2006a).
The leader gene approach, validated by experimental analysis on a
model system, can suggest a more rationale approach to experimental
techniques and methods, as DNA microarray. The application of
bioinformatics studies and the identification of leader genes can predict
the most important genes in a particular cellular process. In this way, it
becomes possible to design smaller microarrays, which display only the
most interesting genes for a specific cellular process and thus are much
easier to interpret.
We experimentally analyze the gene expression of human T
lymphocytes treated with the mitogen compound PHA 24, 48 and 72
hours (“time series analysis”, for a complete reference see Straume,
2004; Willbrand et al., 2005) after the stimulation and compare the
results with independent bioinformatics predictions, in order to give a
further validation to leader gene approach and to identify co-expressions
among genes involved in the cell cycle of human T lymphocytes.
Table 3.1. RNA extraction yield for the different lymphocytes samples (Reprinted with
the permission from Giacomelli and Nicolini, Gene expression of human T lymphocytes
cell cycle: Experimental and bioinformatic analysis, Journal of Cellular Biochemistry 99,
pp. 1326–1333, © 2006, Wiley-Liss, Inc., a subsidiary of John Wiley & Sons, Inc.).
% of % of
Time, in hours,
Number quiescent proliferant Total RNA Fluorochromes
after PHA
of cells cells cells pg/cell utilized
stimulation
(in G0+Q) (in G1,S,G2)
0 107 90 8, 1, 1 30 Cy3 green
24 5 × 106 53 51, 8, 3 30.4 (35,7%) Cy5 red
48 5 × 106 36 – 39.4 (+60%) Cy5 red
72 5 × 106 17 45, 36, 2 38.4 (+72%) Cy5 red
The employed array is the Human Starter Array by MWG Biotech,

chosen on the base of the gene we are interested about (Nicolini et al.,
2006a).
It contains 161 oligonucleotides (designed to be specific for the
respective human gene sequence), 32 replicas and 7 gene specific
Arabidopsis control oligonucleotides, for 200 total spots, disposed in 10
columns and 20 rows, more an exact copy, in total 400 spots for array.
The diameter of one spot is 100 µm and the distance between two near
spots is 250µm.
As previously shown (Nicolini et al., 2006a), in order to obtain total
RNA extraction the cells pellet (minimal 1 x 107 cells) has been dealt
with an extraction kit from Amersham Biosciences containing LiCl,
CsTFA and an extraction buffer. The samples thus obtained have been
conserved at -80 °C. For the estimation of the extracted RNA they have
been used 200 µl RNAsi free cuvettes. In order to avoid contaminations
of genomic DNA, the RNA samples have been subordinates to digestion
with the enzyme Dnase I. For every experiment we obtained a good
RNA total extraction yield, as shown in Table 3.1. Moreover the RNA
spectrophotometrical analysis has evidenced a high purity degree, being
the ratio 260/280 nm always more then 1.9.In total, 32 genes were
identified to be expressed during the 72 hours of analysis. 8 of them
(25%) were included in the list of 238 genes involved in the control of
human T lymphocytes cell cycle (Sivozhelezov et al., 2006a).
Among the 238 genes, we previously identified the 6 “leader genes”
i.e., those showing the highest number of interactions with other genes
(Sivozhelezov et al., 2006a) (Table 3.2). 3 of them (MYC, CDC2,
CDK4) were present on the Starter Array and, as a confirmation of our
prediction; they were all expressed during different experiments.
For instance, MYC is the gene with the highest number of interaction
in our bioinformatic predictions. It is known as a very early gene in the
proliferative response, since it regulates the entrance in G1 phase of the
cell cycle (Oster et al., 2002). It is interesting to notice that MYC is
expressed, in smaller quantities, after 24 hours and 48 hours. Moreover,
the absolute intensity of the corresponding spot on the array decreased
during time, as expected from the entrance in the cell cycle and the
progression along it.
The two other “leader genes” are CDK4 and CDC2. The former is
known to regulate the progression in G1 phase, while the latter is
involved in the G1/S and G2/M transition (Modiano et al., 2000; Kawabe
et al., 2002; Baluchamy et al., 2003).
Table 3.2. Leader genes in human T lymphocytes cell cycle (Reprinted with the
permission from Sivozhelezov et al., Gene expression in the cell cycle of human T
lymphocytes: I. Predicted gene and protein networks, Journal of Cellular Biochemistry
97, pp. 1137–1150, © 2006a, Wiley-Liss, Inc., a subsidiary of John Wiley & Sons, Inc.).
Weighted Apparent
Gene
number Gene description Protein description function in
name
of links cell cycle
MYC 27.81 v-myc myelocytomatosis Myc proto-oncogene Entrance
viral oncogene homolog; protein (c-myc) in G1
determines c-myc mRNA phase
stability; v-myc avian
myelocytomatosis viral
oncogene homolog; v-
myc myelocytomatosis
viral oncogene homolog
CDK2 26.65 cyclin-depen. kinase 2; Cell division protein G1/S
cdc2-related protein kinase 2 (EC 2.7.1.-) phase
kinase; cell devision (p33 protein kinase) transition
kinase 2; p33
CDC2 26.47 Cell division cycle 2, G1 Cell division control G2/M
to S and G2 to M; cell protein 2 homolog phase
cycle controller CDC2; (EC 2.7.1.-) (p34 transition
cell division control protein kinase)
protein 2 homolog; (Cyclin-dependent
cyclin-depen. kinase 1; kinase 1) (CDK1)
p34 protein kinase
CDK4 25.255 cyclin-depen. kinase 4; Cell division protein Progressio
cell division kinase 4; kinase 4 (EC n in G1
melanoma cutaneous 2.7.1.37) (Cyclin- phase
malignant, 3 depen. kinase 4)
(PSK-J3)
CDKN1A 25.08 cyclin-depen. kinase Cyclin-depen. kinase Inhibitor
inhibitor 1A (p21, Cip1); inhibitor 1 (p21) of cyclin-
CDK-inter. protein 1; (CDK-interacting CDK2 or –
DNA synthesis inhibitor; protein 1) CDK4
cyclin-depen. kinase (Melanoma complexes
inhibitor 1A; melanoma differentiation
differentiation associated associated protein 6)
protein 6; wild-type p53- (MDA-6)
activated fragment 1
CDKN1B 23.90 cyclin-depen. kinase Cyclin-depen. kinase Inhibitor
inhib. 1B (p27, Kip1); inhibitor 1B (Cyclin- cyclin-
cyclin-depen. kinase depen. kinase inh. CDK2 or -
inhibitor 1B p27) (p27Kip1) CDK4
Comfortingly CDK4 reaches its maximum expressed after 24 hour

and it is lower after 48 hours. After 72 hours, the corresponding spot
cannot be identified on the array. This can points that, after 24 hours,
human T lymphocytes have entered the cell cycle (MYC and CDK4
expression) and are progressing along the G1 phase. CDC2 encodes for a
member of the Ser/Thr protein kinase family. This protein is a catalytic
subunit of the highly conserved protein kinase complex known as M-
phase promoting factor (MPF), which is essential for G1/S and G2/M
phase transitions of eukaryotic cell cycle (Kawabe et al., 2002;
Baluchamy et al., 2003). Mitotic cyclins stably associate with this
protein and function as regulatory subunits (Olashaw and Pledger, 2002).
This gene regulates the progression from G1 to S and from G2 to M
phase, and therefore represents an important signal of cell cycle
progression (Modiano et al., 2000). Its expression varies during cell
cycle, as shown by our results: it is indeed not expressed in resting cells
and it starts to be expressed only after 24 hours. The expression
decreases, so that cannot be identified, after 48 hours and then it is
already detectable at the end of the experiments. These results and those
derived by MYC and CDK4 expression are compatible with the
expectastions: 24 hours after a mitogen stimulation with PHA resting T
lymphocytes have started cell cycle (MYC), progressed along G1 phase
(CDK4) and are preparing to replicate DNA (CDC2). Also, after 72
hours, T lymphocytes are about to enter mitosis (CDC2). The
bioinformatic-based identification of leader genes as most important
genes for each of the progression points in cell cycle is thus perfectly
confirmed.
Our prediction identified also other genes whose importance was not
as high as “leader genes”, but slightly lower. In fact, we previously
identified “leader genes” using clustering techniques (Sivozhelezov et
al., 2006a). The “leader genes” were selected as the gene belonging to
the cluster with the highest numbers of links (class A), but there were
also other important genes in other high-ranked classes emerging from
the clustering process (classes B, C and D, with decreasing importance).
Of the 8 genes identified on the array and included in the list of
Sivozhelezov et al. (2006a), 3 were of class A (leader genes), 2 of class
B, 2 of class C and only 1 of class D. For instance, the gene encoding for
proliferating cell nuclear antigen, PCNA, belongs to class B (weighted

number of links = 17.59). The protein encoded by this gene is an
auxiliary protein of DNA polymerase delta and appears to be requested
for both DNA synthesis and DNA repair (Ohta et al., 2002). This gene is
present in low amount in resting normal human T lymphocytes and, upon
mitogen stimulation, begins to increase in mid-G1 phase, approximately
12 to 15 hours before entry into S phase (Ohta et al., 2002). PCNA
continues to increase in amount throughout the cell cycle and remains
high in proliferating cultures. This agrees with our experimental data
shown in Figure 3.21. In fact, at the baseline we did not identify PCNA
to be expressed on the array. After 24 hours, PCNA expression has
increased and it reaches its maximum level after 72 hours. Interestingly,
we were not able to identify its expression after 48 hours: this seems in
contrast with the considerations reported above, but probably the missed
identification may be due to experimental problems. This addressed our
attention to have a deeper view of the behavior of this gene, using also
bioinformatics resources.
Figure 3.22. Final map of interactions among 8 high-ranking genes in cell cycle of human
T lymphocytes and their neighbouring. (Reprinted with the permission from Giacomelli
and Nicolini, Gene expression of human T lymphocytes cell cycle: Experimental and
bioinformatic analysis, Journal of Cellular Biochemistry 99, pp. 1326–1333, © 2006,
We used the online available software STRING (von Mering et al.,

2005) to formulate a detailed prediction of PCNA interactions with other
identified genes, not considering text-based interactions. The results are

shown in Figure 3.22, where PCNA is in the center of a complex map of
interactions and is involved in many biochemical pathways. These
interactions can also be identified from our experimental data. For
instance, an interaction was predicted between PCNA and RFC2, a
monomer of a heteropentameric protein complex consisting of the Rfc1,
Rfc2, Rfc3, Rfc4, and Rfc5 subunits (Majka et al., 2004; Majka and
Burgers, 2004). This interaction is confirmed by our experimental data,
which shows that RFC2 has a great level of expression after 72 hours.
Also, other predicted interactions between PCNA and other identified
genes confirm our experimental data, such as the co-expression with
CCNA2 (Vendrell et al., 2004). This protein was identified by us to be a
class B gene for the control of cell cycle in human T lymphocytes
(Sivozhelezov et al., 2006a) (weighed number of links = 15.67). The
protein encoded by this gene belongs to the highly conserved cycling
family, whose members vary in protein abundance through the cell cycle
and act as regulators of CDK kinases. This cycling binds and activates
CDC2 and thus promotes G1/S and G2/M transitions along the cell cycle
(Vendrell et al., 2004). PCNA, CCNA2 and CDC2, which are genes of
great importance in human T lymphocytes cell cycle, are thus bound by a
very close link, which is fully confirmed in our experimental data.
Other high-ranked genes present on the Starter Array are CCNE1,
CCNH (class C) and CHEK1 (class D). CCNE1, another cyclin, is the
gene with the highest absolute intensity value in the 24 arrays
(Sutherland and Musgrove, 2004). It is also expressed after 48 hours and
72 hours, but shows a much lower intensity. A STRING-based
bioinformatics prediction suggests it is linked directly with CDC2
(Figure 3.22). Indeed, its expression reaches the maximum level in
correspondence to one of the process regulated by CDC2, the G1/S
transition after 24 hours. CCNH, also, is highly expressed 24 hours after
the stimulation. In fact, the protein encoded by this gene is known to
phosphorylate CDC2, thus contributing to the G1/S switching (Karan et
al., 2002). The last considered gene, CHEK1, is an essential kinase
required to preserve genome stability. Very recent findings (Syljuasen et
al., 2005) proposed that CHEK1 is required during normal S phase to
avoid aberrantly increased initiation of DNA replication, thereby
protecting against DNA breakage. Our experiments show that CHEK1

expression can be identified 24 hours and 72 hours after the stimulation
with PHA, in correspondence to DNA synthesis and mitosis. This can
strongly confirm the importance of this gene on DNA repair and
preservation mechanisms. Then, we calculated a final map of interactions
among these 8 high-ranking genes in cell cycle of human T lymphocytes,
which is shown in, representing also their neighboring genes. The other
neighboring genes present in the map are not displayed on the Starter
Array. Interestingly, one of them is CDKN1B, which we identified to be
a “leader gene” in the control of human T lymphocytes cell cycle
(Sivozhelezov et al., 2006a; Chang et al., 2004).
The Starter Array displays only 161 genes: therefore data can be
easily analyzed. The use of more complex arrays, displaying a huge
amount of genes (up to 10 thousands) often leads to a difficult and
sometimes misleading analysis, due to the complexity of data. Human
Starter allows a simpler analysis. Anyway, genes to be displayed must be
chosen with a particular care. Presently we have in progress the
construction of DNA chips based on genes identified by bioinformatics,
namely the “leaders” of one particular process and “orphan” needing a
more detailed attention (Sivozhelezov et al., 2006a). In this way, it
becomes possible to create ad hoc arrays, which can guarantee the best
results in analyzing a particular cellular system. The here reported “time
series” experimentation on human T lymphocytes stimulated with PHA
confirm this hypothesis, but are limited by the fact that many interesting
genes involved in the human T lymphocytes cell cycle, including 3
leader genes, are not displayed on the array.
In conclusion, the data on gene expression collected 24, 48, and 72
hours after the mitogen stimulus when compared with our bioinformatics
prediction (Sivozhelezov et al., 2006a) confirmed the theoretical
prediction on leader genes. In particular, 24 hours after the stimulation,
resting T lymphocytes have started cell cycle (MYC), progressed along
G1 phase (CDK4) and are preparing to replicate DNA (CDC2); after 72
hours, T lymphocytes are about to enter mitosis (CDC2). Moreover, we
got a deeper picture of gene expression considering 5 other genes, whose
importance in human T lymphocytes cell cycle was theoretically proven,
even if they were not included in the leader gene class. Our experimental
data show a perfect agreement with theoretical ones, therefore further

validating the leader gene approach, and also point out the importance of
the interaction between PCNA, CDC2 and CCNA2 in controlling cell
cycle and of CHEK1 in regulating DNA repair and preservation. At the
same time the need of ad hoc array was once again confirmed.
The application of the leader gene approach, starting from the
identification of involved genes and their subsequent ranking according
to the number of interactions, should be extended to other cellular
processes, which are known to a lesser extent if compared with human T
lymphocytes cell cycle. Leader genes are defined as the genes with the
highest number of interactions among those involved in a particular
cellular process. By identifying leader genes of a given process, it
becomes possible to design targeted microarray, whose analysis would
allow to describe complex biomolecular pathways thorough the activity
of a few, but highly important genes, which represent the real center of
interactions maps. In this way, an easiest and more rationale approach to
molecular genomics can shed new lights on complex cellular
mechanisms.
3.3.2 Organ transplants
A French group (Jean-Paul Soulillou, Sophie Brouard et al., at Inserm,

Nantes) is systematically studying tolerance of kidney graft. In
particular, they have examined patients tolerating a kidney graft without
any treatment and patients with chronic rejection.
They also performed a microarray analysis and identified a list of
genes able to classify the two different classes. While the French group
data concern 35000 genes of a pangenomic array (Cantrell, 2002), the
bioinformatic analysis being carried by a joint cooperation between
INSERM and Genova University was conducted with two parallel
approaches (Braud et al., 2008, Sivozhelezov et al., 2008) which run
independently and, at the end, compared each other: ab initio analysis
and experimental analysis.
The former concerns the identification of genes involved in kidney
graft tolerance (Figure 3.23) and of their leader genes; the latter concerns
microarray data and clinical data reduction. In the former our completely
ab initio set of genes involved in kidney transplant tolerance will be

indeed used (Cantrell, 2002; Abraham et al., 1980).
Figure 3.23. Classification probabilities of individual patients by Predictive Analysis of

Microarray (PAM) based on the 343 differentially expressed genes between operationally
tolerant kidney graft recipients (TOL) and patients with chronic rejection (CR). Each
patient sample is shown by a bar, as labelled in the X-axis. The colour codes indicate the
probability (0-1, as indicated in Y-axis) that the sample belongs to TOL (red) or CR
(green). (A) Cross-validated probabilities on the 8 TOL and 18 CR that were used to set
up the 2-class (TOL/CR) classification algorithm. Among the 26 patients, 5 samples
(CR013, CR014, TOL01, TOL06 and TOL08) were misclassified. (B) Using the PAM
algorithm defined with 8 TOL and 18 CR patients, 7 serially harvested samples (4 TOL
and 3 CR) at a time interval of more than 1 year after the first sample were classified. The
algorithm correctly classified all samples, with a probability of 100% for TOL and 92.0%
for CR. (Reprinted with the permission from Braud et al., Immunosuppressive drug-free
operational immune tolerance in human kidney transplants recipients: I. Blood gene
expression statistical analysis, Journal of Cellular Biochemistry, in press, © 2008, Wiley-
Liss, Inc., a subsidiary of John Wiley & Sons, Inc.).
This effort is representing a big challenge to our search for automatic

identification of "leader genes" previously described. In particular, it is
important to notice that there is no obvious necessary direct correlation
between leader genes we will identify by ab initio research and the
quantitative changes in expression monitored by experimental analysis.
In a second paper on the nanogenomics of kidney transplants

(Sivozhelezov et al., 2008) we outline a microarray-based identification
of key leader genes associated respectively to rejection and to operational
tolerance of the kidney transplant in humans (Figure 3.24) by utilizing a
non/statistical bioinformatic approach based on the identification of “key
genes”, either as ones mostly changing their expression, or having the
strongest interconnections (Figure 3.25).
Figure 3.24. Genes from the “fullchip” plotted according to their tolerance or rejection
propensity i.e. difference in expression in log scale between RC and TOL genes, with the
56 SAM-identified genes (Braud et al., 2007) marked. Arrows indicate genes included in
the 56 gene dataset but possibly unable to discriminate rejection/tolerance. Lines indicate
thresholds used in selecting genes for “pro-tolerance” and “pro-rejection” leader gene
calculations (Reprinted with the permission from Sivozhelezov et al.,
Immunosuppressive drug-free operational immune tolerance in human kidney transplants
recipients. II. Nonstatistical gene microarray analysis, Journal of Cellular Biochemistry,
in press, © 2008, Wiley-Liss, Inc., a subsidiary of John Wiley & Sons, Inc.).
A uniquely informative picture emerges on the genes controlling the

human transplant from the detailed comparison of these findings with the
traditional statistical SAM analysis of the microarrays and with the
clinical study carried out in the accompanying paper (Braud et al., 2008).
The overall conclusion is that there are many genes in common in the
highest interaction genes derived from individual fullchip and the 343
SAM-gene list. Notably, convergence between the SAM approach and
our non-statistical approach becomes much better for the individual

dataset, in which the CR/TOL fold changes are much lower, compared to
the old dataset.
Figure 3.25. Complete interaction map “no textmining” interaction map calculated for the
“Class 1” reliable genes and filtered by (CR-TOL) amplitude, as obtained from the new
fullchip microarray datasets (see Table 2 for their names and ranking according to their
number of interactions) (Reprinted with the permission from Sivozhelezov et al.,
Immunosuppressive drug-free operational immune tolerance in human kidney transplants
recipients. II. Nonstatistical gene microarray analysis, Journal of Cellular Biochemistry,
in press, © 2008, Wiley-Liss, Inc., a subsidiary of John Wiley & Sons, Inc.).
The primary reason are 1) we use expression levels in normal scale

whereas SAM uses expression levels normalized by their errors (thus
practically reducing the signal to noise level), and 2) SAM uses
permutation (random shuffling) of the data, and then extracts significant
genes by comparing the permuted and non-permuted set. The SAM-
derived genes typically have shown small differences of expression
levels explained by the fact that SAM package operates with ‘‘relative
difference’’ d which is the actual difference D divided by a sum of its
standard deviation s with an arbitrary constant s0, d=D/(s+s0), which
should make equally significant the small but highly reproducible change
and the large but poorly reproducible change in gene expression. Since
physical grounds of such an approach are unclear, we separated the two
parameters, i.e., the magnitude and the reliability, but using two
independent filters, one based on percentages of valid samples, and the
other on amplitude threshold. In this respect, our approach is less
arbitrary because we calculate them using objective clustering and the
actual experimental fluorescence distribution in the microarray. When
proper microarray reliability and proper expression threshold are applied,
we reached the conclusion that it was necessary to acquire and analyze a
new, individual dataset, which proves quite adequate to the task. Poor
compatibility between our approach and the SAM approach in the pool
dataset is apparently caused by the essential difference between the two
approaches in that our two filtering parameters are addressing the
reliability and the amplitude of the expression levels independently.
Indeed, two thresholds are present: one by amplitude, the other by
reliability. Instead, both the SAM denominator parameter and the SAM
significance threshold are related to reliability and amplitude in a
complicated manner. Similarly to our approach, SAM has two adjustable
parameters, namely the above-described arbitrary constant in the
denominator d=D/(s+s0) for relative difference, and the significance
threshold. In this respect, our approach is less arbitrary because we
calculate them using objective clustering and the actual experimental
fluorescence distribution in the microarray. When proper microarray
reliability and proper expression thresholds are applied, the compatibility
between the two approaches is very good (Sivozhelezov et al., 2008).
Furthermore the final leader genes map shed new light in the molecular
mechanisms controlling human kidney transplant. Microarray
experimentation becomes indeed much more targeted and significant, by
comparing gene expression analysis with the analysis of gene networks
and interactions. In this context, we successfully applied different
variants of the leader gene identification algorithm, in order to identify
the ones best representing real gene networks.
All the findings described here, regarding kidney transplant tolerance

but also possibly being extended to other systems, confirm the existence
of a small set of genes, having a higher number of interactions among all
the genes involved in the cellular process and therefore playing a central
role. The identification of most interacting genes can be of great
importance in the systematization and analysis of data, since leader gene,
considering also those largely changing expression in different patients,
form a unique network: the mere changing in expression of a particular
gene is not significant by itself, but only if it is put in a proper
framework. This change can be often considered as a consequence of a
more complex network of events, starting from leader genes, identified
with bioinformatics predictions, which often do not vary their expression
so much to be identified as significant using pangenomic arrays.
However, microarray technology is a necessary confirmation of every
prediction made by theoretical network analysis. On the other hand,
statistically-processed microarray data can serve as the starting point for
network analysis. We introduce a non-statistical approach to processing
microarray data, in which we apply K-means clustering to microarray
data only after independent filtering by both amplitude and reliability
that we define as percentage of valid data for each gene. The need for
non-statistical treatment, in addition to statistical treatment of microarray
data, has been recognized time ago (Affymetrix Inc., 2004) because
“microarrays are the unusual statistical case where the number of tests
greatly exceeds the number of samples, so standard statistical methods
for multiple comparisons are pushed to their limit”. To our knowledge
this is the first step in that direction. Results of the non-statistical
approach of microarray data interpretation are widely different from the
statistical (SAM) approach for the pool dataset, but are similar for the
individual dataset. At the moment, none of the three approaches
(Sivozhelezov et al., 2008), namely the “ab initio” approach, the
microarray-based statistical approach and the microarray-based non-
statistical approach, has proved superior in identifying the key genes
responsible for kidney graft rejection and/or tolerance, and showing that
those approaches must be used in a complementary manner, considering
also that reasons for divergence of those approaches have been
identified. Moreover, Sivozhelezov et al. (2008) showing average
number of pro/tolerance and pro/rejection genes respectively with TOL

and CR patients for the three different microarray-based estimates
provides a basis for combined sets of genes to be used in such
forthcoming studies. Besides, identification of a pathway possibly
important in controlling mechanisms of tolerance and rejection has
demonstrated a high potential for combination of approaches used herein.
Genomics do however suffer many pitfalls (Nicolini et al., 2006a) and
only functional proteomics (LaBaer, 2006) represents the long-range
answer to the basic molecular understanding and to the clinical control of
the human kidney transplants.
3.3.3 Osteogenesis
We tentatively identified 7 leader genes in the osteogenic process

(Covani et al., 2008). They were classified according to their
involvement in osteogenesis subprocesses (cell adhesion and
proliferation, ossification, skeletal development, Calcium ion binding).
On this base we are presently constructing a simple array (2 × 4 × 4
genes, for a total of 16 genes repeated 2 times) using APA technology
and displaying the leader genes and an equivalent number of controls, in
order to begin to study the osteogenetic process at molecular level.
In summary, putative “leader genes” undergo some changing in gene
expression, but the amount of this changing is not necessarily related to
their leader gene status. In fact, leader genes are the most-interacting
genes involved in a process, not necessarily the most varying ones as
apparent also in human T lymphocytes cell cycle where the most
changing gene is CCNH, which is not one of the leader genes. Ab initio
“leader plus class B” genes were scanned against the list of experimental
genes changing their expression levels (quite more numerous than the
leader genes identified ab initio). These very preliminary results show
that text-mining approach is dangerous to use for interpreting microarray
expression data. Indeed “theoretical” and “experimental” leader gene sets
are clearly different and for this reason we are attempting to check links
between the two sets of leaders: links between theoretical and
experimental leader genes using text-mining, and the same without text-
mining. The final results are still in progress and very preliminarily
leader genes identified with text-mining appear to have almost no

interactions shown; the validity of this approach in relating
bioinformatics predictions and experimental data is therefore much lower
than the “no text-mining” approach. Leader genes identified with a
completely independent bioinformatics prediction appear closely
interacting with genes changing expression in experimental analysis. An
important change in expression of these leader genes between the two
conditions is not necessary for their function. This was also proved in our
previous microarray study on T lymphocytes cell cycle: MYC was
overall the most important gene in the whole process (it is necessary to
enter the G1 phase, indeed), but it changes its expression very slightly
from quiescent to replicating cells. Many questions have still to be
answered before we reach a conclusion on this open question, but in any
cell system the changing in expression of a particular gene could be
considered as the ultimate consequence of a complex network of
biochemical interactions, whose most important nodes could be the
“leader genes” as identified with ab initio prediction. Several questions
emerge: which should be considered the true leader gene set, the
theoretical only or theoretical + experimental ? The latter answer proves
correct (Nicolini et al., 2006a; Sivozhelevov et al., 2006a; 2008;
Giacomelli et al., 2006) suggesting that the mere changing in expression
of a particular gene is not meaningful by itself, but only if it is put in a
proper framework. This change can be often considered as a
consequence of a more complex network of events, starting from leader
genes, identified with bioinformatic predictions, which often do not vary
their expression until now identified as significant using pangenomic
arrays. The work in progress in osteogenesis suggest that the leader gene
approach need to be validated by experimental analysis using DNA
microarrays and by independent clinical data.
3.4 Nanoproteomics
Two major lines of nanoproteomics, namely NAPPA Microarray and

Mass Spectrometry, gave independent new results aiding in the
understanding of cell cycle progression.
3.4.1 Cell cycle
Regarding the first variation identified by Mass Spectrometry we

analysed the averaged ESI mass spectrum (300–2000 m/z range)
recorded in the elution range 42.25–42.68 min, shown in the 2nd panel of
Figure 3.26). From the Gaussian deconvolution of this spectrum,
performed by MagTran1.0, we identified three protein species. We
performed first tentative protein identification on the basis of these
weights through a search in Swiss-Prot data bank (Boeckmann et al.,
2003).
Figure 3.26. (A) 1st panel (from the top), HPLC-ESI-MS TIC profile (elution range 41.5-
45.5 min). 2nd panel, averaged ESI mass spectrum (300–2000 m/z range) of CHO-K1
nuclear protein fraction recorded in the elution range 42.25–42.68 min. (B) deconvoluted
spectrum of averaged ESI mass spectrum reported in the second panel. (Reprinted with
the permission from Spera and Nicolini, cAMP induced alterations of chinese hamster
ovary cells monitored by mass spectrometry, Journal of Cellular Biochemistry 102, pp.
473–482, © 2007, Wiley-Liss, Inc., a subsidiary of John Wiley & Sons, Inc.).
We restricted our search to rodent proteins. In Table 3.3, we report

the experimental and theoretical weights and the function of the identify
proteins. Among these proteins, Guanine nucleotide-binding protein
(Gna11) and Myosin heavy chain 10, non-muscle (Myh 10), are
particularly interesting because they are both involved in cellular
regulation. Heterotrimeric guanine nucleotide-binding proteins (G
proteins) are integral to the signal transduction pathways that mediate the
response of the cell to many hormones, neuromodulators, and a variety
of other ligands (Strathmann and Simon, 1990).
Table 3.3: Proteins detected in the CHO-K1 protein nuclear fraction correspondently to
the 42.54 min TIC peak (Reprinted with the permission from Spera and Nicolini, cAMP
induced alterations of chinese hamster ovary cells monitored by mass spectrometry,
Journal of Cellular Biochemistry 102, pp. 473–482, © 2007, Wiley-Liss, Inc., a
subsidiary of John Wiley & Sons, Inc.).
Exp. Th. mass

Protein Function
mass (Da) (Da)
31133 31133 Myh10 Cellular myosin appears to play a role in
(fragment B) cytokinesis, cell shape, and specialized
Myosin heavy chain -B functions such as secretion and capping.
(Fragment), non-muscle
42024 42024 Gna11 Guanine nucleotide-binding proteins (G
Guanine nucleotide- proteins) are involved as modulators or
binding protein, alpha-11 transducers in various transmembrane
subunit signalling systems. Acts as an activator
of phospholipase C.
52136 52134 S61A1 Plays a crucial role in the insertion of
Protein transport protein secretory and membrane polypeptides
Sec61 alpha subunit into the ER. Required for assembly of
isoform 1 membrane and secretory proteins.
Tightly associated with membrane-bound
ribosomes, either directly or through
adaptor proteins.
Nonmuscle myosins play a role in diverse cellular functions, for

instance cytokinesis, proliferation, secretion, and receptor capping. Two
isoforms of the nonmuscle myosin heavy chain (nmMHC), chain A
(nmMHC-A) and chain B (nmMHC-B) have been identified. The
nmMHC-B in particular is involved in cell growth regulation and
transformation (Strathmann and Simon, 1990).
Regarding the 44.90 min TIC peak variation -relative abundance
passed from 26.6 for the untreated cells plot to 12.3 for the cAMP treated
cells plot (Figure 3.27a, 1st panel)- we analyzed the averaged ESI mass
spectra recorded in the elution range 44.40–45.20 min (Figure 3.25a, 2nd
panel). The two spectra are clearly identical: the difference of intensity in
the TIC peaks can be explained by a different amount of the same
proteins in the two samples. We performed a Gaussian deconvolution of
this spectrum and identified three protein species (Figure 3.27b).
Through a search in Swiss-Prot data bank (Boeckmann et al., 2003),
we have been able to identify only the 29628 Da peak as CD82_MOUSE
protein. This protein, associates with CD4 or CD8 glycoprotein delivers
co-stimulatory signals for the TCR/CD3 pathway (Itoh and Adelstein,
1995) involved in apoptosis regulation. For 6632 Da peak and 20739 Da
peak, it was not possible to identify any protein. Moreover we analyzed
seventeen other proteins of the nuclear envelope present in the same
content in the cells before and after cAMP exposure. The relative
molecular weight are 2.4 kDa, 11.9 kDa, 17.3 kDa, 29.55 Da, 35.6 kDa,
41.2 kDa, 46.7 kDa, 52.3 kDa, 52.9 kDa, 58.9 kDa, 64.2 kDa, 70.7 kDa,
60.9 kDa, 82.5 kDa, 93 kDa, 96.2 kDa and 99.3 kDa. It has been possible
to identify none of these proteins only from their molecular weight.
We subsequently performed a series of experiments, coupling HPLC
(Table 2 in Spera and Nicolini, 2007) and MS (Nagira et al., 1994), to
identify all these proteins and to confirm the identity of the proteins via
mass fingerprinting (that allows to identify the protein with a very high
probability).
This second step was conducted on a MALDI-TOF Mass
Spectrometer. For protein fingerprint, the HPLC fractions were digested
and the tryptic digest samples were analyzed by MALDI-TOF MS. Until
now we have obtained from this analysis, the confirm of identification of
31133 kDa protein as “Myosin heavy chain -B (Fragment), non-muscle”,
present only in the CHO-K1 sample (HPLC fraction 3, Table 2 in Spera
and Nicolini 2007).
The study here reported on the effect of cAMP on the protein
expression of the CHO-K1 cells is continuing structural and functional
work started many years ago at the level of nuclei and genes (Nicolini
and Beltrame, 1982; Vergani et al., 1992, 2001). Our RP-HPLC-ESI MS
results, confirmed by HPLC measures, show a different protein content
in the nuclear protein fractions of the cells after exposure to cAMP,

possibly linked to the above early observations.
Figure 3.27. (A)1st panel (from the top), HPLC-ESI-MS TIC profile (elution range 41.5–
45.5 min). 2nd panel, enlargement of averaged ESI mass spectra (300–2000 m/z range) of
CHO-K1 (grey line) and cAMP CHO-K1 (black line) nuclear protein fraction recorded in
the elution range 44.4–45.20 min. The spectra are identical. (B) deconvoluted spectrum
of averaged ESI mass spectra reported in the second panel. (Reprinted with the
permission from Spera and Nicolini, cAMP induced alterations of chinese hamster ovary
cells monitored by mass spectrometry, Journal of Cellular Biochemistry 102, pp. 473–
482, © 2007, Wiley-Liss, Inc., a subsidiary of John Wiley & Sons, Inc.).
In particular we focused our attention on the two main differences in

the TIC plots of the nuclear protein fractions of untreated and treated
cells that show a decrease in the nuclear protein amount after cAMP
treatment Through ESI MS analysis we identified a group of three
proteins, Myh10, Gna11 and S61A1, present only in the nuclear protein
fraction of untreated cells and involved in cellular regulation processes.

The Myh10 protein is involved in the catalysis of movement along a
polymeric molecule such as a microfilament or microtubule, coupled to
the hydrolysis of adenosine 5’-triposphate (ATP), while the Gna11
protein regulates the cascade of processes by which a signal interacts
with a receptor, causing a change in the level or activity of a second
messenger.
Figure 3.28. Mascot results relative to mass fingerprint of HPLC (Reprinted with the
permission from Spera and Nicolini, cAMP induced alterations of chinese hamster ovary
cells monitored by mass spectrometry, Journal of Cellular Biochemistry 102, pp. 473–
482, © 2007, Wiley-Liss, Inc., a subsidiary of John Wiley & Sons, Inc.).
Moreover we identified another group of three proteins present in

both samples but in a double concentration in the nuclear protein fraction
of untreated cells. Through ESI MS analysis we were able to identify
only one of these three proteins, Cd82Mouse, that results involved in
apoptosis regulation processes (Figure 3.27).Other seventeen proteins

present in equal amount before and after cAMP exposure have been
identified.
From preliminary HPLC and MS experiments we had a confirmation
of the inhibition of the nuclear protein expression after exposure to
cAMP; moreover by protein fingerprinting we confirmed the
identification of 31133 kDa protein as Myh10 (Figure 3.28).
The same analysis on the other protein fractions (cytosolic,
membrane and membrane organelle and cytoskeleton fraction) is in
progress to analyze the entire proteome in presence of, to obtain a
comprehensive understanding of the reverse transformation (Spera and
Nicolini, 2007; Spera et al., 2007).
3.4.2 Cell transformation and differentiation
The focus of function-based microarrays is to study the biochemical

properties and activities of the target proteins printed on the array.
Figure 3.29. Different gene collection.

Function-based microarrays can be used to examine protein

interactions with other proteins, nucleic acids, lipids, small molecules
and other biomolecules (LaBaer and Ramachandran, 2005;
Ramachandran et al., 2004).
In addition, function-based microarrays can be used to examine
enzyme activity and substrate specificity associated to cell
transformation and to cell differentiation. These microarrays are
produced by printing the proteins of interest on the array using methods
designed to maintain the integrity and activity of the protein, allowing
hundreds to thousands of target proteins to be simultaneously screened
for function using a wide range of gene collections (Figure 3.29).
Figure 3.30 (A) MALDI-TOF target modification project. (B) MALDI TOF MS
spectrum of Human Kinase NAPPA array acquired after protein synthesis (spot gene NA
7-A 12), low masses region. The arrows indicate the peaks identified also in the Dr.
Fuentes and LaBaer spectrum (namely m/z =711 ± 12, m/z =865 ± 18, m/z =1090 ± 20,
m/z =2900 ± 40, m/z =3540 ± 40, m/z =3860 ± 60) (Reprinted with the permission from
Spera and Nicolini, Nappa microarray and mass spectrometry: new trends and challenges
Essential in Nanoscience Booklet Series, © 2008, Taylor & Francis Group/CRC Press,
http://nanoscienceworks.org).
The list of potential applications of such microarrays is large. A

microarray of a particular class of enzymes such as kinases could be
screened with a candidate inhibitor to examine binding selectivity. A
candidate drug could be used to probe a broad range of enzymes to look
for unintended binding targets that might suggest possible toxicities.
Proteins expressed by pathogenic organisms can be screened with serum
from convalescent patients to identify immunodominant antigens,
leading to good vaccine candidates.
Figure 3.31 In situ protein detection (Reprinted with the permission from Prof. Joshua
LaBaer at Harvard Institute of Proteomics).
Protein interaction networks, including the assembly of multiprotein

complexes, can shed light on biochemical pathways and networks in the
control of cell transformation and cell differentiation. Eventually, it may
even be possible to use with proper inert surface (Figure 3.30) these
high-density microarrays as a MALDI source for mass spectrometry,
allowing users to probe complex samples for binding partners to many
proteins simultaneously.
However, as with the abundance-based microarrays, there still remain
challenges in building and using function based protein microarrays.
First, the notorious lability of proteins raises concerns about their
stability and integrity on the microarray surface. Second, it is time
consuming and costly to produce proteins of good purity and yield, and
many proteins cannot be purified at all. Finally, the methods used to
attach proteins to the array surface may affect the behaviour of the
proteins. Despite these challenges, there has been some success in
building and using function-based protein microarrays in medicine
(LaBaer and Ramachandran, 2005; Ramachandran et al., 2004). In this
Volume we have described different approaches, protein spotting
microarrays and self-assembling microarrays, including their recent most
promising advances utilizing Label Free technologies (Figure 3.31).
3.5 Nanomechanics and Nanooptics
Several new sectors have been emerging in the area of nanomechanics

and nanooptics, which are summarized in this paragraph as an example
of their very promising nature in terms of our basic understandings of
matter and of future challenging new technological applications in health
and science.
3.5.1 Nanocontacts for addressing single-molecules
Of particular interest is how sensitivity, selectivity, and switching may be

improved by directly contacting single molecules (for a review see
Carrara et al., 2005). To achieve this goal the fabrication of electrodes
separated by nanometric sizes gaps that may be bridged by single-
molecules is necessary.
Two approaches to fabricate nanometric-sized contacts were recently
proposed in literature. The first involves the movement of electrodes
pairs. The second relates to the formation of electrodes pairs via etching.
In the field of biosensors they first appeared in the late 1990s, when
papers concerned with the organization of monomolecular layers of
sensing molecules (Bykov, 1996) that either provide stable networks to
benefit electrochemical detection of bound molecules at nanostructured
electrodes (Tiefenauer et al., 1997) or permit single molecules to be
addressed using devices such as Scanning Probe Microscopes (Göpel,
1998) were published. Recently, system based on cantilever
micromechanical technology were proposed (Raiteri et al., 2001) but

more complex nanostructured systems have been employed in biosensors
(Vo-Dinh et al., 2001): for example, carbon nanotubes and fullerenes
have been used to stabilize electrochemical mediators and enzymes
(Sotiropoulou et al., 2003) and gold nanotube structures have been
shown to enhance sensitivity to glucose (Delvaux and Demoustier-
Champagne, 2003). A future goal of nanotechnology in the field of
biosensors must be the development of simple nanoscale devices for
addressing the single molecule. Till now in the field of biosensors such
devices have been built on the micro-scale (Gorschlüter et al., 2002). It
has been demonstrated that it is possible to study DNA molecules that
bridge interdigitated electrodes (Hoölzel et al., 2003). DNA strands,
greater than 5000 base pairs in length, have been investigated by placing
them across a two micrometer gap (see Figure 1(b) in Hoölzel et al.,
2003).
Similarly, sensors based on the change in magnetoresistance when
two micrometer magnetospheres coated with streptavidin are adsorbed in
a six micrometer biotin coated gap have been demonstrated (Graham et
al., 2003). The feasibility of addressing single short chain molecules has
been established in the emerging area of the molecular scale electronics.
Conductivity measurements on single benzene-1,4-ditiol molecules
(Reed et al., 1997) have been performed. It has also been demonstrated
that single molecule conductivity measurements require chemical
binding of the molecule of interest to both contacts (Cui et al., 2001). To
achieve such a configuration requires that facile methods of placing
electrodes at nanometer scale separation be developed. Very recently,
across the field of nanoscience different methodologies of obtaining such
nanocontacts have been proposed.
Literature presents two completely different approaches to the
fabrication of nanocontacts. The first is to mechanically align a pair of
electrodes at nanometer separation. These methods require special
drivers to accurately position the electrodes. The second uses nanometric
level etching of pre-structured conductive materials. The success of this
second approach is dependent on to what degree the etching processes
may be controlled. In the following part of this paper we review these
methods.
In this section we review methods related to the movements of

electrode pairs in order to obtain nanocontacts. The general idea of all
methods presented here is to bring together two electrodes and place
them at nanometer scale separation. García and co-workers (2002)
developed a method in which the tips of two wires are brought in to
contact. In their work, which is primarily concerned with magneto-
conductivity, they describe how nanoscale contacts may be formed
between magnetic and non-magnetic materials. The metals studied
include nickel, copper, aluminum, gold and iron. The nanocontacts were
prepared by first placing two rounded wire tips in the apertures of a
teflon tube (Figure 3.32).
Figure 3.32. Nanocontacts were prepared taking a couple of wires tightly bound within a
Teflon tube. A bias voltage was applied to the wires. The wires were approached till a
current flow is established between them. The resulting contact shows quantum
conductivity.
A bias voltage was then applied to the wires and the wires
approached till a current flow was established. In such a manner,
quantum magneto conductivity was observed between the nanocontacts
at a field of tens of Oe. The as-produced nanocontacts showed quantum
conductivity and the conductivity remained stable even when fields of
more than one hundred Oe were applied (Figure 3b in García et al.
1999). However, the mechanism by which the force was applied to the
wires in order to bring them into contact is not detailed. Further, the fact
that the nanocontacts obtained are housed within a Teflon tube suggests
that the fabrication technique cannot easily be employed to fabricate
biosensors.
Two (La0.7Sr0.3)MnO3 single crystals have been placed at nanometer
separation (Versluijs et al., 2000). The quantum conductance across the
resultant nanoscale gap has been recorded. Initially the crystals were
placed in mechanical contact. They were then pulled apart until quantum
conductivity was established. The authors have employed both a
mechanical relay and a piezo-device to separate the crystals. Quantum
conductance has been realized with both a relay and a piezo-mover. In
the case of piezo-driven separation 20 steps in the conductance versus
time curve were observed. However, even using piezo drivers the
maximum time for which the nanocontact remained stable was of the
order of seconds, precluding the use of this methodology of forming
nanocontacts in biosensor application. It is noteworthy that ceramic
crystals are better suited than metals to nanocontact formation by this
technique as the former undergo brittle fracturing whilst the latter display
plastic deformation. However, temporal nanocontacts have been formed
between vibrating macroscopic wires (Costa-Krämer et al., 1997). Using
a piezo-driver two wires are brought into contact and then set in
vibration. As the wires vibrate a nanocontact is momentarily formed.
Again, the temporary nature of the nanocontact means this method of
production is of limited use in biosensor applications.
A piezo-driver offers the possibility of controlling on the nanoscale
the approach of a wire to a planar macroelectrode. It has been shown
(Facci et al., 1996) that using the tunneling current flowing through the
contact as a feed-back signal it is possible to bring a wire tip to a few
tens of nm from a flat electrode. This is achieved by stopping
piezoelectric movement when the tunneling current reaches a pre-defined
value. Using this technique the distance between electrode and wire has
been controlled to within a nanometer (Carrara et al., 2006). This method
was used for addressing single cadmium sulfide (Facci et al, 1996) and
lead sulfide (Erokhin et al., 1997) nanoparticles. The resultant metal-
insulator-nanoparticle-metal configurations displayed behavior
characteristic of single-electron junctions. It is noted, however, that in
the above configuration it is not possible to realize two chemically
bonded contacts, i.e., the criteria required in order to obtain reproducible

measurements of single molecules conductivity are not met (Cui et al.,
2001).
Alternative approaches to the formation of stable gaps between
conducting materials that are of small enough dimension that they may
be bridged by biological molecules to form nanocontacts are based on
etching. That is, starting from a single piece of conducting material a
nanoscale gap is cut by controlled dissolution of material. The major
constraint to the wide spread use of this approach is the problem of
controlling etching on the nanoscale. In this section we review methods
of fabricating nanocontats in preformed structures. Both physical and
chemical etching is considered.
A chemical method to form nanocontacts is via a combination of
controlled electrochemical etching and electrochemical deposition. The
method has been termed “ELENA” (ELEctrochemical NAnodeposition)
(Céspedes et al., 2002). The technique has been used to fabricate metallic
electrodes with a separation on the nanometer scale. Morpurgo and co-
workers (1999) first developed this two-step process. Using conventional
lithography two electrodes at micrometer separation were prepared, the
electrode pair. At this point the separation of the electrodes was not a
critical parameter. Then, by applying a DC potential relative to a counter
electrode, metal is electrodeposited on to the electrode pair. During the
deposition an AC potential is applied across the electrode pair allowing
the resistance between the electrodes to be measured. As the electrodes
grow towards each other the resistance changes as follows; at large
separation the high resistance of the electrolyte is monitored, at nm
separation a tunnelling current is observed and on contact a step decrease
in resistance is observed. The tunnelling region is characterized by an
exponential relationship between electrode separation and current. At
slow deposition rates, steps may be observed in the resistance versus
time plots at the point when the two electrodes first make contact. The
step features are due to atom-by-atom growth of the contact. A very
important feature of this method is the reversibility of the process. Hence
it is possible to deposit metal until the two electrodes are in contact and
then electro-dissolve the metal and reopen the gap. SEM images of
contacts prepared using this methodology show in Figure 3.33 nanosized

gaps in the range 20 nm to 100 nm.
Figure 3.33. SEM images after the formation of the separation with conventional
lithography (A) and after the electrodeposition (B), electrodes in which the gap was
reopened by electrodissolution, by reversing Vdc following an intentional short circuiting
(contacting) in a previous electrodeposition process (C). In the case of these
measurements the S.E.M. resolution was only 5 nm and, therefore, the gap visualized
could be even smaller (Reprinted with the permission from Morpurgo et al., Controlled
fabrication of metallic electrodes with atomic separation, Applied Physics Letters 74, pp.
2084–2086, © 1999, American Institute of Physics).
Recently it has been shown that using the resistance across the
electrode pair as a feedback signal it is possible to obtain nanocontacts of
pre-defined dimension. Li and co-workers (2000) using e-beam
lithography, prepared two Au electrodes with an initial separation of 60
nm on a silicon dioxide substrate. The structure was covered by
polymeric resist and silicon dioxide. This was to minimize conduction
through the electrolyte and hence permit accurate monitoring of the

feedback signal.
The tunnelling current was observed to increase during Cu
deposition. Comparison with STM experiments indicated that it is
possible to monitor the gap width at a resolution of 0.5 Å resolution.
Thus by switching off etching when a pre-defined tunnelling current is
achieved it is possible to fabricate nanocontacts with sub-nanometer
precision. He and co-workers (2002) have shown that expensive
lithographic pre-etching steps are not required for the ELENA technique.
They demonstrated that starting from a thin copper wire, insulated except
for 1 micrometer region, it is possible to prepare electrode pairs
separated by sub-nanometer gaps. Céspedes et al. (2002) compared the
quality of nanocontacts prepared using the ELENA methodology with
those fabricated using Focused Ion Beam. It was found that nanocontacts
fabricated with the ELENA method were better defined than those
prepared using the FIB method. In addition smaller gap sizes could be
achieved using the ELENA technique (Figure 3.34).
Figure 3.34. In the modified ELENA method, the electrochemical etching or deposition is
controlled by the current flowing through the nanocontacts by monitoring the quantized
conductance through the electrodes in contact with nanometric area or by monitoring the
tunnelling current trough the electrodes gap.
The methods detailed above allow nanosized gaps to be fabricated

between electrodes. In a vacuum a tunnelling current will flow between
such electrodes. The change in current that accompanies bridging of the
gap by an analyte biomolecule will form the basis of future generations

of biosensors.
The physics of conductivity across such a nanocontact remains poorly
understood and the development of biosensors based on this technology
will require further studies in this area. Several models have been
proposed to explain the non-linear I-V characteristics observed in
nanocontacts: blocked conductance channels, parallel metallic and
tunnelling channels and the Luttinger liquid (Versluijs et al., 2000). In
addition to the physics, the chemistry of the molecule-metal interface
must be considered (Cui et al., 2001). For example, it has been
postulated (Hipps, 2001) that the nature of the contact determines
whether a DNA “wire” acts as an insulator, a semiconductor, a conductor
or a super-conductor. In addition, mechanical instabilities related to
fractures (Sotton, 1996) and to rearrangements (Rodrigo, 2002) could
dominate the behavior of nanocontacts.
Above we have considered the fabrication of electrode pairs separated
by nanoscale gaps that may be bridged by analyte molecules. However,
we note that this is not the only nano-electrode configuration that may be
employed in analysis. Most of the fabrication techniques described above
may be used to prepare devices in which two electrodes are in direct
contact, the area of the contact being on the nanoscale. As detailed
elsewhere the conductance of such contacts is quantised. Adsorption of
single analyte molecules on such contacts leads to large changes in the
resistance of the contact, i.e., a large change in signal is observed for a
single molecular event.
For example a conductivity change of 50% has been observed when
mercaptopropionic acid adsorbed on a quantum wire (Bogozi et al.,
2001).
Sensors and biosensors devices need to address single molecules in
order to increase the sensitivity of the devices. Nanotechnology offers
technical solutions to fabricate nanocontacts. The aim of this paper was
to review all the methods for fabrication of nanocontacts that have been
presented recently in literature. Two main classes of methods were
identified. The first is based on the possibility of moving one electrode
close to another at nanometric distances. The second is based on the
etching of a couple of electrodes with gap at nanometric scale. The
second class of methods seems to be the most reliable and return the
most stable nanocontacts. The electrochemical etching and deposition
appears to be the most economic method. This method also allows
control of the electrode gap with a precision of 0.5 Å. The next question
that nanotechnology must answer of the road to nanoscale biosensors is
what are the physical and chemical parameters that must be managed.
3.5.2 Nanofocussing
The Nanofocus extension is now becoming operational at ID13 of ESRF

(Riekel, 2000; Riekel et al., 2000) as the third experimental hutch (EH3).
EH3 will be a dedicated nanofocus facility, offering sub-µm
monochromatic X-ray beams in the energy range 12–13 keV as a matter
of routine.
A pink beam option will be added later. The target beam size once
established will be 50 nm or less. The availability of nano-focused X-ray
beams on the ID13 beam-line will open up many new avenues of
research. Whilst existing scanning X-ray SAXS/WAXS studies will be
enhanced by the higher spatial resolution available, one can expect that
highly coherent nano-beams will find complimentary diffraction and
imaging applications. In addition, many experiments will also benefit
from the higher flux density available for microscopic sample volumes.
EH3 will offer an ultra-stable sample end-station with nm-resolution
translation and tilt options. An integrated rotation axis will allow single-
crystal and texture experiments. It will also provide solutions to the
problems associated with visualizing (and aligning) microscopic
samples. This includes the development of complementary, integrated
nano-tools, calibrated to the X-ray beam's position. The challenges of
preparing and isolating such small samples will be met by
micromanipulation and laser-cutting equipment accessible to users on-
site. The use of dedicated beam-line software will also help ensure the
most efficient use of experimental beam time.
EH3 will offer an ultra-stable sample end-station with nm-resolution
translation and tilt options. An integrated rotation axis will allow single-
crystal and texture experiments. It will also provide solutions to the
problems associated with visualizing (and aligning) microscopic
samples. This includes the development of complementary, integrated

nano-tools, calibrated to the X-ray beam's position. The challenges of
preparing and isolating such small samples will be met by
micromanipulation and laser-cutting equipment accessible to users on-
site. The use of dedicated beamline software will also help ensure the
most efficient use of experimental beam time.
The commissioning of EH3 in February 2007 has herald the start of a
new era for ID13. As an innovative experimental tool, nano-beams
appear to give a new insight into many materials with nm-sized
heterogeneities (Nicolini et al., in preparation; Pechkova et al., in
preparation). They will also ensure that ID13 remains at the cutting edge
of science. The ID13 beam-line will indeed shortly offer in situ micro-
and nano-focus Raman Spectroscopy facilities.
A custom-built MicroRaman system was recently designed in
collaboration with Renishaw PLC. The system consists of a
spectrometer, fibre-optically coupled to a remote probe positioned within
the EH2 sample environment. This coaxially delivers a focused laser spot
to the same position on the sample as the X-ray beam. The laser spot at
the focal position is approximately 1 µm in diameter, comparable to the
X-ray beam size from several different ID13 optics. Using a common
trigger signal between the beam-line control system and spectrometer, it
is possible to simultaneously collect WAXS/SAXS and Raman spectra
from the same position on the specimen at the same time. Raman
spectroscopy and nanofocused X-ray scattering are complementary
techniques on many different levels. They can provide structural
information on a range of different length scales, from molecular bonds
up to tens of nanometers (for SAXS). They complement each other in
their phase- or volume-selectivity, whilst their non-destructive nature
(for many materials) makes them ideal for coupling with other methods.
They therefore have a diverse range of potential applications, from
studying deformation micromechanics and monitoring chemical
reactions to characterizing materials over both macro- and microscopic
length scales.
3.5.3. Optical tweezers
An optical tweezer is a device for the non-contact trapping and

manipulation of micron-sized objects under a microscope using a laser
beam.
Figure 3.35. Optical tweezers. The microscopic sphere is pulled into the brightest part of
the focused laser beam. Schematics showing the principle of optical tweezers based on
ray optics. The ability to trap and manipulate small objects, such as polystyrene beads,
results from light possessing momentum which is in the direction of propagation of the
beam. Here, a bead is illuminated by a Gaussian profiled laser beam. The representative
laser paths are shown as black lines with arrows indicating the direction of beam
propagation. The thickness of the black lines indicates the intensity of laser beam. The
forces are shown as green and blue lines with arrows indicating the direction of forces.
The length of the lines indicates the intensity of forces. (A) Gradient forces which are
generated upon refraction (FG, fa and fb, green lines). The beam is refracted on the
surface of the bead, resulting in the change of momentum of the beam. Gradient forces
(FG) result to compensate the momentum changes on the surface of the bead. The
gradient forces from the inner region (fb) are larger than that from outer region (fa) of the
beam, due to the profile of the laser. Consequently, the net gradient force in the lateral
direction directs particles to center of the beam (FG). (B) Stable 3D trapping. The laser
beam was focused by a lens of high numerical aperture. The scattering forces (Fs, blue
line) are in the direction of propagation of the laser beam (i.e., downward in this figure),
while the gradient forces are directed toward the focused spot. Consequently, the bead is
trapped slightly beyond the focused spot where the gradient force and scattering force are
in equilibrium (Reprinted with the permission from Kimura and Bianco, Single molecule
studies of DNA binding proteins using optical tweezers, Analyst 131, pp. 868–874, ©
2006, Royal Society of Chemistry).
The microscope objective focuses the laser light to a very small spot,
about 1 µm in diameter. The focal spot works like a trap for microscopic
objects, which are pulled into the brightest part of the beam. The
microscopic sphere shown has a higher refractive index than the water
that surrounds it, so it works like a tiny lens bending the rays of light
away from the axis. As the light has momentum, and total momentum
must be conserved, the sphere is then pushed by the light towards the
axis, becoming trapped in the focus. The trapped sphere can then be
moved by moving the laser beam, just as tweezers can be used to pick up
and move small objects.
The magnitude of the force exerted by the laser light is typically a
few pico-newtons (10-12 N), which is comparable to that produced by
biologically interesting molecular motors, and so optical tweezers have
found several applications in interdisciplinary science and biophysics.
Many experiments have been done on trapped biopolymers
commonly used in ultrasound scans to improve the contrast of the image.
However to fully understand their properties single microbubbles must
be studied in order to compare the response to that predicted by theory.
Optical tweezers are the ideal tool for isolating a single bubble from a
sample and observing it while being irradiated with ultrasound, as
recently shown by Sarah Skoff at UCL on the bubble's protein shell,
either to improve the signal in an ultrasound scan, or to make the bubble
break open when exposed to ultrasound waves. This would be useful if
the bubbles were filled with a drug, which could then be targeted to the
exact location needed.
An alternative method for trapping a microbubble is to use a “hollow”
laser beam that has a dark spot on the center, surrounded by a bright ring.
A class of laser beams that has this property is the Laguerre-Gaussian
beams. Most lasers produce a beam with a Gaussian intensity profile -
the brightest part is in the center. In order to make a beam with a dark
center we use a computer-generated hologram which when illuminated
with an ordinary laser beam works like a diffraction grating. The
difference is that the diffracted orders have the dark center characteristic
of a Laguerre-Gaussian beam (see the review by Neuman and Block,
2004). Optical tweezers are a useful and important tool with many
applications in the physical and life sciences. The techniques being used
will help us to understand some of the fascinating physics that controls
biological systems. The technology could one day be used to make self-
assembling electronic components or chemical factories-on-a-chip.
Bernard Yurke and his colleagues (2000) at Bell Laboratories in New
Jersey made a tweezer-like structure out of three strands of DNA. When
a fourth strand is added to the mixture, it joins the loose ends of the
tweezers, pulling them shut. Adding yet another strand of DNA pops the
tweezers open in order to get controllable motion on a nanometre scale.
Single molecule studies of DNA binding proteins using optical tweezers.
Optical tweezers have become a versatile tool in the biological sciences.
Combined with various types of optical microscopy, they are being
successfully used to discover the fundamental mechanism of biological
processes. Recently, the study of proteins acting on DNA was
aggressively undertaken at the single-molecule level, providing detailed
mechanistic insight that could not be revealed, at least not easily, using
bulk-phase or ensemble approaches (Kimura and Bianco, 2006).
3.5.4 Magnetism
Ever since the German physicist Max von Laue's 1913 insight that X-
rays could be used to unravel crystal structure, they have been an
essential tool for the study of matter. Some neighborhoods, however,
have been off limits to X-rays, such as materials' fine-scale magnetic
structure and the fleeting molecular alliances within disordered materials
such as liquids and glasses.
Those have been the domains of neutron beams since the first
research reactors were built in the 1950s. With the advent of third-
generation synchrotron sources, however, X-ray scattering is making
inroads into neutron territory. “The special points are very high
brightness, good-quality polarization, and very high energy x-rays” says
Hiroshi Kawata of the Photon Factory at the KEK high-energy physics
lab in Tokyo. Because of these properties, “you can start thinking about
experiments it would not have been possible to do a few years before”
says physicist Michael Krisch of the European Synchrotron Radiation
Facility (ESRF) in Grenoble, France, the first of the new machines.
Indeed, the third-generation sources are teasing out information about
magnetic properties and disordered materials that neutrons could not
reveal and researchers are beginning to answer what is the real nature of
the magnetization. And the X-rays' brightness and tightly controlled
energy have opened the way to studies of disordered materials that
capture, for example, a high-speed form of sound in water.
Those results are only the first in what is expected to be a torrent,
says David Laundy, an ESRF user from Britain's University of Warwick,
because “X-rays give different information from that of neutrons.”
Neutrons are sensitive to magnetism, for example, because they are
scattered not only by collisions with atomic nuclei, but also by magnetic
interactions with atoms as a whole. Neutrons, although they lack charge,
nevertheless have their own magnetic field. But neutrons cannot
distinguish the two contributions to an atom's magnetic field, which
come from the inherent spin of its electrons and from the magnetic effect
of those electrons orbiting the nucleus. Separating out the spin and
orbital parts really lies at the heart of understanding magnetic properties,
and X-rays offer a way to untangle these two effects. Scattered by an
atom's electrons, x-rays respond mainly to the electrons' electric charge,
but the magnetic part of the photon's electromagnetic wave also interacts
feebly with the electrons' magnetic field if they are aligned
advantageously. This is possible with the new X-ray sources; because
they have beams whose polarization, the alignment of their electric and
magnetic fields, can be controlled. And it turns out that X-rays are also
sensitive to the two different components of magnetism (Suortti at
ESRF).
X-rays offer other advantages over neutrons, tending to be more
sensitive to the surface of the material than neutrons for studying exotic
magnetic structure. X-rays are also flexing their muscles in another
domain that was once the preserve of neutrons, so called inelastic
scattering. Inelastic scattering studies with neutrons have unraveled the
dynamics of a wide range of systems, from liquids to metallic glasses.
But neutron studies often require samples to be made from rare isotopes,
rather than the common ones. Water, for example, has to contain
deuterium rather than hydrogen for neutron studies. Neutron beams are
also dim, and their energy range is limited. ESRF's X-rays, however,
have a wide range of energies and momenta, enabling Francesco Sette
and his colleagues recently to study just this type of fast excitation
process in determining the dynamical properties of disordered materials

in a region that was not accessible before. These transitory get-togethers
by molecules are called collective excitations, and they may affect
everyday properties of a liquid, such as chemical reactions, thermal
properties, and the way sound waves propagate.
However, Sette and colleagues (1996) from ESRF and from the
University of L'Aquila in Italy confirmed the latter effect by setting off
fast sound waves in water with inelastically scattered X-rays.
Solid but disordered materials such as glass are also coming to be
studied by inelastic x-ray scattering. Despite their newfound abilities, x-
rays are not about to replace neutrons as a tool for studying matter, but to
be complementary. Even though x-rays can probe magnetism in ways
neutrons cannot in many situations, neutrons are still the probes of choice
to determine a magnetic structure, at least for the present.
3.6 Cell Nanobioscience
Few key examples are here quoted of the usefulness of

Nanobiotechnology in the study of intact cell component, namely
nucleosome core and proteins in situ.
3.6.1 Nucleosome core
Protein crystallography is currently turning out novel atomic structures

of biological macromolecules at the rate of 400-500 per year
(Hendrickson and Wüthrich, 1997). These structures range in size from
proteins comprising under 100 amino acids to virus particles of several
million molecular weight.
An area of intense interest currently concerns macromolecular
complexes containing multiple components such the combination of
interphase chromosomes named chromatin that must periodically
assemble and disassemble in order to carry out their biological function
in the cell (Baserga and Nicolini, 1976; Diaspro et al., 1991;Kendall et
al., 1977; Nicolini, 1983; Nicolini and Kendall, 1977; Pepe et al., 1990;
Zietz et al., 1983). A classic example in all higher cells is the
nucleosome, which is the fundamental repeating unit of DNA

organization in chromosomes and accounts for the two most fundamental
levels of higher order chromatin structure (Nicolini, 1983; Nicolini et al.,
1975, 1976, 1977, 1977a, 1982, 1983, 1983a, 1984, 1991). Not only do
nucleosomes efficiently package DNA, they are also intimately involved
in the gene expression mechanisms that allow only selected regions of
the vast store of genomic information to be read out as a consequence of
signaling processes. These two functions require about 25 million
nucleosomes in each human cell nucleus. The nucleosome core particle
structure explains in atomic detail how DNA is kept untangled in the cell
nucleus and clarifies the unique role of the nucleosome in maintaining
and controlling the expression of genetic information. Nucleosomes do
not exist as isolated particles in the cell, but are packed into arrays with
an internal repeat of 157–240 bp. The dynamic assembly and
disassembly of the higher-order structures made from these arrays helps
determine the functional and dynamic state of DNA, and this will be
most likely solved by the combined utilization of optical tweezers and
nanofocused beam-line at the Synchrotron Radiation (previously
described) in the study of intact mammalian nuclei and cells. This
endeavor will elucidate the structures of higher-order arrangements of
nucleosomes in the chromatin fiber and to relate this information to the
way these assemblies and their super-coil participate in gene regulation
(Nicolini, 1986).
In 1984, the structure of the nucleosome core particle (NCP), the
larger part of the nucleosome, was published at 7 Å resolution
(Richmond et al., 1984). X-ray data was collected before synchrotron
radiation was generally available for protein crystallography by using a
single detector diffractometer and a rotating anode source. The spatial
resolution of this first structure was limited by the material itself as it
was prepared from whole nuclear chromatin and was therefore
heterogeneous in composition. Eventually, it became technically feasible
to assemble NCP in the test tube from homogeneous components made
individually in bacterial cells (Luger et al., 1997a).
After a long period of experimentation for developing homogeneous
preparations of NCP, crystals were obtained that diffracted to high
resolution. Nevertheless, the Bragg intensities from these crystals are
extremely weak. Fortunately, the ESRF had opened for business with
ID13 and as a result, the structure of the nucleosome core particle was
published in 1997 at 2.8 Å resolution (Figure 3.36) (Luger et al., 1997b).
At 206 kDa, the NCP is the largest and most universal protein/DNA
complex solved in atomic detail.
Figure 3.36. Crystal structure of the nucleosome core particle at 2.8 Å resolution. The
DNA double helix (146 base pairs in two chains: turquoise and brown) is wound around
the protein histone octamer (two copies each of H2A: yellow, H2B: red, H3: blue, and
H4: green) in 1.65 left-handed superhelical turns. This is the form of DNA, which
predominates in higher living cells. The left view is down the superhelix axis. The right
view is orthogonal to the superhelix and overall pseudo-twofold axis (Reprinted with the
permission from Luger et al., Crystal structure of the nucleosome core particle at 2.8 Å
resolution, Nature 389, pp. 251–260, © 1997, Macmillan Publishers Ltd).
Our best nucleosome core particle crystals show Bragg intensities to

1.9 Å and have measurable data to 2.0–2.1 Å spacings (Figure 3.36). The
NCP contains pairs of the four core histone protein molecules named
H2A, H2B, H3, and H4, and a roughly equal mass of DNA in 147
nucleotide pairs (we used 146 bp). Compared to the nucleosome, the
NCP is missing only the “linker histone” H1 and the short stretches of
DNA that connect the nucleosome cores to each other in chromatin
(Nicolini and Kendall, 1976). The core histones are arranged in an
octameric unit around which the DNA is wrapped in 1.65 turns of a left-
handed superhelix (Luger et al., 1997b).
This arrangement necessitates a substantial deformation of the DNA,
bending the 22 Å diameter double helix to a mean radius of 42 Å in the
nucleosomal superhelix.
The histone protein chains are divided into three types of structures:
1) rigid, folded alpha-helical domains named the histone-fold, 2) histone-
fold extensions which interact with each other and the histone-folds, and
3) flexible “histone tails”.
The histone-fold domains are structurally highly conserved between
the four types of core histones and have also been discovered in an
increasing number of other molecules involved in the regulation of gene
read-out or transcription. They form crescent-shaped heterodimers,
which have extensive interaction interfaces in the pairings H3 with H4
and H2A with H2B.
The histone-fold domains are responsible for organizing 121 base
pairs (bp) of DNA in the superhelix, not the entire 147 bp. It is the
responsibility of the extensions just prior to the H3 histone-folds to bind
the first and last 13 bp of DNA.
The flexible tails of the histones reach out between and around the
gyres of the DNA superhelix to contact neighboring particles. About
one-third of these flexible histone tails can be observed in the electron
density map, the remainder is too disordered to be interpreted. The
implication from the structure is that these flexible regions are meant to
make inter-nucleosomal interactions, perhaps facilitating the formation
of nucleosome higher-order structures (HOS).
There are 14 regions of contact between the histone proteins and
DNA: three by each of the four histone-fold dimers and two by histone-
fold extensions. This construction allows the DNA molecule in a single
nucleosome core to come loose over one-half of the superhelix while the
histones maintain their grip on the other half, permitting the genetic
information stored in the DNA to be read out without complete
dissociation of the DNA from the histone octamer.
The nucleosome core was previously thought to be held together
simply by electrostatic attraction: the negatively charged DNA molecule
wound as yarn around a positively charged histone spool.
Although this type of interaction does occur, equally many

interactions of other kinds, such as hydrogen bonds and hydrophobic
interactions, are also important.
3.6.1.1 DNA deformation
The path of the DNA around the histone octamer deviates from that of an
ideal superhelix, displaying strong bends in some regions, while being
nearly straight in others. This path is determined predominantly by the
histone/DNA contacts and is probably largely independent of the DNA
sequence of nucleotides.
The close spatial proximity of the two turns of the DNA superhelix
with a pitch of 24 Å, and the periodic variation of double helix
parameters with a mean of 10.3 bp per turn, result in an alignment of
major and minor grooves from one superhelical gyre to the next (Figure
3.36).
The resulting narrow channels formed by the aligned minor grooves
serve as the exit points for four of the eight basic histone tails, whereas
the large pores formed by the aligned major grooves are, in principle,
free to make base-specific contacts with other proteins.
The Debye-Waller B-factors show that the mobility of the DNA
backbone varies greatly, having low values when it is bound to the
histone octamer and high values when it is facing away from it.
3.6.1.2 Water and ions
The 2.0 Å diffraction data have allowed us to locate a large number of

ions and well-ordered water molecules (see next paragraph). We expect
to gain valuable general information on the role of water molecules in
mediating protein-DNA and protein-protein interactions.
The binding of divalent metal ions, such as manganese or
magnesium, to the DNA appears to favor the distortion of the DNA seen
in the NCP. The presence of ordered water molecules at the interface
between the protein subunits may provide a means to favor their
disassembly and thus could be important to the processes of DNA
transcription and replication.
3.6.2 Protein stability to heat and radiation
Physico-chemical basis of thermal stability of proteins has been a subject

of a large number of both fundamental and applied studies, the former
exploring the many physical mechanisms of thermo-stability and even
linking them to evolutionary relationships between the proteins thus
arriving at physics of evolution, while the latter primarily aiming at
design of thermo-stable enzymes and optimizing their catalytic
properties, not only via rational design but also via directed evolution or
combination of the two approaches.
The general belief is that protein stability is due to many small
effects, and that different factors stabilize different proteins. An
understanding of the molecular motifs of structural resistance in thermo-
stable proteins is pursued by site-directed mutagenesis experiments.
Generally, comparative studies are abundant in this field of research,
which belong to either of the two types. In one, thermo-stable protein is
compared to its mesophilic counterparts in great detail, allowing to
formulate the mechanism(s) of thermal stability for the case of those
particular proteins. In most studies, the specific factors of thermal
stability generally include aminoacid composition, proline residue
occurrence, exposed/buried hydrophobic/hydrophilic areas,
insertions/deletions, disulfide bridges, ion pairs, and hydrogen bonds.
(Pechkova et al., 2007c). However, such approach is usually based on
analysis of individual factors contributions but fail to generalize those
contributions on a unified scale.
The other class spawned by the rapidly growing number of 3D
protein structures resolved using crystallography or NMR, comprises
theoretical or statistical studies of large numbers, tens to hundreds, of
thermostable proteins compared to their homologous mesophilic
proteins. However, the statistical characteristics are in general difficult to
interpret physically, so they mostly remain descriptive. Particularly, the
analysis of Thermotoga maritima/mesophile protein pairs, led the
conclusion that the discriminating parameter is the average number of
atomic contacts within the protein (contact order), which characterizes
protein compactness. Besides, a combination of statistical and simulation
studies suggested that one of thermophilic adaptation mechanism was
related to more compact structure of the globule. In both cases, the

thermophilic/mesophilic discriminating parameter is related to
compactness, and in both cases the authors explicitly specify that it is not
caused by decreased loop contents, i.e., increased secondary structure
contents.
The two other remaining factors that might cause greater compactness
are the following: 1) increased density of the hydrophobic core which
should manifest itself in the change of the structure of the peptide
backbones (however at variance with the classical viewpoint of Argos et
al. (1979); and 2) increased density of the side-chains at the hydrophilic
exterior of the protein that should lead to reduction of the number is the
water molecules bound to the protein surface. From the other hand such a
protein compactness has been observed in the LB film, which shows the
thermo stability of the structure and function of immobilized proteins
(Nicolini, 1997, 1998b), or in crystals growing in presence of Langmuir-
Blodgett (LB) film template (Pechkova and Nicolini, 2004a; Pechkova et
al., 2004), both related to a decrease water contents and a slight increase
the alpha-helix content, maintaining the native folding present in
solution.
To clarify this issue, in this report, we compare the 3D structures in
protein pairs from thermophilic/mesophilic species in terms of overall
fold similarity (via percentage of structurally aligned amino acids) and
average local similarity (via RMS deviations of backbone coordinates).
It is worth to notice that usually the explored properties are
essentially intraprotein and do not explicitly include interactions of the
protein with water, with the exception of exposed areas. The latter,
however, can be misleading because of specific interactions of water
molecules with the highly intricate patterns of protein surface. From our
earlier studies, nanogravimetry revealed a minor content of bound water
in thioredoxin (Trx) from Bacillus acidocaldarius with respect to E. coli
Trx, with the difference of transition temperature of approx. 10 °C.
Moreover, highly ordered protein monolayers with defined structures and
function, generated using appropriate modifications of original
Langmuir-Blodgett method, shows the thermostability of the structure
and function of immobilized proteins (Nicolini, 1997, 1998b). It is well
known, that the water content in the LB film inside the molecule is low.
Thus, the mechanisms by which LB film can induce protein stability are
via altering patterns of aqueous environment of the given protein. This is
in agreement with the fact that smaller amount of water should enhance
thermo stability, taking in the consideration that the limited availability
of water hinders thermal denaturation. Several additives decreasing the
amount of water such as charged polymers (Foremant et al., 2001) were
found to increase protein thermo stability. Thus, our previous data and
the large body of literature data suggest a marked role of aqueous
environment in protein thermal stability. This prompted us to compare
the number of water molecules present in the thermophilic
species/mesophilic species protein pairs in crystals, and to compare the
3D backbone structures in protein pairs from thermophilic/mesophilic
species in terms of overall fold similarity (via percentage of structurally
aligned amino acids) and average local similarity (via RMS deviations of
backbone coordinates).
3.6.2.1 Bioinformatic analysis
The selection of the first set (representatively-based) constructed with

only wild-type proteins, was based on statistical examination of sequence
and structural parameters in families of homologous thermophilic and
mesophilic proteins.
The conditions used were as follows. Each pair consisted of a protein
from a thermophilic or hyperthermophilic species and its most similar, in
sequence and structure, mesophilic homolog. The other condition was
the maximum dissimilarity within each of the thermophilic proteins
dataset. Therefore the proteins for thermophilic species were selected so
as to be maximally dissimilar from each other, which was used a
criterion for representatively of the data set. Among those, only high-
resolution (R<2.5 Å) crystal structures for wild-type proteins available in
the RCSB Protein Data Bank (PDB). Selection of the other dataset of
protein pairs (filter-based) initially was formulated starting from the
growth temperatures of microorganisms. Those with growth
temperatures above 45 °C were defined as thermophilic (Pechkova et al.,
2007c). The corresponding structures from PDB from these source
organisms were filtered with respect to resolution and nativity, so that
only the ones with highest resolution and non-mutant proteins were
retained. For each of the proteins from thermophilic species, their
mesophilic homologs were found using the FSSP database (Holm and
Sander, 1996). FSSP entries containing the thermophilic proteins also
contain all their structural homologues by definition. Pairs that did not
contain proteins from mesophilic species were excluded. The next filter
was identity of the sequence for which the cut off was set at 30%, which
always selects proteins with the same fold. Also excluded were structure
with missing atoms and chain breaks. The final filter was
crystallographic data quality from the PDBREPORT database. Pairs with
resolution of 2.5 Å or worse, as well as those qualified as ‘bad’ in the
quality report, were removed.
Finally, the pairs with no direct experimental evidence for thermal
stability of the thermophilic-species protein were excluded using the
ProTherm database, resulting in 20 filter-based protein pairs. Some of the
pairs found using the above-described procedures were manually
excluded on a case-by-case basis. Such cases included firstly the falsely
detected homologues as in ferredixins whose homology is limited to the
immediate vicinity of the iron-sulfur cluster, which is the mail structure-
forming element.
Secondly, multidomain proteins were excluded, but only those for
which relative positions of domains very strongly varies in the course of
function and consequently in crystallographic structures, as exemplified
by the pair of elongation factors Tu (1EFT) from T. aquaticus versus Ef-
Tu from E. Coli (1EFU). Other excluded cases contained strains of B.
subtilis as the mesophilic source of protein. For B. subtilis, both
mesophilic and thermophilic strains were reported. We failed to find
experimental evidence that, even though the particular strain is
mesophilic, the corresponding protein is not thermally stable. The
relations between thermal stabilities from various strains of Bacillus
subtilis therefore requires further study. However, an indirect evidence
that the “mesophilic” proteins can possess thermo-stability comes from
the fact that there are as much as 217 examples of various B. subtilis
proteins showing marked thermophilicity according to the ProTherm
database (see Pechkova et al., 2007c for further details and references),
while no such data is available for other mesophilic bacterial species.
The eventual protein pairs from the two datasets combined are presented
in Figure 3.37, and are separately shown in Figure 3.38, top, where their
water contents are shown.
Figure 3.37. Similarity of the overall fold (top) and local geometries (bottom) depending
on sequence homology (in terms of sequence identity %) between the thermostable and
mesophilic proteins. Each point represents a thermostable/mesophilic pair and numbered,
with numbers encoding the following PDB ids: 1-1PCZ/1VOK, 2-1BDM/4MDH, 3-
1BMD/1B8P, 4-1CAA/8RXN, 5-1CIU/1CDG, 6-1CYG/1CDG, 7-1EBD/1AOG, 8-
1GTM/1HRD, 9-1HDG/1GAD, 10-1LDN/1LDG, 11-1LNF/1NPC, 12-1OBR/1AYE, 13-
1QEZ/1OBW, 14-1Q9H/1GPI, 15-1THM/1BH6, 16-1TMY/3CHY, 17-1VJW/1FXD, 18-
1WB8/1JA8, 19-1WL7/1UV4, 20-1XGS/1BN5, 21-1XYZ/1CLX, 22-1YNA/1XNB, 23-
1YNA/1ENX, 24-1ZIP/1AKY, 25-2BMM/1NGK, 26-2PRD/1SXV, 27-3MDS/1D5N,
28-3TGL/1LGY, 29-4PFK/1PFK, 30-1YNR/451C. (Reprinted with the permission from
Pechkova et al., Protein thermal stability: the role of protein structure and aqueous
environment, Archives of Biochemistry and Biophysics 466, pp. 40–48, © 2007c,
Elsevier).
3.6.2.2 Structural comparisons
The protein parameters were computed using VAST software, and

described below. Namely, the percentage identity refers to residues in the
aligned sequence region. This is a raw measure of sequence similarity in
the parts of the proteins that have been superimposed.
The RMSD signifies the root mean square superposition residual in
Angstroms. This number is calculated after optimal superposition of two
structures, as the square root of the mean square distances between
equivalent Cα atoms. Note that the RMSD value scales with the extent of
the structural alignments, so RMSD number should always be used in
combination with Aligned Length, as done in this study.
The Aligned Length parameter is the number of structurally
equivalent pairs of Cα atoms superimposed between the two structures.
In other words, this is the maximal number of residues possible to
superimpose for two proteins.
In this study the percentage Aligned/Total is used as the characteristic
of the overall fold similarity, since obviously Aligned Length scales
quasi linearly with the total length. Incidentally, we calculated also the
measure Loop Hausdorff Metric (LHM), which is a loop similarity
measure showing how well two structures conform to each other in the
loop regions, once structurally superposed, loop regions being the parts
of the structures between aligned secondary structure elements (helices
and strands).
The LHM value is in Angstroms, with a smaller value showing
greater similarity. Similar arrangements of loops typically have LHM
smaller than 3 Angstrom, while considerable differences in loops have
LHM over 5 Å. The loop similarity may be undefined if there are too
many residues with missing coordinates in the loops, which never
occurred in this study.
3.6.2.3 Structural comparisons of homologous thermophilic/mesophilic

pairs
Structural similarity parameters of protein pairs from the representative

thermophilic/mesophilic species are shown in Table 3.4.
Here we aimed to characterize it qualitatively using the fraction of

structurally aligned residues in the entire sequence of each protein, in
addition to RMSD comparison of the protein backbones.
Table 3.4. Numbers of water molecules per subunit in crystals of several thioredoxin.
(Reprinted with the permission from Pechkova et al., Protein thermal stability: the role of
protein structure and aqueous environment, Archives of Biochemistry and Biophysics
466, pp. 40–48, © 2007c, Elsevier).
PDB id Organism Waters molecules per subunit

1thx Mesophilic E. coli 222
1t00 Mesophilic S. coelicolor 150
2cvk Thermophilic T. thermophilus 43
1nw2, Thermophilic A. acidocaldarius mutanta 72
1nsw
2trx E. coli with artificial water removal 70
a
Thermophilic but with mutations introduced canceling thermophilicity (Bartolucci
et al., 2003): average value is shown.
b
Mesophilic but crystallized in presence of MPD, which is known to displace water
from protein surface (Anand et al., 2002)
Besides, since structural alignment requires that amino acids are

superimposed in 3D, the RMSD parameter does not include the amino
acids that cannot be aligned, as for example in the case of large inserts.
As seen from Figure 3.37, most pairs have the “crude” similarity
parameter above 80%. Besides, no correlation is observed between
sequence identity/similarity and structural similarity, suggesting that the
overall fold remains the same in the thermophilic/mesophilic pairs for
both higher and lower sequence identity pairs. Loop lengths were
compared using the above-quoted VAST software, and it was found that
the differences were marginal (data not shown), with one exception of 26
extra amino acids in the single insertion loop in the mesophilic protein,
which is unlikely to affect thermal stability since it is presence of
multiple requires multiple extra loops rather than a single long loop that
strongly affects (thermo)dynamics of protein folding. As seen from
Figure 3.37, the RMSD ranges from 0.6 Å (practical identity, in case of
rubredoxins) to 2.3 Å (hydantoinases) which indicates significant
similarity, considering also that the thermo-stable hydantoinase is L-
hydantoinase while the mesophilic is D-hydantoinase. The RMSDs are
plotted against sequence identity, but only a weak correlation is observed

(Figure 3.37). There are at least as many protein pairs not following the
expected correlation pattern, which is high sequence identity for lower
RMS, as the ones following it. Overall, relation between two parameters,
the percentage of aligned residues and the RMSD values, allows to
conclude that the conformations of loops were different in the pairs,
loops were further compared using the new option LHM in the pairs
using the newly available option of the VAST software, but no
significant difference within the mesophilic species protein/thermophilic
species protein was observed.
As seen from Figure 3.37, the RMSD ranges from 0.6 Å (practical
identity, in case of rubredoxins) to 2.3 Å (hydantoinases) which indicates
significant similarity, considering also that the thermo-stable
hydantoinase is L-hydantoinase while the mesophilic is D-hydantoinase.
The RMSDs are plotted against sequence identity, but only a weak
correlation is observed (Figure 3.37). There are at least as many protein
pairs not following the expected correlation pattern, which is high
sequence identity for lower RMS, as the ones following it. Overall,
relation between two parameters, the percentage of aligned residues and
the RMSD values, allows to conclude that the 3D structures of proteins
from thermophilic and mesophilic species remain the same, regardless of
their sequence identities.
3.6.2.4 Water comparisons of homologous thermophilic/mesophilic pairs
For the representatively-based dataset, overall numbers of water

molecules per protein molecule scaled by the water-accessible surface
area are shown in Figure 3.38, top left. It is evident that the number of
water molecules in all cases is larger in the mesophilic species proteins
than in their thermophilic homologs, and several cases is drastically
larger.
In all cases, resolution is similar in the pairs, typically within 0.2 Ǻ,
with the only exception of the 2PRD/1SXV pair, where the difference is
0.6 Ǻ, but the 2PRD/1SXV pair from the same dataset has the difference
of 0.3 Ǻ, but shows practically the same difference in water content.
Figure 3.38. Numbers of water molecules scaled by water-accessible surfaces of protein

subunits contained in a unit cell in crystals of thermophilic species protein versus
mesophilic species proteins (light gray). Top left, the representative dataset taken from
Kumar and Nussinov (2001). Top right, dataset verified by experimentally determined
thermal stability of all proteins studied (Szilágyi and Závodszky, 2000). Bottom,
Dependence of scaled number of water molecules on crystallographic resolution.
(Reprinted with the permission from Pechkova et al., Protein thermal stability: the role of
protein structure and aqueous environment, Archives of Biochemistry and Biophysics
466, pp. 40–48, © 2007c, Elsevier).
To clarify the issue, we performed the same analysis of the water

content for the dataset in which all protein pairs were verified by
experimentally determined thermal stability for both the “thermophilic”
and the ”mesophilic” members of the pair (Szilágyi and Závodszky,
2000). Results (Figure 3.38, top right) show the same pattern as our
original dataset. Considering that the number of crystallographically
detected water molecules essentially depends on resolution of the
crystallographic structure, we also plotted the dependence resolution on

water molecules scaled by protein surface, and found very weak
correlation (Figure 3.38, bottom) for both thermo-stable and mesophilic
proteins. Therefore our results, either from the representativity-based or
filtering-based datasets, are not likely caused by a crystallographic
artifact.
Comfortingly the number of internal (buried) water molecules, even
if not found in several protein pairs present in PDB, is far larger in the
mesophilic species proteins than in their thermophilic homologs (Figure
3.39) for the vast majority of the protein pairs selected by
representatively (mostly) or by experimental verification. The plot in
Figure 3.39 includes only data for those protein pairs, scaled to show the
numbers per 10 kDa of molecular weight of proteins, which is 0.18%
w/w.
Figure 3.39. Numbers of internal water molecules per 10 kDa molecular weight of
protein in crystals of thermophilic (red) versus mesophilic proteins (green). (Reprinted
with the permission from Pechkova et al., Protein thermal stability: the role of protein
structure and aqueous environment, Archives of Biochemistry and Biophysics 466, pp.
40–48, © 2007c, Elsevier).
The data with two and less water molecules are probably insignificant
and thereby not included. Conditions of crystallization and data
acquisition were similar in each protein pair with respect to the following
parameters potentially able to affect the quantity of crystallization water:
concentration of amphiphiles (although not the exact formula of
amphiphiles), ionic strength (not exact composition of the buffer),
presence of organic solvents (never used), and temperature (room
throughout the datasets). The data collection parameter, apart from the
parameters that are accumulated into the resolution, also affecting the
number of water molecules, is the electronic density threshold for water
detection, but this parameter is very seldom reported or varied during
data processing. Wherever it was reported, it was the same within the
pairs.
3.6.2.5 Detailed comparison of mesophilic versus thermophilic

thioredoxin
One special case, for which two mesophilic homologs correspond to two
thermophilic homologs, is bacterial thioredoxin. Particularly, thioredoxin
from Alicyclobacillus acidocaldarius (BacTrx) is homologous to
thioredoxins of T. termophilus (both thermophilic), E. coli and S.
coelicolor (both mesophilic).
Their water contents are shown in Table 3.4. The highest water
contents are shown by two mesophilic proteins thioredoxins from E. coli
and S. coelicolor. If thioredoxin from E. coli is crystallized in presence
of a strong protein dehydration agent, MPD (2-methyl-2,4-pentanediol) ,
then the number of water molecules drops to 70 (Line 5 of the Table 3.4).
Similar water content is shown also by crystals of thermophilic
thioredoxin from A. acidocaldarius. Those crystals, however, contain the
protein that has been mutated in such a manner as to reduce thermo-
stability (about 10 degrees drop in denaturation temperature). Finally,
thioredoxin from T. termophilus shows the lowest water content. As
follows from Table 3.4, water contents in bacterial thioredoxins can be
arranged in the following row: mesophilic > mesophilic with artificially
reduced hydration ≈ thermophilic with mutation-reduced thermal
stability > thermophilic, which is in agreement of our analysis of water
content in crystals.With respect to thioredoxin, we also measured the
water contents experimentally.
The thioredoxin from Alicyclobacillus acidocaldarius BacTrx

purified by anion-exchange chromatography (11577 Da) has in primary
structure an identity ranging from 45 to 53% with all sequences of
known bacterial Trxs. Nanogravimetry experiments (Figure 3.40.)
showed a lower content of bound water in BacTrx than in E. coli Trx,
and a transition temperature approx. 13 °C higher for BacTrx either in
solution or in non-oriented self-assembled film, while in LB highly
oriented film the same E. coli Trx, displays a transition temperature more
than 25 °C higher with a dramatic decrease in desorbed water (Figure
3.40).
Figure 3.40. Desorbed water versus temperature for non-oriented self-assembled film 7 of
E. coli TrxEc (black squares), BacTrx in non-oriented self-assembled (white squares) and
LB (gray squares) film. The water amount has been normalized to the initial protein
amount in the sample. Thioredoxin samples were deposited on the quartz crystals in 50
mM sodium phosphate ph 5.8 and dried under vacuum for 30 minutes (Bartolucci et al.,
1997; Facci et al., 1994b). (Reprinted with the permission from Pechkova et al., Protein
thermal stability: the role of protein structure and aqueous environment, Archives of
Biochemistry and Biophysics 466, pp. 40–48, © 2007c, Elsevier).
Comfortingly, the circular dichroism signal at 193 nm versus

temperature, as shown earlier by differential scanning calorimetry,
demonstrated that BacTrx in self-assembled non oriented film is
endowed with a higher conformational heat stability than the self-
assembled film of Trx from E. coli, which instead when immobilized in
LB film displays an even more dramatic increase in heat stability up to
200 °C. As indeed shown earlier in solution, E. coli Trx displayed a
melting temperature at 81 C against 94 C for BacTrx, which in turn is

much lower than the 200 °C shown here for E. coli Trx in LB film
(Figure 3.41). Incidentally, BacTrx in LB film displays the same
dramatic increase in heat stability up to 200 °C (not shown) and a similar
dramatic decrease in desorbed water (not shown).
Figure 3.41. Temperature dependence of circular dichroism signal at 193 nm LB film

(black line) and in non-oriented self-assembled film of BacTrx (light gray line).
Measurements have been taken in 50 mM sodium phosphate ph 5.8 The CD signal for
non-oriented self-assembled film of E. coli EcTrx displays a melting temperature about
13°C lower than the corresponding BacTrx one (dark gray line). Instead, the LB film of
E. coli EcTrx even after heating up to 200°C does not display a melting transition (black
line). The CD signal has been normalized to the initial molar ellipticity value of the
BacTrx self-assembled film sample at 25°C (Reprinted with the permission from
Pechkova et al., Protein thermal stability: the role of protein structure and aqueous
environment, Archives of Biochemistry and Biophysics 466, pp. 40–48, © 2007c,
Elsevier).
Note that the water desorption rate which is calculated relative to the
weight of protein is used herein as a parameter characterizing thermal
phase transition of protein with increasing protein, the main parameter
being the transition temperature. The empirical equation used for curve
fitting and thus determining transition temperatures (end of Methods)
was derived from the general phase transition theory, implicitly assuming
that the transition in question in protein unfolding. It does indeed follow
from comparing Figures 3.40 and 3.41 that water desorption and protein
unfolding are closely correlated. However, the actual amount of water

contained in the film per protein weight, which is roughly proportional to
the height of each desorption curve, is progressively decreasing in the
row “self assembled mesophilic ” – “self assembled thermophilic” – “LB
mesophilic”, in perfect agreement with the discussed correlation of
increased thermal stability and lower water contents.
Figure 3.42. Positions of internal and first hydration shell water (red) around the B.
acidocaldarius thioredoxin (top) and the E. coli thioredoxin (bottom). Left and right
panels differ by 180° rotation around a horizontal y-axis. Hydrophilic surface is in green,
hydrophobic surface is in white (Reprinted with the permission from Pechkova et al.,
Protein thermal stability: the role of protein structure and aqueous environment, Archives
of Biochemistry and Biophysics 466, pp. 40–48, © 2007c, Elsevier).
In an attempt to capture atomic-level mechanisms of the relationship

between thermal stability and water content, we compared atomic-level
structures of E. coli and B. Acidocaldarius thioredoxins (Figure 3.42)
where we show positions of internal first hydration shell water over the
surface of the protein, relative to positions of hydrophilic and
hydrophobic surfaces of the protein. It is evident that not only the overall
quantities but also the first hydration shell contains fewer water
molecules in the case of a thermo stable protein. Note that, in the case of
the thermo stable protein, water molecules do not assemble into clusters.
Lower water content is consistently apparent in thermophilic proteins
with respect to their mesophilic counterparts possibly just a consequence
of the well-known mechanisms of stabilization as electrostatic
interactions or compactness. We infer from combining the representative
PDB data with the fact that the protein backbone remains unchanged in
the mesophilic/thermophilic protein pairs that the compactness, when
determining the protein thermal stability, is related as to the protein inner
aqueous environment. Relation between protein stability and water
content is confirmed by the dramatic thermal stability induced in
thioredoxin by the LB immobilization, which strikingly correlates with
the similarly dramatic decrease in the amount of inner water.
The other possibility could be related to decreased sizes of water-
accessible cavities in the protein, even if no water is crystallographically
observed therein. To clarify, we applied two different compactness
criteria, one known as packing scores, and the other being the volume of
water accessible cavities in the protein. Neither was found to correlate
within the thermostable/mesophilic protein pairs. Interestingly the three-
dimensional model of the two thioredoxins displays minor differences in
the tertiary atomic structure. Indeed, a comparison with the EcTrx
structure and analysis derived by X-ray crystallography of stabilizing
factors in terms of the global folding indicates that the BacTrx overall
fold is very similar to that of EcTrx and only small differences in the
molecular architecture can be observed.
The minor NMR structural differences suggested that protein stability
could be due to cumulative effects, the main factor being an increased
number of ionic interactions cross-linking different secondary structural
elements and clamping the C-terminal alpha-helix to the core of the
protein. The superposition of all the backbone atoms (N, Cα, C)
(residues 5–104) of the 20 final BacTrx structures onto the E. coli crystal
structure yields an average RMSD of 0.14 nm, while the RMSD is
reduced to 0.12 nm if the secondary structure elements are compared.
This indicates that some external factor is responsible for the thermal
stability of thioredoxin, which could well be related to smaller amount of

water contained in the BacTrx compared to the E. coli Trx.
In an attempt to capture atomic-level mechanisms of the relationship
between thermal stability and water content, we compared atomic-level
structures of E. coli and B. acidocaldarius thioredoxins where we show
positions of internal first hydration shell water over the surface of the
protein, relative to positions of hydrophilic and hydrophobic surfaces of
the protein. It is evident that not only the overall quantities but also the
first hydration shell contains fewer water molecules in the case of a
thermo stable protein. Note that, in the case of the thermo stable protein,
water does not assemble into clusters. This allows to suggest that thermo
stable proteins have more self-neutralized charge distributions over its
surface, which would be related to lower contents of crystallization
water.
Another pertinent observation being made recently, compatible with
the above data and with Pyrococcus furiosus chromosome reassembly
following gamma irradiation and with the heat shock enhancing radiation
resistance, revealed that the synchrotron radiation resistance versus time
of Lysozyme crystal prepared by LB nanotemplate was larger than the
corresponding classical vapour diffusion, as previously discussed. Here
we find that lower water contents also correlates with higher thermal
stability, while placing a mesophilic protein in a LB film leads to both
lower water content and higher thermal stability, thus possibly linking
together water contents, thermal stability, and LB film effects. Since
entropy-driven water release, as recently shown, is the main
thermodynamic driving force of protein crystallization, our data indicate
that thermophilic proteins possibly have better structured protein/water
interface than their mesophilic homologs.
Considering that structuring of water caused by increased
hydrophobic group exposure has been traditionally assumed to be the
cause of heat capacity increase upon protein unfolding, our data indicate
that, for thermophilic proteins, the variation in Cp should be lower than
those for their mesophilic homologs, which is the tendency in fact
observed. Alternatively since solvent water structuring can result either
from the hydrophobic effect or from more regular occurrence of polar
protein groups along the protein-water boundary leading to higher
hydrogen bond propensity, our finding is in agreement of the two

apparently conflicting concepts.
The very well known phenomenon of increased numbers of salt
bridges in thermo-stable proteins (even if apparently not sufficient to
justify the increased thermal stability) could also lead to higher
compactness of protein structure not necessarily via a denser
hydrophobic core. If these considerations are true, some charged amino
acid residues may have exceptionally high contributions to the free
energy of the thermo stable protein’s electric field so their mutation may
lead to drastic effects, which apparently follows from the above quoted
work. We have shown, by data mining, the systematically lower water
contents in crystals of thermophilic protein compared to their mesophilic
counterparts, especially for the case of thioredoxin, for which we also
experimentally show that the low water content in the thermophilic
homolog and in the LB film of mesophilic homolog corresponds to their
higher thermal stability. In combination with our earlier data, this
indicates that the decrease in surface or inner bound water may be the
factor connecting experimentally observed changes in protein stability
made in solution (as shown in all papers here cited and here), thin
Langmuir film and nanotemplate-grown crystals, namely of the thermal
stability in thermophiles, the thermal stability and storage stability for
LB film, and the radiation resistance in nanotemplate-grown crystals. As
shown before, indeed the significant radiation resistance of protein
crystal derived from the protein thermo-stable LB patches acting as
nucleation centers. Water content in LB-based protein crystal indeed
decreases with respect to “classical” along with increase in alpha helix
content, thereby suggesting that decrease in bound water is one the
sources of thermal stability in thermophiles, thermal and storage stability
in LB films, and radiation resistance in crystals (Table 3.4).
Chapter 4
Nanoscale Applications in Industry and

Energy Compatible with Environment
This chapter overviews the present status of nanoscale applications of

organic and biological nanotechnology to industry and energy, namely of
those capable so far to yield a potential technological progress to
electronics, energy and catalysis.
Particular emphasis is placed on what has been accomplished in our
laboratory in the last eight years, whereby the references to numerous
other groups can be found in recent complete reviews (Nicolini et al.,
2001; 2005; Nicolini and Pechkova, 2006).
The data presented here point to the successful engineering of
nanotechnology based on supramolecular layer engineering of potential
industrial relevance. In fact, as emphasized, the filmation process was
able to induce high thermal stability and associated high lifetime and
recycling, which represent a prerequisite for several processes of
industrial interest.
Therefore, although work is still in progress to further optimize the
parameters and to evaluate in more detail, case by case, the temporal
stability of thin layers within required cost effectiveness and the
reproducibility within a highly competitive industrial context, this
methodology clearly represents a promising general-purpose tool for the
design of new industrial products and processes.
219
220 Nanobiotechnogy and Nanobiosciences
4.1 Nanobioelectronics
This subchapter of the volume on Nanobiotechnology comes ten years

after a comprehensive and well received treaty on Molecular
Bioelectronics (Nicolini, 1996a) and eleven years after a special issue of
Bioelectronics and Biosensors (volume 10, 1995) as a result of a
Workshop called in Bruxelles by the European Commission to launch a
Program on Bioelectronics, comprehensive of neural VLSI chips,
engineered proteins, molecular manufacturing and biomolecular
electronic devices (Nicolini, 1995).
Waiting then for a new book on the very same subject to be done in
the near future, this section intends to summarize the most recent
pertinent to Nanobiotechnology and promising applications to industry
and energy in the field of organic and biomolecular nanoelectronics.
4.1.1 Nanosensors
Nanosensors represent by far the most active area in the field of

nanoelectronics with interesting and promising applications in the short
term, which will be here summarized in key examples representative of
organic and protein-based nanosensors (for a recent review see Nicolini
et al., 2006).
This subchapter summarizes with few key examples the significant
potential of nanostructured organic matrices for developing intelligent
sensors for gas and liquid having potential impact to health care and
environment control (Nicolini et al., 2001a; 2006).
4.1.1.1 Protein-based nanosensors
Light sensitive proteins and metallo-proteins are the most frequently

used proteins in sensor technology in liquid and in air.
Starting from the effects of volatile anaesthetics on the structure of
bacteriorhodopsin (bR) in the purple membrane in solution this work
tries to investigate the interaction of ether and hydrocarbon type
anesthetics vapors with self-assembled bR thin films (Figure 4.1). A
dedicated constant flux chamber has been built to maintain the sample in
Nanoscale Applications in Industry and Energy Compatible with Environment 221
a rather constant atmosphere of anaesthetics, during the absorption

measurements. The kinetics of absorption and desorption have been
determined. The results obtained have been compared with those of bR
in solution (Maccioni et al., 1996).
Figure 4.1. (a) The absorption spectra of the film pure and treated with diethylether
during different periods of time and after relaxation. (b) The absorbance profile at 570
nm of a self-assembled film of bacteriorhodopsin upon exposition to vapors of diethyl
ether by increasing pressure (A, B, C, D) and upon exposition to a compressed air stream
for the desorption of the anesthetic (regions 1.2.3). (Reprinted with the permission from
Maccioni et al., Bacteriorhodopsin thin film as a sensitive layer for an anaesthetic sensor,
Thin Solid Films 284–285, pp. 898–900, © 1996, Elsevier).
The suspensions of PM have been obtained by dilution of the stock

suspension (5 mg ml-1) with distilled water. The concentration was
checked spectrophotometrically, in particular the peak of proteins (at 280
nm) should not show the characteristic scattering signal. The

concentration chosen for the experiments was 0.3 mg ml-1. In the case of
bR in a solution, the anaesthetic solution was just added to the bR
solution and stirred for 10 min in order to equilibrate the system. In the
case of chloroform, not mixable with water, the cuvette was shaken in
order to promote the contact of PM with the solvent. The amount of the
income anaesthetic vapours was qualitatively monitored injecting by a
syringe different amounts proceeding from the heated vessel. In Figure
4.1a, a self-assembled film of bR in PM was treated with diethylether
vapours: (a) the absorption spectra of the film pure and treated with
diethylether during different periods of time is showed; (b) the
decreasing of the absorbance at 570 nm was recorded during the
injection of successive amounts of vapours of anaesthetic. In Figure 4.1a
the absorbance maximum of the PM thick film appears at 563 nm and the
intensity of absorbance decreases with the treatment with anaesthetic.
At the same time a broad band occurs at about 400 nm during the
treatment and disappears after relaxation. It is difficult to give an
interpretation of such a band, which cannot be precisely attributed to the
anesthetic induced bR480, and bR380 forms further experiments will
probably clarify the role of this band. Nevertheless this figure reveals
either the functionality of the protein or the reversibility of the process
qualitatively comparable with that obtained in solution.
Zones A, B, C, D are evident in Figure 4.1b, each of them
corresponds to a progressive increase of the anaesthetic pressure: zone
A is more wide than the others, this could be due to a higher pressure of
the vapour injected in the cell or to an effect of saturation of the sample
that makes more difficult the successive binding in the other zones. After
the decrease of the absorbance of 0.1 units compressed air was injected
in three steps (regions 1, 2, 3), obtaining almost the total recovering of
the structure after 15 min. The work pointed out the possibility to use the
self-assembled bR films as sensitive layers for optical biosensors for
anesthetics. The important point is that the optical properties of the layer
are reversible not only for ether type anesthetics but also for hydrocarbon
type as chloroform. It allows one to consider such films as good
candidates for biosensors of continuous monitoring of anesthetics.
4.1.1.1.1 P450’s Langmuir films for clozapine, styrene and cholesterol

Recombinant cytochrome P450s consist of a large and highly diverse
enzymes family that play a pivotal role in the metabolism of a wide
variety of xenobiotics and drugs. For these attractive properties we have
performed several studies to obtain nanostructures for device
applications.
For each enzyme we have carried out structural and functional
characterizations in order to optimize the immobilization process
(Nicolini et al., 2001; Ram et al., 2001, Paternolli et al., 2002a; Antonini
et al., 2003) sensing we have used several immobilization techniques
including the layer-by-layer, the Langmuir-Blodgett, a gel-matrix and the
self-assembly (solution spreading) and several forms of the given
cytochromes including the wild type, the native recombinant and the
GST-fused P4501A2, P4502B4 and P450scc cytochromes.
The conclusive new insights respect to all our previous papers
published is the comparative undertaking capable to identify Langmuir-
Blodgett among all various immobilization technologies as the one
yielding the optimal sensing results as long recombinant P450 of high
grade is utilized (Paternolli et al., 2004, 2007).
Figure 4.2. Graph of the spin state equilibrium index of cytochrome P4502B4 in solution
and in LB film (30 layers). The data were acquired at different exposure times to styrene
atmosphere, pointing to a shift to higher spin value (Reprinted with the permission from
Paternolli et al., Recombinant cytochrome P450 immobilization for biosensor
applications, Langmuir 20, pp. 11706–11712, © 2004, American Chemical Society).
LB proves indeed superior to all other approaches being tested with

the only exception of APA (see later). This has been generalized to all
P450-based sensors being tested (Nicolini et al., 2001, 2006; Paternolli et
al., 2002a, 2007), namely that the sensing can be optimized in terms of
performance, stability, reusability and efficiency whenever LB
immobilization and proper mutant are utilized.
Table 4.1. Substrates and primary products of the cytochrome P450s. (Reprinted with the
Society).
Cytochrome Reaction Substrate Product

P4501A2a H3C HN
Demethylation
N
N
N
N Cl
N N
H
Cl
N-desmethylclozapined
N
H
Clozapine
P4502B4 b Epoxydation O
Epoxystyrene
Styrene
P450scc c Hydroxylation O
HO
HO
Cholesterol Pregnenolone e
a
Brosen, 1993; Brosen et al., 1993, Pirmohamed et al., 1995.
b
Miller, 1988; Vaz et al., 1998.
c
Nicolini et al., 2001; Ortiz de Montellano, 1986;Waterman and Simpson 1985.
d
Cytochrome P4501A2 metabolizes the clozapine to produce N-desmethylclozapine
and, secondarily, N-oxideclozapine.
e
Cytochrome P450scc converts the cholesterol into pregnenolone and
isocapraldehyde.
Spectroscopic biosensor for styrene is based on P4502B4 LB,

whereby the characteristic Soret peak of the cytochrome P4502B4 free
and complexed with its substrate was monitored in LB film.
In fact, it is known that the interaction between cytochrome and the

substrate changes the protein spin state. The results are summarized in
Figure 4.2.
The indexes of P4502B4 spin state equilibrium were calculated from
the absorbance spectra acquired at different times of exposition to the
styrene atmosphere. It was found that the exposure of cytochrome
P4502B4 to a styrene saturated environment determines the progressive
shift to higher spin value following the P4502B4 molecules binding to
their substrates in both solution and film, as it is the case going from
solution to LB film without styrene (Table 4.1).
Figure 4.3. Cyclic voltammetry of cytochrome P450scc LB film with addition of

cholesterol (Reprinted with the permission from Paternolli et al., Recombinant
cytochrome P450 immobilization for biosensor applications, Langmuir 20, pp. 11706–
11712, © 2004, American Chemical Society).
The styrene detectable quantity found is limited by the home-made

measurement system, in which the control of the working volume is
difficult (Table 4.2).
Amperometric sensors for cholesterol based on P450scc in LB films.
Protein studies utilizing electrochemical techniques such as cyclic
voltammetry have provided insight into the functional properties of
redox-active centers (Paternolli et al., 2004). Electrochemical
measurements are thereby performed in order to study the binding
between cholesterol and cytochrome P450scc. The current values of
P450scc LB film are plotted as a function of cholesterol concentration

additions in Figure 4.3.
Table 4.2. Physiological and minimum detectable concentrations of clozapine and

cholesterol in the blood and of the styrene in the atmosphere. (Reprinted with the
biosensor applications, Langmuir 20, pp. 11706-11712, © 2004, American Chemical
Society).
Detected metabolite Physiological Minimum

concentration range detectable concentration
(P450 µg in the LB per
electrode
clozapinea 50–600 ng/mL 50 ng/mL
styreneb 0–85.2 mg/mc 500 mg/mc (4 µg P4502B4 in
40 LB
cholesterolc 100–220 mg/dL 11.6 mg/dL (5 µg P450scc in
40 LB)
a
Haring et al., 1989; Buur-Rasmussen and Brosen, 1999; Rahden-Staron et al.,
2001.
b
American Conference of Governmental Industrial Hygienists, Inc., ACGIH, 1997.
c
Report of the National Cholesterol Education and Treatment of High Blood
Cholesterol, 1988.
Each addition consists of 50 µl of cholesterol solution (10 mM in

TritonX-100) until reaching a final concentration of 100–750 µM. The
time of the process is estimated to be about 3 minutes. The data show
that the electrochemical process itself gives cytochrome P450scc
electrons allowing it to react with cholesterol. The kinetics of the
absorption and reduction process might be the result of the ion diffusion
controlled process.
Moreover, the data plot suggests that the steady-state current is
directly proportional to the cholesterol concentration until the beginning
of the saturation trend. Amperometric sensor for clozapine based on
P4501A2 was performed in gel matrix.
The electrochemical studies proved, however, that while the behavior
of P4502B4 is comparable to P450scc in LB films (data not shown), the
cytochrome P4501A2 LB films cause some problems with the cyclic
voltammetry particularly in the study of the interaction between this
enzyme and its substrate (clozapine), apparently due to the low pure
grade (as shown in materials and methods). In fact, it is well known that
molecular impurities on the electrodes may impede electron transfer and
prevent enzyme-electrode electrical communication (Joseph et al., 2003).
For this reason we immobilized P4501A2 in a gel-matrix (Figure 4.4)
as described in the materials and methods, and we employed
chronoamperometry to verify the possibility of producing an
amperometric sensor to detect clozapine. Aliquots of clozapine (40 µM
in methanol) were added to the working mixture in order to obtain an
amperometric response curve shows the resulting current as a
consequence of constant potential.
Figure 4.4. Current response of cytochrome P4501A2 gel-matrix as a function of the

clozapine concentration by rhodium-graphite s.p.e. Working solution is 10 mM K-
phosphate buffer, pH 7.4, The potential electrode was poised at -600 mV (Reprinted with
the permission from Paternolli et al., Recombinant cytochrome P450 immobilization for
Society).
It can be observed that raising the clozapine concentration increased

the response current, which appear stable at room temperature for over
30 days to 60% of its original value. Because the therapeutic range of
clozapine in plasma is between 0.16 and 1.83 µM (50–600 ng/ml)
(Haring et al., 1989; Buur-Rasmussen and Brøsen, 1999; Rahden-Staron
et al., 2001) we conclude that the sensitivity extrapolated by our
experiments is sufficient for routine measurements, even if the temporal
stability of gel matrix appears quite less than that of the LB method
(Table 4.2). By the combination of proper immobilization (LB),
transducer and nanostructured mutants of high-grade stable and selective,
P450-based sensors appear capable to detect the interaction with a wide
range of organic substrates such as fatty acids, drugs, and toxic
compounds. Only in the presence of low purity grade protein, as in the
case of our preparation of P4501A2, is necessary to use a gel-matrix to
warrant the optimal clozapine sensing (Figure 4.4).
4.1.1.1.2 Direct electron-transfer with gold nanoparticles

As shown in chapter 1, nanoparticles of metals have been investigated
due to their novel material properties which differ from the bulk
materials (Penn et al., 2003; Han et al., 2002). The gold nanoparticles
display electronic, chemical and physical properties advantageous for
application in bioelectrochemistry. Colloidal gold nanoparticles can
adsorb proteins (Hu et al., 2003).
Figure 4.5. Cyclic voltammograms of screen-printed rhodium-graphite electrode with Au

nanoparticles (1), with cytochrome P450scc and Au nanoparticles (2) and bare electrode
with cytochrome P450scc (3). Experiments were performed in aerobic 100 mM
phosphate buffer, 50 mM KCl, pH 7.4. Scan rate 50 mV/s-1. Gold nanoparticles were
prepared in the presence of dodecanthiol (Reprinted with the permission from
Shumyantseva et al., Direct electron transfer between cytochrome P450scc and gold
nanoparticles on screen-printed rhodium-graphite electrodes, Biosensors & Bioeletronics
21, pp. 217–222, © 2005, Elsevier).
Bio-composites of gold nanoparticles and glucose oxidase were

electrodeposited on indium tin oxide glass electrode for fabricating
glucose biosensor (Bharathi and Nogami, 2001).
Gold nanoparticles was here utilized in the direct electron transfer
from electrode to cytochromes P450scc, namely between cytochrome
P450scc and Au colloid modified screen-printed rhodium graphite
electrode (Shumyantseva et al., 2005). To construct a biosensor on the
basis of cytochrome P450scc the amperometric response on the
cholesterol addition has been measured (Figure 4.5).
The combination of bioelectrochemistry and nanobiotechnology has
permitted thereby to construct a high sensitive amperometric biosensor
for cholesterol measurements.
At vertical position of electrode in 1 ml electrochemical cell
voltammograms were observed only for the first scan. Then enzyme was
dissolved in solution. Vertical regime needs additional covalent binding
of enzyme onto the electrode surface.
In planar regime only a small volume (20–60 µl) of buffer is needed.
Integration of the reduction peak permits calculating the charge and thus
the concentration of electro-active molecules on the surface of the Au-
nanoparticles-electrode.
For the P450scc a value of 2.57 pmol (16.1 pmol/cm2, when
assuming a plane surface) was estimated, corresponding to 5.14% of the
total amount of the loaded enzyme to be electro-active.
Electrodes with gold nanoparticles and P450scc were used for
measurements of cholesterol concentration in solution. Figure 4.6 shows
the steady-state current response of the cytochrome P450scc electrodes
to cholesterol addition. In these experiments 100 µM cholesterol stock
solution in 0.3% sodium cholate was used.
Aliquots of cholesterol were added to the analyzed solution. The
sensitivity of this type of biosensor is 0.13 µA µM-1 and the detection
limit is 70 µM of cholesterol in the presence of sodium cholate as
detergent. This type of P450scc-electrode is quite sensitive and needs
small volumes of analyzed solutions.
Figure 4.6. The amperometric response of screen-printed rhodium-graphite Au-P450scc

electrode to increasing cholesterol concentration: 100 µM stock solution in 0.3 % sodium
cholate, with 10 µM cholesterol being repeatedly added. Total volume of electrolyte was
60 µl. Current was measured at constant potential of -400mV (vs. Ag/AgCl) (Reprinted
with the permission from Shumyantseva et al., Direct electron transfer between
cytochrome P450scc and gold nanoparticles on screen-printed rhodium-graphite
electrodes, Biosensors & Bioeletronics 21, pp. 217–222, © 2005, Elsevier).
It is worth to notice that the solution casting nanostructured

electrodes (Shumyantseva et al., 2005) have an increased electron-
transfer rate with respect to the clean rhodium-graphite electrodes; this
fact may be due to the increased diffusion of the electrolyte in the
nanostructured electrodes (Akiyama et al., 2003), making this type of
electrodes suitable for the detection of cholesterol in a small volume of
electrolyte.
4.1.1.1.3 Improved mechanical stability and optimal performance with

anodic porous alumina
To further improve the mechanical stability of electrodes based on
P450scc for LDL-cholesterol detection and measure, anodic porous
alumina (APA) was recently used (Stura et al., 2007).
This inorganic matrix, which pores can be tuned in diameter
modifying the synthesis parameters (see chapter 1), was realized with
cavities 275 nm wide and 160 micron deep (as demonstrated with the
AFM and SEM measurement shown in chapter 1), to allow the
immobilization of P450scc macromolecules preserving their electronic

sensitivity to its native substrate, cholesterol. Even if the sensitivity of
the APA+P450scc system was slightly reduced with respect to the pure
P450scc system, the readout was stable for a much longer period of time,
and the measures remained reproducible inside a proper confidentiality
band, as demonstrated with several cyclic voltammetry measures (Figure
4.7).
Figure 4.7. I–V current–voltage curves of s.p.e. of APA-P450scc. Electrode in presence

of substrate LDL-cholesterol. The s.p.e. of APA-P450scc electrode was tested, after a
month, in a 10mM K-phosphate buffer pH 7.4 in presence of LDL. (a) P450, (b) LDL 0.5
mg/ml, (c) LDL 1.1 mg/ml and (d) LDL 1.6 mg/ml. Results are representative of one of
three similar experiments. (Reprinted with the permission from Stura et al., Anodic
porous alumina as mechanical stability enhancer for LDL-cholesterol sensitive
electrodes, Biosensors & Bioeletronics 23, pp. 655–660, © 2005, Elsevier).
To optimize the adhesion of P450scc to APA, a layer of poly-l-lysine,

a poly-cathion, was successfully implemented as intermediate organic
structure (Figure 4.7). The functionality of a rhodium-graphite screen-
printed electrode (s.p.e.) modified with anodic porous alumina, and the
reported results prove the achievement of the optimal immobilization of
cytochrome P450scc onto the modified electrode in both vertical and
horizontal position, achieving far better results with respect to previously
ones obtained in our laboratories (Shumyantseva et al., 2005); and the
optimization of the electron transfer’s stability leading to the optimal

detection of cholesterol in the clinical range concentration for longer
times of use.
Table 4.3. Comparison among the results of the works about cholesterol detection using
P450scc published by our group in the last years (Reprinted with the permission from
Stura et al., Anodic porous alumina as mechanical stability enhancer for LDL-cholesterol
sensitive electrodes, Biosensors & Bioeletronics 23, pp. 655–660, © 2005, Elsevier).
P450scc Response in Time Temperature Range of

current stability stability cholesterol
[nA µM-1] detectability
[µM/cm2]
In LS film (30 0.4 90 days Room 50–750
layers)1 temperature
With gold 130 2–3 days 4 °C 40–300
nanoparticles
optimization2
In Gel matrix3 400 10 days Room 50–1000
temperature
In APA+PLL 300 >5 months Room 50–1000
matrix4 temperature
1
Nicolini et al., 2001
2
Shumyantseva et al., 2005
3
Antonini et al., 2004
4
Stura et al., 2007
In comparison with all other methods previously described (Table

4.3) the APA-P450scc electrode represents the most promising
alternative for the existing amperometric biosensor, quite more stable
and quite independent from its working position (either vertical or
horizontal).
4.1.1.1.4 BR-based light adressable potentiometric sensor

Thin-film technologies (Ulman, 1991; Nicolini, 1997) allow the
assembly of biological materials as bacteriorhodopsin (bR) in a 2D
system for photocells (see later and Paternoli et al., 2008; Nicolini et al.,
1999) and for Light Addressable Potentiometric Sensor (LAPS)
development (Nicolini et al., 1998; Fanigliulo et al., 1996; Bousse et al.,
1994; Sartore et al., 1992a,b,c).
For these devices, LB seems to be one of the most promising, due to

its ability to form molecular systems having high packing degree and
molecular order.
Therefore, the ability of bR to form thin films with excellent optical
properties and the bR intrinsic properties themselves make it an
outstanding candidate for use in optically coupled devices. bR thin layers
have been widely studied (Hwang et al., 1977a,b); Ikonen et al., 1993;
Shibata et al., 1994; Méthot et al., 1996; Sugiyama et al., 1997) because
they exhibit bi-stability in optical absorbance and they provide light-
induced electron transport of protons through the membrane.
Furthermore, their extremely high thermal and temporal stability also
allows considering them as sensitive elements for electro-optical devices
(Erokhin et al., 1996; Miyasaka et al., 1991). However, in order to use
bR properties to provide photo-voltage and photocurrent, it is necessary
to orient all the molecules in such a way that all the proton pathways are
oriented in the same direction. The LB technique in its usual version
does not allow this quite efficiently. When bR-containing membrane
fragments are spread at the air/water interface, they orient themselves
rather randomly in such a way that the proton pathway vectors are
oriented in opposite directions in different fragments. Nevertheless, a
technique of electrochemical sedimentation is known that allows the
deposition of highly oriented bR layers. However, the layers deposited
with this technique are rather thick and not well controllable in thickness.
LB technique allows one to form a monolayer at the water surface and to
transfer it to the surface of supports. Formation of the bR-containing
membrane fragments monolayer at the air/water interface, however, is
not a trivial task, for it exists in the form of membrane fragments. These
fragments are rather hydrophilic and can easily penetrate the subphase
volume.
In order to decrease the solubility, the subphase usually contains a
concentrated salt solution. The efficiency of the film deposition by this
approach (Sukhorukov et al., 1992) was already shown. Nevertheless, it
does not allow one to orient the membrane fragments. Because the
hydrophilic properties of the membrane sides are practically the same,
fragments are randomly oriented in opposite ways at the air/water
interface. Such a film cannot be useful for this work, because the proton
pumping in the transferred film will be automatically compensated; i.e.,

the net proton flux from one side of the film to the other side is balanced
by a statistically equal flux in the opposite direction. On the other hand,
the technique of electrochemical sedimentation is known allowing the
formation of rather thick bR films by orienting them in the electric field.
Therefore, the following method was realized the results obtained
scheme shown in Figure 4.8. A 1.5 M solution of KCl or NaCl (the effect
of preventing bR solubility of these salts is practically the same) was
used as a subphase. A platinum electrode was placed in the subphase. A
flat metal electrode, with an area of about 70% of the open barrier area,
was placed about 1.5–2.0 mm. above the subphase surface. A positive
potential of 50–60 V was applied to this electrode with respect to the
platinum one. Then bR solution was injected with a syringe into the
water subphase in dark conditions.
The system was left in the same conditions for electric field-induced
self-assembly of the membrane fragments for 1 h. After this, the
monolayer was compressed to 25 mN/m surface pressure and transferred
onto the substrate (porous membrane). The residual salt was washed with
water. The water was removed with a nitrogen jet.
Figure 4.8. Surface pressure as a function of time in the electric-field assisted bR for the
construction of a new kind of Light Addessable Potentiometric Sensor. The photosignal
(see Table 4.4) has a similar dramatic enhancement with application of the electric field
(Reprinted with the permission from Nicolini et al., Towards light-addressable transducer
bacteriorhodopsin based, Nanotechnology 9, pp. 223–227, © 1998, IOP Publishing
Limited).
The dependence of the surface pressure upon the time with and
without applied electric field is shown in Figure 4.8. It is clear that the
electric field strongly improves the ability of the membrane fragments to
form a monolayer at the water surface and thereby to enhance
significantly the photosignal. X-ray measurements of the deposited
multi-layers revealed practically the same structure in films prepared
with the usual LB technique and electric field-assisted monolayer
formation. This finding does not seem strange. In fact, an electric field
only aligns the fragments at the air/water interface, providing equal
orientation of the proton pathways. The layered structure in this case
remains the same. X-ray curves from both types of samples revealed
Bragg reflections corresponding to a spacing of 46 Å, which is in a good
correspondence with the membrane thickness. In order to control the
degree of bR orientation, photo-induced current was also measured.
Photosignal was also measured, as a function of the illumination
wavelength (Nicolini et al., 1998). Moreover, one monolayer of bR was
deposited onto the porous membrane.
Table 4.4. Photocurrent observed in a system using porous membranes covered with bR
film deposited by the usual LB technique and electric field-assisted technique. A standard
error of about 10% is observed over five independent positive measurements (Reprinted
with the permission from Nicolini et al., Towards light-addressable transducer
bacteriorhodopsin based, Nanotechnology 9, pp. 223–227, © 1998, IOP Publishing
Limited).
Light-on current [pA] Light-off current [pA]

Usual LB technique 15 10
Electric field-assisted 820 10
monolayer formation
The results are summarized in Table 4.4. It is clear that the

photoresponse in the case of electric field-assisted monolayer formation
is much higher compared to that after a normal LB deposition (in the last
case the signal value is comparable with the noise, indicating a mutually
compensating orientation of the membrane fragments in the film).
The observed results allow the conclusion that the suggested method
of electrically assisted monolayer formation is suitable for the formation
of bR LB films, where the membrane fragments have preferential
orientation.Since electric field-assisted monolayer formation at the

air/water interface turned out to provide the possibility of highly oriented
bR LB film formation, it was possible to suggest another application of
bR films for transducing purposes.
The principles of device realization are described next. The scheme of
the proposed device is presented in Figure 4.9. Porous membrane with
deposited bR film separates two chambers with electrolytes and two
platinum electrodes.
Figure 4.9. Schematic view of the measuring chamber used for the experiment with the
bR membrane. Porous membrane with deposited bR film is separating two chambers with
electrolytes. Light fibre is attached to the X-Y mover, which allow to illuminate the
desirable parts of the membrane (Reprinted with the permission from Nicolini et al.,
Towards light-addressable transducer bacteriorhodopsin based, Nanotechnology 9, pp.
223–227, © 1998, IOP Publishing Limited).
A light fiber is attached to the X-Y mover, which allows illuminating

desirable parts of the membrane. Illumination of the membrane part will
result in the proton pumping through it, carried out by bR. Therefore, a
current between the electrodes will appear. This current must depend
upon several factors, such as light intensity, pH of the electrolytes, and
gradient of the pH on the membrane. One of the possible applications of
the suggested device is mapping of 2D pH distribution in the measuring
chamber, which can result from the working of enzymes immobilized in
this chamber. By scanning the light over the membrane it will be
possible to obtain the current proportional to the pH gradient at the
illuminated point, and by maintaining the pH value fixed in the reference

chamber it will be possible to calculate absolute pH values at different
points over the whole membrane surface (Nicolini et al., 1998;
Fanigliulo et al., 1996; Bousse et al., 1994; Sartore et al., 1992a,b,c). If
different types of enzymes, producing or consuming protons during their
functioning, are distributed over the area close to the membrane, the
device will allow one to determine the presence of different substrates in
the measured volume, performing, therefore, as a multiple enzymatic
biosensor. Space resolution of the transducer is extremely high. Because
each bR molecule performs proton pumping, it will be comparable with
the protein size (about 2 nm). In practice, however, it will be limited by
the possibility of focusing the light beam, which as shown separately can
be now focused to nanosize range. But, in any case, it will be more than
in existing transducers, subject to technological problems.
4.1.1.2 Organic nanosensors
Over the last decade the Biophysics Institute of the University of

Genova and the Fondazione EL.B.A. in close cooperation with the
Scientific and Technological Park of the Elba Island and with numerous
leading international companies (STM, ABB, Edison, FIAT, Elsag-
Bailey) have been quite active in developing neural sensors (Adami et
al., 1996a,b), based on organic materials (Ding et al., 2001, 2002b; Ram
et al., 1997, 1997a, 1997b, 1997c; Bavastrello et al., 2002, 2004, 2004a;
Valentini et al., 2004a) and utilizing a wide range of transducers, either
amperometric (Antonini et al., 2004; Shumyantseva et al., 2004),
potentiometric (Adami et al., 1995a, 1996a,b, 2007), conductimetric
(Valentini et al., 2004a; Bavastrello et al., 2004a), nanogravimetric
(Nicolini et al., 1997; Adami et al., 1996a,b), spectrophotometric
(Paternolli et al., 2002a) and fluorometric (Paddeu et al., 1995b). Several
immobilizing techniques were also employed over the time, ranging from
self-assembly (Shumyantseva et al., 2004; Paternolli et al., 2002a), to
layer-by-layer (Ram et al., 2001b; Shumyantseva et al., 2001) and
Langmuir-Blodgett (Valentini et al., 2004b; Ghisellini et al., 2004; Ding
et al., 2002a; Ram et al., 2001a). We intend here to summarize the
present state of the art with emphasis on the recent nanostructuring of
sensing organic matrices and to critically assess the potential industrial

relevance of emerging intelligent sensors for a wide variety of
applications for health and environment.
4.1.1.2.1 Intelligent sensing for metals in the environment

In 1976 a new technique, derived from polarographic methods, was
proposed, the Potentiometric Stripping Analysis (PSA).
Figure 4.10. Schematic description of a PSA experiment. (A), part 1, the electrochemical
process taking place during the first step is illustrated: the working electrode polarization
causes the deposition of the metal ions on it; part 2 shows the circuital schematics to
drive this phase. (B) describes, in the same manner, the second step of the analytical
technique. (Figure 1 from Nicolini et al, 2006). (C) the data analysis software transforms
and plot the acquired data E(t) in the inverse derivative form dt/dE as a function of the
recorded potential, evidentiating the plateau of the stripping potentiogram as peaks in the
derivative potentiogram, whose area is a linear function of the concentration of the metal
in solution (A,B: Reprinted with the permission from Nicolini et al., Nanostructured
organic matrices and intelligent sensors, in Smart Biosensor Technology, (G. Knopf &
Bassi A.S. eds), CRC Press, pp. 231–244, © 2006, Taylor & Francis Group LTD; C:
Reprinted with the permission from Adami et al., A potentiometric stripping analyzer for
multianalyte screening, Electroanalysis 19, pp. 1288–1294, © 2007, Wiley-VCH Verlag
GmbH & Co. KGaA).
This chemical method (Estela et al., 1995) is very sensitive for the
detection of metal ions in aqueous samples and consists of two steps: the
ions are first electrolytically concentrated by deposition on a working
electrode (preconcentration, or plating stage), then a metal stripping
phase follows, during which no control is performed on the potential.
The latter step is accomplished, usually, with a chemical oxidant in
solution (Hg++ or dissolved oxygen).
After the preconcentration phase, the analytical signal (the potential
of the working electrode) is recorded as a function of time and then
utilized to obtain quantitative information about the metal ions in
solution. A recent paper (Adami et al., 2007) demonstrates the possibility
to design and realize a simple and low-cost instrument for monitoring
environmentally significant metals by implementing the PSA technique
(Figure 4.10), connecting a cheap electronics to a computer.
Figure 4.11. Intermetallic formation during the multianalyte determination of lead and
copper (A) and zinc and copper (B). This effect, related both with the analytes and with
the technique, affects the performance of the analyzer in terms of sensitivity and
specificity (Reprinted with the permission from Adami et al., A potentiometric stripping
analyzer for multianalyte screening, Electroanalysis 19, pp. 1288–1294, © 2007, Wiley-
VCH Verlag GmbH & Co. KGaA).
Such analyzer, suitable for heavy metals analyses is based on the

Constant Current Potentiometric Stripping Analysis and on a very simple
electrochemical cell. In the first step a potentiostat (Jagner et al., 1993)
drives the electrochemical cell through the counter electrode ensuring the
proper biasing voltage Vref/wk. In the second step, a constant oxidizing
current (1–100 µA) is fed into the working electrode and the metal is
forced to move again into the solution, producing a potential drop
between reference and working electrodes. The system becomes a
galvanostat and the potential drop Vref/wk is now recorded as a function of
time.
Figure. 4.12. A) NN response after the training phase. During this phase the net knows
the final concentrations and is let running in order to detect the optimal node weights to
obtain the desired result. Once finished, the net is well trained if the results (red) fit with
the expected concentrations (—) for most samples. The figure shows both lead and
copper results for the same set of samples, where there was simultaneous presence of
both ions. B) NN response after the test phase. Here the net has fixed weights (those
calculated and optimized in the previous step) and must detect the correct ion
concentrations as the output of the net itself. (—) is again the correct ion concentration,
while (red) are the detected values for all the samples (Reprinted with the permission
from Adami et al., A potentiometric stripping analyzer for multianalyte screening,
Electroanalysis 19, pp. 1288–1294, © 2007, Wiley-VCH Verlag GmbH & Co. KGaA).
The software tools, user-friendly and easy to use, allow to detect the
analytical signals and to extract the information data. With synthetic
sample we obtained enough sensitivity to propose a first-screening

apparatus but, when the sample is a real matrix, it can contain some
different components, in particular some different metal ions, each one
acting as a possible interferant (Figure 4.11). In order to overcome this
problem (Adami et al., 2007), we integrated in the proposed system a
Neural Network Algorithm, the Multi Layer Perceptron (MLP), which
appears able to reveal the real concentrations of the different metal ions,
after a severe training (Figure 4.12).
From the results described it appears that sophisticated software
implementation, such as those based on NN algorithm could be utilized
for recovering an analytical signal derived from a multisensing
application. Many examples were proposed for gas sensing but not so
many for ions screening in solution. We are optimizing our NN for a
better discrimination of metal traces, by changing some parameters and
some strategies and we hope that a software solution will eliminate the
need to introduce modifications in the proposed analyzer, such as the use
of a modified working electrode (with a mercury layer or with some
chemical agents, such as chelant compounds or resins) or of some sample
treatments or of more sophisticated electronics.
4.1.1.2.2 Nanocomposites sensing for inorganic vapours

Various nanostructures have been investigated to determine their
possible applications in biosensors. These structures include nanotubes,
nanofibers, nanorods, nanoparticles and thin films (see chapter 1). A gas
sensor, fabricated by selective growth of aligned carbon nanotubes
(CNTs) by pulsed plasma on Si3N4/Si substrates patterned by metallic
platinum, was recently presented for inorganic vapor detection at room
temperature (Valentini et al., 2004a).
Poly(o-anisidine) (POAS) deposition onto the CNTs device was
shown to impart higher sensitivity to the sensor (Valentini et al., 2004a).
Upon exposure to HCl the variation of the CNTs sensitivity is less than
4%, while the POAS-coated CNTs devices over a higher sensitivity (i.e.
28%). The extended detection capability to inorganic vapors apparent in
Figure 4.13 is attributed to direct charge transfer with electron hopping
effects on intertube conductivity through physically adsorbed POAS
between CNTs. The CNTs thin film was grown using a radio frequency
pulsed plasma enhanced chemical vapor deposition (RF-PECVD) system

(Gonzalez, 1992). Prior to the nanotube growth, a Si3N4/Si substrate was
patterned with platinum film (60 nm thick) by vacuum deposition
through shadow masks, containing rectangular stripes 30 µm wide and a
back deposited thin film platinum heater commonly used in gas sensor
applications. A thin film (3 nm) of Ni catalyst was deposited onto the
Si3N4/Si substrates using thermal evaporation.
Figure 4.13. (a) I-V curves at room temperature of POAS coated devices to HCl vapor.
The inset shows I-V curves of the CNTs device at room temperature to HCl 100 ppm. (b)
The time-dependence change of the normalized resistance (Rt0 is the initial resistance of
the sample) of POAS coated device and CNT film at room temperature to HCl 100 ppm.
(c) Sensitivity vs. HCl concentrations of POAS coated device and CNT .lm at room
temperature (Reprinted with the permission from Valentini et al., Sensors for inorganic
vapor detection based on carbon nanotubes and poly(o-anisidine) nanocomposite
material, Chemical Physics Letters 383, pp. 617–622, © 2004a, Elsevier).
Figure 4.13 shows two distinct groups of I-V curves resulting from
nanotubes and polymer POAS coated nanotubes. Each group consists of
results from several sensors. It can be seen (Valentini et al., 2004a;
Nicolini et al., 2006) that the conductivity (slope) is almost the same for
CNTs and POAS-coated CNTs, but it varies for the device when exposed
to HCl. In particular, it shows that the conductance of nanotubes slightly
increases), while the POAS coated device shows a significant
conductance increment. It is interesting to note that the response transient
of POAS coated device is a few seconds, while sensor based on
resistance changes of POAS exhibits a poorer response time. The
circumstance to maintain film resistance below 1 kΩ, which is significant
lower with respect to that reported for POAS sensors (about 50 MΩ),
makes POAS coated CNTs films integration in electronic circuitry easier
and cheaper, since lower DC voltages are required to drive the sensor
response. The doping process of poly-anilines is always associated to
conformational modifications of the polymer chains, due to the local
distortions created by the addition of H+ ions to the basic sites and
usually provides stable systems. It means that the conducting polymer in
the doped form can be maintained in this state for long periods of time
till the material reacts with basic reagents and strongly changes its
chemical-physical properties. In other words, the reversibility of the
process is not spontaneous. If we define sensor sensitivity (S) as the ratio
S =[(RA – RG)=RA] x 100, where RA represents the resistance in air and
RG the resistance in vapor, the gas sensitivity increases from S = 3:0% to
27.9%. It reveals that by selecting proper polymer functionalization
sensor sensitivity to HCl may be improved. In conclusion, CNTs thin
films prepared by pulsed RF PECVD demonstrated their potentiality as a
new class of materials for HCl detection for environmental applications,
with polymer functionalization enhancing their sensitivity.
4.1.1.2.3 Organic gas sensing

Whenever a nanocomposite of multiwalled carbon nanotubes (MWNTs)
embedded in poly(2,5-dimethylaniline) (PDMA) was synthesised by
oxidative polymerisation, the nanocomposite (PDMA-MWNTs) showed
a progressive spontaneous undoping process along the time associated to
the instability of the doping agent, constituted by HCl, inside the
polymeric matrix (Bavastrello et al., 2004a,b).
The study of the undoping process revealed that this phenomenon is
related to the synergetic effect of the sterical hindrance of the
substituents on the aromatic rings and the presence of MWNTs inside the
polymeric matrix. The conducting properties connected to a doping-
undoping equilibrium in the presence of the doping agent were also
investigated. The instability of the doping process allowed us to fabricate
a spontaneous reversible sensor for acid vapours by setting up a
comparative potentiometric circuit and engineering the sensitive element
directly on the circuit board. The fabricated devices were connected to
the electrometer by means of silver wires and silver paint, as shown in
the schematic of Figure 4.14.
Figure 4.14. Schematic of devices employed for the determination of the specific
resistance (Reprinted with the permission from Bavastrello et al., Poly(2,5-
dimethylaniline-MWNTs nanocomposite: a new material for conductometric acid vapors
sensor, Sensors and Actuators B 98, pp. 247–253, © 2004b, Elsevier).
The instability of the doping process allowed us to fabricate a

spontaneous reversible sensor for acid vapors (Bavastrello et al., 2004b)
by setting up a comparative potentiometric circuit and engineering the
sensitive element directly on the circuit board. In order to test the
response of the fabricated device after exposing to HCl vapors, an
experimental set up was realized as following. The device was placed
inside a container of 120 ml and thus sealed by means of a parafilm
layer, letting the wires out through it to be connected to the electrometer.
Volumes of 0.2 and 0.5 ml of saturated vapors of HCl were injected
through the parafilm membrane. Current versus time measurements at a
fixed bias of 10 V was then carried out. The relative results obtained
from the experiments are shown in Figure 4.15, which illustrates the V-I
characteristics of PDMA-MWNTs nanocomposite in the undoped and
doped forms evidenced with curves 1 and 2, respectively.
Figure 4.15. V-I characteristics of PDMA-MWNTs nanocomposite in both the undoped

(curve 1) and doped (curve 2) forms. The material showed a quasi-linear behaviour.
(Reprinted with the permission from Bavastrello et al., Poly(2,5-dimethylaniline-
MWNTs nanocomposite: a new material for conductometric acid vapors sensor, Sensors
and Actuators B 98, pp. 247–253, © 2004b, Elsevier).
It can be observed that the system tends to return to the initial

conditions of resistance after about 3 min since the injection of HCl
vapors. This behavior is the consequence of the instability of EB/ES
equilibrium, which can be shifted to the stable formation of the ES form
only by means of a continuous dynamic inflation of acid vapors. Curve 1
represents the saturation level of the sensor device valuable in a
concentration of 4200 ppm of HCl vapors, since higher levels of
concentration gave the same results. Curve 2 shows the data obtained for
an intermediate concentration between the absence and the saturation of
vapors, and the device evidenced a response in function of HCl vapors
concentration. The experiment was carried out by quickly injecting the
acid vapors into the system containing the sensor device and the
acquisition of data started immediately after the complete injection
(Bavastrello et al., 2004b). Other nanocomposites are now synthesized
by using different monomers and concentrations of MWNTs.Ts inside
the polymeric matrix for a wide range of gas sensor applications.
4.1.2 Passive elements
Electronic components are normally divided in three classes: passive

electronic components (paragraph 4.1.2), active electronic components
(paragraph 4.1.3) and energy electronic components (paragraph 4.2). The
fist two classes consider the devices that don’t generate (or store) electric
power for supplying purposes; the third class contains all the elements
dealing with generation or storage of electric energy.
Molecular electronics and nanostructuring of polymers for industrial
applications are a challenge for many research groups across the world
(Nicolini, 1996a,b). In particular, the development of discrete devices is
a problem involving many difficulties, including the connection of the
active elements and the circuit. In modern electronics, technical
applications require capacitors of high value, so the aim of the research
for passive electronic elements is to obtain a dispositive based on
electrolyte with sufficient characteristics of breakdown voltage and
capacity. An alternative way to silicon electronics (Figure 4.16) is the
developing of circuital elements based on organic materials and low-cost
technologies, allowing the production of lightweight, thin, flexible
dispositives.
Figure 4.16. Silicon-based devices process steps, which for their difficulty provide a
rationale for a new organic electronics.
International industries (NEC, Bayer and Kemet) are already

developing the first commercial capacitors based on organic elements,
reaching considerable results in capacity achievement but low applicable
voltage. In the case of capacity typical problems of commercial organic
capacitors are the excessive variability at different voltage ranges, and

complicated and expensive oxidation of electrodes. The objective is to
achieve a satisfying result with cheaper technology for producing both
resistance and capacitance.
4.1.2.1 Resistors
Nanoparticles of CuS are typically formed by exposing the deposited LB

films of copper stearate to the H2S atmosphere for at least 12 hours
(Erokhina et al., 2003). The aggregation of the nanoparticles into thin
layers was performed by washing the sample with chloroform after the
reaction for removing stearic acid molecules. Thin layers of CuS were
formed by aggregation of nanoparticles formed in LB precursor layer of
copper stearate. Very thin layers are not uniform and can be represented
as conductive aggregated “islands” with insulating gaps between them
(Figure 4.17).
Figure 4.17. Conducting nanostructure based on CuS islands nanoparticles bridged by

conducting polymer.
Once the electrical properties of CuS layers of different thickness

were examined, the dependence of an electrical conductivity upon the
frequency was observed. The electrical conductivity appears strongly
dependent upon the frequency when the thickness of the precursor
copper stearate LB films is less than 30 bi-layers. For such thickness the
resultant aggregated layer is not continuous one and can be considered as
a film of “islands” separated one from the other. This fact is responsible
for the increase of conductivity with frequency. When the thickness of
the initial precursor layer is more than 30 bi-layers, the aggregated layer
becomes uniform and displays ohmic conductance (Figure 4.17).
Interestingly the heterostructure of less than 30 nm showed ohmic
conductance only when is formed by CuS semiconducting nanoparticles
and a conjugated polymer synthesized by oxidative copolymerization of
3-thiopheneacetic acid and 3-hexylthiophene leading to an amphiphilic
polythiophene that allows the formation of a stable polymer layer at the
air-water interface (Narizzano et al., 2005).
Figure 4.18. (A) The structure contains 30nm of CdS (red) and 30 nm of CuS (blue); (B);
rectifying behavior of Cds-CuS heterostructure; (C) dependence of the specific resistance
of aggregated CuS layers on the number of bilayers of copper arachidate LB precursor
(Part C: Reprinted with the permission from Erokhina et al., Microstructure origin of the
conductivity differences in aggregated CuS films of different thickness, Langmuir 19, pp.
776–771, © 2003, American Chemical Society).
Finally, when the thickness of the initial precursor LB layers was

more than 25 bi-layers, the resulting aggregated films were uniform and
their electrical conductivity was high (Erokhina et al., 2003). Linear
voltage-current characteristics with clear rectifying behavior and with
practically no dependence of the conductivity on frequency were
measured for the films obtained from more than 30 bi-layers of the
precursor (Figure 4.18 right below). Typical resistivity of such layers
was less than 1.0.
Such low value of the specific resistance together with small
thickness of the layers (21 nm) allows considering this material as very
perspective for applications in electronics (Erokhina et al., 2002). It is a
well-known fact that for very thin metal films it is possible to observe a
nonmetallic temperature dependence of the conductivity similar to that
found in semiconductors, i.e., an increase of conductivity with
temperature. The structure of thin, aggregated layers of CuS
nanoparticles, grown in Langmuir-Blodgett film precursors was
investigated with atomic force microscopy (Erokhina et al., 2003) along
with the study of their electrical conductivity.
Very thin layers revealed an essentially insulating behavior. These
layers were composed of isolated particle aggregates that had a mean
thickness corresponding to the average particle diameter. The increase of
the film thickness resulted in the formation of conducting pathways
formed by the aggregates in the layer plane. Such samples revealed an
increased conductivity. When the thickness of the initial precursor LB
layers was more than 25 bi-layers, the resulting aggregated films were
uniform and their electrical conductivity was high (Erokhina et al.,
2003). Finally, it is worth of notice an in-plane patterning process of
aggregated nanoparticle thin layers of different inorganic conducting and
semi-conducting capable to develop passive resistors produced in
Langmuir-Blodgett precursors using film irradiation with an electron
beam (Erokhin et al., 2002a).
In conclusion, nanoparticles, formed in LB precursors, can be
aggregated into thin inorganic layers to yield organic resistors of unique
electrical properties at the nanoscale. When this thickness is low (one bi-
layer of precursor), the particles form aggregates with the lateral size of
about 70–80 nm and with a thickness of one individual particle diameter
(2.3 nm). The layer is not uniform and homogeneous the film volume is
only 8.5% filled by particle aggregates. Such “porosity” of the film
determines its practically insulating behavior. Increased thickness of the
precursor film results in significant changes of the aggregated layer
structure and properties. Particles of the upper precursor layers tend to
occupy empty spaces of the sub-layers during the aggregation process,

resulting in the decrease of the height non-uniformity in the final film.
Increased filling of the film volume by particle aggregates provides the
formation of conducting pathways and a significant decrease of the film
specific resistance. Further increase of the precursor film thickness
results in the increase of the height non-uniformity of the aggregated
film, providing a further increase of the filling of the film volume by
particle aggregates, resulting in a pronounced decrease of the film
specific resistance. The results obtained have demonstrated that
effectively conducting thin inorganic layers can be prepared by the
aggregation of CuS nanoparticles, produced in LB films, when the initial
precursor thickness is more than 25 bi-layers. In this case, the layers are
rather homogeneous. The variation in the film roughness is about 3 nm,
corresponding to a variation of 15% of the average thickness, and
electrical properties of these layers are stable and reproducible.
4.1.2.2 Capacitors
Electrolyte capacitor is a type of device offering intermediate

characteristics between those of battery and simple dielectric capacitor.
Usually, solid electrolyte capacitors are composed of an electrode (e.g.
tantalum or aluminum), an electrolyte layer (e.g. manganese dioxide) and
a very thin insulator oxide (0.01–1 Am) realized at or above
approximately 275 °C.
This is a relatively violent reaction, which often damages the
capacitor structure rendering it unuseful. Furthermore, deposition and
pyrolysis have to be repeated several times since nitrates react with the
oxide dielectric. In order to eliminate these drawbacks polymer
electrolytic capacitors with a cathode of conducting polymer appears an
adequate substitute for manganese dioxide (Figure 4.19). In this case, the
production is quite simple: after the formation of a thin metal oxide by
anodic oxidation, the tantalum or aluminum anode is immersed in the
conducting polymer solution (Erokhin et al., 2002). The use of a
conducting polymer instead of manganese dioxide offers another
advantage. The conductivity of manganese dioxide is quite low, thus, in
order to increase the performance at high frequencies, it is better to use a
polymer characterized by a higher conductivity than MnO2. The rapid

development of portable electronic devices has increased the demand for
compact, lightweight, high capacity batteries. Polymers such as
poly(ethylene oxide) (PEO) are widely studied due to their significant
potential as “solid” polymer electrolytes (SPEs) in secondary (i.e.,
rechargeable) lithium/polymer batteries. Its characteristics can be
considered as an advantage for the realization of high-value electrolytic
capacitors (Erokhin et al., 2002). Al foil is used as a first electrode and
since it presents a few nanometers thin native oxide layer, the
manufacturing process is a very easy one since there is no
electrochemical deposition of an additional oxide layer.
Recent work realized and tested such high value organic capacitors
by using easy and low-cost fabrication techniques with solid solutions of
Li salts in poly(ethylene oxide) (PEO), deposited by solution casting
(Figure 4.19).
Figure 4.19. High value organic capacitors with poly(ethylene oxide) (PEO) deposited by
solution casting, with LiCl salt content of 20% (right) and LiClO4 salt content of 50%
(left) at a frequency of 20 Hz.
With electrodes realized from different materials, the electrical tests

of the elements were performed in order to check the excellent stability
of properties in time and the high performance reproducibility with
polymer electrolytic capacitors being formed by electrolytes of PEO/LiCl
and PEO/LiClO4 and by electrodes of Al and silver paint, without

electrochemically deposited oxide layer (Erokhin et al., 2002). Since the
solubility of LiCl in PEO solution turned out to be rather restricted, it
was decided to study in a more detailed manner the element also with
LiClO4 as an ion source. With LiCl, the best capacity value of about 0.34
µF/cm2 (specific value 8 µF/cm2) was obtained with a salt content of
20% at a frequency of 20 Hz. With LiClO4, the best capacity value of
about 0.8 µF (specific value 20 µF/cm2) was obtained with a salt content
of 50% at a frequency of 20 Hz (Figure 4.19).
Figure 4.20. Breakdown voltage test on hybrid capacitor (Reprinted with the permission
from Stura et al., Hybrid organic-inorganic electrolytic capacitors, IEEE Transaction on
Nanobiosciences 1, pp. 141–145, © 2002, IEEE).
Concerning LiClO4-based capacitors, there is a clear evidence of only

a very low dependence of the capacity on the salt concentration, which is
very interesting from an industrial point of view, as it can simplify the
fabrication process. Capacity was found to be quite stable in time even if
the measurements made day by day show small oscillations probably due
to the environmental humidity variations. Both reproducibility and
stability could be optimised by encapsulating the device with, for
example, hydrophobic silicone elastomer coating. Finally, capacity
measurements revealed a low-temperature dependency at a frequency of
50–60 Hz, which is also very important from the industrial applications
point of view (Erokhin et al., 2002).
Organic capacitors are then among the most significant applications

of conductive polymers, allowing the realization of lightweight and
flexible dispositives. The main drawback of completely organic
capacitors is the very low dielectric rigidity, due to irregular surface of
the polymer-polymer interface. To overcome this, Stura et al. (2002)
have used a hybrid solution using aluminum electrodes (both anode and
cathode), with the very important characteristic of easy connection
between contacts of the discrete dispositive and electrodes.
Figure 4.21. A) Standard capacitor breakdown; B) Hybrid capacitor self-healing

(Reprinted with the permission from Stura et al., Hybrid organic-inorganic electrolytic
capacitors, IEEE Transaction on Nanobiosciences 1, pp. 141–145, © 2002, IEEE).
Chemical, technological, and functional tests have been conducted to

estimate the effective values of the breakdown voltage of the hybrid
capacitor (Figure 4.20), and its natural properties of self-healing and non-
polarity with respect to standard capacitor has been investigated (Figure
4.21). Poly-ethylene oxide (PEO) deposited by solution casting is the
matrix in which various salts have been solved, that separate in ions with
the role of charge carriers. Our hybrid solution using aluminum
electrodes appears to have the very important characteristic of easy
connection between contacts of the discrete dispositive and electrodes.
4.1.2.3 Wires
Among the most challenging recent developments in molecular

electronics is the construction of molecular wires of exceptional
electronic properties from the viewpoint of both the chemical synthesis
of conducting polymer (Reed et al., 1997; Chen et al., 1999; Collier et
al., 1999) and new physical means.
This latter approach has been strongly developed using scanning
tunnelling microscopy that offers the possibility of both observation and
manipulation of single atoms or molecules as shown by Bumm et al.
(1996). Another approach has been developed by Reed et al. (1997) by
simply connecting the benzene 1,4 dithiol self-assembled molecules onto
two facing gold electrodes of mechanically controllable break junction to
understand the charge transport through the molecules. Molecular logic
gates were fabricated from an array of configurable switches from
monolayer of redox-active rotaxanes sandwiched between metal
electrodes as shown by Collier et al. (1999). It is necessary to have π-
conjugated system organic molecule to the realization of the molecular
wire and of many other potential applications.
Among the infinite possibilities of synthetic organic chemistry,
linearly π-conjugated systems with nanometer dimensions can be
synthesized as summarized by Ellenbogen and Love (2000). The
molecular wire of chain molecule composed of repeating units bound
together by conjugated π-bands in ethyl substituted 4,4-di(phenylene-
ethynylene)benzenethiolate was already shown in literature (Pearson,
1994; Wu et al., 1996). In this context, extended π-conjugated oligomers
based on phenyleneethynylene and thiopheneethynylene have also been
proposed as potential molecular wires (Reed, 1999). The introduction of
alkyl chain in thiophene will allow us chain dimensions close to the
present limits of the nanolithography (10 nm).
4.1.3 Active elements
A new generation of active electronic elements have been recently

constructed by nanobiotechnology utilizing processes occurring at the
nanoscale and based on LB-induced assembly and/or self-assembly of
conductive polymers and biopolymers.
An example of the latter appears alternative to molecular beam
epitaxy technology allowing the thickness resolution of 0.5 nm (Figure
4.22) whereby ultrathin semiconductor layers of different aggregated
semiconductors are fabricated by a process of self-aggregation of
different nanoparticles formed in organic layer.
Figure 4.22. SEM image of PbS-CdS-PbS superlattice. it is clearly possible to distinguish

layers about 60 nm thick (Reprinted with the permission from Erokhin et al., Preparation
of semiconductor superlattices from LB precursor, Thin Solid Films 327-329, pp. 503–
505, © 1998a, Elsevier).
While molecular beam epitaxy is a complicated and expensive

technique carried out at 10-10 vacuum with extra-pure materials, in this
technique the layers are synthesized at normal conditions and fabrication
of devices is based on these superlattices being a resonant tunneling
diode with the quantum surrounded by two quantum barriers, or a
semiconductor laser with the transitions of electrons through resonant
levels within quantum wells in a semiconductor superlattice. The
advantage is that in processes occurring at normal condition this
technology is not expensive, without toxic waste and with low energy
consumption. Today, the exploration of biopolymers and of neutral or
pristine conjugated polymers for semiconductor device applications such

as photovoltaic cells, field effect transistors, light-emitting diodes
(LEDs), Schottky diodes and monoelectronic transistors have become the
major point of interest.
4.1.3.1 Schottky diode
The Schottky diode has been fabricated using various classes of poly-
aniline films. In this case a low work-function metal (Al, In, Sb etc.) is
deposited on one side of poly-aniline film and the other side is vacuum
deposited by high work-function metal electrode (Au, Ag etc.).
The possibility to utilize the PANI as Schottky diode was already
established due to the semiconductor-like behavior of such polymers
(Pandey et al., 1997) summarizes the Schottky diode parameters realized
on various types of poly-aniline films. In later work we made an attempt
to study the Schottky diode characteristics on a Langmuir-Blodgett
monolayer film of poly(ortho-anisidine) (POAS) conducting polymer.
The Schottky single junction was made by depositing POAS LB film on
a flat graphite electrode, and subsequently approached by a second sharp
electrode (Figure 4.23)
Figure 4.23. Experimental set-up for fabricating Schottky junctions. The positioning of a
second point contact tungsten electrode on the POAS film, deposited onto a graphite flat
electrode, is realised enabling a tunneling current to pass between the tungsten and the
graphite electrodes. (Reprinted with the permission from Ram and Nicolini, Thin
conducting polymeric films and molecular electronics, in Recent Research Development
in Physical Chemistry 4, pp. 219–258, © 2000, Transworld Publishing).
Scanning tunnelling microscopy and spectroscopy on polymer films

assured a device configuration similar to one for point-contact diodes
that has been suggested to solve such problems (Pandey et al., 1997). We
made the point contact electrode positioned with a mono-dimensional
piezo-mover until a tunnel current was achieved in order to avoid pine-
hole between the flat electrode and the tip. Once realized, the Schottky
junctions were tested in working devices (Figure 4.23).
The contact between the tungsten electrode and the POAS film was
obtained with a feedback system and the piezomover was stopped, once
reached to “a priori” imposed tunneling current between the graphite and
the tungsten electrode.
The feedback system was switched off when the tunneling current
was obtained in order to record possibly current-voltage (I-V)
characteristics of the junctions. Figure 4.23 depicts I-V characteristics of
Schottky junction when the tunneling current equals 0.9 nA set by the
feed-back system at applied bias voltage of 4V.
The figure exhibits a pronounced rectifying effect from -0.3 V to +0.3
V. Potential increase above 4 V causes a change in current magnitude,
giving also rise to high differential conductivity, indicating a good
switching current passing through the barrier voltage.
Typically only 64% of the junctions have been found to be of the
Schottky type while 36% of junctions characteristics differed somehow
from Schottky junction behavior. Anomalous behavior can also be
related to defects present in POAS films, like impurity or polymer break
region or empty regions, or in the point contact electrode etching.
4.1.3.2 Led
Light-emitting diodes (LEDs) based on the electro-luminescent

conjugated polymers (Figure 4.24) have attracted significant attention,
both in academic research and industrial development, and are now on
the stage of commercialization.
Polymer LEDs require properties such as shown in Table 4.5 and
efforts have been made to design and synthesize electroluminescent
polymers, tailor their properties, investigate the device physics, and
engineer light-emitting diodes.
Figure 4.24. Schematic for producing polymer LEDs. The accomplishment of polymer
LEDs scheme where, Al - 2000 Å, Emissive polymer layer =1500 Å, ITO work function
value = 4.7 (positive electrode), Al work function value = 4.2 to 3.76 (negative electrode)
(Reprinted with the permission from Nicolini et al., Supramolecular layer engineering for
industrial nanotechnology, in Nano-surface chemistry, pp. 141–212 © 2001a, Marcel
Dekker/Taylor & Francis Group LTD).
Among various conducting polymers, aromatic conducting polymers

such as polythiophene, poly(p-phenylene vinylene), poly(pphenylene),
poly(phenylene sulphide), and their derivatives have been used as the
emission layer in polymer LED devices. A low voltage is applied over a
thin film of the polymer. Subsequently the electrons and holes are
injected from the electrodes; when a hole and an electron collide in the
polymer, a local excited state can be formed that emits light (electro-
luminescence), as shown in Figure 4.24. The basic requirements for the
choice of electro-luminescent polymers are:
(1) the polymer should have good film-forming properties (smooth
surfaces, no pinholes, minimum thickness of 50 nm);
(2) the polymer film should have good thermo-mechanical stability;
(3) the polymer films should be transparent;
(4) the polymer should be amorphous;
(5) the polymer should exhibit excellent heat, light, and environmental
stability;
(6) special requirements for light-emitting polymers are light emission in
the visible region and color tunability.
Table 4.5. Physical properties of interest for LED materials (Reprinted with the
permission from Ram and Nicolini, Thin conducting polymeric films and molecular
electronics, in Recent Research Development in Physical Chemistry 4, pp. 219–258, ©
2000, Transworld Publishing).
Polymer Properties Notes Dopant

compounds
poly(phenylene Bandgap = 2.5÷2.6 eV See forward and iodine, FeCl3, K,
vinylene) Intrinsic electrically reverse potential Ca, Mg, or acids
(PPV)) conductivity 10-3 S/cm.
Emission range 1.8–2.5
eV. Ionization potential
= 5.1 eV Electron
affinity = 2.6 eV
poly(2-methoxy, Emission peak = 605 nm Structure: p- doping by
5-(2'- Es PANI sulfuric acid; n-
ethylhexyloxy)- electrode / type by sodium
1,4-phenylene MEH- Iodine (I2)=
vinylene) PPV/Calcium electron
(MEH-PPV) Electrode acceptor →
oxidizing agent
To control the preceding parameters, Langmuir-Blodgett and layer-

by-layer adsorption techniques for the preparation of PPV conjugated
polymer films have recently been developed. These demonstrate the
fabrication of optically transparent PPV-containing multi-layers of
precursor PPV, which is converted to PPVs by thermal elimination. The
PPV family of polymers serves as a prototypical conjugated polymer
class for application as well as for fundamental understanding of the
electronic processes in conjugated polymers. Ohmori et al. (1997)
showed that other conjugated polymers like poly(3-alkylthiophene) are
also useful as the emitting species in LEDs (Granstrom, 1997). Recent
advances for both nonpolar, i.e., “molecular device”, and polymer LEDs
indicate a great potential of organic-base diodes.
A survey of the preparation of electro-luminescence conjugated
polymers evidences that the advances in synthetic methodologies for the
preparation of thin films and fibers of PPVs enable them to be considered
in various applications. The overall methodology can be roughly divided
into three categories: precursor approach, side-chain derivatization and in
situ polymerization. The sulfonium precursor route (SPR) to PPV is
particularly well known and involves the polymerization of p-xylene bis-

(tetrahydronium thiophenium chloride) or one of its analogues or
derivatives, in the presence of water or methanol to give the
corresponding sulphonium precursor polymer. Table 4.5 shows the
common LED materials and their physical properties. The poly(2-
methoxy-5-(2’-ethyl-hexyloxy) phenylene vinylene) (MEH-PPV), P-
PPV, etc. have been synthesized by Gilch route. Furthermore, a
modification of the Gilch route, namely the chlorine precursor route
(CPR) was introduced in 1990 in order to avoid polymer precipitation.
DP-PPVs have been prepared by this route in 1993. The overall
impression is that CPR is very simple, general, versatile and reproducible
and superior to the SPR and Gilch route for the preparation of PPV
derivatives but SPR remains the best approach for the fabrication of PPV
thin films. However, improvement in the luminance efficiency and
durability of device remain unsolved. The processability of PPVs
compounds has recently been increased for the fabrication of thin films
for real device application. To increase the efficiency of devices, electron
injection has also been significantly boosted. Green emission with a
luminance of 100 Cd m2 and an external quantum efficiency of 0.4 % at a
driving voltage of 4 V has been obtained from a device using PPV.
Poly(3-alkylthiophene)s provide a good example of how the colour of
emission can be varied in polymer LEDs by modifying both the polymer
structure and the design of the devices colour control by alteration of
substituents and regio-regularity is also illustrated by the pyridine-based
analogue of PPV and its derivatives.
The overall photoelectrical properties of PPV resemble closely those
of aromatic dyes and pigments, which are the first generation of organic
semiconductor materials. However, PPVs are much less stable than
organic pigments, mainly because the vinylene groups are highly
susceptible to photoxidation and it limits the lifetime of the device.
However a different synthetic route may result in PPV with different π-
conjugated chain lengths and chain configurations. These factors may
affect the electronic and optoelectronic properties of the PPV. The one
remaining obstacle to the widespread use of polymer LEDs is that, to
date, their lifetimes remain lower than the best devices using inorganic or
small-molecule organic compounds, although this gap is rapidly being
closed. Singlet oxygen has been implicated in the cleavage of vinylene

groups in MEH-PPV and bis-(cholestanyloxy)-PPV, leading to ester or
aldehyde groups. A study of PPV-based LEDs revealed that exposure to
air produced a drop in efficiency and an increase in threshold voltage,
while degradation of the polymer is one of the primary processes
involved in the breakdown of MEH-PPV-based LEDs. These results
suggest that suitable protection from aerial oxidation may be a significant
factor in improving the lifetime and efficiency of LED devices. Work is
in progress about the implementation of this technology for LED
operating in the infrared.
4.1.3.3 Optical filtering and holography
Optical information storage and processing utilized up to now only

synthetic, photo-chromic and refractive materials as reversible recording
media. However, the effect of side reactions on photochemical processes,
the complicated crystal growth procedure (which is usually also
expensive) impeded the realization of several important applications. The
use of light-sensitive proteins, such as photosynthetic reaction centers
and rhodopsins of various origins is the object of intensive studies in the
recent years.
Bacteriorhodopsin, a retinal protein, the main source of which is
Halobacterium salinarium (formerly Halobacterium halobium), is one of
the most widely used in the experiments aimed at the application of
proteins for the creation of alternative dynamic recording material
instead of conventional synthetic ones (Hampp, 1993, 2000, Hampp and
Zeisel, 2003; Hampp and Juchem, 2004). The extraordinary features,
maturated during the long natural selection process, include, among
others, very high stability, and reversibility of physicochemical
processes, in the form of optically homogenous thin films. The
possibility of gene engineering manipulations of bacterial strains that
produce the protein permits the creation of mutants with optimal
photochromic characteristics for specific applications such as optical
filtering, re-writeable media and holography. According to the
mechanism of functioning of bacteriorhodopsin, the absorption of a
photon by a protein molecule leads to a fast (500 fs) trans-cis photo-
isomerization of retinylidene around the 13, 14 double bond with a

quantum yield of 0.64. The subsequent steps involve thermal relaxation
of several amino acids and of Schiff base linkage, involved in the proton
transport, plus the backbone conformational changes. The states of
bacteriorhodopsin are characterized by the retinylidene conformation and
the initial state is the B-state with an all-trans configuration. In the dark
conditions, there is an equilibrium between the D-state (dark-adapted,
with 13-cis configuration) and the B-state. The proton adsorption
converts the bR molecule into light-adapted B-state (all-trans retinal
configuration). The subsequent conformational changes bring the protein
into the L550-state via the intermediate K590-state by a thermal relaxation
process. The subscript indices refer to characteristic wavelengths of the
absorption spectra.
These features of photochromism are the most interesting ones in the
determination of the applicability of bR to optical storage and
information processing. However, an important point to be kept in mind
is the operational lifetime of such media, evaluated in this case by the
number of write/erase cycles that can be effected. In a holographic film
the intensity and phase distribution of an object is recorded. The resulting
hologram is a diffractive element which generates the original object
wave in amplitude and phase when illuminated with the same reference
beam as was employed during recording. The same happens with bR-
films, but since they are reversible recording materials, these holograms
can be either erased at any time or decay with the time constant of the M-
lifetime (Hampp, 2000; Hampp and Juchem, 2004). The bR-film is used
as short time storage for the reference hologram. With BRD96N-films, a
lifetime of the reference hologram of up to several minutes is obtained.
After this time, a new reference hologram has to be recorded. The
interference of the holographic image and the directly transmitted light
can be described as a processing of two images that correspond to
different times but originate from the same location.
The other example of bR-based image processing is holographic
pattern recognition. This technique allows the correlation of two images
that exist at the same time but are spatially separated. Common features
of both patterns are detected. Since this technique is an analog computing
method, signal-to-noise ratio is of central importance. Due to the
polarisation recording properties of bR, an effective suppression of noise

can be realized. Bacteriorhodopsin films counting the variant BRD96N,
which differs from the wild type bRWT by a single amino acid exchange,
Asp96→Asn, show significantly higher holographic diffraction
efficiencies (η) than bRWT films (Table 4.6).
Table 4.6. Holographic properties of bacteriorhodopsin films.
Resolution ≥ 5000 lines/mm

Optical density 1–20 (OD570)
Bleaching 90–100 percent (e.g., BRD96N)
Index of refraction 1.47
Refraction index change 10-3–10-2
Diffraction efficiency 1–7 percent (type 2-3 percent)
Light sensitivity 1–80 mJ/cm2 (B-type)
30 mJ/cm2 (M-type)
Polarization recording possible
Reversibility ≥ 106 cycles
Thickness 10-500 µm (type 20 mm)
Speed msec-sec
Aperture Unlimited
A good correlation of the theoretically derived and experimentally

measured values of the refractive index changes was found, indicating
that the chromophore system of bacteriorhodopsin, which is formed by
the retinal molecule, its Schiff base linkage to the protein moiety, and an
inner shell of amino acids, behaves like an almost undisturbed
chromophore with respect to the photo refractive properties at low actinic
light intensities, despite the fact that all components of the chromophoric
system are covalently linked to the amino acid matrix.
Bacteriorhodopsin films are however made with intact purple
membranes, which contain about ten lipid molecules for one
bacteriorhodopsin.
Advantage to use rhodopsins from bovine (Maxia et al., 1995) or
octopus (Paternolli et al., 2008) to make thin films rather than
bacteriorhodopsin is that rhodopsin can be extracted by detergent from
the membrane without loosing its properties while isolated molecules of
bacteriorhodopsin did no longer behave like a proton pump (Fisher and
Oesterhelt, 1979).
We show furthermore that thermal stability is also a property of

bovine rhodopsin in LB films, which can reach, in fact, a temperature up
to 175 °C with a negligible loss of secondary structure (Maxia et al.,
1995).
In a recent work (Paternolli et al., 2008) a new biomaterial resulting
from the isolation of octopus rhodopsin starting from octopus
photoreceptor membranes was obtained.
Figure 4.25. Reversibility of the octopus rhodopsin thin film (Reprinted with the
permission from Paternolli et al., Photoreversibility and photostability in films of octopus
rhodopsin isolated from octopus photoreceptor membranes, Journal of Biomedical
Materials Research Part A, in press, © 2008, Wiley Periodicals, Inc., a Wiley Company).
Mass spectroscopic characterization was employed in order to verify

the presence of rhodopsin in the extract, and photo reversibility and
photo-chromic properties were investigated utilizing spectrophotometric
measurements and pulsed light.
Thin films of octopus rhodopsin were realized, utilizing the gel-
matrix entrapment method in polyvinyl alcohol solution. The results
indicate that the photo-reversibility and the photo-stability of the octopus
rhodopsin in gel-matrices are maintained in time and at room
temperature up to few days (Figure 4.25) and after the exposure for
several hours at room temperature.
4.1.3.4 Displays
Electrochromism is the property of a material or a system to change

colour reversibly in response to an applied potential. There has been a
growing interest in electrochromic materials for use in practical
electrochromic displays. Oxides of W, Mo, V, Nb, Ti and a number of
conducting polymers PPY, polythiophenes and PANIs are known to be
electrochromic materials, the latter being considered as the most
promising for electro-chromic displays (Figure 4.26).
The use of solvents plays an important role in the performance of the
electro-chromic displays while problems associated with liquid
electrolytes in such devices are due to their high degree of hydration,
rapid proton insertion and stability against any chemical or
electrochemical corrosion. A cell based on PANIs can be only used a
thousand times due to the liquid solvent used, which is the disadvantage
with respect to life-time of the electro-chromic cell. In practical
applications it is preferable to employ solid-state materials for electro-
chromic displays in order to minimize the problems of sealing using any
hazardous liquids.
Electro-chromic displays are usually required to have thin layers
configuration. The prospects for solid polymer electrolyte and
conducting polymer look promising for the development of practical
electro-chromic displays. The solid polymer electrolyte and conducting
polymer form an ideal medium for a wide range of electrochemical
processes, paving the way to easy fabrication processes. A solid polymer
electrolyte (i.e. poly(ethylene oxide (PEO)) and its complexes) based on
a conductive polymer would eliminate the use of toxic solvents. We have
focused our attention on PEO, the use of which has been attempted as a
solid electrolyte for the fabrication of electro-chromic displays, due to
the recent synthesis of new PEO-complexes with improved low
temperature electrical properties for the fabrication of electro-chromic
displays based on conducting PANIs. The films of PANI, and its
copolymer films of poly(aniline-co-o-toluidine) (PAOT) and
poly(aniline-co-o-anisidine) (PAOA) have been electrochemically
obtained on indium-tin-oxide (ITO) glass plates and silicon, respectively.
The ultra thin films of substituted PANIs were fabricated by Langmuir-
Blodgett (LB) technique aiming towards their potential application in

electrochromic cell. The current transients for coloration and
decoloration have been analyzed in order to understand the charge
transport for PANI and its conducting copolymer films of PAOT and
PAOA. The electrochromic switching response time in different protonic
acid media was studied on such poly(ortho-anisidine) POAS LB films.
Figure 4.26. Reaction mechanism for PANI (X= H), poly(aniline –co-o-anisidine) (x =
CH3) (POAT) and poly(aniline-co-o-anisidine) (PAOA) (Reprinted with the permission
from Ram and Nicolini, Thin conducting polymeric films and molecular electronics, in
Recent Research Development in Physical Chemistry 4, pp. 219–258, © 2000,
Transworld Publishing).
We have performed electrochemical studies for each electrochromic

cell based on PANI, POAT and PAOA films. In evaluating the
performance of the electro-chromic phenomenon of cells, we focused on
the aspects mentioned, namely, switching time (t1/2), the dependence of
the switching time on the ionic diffusion process (Do), the applied
voltage required for the cell and the stability of the cell in the repeated
cycling. In fact, the mechanism of PANI switching has an important
bearing on these aspects. Bearing in mind that PANI system has a
switching response; we applied a frequency from the functional
generator coupled to electrochemical interface for recording the current
versus time plot.
The electro-chromic cells have been studied at a frequency of 10-1 Hz

obtained from the function generator. The applied potential for the
switching reaction is listed in Table 4.7 for PANIs.
Table 4.7. Electrochromic parameters of conducting polymer and copolymeric films.

(Reprinted with the permission from Ram and Nicolini, Thin conducting polymeric films
and molecular electronics, in Recent Research Development in Physical Chemistry 4, pp.
219–258, © 2000, Transworld Publishing).
Conducting Applied Response Slope (n) Slope (n) Diffusion Process

polymers voltage time reduction oxidation coefficients and life
(V) (ms) (D0) 10-10 cycle
t1/2 cm2/sec
Polyaniline -0.4–0.8 205 0.51 0.44 5.39 Diffusion
104
Poly(aniline -1.0–1.2 299 0.13 0.37 0.40 Diffusion
-co-o- 104<
toluidine)
Poly(aniline -0.6–1.0 143 0.07 0.06 18.95 Diffusion
-co-o- >105
anisidine)
The oxidation and reduction of the conducting polymer and

copolymer films take place under steady-state conditions. The life-time
of PAOA has been estimated to be 105 cycles.
4.1.3.5 Monoelectronic transistors
Inorganic semiconductor nanostructures formed inside fatty acid films,

such as cadmium sulphide nanoparticles as small as 50 Å (see chapter 1),
were found first by Smotkin et al. (1988) exposing cadmium arachidate
LB films to atmosphere of H2S, suggesting the idea that V-I
characteristics out of them could display monoelectron behaviors
(Devoret et al., 1992) even at room temperature. During the reaction, the
head groups of arachidic acid were protonated, and CdS was produced
according to the following reaction:
[CH3 (CH2)18 COO]2 Cd + H2S → 2CH3 (CH2)18 COOH + CdS
Monoelectron junctions are of paramount importance in
nanoelectronics because they could represent the basic elements for
electronic chips exploiting an integration scale up to 104 folds the

nowadays submicron limit, they could allow to implement a multi-state
logic and function with very low power consumption.
Taking into account the actual particle size of CdS nanoparticles
(Figure 4.27), it is indeed found monoelectron phenomena on CdS
granules at room temperature (Facci et al., 1996).
Figure 4.27. STM image of CdS granules inside a LB film of cadmium arachidate.
Imaging parameters Vt=0.6 V (tip positive), I=1.5 nA, scanning speed 12Hz, image size
51.2 × 51.2 nm (Reprinted with the permission from Erokhin et al., Observation of room
temperature mono-electron phenomena on nanometre-sized CdS particles, Journal of
Physics D: Applied Physics, 28, pp. 2534–2538, © 1995a, IOP Publishing Limited).
Such kind of behavior, i.e. Coulomb Blockade (Devoret et al., 1992)

was previously observed at low temperature on particles of larger size
(Mullen et al., 1988), and by STM on metal granules even at room
temperature (Shönenberger et al., 1992a,b).
This phenomenon, which can take place when a conductive or
semiconductive granule is separated from two electrodes by two
tunnelling junctions, consists in the quantified increase of the average
number of electrons occupying the granule upon the bias voltage through
the described structure (Devoret et al., 1992).
The presence of electrons in the granule provides an electric field,
which prevents a further incoming electron to enter the granule until a
suitable bias voltage is applied through the junction.
Each quantized increase of the average number of electrons takes

place whenever the voltage through the structure changes of a quantity
given by:
e
∆V =
C
where e is the electron charge and C the capacity of the structure
Coulomb Blockade appears when the following inequality (Averin and
Likharev, 1986) holds true:
e2
> kT
2C
where e is the electron charge, C is the capacity of the structure, k is
the Boltzmann constant and T the absolute temperature (i.e. when the
effect of the thermal excitation is negligible with respect to electrostatic
repulsion energy).
The classical approach to make the above inequality valid is to
decrease the value of T, as there are technological limits in decreasing
the value of C. CdS nanoparticles, however, seem to be right candidates
for facing the problem of making the above formula valid by decreasing
the value of C and, therefore, achieving high temperature Coulomb
Blockade. Besides, as the sizes of CdS particles can be of the order of
magnitude of tens of an Å, rough estimations foresee capacities as small
as 10-18 F, which should display room temperature Coulomb Blockade
(Facci et al., 1996; Nicolini, 1996a).
The simplified scheme of the measuring set-up is shown in Figure
4.28, where arachidic acid monolayers were formed onto the surface of
water containing 10-4 M of CdCl2.
One bi-layer of cadmium arachidate was deposited with LB trough
(MDT, Moscow) onto graphite substrate at the surface pressure of 28 mN
m-1 by LB technique.
The samples were exposed to an atmosphere of H2S for 15 minutes
(Figure 4.28A) and the V-I characteristics measured on these samples
(Figure 4.28B).
Figure 4.28. (A). The simplified scheme of the measuring set-up for tunnelling. (B).
Voltage-current characteristics with single-electron conductivity. (A: reprinted with the
permission from Erokhin et al., Observation of room temperature mono-electron
phenomena on nanometre-sized CdS particles, Journal of Physics D: Applied Physics, 28,
pp. 2534–2538, © 1995a, IOP Publishing Limited; B: reprinted with the permission from
Nicolini et al., Supramolecular layer engineering for industrial nanotechnology, in Nano-
surface chemistry, pp. 141–212 © 2001a, Marcel Dekker/Taylor & Francis Group LTD).
To achieve a deeper understanding of the process at issue and in order

to give one more proof of the appearance of Coulomb Blockade, it was
performed a theoretical simulation of the V-I characteristics expected
from structures like those above described, by means of a semi empirical
model (Nicolini, 1996a); the average electron occupation number in the
island is estimated by means of the Boltzmann statistics:
+∞ E (V )
− n
∑ ne kT
< n >= n = −∞
+∞ E n (V )
−
∑e
n = −∞
kT
where n is the electron occupation number of the granule and En(V) is

the energy of the electron inside the granule. Within this frame the
observed phenomena was corroborating the hypothesis that the behavior
of V-I characteristics we observed on the wells inside the fatty acid film
is really due to Coulomb Blockade at room temperature and that inside
the wells are really present granules of nanometer sizes (Nicolini, 1996).
The experiment hereby described represents only the proof of
principle and still many difficulties remain to overcome the possibility of
exploiting phenomena such as the Coulomb Blockade from a

technological point of view opening the way to a revolution in the
concept of electronics. It was then comforting to find many years later
unexpectedly similar mono-electronic transitions also in composite
material (Figure 4.29) made by a multinational company on solution
casted POAS for silver paint containing conductive nanoparticles of
similar size as apparent by the associated atomic force microscopy.
Figure 4.29. Unexpected results displaying Coulomb staircase phenomenon in composite

nanomaterials characterized electrically and morphologically (private communication,
2003).
Towards mono-electronic applications, we can say that these results

represent only the first steps. Further steps toward the realization of
stand-alone mono-electron devices involve the formation of a network of
connections capable of addressing the single mono-electron junctions,
which seems to be still far away.
4.1.4 Quantum dots and quantum computing
Quantum phenomenon appears already in a nanometric sized wire, where

the conduction electrons meet ballistic transport and quantum nodes in
the transverse direction (Figure 4.30).
This quantum conductivity originates in a nanometric sized wire due
to the decreasing conductance with the decreasing size, resulting in the
characteristic stair-like conductance decreasing which demonstrates that
quantum conductivity is taking place. In comparison, in a normal size

metallic wire the conduction electrons have multiple classical collisions
through the wire.
Figure 4.30. The quantum conductivity in a nanometric sized wire (A) is associated to a
stair-like decreasing conductance (B), while in a classical metallic wire the conduction
electrons have multiple collisions through the wire.
An other example of quantum phenomenon is exemplified by

microtubule and tubulin (Hameroff et al., 2002), where tubulin can
undergo a conformational change from black to the white basis state
depending on the localization of electrons in its hydrophobic pocket. A
schematic representation of the superposed state is shown in (Figure
4.31).
Finally also single-electron phenomena were linked to the concept of
quantum devices and of quantum dots (Glazmann and Shekhter, 1989).
In particular, considering a ballistic model for the charge transport
through a dot, it was possible to demonstrate that the current through it
should be represented as a series of equidistant peaks whose positions
correspond to the steps in the coulomb staircase. Moreover, the
possibility of considering single-electron phenomena in a frame of a dot-
based system theory allows consideration of even semiconductor
nanoparticles as quantum dots, useful for single-electron junctions
(Averin et al., 1991).
Figure 4.31. Left: microtubule, a cylindrical lattice of tubulin protein molecules. Right:
Each tubulin molecule may occupy two classical conformations (top) or exist in quantum
superposition of both conformational states (bottom), with each conformation coupled to
position of a pair of electrons in an internal hydrophobic pocket. A tubulin may thus act
as a classical bit (top) or as a quantum bit, or ‘qubit’. The difference between the two
conformations of tubulin, as well as the size of the hydrophobic pocket, have been
exagerated for illustrative purposes.
The modeling of junctions based on these semiconductor quantum

dots reveals that their behavior in terms of single-electron phenomena
can result in current-voltage characteristics with differential negative
resistance regions (Gritsenko and Lazarev, 1989). This fact was
connected to the possibility of resonating tunneling through quantized
energy levels inside the dot (Guinea and García, 1990; Beenakker, et al.,
1991; Sumetskii, 1993; Groshev et al., 1991).
On the other hand, some work on the topic considers the presence of
negative differential resistance in the current-voltage characteristics and
the possibility of a coulomb staircase as different output of the very same
phenomenon and, therefore, has tried to consider both of them in the very
same conceptual frame (Beenakker, 1991; Stone et al., 1992; Prigodin et
al., 1993).
This approach seems to be successful; in fact, it was possible to see
models describing current-voltage curves presenting both stairs and
negative resistance along them (He and Dassarma, 1993; Carrara et al.,
1996). The first stand-alone room-temperature single electron junction
was made by depositing a semiconducting particle directly onto the tip of
a very sharp electrode, avoiding in this case the use of an STM
microscope, and it was possible to observe the coulomb staircase in such
a system (Facci et al., 1996).
In addition to the mainstream of element formation, several non
traditional technological approaches were carried out for the formation of
elements with nanometer sizes and their utilization for construction of
single-electron elements (Wilkins et al., 1989; Shónenberger et al.,
1992a; Dorogi et al., 1995; Erokhin et al., 1995a). Several possible
applications of the phenomenon were discussed. The easiest one was to
consider it an analog-digital transducer. In fact, continuous sweeping of
the voltage applied to the junction results in the digital output of the
current, providing, therefore, a fixed value of the current to the different
voltage intervals.
Moreover, it is possible to vary the unit step of the digitization, taking
a granule of different sizes. The next steps in the practical realization of
such a transducer will be in a synthesis of the granule between preformed
sharp metal electrodes. The electrodes can be prepared using the
selective etching technique of the thin and narrow metal strips deposited
onto insulating substrates. Several possible applications are proposed for
systems, using three electrodes. In this case, two of them with a granule
between them serve as analogs of the source and the drain in a field
effect transistor.
The third one, the analog of the gate electrode, serves for the
application of the electric field to the granule, which varies the character
of the current flow between the source and the drain. Apart from
transistor-like devices, single-electron junctions can also be useful for
sensor applications.
The simplest one is the monitoring of H2S-. Since the formation of
CdS nanogranules takes place when an initial cadmium arachidate layer
is exposed to this gas, we can expect the appearance of single-electron
conductivity only when it is present in the atmosphere.
4.2 Nanoenergetics Compatible with Environment
The recent upgrade in the new sources of renewable energy based on

Nanobiotechnology represent the core of this subchapter.
Photovoltaic cells based on both polymers and biopolymers, organic
batteries, hydrogen production and storage, and fuel cells represent most
likely (considering the insurmountable problems of nuclear energy) the
only possible solution to the dramatic consequences of Serra effect for
the human race that in 200 years has burned nearly all fossils being
accumulated in five millions years on the Earth planet.
4.2.1 Photovoltaic cells
Photovoltaic (PV) solar cells, which convert incident solar radiation

directly into electrical energy, today represent the most common power
source for Earth-orbiting spacecraft, such as the International Space
Station, where a “photovoltaic engineering tested” (PET) was actually
assembled on the express pallet.
The solid-state photovoltaic, based on gallium arsenide, indium
phosphide, or silicon, proves to be capable, even if to different extents
and with different performances, of operating in a reliable fashion at less
than the 10-KW low-power range typical of the missions orbiting the
Earth (Table 4.8); the electrical power generated over many orbital
cycles supports both the electrical loads and the recharge of batteries.
Sunlight is practically an inexhaustible energy source, and increasing
energy demand makes it a primary source of renewable energy. Sunlight
possesses a very high energy potential and is an ecologically pure and
easily accessible energy source.
The electrical power obtained from solar energy conversion is widely
used in spacecraft power supply systems (the latest very important
example is the International Space Station-ISS within the framework of
Italian and European Space Agencies) and in terrestrial applications to
supply autonomous customers with electrical power (portable equipment,
houses, automatic meteostations, etc.).
Table 4.8. Photovoltaic parameters of various tested materials. (Reprinted with the
Cell Efficiency Notes

Inorganic-based cells 14% Hydrogenated amorphous silicon
Single junction thin-film (a-SM); cadmium Telluride
polycrystalline cell (CdTe); copper indium diselenide
(CulnSe2)
Single-junction single 30%
crystal
Multijunction cells > 30%
Ga/As/CuInSe2 21.3% Year 1977/1988
GaAs/Si 31% 1988
AlGaAs/GaAs 24–28% 1988/1989
a-Si:WcuInSe2 15.6% 1988
a-Si:H/a-Si:Ge:H 13.6% 1989
GaInP2/GaAs 25% 1989
n-CdS/p-CdTe 15.8% 1993-heterojunction
Cr/chlorophyll-,affig 0.016% λ = 745 run
junction (monochrom. eff.)
The irregular incidence of sunlight on the Earth (daily and seasonal

variations) represents one of its disadvantages, together with its low
energy density. For these reasons, there is need to cover large areas with
expensive semi-conducting solar cells, and consequently, costs are
increased. Thus the electrical energy obtained in such a way is more
expensive than that from conventional methods. Although reduction of
pollutant emission is a key factor in the preservation of the environment
and subsequently the quality of the life itself, the increase in costs retards
the development of a large-scale solar power industry. Nowadays, solar
cells are based on inorganic semi-conducting materials, namely,
amorphous silicon (efficiency about 12%), multicrystalline silicon
(18%), and CdTe (16%), and yield an average energy cost of about $5
per watt.
Given the present scenario, one can state that the emerging field of
nanotechnology represents a new effort to exploit new materials as well
as new technologies in the development of efficient and low-cost solar
cells. In fact, the technological capabilities to manipulate matter under
controlled conditions in order to assemble complex supramolecular

structures within the range of 100 nm could lead to innovative devices
(nano-devices) based on unconventional photovoltaic materials, namely,
conducting polymers, fullerenes, biopolymers (photosensitive proteins),
and related composites. Among such techniques, the most promising
seems to be the Langmuir-Blodgett one.
Figure 4.32. Photoelectrochemical response of ITO/(PDDA/CuTsPc-capped TiO2)15 thin

films in an electrolyte of 0.1 mol dm-3 TBA TFB acetonitrile solution. The potential of
the working electrode was set at 0.0 V versus the Pt counterelectrode (Reprinted with the
permission from Ding et al., Ultrathin films of tetrasulfonated copper phthalocyanine-
capped titanium dioxide nanoparticles: fabrication, characterization and photovoltaic
effect, Journal of Colloid and Interface Science 290, pp. 166–171, © 2005, Elsevier).
As far as organic materials for photovoltaic applications, such as

conducting polymers (Figure 4.32), we note that present research is
focused on understanding the physicochemical phenomena that underline
the applicability of such new materials. Several research groups around
the world are trying to develop new photovoltaic cells based on
unconventional materials, particularly sunlight-converting solar cells
based on conducting polymers and on composites. Photo-excitation,
charge injection, and/or doping induce local electronic excitations
necessary for charge transport. Among them, only the doping process
(intercalation) is able to induce a permanent transition to a conductive
state. As far as the photo-excitation process is concerned, we note that
excited states produced by photon absorption, namely, excitons, have

high relatively binding energies and do not dissociate themselves to give
electrons and holes. Therefore, the entire process of exciton ionization is
not a promising method for the development of an organic PV device.
The correct energetic, which allows charge separation, can be provided
either by the interfaces between molecular semiconductors or with
electrodes.
4.2.1.1 Reaction centers-based
An alternative approach is the construction of photocell using

photosynthetic bacterial membranes. The work presents experimental
data, which allow making a conclusion about the possibility of
constructing such type of cells using biological materials. To optimize
performance we plan to try different types of LB deposition, namely
orientation by pressure, electric field and different types of RC: from
Rhodobacter sphaeroides, Rhodopseudomonas viridis and Chromatium
minutissimum. Next step will be the use of proteins instead of membrane
fragments to yield an increase in the efficiency of the light conversion.
The recent progress in research indicates that the disentanglement of
photosynthesis will give strong impulse to the application of solar cells.
Moreover, as far as the bioconversion of sunlight is concerned, it is
known that photosynthesis starts with a charge separation process in the
photosynthetic reaction centers (RCs): a photoactive bacterial protein.
Therefore, several experiments have been carried out to test the
possibility of the conversion of light to electrical energy by
photochemical cells (or simply photocells) containing such
photosynthetic bacterial proteins. The photocells developed have
different geometries, and therefore it is very hard to classify the
reliability of such devices. Nevertheless, the results indicated that the
efficiency of quantum energy conversion is practically 100% (all the
light energy was converted into the charge separation). Nevertheless, the
energy conversion efficiency of the realized photosensitive units
crucially depends on molecular orientation and is very difficult to
estimate, because articles usually contain incomplete information.
Usually, such proteins are immobilized by thin-film fabrication

techniques, such as self-assembly, electrical sedimentation, Langmuir-
Blodgett, polymer matrix, or gel entrapment. Among such techniques,
the most promising seems to be Langmuir-Blodgett. In fact, this
technique allows the protein molecules to be organized in an ordered LB
array. There are many publications on films of RCs. The films were
dually characterized from different points of view. The discovery of heat
and temporal stability had opened big possibilities in using the protein in
devices (Nicolini et al., 1993).
In the literature, Japanese authors (Yasuda et al., 1993) suggested the
making of a photo-device, using an RC LB film sandwiched between two
electrodes (one of them transparent). Photo-voltage registered in this
work was 4–5 mV for the film, which contained 44 layers. In our work
(Facci et al., 1998) we have shown that it is possible to adjust a tilt of RC
molecules in the layer by controlling the surface pressure. On the other
hand, for bR the possibility of increasing anisotropy by electric field
application was shown. We can hope that by applying our technique of
electric field-assisted deposition we will be able to increase this number
(according to existing estimations, there is only about 12% of prevalent
orientation with respect to the opposite one; with our technique, we can
hope to increase this anisotropy). The other possible way of increasing
the film anisotropy was suggested by Miyake et al. (1998) in LB8
conference. They voltage-biased the substrate during deposition with
respect to the water subphase. He reported that even in this case the
anisotropy of the film was improved. Thus, the films can be useful for
the construction of devices for converting light to electron energy,
working in the range of visible-near-IR spectrum (Nicolini, 1997).
4.2.1.2 Purple-membrane based
In recent years our understanding of the structure and function of several

biological systems has grown rapidly. The study of bacteriorhodopsin
(bR) protein and the elucidation of its function as a light-driven proton
pump represent one of the most interesting examples (Oesterhelt et al.,
1991; Brauchle et al., 1991; Birge, 1990).
BR is a light-transducing protein in the purple membrane (PM) of

Halobacterium Halobium.
Its features allow one to identify and design several potential
bioelectronics applications aimed at interfacing, integrating, or
substituting for the silicon-based microelectronics systems, as well as
developing molecular devices (Birge, 1992).
Figure 4.33. Photosignal measured for bR photo-induced current as function of time in

the structure as shown above (Reprinted with the permission from Nicolini et al.,
Supramolecular layer engineering for industrial nanotechnology, in Nano-surface
chemistry, pp. 141–212 © 2001a, Marcel Dekker/Taylor & Francis Group LTD).
BR is a notable exception as compared to the usual biological

molecules, being mechanically robust and chemically and functionally
stable in extreme conditions, such as high temperatures (Hampp, 1993,
Shen et al., 1993; Zeisel and Hampp, 1996), which usually represents
one of the key parameters of working conditions.
Furthermore, it possesses remarkable photonic and photovoltaic
properties, which have been exploited for molecular device construction.
For these reasons, bR has been adopted as a building block for a number
of experimental prototypes previously discussed (Birge, 1990; Oesterhelt

et al., 1991; Fukuzawa et al., 1996; Fukuzawa, 1994, Miyasaka et al.,
1991, Storrs et al., 1996; Maccioni et al., 1996; Chen and Birge, 1993),
and particularly for photovoltaic cells (Bertoncello et al., 2004; and
Figure 4.33).
Figure 4.34. Photovoltaic cell fabrication process. (Reprinted with the permission from
Bertoncello et al., Bacteriorhodopsin-based Langmuir-Schaefer films for solar energy
capture, IEEE Transactions on Nanobioscience 2, pp. 124–132 © 2003, IEEE).
The fabrication process was also optimized for the photovoltaic cell
giving watt per area and per weight rather reproducible and utilizable for
several application (mainly space), but in constant progress and further
optimization using a combination of Gratzel cell and nanocomposite
materials of inorganic, organic and biological origin and manufacturing
(Stura et al., in preparation).
4.2.2 Batteries
The batteries called “rocking-chair” systems are one of the most

promising electrochemical energy storage systems, and they have a
tremendous role in industrial applications of nanobiotechnology.
Mounting concern regarding the environmental impact of throwaway
technologies has caused a discernible shift away from primary batteries
and toward rechargeable systems.
The secondary batteries (“rechargeable systems”) have the advantage
of being able to operate for many charge cycles without significant loss
of performance. With technologies emerging today, an even higher
demand for rechargeable batteries with high specific energy and power is
expected.
Technological improvements, allowing to manipulate and investigate
the properties of nanomaterials, are nowadays changing the approach to
the energy storage and power supply vision. Modern nanoscale
techniques led the market in the realization of nanostructured inorganic
and organic materials increasing the efficiency of different devices, like
lithium batteries, one of the most promising energy storage elements,
obtaining everyday higher values of capacity, cyclability and
environmental resistance. Each part of the battery, the anode, the cathode
and the electrolyte, are here described analyzing the nanomaterials used
for their realization.
Energy electronic components are elements designed in a power
supply context, so oriented to the generation (conversion) or storage of
energy in electrical form. Nanotechnology can help in the realization of
materials with particular characteristics that make them suitable for the
use in the energy field, particularly for the photovoltaic power generation
and for the realization of parts of lithium-ion batteries, object of this
review. The constant development of technological electronic devices is
leading circuits to very small dimensions. The power supply must
provide enough energy for the proper functionality of these structures, so
it should be based on a material with high-energy storage/weight ratio.
Energy storage/volume ratio is also very important to obtain valid small
stand-alone devices. Lithium ion electrolytic cells for batteries are
nowadays considered the best dealing with these two characteristics

(Winter and Brodd, 2004).
A number of improvements of electrolytic lithium-ions cells for
hybrid organic batteries were studied. Each part of the system is object of
many studies, the cathode, the anode, and the electrolyte (Figure 4.35),
that can be realized in liquid, gel or solid form. Another important
characteristic to consider for developing a lithium ions battery is the cost
of the materials involved, and it is one of the reasons for the use of
polymers, spinels and other low cost materials.
Figure 4.35. Physical layout of the hybrid organic battery. 1: Negative current collector
(stainless steel). 2: Cathode 3: Electrolytic solution (LP30 pregnated porous membrane).
4: Anode (metallic lithium) 5: Positive current collector (stainless steel). 6: Hermetic
plastic container (Reprinted with the permission from Stura and Nicolini, New
nanomaterials for light weight lithium batteries, Analytica Chimica Acta 568, pp. 57–64
© 2006, Elsevier).
The overall effectiveness of different kinds of nanocomposite

batteries is given in Table 4.9. The demand for high performance of
lithium ion batteries generated strong incentives for the promotion of
first-rate basic studies in different materials sciences, surface science,
crystallography, spectroscopy, microscopy and electrochemistry.
Many worthy results have been achieved, among these the
development of novel, high capacity negative electrodes based on
amorphous silicon (Graetz et al., 2003), different kind of tin alloys (Kim
et al., 2003), nanoparticles based on transition metal oxides (Nazar and
Crosnier, 2004), inter-metallic compounds (Yin et al., 2004) and new
materials based on carbon (Li et al., 2003). Intensive efforts are now in
progress in order to develop new electrolyte solutions with non-

flammable solvents (Zhang et al., 2003), for similar safety reason also
salts such as Li-bi oxalato-borate (LiBOB) are topic of many worldwide
researches (Xu et al., 2002) and, to assure a safe operation, over charge
protection (Adachi et al., 1999).
Table 4.9. Overall effectiveness of different kinds of nanocomposite batteries (Reprinted

with the permission from Stura and Nicolini, New nanomaterials for light weight lithium
batteries, Analytica Chimica Acta 568, pp. 57–64 © 2006, Elsevier).
Battery Energy density: Energy density: Maximum Nominal

type (Wh kg-1) (Wh l-1) charge/discharge voltage
cycles
Ni-Cd 34 80 ~1000 1.2
Ni-H 54 64 ~5000 1.2
Pb acid 38 78 ~500 2.0
Li-ion 152 305 ~3000 3.3–4.0
Many efforts are spent to develop new materials for separators

(Bohm, 1999) and new solid-state electrolytes, both polymeric (Scrosati,
2002) and ceramic (Birke and Weppner, 1999).
There also are highly intensive efforts developing new cathode
materials with high redox potential (Wang et al., 2004), olivines
(Pasquadi et al., 2004), and LiMn2-xMxO4 spinel compounds.
The efficiency of different materials changes drastically between the
raw form and nanostructured form, the same molecules organized in
different spatial position (pellets, nanoparticles, nanotubes, high surface
ratio elements) can change their macroscopical behaviour and electrical
characteristics, because of their chemical and physical parameters (like
surface energy).
To understand all the details regarding the structure of these materials
and their surface chemistry other works are now in progress. The
correlation between these parameters and the electrochemical behavior of
electrode materials are still not completely defined.
The use of different analytical chemistry techniques such as neutron
diffraction (Kim and Chung, 2004a), X-ray absorption near edge
structure (Kim and Chung, 2004b), extended X-ray absorption fine
structure (Okada et al., 2003) and in situ techniques like Raman
spectroscopy (Dokko et al., 2003), infrared spectroscopy (Santner et al.,

2004), various kind of atomic force and tunnelling microscopies
(Aurbach et al., 2002), X-ray diffraction (Hatchard and Dahn, 2004) and
mass spectroscopy (Lanz and Novak, 2001) contribute to obtain clear
relation between the chemical-morphological aspect and their behavior.
The use of electrochemical impedance spectroscopy is also an important
tool for the characterization of batteries and fuel cells. This technique
yields quantitative information on a diverse range of processes including
the analysis of state of charge, study of reaction mechanisms, film
aestivation and corrosion processes.
4.2.2.1 Lithium ion batteries elements
The electrolytic cells used to test our electrodes (i.e., the cathode) are
typically assembled as shown in Figure 4.35.
An ABS plastic cylindrical box was used as container for the
electrochemical elements, avoiding the dripping of the electrolytic
solution. The anode is obtained from a foil of the material tested using a
punch with 0.7 cm2 circular head, the electrolyte is based on three disks
of porous material with a surface of 1 cm2 pregnated with the liquid
electrolyte, if a solution is used, or a mass of plasticized gel if a
polymeric electrolyte is used. The cathode is obtained using the same
punch used for the anode, piercing a foil of the amalgamated materials.
These three elements are put in close contact using the stainless steel
cylindrical current collectors. The last elements also served as blocking
objects to close the electrolytic cells.
4.2.2.2 The cathode
A considerable variety of cathode materials have been studied to develop

efficient elements with high energy density: metallo-phosphate, lithium
spinel materials, metallo-phosphates (LiFePO4, LiCoPO4, LiMnPO4 and
LiNiPO4) with olivine-type structure attracted a noticeable attention as
possible intercalation materials for the Li+ ion (Padhi et al., 1997;
Garcìa-Moreno et al., 2001; Yamada et al., 2001).
This interest is focused mostly on the lithium iron phosphate

(LiFePO4) because of the high discharge voltage of 3.6V versus metallic
lithium and the environmental compatibility of iron. Different
approaches for enhancing the electrochemical performance of this
compound were studied (Yamada et al., 2001; Huang et al., 2001;
Franger et al., 2002) in order to get small particle size and good
electronic contact between the particles of the sample. The mechanism of
lithium extraction–insertion from LiFePO4 is properly proved with ex-
situ and in-situ X ray diffraction and electrochemical techniques (Padhi
et al., 1997; Andersson et al., 2000; Takahashi et al., 2002). As the
lithium extraction from LiFePO4 proceeds for electrical field reasons, a
new material, FePO4, is formed. The dual phase characteristic of the
electrochemical reaction seems to provoke low rate capability of this
compound due to the hindered lithium diffusion through the interface
between LiFePO4 and FePO4 (Padhi et al., 1997). Differently from what
happens with FePO4 with olivine-type structure, the existence of CoPO4
and MnPO4 crystallizing in the same space group is not proved yet.
However, a two-phase mechanism of lithium deinsertion from LiMnPO4
and LiCoPO4 seems to take place also in this case (Li, et al., 2002;
Delacourt et al., 2004; Amine et al., 2000; Okada et al., 2001). Delacourt
et al. (2004) showed that the electrochemical deintercalation of the Li+
ion from LiMnPO4, prepared by a new precipitation mean, leads to the
disappearance of LiMnPO4 and to the formation of the new material with
the same structure, but with lower cell volume, probably is MnPO4.
Bramnik et al. (2004) showed that LiCoPO4 samples prepared by solid-
state reaction at high temperature gives unsatisfactory electrochemical
performance. The reversibility of lithium intercalation and
deintercalation from LiCoPO4 was improved by an artificial method
based on the precursor NH4CoPO4·H2O (Lloris et al., 2002), even if the
cyclability of the material is not good. Diffraction patterns of LixCoPO4
sample charged to x = 0.05 (from the electrochemical data) by refining a
two-phase model were analyzed (Bramnik et al., 2004). The difference in
the cell volumes for both iso-structural phases was found to be much
lower in comparison with the data reported for manganese metallo-
phosphate. Another interesting material for batteries cathodes is
LiMn2O4, that attracted significant interest mostly because it has 4Vas
voltage versus metallic lithium. Its reversible capacity (110–120mAh g-1)

and its characteristics are similar to the ones of the currently used
LiCoO2. The manganese spinel material is cheaper and more
environmentally compatible, and undergoes a phase transition at 290 K,
transforming itself from a cubic phase (at high temperature) to a
orthorhombic phase (at low-temperature). This transition takes place for
the critical concentration of Mn3+ ions (Jahn-Teller ions). This is due to
the presence of Mn3+ ions, and this transition is probably responsible of
the limited cyclability (Tarascon et al., 1994; Shimakawa et al., 1997).
LixMyMn2−yO4 (where M is Al, Mg, Ti, V, Cr, Fe, Co, Ni, Cu or Zn)
spinels are also currently investigated as possible cathode materials (de
Koch et al., 1998; Shao-Horn et al., 2001; Kumagai et al., 1997; Iwata et
al., 1999; Thirunakaran et al., 2001; Tarascon et al., 1991) and the
results look encouraging. These studies, however, are limited to
determining lattice parameters and electrochemical characteristics. For
different dopants, the charging curve drastically changes its shape,
suggesting a variation in the electronic structure of the manganese spinel.
At this moment a very few research results on the electronic transport of
doped manganese spinel are available, it is only known that its value of
conductivity is low (10-4 S/cm) because of its small polaron mechanism
(Marzec et al., 2002). Molenda et al. (1999, 2003) and Swierczek et al.
2003) noticed that the polaron mechanism of charge transport, in the
manganese spinel is very stable.
Recent results in the cathode research introduced LiNi0.5Mn0.5O2,
providing over 30 cycles of charge/discharge with a capacity of 200 mAh
g-1 (Makimura et al., 2003). Additional research has shown that the redox
center is the divalent nickel, which becomes tetravalent due to the
oxidation occurring during charging, and is then reduced to divalent
nickel again during discharging at a 4V (Yoon et al., 2003; Nakano et
al., 2003; Johnson et al., 2003). Manganese remains tetravalent during
cycles at the 4V plateau, and the absence of trivalent manganese
contributes to its structural stability. Another characteristic of
LiCo0.5Mn0.5O2 is its first charge and discharge capacities of respectively
220 mAh g-1 and 125 mAh g-1, that makes it suitable for non
rechargeable batteries too (Tsuda et al., 2005).
In the last ten years there is a particular attention to TiO2

nanoparticles and their electronic properties (as shown earlier for
photovoltaic cells). This material can attract interest for the realization of
low voltage batteries, since its discharge voltage is 1.8 V versus Li/Li+.
However the insertion of 1 Li per TiO2 unit corresponds to a capacity of
335 mAh g-1. For the best results in the realization of cathodes for
lithium batteries, 20 nm nanoparticles are normally used, since it has
been reported that a sol-gel sample prepared starting with these
nanoparticles undergoes an optimal partial phase transformation around
600 °C that generates a favourable material with good electrochemical
characteristics. The insertion coefficient in anatase (TiO2) is usually
close to 0.5 (Kavan et al., 1996), making these nanoparticles interesting
materials for batteries designed for particular purposes.
4.2.2.3 The anode
Carbon-based materials are currently commercially used as anode

materials due to the flat charge and discharge plateau and excellent
cycling stability.
However, their theoretical maximum capacity is limited to 372 mAh
-1
g , corresponding to the formation of LiC6 (Fong and Sacker, 1990).
Since the introduction of tin-based oxide composite by Fuji Photo Film
Celltec in early 1997, great interest has been turned to metal, inter-
metallic compounds, alloy anodes due to their extremely larger capacity
compared to those of carbon-based materials. Also organic salts and
polymers attracted interest lately, for the good mechanical and
technological properties of organic matter. In the new battery technology
there is a renewed interest in metal alloys and inter-metallic compounds
for replacing graphitic carbon as the anode of choice in lithium-ion
batteries.
The lithiation voltage of these materials is enough positive versus
metallic lithium to minimize lithium-plating problems (occurring in case
of overcharge). Furthermore, these materials offer a higher volumetric
capacity than graphite. For example, the theoretical volumetric capacity
of InSb is 1904 mAh cm-3 (assuming a constant electrode density of 5.6 g
cm-3 throughout charge and discharge) compared with a theoretical 818
mAh cm-3 for graphite (ρ =2.2 g cm-3). Two reactions can happen
between inter-metallic compounds and lithium: insertion of lithium with
no extrusion of metal atoms from the host structure, or insertion of
lithium into the structure. For the first reaction typical materials are
Cu6Sn5 and MnSb that, on lithiation, form Li2CuSn (Kepler et al., 1999)
and LiMnSb (Fransson et al., 2003), respectively. In the second case, the
inter-metallic compound can be comprised entirely of elements that react
with lithium, such as in SnSb (Rom et al., 2001), InSb (Tostmann et al.,
2002) or Ag3Sb (Vaughey et al., 2003) when discrete LixSn, LixSb,
LixIn and LixAg phases are formed. In other cases, the intermetallic
compound can consist of two elements, and only one of these reacts with
lithium, as in FeSn2 (Mao et al., 1999a), Cu2Sb (Fransson et al., 2001)
and CoSb3 (Alcantara et al., 1999) in which case the LixSn or LixSb
phases are cycled within electrochemically inactive Fe, Cu or Co metal
matrixes (Sarakonsri et al., 2005).
Although alloys have much higher capacities than that of graphite,
they undergo severe volume expansion/contraction and pulverization.
The effect of these events limits the lifecycle of the anode and of the
whole battery. The performance of an alloy anode can be improved if the
active alloy is supported by inactive components, which provide
structural stability during cycling (Mao et al., 1999b; Beaulieu et al.,
2000).
Sn-Cu intermetallic compounds have been suggested as promising
alternative anode materials (Thackeray et al., 2002; Wachtler et al.,
2001; Winter and Besenhard, 1999). Kim et al. (2002) prepared nano-
sized Cu6Sn5 electrodes by chemical reduction and found that their
cyclability was significantly enhanced as compared with the same
material prepared by sintering or mechanical alloying. Even though, the
above preparation methods are not suitable for large-scale, low cost
production of alloy electrode materials (Pu et al., 2005).
Among alloys, which can be potential anode materials for secondary
Li-ion batteries, the Sb-based intermetallic compounds received much
interest lately. Alcantara et al. (1999), first reported CoSb3 as possible
anode materials for Lithium ion batteries. Thereafter, many Sb-based
intermetallic compounds, such as CrSb2 (Fernandez-Madrigal et al.,
2001), TiSb2 (Larcher et al., 2000), Cu2Sb (Fransson et al., 2001a),
Mn2Sb (Fransson et al., 2003), Co1–2yFeyNiySb3 (Monconduit et al.,

2002), SnSb (Besenhard et al., 1999) etc., were investigated. Although
these compounds show slight lower capacity than pure Sb (660 mAh g-1),
they show improved cycling behavior. However, the long-term
cyclability cannot meet the requirement for the practical application of
these materials as anode in commercial Li-ion batteries. The main
drawback of these compounds is the rapid capacity fade after repeated
cycling, resulting from the large volume changes (expansion and
compression) during the charge and discharge phases (Xie et al., 2005).
Figure 4.36. Comparison among different anode nanomaterials: density versus capacity
(mAh g-1) (Reprinted with the permission from Stura and Nicolini, New nanomaterials
for light weight lithium batteries, Analytica Chimica Acta 568, pp. 57–64 © 2006,
Elsevier).
The electrochemical application of organic ionic liquids (room

temperature molten salts) that show attractive properties such as high
ionic conductivity (~10-3 S cm-1), wide electrochemical window (wider
than 3.0 V), and good thermal and chemical stability, has been explored
in the fields of photo-electrochemical cells (Nazeeruddin et al., 1993);
Papageorgiou et al., 1996), lithium secondary batteries (Fuller et al.,
1997,1998; Koch et al., 1996) and electrochemical capacitors
(Nanjundiah et al., 1997). Among organic materials, dialkylimidazolium
based ionic liquids with the weakly complexing anions (e.g. PF6-, BF4-,
or CF3SO3-, (CF3SO2)2N-) seem to be the most stable and conductive to
date (Koel, 2000; Hagiwara and Ito, 2000; Bonhote et al., 1996;
McFarlane et al., 2000). The 1-butyl-3-methylimidazole based ionic
liquids with PF6- (Fuller et al., 1998) and (CF3SO2)2N- (Bonhote et al.,
1996) have high hydrophobicity besides other good properties (Fuller et
al., 1998). These materials are particularly useful for the realization of
anodes for specific applications like lithium/seawater batteries (Zhang et
al., 2005). In Figure 4.36 is given the comparison among the anode
nanomaterials: density versus capacity (mAh g-1). This figure gives an
idea of how certain materials, universally considered “good” in reality
need large volumes to reach few milligrams, while others considered
“mediocre” need instead small volumes to obtain large masses.
4.2.2.4 The electrolyte
Actually most industrial electrolytes are mixtures of ethylenecarbonate,

diethylcarbonate and dimethylcarbonate with lithium
hexafluorophosphate (LiPF6) as salt. This type of electrolyte permits a
great number of charge–discharge cycles without noticeable loss in
capacity but the search of new electrolytes with higher thermal stability
is of great importance (Botte et al., 2001; Zhang et al., 1998; von Sacken
et al., 1994; Dahn et al., 1994; Du Pasquier et al., 1998; Gee and Laman
1993; Hong et al., 1998; Richard and Dahn, 1999; Ohta et al., 1995;
Roth, 1999; Kumai et al., 1999).
Among polymeric electrolytes, poly(ethylene oxide)-lithium salt
complexes are promising candidates as electrolytes for lithium polymer
battery applications (Armand et al., 1989; Gray, 1997; Gray and
Armand, 2000; Lightfoot et al., 1993; Vincent and Scrosati, 1993). Large
research efforts were spent for the development of poly(ethylene oxide)
electrolyte solutions allowing to combine high conductivity, good
interfacial stability with lithium metal anode and good mechanical
properties (Wieczorek et al., 1989; Borghini et al., 1995; Appetecchi et
al., 2000). A common approach is the use of a lithium salt having a very
large counter-ion, able to interfere with the crystallization process of the
polymer chains (Appetecchi et al., 2001a; Rossi Albertini et al., 1997),
promoting amorphous regions and increasing the lithium ion transport

across the polymeric electrolyte (Gray, 1997; Lascaud et al., 1994;
Feuillade and Perche, 1975). Following this approach, Appetecchi et al.
(2001b) have shown that the use of a lithium salt whit large anion,
N(SO2CF2CF3)2-, enhances the conductivity of poly(ethylene oxide)
based polymer electrolytes. In addition, these polymer electrolytes
develop a very stable interface with lithium metal anode both under rest
conditions and current flow (Appetecchi and Passerini, 2000).
Room temperature ionic liquids (RTIL) can also be used as safe
electrolytes in electrochemical applications owing to their wide thermal
stability, wide liquid-phase range, non-flammability and very low vapour
pressure (Hu et al., 2004; Ngo et al., 2000). These RTIL are composed of
a cation like quaternary ammonium (Sun et al., 1998), alkylpyridinium
(Chum and Osteryoung, 1981), alkylpyrrolidinium (MacFarlane et al.,
1999), alkylpyrazolium (Caja et al., 1999), alkyltriazolium (Vestergaard,
et al., 1993), alkylphosphonium (Holbery and Seddon, 1999) and
alkylimidazolium (Blanchard et al., 1999), combined with a variety of
large anions having a delocalized charge (PF6-, BF4-). All RTILs show a
high viscosity and therefore a relatively low conductivity. In order to
decrease the viscosity and increase the conductivity, aprotic dipolar
organic solvent may be added to RTIL, experimental results obtained by
Chagnes et al. (2005) using butyrolactone confirm this.
Recently, to avoid liquid electrolytes and the related hydraulic
insulation issues, some efforts were spent in the research of solid state or
gel electrolytes. Ion-conducting polymer electrolytes have contributed to
the development of lithium battery technology by replacing the liquid
electrolyte and thereby enabling the fabrication of flexible, compact, and
laminated solid-state structures free from leaks of the electrolyte (Croce,
et al., 1998). Among these, solvent free polymer electrolytes formed by
complexes of a lithium salt with a polyether such as poly(ethylene oxide)
received considerable attention for their advantages in terms of the ease
of fabrication, flexibility in dimensions, good mechanical properties,
safety features, and good stability at the lithium interface (Ulrich et al.,
2002; Gadjourova et al., 2001; Kim, 1998; Appetecchi et al., 1998).
However, their low ionic conductivities have been the reason for them
not being used in practical applications in rechargeable lithium batteries
that require a value of above 10-4 S cm-1 at room temperature (Jeon et al.,
2005). It is only above the melting temperature of crystalline
poly(ehtylene oxide) – lithium salt complexes (~60 °C) that significative
conductivity values (σ >10-4 S/cm) are measured (Fauteux et al., 1995).
Many efforts aimed to the lowering of operation temperatures of
poly(ehtylene oxide) – lithium salt systems to the room temperature
region have focused on the development of copolymerization (Fauteux et
al., 1995; Soo et al., 1999; Allcock et al., 1986; Abraham et al., 1988;
Tonge and Shriver, 1987; Xia and Smid, 1984; Cowie et al., 1985a,b;
Gray et al., 1988) or cross-linking (Maccallum et al., 1984; Watanabe et
al., 1986; Andrei et al., 1994; Killis et al., 1982; Cheradame et al., 1987)
strategies and the use of suitable plasticizers (Gray, 1991; Abraham,
1993; Kelly et al., 1985) to create completely amorphous systems,
therefore with enhanced conductivity. Incorporation of inorganic
particles in the polymer matrix to obtain mechanical stability (Weston
and Steele, 1982), and enhance interfacial properties (Capuano et al.,
1991) and conductivity by suppressing crystallization of the PEO host
has also been investigated (Croce et al., 1998, 1999; Wieczorek et al.,
1995; Krawiec et al., 1995; Best et al., 1999; Capiglia et al., 1999).
An alternative strategy for creating polymer electrolyte systems with
improved electrical and mechanical properties is through fabrication of
polymer silicate nanocomposites. These materials are a class of
compounds in which nanoscale clay particles are molecularly dispersed
within a polymeric matrix (Yano et al., 1993; Messersmith and Stupp,
1992; Kojima et al., 1993; Krishnamoorti et al., 1996; Shi et al., 1996;
Wang and Pinnavaia, 1998a,b). Recent commercial interest in these
nanocomposites is derived from the fact that they show significant
increases in tensile strength (Kojima et al., 1993), heat resistance
(Messersmith and Stupp, 1992) and solvent resistance (Burnside and
Giannelis, 1995) as well as decreases in gas permeability when compared
with the bulk polymer (Messersmith and Stupp, 1992). These
characteristics are useful also developing lithium batteries.
By now, lithium ions batteries are the most used devices in almost
any kind of mobile devices, both for industrial and for commercial
applications, overcoming most part of the issues typical of the previous
technologies in batteries. In the last years, engineered polymers were
implemented in the realization of lithium batteries, reducing the

complexive weight of the power supply element providing acceptable
values of capacity. Most industries included in their next future research
plans studies of better nanomaterials for the application in lithium ion
batteries.
Particular attention is oriented nowadays to organic cathodes, not for
their capacity values but for the low cost of production, ease of synthesis
and good technological properties like material modelling and solubility
in organic solvents.
Figure 4.37. Chemical intercalation of the lithium ions in the polymer. (Reprinted with
the permission from Stura and Nicolini, New nanomaterials for light weight lithium
batteries, Analytica Chimica Acta 568, pp. 57–64 © 2006, Elsevier).
Our research group obtained acceptable values (130 mAh/g-1) for a

nanocomposite material based on poly(ortho-anisidine) and titanium
dioxide nanoparticles, taking benefit from the optimization of the
polymer chain occurring in presence of TiO2 nanoparticles. This
nanocomposite showed a particularly higher capacity value in the first
charge and discharge cycle than in the following ones. When Li+ ions
reach the polymeric matrix, they dope the polymer taking the place
usually occupied by the H+, tied to the imines group. In this operation, a
charge transfer occurs, and the difference of potential between the anode
and the cathode changes.
The opposite process happens when the Li+ ions leave the polymeric
material and are released into the electrolytic solution, moving charge in
the opposite direction and undoping the organic material (Stura et al.,
2004). Cathodes based on the above polymeric material and

nanocomposite material (polyorthoanisidine with titanium dioxide
nanoparticles) are typically intended for a low power and long life power
supply, and tested in classical lithium-ion rechargeable batteries.
The results are promising considering that multiple cycles of charge-
discharge tests result in sufficient performance of the synthesized
materials that after 20 cycles tend reach a constant value of capacity, and
can be readily explained in terms of the motion of lithium ions (Li+) in
the electrolyte, undergoing, when the electrical field is present (in the
charge phase), an intercalation process in the polymer matrix, thereby
doping the polymer similarly for what happens when the polymer is in
presence of acid agents. A schematic of this process is presented in
Figure 4.37. The present status of new materials by organic
nanotechnology and their applications to molecular electronics has been
recently overviewed (Nicolini et al., 2005), with respect to the
development of organic nanotechnology capable to yield new materials
for a variety of technological applications. Particular emphasis has been
placed on what has been accomplished in our laboratory in the last few
years (Narizzano and Nicolini, 2005; Valentini et al., 2004a; Carrara et
al., 2005; Stura et al., 2002), whereby can be found in earlier papers the
details on the supramolecular layer engineering and its application to
industrial nanotechnology (Nicolini et al., 2001a) and to molecular
electronics (Nicolini, 1996b; Facci et al., 1996). As previously pointed
out (Nicolini et al., 2005) the major drawback of the polymeric materials
is the multi-step synthesis required for the functionalization and the
stringent process requirements of the condensation polymerization. It
was then our efforts in the last few years therefore to find shorter
synthetic routes to process the polymers with predictable absorption
wavelengths of light. Several types of polymer poly(p-
phenylenevinylene) (PPV), derivatives have been synthesised in our
laboratory, namely poly(2-methoxy-5-(2’-ethyl)hexyloxy-p-
phenylenevinylene) (MEHPPV), whereby the Gilch route has been
modified in order to increase the processability for specific device
application.
The data here presented point to the successful engineering of organic
nanotechnology-based materials and using polymer chemistry having
potential industrial relevance in the area of lightweight lithium batteries.

Figure 4.38 shows indeed the significant cyclability of present
nanomaterials for lithium ion batteries, grouped in four large families.
Figure 4.38. Ciclability of nanomaterials for lithium ion batteries (Reprinted with the
permission from Stura and Nicolini, New nanomaterials for light weight lithium batteries,
Analytica Chimica Acta 568, 57–64 © 2006, Elsevier).
We may then conclude that, although the work is still in progress in

order to further optimize the parameters and to evaluate in more needed
details, case by case, the optimal implementation of this technology
within the required cost effectiveness, and the reproducibility within an
highly competitive industrial context, it is conservative to conclude that
light weight lithium methodology represents a promising general purpose
tool for the design and production of new batteries.
4.2.3 Hydrogen storage and fuel cells
The main impediment to the use of hydrogen as a transportation fuel is

the lack of a suitable storage system (Figure 4.39).
Compressed-gas storage is bulky and requires the use of high-strength
containers. Liquid storage of hydrogen requires temperatures of 20 K and
efficient insulation. Solid-state, storage offers the advantage of safer and
more efficient handling of hydrogen, but promises at most 7% hydrogen

by weight and more typically 2%.
Figure 4.39. Hydrogen storage (Reprinted with the permission from Stura et al.,
Hydrogen storage as stabilization for wind power: completely clean system for insulated
power generation, Chemical Engineering Transactions 4, pp. 317–323 © 2004, AIDIC,
http://www.aidic.it).
Various materials, such as palladium (Pd), palladium alloy,

palladium-ruthenium alloys, nanocrystalline FeTi, mechanically alloyed
amorphous Ni1-x, Zrx, alloys, carbon nanofibers, and carbon nanotubes,
are employed for the storage of hydrogen. There have been reports that
certain carbon graphite nanofibers are able to absorb and retain 67 wt%
hydrogen gas at ambient temperature and moderate pressure, i.e., up to
23 standard liters (2 grams of hydrogen per gram of carbon at 50–150
bars). The lowest hydrogen adsorption reported for any graphite fiber
microstructure was shown to be 11 wt%. Approximately 90% of the
adsorbed hydrogen can be desorbed at ambient temperature by reducing
the pressure, while the balance is desorbed upon heating.
Such claims are especially noteworthy, given that up to this point the
typical best value of hydrogen adsorption in carbon materials has been
4%, or 0.5 H/C. A large number of research Institutes and various
companies are involved in the storage of hydrogen and production of full
cells based on hydrogen: the Electric Power Research Institute, the
American Gas Association, the Gas Research Institute, International Fuel
Cells, Energy Partners, Ballard Power Systems, the Energy Research
Corporation, MC Power, Westinghouse Electric Corp, Daimler-Benz,
BMW, Volkswagen, Volvo, Renault, Peugeot, Siemens, Toyota, Honda,
Toshiba, Mitsubishi, Fuji, and Sanyo. Fuel cell-powered cars (Figure
4.40B) and field emission by carbon nanotubes in spacecraft (Figure
4.40C) are being researched and tested.
Figure 4.40. Chemical vapor deposition for carbon nanotubes manufacturing (A) in
automotive fuel cell (B) and spacecraft field emitter (C).
Hydrogen is excellent for storage and would make certain sources

more feasible. This would open doors to many alternative resources and
begin to shift our use away from fossil fuels. Still, electrolysis and
cryogenic cooling are both very expensive. Hydrogen storage is
economically viable only when it is sent over very long distances, where
piping hydrogen would be more efficient then sending electricity, or

when a storage system is necessary, as in the case of solar or wind power
(Stura et al., 2004). The use of Pd has revealed the restriction in storage
capability due to the change in structures upon a few cycles of the
adsorption-desorption process. The Pd becomes disordered after a few
cycles of adsorption. The Pd-Ru structure remained almost unaltered
after cycling, but the disadvantage could be that the efficiency of
adsorption decreases during alloy formation. Several graphite
nanostructures were prepared using Fe-Cu catalysts of different
compositions, in order to generate a range of fiber sizes and
morphologies. The hydrogen desorption measured from these materials
was found to be less than the 0.01 R/C atom, compared to the other
forms of carbons. The hydrogen exposed in the metal alloy Ni4-nZrn, has
shown that hydrogen resides in Ni4-nZrn (n = 4, 3, 2) tetrahedral
interstitial sites, with a maximum hydrogen ratio of 1.9. Carbon
adsorption techniques rely on the affinity of carbon and hydrogen atoms.
Hydrogen is pumped into a container with a substrate of fine carbon
particles, where molecular forces hold it. This method is about as
efficient as metal hydride technology but is much improved at low
temperatures, where the distinction between liquid hydrogen and
chemical bonding needs to be considered. One of the most exciting
advances recently has been the announcement of carbon nanofiber and
carbon nanotube technologies. There is also the claim that up to 10 wt%
was achieved for hydrogen storage in single-wall nanotubes. Owing to
the potential importance of new materials with high hydrogen storage
capacity for the worldwide energy economy, transportation systems, and
interplanetary propulsion systems, carbon nanotubes could play an
important role in hydrogen storage.
Iijima (1991) has focused much attention on both fundamental and
applied research on carbon nanotubes (see CVD in Figure 4.40A) since
the discovery of multiwall carbon nanotubes (MWNTs) in 1991. In
particular, recent progress in research on the properties of single-wall
carbon nanotubes (SWNTs), such as their atomic structure and electronic
properties, hydrogen storage properties, mechanical properties, and
property enhancement through nanotube modification, has been
outstanding, due mainly to the availability of sufficient quantities of
SWNTs that can be obtained using the pulsed laser vaporization method
and the electric are technique. It has been both predicted theoretically
and demonstrated experimentally that SWNTs have many interesting
properties. Pores of molecular dimensions can adsorb large quantities of
gases, owing to the enhanced density of the adsorbed material inside the
pores, a consequence of the attractive potential of the pore wall. Dillon et
al. (1997) have shown that a gas can condense to high density inside
narrow SWNTs. Simonyan et al. (1999) described the adsorption of
molecular hydrogen gas onto charged single-wall nanotubes by grand
canonical Monte Carlo computer simulation. The present availability of
various fullerene structures points up a large gap in the intermediate size
range between small, highly tangled ropes of nanotubes that are currently
available in short lengths. Recently, laser vaporization and electric arc
methods have best for even for obtaining a continuous process for
SWNT production on a commercial scale. Therefore, from an
applications standpoint, emphasis is given to the production of high-
purity, high-yield, low-cost, large-scale, and easily handled SWNTs for
the storage of hydrogen. Recently, a novel method for synthesizing
SWNTs reported the catalytic hydrocarbon decomposition method, in
which benzene is catalytically decomposed at 1100–1200°C, yielding
SWNTs that are similar, on a nanometer scale, to those obtained by laser
vaporization and electric-arc techniques. This growth method allows
lower growth temperatures, permits semi-continuous or continuous
preparation, and produces a large quantity of SWNTs at relatively high
purity and low cost. However, subsequent experiments showed that the
ends of the tubes remained open during the growth process, with highly
reactive dangling bonds located around the tube ends.
In the near future, the possible synthesis of nanotubes with solid-gas
potential will be more favorable to adsorption. The effect of hydrogen
overpressure on the stability of adsorbed H2 needs to be verified in the
near future. The high-purity nanotube produced by laser vaporization,
catalytic decomposition, or other techniques as chemical vapor
deposition (Figure 4.40A) should be investigated. It is noteworthy that
the synthesis of the SWNT with defined diameters and distances between
the walls is difficult to perform at present, but future synthesis routes will
allow more hydrogen adsorption in the SWNT. Some theoretical
calculations, such as Monte Carlo simulation, were performed for the

adsorption of hydrogen with carbon nanotubes, but the real mechanisms
of adsorption and desorption are still unknown. Control of these
parameters, coupled with improvements in production, purification, and
alignment of SWNTs, may lead to a new technology for hydrogen
storage.
4.3 Nanobiocatalysis
The Langmuir-Blodgett (LB) technique was successfully applied for the

deposition of thin protein layers (Langmuir and Schaefer, 1938; Tiede,
1985); Lvov et al., 1990). LB organization of enzymes in film of
different number of monolayers (see Figure 4.41 for the GST enzyme)
not only preserved the structure and functionality of the molecules, but
also resulted in the appearance of new, useful properties, such as
enhanced thermal stability as shown earlier in this Volume and by
Nicolini et al. (1993) and Erokhin et al. (1995).
The enzymatic activity of GST was evaluated spectrophotometrically
following the conjugation of glutathione (GSH) thiol group to 1-
chloro2,4-dinitrobenzene (CDNB) at a wavelength of 340 run (Habig et
al., 1974) by a double-beam spectrophotometer (Jasco 7800). The GSH
and CDNB concentrations were 2.5 mM and 0.5 mM, respectively. Ten
covered spheres were placed into the cuvette. The diffusion effects
(Antolini et al., 1995b) were avoided by carrying out the measurements
under continuous stirring with a magnetic microstirrer (Bioblock
scientific) at a speed of 600 rpm.
The deposition procedure described earlier allows one to obtain
protein films chemically bound to the activated surface of spherical glass
particles. Subsequent compression of preformed protein monolayer with
these particles permitted to coverage of the particle area that initially has
not come in contact with the monolayer. Even if such a procedure does
not initially result in deposition of strictly one monolayer, this fact does
not seem to be critical, because only the monolayer chemically attached
to the surface remains after washing as can be seen from the rather
constant functional activity remaining with increasing number of layers
(Figure 4.41). Indeed, whenever the single protein monolayer is properly

formed by Langmuir-Blodgett technique at the saturating surface
pressure (see earlier sections), the enzymatic functional activity appears
nearly independent of the number of layer as proven for GST (Antolini et
al., 1995b) and alkaline phosphatase (Petrigliano et al., 1996) enzymes.
Figure 4.41. Dependence of GST activity after washing as function of the number of
monolayers. For each point is given the error (for a confidential level of 95%). The
reaction volume is 2 mL. (Reprinted with the permission from Antolini et al., Heat-stable
Langmuir-Blodgett film of glutathione-S-transferase, Langmuir 11, pp. 2719–2725 ©
1995b, American Chemical Society).
Enhanced thermal stability enlarges the areas of application of protein

films. In particular it might be possible to improve the yield of reactors in
biotechnological processes based on enzymatic catalysis, by increasing
the temperature of the reaction and using enzymes deposited by the LB
technique. Nevertheless, a major technical difficulty is that enzyme films
must be deposited on suitable supports, such as small spheres, in order to
increase the number of enzyme molecules involved in the process, thus
providing a better performance of the reactor. An increased surface-to-
volume ratio in the case of spheres will increase the number of enzyme
molecules in a fixed reactor volume. Moreover, since the major part of
known enzymatic reactions is carried out in liquid phase, protein
molecules must be attached chemically to the sphere surface in order to
prevent their detachment during operation.
The aim of the work was the development of a technique to deposit

enzyme LB films on the surface of small glass spheres and to test the
enzymatic activity of such samples before and after thermal treatment.
The experiments were carried out with two enzymes, urease and
glutathione-S-transferase (GST). Urease catalyses the hydrolysis of urea,
while GST is an enzyme that catalyzes the reduction of compounds such
as alkylants with a nucleophilic addition of the thiol of glutathione to
electrophilic acceptors (e.g., aryl and alkyl halides, quinones, organic
peroxides) (Pickett and Lu, 1989). Both enzymes were chosen, since
their activity can easily be tested by spectrophotometric measurements.
The LB technique was chosen for covering the spheres because it was
shown to provide enhanced thermal stability of many types of proteins in
deposited layers (Nicolini et al., 1993; Erokhin et al., 1995, 1995a),
which no other technique is able to achieve. Since only the upper protein
layer is involved in the catalytic activity, no special attention was paid to
check whether the deposited layer is a monolayer or multilayer.
However, the samples were thoroughly washed to remove protein
molecules not bound covalently to the sphere surface, since during the
functional test these molecules could contribute to the measured apparent
catalytic activity. Borosilicate glass spheres with a diameter of 2 mm
were used as substrates for the deposition.
The surface of the spheres was activated in the following way.
Spheres were treated with boiling chloroform, rinsed on a glass filter,
and dried under nitrogen, to be subsequently silanized with
3-glycidoxypropyl trimethoxysilane following the technique proposed by
Malmquist and Olofsson (1989). Silanization of the spheres was
performed in nitrogen flux in order to prevent reciprocal attachment of
spheres and to activate their surface homogeneously.
The essential steps of the deposition procedure, which are the same
for both enzymes, are illustrated in Figure 4.42. The protein solution was
spread over the water subphase, and the monolayer was compressed up
to 25 mN/m. Activated spheres were distributed over the monolayer in
the following way: A plate with spheres over it was moved along the
monolayer, while the weak nitrogen flow was used for transferring the
spheres from the plate to the layer. It is important to have the plate in
close vicinity to the water surface in order to keep all the particles
floating over the monolayer. The spheres were kept at the surface for 30
minutes in order to provide chemical linking of the monolayer to
activated surface. After this time the feedback system was switched off
and the Wilhemy plate was removed from the water. The layer with
particles was compressed until the minimum area (20 cm2), which
corresponds to the collapse of the monolayer, was reached. Even though
this compression yields a multilayer film, such action seems to be
necessary, since otherwise only half of the sphere surface was covered
with protein monolayer, while compression induced the motion both of
spheres and the monolayer, covering other regions of the spheres. The
spheres were collected, washed with substrate buffer in order to remove
parts of the monolayer not attached chemically to the sphere surface, and
dried.
Figure 4.42. Urease activity test at different temperature (Reprinted with the permission
from Nicolini, Heat-proof enzymes by Langmuir-Blodgett technique, Annals New York
Academy of Science 799, pp. 297–311 © 1996, Blackwell Publishing).
The activity of enzymes in the film was estimated in the following

way: In order to test the activity of urease, we utilized a calorimetric
assay based on urea hydrolysis; the enzymatic reaction was followed at
590 nm, the suitable wavelength for bromcresol purple (Chandler et al.,
1982). Urea concentration was 1.67; ts 10-2 M. From the results of the
urease activity test summarized it is clear that the deposition procedure
preserved to a certain extent the enzyme catalytic activity. Heating the
sample before testing decreased the enzyme in the film by about 30% but
did not eliminate it completely. The results of the activity test of two
samples are summarized in Table 4.10 together with reference values for
a spontaneous reaction without enzyme.
Table 4.10. Slopes of activity for two LB samples of urease and for spontaneous reaction.
(Reprinted with the permission from Nicolini et al., Supramolecular layer engineering for
industrial nanotechnology, in Nano-surface chemistry, pp. 141–212 © 2001a, Marcel
Dekker/Taylor & Francis Group LTD).
Sample 1 7.01 × 10-4

Sample 2 5.97 × 10-4
Average 6.49 × 10-4
Mean error 7.4 × 10-5
Spontaneous reaction 2.49 × 10-6
It is necessary to underline that enzymatic activity on spherical

supports was higher than the respective value in “flat” films, which could
indicate that apparent catalytic efficiency was improved due to an
increased area-to-volume ratio.
As evident from results of the GST activity test presented in Figure
4.41, the enzyme is active in LB films. Comparison of these catalytic
activity values with the balance values of GST LB film deposited onto
flat silanized surfaces (Antolini et al., 1995b), and presuming a linear
dependence of activity on enzyme concentration, gives an effective
amount of 2.44 pmol in the sample. Knowing the total area of spheres in
the reaction medium (10 spheres with total area of about 125 mm2) and
taking into account that only the upper layer is involved in the reaction,
we can estimate the surface density of the enzyme in the layer. The area
per molecule was found to be 83 nm2. On the other hand, the area per
molecule can be estimated from strictly geometric considerations of
protein sizes approximations taken from the RCSB Protein Data Bank
(Berman et al., 2000), and such calculations yield a value of 34 nm2.
Thus, comparing these values the conclusion can be reached that the
amount of active enzyme in the film is about 41% of a maximum
possible in the close-packed layer. The resultant activity is reported in
Figure 4.31 together with the value for a spontaneous reaction.
The comparison of the results on enhanced thermal stability of
proteins in LB films reported here and those already published again
underlines that molecular close packing is a critical parameter

responsible for this phenomenon.
The described procedure allows one to deposit protein, in particular,
enzyme, LB films onto the surface of small spheres. Deposited
multilayer film was washed in order to leave at the surface only a layer
covalently attached to the activated surface.
The enzyme in the film preserves its catalytic activity and
demonstrates highly increased thermal stability. The procedure can be
useful for the fabrication of heat-proof active elements for bioreactors
based on enzymatic catalysis.
4.3.1 Bioreactors
In recent time, Pastorino et al. (2004) showed the production of new and
efficient catalytic biomaterials was analyzed also on lipase, investigating
in toluene and comparing self-assembled lipase from Candida rugosa,
Mucor miehei and Rizhopus delemar. Of these ones, M. miehei lipase
resulted in the highest degradation yield and was used in further
experiments.
Figure 4.43. SEC profiles of (a) PCL and (b) degradation products of one-hour lipase-
catalyzed PCL hydrolysis under the optimal conditions (Reprinted with the permission
from Pastorino et al., Lipase-catalyzed degradation of poly(epsilon-caprolactone),
Enzyme and Microbial Technology 35, pp. 321–326 © 2004, Elsevier).
Almost 74% of PCL (starting polyε-caprolactone, Mn ≅ 87000 Da)

was hydrolyzed at 40 °C by this lipase within 24 h, while a lower yield
was observed with the other two lipases. Figure 4.30. illustrates the time
course of the degradation of PCL catalyzed by M. miehei lipase under the
optimal conditions (protocol A, 40°C). The peak due to PCL after one-
hour reaction shifted to lower molecular weights and its area had
increased, thus pointing out a wide distribution of the obtained molecular
weights. Moreover the profile of the degradation products showed two
peaks due to the simultaneous presence of both the dimer and the
monomer. We found that the relative concentrations of reactants, the
lipase source and the temperature all remarkably influenced the yield of
the enzymatic hydrolysis of PCL. Moreover, the data obtained in this
study provided indirect confirmation that the water content in the
reaction mixture could dramatically influence the enzyme stability and
activity. The stability of lipase in toluene at different temperatures proves
good and the time course of the degradation reaction was relatively fast.
It must be noticed that in water instead also for the lipase the optimal
immobilization and biocatalytic process was achieved only with LS and
protective plate nanostructured films.
Hans Kuhn was the first to design and make molecular machines on
the basis of LB assembly (Kuhn, 1983). However, only recently
(Troitsky et al., 1996b, 2003) variations in the assembly composition and
sequence of enzyme layer alternation were shown to influence the
properties of the biocatalytic film, i.e., its activity and structural stability.
These studies have allowed us finally to produce biocatalysts with
enhanced performance based on enzyme penicillin G-acylase (PGA).
Penicillin acylases catalyse the hydrolysis of the side chain amide bond
of penicillin (Figure 4.44). PGA preferentially hydrolyses penicillin G to
give 6-aminopenicillanic acid (6-APA) and the side chain phenylacetic
acid (Shewale and Sivaraman, 1989). 6-APA serves as a backbone for
the synthesis of semisynthetic penicillins, providing a range of penicillin
variants with differing antibiotic characteristics. The chemical methods
for producing 6-APA are environmentally burdensome and require the
use of hazardous chemicals. Current processes use penicillin acylases to
remove the side chain from penicillin G/V, providing a green route to 6-
APA (Parmar et al., 2000). Immobilized PGA technology has had major
success in making the enzymatic production of 6-APA economically

viable. In those industrial process the immobilization is carried out in
different ways; the most frequently used methods are based on
membrane entrapment, chemical surface activation and physical
absorption. However, these techniques do not provide control of the
immobilization process or of the properties of the biocatalytic medium
(Troitsky et al., 1996b).
Figure 4.44. PGA model structure model of film structure obtained in the presence of an
adsorbed layer of gluteraldehyde near the surface of the solid support. A frame-like film
of PGA molecules cross-linked by GA (c) is formed over the sublayers of GA (b) and
polymer p-DADMAC (a) and is protected by the monolayer of stearic acid (d).
(Reprinted with the permission from Troitsky et al., A new approach to the deposition of
nanostrructured biocatalytic films, Nanotechnology 14, pp. 597–602 © 2003, Elsevier).
Manipulation here described with LB monolayers and adsorbed

layers without any lateral patterning is a promising methodology for the
development of efficient biocatalytic films with a predetermined
structure. Films of different structure and composition were investigated
in order to demonstrate that the structure determines the functional
properties of the biocatalytic film. The general approach can be
described dividing the film into three hypothetical functional blocks
(Figure 4.44). The functions of the bottom block are to provide high
adhesion of the film to the surface of the solid support, to bind the
enzyme molecules, and to orient them in a proper way. The function of
the middle block is to catalyse the reaction while the top block provides
protection for the enzyme layer. The purpose of depositing such a
protective coating is to facilitate the storage of the biocatalyst and to
increase the period of preservation of enzyme activity. The deposition
procedure resulted in a considerable increase in the activity of the film
per unit of the surface and the addition of a small amount of cross-linker
near the surface where the adsorption of PGA should take place appeared
to yield striking results. The enzyme activity, per unit of film surface,
was immediately increased at least 20 times compared with the activity
reported above for one closely packed monolayer. By further
optimization of the film structure the best results were obtained for films
with a poly(diallyldimethylammonium) chloride (p-DADMAC) sublayer
in the bottom block (Figure 4.44).
4.3.1.1 From lab scale to industrial scale
For the purpose of industrial applications, the biocatalytic film has to be

deposited on to supports of very large effective area to be utilized in
bioreactors. For this reason, we are presently developing methods for
technological realization of a new-patented procedure for large-scale
nanobiocatalysis (Stura and Nicolini, 2006).
For practical applications it is indeed not enough to obtain just high
efficiency and stability of the biocatalytic medium. It is also necessary to
provide rather fast film deposition onto supports of very large effective
area to be used in industrial bioreactors (Dubrovsky and Nicolini, 1994).
Thus, we have performed a modification of the “protective plate”
technique in order to carry out continuous deposition onto a flexible
support (e.g. polymeric tape) of practically unlimited length. If the film
containing the enzyme is deposited on to such tape, it is easy to make
from the latter a compact roll with small gaps between the turns. The
rolls with the immobilized biocatalyst can be used in bioreactors of
different types. The idea behind the solution to the problem is to provide
the possibility of transferring the flexible support from one compartment
to another through the slits, which are located over the levels of liquids in
the adjacent compartments, while holding the solutions inside the gap by
capillary forces. The main time-consuming process is the deposition of
LB monolayers.
Figure 4.45. LB-based large scale bioreactor prototype (Reprinted with the permission
from Nicolini C., Engineering of enzyme monolayer for industrial biocatalysis, Annals
New York Academy of Sciences 864, pp. 435–441 © 1998b, Blackwell Publishing).
However, coating a 20–30 m long tape during the working day is

quite possible (Nicolini, 1998b) (Figure 4.45). The other problems which
arise when one tries to substitute small solid supports by long polymeric
tape and to adopt a continuous process of deposition are the depletion of
protein solution during the film deposition and transferring the cross-
linker solution with the tape into the volume for protein adsorption. The
calculations showed that either big volumes of protein solution should be
used or a continuous supply of protein should be provided into the
compartment, which should be of small volume. Transfer of cross-linker
through the slit can be in principle be limited by careful elaboration of its

design up to such a level that during the whole working day coagulation
of protein in the volume will not occur. At present, development of the
laboratory prototype of an apparatus based on this principle is in
progress. Its design and the results of testing will be reported in a
separate publication (Stura and Nicolini, 2006).
Figure 4.46. Prototype of industrial reactor, consiting of a rotating pilot reactor at

laboratory level but displaying deposition industrially feasable (Reprinted with the
of Nanoscience and Nanotechnology 6, pp. 2209–2236 © 2006a, American Scientific
Based on the obtained results we can conclude that the methods of

monolayer engineering are suitable for application in the field of
biocatalysis. Although laboratory developments of the deposition
technique do not prove the possibility of elaboration of the technology as
well as its efficiency we do not see strong objections to this work. The
main problem is working out a fast continuous technological process of
biocatalyst deposition on to the surface of very long polymeric tape. At
the same time, it seems that the problems of structural stability and
preservation of enzymatic activity can be successfully solved. In
conclusion, industrial applications, which already found their
implementation in biocatalysis are gene engineering-based production of
biologically essential macromolecules and the stereo-specific
biosynthesis with the utilization of specific enzymes such as shown here
for PGA and other enzymes (i.e., urease, alcohol dehydrogenase, alkaline
phosphatase, glutathione-S-transferase and DNA polymerase).
4.3.2 Bioactuators
The application of biological materials to different branches of modern

technology has expanded enormously to bioactuators during the recent
period. Examples of heat-proof and stable bioactuators are LB films
based on the phenol oxidases and the laccase from animals which are
specific for bioactive phenols (Tyr, DOPA, dopamine, adrenaline and
noradrenaline), while enzymes from fungi and higher plants are active
towards a wider spectrum of substrates (mono and polyphenols).
Laccase (p-diphenol: oxygen oxidoreductase) is a ubiquitous enzyme,
which catalyzes:
O
OH
+ 1 /2 O _ _ _ _ >
2 + H O
2
O
OH
while the enzyme cathechol oxidase (o-diphenol: oxygen

oxidoreductase) catalyzes a similar reaction:
O
OH
O
OH
+ 1 /2 O _ __ _ > + H 2O
2
These two enzymes have been used:

• as bioelements in amperometric biosensors for the selective
determination of neurotransmitters;
• for the construction of bioreactors for the enzymatic degradation of
phenolic compounds in waste water.
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Index
adrenodoxine, 10 Brewster angle microscopy (BAM), 36;

AFM, see atomic force microscopy 70-71
alcohol dehydrogenase, 311
alkaline phosphatase, 302; 311 cadmium arachidate, 34-35; 66;
amperometric sensor, 226-227 267-269; 274
anodic porous alumina (APA), 48-53; cadmium sulphide, 267
81, 230-232; 307 cAMP, 101; 103-104; 175-180
APA, see anodic porous alumina cantilever, 183
archaea, 16 capacitor, 246; 250-253; 290
atomic force microscopy (AFM), 54; carbon nanotubes (NT), 148-151; 184;
58-65; 88; 115-116; 118; 127; 230 241; 299; 301
ATP, 179 cardiomyocytes, 148-149; 151
cardiovascular cells, 148
bacteriorhodopsin (bR), 6-7; 13; 43-45; CCD, 36; 159
93-95; 97; 100; 110; 220-222; CD, see circular dichroism
232-236; 261-263; 279-281 CdA, 36
BAM, see Brewster angle microscopy CdS, 18; 35; 66; 248; 255; 267-270;
batteries, 251; 275; 282-290; 292-296 274; 276
bioactuators, 312 CHO-K1, 2-3; 5; 101-104; 175-178
biocatalysis, 310; 311 cholesterol, 72-73; 223-226; 229;
bioinformatics, 67; 90-91; 93; 158; 230-232
160; 164-166; 172; 174 chromatin, 197-199
biomaterials, 1; 306 chromatography, 5; 11
biosensor, 183; 186; 190; 223-227; circular dichroism, 44-45; 61-62; 69-70
229; 232; 237 CK2α, see human kinase CK2α
Bone Morphogenetic Proteins (BMP), clustering algorithms, 91
14 conducting polymer, 18-19; 25; 27-31;
bovine rhodopsin, 13; 93; 95; 97 38; 67; 258; 265; 277
bovR, see bovine rhodopsin contact mode, 58-59
Bragg reflections, 108; 111; 235 Coulomb Blockade, 268-271
363
crystal growth, 113; 115 G2, 159-163; 165

crystallization, 59; 93; 95-98; 100; gel electrophoresis, 12
113-116; 118; 121; 123-124; gene expression, 158-160; 166; 168;
126-127; 129-132; 134; 136-137; 171; 173; 198
141-145; 211; 217 glutathione-S-transferase (GST), 11;
crystallography, 118; 131-132; 136; 13; 223; 301-303; 305; 311
157; 197-198; 202; 216; 283 gold nanoparticles, 228-230; 232
CV, see cyclic voltammetry; 72 GST, see glutathione-S-transferase
cyclic voltammetry (CV), 72-73;
225-226; 231 H9c2, 2-3; 148-151
cytochrome C, 74-75 HeLa, 2
histone, 199-200
dental implants, 152 homology modeling, 64; 93-94; 97-98
differential scanning calorimetry HPLC, 10; 101-104; 175; 177-180
(DSC), 213 human kinase CK2α, 77; 109; 120;
diffraction patterns, 96; 109-110; 119; 125; 129-131
130; 144 human T lymphocytes, 4; 84; 91-93;
diffractometry, 108 158-167; 173
DNA microarray, 15; 51; 81-83; 93; hydrogen storage, 296-298
158-159; 174
DNASER, 82-83; 88; 159 infrared spectroscopy (IR), 73; 285
IR, see infrared spectroscopy
ELEctrochemical NAnodeposition
(ELENA), 187; 189 kidney transplant, 84
electron spray ion (ESI), 101-104; 175; Kiessig fringes, 108; 111
177-179
ELENA, see ELEctrochemical Label Free, 65
NAnodeposition Langmuir-Blodgett (LB), 2; 13; 34-35;
ellipsometry; 71 37-47; 54-55; 57; 59-61; 63; 66; 69;
ESI, see electron spray ion 72-73; 75, 77-78; 97; 100; 107-108;
111; 116 124-125; 127; 135; 145;
FIB, see focused ion beam 203; 213-218; 223-226; 228; 233,
focused ion beam (FIB), 189 235; 237; 247-249; 255-256; 259;
Fourier transform infrared spectroscopy 264; 266-269; 277-279; 301- 308;
(FTIR), 74-76 310; 312
FTIR, see Fourier transform infrared Langmuir-Schaefer (LS), 24-25; 29;
spectroscopy 36; 39-41; 47; 70-71; 73; 76; 137;
fuel cells, 275; 285; 296 141;
fullerene, 17; 18; 277; 300 layer-by-layer (LBL), 37-38; 223; 237;
259
G0, 159-160 LB, see Langmuir-Blodgett
G1, 159; 161-164; 166; 174 LBL, see layer-by-layer
Index 365
LDL-cholesterol, 53 nanocrystals; 112; 114; 117-118; 138

leader genes, 91-93; 158-163; 166-169; nanoelectronics; 220; 267
171-173 nanogenomics, 4; 158
LED, see light emitting devices nanogravimetry, 118; 127; 203
light emitting devices (LED), 19; 25; nanomaterials, 1-2; 6; 37
256-261 nanomechanics, 183
lipase, 306-307 nanomedicine, 148
LS, see Langmuir-Schaefer nanooptics, 183
lysozyme, 13; 59-61; 63; 76; 102; nanoparticles, 17-18; 34-36; 38; 74;
124-125; 127; 133; 135; 139; 142; 118; 132-134; 137; 186; 228-229;
145-147; 217 241; 247-249; 255; 267-269;
271-272; 277; 283-284; 288;
MALDI TOF, 87; 89; 102; 105-106; 294-295
177; 181 nanoscale, 219; 249; 255; 282; 293
mass spectrometry (MS), 5-6; 87-90; nanosensors, 220; 237
93; 101-106; 174-175; 177-181 nanostructures, 1; 41; 132-133
metalloproteins, 6; 8; 10; 13 nanotechnology, 1; 12; 17-19; 21;
micro Grazing Incidence Small Angle 23-25; 27; 30-31; 118; 128; 132;
X-ray Scattering (µGISAXS), 54; 136; 184; 191; 219; 258; 270; 276;
120; 131; 135-137; 140-143; 280; 295; 305
145-147 nanotemplate, 59; 98; 113-114; 118;
microarray, 50-51; 65; 81-82; 84-89; 124-125; 132; 135-137; 140-143;
91-93; 105-106; 158; 160; 167; 145-147; 217-218
169-173; 180-182 nanotubes (NT), 3; 17; 25-32; 34;
microcrystals; 63; 96; 98; 109-111; 148-149; 151; 241-243; 284;
116; 119-120; 130; 136; 139; 143 297-300
microfocus; 98; 109-110; 119-120; NAPPA, see Nucleic Acid
127; 129-131; 136; 137; 145 Programmable Protein Array
molecular modelling, 78; 90; 93 NMR, see nuclear magnetic resonance
MS, see mass spectrometry non-contact mode, 58
multi-walled nanotubes (MWNT), 26; NT, see nanotubes
243; 299 nuclear magnetic resonance (NMR),
12-14; 67-68; 202; 216
nanoarrays, 132 Nucleic Acid Programmable Protein
nanobiofilm, 127; 130; 132; 143; 147 Array (NAPPA), 65; 87-90;
nanobiotechnology, 10; 90; 93; 100; 105-106; 174; 181
220; 229; 255; 275; 282 nucleosome, 197-200
nanocomposites, 17; 25; 27
nanocontacts, 184-190 octopus rhodopsin (octR), 13; 93-95;
nanocrystallography; 77; 109; 118-120; 97; 100
131; 136 octR, see octopus rhodopsin
optical tweezer, 193-195 radiation damage; 110-111; 115; 120;

opto-electronics, 7; 18 128
osteogenesis, 91; 173-174 Raman spectroscopy; 192; 285
RC, see photosynthetic reaction centers
P450, 56-58; 69; 72-73; 98; 223-228; reflectometry, 108
231 rhodamine B, 153-155
P4501A2, 12; 13; 223-228 rhodopsin, 93-95; 97; 100; 261;
P4502B4, 11-13; 96; 99; 223-226 263-264
P450scc, 8-10; 12-13; 51-53; 63; 69;
72-73; 77; 93; 96; 98; 100; 107-108; scanning probe microscopes (SPM),
116: 120; 125; 131; 135-137; 183
140-144; 223-226; 228-232 Schottky diode, 256
PANI, 256; 259; 265-266 SEM, 230; 255
PAOA, 265-267 semiconductor, 255-256; 260; 267;
PAOT, 265 272-273
penicillin G-acylase, 307-309; 311 sensors, 26; 29; 30
PGA, see penicillin G-acylase SFM, 137
photocells, 232; 278 STM, 17; 46; 65; 66; 189; 254;
photosynthetic reaction centers (RC), 6; 257
13; 43; 46 57; 278-279 surface potential, 54-57
photovoltaic cells, 256; 275; 277; 281; synchrotron radiation, 108; 111; 112;
288 116; 118-120; 127; 129; 131;
POAS, 241-242; 256-257; 266; 271 135-136; 140-141; 143; 146-147;
POAT, 266 195; 198; 217
polyaniline, 27; 29-30; 37
potentiometric stripping analysis tapping mode; 58; 59; 60; 62; 63
(PSA), 238-240 thermal stability; 43-45; 47; 202;
PPV; 259-260; 295 204-206; 208; 210-215; 217-219;
PPY; 265 264; 291- 292; 301-306
protective plate; 38; 41-43 thin films; 118; 124-125; 136-137; 139;
protein microcrystals; 120; 126; 128; 145; 220; 232-233; 241; 243; 259;
131; 135; 140-141; 143; 146-147 261; 263; 265; 277
protein-chip; 81 thioredoxin; 13; 203; 208; 212-213;
proteomics; 77; 81; 109-110; 118; 120; 215-218
127-128; 173
PSA, see potentiometric stripping urease; 303-305; 311
analysis
purple membrane, 94; 97; 220; 280 X-ray; 17; 54; 61; 63; 66; 107;
108-112; 115-116 119-121; 128;
QC, see quantum computing 131-132; 136-137; 139; 143-145;
quantum computing (QC), 100 157; 191-192; 195-198; 216; 235;
quartz crystal balance; 77 284
Index 367
Yoneda, 113;-114; 135; 139-140; µGISAXS, see micro Grazing

142-143; 144-145; 147 Incidence Small Angle X-ray
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Nanobiotechnology

V020tp.indd 1 11/6/08 4:05:24 PM

Pan Stanford Series on NANOBIOTECHNOLOGY

V020tp.indd 2 11/6/08 4:05:26 PM

British Library Cataloguing-in-Publication Data

Pan Stanford Series on Nanobiotechnology — Vol. 1

Rhaimie - Nanobiotechnology.pmd 1 6/19/2008, 3:00 PM

This volume introduces in a coherent and comprehensive fashion the

I am particularly grateful to all numerous members of the Nanoworld

Genoa, 30 March 2008

2.3 Nuclear Magnetic Resonance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 67

3. Nanoscale Applications in Health and Science 118

3.4.2 Cell transformation and differentiation . . . . . . . . . . . . . . . . . . 180

4. Nanoscale Applications in Industry and Energy Compatible with

4.2.2.1 Lithium ion batteries elements . . . . . . . . . . . . . . . . . . . 285

This chapter overviews the present status of new materials by organic

the last years are here summarized to exemplify potentially useful

1.1 Produced Via LB Technology

Several well-established cell culture systems, like mammalian CHO-K1,

and viability. Digital photomicrographs were recorded and the number of

Recombinant protein expression and purification is the single most

were examined by a phase contrast microscope (Wilovert, Wesco). The

1.1.2.1 Light sensitives

Photosynthetic reaction centers from Rhodobacter sphaeroides and

code facilitates manipulations of organisms that can produce these new

Therefore, the price of such products will be affordable. An intensive

filtering; a similar route is presently being pursued with metallo-proteins

Expression and purification of a typical metal-protein, i.e. recombinant

and functional alterations, is consistent with the hypothesis that the

In the latter, the cDNA of the cytochrome P450scc obtained from

βol), styrene (99% pure grade), clozapine (8-chloro-11-(4’-

The cDNA encoding for another mature form of cytochrome called

1996). The purified cytochrome P4502B4 recombinant was in a 10 mM

Other different proteins, both water-soluble and membrane-bound, have

only minute amount of protein is necessary to make 3D atomic structure

Protein Source Method Reference

As model system for membrane-bound proteins we have chosen

Morphogenetic Proteins representing a family of 13 proteins capable to

1.1.3 Genes and Oligonucleotides

To analyze either a fluorescently labelled DNA sample of unknown

DNA microarray (see chapter 3) allows the study at the nanoscale of

lyophilized and resuspended in an opportune volume (120 µl) of

1.1.4 Lipids and Archaea

Archaea, a philogenetically coherent separate group of microorganisms,

1.2 Produced Via Organic Chemistry

Among the numerous organic compounds produced via chemical

The major drawback of the polymeric materials is the multi-step

1.2.1. Conductive polymers

The structures of polymeric nanoscale materials are summarized in

1996a). From 1977, the dream of combining the mechanical and

A central topic in the physic of the π-conjugated polymers (and their

electronic structure of these polymers. In this contest the understanding

State Chemical term Charge Spin

Reassuming, the band gap value (Eg) in the conjugated polymers is

Esub: effects of substituents – inductive and mesomeric electronic