Chitosan: An Integrative Biomaterial For Lab-On-A-Chip Devices

CRITICAL REVIEW
www.rsc.org/loc | Lab on a Chip
Chitosan: an integrative biomaterial for lab-on-a-chip devices

S. T. Koev,a P. H. Dykstra,a X. Luo,b G. W. Rubloff,c W. E. Bentley,b G. F. Payneb and R. Ghodssi*a
Received 24th May 2010, Accepted 17th August 2010
DOI: 10.1039/c0lc00047g
Chitosan is a naturally derived polymer with applications in a variety of industrial and biomedical
fields. Recently, it has emerged as a promising material for biological functionalization of
microelectromechanical systems (bioMEMS). Due to its unique chemical properties and film forming
ability, chitosan serves as a matrix for the assembly of biomolecules, cells, nanoparticles, and other
substances. The addition of these components to bioMEMS devices enables them to perform functions
such as specific biorecognition, enzymatic catalysis, and controlled drug release. The chitosan film can
be integrated in the device by several methods compatible with standard microfabrication technology,
including solution casting, spin casting, electrodeposition, and nanoimprinting. This article surveys the
usage of chitosan in bioMEMS to date. We discuss the common methods for fabrication, modification,
and characterization of chitosan films, and we review a number of demonstrated chitosan-based
microdevices. We also highlight the advantages of chitosan over some other functionalization materials
for micro-scale devices.
1 Introduction
a
Department of Electrical and Computer Engineering, Institute for Systems
Research (ISR), University of Maryland, College Park, MD, 20742, USA.
E-mail: [email protected]
b
Fischell Department of Bioengineering, Center for Biosystems Research,
University of Maryland, College Park, MD, 20742, USA
c
Department of Materials Science and Engineering, Institute for Systems
Research (ISR), University of Maryland, College Park, MD, 20742, USA
These authors contributed equally to this work.
Currently address: Center for Nanoscale Science and Technology,
NIST, Gaithersburg, MD, 20899, USA
such as DNA, enzymes, or antibodies to operate with sufficient

specificity and sensitivity. However, the harsh fabrication techniques and materials involved in traditional MEMS fabrication
are incompatible with the labile biological components.
Specialized materials and processes are needed to allow for
seamless integration of biology into microdevices. Several
approaches toward this goal have been demonstrated based on
the use of self-assembled monolayers or surface-immobilized
polymers.1,2 The polymer chitosan is one of the most promising
candidates for interfacing biology and microdevices, and it is the
subject of this review paper.
Chitosan is a polysaccharide derived from naturally occurring
chitin. Its unique properties make it attractive for many industrial and biomedical applications. Due to its pH dependent
solubility, it forms stable films on various surfaces under neutral
and basic pH conditions. Its amine groups serve for covalent
Stephan Koev was born in Bulgaria in 1981. He received a BS

degree in Electrical Engineering
from the US Naval Academy in
2004, and MS and PhD degrees
in Electrical Engineering from
the University of Maryland in
2007 and 2009, respectively. His
doctoral research was in the area
of MEMS for biomedical applications. His interests include
micro- and nano-fabrication,
integrated optics, bio-device
S: T: Koev
interfaces,
and
MEMS
metrology. Currently, he is
a postdoctoral research associate with the Center for Nanoscale Science and Technology at the
National Institute of Standards and Technology, Gaithersburg,
MD, USA.
Peter Dykstra received his B.S.

and M.S. degrees in Electrical
Engineering from Bucknell
University in 2006 and the
University of Maryland in 2008,
respectively. He is currently
pursuing his Ph.D. in Electrical
Engineering while working at
the MEMS Sensors and Actuators Lab (MSAL) at the
University of Maryland. His
masters research involved the
use of the biopolymer chitosan in
P: H: Dykstra
a microfluidic biosensor while
his current research focuses on
an electrochemical microfluidic
DNA array for protein sensing. His research interests include
biological and chemical sensors, micro fabrication, microfluidics,
and electrochemistry.
One of the main challenges in the development of miniaturized

sensors and systems for life science applications continues to be
the integration of biological components. These types of microdevices typically need to be functionalized with biomolecules
3026 | Lab Chip, 2010, 10, 30263042
This journal is The Royal Society of Chemistry 2010
attachment of biomolecules, and it can be co-deposited with

other polymers or nanoparticles. Several review papers survey
the diverse applications of chitosan,311 including controlled drug
release, wound healing, nutrition supplements, water purification, removal of toxins, scaffolds for tissue engineering, and
semipermeable membranes.
Recent advances in chitosan fabrication have allowed this
material to be integrated into lab-on-a-chip devices.6,1214 The
primary role of chitosan in the microdevices is to immobilize
biomolecules, cells, or nanoparticles. This enables the devices to
perform advanced functions such as specific recognition of
analytes and enzymatic conversions. The present article starts
with a brief review of chitosans properties. Next, we describe the
common methods for microfabricating chitosan films, for
modifying them with other substances, and for characterizing
them. This is followed by a survey of reported microdevices that
utilize chitosan. We conclude by summarizing the advantages of
chitosan mediated assembly and by discussing possible future
developments in the field.
Xiaolong Luo received his B.S.E. from Zhejiang University in

China in Mechanical & Electrical Engineering in 2001, M.S.E.
from Temple University in Mechanical Engineering in 2003 before
he obtained his Ph.D. in Bioengineering from University of
Maryland in 2008.He is currently a post-doctoral research associate at University of Maryland working on biofabrication and
novel applications of 3D biopolymer membranes in microfluidics.
His research interests are in microfluidics and microfluidic platforms for signal-guided assembly of biomolecules and cells, metabolic engineering, cell-cell communication, bacterial quorum
sensing, and antimicrobial drug discovery.
Gary Rubloff received his PhD in physics from the University of
Chicago in 1971. After a postdoc in physics at Brown University, he
joined IBM Research in Yorktown Heights in 1973 where he did
fundamental surface science, electronic materials and processing
science, silicon technology and manufacturing research, as both
researcher and manager. He has been Professor in Materials
Science and Engineering and the Institute for Systems Research
(ISR) at the University of Maryland since 1996, serving previously
as ISR Director, and currently as founding Director of the
Maryland NanoCenter, Minta Martin Professor of Engineering,
and Director of Nanostructures for Electrical Energy Storage,
a DOE Energy Frontier Research Center.
William E. Bentley is the Robert E. Fischell Distinguished
Professor of Engineering and founding Chair of the Fischell
Department of Bioengineering. He is also appointed in the
Department of Chemical and Biomolecular Engineering at the
University of Maryland, College Park and the Institute for
Bioscience and Biotechnology Research. He received his undergraduate (BS, 82) and Master of Engineering degrees (83) from
Cornell University and his PhD (89) from the University of
Colorado, Boulder. His recent interests are on deciphering and
manipulating signal transduction pathways, including those of
bacterial communication networks, for altering cell phenotype and
constructing devices via principles of biofabrication for interrogating biological signaling processes.
Properties of chitosan
Chitosan is obtained by the deacetylation of chitin. Chitin is the

structural material of the shells of crustaceans and insects, and it
is the second most widespread natural polymer on earth after
cellulose. Therefore, chitosan is abundant, biocompatible and
biodegradable. Its degradation products are harmless natural
metabolites, and it is non-antigenic (it does not cause an immune
response in an organism).
The structural formulas of chitin and chitosan are shown in
Fig. 1 Many of chitosans properties are explained by the presence of its primary amine groups with pKa 6.5.6 At pH lower
than 6.5, the amines are positively charged and chitosan is
soluble. At higher pH, the amines are increasingly deprotonated,
and it becomes insoluble. This pH dependent solubility allows
stable chitosan films to be deposited on device surfaces through
several methods that will be reviewed in section 3. Importantly,
the film can be dissolved and removed by a mildly acidic wash,
and the device can be re-used multiple times. The structure of the
film depends on the deposition conditions, the chitosan molecular weight, and its degree of deacetylation.
Chitosan films immersed in liquid experience strong pH
dependent swelling.15,16 The pH of the solution determines the
Gregory F. Payne received his B.S. and M.S. degrees in

Chemical Engineering from Cornell University in 1979 and 1981,
respectively. He received his Ph.D. in Chemical Engineering from
The University of Michigan in 1984. After completing his Ph.D., he
returned to Cornell to do post-doctoral work with Michael Shuler
in biochemical engineering. In 1986 he joined the faculty of the
University of Maryland where he is currently a Professor jointlyappointed in the Center for Biosystems Research and the Fischell
Department of Bioengineering. His research is focused on biofabricationthe use of biological or biomimetic materials and
processes for construction. Specifically, his group biofabricates
using enzymes and biologically-derived polymers such as chitosan.
Reza Ghodssi received his Ph.D.
in Electrical Engineering from
the University of Wisconsin
Madison in 1996. He joined the
faculty of the University of
Maryland in 2000. Currently, he
is the Director of the Institute
for Systems Research, Herbert
Rabin Distinguished Professor
of Electrical Engineering, and
Director of the MEMS Sensors
and Actuators Lab. He is also
affiliated with the Fischell
R: Ghodssi
Department of Bioengineering,
Maryland NanoCenter, UMD
Energy Research Center, and
the Materials Science and Engineering Department at UMD. His
research interests are in the design and development of microfabrication technologies and their applications to micro/nano
devices and systems for chemical and biological sensing, smallscale energy conversion, and energy harvesting.
Lab Chip, 2010, 10, 30263042 | 3027
Methods for chitosan microfabrication
Chitosan is normally purchased in the form of dried flakes. These

flakes are dissolved in an acidic solution, typically dilute
hydrochloric or acetic acid, which is then filtered to remove
particulates. The preparation of chitosan solutions is presented
in detail in many articles.1214,23,28 Several different methods have
been developed to deposit and pattern chitosan films that are
compatible with bioMEMS fabrication.
3.1 Solution casting
Fig. 1 Structural formulas of chitin and chitosan showing the three

possible states of the amine group: (a) chitin, amine group acetylated; (b)
chitosan, amine group free; (c) chitosan, amine group protonated.
amount of positive charge on the chitosan chains, which in turn

affects the electrostatic repulsion between the chains and the
volume of the polymer. This swelling behavior can be utilized for
actuation17 or for controlled drug release.18
Perhaps the most significant property of chitosan is its ability
to be modified with other substances. The amine groups can be
used as sites for covalent attachment of various biomolecules,
including enzymes, DNA, and antibodies. The modification can
also be performed by physical interaction (e.g. surface absorbance, entrapment) instead of chemical bonding, especially in the
case of negatively charged substances. The types of chitosan
modifications that have been demonstrated are discussed in
section 4. The chitosan chains can also be covalently crosslinked
with each other instead of with biomolecules. As explained in
section 3.5, this crosslinking improves the strength and chemical
resistance of the film.
Unmodified chitosan films are transparent and therefore
suitable for use in optical sensors.1921 Crosslinking of the chitosan somewhat increases its optical absorbance in the UV
region, but it still remains low in the visible range.22 The electrochemical properties of chitosan are also favorable. Wet chitosan films are porous and highly permeable to ions.23 This
enables the construction of electrochemical sensors with chitosan
coated-electrodes.2426
The mechanical properties of chitosan vary widely with the
preparation method. For example, its Youngs modulus has been
reported to be 9 MPa22 and 3.50 GPa,14 both for unmodified
films. Note that the mechanical properties of chitosan do not
significantly affect the design of bioMEMS devices since it is
normally used as a coating rather than a structural material. The
thickness of the chitosan is typically smaller than that of the
structural material, and its stiffness is much lower (for example,
the Youngs moduli of Si, Si3N4 and SiO2 are on the order of 100
GPa). The reported residual stress of dried electrodeposited
chitosan films is approximately 60 MPa27 (in comparison, the
residual stresses of Si3N4 and SiO2 are normally hundreds of
MPa). Therefore, the addition of chitosan is likely to have
a minimal impact on the mechanical characteristics of the
microdevice.
3028 | Lab Chip, 2010, 10, 30263042
The simplest fabrication method consists of coating the surface

with chitosan solution and evaporating the solvent. This leaves
behind a solid chitosan film with a thickness dependent on the
solution concentration. The coating procedure can be performed
with a pipette or with a spin coater (if a highly uniform film is
needed). Elevated temperatures can be applied to the substrate to
speed up the solvent evaporation.
Solution casting is the predominant method for applying chitosan to macroscale devices. However, it does not inherently
provide the spatial control necessary for use in bioMEMS. For
this reason, several approaches have been demonstrated to
pattern the cast film. These include a containment method in
which chitosan droplets are laterally constrained24 and a thermolithography method in which areas of the chitosan film are
masked with a heat sensitive thermoresist.29 More recently, spincast chitosan films were patterned by conventional photolithography by masking them with photoresist and etching them
with oxygen plasma14 (Fig. 2). In another work, chitosan was
mixed with hydroxyethyl methacrylate (HEMA) and a photoinitiator, making it UV-curable.30 This suggests that photopatternable formulations of chitosan may be developed in the
future, which will make the solution casting method more
applicable to bioMEMS integration.
Unpatterned solution-cast films are quite useful for fabricating
chitosan test samples and simple glass slide based optical sensors.
Fig. 2 Photolithographically patterned chitosan features. (a) SEM of

matched comb structure at a 50r tilt. (b) SEM of 2-mm-wide lines with
4-mm pitch. (c) SEM of serpentine spring structures. (d) SEM of
a sawtooth structure. Note: All patterns are in a 2-mm-thick chitosan
layer. Reprinted with permission from ref. 14. 2008 IEEE.
The film is deposited onto a transparent substrate, and its

absorbance is measured with a spectrophotometer.19,3133 This
approach can be used either to determine the optical properties
of the chitosan or detect an analyte bound to it. Films of sufficient thickness (typically greater than 10 mm) can be completely
peeled off the substrate after drying and subjected to further
optical and mechanical testing.22,34 Common chitosan characterization methods are discussed in section 5.
An interesting extension of the solution casting method is the
layer-by-layer assembly (LBL) technique. The substrate is
exposed to alternating washes in the cationic chitosan solution
and a solution of some anionic species. Due to electrostatic
attraction, a multi-level composite film is produced. This method
has been used for the entrapment of enzymes,3538
nanoparticles,3941 DNA42 and cells.43 The modifications of chitosan with other substances are described further in section 4.
3.2 Printing
Both nanoimprinting and microcontact printing have been
successfully demonstrated for patterning chitosan films with
nanoscale resolution. These methods make use of a patterned
mold or stamp, which is typically a flexible polymer such as
PDMS. For nanoimprinting, the PDMS mold is placed over
a substrate coated with chitosan solution while applying heat and
pressure. After cooling, the PDMS is peeled off, and molded
structures of chitosan remain patterned on the substrate. Park
et al. demonstrated the use of nanoimprint lithography to create
nanowire and nanodot chitosan structures.13 Tan et al. added
plasticizers to the chitosan to allow for imprinting at reduced
temperature and pressure.44 For microcontact printing, the chitosan solution is applied to the PDMS stamp surface, which is
then contacted with the substrate. The pattern formed on the
substrate corresponds to the geometry of the raised features on
the stamp. Feng et al. demonstrated co-patterning of chitosan
and bovine serum albumin on the same substrate using microcontact printing.45
Printing methods can easily create arrays of chitosan structures for spatially resolved functionalization. However, both
nanoimprint lithography and microcontact printing are inherently planar processes and cannot be used to pattern chitosan
films on non-planar surfaces (such as microchannels or sidewalls). Furthermore, alignment of the mold or stamp to preexisting features on the substrate can be difficult.
3.3 Electrodeposition
A very promising method for chitosan integration in microdevices is electrodeposition.12,28,4648 It allows for both spatial and
temporal control of the chitosan film location and thickness, and
it can be used with a variety of device geometries. Electrodeposition takes advantage of chitosans pH dependent solubility. At
pH lower than 6.5, chitosan is soluble and cationic due to its
protonated amino groups. At higher pH values, chitosan
becomes deprotonated and is no longer soluble. When an anode
and cathode are immersed in a chitosan solution and a voltage is
applied, electrochemical reactions lead to a locally high pH
adjacent to the cathode surface. While Fig. 3 illustrates these
cathodic reactions as a net consumption of hydrogen ions, we
Fig. 3 Schematic demonstrating chitosan film electrodeposition.

Adapted with permission from ref. 6. Copyright 2005 American Chemical
Society.
should note that the mechanistic details are more complex and
likely involve the reduction of oxygen ions as well.23 Chitosan
forms a thin film over the cathode surface as a result of the high
pH there. The rate of the chitosan deposition is influenced by
many factors: the molecular weight of the chitosan, the pH of the
solution, the sizes and separation of the anode and cathode, and
the applied voltage. Buckhout-White et al. has extensively
characterized the spatial resolution of electrodeposited chitosan
films.49 Recent in situ visualization and characterization of electrodeposited chitosan done by Cheng et al. further proved that
the gelation and immobilization of chitosan onto the cathode are
due to the electrochemically generated pH change by OH ions
and the density distribution of deposited chitosan hydrogel is
electric field-dependent.50 Further dependence of electrodeposition rates on applied stimuli has been investigated in several
articles.6,23,28,47,51
The electrodeposition method inherently provides a patterning
capability. The resulting chitosan film pattern is identical to the
electrode geometry, which can be easily defined by standard
microfabrication techniques. Unlike other chitosan deposition
methods such as solution casting or printing, electrodeposition
does not require an exposed, planar surface. This allows for the
deposition of chitosan films on a variety of micro-scale geometries such as released structures,27,52 out-of-plane surfaces,20,5355
and sealed microchannels.5658 Fig. 4 shows electrodeposited
chitosan on the side of a thick polymer film (SU-8), highlighting
the ability of this method to coat non-planar surfaces.
Importantly, chitosan can retain its pH responsive and electrodeposition properties when combined with other substances.
The electrical co-deposition of chitosan with several proteins46,5961
and nanoparticles has been successfully demonstrated.62,63 This
facilitates the fabrication of modified chitosan films.
3.4 Other methods

Several other methods for chitosan fabrication have been
reported that could be viable for use in bioMEMS devices. Very
thin chitosan films can be formed by starting with a self-assembled monolayer of cysteamine on a gold surface.64 Chitosan
chains are then anchored to the cysteamine with a glutaraldehyde
cross-linker, forming a chitosan monolayer. Chitosan microstructures other than thin films have also been developed. For
Lab Chip, 2010, 10, 30263042 | 3029
Fig. 4 SEM of chitosan film electrodeposited on the sidewall of

a microchannel. A thin film of patterned indium tin oxide acted as the
cathode surface for the deposition. Reprinted with permission from ref.
162.
example, chitosan fibers with an average diameter of 100 mm

were used as pH sensors.17 These fibers are created by a wet
spinning method, in which the chitosan solution is ejected
through a small nozzle into a coagulation solvent of NaOH. The
fibers can be further cross-linked using glutaraldehyde and
conjugated with polyaniline or carbon nanotubes to improve
their strength and conductivity.65,66
Porous 3D chitosan scaffolds have been developed to aid the
attachment and proliferation of cells for tissue engineering.
Tangsadthakun et al. fabricated such structures by freeze drying
a collagen/chitosan blend and crosslinking it.67 Ang et al. constructed a micropatterned scaffold of chitosan-hydroxyapatite
using a robotic dispensing system.43 Slavik et al. used a PDMS
mold to form chitosan fibers, which were coagulated to create
a scaffold.68
Luo et al. recently utilized chitosans pH responsive solubility
to form freestanding chitosan membranes in a microfluidic
channel.69 A pH gradient is formed at the interface between two
converging flows of slightly acidic chitosan and slightly basic
buffer solution. Due to laminar flow conditions, the two solutions do not mix. A chitosan membrane 3060 mm thick forms at
the solution interface with pore size in the range of a few nanometers.
In one particularly interesting demonstration, chitin
membranes were obtained directly from the carapaces of blue
crabs and used to construct a glucose biosensor.70 Although this
approach requires extensive manual manipulation and is not
applicable to microdevices, it highlights the versatility of chitosan fabrication methods.
3.5 Chemical compatibility and adhesion of chitosan
Once deposited, the chitosan films must be kept at a pH above
6.5 to prevent them from dissolving. They are typically washed
with a basic solution after deposition in order to neutralize any
remaining acid and reduce the positive charge on the amine
groups. The films can be crosslinked with reagents such as
phenols,22,34 DTBP,71 or glutaraldehyde5,6 to make them more
chemically resistant and physically harder. However, the crosslinking consumes chitosans amine groups and may limit the
ability to modify the film with other substances.
3030 | Lab Chip, 2010, 10, 30263042
Chitosan films are fully compatible with the near-neutral

physiological conditions encountered during their use. However,
they can be damaged by any strong acids, solvents, plasmas, or
extreme temperatures commonly used in MEMS or bioMEMS
fabrication. Therefore, it is a better approach to deposit chitosan
in the device after the fabrication is completed.57
The adhesion of the chitosan to the substrate can vary greatly
with the deposition conditions and surface properties. Films with
high water content (hydrogels) have very poor adhesion and may
be removed even by rinsing the substrate.47 More compact films
have excellent adhesion and withstand the high shear forces in
microfluidic channels.57,58 It is believed that electrodeposition
produces films with better adhesion than the other deposition
methods.12,28 The attraction of the positively charged chitosan
chains to the electrode can lead to tightly packed films with low
water content. The density of the film can be varied by adjusting
the applied voltage to produce either compact films or hydrogels.
Although the mechanism of chitosan adhesion to substrates is
not entirely clear, problems with the adhesion have generally not
been encountered.
Strategies for chitosan modification
Chitosan films can be readily functionalized with other

substances such as polymers, nanostructures, biomolecules, or
dyes. The purpose of these modifications is typically to improve
the films mechanical properties, to make it selectively responsive
to certain stimuli, or to immobilize biomolecules. Table 1 lists
common modifications of chitosan that have been reported in
literature and their applications. Many of these examples come
from work on macroscale devices. However, the same modifications should be readily applicable to microdevices as well. The
table aims to show the versatility of chitosan and is by no means
exhaustive.
Multiple different methods have been demonstrated for chitosan modification. The modifying substance can be dissolved in
the chitosan solution and co-deposited with the chitosan to form
a composite film. Alternatively, a pure chitosan film can be
deposited first, followed by attachment of the modifying
substance. The chitosan deposition can be performed by solution
casting, printing, or electrically. Therefore, a large variety of
chitosan films can be formed by choosing a different modifying
substance, modification method, and deposition method.
4.1 Assembly of biomolecules
Due to its porous structure and dense amine groups, chitosan is
well-suited for attachment of biomolecules. Chitosan films have
been modified with proteins, enzymes, antibodies, and DNA
(Table 1). The typical application of chitosan films with
biomolecules is selective coatings for biosensors.27,62,64,79,82,103
When a matching element (enzyme substrate, antigen, or
complementary DNA) binds to the coating, a measurable
physical signal is generated that is transduced by the sensor.
Chitosan films modified with enzymes can also be used for
catalysis in microfluidic reactors.58 The modification of chitosan
films with certain structural proteins such as gelatin or hemoglobin improves their mechanical properties, making them useful
as a cell scaffold67 or pH responsive material.72,73
Table 1 Survey of chitosan modifications reported in literature

Modifying substance
Proteins
Collagen
Gelatin
Gelatin
Green fluorescent protein (GFP)
Hemoglobin
Myoglobin
Protein A
Protein G
Enzymes
Acetylcholinesterase
Alcohol oxidase
Catalase
Creatinine iminohydrolase
Glucose dehydrogenase
Glucose oxidase
Glutamate oxidase
Horseradish peroxidase
Laccase
Lactate oxidase
Lipase
Polyphenol oxidase
S-adenosylhomocysteine
nucleosidase (Pfs)
Sulfite oxidase
Tyrosinase
Urease
Uricase
Antibodies
Alginate-factor B
Anti-CEA
Carbohydrate antigen 19-9
Immunoglobulin G
DNA
dsDNA
ssDNA
Nanostructures
Carbon nanotubes
Carbon nanotubes
Carbon nanotubes
Carbon nanotubes
Fe3O4 nanoparticles
Gold nanoparticles
Gold nanoparticles
Gold nanoparticles
Metallophthalocyanines
Tobacco mosaic virus
Vesicles (liposomes)
Polymers
Cellulose
Poly(acryl amide)
Poly(diallyldimethylammonium
chloride)
Poly(o-ethoxyaniline) and
poly(methacrylic acid)
Polyaniline
Polyvinyl alcohol (PVA)
Dyes
Acetyl yellow 9
Azure C
Lucifer yellow VS
4-(2-Pyridylazo)resorcinol
Procion brown, procion green
Prussian blue
Purpose of modification
References
Scaffolds for cell assembly

Used as a model protein
pH responsive hydrogels
Used as a model protein
Direct electrochemistry of proteins
Direct electrochemistry of proteins
Attachment of antibodies
Attachment of antibodies
67
61
72,73
29,46,57,59,61
74,75
74
76
77
Pesticides detection
Ethanol detection
Hydrogen peroxide detection
Creatinine detection
Glucose detection
Glucose detection
Glutamate detection
Catechol detection
Lactate detection
Processing of lipids
Phenol detection
Studies of bacterial signaling
25,62
78
74,79
80
81
35,36,38,63,8289
90
26,74,9195
19
96
97,98
99
58,60
Sulfite detection
Phenol detection
Urea detection
Uric acid detection
30
31,33,100
101
102
Detection of factor B in blood

plasma
Detection of carcinoembryonic
antigen
Detection of carbohydrate antigen
Used as model antibody
64
103
104
76
Effect of drugs on dsDNA

Sequence specific detection of DNA
(hybridization)
42
27,37,61,105,106
Glucose detection
Fabrication of composite films
Insulin detection
Phenol detection
Pesticides detection
Glucose detection
Dopamine detection
Assembly of nanoscale materials
Drug delivery, chemical signaling
17,66
63,88,107
108
109
94
62
35
92
41
110
111
Electroactive polymers for

actuation
pH responsive hydrogels, drug
delivery
Temperature and pH responsive
hydrogels
Impedance spectroscopy for copper
ion detection
Electroactive polymers for
actuation
112,113
Permeability control
NADH detection
Permeability control
Detection of Cobalt
Affinity ligand for lysozyme
purification
18,73,114
16
39
17,65
15
86
115
116
117
118120
121,122
Lab Chip, 2010, 10, 30263042 | 3031
Table 1 (Contd. )
Modifying substance
Purpose of modification
References
Redox salts
Thionine
Toluidine blue O
Tungsten oxide (WO3)
Detection of glucose, glutamate,

galactose
Glucose detection
Optical pH sensor
NADH detection
Electrochromic films
123
124
125
40
The simplest method for attaching biomolecules to chitosan is

by surface adsorption. The deposited chitosan film is exposed to
a solution of biomolecules, and some of them bind to the
surface.25,33,37,38,42,62,64,80 Since the chitosan chains are positively
charged, this approach works particularly well for negatively
charged molecules due to the electrostatic interaction.38,42,64 The
biomolecules can also be co-deposited with the chitosan by mixing them in the chitosan solution prior to deposition.19,31,74 Since
biomolecules are normally large, they are trapped in the chitosan
matrix even if there is no chemical bond or electrostatic attraction. The content of biomolecules in the film can be varied by their
concentration in the initial solution. Both the surface adsorption
and physical entrapment methods are straightforward, but they
might not provide stable immobilization. The biomolecules can
leak out over time since the interaction with the chitosan is weak.
An improved method for attaching biomolecules to chitosan is
covalent bonding. For this purpose, the biomolecule of interest
must have a functional group that can be linked to chitosans
amines. For example, proteins56,57 and DNA27,61,105,106,126 tagged
with amine groups can be attached to the chitosans amines by
glutaraldehyde. The glutaraldehyde molecule has aldehydes on
both sides that covalently bind to amines, therefore establishing
a strong link between the chitosan and the biomolecules.
Biomolecules tagged with tyrosine can be bound to the chitosan
by first activating them with tyrosinase. This enzyme converts the
tyrosine residue into a reactive o-quinone, which binds to the
amine groups of chitosan.29,58,59,61 The covalent binding of
biomolecules to chitosan can be performed after a plain chitosan
film is deposited,56,57,105,106 resulting in biomolecule attachment to
the film surface only. It can also be performed prior to the
deposition by conjugating the biomolecules to chitosan chains in
solution.46,58,61 This produces a composite chitosan film with
biomolecules dispersed throughout its volume. We should note
that since the chitosan deposition solution is mildly acidic (pH less
than about 6) and deposition takes a few minutes, extremely acidlabile biomolecules may not be good candidates for co-deposition. For such labile biomolecules, the first approach would be
more appropriate. Once a chitosan film is deposited, it is neutral
and the biomolecules are not exposed to harsh conditions.
The covalent bonding approach results in more stable
attachment of biomolecules than the surface absorption and
physical entrapment methods. It also provides some control over
the spatial orientation of the biomolecule, which is attached to
the chitosan matrix at a known site. However, the covalent
bonding is more complicated, and it requires the biomolecule to
have accessible functional groups. Ultimately, the choice of
coupling approach depends on the biomolecule in use and on the
application of the sensor.
3032 | Lab Chip, 2010, 10, 30263042
4.2 Modification with nanostructures

Chitosan films have been modified with various nanostructures
such as carbon nanotubes, metallic nanoparticles, vesicles, and
even viruses (Table 1). The modifications with carbon nanotubes
are aimed at increasing the mechanical strength of chitosanbased pH responsive hydrogels and to improve the films
conductivity, resulting in better electrocatalytic activity of chitosan-coated electrodes for amperometric sensors.17,66,88,107,126,127
Similarly, chitosan films modified with gold nanoparticles have
been used in enzyme-based amperometric biosensors.35,62,92 In
addition to improving the electron transfer between the electrode
and the solution, the nanoparticles have been shown to enhance
the specificity of the sensor due to their interaction with the
analyte. Nanoparticles also increase the chitosan surface area
available for enzyme immobilization and thus improve the
overall catalytic activity.94
Chitosan films have been successfully modified with vesicles,
including both synthetic surfactant vesicles and liposomes.111
The vesicles can be filled with other substances such as drugs or
signaling chemicals, and they can be released from the chitosan
by dissolving it in a low pH solution. Therefore, chitosan films
with vesicles can serve for storage and controlled release of
substances.
The modification of chitosan with nanostructures can be performed either by co-deposition88,107,108,111 or layer-by-layer
deposition (LBL).35,39,41 The success of the co-deposition
approach depends on how the nanoparticles impact the filmforming properties of chitosan. Sonic agitation can be used to
disperse the nanotubes or nanoparticles in the chitosan solution
if they aggregate.66 The LBL approach relies on ionic interactions
between a deposited chitosan film exposed to a solution of
nanoparticles, and it works mainly for particles with negative
charges (since chitosan is cationic).
Improved control over the assembly of the nanostructures in
chitosan can be achieved if they contain a specific recognition
element. For example, Yi et al. demonstrated assembly of viruses
on chitosan functionalized with DNA.110 Exposed RNA at one
end of the virus hybridized with the chitosan-bound DNA,
thereby anchoring the virus on the chitosan film. The viruses
attach only to chitosan with the correct DNA sequence, allowing
for spatial selectivity of the assembly.
4.3 Codeposition with other polymers

Chitosan can be co-deposited with other polymers to alter its
mechanical properties (Table 1). Chitosan hydrogels with large
water content are very sensitive to pH and are useful for
controlled drug release or actuation; however, they have low

mechanical strength. It has been shown that combining the chitosan with polymers such as polyvinyl alcohol (PVA)15 or
poly(acryl amide)18 considerably increases the fracture strength
and the elastic modulus of the films. The combination of chitosan
with other polymers also alters its micropore structure and
allows for tuning its swelling behavior.114,128
Chitosan modified with cellulose has been used to make
actuator films, also called electroactive paper.112 Cellulose
exhibits a piezoelectricity effect, which is conferred to the chitosan-cellulose composite. Chitosan modified with polyaniline
has also been demonstrated as an actuator.17 In this type of
composite material, two modes of actuation are possible.
Changing the solution pH causes large strains due to chitosans
swelling, while redox reactions of the polyaniline cause smallscale strain adjustments.
4.4 Modification with dyes
Chitosan films have been successfully functionalized with several
different dyes (Table 1). Dyes that act as chromogenic agents
undergo an optical absorbance change due to certain chemical or
physical conditions and are useful for optical sensing. For
example, chitosan modified with 2-(4-Pyridylazo)resorcinol is
capable of detecting cobalt ions.117 Chitosan modified with thionine undergoes a color change with pH variation. Chitosan
modified with tungsten oxide changes its color due to redox
reactions.40 Other types of dyes are added to chitosan to exploit
their chemical properties and not necessarily their color. For
example Prussian blue has catalytic capabilities for hydrogen
peroxide, and it has been used with chitosan in amperometric
glucose sensors.121,122 Procion brown and procion green dyes are
ligands for certain proteins and have been combined with chitosan to make affinity membranes for lysozyme purification.118120
5 Chitosan characterization methods

A variety of methods have been employed to determine the
physical and chemical properties of pure and modified chitosan.
Some of these methods are not applicable to chitosan deposited
in lab-on-a-chip devices, and specialized samples need to be
fabricated (e.g. freestanding films or films on transparent
substrates). Advanced characterization procedures, such as
atomic force microscopy (AFM), Fourier Transform Infrared
Spectrometry (FT-IR), and X-Ray Diffraction (XRD) are performed to investigate subtle structural or compositional changes
in the chitosan film following fabrication or modification with
other substances. The results from relatively simple characterization techniques such as absorbance measurements, tensile
testing, or cyclic voltammetry can also be quite valuable, especially for the development of sensors. As discussed in section 6.2,
the operation of most chitosan-based sensors is based on
a change in optical absorbance, mechanical strain, or electrical
conductivity.
5.1 Fluorescence microscopy
Fluorescence microscopy is commonly used to confirm the
localized patterning of chitosan films.12,47 Unmodified chitosan is
transparent and is difficult to image with a standard optical
microscope. However, it can be readily conjugated with fluorescent dyes and imaged with a fluorescent microscope. Fig. 5 is
a micrograph of electrodeposited chitosan labeled with fluorescein, demonstrating the selective pattering of the film.
Fluorescence microscopy is also used to verify the presence of
fluorescently active biomolecules in a deposited chitosan film.61
For example, green fluorescent protein (GFP) has been used to
demonstrate the capability of chitosan to bind proteins.57 Fluorescent intensity images of chitosan films were used to confirm
the entrapment of vesicles filled with fluorescent substances.111
The fluorescence microscopy method is compatible with nearly
all types of bioMEMS devices, and it does not require any
specialized sample fabrication.
5.2 Fourier transform infrared (FT-IR) and UV-VIS
spectroscopy
FT-IR spectroscopy identifies the molecular structures that are
present in a substance based on their respective absorption bands
in the infrared spectrum. Chitosan exhibits characteristic
absorption bands at 3400, 2886, 1643 and 1593 cm1. These
features correspond to the stretching or bending vibrations of
chitosans hydroxyl groups (OH), protonated amine groups
(NH3), carbonyl groups (NHC]O), and amine groups
(NH2), respectively.
Cheng et al. and Ma et al. performed FT-IR measurements to
verify that their respective fabrication techniques with chitosan
did not alter its chemical structure.14,129 Similarly, Kam et al.
used FT-IR to characterize any change to chitosan that may
occur due to storage conditions.130 The results of chemical
reactions, including covalent bonding of molecules to chitosans
amine groups or enzyme-catalyzed hydrolysis, can also be
observed by FT-IR.22,83 Therefore, this method is commonly
used to confirm that certain proteins or nanoparticles are
Fig. 5 Resolution of spaces between 500 mm wide gold lines after chitosan deposition and subsequent reaction with NHS fluorescein. The widths of
spaces were 1000, 500, 200, and 50 mm. Reprinted with permission from ref. 12. Copyright 2003 American Chemical Society.
Lab Chip, 2010, 10, 30263042 | 3033
immobilized within the chitosan and that they have retained their
structure following deposition.25,30,41,62,86,104,131133 The co-deposition of chitosan with other polymers can also be studied with
FTIR.15,67,134,135 For example, Cai et al. added cellulose to
chitosan to decrease the blends sensitivity to humidity and used
FT-IR to characterize the effect of blending different ratios
together.113
UV-VIS spectroscopy measures the light absorbance of the
sample in the UV and visible ranges. Traditionally, it has been
used along with the BeerLambert law to measure the concentration of an absorbing species in liquid, but it is also suitable for
transparent solids. Many research groups have used UV-VIS
measurements to study changes that occur in chitosan upon
conjugation with other substances such as enzymes or metallic
nanoparticles.41,62,86,92,103 For example, Wu et al. noted a large
absorption increase around 420 nm when chitosan is subjected to
oxidized catechol molecules.22 This effect has been used to make
absorbance-based optical sensors for catechol detection,54 which
are discussed in section 6.2.
5.3 X-Ray diffraction
X-Ray diffraction (XRD) can yield further information
regarding the crystalline structure of the solid. Chitosan exhibits
a fairly broad peak due to its amorphous structure at 2q of 20 .
Studies have suggested that neutralization of chitosan by NaOH
induces crystalline formations as evidenced by the creation of
peaks at 2q of 10 and 15 .130 Ma et al. performed XRD
measurements to characterize structural changes in the chitosan
resulting from different degrees of chitin deacetylation.129 Ogawa
et al. used XRD data to directly compare the molecular and
crystallographic differences between chitin and chitosan.136
Characterizing the changes in chitosans crystalline structure
may be useful for determining the density of exposed surface
amine groups. These amine groups are crucial for the conjugation of biomolecules to the chitosan and are what make it
a functionalization layer in sensors.
5.4 Atomic force microscopy (AFM)
Nanometer changes in the morphology of the chitosan film can
be determined using atomic force microscopy (AFM). A microscale cantilever with a sharp probe tip is scanned over the chitosan surface. The minute deflections of the cantilever are
measured to create a surface topography map. Cai et al. used
AFM to study the morphology of adsorbed chitosan films on
glass slides.137 AFM measurements are also often used to confirm
cross-linking reactions at the chitosan surface and to verify
successful conjugation of chitosan with other materials such as
metallic nanoparticles. Lin et al. observed an increase in thickness and surface regularity for crosslinked chitosan vs. noncrosslinked chitosan.104 Du et al. used AFM imaging to ensure
that gold nanoparticles were embedded in the chitosan film
following the chemical reaction between tetrachloroauric (III)
acid, acetic acid and chitosan.62 Similar studies have been performed on chitosan films containing metallic nanoparticles.94
The morphology of chitosan films that have been patterned using
either nanoimprint lithography or microcontact printing is also
commonly examined using AFM.13,44,45 Example images of
3034 | Lab Chip, 2010, 10, 30263042
Fig. 6 Examples of AFM images taken of (a) printed chitosan squares

6.7 mm across and (b) chitosan nanodots with 150 nm width and 400 nm
pitch. Reprinted with permission from ref. 13. Copyright 2007, American
Institute of Physics.
chitosan patterned using nanoimprint lithography can be seen in

Fig. 6
5.5 Cyclic voltammetry
Cyclic voltammetry (CV) is an electrochemical method for
quantifying redox reactions of target species within the chitosan
films. The redox activity of compounds such as thionine, DNA,
dopamine and other proteins conjugated with chitosan have been
reported.37,74,124 Siqueira et al. utilized cyclic voltammetry
measurements to confirm the number of bilayers and the types of
metallic nanoparticles embedded in layer-by-layer chitosan
films.41 Since the electrodeposition of chitosan involves a reduction reaction at the cathode, cyclic voltammetry has been utilized
to characterize deposition conditions and the permeability of
chitosan films to various redox species.23 By detecting the onset
of hydrogen gas evolution at the cathode due to increasing
potential, the electrodeposition conditions of chitosan can be
more accurately controlled with regard to film thickness and
uniformity.
Although most of these studies involve large electrodes placed
in a beaker of solution, the CV method can be implemented on
the microscale through standard fabrication techniques. Many
groups have already demonstrated CV measurements for film
characterization or analyte detection with patterned micro-scale
electrodes.138140
5.6 Tensile testing
Understanding the mechanical properties of chitosan and their
dependence on environmental conditions aids in the fabrication
of stronger or more flexible films. The stiffness and strength of
chitosan can be determined by tensile testing. This requires
preparation of a freestanding film or fiber that is significantly
longer in one direction compared to the other two. The sample is
stretched with increasing tensile force until it breaks. The
Youngs modulus can be extracted from the resulting stressstrain curve; it has been measured for several different
fabrication methods and for blends of chitosan with other
materials.14,22,66,113 Changes in chitosans mechanical properties
can be used to characterize structural variations in the chitosan

film as well as fluctuations in environmental conditions. For
example, Cai et al. observed a decrease in Youngs modulus for
chitosan/cellulose blends with increasing chitosan content and
attributed this to a decrease in crystallinity combined with an
increase in moisture uptake caused by the chitosan.113 Kim et al.
also used a chitosan/cellulose blend to investigate the effect that
free ions such as Cl and NO3 have on the mechanical behavior
of the film.112 Chitosan fibers have also been shown to undergo
strain variations with the pH of the solution.17,65
As expected, crosslinking the chitosan chains yields an increase
in the mechanical stiffness.22,34,71 Adekogbe et al. observed this
effect after crosslinking chitosan with DTBP and also found that
tensile strength increases for higher degrees of chitosan
deacetylation.71
6 Microscale devices using chitosan

The majority of chitosan-based devices reported in literature to
date are macroscale. However, significant progress has been
made in the area of microfabrication with chitosan as discussed
in section 2, and the number of chitosan-based microscale
devices is rapidly increasing. The unique properties of chitosan
and its multiple possible modifications make it particularly useful
for bioMEMS applications. Here, we review recent demonstrations of microdevices functionalized with chitosan. These
examples fall under two broad categories: platforms for assembly
of other substances and sensors.
6.1 Immobilization platforms

Chitosan can serve as a matrix for immobilizing other substances
such as enzymes, drugs, and even living cells. This capability is
essential in microdevices used for biocatalysis, drug delivery, and
cell studies.
Xie et al. reported a microneedle array for transdermal
transport of drug compounds dispersed in chitosan films.141 The
model drugs calcein and bovine serum albumin (BSA) are dissolved in a chitosan solution. A thin chitosan film is then
deposited on a silicon microneedle array by solution casting.
When pressing the array on rat skin, the chitosan-coated
microneedles penetrate the outer protective layer of the skin and
increase its permeability to the drugs. The permeation rate of the
drugs can be regulated by the chitosan film thickness and by the
concentration of drugs in the chitosan. This device provides
a way for controlled transdermal delivery of therapeutic agents
as an alternative to oral administration.
Co et al. used chitosan to micropattern two different cell types
on the same substrate sequentially.142 First, the chitosan-coated
substrate is patterned with a cell-resistant polyelectrolyte by
microcontact printing. The first set of cells adhere only to the
exposed chitosan. Then, the cell-resistant polyelectrolyte is
covered with fresh chitosan, making these regions adhesive to
cells again. The substrate is exposed to a second type of cells,
which adhere to the new chitosan but not to the regions already
occupied by cells. This approach can be used for incorporating
different cell types into microdevices with sub-cell patterning
resolution.
Kastantin et al. constructed a polymeric microfluidic device

capable of patterning proteins by means of chitosan electrodeposition.56 It consists of SU-8 channels with a PDMS capping
layer and polypyrrole electrodes. Chitosan is deposited on the
electrodes inside the fluidic channel, and the model protein GFP
(green fluorescent protein) is assembled in the device by glutaraldehyde covalent bonding. The GFP retains its fluorescence,
indicating that the structure of the protein is preserved. The
chitosan deposition and GFP attachment are addressable, and
they occur only at the electrodes on which a voltage is applied.
This demonstrates for the first time the feasibility of chitosanmediated protein assembly inside microfluidic devices with
spatial selectivity and electrical control.
Park et al. developed another microfluidic device for biomolecule assembly using electrodeposited chitosan.57 This implementation consists of SU-8 channels, gold electrodes, a PDMS
gasket, and a Plexiglass compression package for reversible
sealing. The package can be disassembled after use, allowing the
chitosan film thickness inside the device to be measured by
contact profilometry. As seen in Fig. 7, the model protein GFP is
assembled only on electrodes where a voltage is applied. Traditional protein patterning methods such as microcontact printing
require physical access to the device surface and must be performed before packaging. However, the chitosan electrodeposition method enables the biomolecule patterning to be performed
after packaging and therefore decouples the manufacturing of
the device from its use. The microfluidic device can be completely
fabricated, packaged and stored without any perishable
biomolecules. The biomolecules can be later added by chitosan
mediated assembly when the device is ready for use.
The microfluidic platform demonstrated by Park et al. was
further developed by Luo et al. and adapted to enzyme
assembly.58 The device in this work is aimed at studying a bacterial metabolic pathway. The enzyme S-adenosylhomocysteine
nucleosidase (Pfs), which participates in the synthesis of bacterial
signaling molecules, is tagged with a tyrosine residue and is
conjugated with chitosan in solution. The chitosan-Pfs conjugate
is electrodeposited on electrodes inside the microfluidic channel
after complete packaging of the device as shown in Fig. 8 It was
shown that the chitosan-immobilized Pfs retains its catalytic
activity and even that it has better long-term stability than the free
enzyme in solution. The chitosan can be dissolved with a mildly
acidic wash, allowing the device to be reused multiple times.
Fernandes et al., in collaboration with Luo, used a similar
microfluidic device to conjugate biological nanofactories to an
immobilized chitosan film.143 These nanofactories shown schematically in Fig. 9 consist of linked enzymes for substrate
conversion as well as antibodies for cell entrapment. In this
study, Escherichia coli was captured by the nanofactories while
their response to an autoinducer signaling molecule (AI-2) was
observed via the production of GFP by the bacteria. The chitosan-provided spatial control of the biological nanofactories
allows for the response of the captured bacteria to be observed as
a function of outwardly controlled parameters such as pH,
temperature or flow rate.
Luo et al. continued the work involving enzyme assembly with
chitosan by improving the microfluidic packaging in order to
optimize the signal-to-noise ratio of the enzymatic conversion.144
The device is improved by using an alignment scheme for the
Lab Chip, 2010, 10, 30263042 | 3035
Fig. 7 Chitosan mediated assembly of GFP in micro fluidic device. (a) Fluorescence image of the assembly site after glutaraldehyde reaction; (b) after
GFP reaction; (c) negative control (no bias); (d) GFP conjugated chitosan film; (e) Image J fluorescence surface plot.57Reproduced by permission of
the Royal Society of Chemistry.
fluidic interconnects which reduces the dead volume created by

reservoirs at the inputs and outputs. In addition, a cross channel
design over the sensor site allows the user to separate the path of
the enzyme flow from that of the substrate. This separation
reduces parasitic enzymatic conversion that may result from nonspecifically bound enzymes on the channel walls. By implementing both improvements into the design of the device, the
signal-to-noise ratio of the enzymatic conversion was improved
from 0.72 to 2.43.
6.2 Sensors
The other category of microdevices that employ chitosan are
sensors. Typically, in this case the chitosan is modified with
probe biomolecules that serve as specific recognition elements.

When a target biomolecule in the sample binds to the probe,
a physical signal is produced such as a change of mass, strain,
optical absorbance, or electrical conductivity. This signal is then
detected by mechanical, optical, or electrochemical means.
Zhu et al. reported micromachined amperometric sensors for
glucose, glutamate, and galactose using chitosan membranes.24
The device has a pyramidal chamber array etched in silicon
anisotropically (Fig. 10). Each chamber contains a platinum
electrode at the bottom covered with a solution-cast chitosan
membrane. The membranes are modified with the enzymes
glucose oxidase or galactose oxidase by means of glutaraldehyde
crosslinking. The enzymes perform highly specific conversion of
the samples, and the products of the conversion are detected by
Fig. 8 Schematic of chitosan conjugation and electrodeposition in a microfluidic channel. (a) Prefabricated device, (b) conjugation between chitosan
and the enzyme Pfs, (c) electrodeposition of Pfs-chitosan conjugate, (d) enzymatic small molecule detection, (e) mild acid wash removes chitosan and
refreshes electrode surface.58Reproduced by permission of the Royal Society of Chemistry.
3036 | Lab Chip, 2010, 10, 30263042
nanotubes for enhanced electrical conductivity and deposited

over an electrode by solution casting. The bacteria Pseudomonas
fluorescens and Pseudomonas putida are then allowed to adhere to
the chitosan film over a period of 30 min. The reduction of the
dissolved oxygen produced by bacterial respiration in the presence of glucose is electrochemically detected. The biosensor is
used to test the effect of varying pH, temperature, cell density,
and sugar type to the respiratory activity of the bacteria. This
device demonstrates how the biocompatible nature of chitosan
can be used to help analyze biomolecules in vitro.
Powers et al. developed a multipurpose optical detection
platform with an electrodeposited chitosan film.20 The device
consists of an SU-8 microfluidic channel and an optical waveguide defined on a Pyrex substrate as shown in Fig. 11. One of the
facets of the optical waveguide is located at the sidewall of the
fluidic channel and is covered with a transparent ITO electrode
for chitosan deposition. This allows a chitosan film to be formed
in the path of light passing through the waveguide. Molecules
attached to the chitosan film are detected by the change in the
output light spectrum, which is analyzed with an optical fiber
coupled spectrometer. The concentration of analytes attached to
the chitosan can be much higher than their concentration in the
solution. Therefore, the chitosan film acts as a pre-concentrator
and enhances the optical detection signal. Furthermore, the
ability to deposit the chitosan film out-of-plane (i.e. perpendicular to the substrate surface) and the use of in-plane optics allows
for dense integration of the sensors. This demonstrated chitosan
Fig. 9 Assembly and manipulation of quorum sensing (QS) bacteria in
a bioMEMS device via biological nanofactories. (a) Components of
a biological nanofactory: cell targeting module (antibody); sensing,
synthesis and assembly modules (HGLPT nano-construct). (b) The
nanofactories are spatially assembled onto chitosan electrodeposited
within the device; they capture targeted bacteria and manipulate their QS
response.143Reproduced by permission of the Royal Society of
Chemistry.
cyclic voltammetry. In addition to enzyme immobilization, the

chitosan membrane also has a second role. Due to its selective
permeability, it reduces the penetration of interferents such as
uric acid or ascorbic acid to the electrode and improves the
specificity of the sensors.
Odaci et al. demonstrated the use of chitosan for the immobilization of microbial cells and further sensing of their respiratory activity.145 Chitosan solutions are mixed with carbon
Fig. 11 Perspective view of optical waveguide sensor with chitosan film

on sidewall. The waveguide is shortened to show fiber coupling. The blue
arrow represents the optical excitation signal while the red arrow represents the collected fluorescence signal.20Reproduced by permission of
the Royal Society of Chemistry.
Fig. 10 Schematic of micromachined amperometric sensor. 1. Pyrex glass down-substrate, 2. Pt WE, 3. Conduction line, 4. Pads, 5. Up-substrate made
of silicon, 6. Quadratic hole, 7. Chitosan membrane with cross-linked enzyme, 8. Ag/AgCl RE. (a) Down-substrate, (b) up-substrate, (c) bonding
between up- and down-substrates, (d) cross section along A-A0 . Redrawn with permission from ref. 24.
Lab Chip, 2010, 10, 30263042 | 3037
sidewall patterning technique is crucial for the design of future

optical, in-plane, lab-on-a-chip sensors.
The same optical platform was applied to detection of DNA
hybridization.21 In this work, the chitosan film is functionalized
with amine-tagged probe DNA by glutaraldehyde crosslinking.
It is then exposed to fluorescently labeled target DNA with two
different sequences. The spectrum changes of the output light
confirm that sequence specific binding of the target to the probe
occurs. The device is also used for detection of phenols after
some modifications to the microfluidic design.54 The microfluidic
channel is enclosed using a capping layer of PDMS while metal
capillaries and plastic tubing allow for continuous fluid flow
through the device as shown in Fig. 12. It is shown that the
products of phenol electrochemical oxidation readily bind to the
amine groups of the chitosan film and cause a measurable change
in its optical absorbance. Various phenol concentrations are
detected and a dramatic increase in the absorbance signal is
observed for devices that include the chitosan film vs. those that
do not. Common interferents such as ascorbic acid do not react
with the chitosan, which results in good selectivity of the phenol
sensor.
Some other demonstrations of optical sensors using chitosan
have also been reported. Lin et al. constructed a chemiluminescent immunosensor for Carbohydrate Antigen 19-9 (CA19-9)
containing a chitosan membrane.104 The membrane is functionalized with CA19-9 and placed inside a fluidic channel monitored
by a photomultiplier tube. For detection, the CA19-9 sample is
mixed with enzymatically labeled CA19-9 antibodies that
produce a chemiluminescent signal when injected into the
channel. The concentration of CA19-9 in the sample is determined indirectly from the power of that signal. Yusof et al.
reported an optical sensor for measuring cobalt ion concentration in solution.117 A chitosan membrane doped with the chromogenic reagent 4-(2-Pyridylazo) resorcinol (PAR) is prepared
and placed inside a flow cell. The PAR undergoes a color change
when exposed to cobalt. Optical fibers are used to shine light on
the membrane, collect the reflected light, and guide it to a spectrometer. The cobalt concentration can be reliably determined
from the measured membrane reflectance.
Resonant sensors using chitosan have also been demonstrated.
Deng et al. reported a piezoelectric immunosensor for the
detection of the enzyme factor B in human serum.64 A piezoelectric quartz crystal (PQC) with gold electrodes is first coated
with a self assembled monolayer of cysteamine and then with
chitosan by glutaraldehyde crosslinking. Factor B antibodies
conjugated with a negatively charged polymer, alginate, are
Fig. 12 Schematic of the microfluidic sensor for phenol detection. Both

waveguides patterned perpendicular to the channel allow for in-plane
light interrogation. Reprinted from ref. 54 with permission from Elsevier.
3038 | Lab Chip, 2010, 10, 30263042
adsorbed on the chitosan. When the device is exposed to samples

containing factor B, its resonant frequency decreases in response
to the mass loading caused by the immunorecognition. Experiments without chitosan were also performed, in which the antibodies were attached to the cysteamine monolayer directly by
glutaraldehyde crosslinking. It was discovered that the use of
chitosan significantly improves the response of the sensor,
presumably by better preserving the biological activity of the
antibodies. The chitosan mediated assembly also allows the
sensor to be more easily regenerated by washing off the alginateantibody complex with a saline solution.
Another chitosan-based PQC sensor was developed by Zhao
et al. for determination of fish freshness.146 The device surface is
coated with a chitosan film containing pimelic acid. The sensor
exhibits a specific response to trimethylamine (TMA) vapor,
which is a byproduct of fish decomposition and can be used as an
indicator of its freshness. TMA concentrations down to 50 ppm
are reliably detected, and the response to interfering gases such as
water vapor is shown to be negligible.
A different type of chitosan-coated resonator was constructed
by Gao et al. for the detection of glucose in urine.83 A 28 mm thick
magnetoelastic element is covered with a pH responsive polymer
and with chitosan conjugated to glucose oxidase. Any glucose in
the sample is converted by the enzyme into gluconic acid and
reduces the pH of the solution, increasing the mass of the pH
responsive polymer. The magnetoelastic element is excited by
a time-varying magnetic field to vibrate at its resonant frequency,
which depends on the mass loading of its surface. As the element
vibrates, it generates a secondary magnetic filed that is measured
by a pickup coil and used to determine the resonant frequency.
Therefore, the concentration of glucose can be measured wirelessly by the resonant frequency shift.
Koev et al. has demonstrated microcantilever sensors functionalized with electrodeposited chitosan.27 These devices are
micromachined beams (Fig. 13) that can be used for detection
either in dynamic (resonant) or static mode. In the dynamic
mode, the cantilever is actuated electrostatically, and its resonant
Fig. 13 (a) Schematic of microcantilever with chitosan used for detection of DNA hybridization and for detection of dopamine electrochemical oxidation. (b) SEM of fabricated cantilever. (c) Optical
micrograph of cantilever after chitosan electrodeposition. The chitosan is
deposited everywhere except at the electrically isolated tip. (d) Contact
profiler scan of chitosan film along dashed line in C.27Reproduced by
permission of the Royal Society of Chemistry.
frequency is measured. Any change of mass on the surface causes

a resonant frequency shift. In the static mode, the position of the
cantilever tip is measured. Substances that bind to the surface
create a stress and deflect the beam. The cantilevers are functionalized with a chitosan-based coating to make them specific,
and are used for the detection of DNA hybridization or phenols.
For the DNA hybridization experiments, the coating consists of
probe DNA immobilized on chitosan by glutaraldehyde crosslinking. Sequence-specific detection of target DNA was demonstrated by both the dynamic and static methods. The measured
sensor response was significantly higher than values reported in
literature for similar sensors without chitosan.
For the phenol detection experiments, the cantilever coating is
an unmodified chitosan film.27,52 The phenol samples are electrochemically oxidized; the products of the oxidation are known
to crosslink chitosan and change its structure. The crosslinking
causes a stress in the film and bends the cantilever, allowing for
static-mode detection. This chitosan mediated detection was
shown to be specific to phenols. For example, the oxidation of
ascorbic acid did not cause chitosan crosslinking and cantilever
bending.
Mao et al. used microcantilever sensors to characterize the
swelling behavior of chitosan/gelatin hydrogels.72 Due to
protonation and deprotonation of the amine groups in the chitosan, the hydrogels undergo volume changes when the pH is
varied and can be useful for controlled release devices. Cantilevers are coated with mixtures of chitosan and gelatin, and their
deflections under different pH conditions are measured. It was
shown that the swelling depends on the ratio of chitosan to
gelatin and the content of the crosslinking agent. The device
demonstrated in this work can potentially be used for biosensors
by incorporating enzymes that cause pH changes.
The examples of devices presented so far used chitosan as
a coating layer but not as a structural material. Cheng et al.
reported a cantilever made entirely of chitosan.14 The device is
fabricated by spin casting of chitosan, conventional photolithography with photoresist, etching of the chitosan with oxygen
plasma, and etching the Si substrate under the chitosan with
XeF2. An SEM of the released cantilever is shown in Fig. 14. The
ability to construct devices entirely of chitosan can potentially
simplify the fabrication process and lower the cost.
Fig. 14 Partially released chitosan cantilever on silicon with dimensions

240 25 2 mm. Chitosan serves as a sufficient structural material to
support itself. Reprinted with permission from ref. 14. 2008 IEEE.
Conclusions
The polysaccharide chitosan provides a unique interface between

microdevices and biology. It offers the ability to realize lab-on-achip devices with spatiotemporal control of biomolecules and
other organic compounds. In reviewing the properties of chitosan, it is instructive to discuss briefly other biofunctional materials used in MEMS. These alternative approaches can be
broadly divided into self assembled monolayers and surface
immobilized polymers.
Self-assembled monolayers (SAMs) are organized layers of
molecules with a head group and a tail.147150 The head group,
typically silane or thiol, has a binding affinity for the substrate;
the tail has a functional group, to which biomolecules can be
anchored. A variety of SAM systems have been demonstrated.
They provide very strong biomolecule attachment to the surface,
and they are used extensively in the area of DNA microarrays.151
Although SAMs have many excellent properties, chitosan has
some potential advantages. It has been shown that chitosan
provides a stronger response27,64 and better long-term
stability58,100 of sensors with immobilized biomolecules than the
ones bound directly to the surface by SAMs. This may be due to
the large water content in the chitosan film, which provides
a favorable microenvironment for the biomolecules and
preserves their three-dimensional structure. In addition, the high
density of amine groups on the chitosan network and its large
surface area allows for high loading of the film with biomolecules. These effects are still not completely understood and need
further study.
Another potential advantage of chitosan over SAMs is its
robustness. SAMs require atomically clean surfaces and fail to
form even if there is minor contamination.148,149 They require
rigorous cleaning procedures with piranha solution or oxygen
plasma, which are typically not feasible for already-packaged
devices and for microfluidic channels. In contrast, chitosan is
much more tolerant to the properties of the device surface
because it does not form a chemical bond with it. A mildly acidic
wash is sufficient for cleaning the surface and even removing
previous chitosan to allow for reusing the device multiple times58
The other approach for functionalization of bioMEMS is
based on surface immobilized polymers. A wide variety of
polymers in addition to chitosan have been used in microdevices,
including alginate,152,153 cellulose,154 collagen,155 gelatin,156 polyethylene glycol,157 polypropylene glycol,152 polylysine,158,159
PLGA,160 and even spider silk.161 The functions performed by
these polymers are similar to the ones performed by chitosan:
drug release,152,160 cell culture adhesion,155,156,158,159 structural
support and actuation,154,161 scaffolding for tissue engineering,153
and biomolecule entrapment.157
There is insufficient data at present to compare the properties
of chitosan comprehensively with the other polymers. Depending
on the application, some of the polymers may offer better
performance than chitosan. For instance polylysine is more
popular for cell culture adhesion,158,159 and the mechanical
properties of spider silk are superior to those of chitosan.161
However, in general, chitosan has two advantages over other
biofunctional polymers in MEMS: the availability of dense
amine groups for biomolecule attachment and fabrication
through electrodeposition. Most other polymers can entrap
Lab Chip, 2010, 10, 30263042 | 3039
biomolecules only by physical absorption, which is weak and

may allow them to leak out over time. Chitosans amines enable
for strong covalent attachment as described in Section 4.1 and
improved long-term stability.
Most of the fabrication techniques used for chitosan have also
been demonstrated for other biopolymers as well: solution
casting,152,159 spin casting,161 printing,154 molding,160 and
photolithography.156,157 However, the electrodeposition method
is unique to chitosan, and it allows improved pattern control and
more fabrication flexibility. The other patterning methods
(printing and lithography) require physically accessible planar
surfaces. In contrast, chitosan can be electrodeposited on any
surface geometry (including vertical sidewalls20 and cantilever
beams27) and inside enclosed fluidic devices.57 Therefore, the
functionalization can be performed after complete packaging of
the device and just prior to its use.57 In addition, chitosan electrodeposition is (i) reagentless (no requirement for complex
conjugation chemistries or extensively-cleaned surfaces) and (ii)
reversible with a change in conditions (potentially, reversibility
allows recovery of the biomolecule and enables post-fabrication
biofunctionalization).
While the reviewed demonstrations of chitosan functionalization have been successful, many opportunities for future
improvement exist. Only a small portion of the possible chitosan
modifications have been employed in bioMEMS. We expect that
more of the knowledge generated by macroscale applications of
chitosan will be used for microdevices. In addition, new modification methods will be developed to further enhance the functionality of chitosan based devices. We also believe that chitosan
biofabrication methods will be improved, possibly by transitioning toward 3D shaped structures instead of 2D thin films.
This will provide more area for surface immobilization of
biomolecules, cells and other substances.
Acknowledgements
This work was supported in part by the National Science
Foundation (EFRI), the R. W. Deutsch foundation and the
Laboratory for Physical Sciences. The authors also appreciate
the support of the Maryland NanoCenter and both its Fablab
and NispLab. The NispLab is supported in part by the NSF as
a MRSEC Shared Experimental Facility.
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Chitosan: An Integrative Biomaterial For Lab-On-A-Chip Devices

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CRITICAL REVIEW

www.rsc.org/loc | Lab on a Chip

Chitosan: an integrative biomaterial for lab-on-a-chip devices

such as DNA, enzymes, or antibodies to operate with sufficient

Stephan Koev was born in Bulgaria in 1981. He received a BS

Peter Dykstra received his B.S.

One of the main challenges in the development of miniaturized

3026 | Lab Chip, 2010, 10, 30263042

This journal is The Royal Society of Chemistry 2010

attachment of biomolecules, and it can be co-deposited with

Xiaolong Luo received his B.S.E. from Zhejiang University in

Chitosan is obtained by the deacetylation of chitin. Chitin is the

Gregory F. Payne received his B.S. and M.S. degrees in

Methods for chitosan microfabrication

Chitosan is normally purchased in the form of dried flakes. These

3.1 Solution casting

Fig. 1 Structural formulas of chitin and chitosan showing the three

amount of positive charge on the chitosan chains, which in turn

The simplest fabrication method consists of coating the surface

Fig. 2 Photolithographically patterned chitosan features. (a) SEM of

This journal is The Royal Society of Chemistry 2010

The film is deposited onto a transparent substrate, and its

Fig. 3 Schematic demonstrating chitosan film electrodeposition.

3.4 Other methods

Fig. 4 SEM of chitosan film electrodeposited on the sidewall of

example, chitosan fibers with an average diameter of 100 mm

Chitosan films are fully compatible with the near-neutral

Strategies for chitosan modification

Chitosan films can be readily functionalized with other

Table 1 Survey of chitosan modifications reported in literature

This journal is The Royal Society of Chemistry 2010

Scaffolds for cell assembly

Detection of factor B in blood

Effect of drugs on dsDNA

Electroactive polymers for

Lab Chip, 2010, 10, 30263042 | 3031

Detection of glucose, glutamate,

The simplest method for attaching biomolecules to chitosan is

4.2 Modification with nanostructures

4.3 Codeposition with other polymers

controlled drug release or actuation; however, they have low

5 Chitosan characterization methods

This journal is The Royal Society of Chemistry 2010

Lab Chip, 2010, 10, 30263042 | 3033

Fig. 6 Examples of AFM images taken of (a) printed chitosan squares

chitosan patterned using nanoimprint lithography can be seen in

can be used to characterize structural variations in the chitosan

6 Microscale devices using chitosan

6.1 Immobilization platforms

Kastantin et al. constructed a polymeric microfluidic device

fluidic interconnects which reduces the dead volume created by

probe biomolecules that serve as specific recognition elements.

3036 | Lab Chip, 2010, 10, 30263042

This journal is The Royal Society of Chemistry 2010

nanotubes for enhanced electrical conductivity and deposited

cyclic voltammetry. In addition to enzyme immobilization, the

Fig. 11 Perspective view of optical waveguide sensor with chitosan film

This journal is The Royal Society of Chemistry 2010

Lab Chip, 2010, 10, 30263042 | 3037