Surface-enhanced Raman spectroscopy biosensors:

excitation spectroscopy for optimisation of

substrates fabricated by nanosphere lithography
X. Zhang, C.R. Yonzon, M.A. Young, D.A. Stuart and R.P. Van Duyne

Abstract: In the 28 years since its discovery, surface-enhanced Raman scattering (SERS) has
progressed from model system studies of pyridine on a roughened silver electrode to state-of-the-art
surface science studies and real-world sensing applications. Each year, the number of SERS
publications increases as nanoscale material design techniques advance and the importance of trace
analyte detection increases. To achieve the lowest limits of detection, both the relationship between
surface nanostructure and laser excitation wavelength and the analyte–surface binding chemistry
must be carefully optimised. This work exploits the highly tunable nature of nanoparticle optical
properties to establish the optimisation conditions. Two methods are used to study the optimised
conditions of the SERS substrate: plasmon-sampled and wavelength-scanned surfaced Raman
excitation spectroscopy (SERES). The SERS enhancement condition is optimised when the energy
of the localised surface plasmon resonance of the nanostructures lies between the energy of the
excitation wavelength and the energy of the vibration band of interest. These optimised conditions
enabled the development of SERS-based sensors for the detection of a Bacillus anthracis biomarker
and glucose in a serum–protein matrix.

1 Introduction application in materials characterisation, biochemistry and

even art conservation, owing to the rich spectral informa-
Inelastic photon scattering was first witnessed by C.V. tion and the ability to obtain spectra for non-transparent
Raman and K.S. Krishnan in 1928 [1], a process later to solids or aqueous samples. The advent of micro-Raman
bear the name Raman scattering and to garner its techniques permits characterisation of small quantities of
discoverer a Nobel Prize in 1930. Raman scattering is a materials with high spatial resolutions [2]. Recently, the
very inefficient process, in that approximately one photon combination of laser trapping [3, 4] with Raman micro-
is inelastically scattered for every 1010 elastically scattered scopy has enabled the spatially localised study of chemical
photons. In a simple classical view of the Raman effect, species and reactions in single micrometre-sized [5] or even
incident light is considered as an electromagnetic wave that nanometre-sized volumes [6]. Raman spectroscopy is no
induces polarisation in a molecule. Then, the induced dipole longer an under-utilised, but information-rich, spectroscopic
emits or scatters light at the optical frequency of the incident technique; rather, it is a powerful tool for the fundamental
light wave, plus or minus the energy of a molecular study of molecular structure, dynamics and analysis.
vibration. The magnitude of the induced polarisation, P, is As the Raman effect is inherently weak, it is usually
related to the molecular polarisability, a, and the magnitude difficult to use normal Raman scattering either for trace
of the incident electric field, E, by [2]: analysis or for the study of highly fluorescent samples [2].
One way to overcome this problem is to use surface-
P ¼ aE ð1Þ enhanced Raman spectroscopy. SERS is a plasmonic
The molecular polarisability describes the ease with which phenomenon whereby molecules at or near a noble metal
the electron cloud around a molecule can be distorted by surface with nanoscale structural features experience a
the electric field. The strength of E is dependent on the dramatic increase in the incident electromagnetic field,
power and wavelength of the incident electromagnetic field. yielding high Raman intensity. SERS can increase the
In surface-enhanced Raman scattering (SERS), the E field is Raman intensity of a molecule in proximity to a nanoscale
increased by a factor of 10–1000 by the nanoscale optical roughened surface by 108 [7, 8], and up to 1014 times under
special circumstances [9, 10]. The mechanism responsible for
properties of the enhancing substrate, as described below.
this large enhancement in Raman intensity continues to be
Several technological developments, such as laser light
the subject of considerable research [11–16]. However, it is
sources for excitation, holographic notch filters and
gratings, and array-based detectors, led to a renaissance in generally agreed that two mechanisms contribute to the
overall enhancement: a chemical effect (CHEM) and an
Raman spectroscopy. Raman spectroscopy finds increasing
electromagnetic enhancement (EM).
The theory of chemical enhancement was invoked to
r IEE, 2005
account for the discrepancy between the enhancements
IEE Proceedings online no. 20050009
predicted in the initial electromagnetic models (B104) and
doi:10.1049/ip-nbt:20050009
the empirically determined enhancement (B106). It is
Paper first received 17th May and in final revised form 4th November 2005
thought that ‘active sites’, either crystal defects or adatoms
The authors are with the Department of Chemistry, Northwestern University,
Evanston, IL 60201, USA
on the surface of the metal, undergo a charge-transfer
Email: [email protected] process enabling the CHEM enhancement. The dependence

IEE Proc.-Nanobiotechnol., Vol. 152, No. 6, December 2005 195

of the optimum SERS excitation frequency on electrode is desirable to be able to produce SERS substrates that not
potential suggests that new electronic states may arise upon only provide the optimum EF, but also are stable from
adsorption of the analyte, which leads to resonant substrate to substrate and from experiment to experiment.
intermediate states for Raman scattering [17]. The CHEM The first SERS experiments generated largely random
enhancement has been investigated, particularly the effect of structures, and enhancement factors could vary wildly.
halide ions on increasing SERS [16, 18]. However, because Modern synthetic techniques allow exquisitely precise
the chemical environment of a given sample may be largely control over nanoscale features and thus over the nanoscale
outside experimental control (e.g. in biological media), optical properties upon which SERS intimately depends.
or determined by other factors (e.g. in ultra high vacuum The choice of substrates with specific, well-defined and
systems), it is often difficult to exploit the CHEM controlled LSPR and laser excitation wavelength, lex, is the
enhancement mechanism. Furthermore, because electro- critical factor that affects the resulting SERS EF and,
magnetic enhancement is the dominant contributor to accordingly, the analytical limit of detection (LOD). This
SERS, and because of the large variety of substrate review will demonstrate how the SERS enhancement factor
materials and geometries available, maximising the electro- depends on these characteristics.
magnetic enhancement provides the most convenient and The remainder of this review will be organised into three
effective handle to optimise SERS in a typical empirical parts. First, we compare the available methods for
setting. Modern advances in both experimental science fabrication of SERS-active surfaces. Secondly, we discuss
and theoretical modelling allow better understanding of, techniques designed to optimise the EM enhancement [7, 8].
and control over, SERS than ever before. Specifically, we describe both wavelength-scanned and
The discovery of single-molecule SERS (SMSERS) by plasmon-sampled SERS excitation spectroscopy. Thirdly,
Emory and Nie [9] and Kneipp et al. [10], in 1997, has we review the most recent progress in the SERS-based
necessitated a re-examination of the EM mechanism. The sensors for two important target molecules: a Bacillus
EM mechanism is based on the significantly amplified anthracis biomarker [21, 39] and glucose in a serum protein
electromagnetic fields (E) generated by the localised surface mixture [36, 38].
plasmon resonance (LSPR) of nanoscale surface roughness
features [12]. It is the intricate relationship between the 2 SERS-active surfaces
surface structure and the structurally dependent optical
characteristics that determines the magnitude of the EM SERS is usually performed on Ag [13], Au [40] or Cu [41]
portion of SERS enhancement [7, 8]. In the so-called surfaces, because these metals have appropriate values of
‘lightning rod effect’, nanoscale features such as sharp the real and imaginary parts of the dielectric constant [42]
points or interparticle junctions can generate intense EM and can be easily handled in ambient, electrochemical
fields that can amplify both the incoming excitation photons and ultra-high vacuum environments. With the variety of
and the exiting Stokes shifted photons by orders of magni- SERS-active surfaces available today, e.g. colloids, colloid
tude [12, 19, 20]. These factors must be well-controlled for in sol-gel [43], electrochemically roughened electrodes
SERS to be used effectively [21]. [11, 13], vapour-deposited metal island films [44] and
The EM mechanism has several experimental signatures, lithography-produced nanostructures [45], researchers are
such as long-range (i.e. a few nanometres) distance able to select the SERS substrate architecture that best
dependence [22–24], the weak dependence of the enchant- matches their experimental needs.
ment factor (EF) on the chemical identity of the adsorbate The preparation of noble metal colloids has been studied
[25] and the existence of surface-enhanced second harmonic since ancient times [46]. Colloidal substrates can be
generation (SESHG) [26–28] SMSERS. The improved prepared in many different shapes [47, 48], such as spheres,
understanding of the nature of the EM enhancements has rod-shaped [49, 50], cubes, triangles [51, 52] and shells
generated renewed interest in the use of SERS as an [53, 54]; they can be dispersed throughout the object of
analytical technique. Many groups continue to generate study and even inside macrostructures [55], such as cells [56]
new insights as new mathematical models are coupled and tissue samples [57]. However, the distribution of shapes
to more accurate characterisation in the nanoscale regime and sizes and the ease of aggregation will influence the
[29, 30]. surface-enhancing properties.
SERS possesses many desirable characteristics as a tool The nanoparticle synthesis methods discussed above
for the chemical analysis of interfacial molecular species, create suspensions of nanoparticles in solution. Another
including high specificity attomole to high zeptomole mass class of fabrication techniques yields surface-bound nano-
sensitivity [9, 10], micromolar to picomolar concentration structures. The standard approach for making surface-
sensitivity [21] and interfacial generality [31, 32]. Other bound nanostructures is electron beam lithography (EBL).
examples include concentration-jump or potential step In EBL, the desired pattern is serially produced by the
studies of adsorption/desorption kinetics at electrode or exposure of a thin layer of photo resist to high-energy
colloid surfaces [33] and the use of ultra fast, pulsed laser electrons, followed by chemical development and deposition
excitation to probe vibrational relaxation, energy transfer or of the noble metal. Several research groups have focused on
electron dynamics at surfaces [34, 35]. Recently, SERS has the optical properties of two-dimensional arrays to utilise
been used in the quantitative detection and analysis of these nanoparticle assemblies as surface-enhanced spectro-
bio-related molecules [21, 36–38]. The variation in surface scopy substrates [30, 58, 59]. Although EBL provides
enhancement is easily corrected for by addition of an exquisite control over interparticle spacing and can fabricate
internal standard to the samples [21]. Ideally, the signal arbitrary shapes, it is an expensive and slow, serial form of
intensities from the internal standard should be comparable lithography.
with those of the analyte, and one or more bands should be An alternative method for the large-scale production
in spectrally silent regions. Internal standards with only a of surface-bound nanoparticle arrays is nanosphere litho-
few spectral bands are preferable for easier data evaluation. graphy (NSL). NSL is a powerful fabrication technique that
However, in many instances, the addition of internal inexpensively produces nanoparticle arrays with controlled
standards is either impossible, or would unduly interfere shape, size and interparticle spacing [60]. Every NSL
with the analyte or process under observation. Therefore it structure begins with the self-assembly of size-monodisperse
196 IEE Proc.-Nanobiotechnol., Vol. 152, No. 6, December 2005
nanospheres of a uniform diameter, D, to form a two- to large temperature [64] and electrochemical potential
dimensional colloidal crystal deposition mask [60–63]. As excursions [39].
the solvent evaporates, capillary forces draw the nano-
spheres together, and the nanospheres crystallise in a
3 Optimisation of SERS by surface-enhanced
hexagonally close-packed pattern on the substrate (Fig. 1).
Raman excitation spectroscopy
As in all naturally occurring crystals, nanosphere masks
include a variety of defects that arise as a result of 3.1 Plasmon-scanned surface-enhanced
nanosphere polydispersity, site randomness, point defects, Raman excitation spectroscopy
line defects and polycrystalline domains. Typical defect-free Plasmon-sampled surface-enhanced Raman excitation spec-
domain sizes are in the 10–100 mm range. Following self- troscopy (PS SERES) was developed to explore the
assembly of the nanosphere mask, a metal or other material relationship between nanoparticle optics and the resulting
is then deposited by thermal evaporation, electron beam EF [65]. In the PS SERES experiment, a Raman-active
deposition or pulsed laser deposition to a target metal molecule is adsorbed onto the substrate of choice, and then
thickness, dm. After metal deposition, the nanosphere mask the correlated SERS and LSPR spectra of that adsorbate
is removed, leaving behind surface-confined nanoparticles are captured from a microscale domain using a single laser
with triangular footprints (Fig. 1a). At mass thicknesses excitation wavelength. This procedure can be repeated on a
exceeding 100 nm, a continuous film of metal is deposited single substrate until all the different LSPR domains have
on the nanospheres (Fig. 1b). These metal film over been sampled. The use of NSL fabricated arrays permits
nanosphere (MFON) substrates are some of the most agile frequency tuning of the LSPR based on the nano-
robust SERS substrates available in terms of spectral sphere mask dimensions, metal thickness and metal type.
stability, sample-to-sample uniformity and mechanical The intensity or enhancement factor of a specific Raman
ruggedness [45]. The diameter of the nanosphere cores band is plotted against the LSPR maximum to reveal the
and the thickness of the metal film shell determine the size optimised experimental conditions. There are multiple
distribution of the roughness features and, hence, the advantages in performing the PS SERES profiling:
optical response. Even though the nanoscale roughness
features are not homogeneous in size but, instead, are (a) the number of data points in the PS SERES profile is
driven by the larger-scale templating, they are sufficiently only limited by the number of microscale sample
uniform to generate a relatively narrow surface plasmon regions (and fabricated substrates) with different LSPR
peak (FWHM B200 nm) [21]. Recent experiments have characteristics
conclusively demonstrated that MFON substrates are (b) the experiment requires only a single laser excitation
stable for months [21] (unlike many other nanostructured wavelength, a single notch filter and a single stage
surfaces) and remain SERS-active even when exposed monochromator

assemble
mask deposit
metal

remove deposit
mask metal
125 cm

particle film
array over
nanosphere

500 nm

a b

Fig. 1 Nanosphere lithographic fabrication of triangular particle array and metal film over nanosphere (MFON) substrates
Carboxylate modified polymer microspheres of uniform diameter are self-assembled into hexagonally close-packed array on supporting substrate.
Metal (typically Ag or Au) is deposited through sphere array, which serves as deposition shadow mask
a Array of triangular-shaped particles is formed in interstices between spheres, which can be removed by sonication
b If 4100 nm of metal is deposited, continuous film covers spheres, forming FON surface

IEE Proc.-Nanobiotechnol., Vol. 152, No. 6, December 2005 197

(c) the information gained gives the general guidelines for a λmax =
achieving an optimised EF on any substrate with a 529.5 nm
λex = λex =
narrow LSPR. 514.5 nm 559.9 nm

NSL-fabricated Ag nanotriangles were used as SERS 610 nm

substrates for the PS SERES experiments. The size/shape
of these Ag nanotriangles can be tuned systematically by
b λmax =
changing the nanosphere size in the deposition mask or the 541.8 nm
deposition thickness [66, 67]. Slight variations in the size or λex = λex =
532 nm

extinction
shape of these nanoparticles yield significant shifts in the 580.7 nm
narrow LSPR spectrum.
Even though the structural variations among the
nanoparticles on a given NSL sample are small, they
λmax =
generate varying LSPR properties between microscale 654.5 nm
domains. Although it is possible to eliminate much of this c λex =
632.8 nm λex =
variation if necessary, the PS SERES experiment exploits 702.9 nm
the distribution of optical properties to maximise the 0.04
number of data points in the excitation profile. In all cases
to date, PS SERES experiments have explored adsorbates
that bind irreversibly to the silver nanoparticles to simplify
data collection and interpretation.
Below we present three separate PS SERES profiles 400 500 600 700 800
that were constructed using benzenethiol adsorbed onto LSPR wavelength, nm
Ag nanoparticles. Each profile was measured with a
different laser excitation wavelength (514.5, 532.0 or d 1575
19.05 adu mW−1 s−1
632.8 nm). The correlated LSPR and SERS spectra for

1032
1009
the optimised enhancement factor in each case are shown 1081

1476
in Fig. 2, and the PS SERES profiles are shown in Fig. 3.
The profiles clearly demonstrate that the maximum 1116
enhancement factor occurs when the LSPR lmax lies
between the energy of the excitation wavelength (solid
lines) and the vibration mode (broken lines). It is interesting e
47.62 adu mW−1 s−1
to note that the enhancement factor does not vary by more
than a factor of 10 once the signal is measurable above
intensity

background.
To ensure that the results observed are not an artifact of
the molecule adsorbed on the surface, more PS SERES
profiles were constructed with varied molecular adsorbates
(3,4-dichlorobenzenethiol, 1,4-benzenedithiol, and Fe(bpy)2+
3 ), f 27.78 adu mW−1 s−1
and similar results were achieved in all cases. The two non-
resonant molecules yielded maximised enhancement factors
of 2.3
107 and 1.4
108, and the maximum enhancement
factor for the resonant molecule Fe(bpy)2+ 3 was 7.1
109.
To verify that various vibration modes adhere to the
demonstrated pattern, PS SERES profiles were constructed
for five different vibration modes of 3,4-dichlorobenzene- 2000 1600 1200 800
thiol, including in-plane ring deformations, a ring-breathing wavenumber shift, cm−1
mode, the C-S stretch and the C-Cl stretch. Again, the
maximised enhancement factors occurred when the LSPR Fig. 2 Correlated, spatially resolved LSPR extinction and SERS
spectra of benzenethiol-dosed NSL substrates with maximised
lmax was between the excitation wavelength and the
enhancement factors
wavelength of the vibration mode. a and d Measured from D ¼ 280 nm, mass thickness of Ag film
Thus, for practical application, the PS SERES technique dm ¼ 36 nm, Pex ¼ 0.7 mW, lex ¼ 514.5 nm
has revealed a general rule for optimising EF when b and e Measured from D ¼ 280 nm, dm ¼ 36 nm, Pex ¼ 0.7 mW,
substrates with narrow LSPR spectra and a single laser lex ¼ 532.0 nm
excitation source are used. The largest EF is achieved when c and f Measured from D ¼ 400 nm, dm ¼ 56 nm, Pex ¼ 0.7 mW,
the energy corresponding to the narrow LSPR lmax falls lex ¼ 632.8 nm
within a B120 nm window that includes the energy of All Raman spectra were captured with integration time of 30 s.
the excitation wavelength and that of the Raman-shifted Reproduced with permission from [7]
scattered photons. The LSPR variation on the NSL Copyright 2003, the American Chemical Society
substrates used in the work presented above can be easily
exploited to find the microscale domains with the best
optical properties for a given experiment, thus maximising
the enhancement factor and lowering the detection limit. 3.2 Wavelength-scanned surface-enhanced
Although the reported enhancement factors from NSL Raman excitation spectroscopy
microdomains are significantly smaller than those measured For all SERS sensing applications, sensitivity is of primary
in single-molecule SERS studies, these ensemble-averaged importance. To this end, we have undertaken a study of the
values can be regularly achieved, are temporally stable fundamental mechanism of SERS to ascertain the specific
and are more readily understood. conditions necessary for maximising the enhancement of the
198 IEE Proc.-Nanobiotechnol., Vol. 152, No. 6, December 2005
 10 unknown or poorly characterised distribution of roughness
features. In the few cases where the surfaces are carefully
8
characterised, it has been shown that there is a wide
distribution of roughness feature sizes [31, 71]. Other studies
EF, 1575 cm−1 × 107

do not include characterisation of the LSPR of the sub-

6 strate [69, 70], which prevents any direct comparison of
the excitation profiles with the spectral location of the
4
wavelength of maximum LSPR extinction, lmax.
The most common substrates historically employed in
SERES experiments are Ag island films and Ag colloidal
2 solutions. In these cases, most of the SERS excitation
profiles peak at excitation wavelengths (lex,max) near
500–600 nm [71–75]. The peaks of the excitation profiles
480 560 640 480 560 640 600 680 760 have been shown to shift to longer wavelengths with
a b c increased aggregation [68, 71, 73, 74], which is a qualitative
LSPR λmax, nm result predicted by the EM mechanism. With these types of
substrate, it is difficult to make a direct comparison between
Fig. 3 PS SERES for n8a (1575 cm1) band of benzenethiol with the LSPR of the substrate and the SERS excitation profile,
three different excitation wavelengths
because the LSPR of the substrate is a superposition of
a lex ¼ 514.5 nm
b lex ¼ 532.0 nm
a wide variety of LSPR wavelengths corresponding to the
c lex ¼ 632.8 nm different roughness features caused by morphological (size
- - - - - - - - - Wavelength location of excitation and shape) polydispersity between islands or colloidal
- - - - - - - Wavelength location of scattering nanoparticles.
Overlaid line represents bin-averaged values of LSPR lmax and EF Two exceptions to the above statements regarding
roughness features are the well-known experiments by Liao
and co-workers on microlithographically prepared Ag posts
Raman scattering. There are three principle factors that [76] and recent work by Felidj and co-workers on EBL-
determine the sensitivity of a Raman experiment: produced arrays of gold elongated nanoparticles [77]. The
(i) the frequency to the fourth (n4) scattering dependence of former ground-breaking work demonstrated excitation
Raman photons profiles where lex,max shifted to longer wavelengths with
(ii) the wavelength-dependent efficiency of the detection increased particle aspect ratio and with increased dielectric
system constant of the medium surrounding the particles. These
results qualitatively agree with the EM mechanism, but the
(iii) the relationship between the LSPR of the substrate and
LSPR of these substrates was not measured for a direct
the maximum SERS enhancement.
comparison. In the latter work, the SERS enhancement was
The first factor would indicate that shorter excitation shown to peak halfway between the excitation wavelength
wavelengths are always best, but that is not necessarily the and the wavelength of the Raman scattered photon. This
case. For example, in many biological applications, it is important experiment was the first observation of precisely
often desirable to use near IR excitation to avoid damaging what is predicted by the EM mechanism. Unfortunately,
the sample or exciting sample autofluorescence, even at the this result was only obtained on one sample with a profile
cost of some signal loss due to n4. The second factor is consisting of only three data points.
straightforward and is simply a function of the configura- Herein, we describe the utilisation of a broadly tuneable
tion of the spectrograph and detector. Consultation of the Raman system to measure excitation profiles with the
efficiency curves of the dispersive elements and the detector greatest number of data points ever achieved in a WS
are readily available from manufacturers and should factor SERES experiment. A broadly tuneable laser system, a
into purchase planning and experimental design. For versatile detection system and a well-characterised surface-
example, most systems are configured with gratings and enhancing substrate are all employed to surmount the
detectors optimised in the visible region of the spectrum, traditional shortcomings of WS SERES experiments. The
decreasing their efficacy in the UV or NIR region. The third use of a CW mode-locked Ti : sapphire and its harmonics
factor, the relationship between the LSPR of the substrate allows for continuous tuneability over the spectral ranges
and the maximum SERS enhancement, is more complex 350–500 nm and 700–1000 nm. The visible region not
and interesting, yet less well understood or utilised. We covered by the Ti : sapphire system was augmented with
have explored this third factor using a technique called the use of a solid-state laser and a tuneable dye laser. A
wavelength-scanned surface-enhanced Raman excitation triple spectrograph equipped with a CCD camera allows
spectroscopy (WS SERES). for rapid, multichannel spectral acquisition with efficient
WS SERES involves the measurement of SERS en- rejection of Rayleigh-scattered photons. The SERS sub-
hancement for several laser excitation wavelengths, lex. This strates used in this work are triangular nanoparticle arrays
technique was recognised as a useful tool for exploring fabricated by NSL. These substrates have been well
the EM mechanism soon after the discovery of SERS. An characterised previously [61, 66, 67]. They present a
obvious limitation of this technique is that the number of significant advantage over many of the traditional SERS
data points is determined by the tuneability of the excitation substrates for SERES studies, because NSL-fabricated
laser and detection system. These substantial instrumental triangular nanoparticles exhibit extremely narrow size
requirements have led to the majority of SERES publica- distributions, making them an indispensable tool for
tions suffering from low data point density and/or limited probing the fundamental characteristics of SERS. Tune-
spectral coverage [31, 68–70]. These limitations prevent the ability of the LSPR lmax of these nanoparticles throughout
establishment of conclusive generalisations from SERES the visible and NIR wavelengths can be achieved by sys-
data. Additionally, most SERES experiments have been tematic variation of the dimension of the nanoparticles [66].
performed using surface-enhancing substrates with an Even though the surface coverage of these nanoparticles is

IEE Proc.-Nanobiotechnol., Vol. 152, No. 6, December 2005 199

B7%, strong SERS intensities are observed from analytes wavelength, nm
adsorbed to these substrates, owing to the strong enhance- 600 500 400
6.0E+5
ment NSL-fabricated arrays exhibit [65]. 0.16
For the work presented below, the intensity of the
5.0E+5
1575 cm1 band of a benzenethiol monolayer adsorbed to

enhancement factor
the Ag nanoparticles was measured at many different
0.12 4.0E+5
excitation wavelengths, lex. Benzenethiol is used as a model

extinction
molecule in both this and the following sections because it:
(a) binds via the thiol group to the metal surface 3.0E+5
0.08
(b) has a high Raman scattering cross-section
2.0E+5
(c) lacks electronic transitions in the visible range, rendering
it non-resonant at the excitation wavelengths used
0.04 1.0E+5
(d ) has a well-resolved spectrum. 16 000 18 000 20 000 22 000 24 000
a
To account for any variation of the SERS intensity not due wavelength, nm
to the enhancement by the substrate, the 1444 cm1 normal 800 700 600 500
Raman scattering band of neat cyclohexane was used as an 1.2E+7

intensity standard. This standard was used to correct for 0.16

the inherent n4 behaviour of Raman scattering, spectral 1.0E+7
dependence of the detection system and differences in the

enhancement factor
illumination power. 8.0E+6

extinction
0.14
Figure 4 shows four excitation spectra for the 1575 cm1
peak of benzenethiol, each with an LSPR lmax at a 6.0E+6
distinctly different location. The SERES spectrum in Fig. 4a
0.12
is measured over the spectral range 420–500 nm. Because
4.0E+6
the formation of a monolayer of benzenethiol on these
nanoparticle arrays results in a large red shift in the position
of the LSPR lmax, it was necessary to anneal this sample at 0.10 2.0E+6
14 000 16 000 18 000 20 000 22 000
3001C for 1 h prior to benzenethiol addition, to achieve a b
final LSPR lmax at a wavelength shorter than 500 nm. It has
wavelength, nm
been previously shown that annealing NSL-derived samples 1000 900 800 700 600 500
results in a large blue shift of the LSPR owing to the shape 1.6E+7
0.18
of the nanoparticles being changed [66]. The LSPR lmax of
this substrate was measured to be 489 nm (20 450 cm1).
0.16 1.2E+7
The largest SERS enhancement occurs at lex ¼ 485 nm.

enhancement factor
Fitting a Gaussian line shape to the data reveals that the extinction
peak of the excitation profile, lex,max, is 480 nm 0.14
8.0E+6
(20 833 cm1). The peak enhancement factor (EF) value
for this sample was calculated to be 5.5
105. This value is 0.12
low in comparison with the values determined for the other 4.0E+6
samples, because the shape of the nanoparticles is made 0.10
more ellipsoidal by annealing. In addition to shifting the
LSPR lmax to shorter wavelengths, this change decreases 0.08 0E+0
the intensity of the electromagnetic fields at the nanoparticle c
surfaces by removing the sharp corners that are calculated wavelength, nm
1000 900 800 700 600 500
to possess the greatest field enhancements [12]. 1.0E+8
The SERES spectrum in Fig. 4b was measured over the 0.3
spectral range 532–690 nm. The LSPR lmax of this substrate 8.0E+7
was measured to be 663 nm (15 083 cm1). The largest
enhancement factor

SERS enhancement occurs for lex ¼ 625 nm. The maximum 0.2 6.0E+7
extinction

of a Gaussian line shape fit to the data is 625 nm

(16 000 cm1). The peak EF value for this sample is
1.2
107. The SERES spectrum in Fig. 4c is measured 4.0E+7
over the spectral range 532–740 nm. The LSPR lmax of 0.1
this substrate was measured to be 699 nm (14 306 cm1). 2.0E+7
The largest SERS enhancement occurs for lex ¼ 670 nm.
The maximum of a Gaussian line shape fit to the data is 0 0E+0
671 nm (14 903 cm1). The peak EF value for this sample is 10 000 12 000 14 000 16 000 18 000 20 000
1.4
107. The SERES spectrum in Fig. 4d is measured over wavenumbers, cm−1
the spectral range 630–800 nm. The LSPR lmax of this d
substrate was measured to be 810 nm (12 346 cm1). Fig. 4 Surface-enhanced Raman excitation spectra of 1575 cm1
The largest SERS enhancement occurs for lex ¼ 770 nm. peak of benzenethiol with cyclohexane as intensity standard
The maximum of a Gaussian line shape fit to the data is a Substrate annealed at 3001C for 1 h. LSPR lmax ¼ 489 nm; profile fit
765 nm (13 072 cm1). The peak EF value for this sample is maximum at lex,max ¼ 480 nm
9.3
107. b LSPR lmax ¼ 663 nm; profile fit maximum at lex,max ¼ 625 nm
If the peak in the SERS enhancement occurs when the c LSPR lmax ¼ 699 nm; profile fit maximum at lex,max ¼ 671 nm
LSPR lmax of the sample is equal to (lex+lvib)/2 (where lvib d LSPR lmax ¼ 810 nm; profile fit maximum at lex,max ¼ 765 nm

200 IEE Proc.-Nanobiotechnol., Vol. 152, No. 6, December 2005

denotes the wavelength of the Raman-shifted peak of shown, and the separation in wave numbers between the
interest), then lex,max should be different for the various LSPR lmax and lex,max is 488 cm1.
Raman bands of benzenethiol on a single sample. It is These data demonstrate the qualitative trend whereby the
expected that lex,max will have a larger separation from the lex,max in the excitation spectra of larger Raman-shifted
LSPR lmax for a large Raman shift than for a small shift. bands yield a larger separation from the LSPR lmax than
Excitation spectra for three benzenethiol peaks on a single those of smaller Raman-shifted bands. These data lend
substrate are shown in Fig. 5. For this substrate, the LSPR validation to the EM mechanism as well as highlighting
lmax is 729 nm. Figure 5a shows the SERS excitation profile another factor that must be kept in mind in the design of an
for the 1575 cm1 peak of benzenethiol, normalised to SERS-based sensor. The relationship between the peak in
the 1444 cm1 peak of liquid cyclohexane. The separation the SERS enhancement factor and the extinction maximum
in wave numbers between the LSPRs lmax and lex,max is of the substrate’s LSPR has been shown to be dependent
734 cm1. In Fig. 5b, the excitation profile for the 1081 cm1 upon the magnitude of the Raman shift.
benzenethiol peak (normalised to the 1028 cm1 peak of The work above demonstrates the most thorough WS
cyclohexane) is shown. The separation in wave numbers SERES experiments heretofore performed on optically and
between the LSPRs lmax and lex,max is 569 cm1. Finally, in topographically characterised SERS substrates. The experi-
Fig. 5c, the excitation profile for the 1009 cm1 benzenethiol mental apparatus utilised has been proven effective for the
peak (normalised to the 1028 cm1 peak of cyclohexane) is measurement of relative SERS enhancements that vary by
three orders of magnitude. This work demonstrates that the
relationship between the substrate LSPR and the SERES
wavelength, nm
profile for size-homogenous nanoparticles is consistent
900 800 700 600 500
throughout the visible range. In all cases, the experimentally
2.0E+7 observed results are consistent with those predicted by the
0.24 νvib = 1575 cm−1 EM mechanism. Specifically, the maximum SERS enhance-
shift = 734 cm−1 ment always occurs when the substrate LSPR lmax is
1.6E+7
located between the laser excitation wavelength, lex, and the
enhancement factor

0.20
wavelength of the Raman-scattered photon, lvib. Under
extinction

1.2E+7 these conditions, both the incident and scattered photons

0.16
experience strong enhancement by the LSPR. Each
0.12
8.0E+6 substrate exhibits a WS SERES profile that has a similar
line shape to the extinction spectrum of the substrate.
0.08 4.0E+6 The largest EF measured was B108 for the triangular
nanoparticle arrays.
3.0E+7
Ultimately, refinement of the experimental apparatus and
a optimisation of SERS enhancement will allow SERES to be
3.0E+7
performed using single nanoparticle substrates. This level
0.24 provides the best possible case in terms of reducing sample
νvib = 1081 cm−1
2.5E+7 heterogeneity, in that it will remove the effects of ensemble
shift = 569 cm−1 averaging or particle coupling. These experiments are
0.20
enhancement factor

2.0E+7 expected to provide key information to validate the EM

mechanism of SERS and will present an additional
extinction

0.16
1.5E+7 technique that can be used to study the SMSERS effect.
0.12
Our detailed WS SERES studies unequivocally demon-
1.0E+7 strate the importance of designing SERS experiments such
that the LSPR lmax of the substrate is located in between
0.08 5.0E+6 the laser excitation wavelength and the wavelength of the
Raman peak of interest. One very attractive feature of
3.0E+6
b the NSL-derived nanoparticle substrates is the tuneability of
the LSPR lmax spectral position. Therefore, by using these
3.0E+6
0.24
substrates for SERS-based sensing, we can optimise the
νvib = 1009 cm−1 sensor by using an excitation wavelength that utilises the n4
2.5E+6
shift = 488 cm−1 dependence of Raman scattering while taking into account
0.20
enhancement factor

2.0E+6
other factors of the particular application, assembling a
detection system that is optimised for highest throughput
extinction

0.16
1.5E+6 at the wavelength of interest, and fabricating nanoparticle
arrays with an LSPR lmax precisely located in between lex
0.12
1.0E+6 and lvib.
0.08 5.0E+5 4 Biological applications of surface-enhanced
Raman scattering
0.04 0.0E+0
12 000 14 000 16 000 18 000 20 000 4.1 Rapid anthrax biomarker detection
wavenumbers, cm−1 based on SERS
c Anthrax is an infectious disease caused by the spore-
Fig. 5 Effect of Stokes Raman shift forming bacterium Bacillus anthracis [78]. Currently, the
a Profile of 1575 cm1 vibration mode of benzenethiol. Distance demand for better field tests for B. anthracis spores has
between LSPR lmax and excitation profile fit line lex,max ¼ 734 cm1 increased as a consequence of contaminated mail and false
b 1081 cm1 vibration mode, shift ¼ 569 cm1 alarms. Several techniques have been developed for real-
c 1009 cm1 vibration mode, shift ¼ 488 cm1 time detection of Bacillus spores [79, 80]. Examples include

IEE Proc.-Nanobiotechnol., Vol. 152, No. 6, December 2005 201

fluorescence detection using portable devices, which 104 spores

I1020 / I1050
0.6

1050

1020
is sensitive enough to detect B103 spores in 7 min, but

1050
0.4

826
860
889
has the potential to give false positive readings [79]. In

Raman intensity

666
0.2

contrast, SERS possesses highly specific chemical informa- 0 15 3045

1020
day
tion content and therefore is capable of uniquely identifying

824
CaDPA

1595

1020
1393
target analytes, thereby limiting false positives [2]. We have

826
1050
developed a procedure for the rapid extraction of CaDPA

850
889
1194

666
700
638
from B. subtilis spores (simulants for B. anthracis spores),
followed by SERS detection on AgFON substrates [21].
These substrates were designed and optimised using the 1600 1200 800 1000 800 600
methods described in the preceding sections. a b
Prior work demonstrates that when the localised surface wavenumber, cm−1
plasmon resonance maximum of a AgFON substrate Fig. 7 SERS spectra
closely matches the laser excitation wavelength, the max- a SERS spectrum of 2.1
1014 M spore suspension (2.6
103 spores
imum SERS signal intensity results. As AgFONs are not in 0.2 ml, 0.02 M HNO3) on AgFON. Inset shows intensity ratio (I1020/
optically transparent, the reflectivity minimum was used to I1050) variation with time. lex ¼ 750 nm, Pex ¼ 50 mW, acquisition
locate the LSPR maximum. AgFON surfaces fabricated time ¼ 1 min, D ¼ 600 nm, and dm ¼ 200 nm
using 600 nm spheres show a reflectivity minimum at b SERS spectra obtained by portable Raman spectrometer. Upper
753 nm (Fig. 6a). In fact, further SERS experiments using spectrum is 8.3
1014 M spore suspension (1.0
104 spores in
AgFON substrates with different polystyrene nanosphere B0.2 ml, 0.02 M HNO3) on 30-day-old prefabricated AgFON. Bottom
diameters demonstrate that the largest SERS enhancement SERS spectrum is 104 M CaDPA in 0.2 ml 0.02 M HNO3 on 30-day-
old pre-fabricated AgFON substrate. lex ¼ 785 nm, Pex ¼ 35 mW,
for 750 nm laser excitation was obtained from the AgFON
acquisition time ¼ 5 s, resolution ¼ 15 cm1, D ¼ 600 nm, and
where D ¼ 600 nm [21]. The use of NIR laser light in dm ¼ 200 nm
Raman spectroscopy is advantageous because it causes less
photochemical damage to the biological samples and results
in much less fluorescence background in the Raman
spectrum. Therefore this AgFON substrate was chosen as indicating the laser power [85]. In contrast, the SERS LOD
optimum for the bacillus spore detection experiments that of CaDPA is B3.1
106 M using the optimised NSL
follow. substrate and laser excitation at 750 nm [21]. It should be
Calcium dipicolinate (CaDPA) was extracted from spores noted that the intensities of Raman spectra with 750 nm
by sonicating in 0.02 M HNO3 solution for 10 min. Figure excitation are expected to be about a quarter of those
6b shows the SERS spectrum of 3.7
104 spores in 0.2 ml, measured with 532 nm excitation, because the Raman signal
0.02 M HNO3 on a AgFON substrate (D ¼ 600 nm, intensities are inversely proportional to the fourth power of
dm ¼ 200 nm), which is dominated by bands associated the laser wavelength (see above) [2]. Therefore we calculate
with CaDPA, in agreement with the previous Raman an LOD of CaDPA is B30 times more sensitive than
studies on CaDPA and bacillus spores [81–84]. The peak at literature values.
1050 cm1 in Fig. 6b arises from the symmetrical stretching Previous research has demonstrated that bare AgFON
vibration of NO 3 . This peak is used as an internal standard surfaces display extremely stable SERS activity when
to reduce the sample-to-sample deviations, because of its challenged by negative potentials in electrochemical experi-
high intensity and spectral isolation. ments [39] and high temperatures in ultrahigh vacuum
We show in Fig. 7a an LOD of 2550 anthrax spores with experiments [86]. In the present example, the intensity ratios
a data acquisition period of 1 min and a laser power of between the strongest CaDPA peak at 1020 cm1 and the
50 mW, well below the level believed to trigger an infection NO 3 peak at 1050 cm
1
(I1020/I1050) were measured so that
in humans. To put these results into context, previously prefabricated AgFON substrates of different ages could
published SERS studies of bacillus spore detection were 200 be compared quantitatively (Fig. 7a, inset). The CaDPA
times less sensitive and required three times more laser intensity remained constant over the course of 40 days,
power [83]. Recently, Bell and co-workers reported a SERS indicating the temporal spectroscopic stability of AgFON
LOD of B4 parts per million of dipicolinic acid, i.e. substrates over a period of 40 days.
2.4
105 M, using laser excitation at 532 nm without Finally, a portable, handheld SERS device successfully
produced a SERS spectrum from 104 spores in 5 s using
a one-month-old prefabricated AgFON substrate (Fig. 7a).
The SERS peak positions and intensity pattern for the
spore sample were similar to those of CaDPA recorded
1020
1050 (NO3)
Raman intensity

824

utilising the same device (Fig. 7b). This represents the first
reflectance

reported use of a compact vibration spectrometer for the

1393
1595
1575

detection of bacillus spores. Coupling the portability and

1438

1194

666

ease of use of this type of device to the molecular specificity

and spectral sensitivity inherent in SERS, a range of
λex = 750 nm possibilities become available in the area of detecting bio-
agents and other chemical species, such as environmental
600 750 900 2000 1500 1000 500 pollutants, toxic industrial chemicals, process reaction
wavelength, nm wavenumbers, cm−1 streams, food products etc.
a b

Fig. 6 Reflectance and SERS spectra 4.2 SERS-based measurement of glucose

a UV-vis diffuse reflectance spectrum of AgFON substrate in air Although, the CaDPA biomarker in Bacillus subtilis spores
b SERS spectrum of 3.1
1013 M spore suspension (3.7
104 spores directly binds with the silver surface, which is required for
in 0.2 ml, 0.02 M HNO3) on AgFON substrate. lex ¼ 750 nm, SERS detection, many important molecules (e.g. glucose)
Pex ¼ 50 mW, acquisition time ¼ 1 min, D ¼ 600 nm, dm ¼ 200 nm do not have any natural binding affinity for the silver
202 IEE Proc.-Nanobiotechnol., Vol. 152, No. 6, December 2005
surface [37]. Although the normal Raman cross-section of actual concentration, mM
glucose should provide sufficient signal to register on most 5.0 10.0 15.0 20.0
detection systems [2], the inability to observe glucose using
SERS must be attributed to the weak or non-existent normal
400 A
interaction of glucose with bare noble metal surfaces. This pre-
A
section demonstrates quantitative glucose detection by diabetes 20.0

predicted concentration, mg/dL

predicted concentration, mM
tailoring a AgFON substrate similar to that used above 320 E C
with a self-assembled monolayer (SAM) to partition glucose
within the range of the enhanced electromagnetic fields on 15.0
the AgFON surface (Fig. 8) [37], in a manner analogous 240 B
to that used in high-performance liquid chromatography
(HPLC) [87–91]. Functionalising the AgFON substrate 10.0
with a partition layer has three advantages: the SAM 160
D D
stabilises the Ag surface against oxidation; the SAM is
exceedingly stable; and pre-concentration functionality is 80 5.0
B
built in and can be tailored by synthetic control of the
partition layer. Such chemical functionalisation of the C
E
SERS substrate enables the extension of SERS to
previously inaccessible molecules and provides an additional 0 80 160 240 320 400
parameter that can be controlled in the development of actual concentration, mg/dL
SERS-based experiments [23]. Fig. 9 Clarke error grid of LOO-PLS predicted glucose concen-
tration against actual glucose concentration (3 loading vectors)
AgFON samples were fabricated (D ¼ 390 nm, dm ¼ 200 nm), incu-
HOH2C bated for B16 h in 1 mM EG3 solution, and dosed in glucose solution
HO O (range: 0–450 mg/dl, 0–25 mM) for 10 min. Each SERS measurement
HO OH
OH was made in flow cell under saline with pH ¼ 7.4, using lex ¼ 632.8 nm,
Pex ¼ 1.0 mW, t ¼ 30 s
partition All SERS measurements were taken in single spot Reproduced with
permission from [32]
departition
Copyright 2003, American Chemical Society

AgFON AgFON

Fig. 8 Schematic demonstration of hypothetical glucose concen- point, affecting the localised surface plasmon resonance
tration gradient created by EG3 partition layer and, accordingly, the EF.
Alkanethiol portion of EG3 helps form stable SAM, and glycol Although quantitative detection is an important char-
portion provides hydrophilic environment amenable to glucose acteristic of a viable biosensor, the glucose sensor must also
salvation be effective in the presence of interfering proteins. Serum
albumin was used to mimic blood serum protein as a
challenge to the glucose sensor. The EG3-functionalised
AgFON substrate was installed in a flow cell under a saline
Straight chain alkanethiols and tri(ethylene glycol) environment, and the SERS spectrum was obtained
terminated alkanethiol (EG3) were found to be effective (Fig. 10a). Then, the BSA solution was injected into the
partition layers. Of the many SAMs tested to determine flow cell, and the SERS spectrum was collected throughout
their effectiveness as a partition layer, EG3 was chosen as the 240 s incubation (Fig. 10b). Finally, the sample was
a partition layer because of its ability to reject non-specific exposed to 100 mM glucose, and the SERS spectrum was
binding by background proteins [92–95] and its biocompat- collected (Fig. 10c). Figure 10d is the difference spectrum
ibility [96, 97], with the goal in mind of progression towards between the sensor under saline and the same surface
fabrication of an implantable SERS-based glucose sensor. exposed to the BSA solution, demonstrating that BSA does
Each EG3-modified AgFON sample was incubated in not have a measurable SERS spectrum on the EG3-
saline solution with glucose (0–25 mM; 0–450 mg dl1) at a modified surface. The lack of SERS bands from BSA is
physiological pH of 7.4. Then, the samples were placed in attributed to inefficient adsorption of BSA to the EG3
an environmental control flow cell under saline, and SERS partition layer. The data in Fig. 10e confirm that the SERS
spectra were collected. The spectra were normalised using glucose sensor is still effective after substrate exposure to an
EG3 peak intensities, followed by partial least squares interfering protein and that the peaks correspond to the
analysis [36, 37]. The resulting cross-validated glucose crystalline glucose peaks shown for comparative reference
concentration predictions are presented in the Clarke error in Fig. 10f. This experiment clearly shows that glucose
grid (Fig. 10). Clarke and co-workers established the error partitioning into EG3 is not influenced by the presence of
grid as a metric for evaluating glucose sensor efficacy in the large molecules such as serum albumin. It is interesting to
clinically relevant concentration range [98]. note that the peak at 695 cm1 (Fig. 10a) shifts to 710 cm1
The EG3-modified AgFON sensor allows quantitative (Fig. 10c) in the presence of glucose. The rearrangement of
detection of glucose in the physiological range with a the SAM when the glucose molecules partition into EG3
corresponding prediction error of 82 mg dl1 (4.5 mM). may cause this shift. The observed shift in this peak further
In Fig. 9, 94% of the predictions fall in zones A and B, corroborates the hypothesis of glucose penetrating deeply
whereas a few data points overlap in zone D within the into the EG3 monolayer, affecting even the character of the
hypoglycaemic area (o70 mg dl1, o3.9 mM). The error of C–S bond.
82 mg dl1 (4.5 mM) can be partially attributed to slight In addition to the demonstration of quantitative glucose
variation in the EF between different AgFON samples. The measurement in a clinically relevant concentration range
nanostructure on a AgFON substrate varies from point to and detection of glucose in the presence of other interfering

IEE Proc.-Nanobiotechnol., Vol. 152, No. 6, December 2005 203

 a 5 Conclusions
10∗
SERS is a powerful technique for the sensitive and selective

1108
detection of low-concentration analytes. To achieve the

1127
695

1295
1434

1061

882
1340
lowest limits of detection, both the relationship between

834
1001
surface nanostructure and laser excitation wavelength,
as well as the analyte–surface binding chemistry must be
carefully optimised. This work exploits the highly tuneable
nature of nanoparticle optical properties to establish the
b

1108
first set of optimisation conditions. We conclusively

1127
1296
1437

1064
intensity

demonstrated that the SERS enhancement factor is

885
698
1341

optimised when the energy of LSPR lies between the energy

836
1002
of the excitation wavelength and the energy of the vibration
band of interest. With the narrow LSPRs used in this work,
it is straightforward to achieve an EF of B108. We also
detailed the use of the PS SERES technique to aid in the
1108

c design of experiments that are unable to exploit a broadly

1297
1437

1077
855 tuneable laser and detector system, i.e. those most routinely
888
1341

1061 835 710

available.
1002

The experience garnered in the development of the

SERES work was applied to the analytically challenging
task of detecting biomarkers for bacillus spores. This review
also covered the exploitation of chemical modification
1600 1400 1200 1000 800
of SERS substrates to improve the interaction between
wavenumber shift (cm−1) the surface and target analytes. Specifically, we used the
measurement of glucose, a molecule with no detectable
d SERS signal under normal circumstances, to illustrate how
5∗
effective surface functionalisation can be for expanding the
B A
potential role of SERS.

6 Acknowledgments

The authors gratefully acknowledge the financial support of

e C B the Air Force Office of Scientific Research MURI program
1077

1108 (F49620-02-1-0381) and the National Science Foundation

1433

855
1291
1449

1059 710 (DMR-0076097). Any opinions, findings and conclusion or

1339
intensity

704
recommendations expressed in this material are those of the
authors and do not necessarily reflect those of the National
1195

Science Foundation. This project was also supported by the

Institute for Bioengineering and Nanoscience in Advanced
698 Medicine at Northwestern University, and the National
Institutes of Health (EY13002 and EY13015). The
1116

f
840

authors wish to acknowledge the following individuals

1070

1342
914

1.3∗
1427

for their experimental assistance, materials or expertise:

1146
1270

1050
1456

J.A. Dieringer, L. Chang, M. Glucksberg, C.L. Haynes,

1201

E. Jeoung, M. K.all, O. Lyandres, M. Mrksich, C. Murphy,

A.D. McFarland, S. Nie, J.C. Riboh, G.C. Schatz, K.
Shafer-Peltier, J. Walsh and S. Zou.
1600 1400 1200 1000 800
wavenumber shift (cm−1)
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