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SERS biodetection
SERS-Based Diagnosis and BiodetectionRamon A. Alvarez-Puebla* and Luis M. Liz-Marzan*
Keywords:� biodetection
� nanoparticles
� sensing
� SERS
� surface plasmon resonance
Surface-enhanced Raman scattering (SERS) spectroscopy is one of the
most powerful analytical techniques for identification of molecular species,
with the potential to reach single-molecule detection under ambient
conditions. This Concept article presents a brief introduction and discussion
of both recent advances and limitations of SERS in the context of diagnosis
and biodetection, ranging from direct sensing to the use of encoded
nanoparticles, in particular focusing on ultradetection of relevant
bioanalytes, rapid diagnosis of diseases, marking of organelles within
individual cells, and non-invasive tagging of anomalous tissues in living
animals.
1. Introduction
The prompt, sensitive and accurate response of analytical
techniques to resolve detection issues, in particular those
related with health, has always been a key aspect in (applied)
science. To date, many analytical tools based on different
physical, chemical, and biological phenomena have been
developed for structural characterization of biomolecules,
biosensing, biodiagnosis, and biomedical imaging, including
mass spectrometry, fluorescence spectroscopy, and techniques
based on specific recognition events such as enzyme-linked
immunosorbent assay (ELISA), fluorescence immunoassay
(FIA), or radioimmunoassay (RIA). However, none of these
techniques has been able so far to fulfill all the expectations of
modern biomedicine because they are time consuming, have
relatively low detection limits, and/or require special environ-
ments, far away from biological conditions. Recently, mainly
driven by the significant advances in optics, laser technology,
detection devices, and nanofabrication, surface-enhanced
Raman scattering (SERS) has arisen as a versatile tool that
offers sensitivity, together with structural information in
biological media.
[�] Dr. R. A. Alvarez-Puebla, Prof. L. M. Liz-Marzan
Departamento de Quimica-Fisica and
Unidad Asociada CSIC-Universidade de Vigo
36310 Vigo (Spain)
E-mail: ramon.alvarez@uvigo.es; lmarzan@uvigo.es
DOI: 10.1002/smll.200901820
� 2010 Wiley-VCH Verla
SERS spectroscopy is one of the most powerful analytical
techniques for identification of molecular species, with the
potential of reaching single-molecule detection under ambient
conditions.[1] SERSprovides complete vibrational information
of the molecular system under study and, since the output is
essentially a Raman scattering spectrum, it is highly sensitive
toward conformational changes.[2] On the other hand, and due
to surface selection rules,which further increase the intensity of
the vibrational modes perpendicular to the surface while
maintaining parallel modes constant, the orientation of the
molecule on a given support can be readily extracted from
the acquired spectrum.[3,4] All of these features together make
SERS not only the tool of choice for a number of analytical
problems comprising molecules but also an extremely inter-
esting technique for the study of biomolecules, pathogens, and
disease markers.
SERS is purely a nanoscale effect, deriving from localized
surface plasmon resonances (LSPR) in nanostructured metals,
which give rise to huge electromagnetic fields at the nanometal
surface.[5] The enhancement of the Raman signal is mainly
achieved by coupling of the vibrational modes of the analyte
molecule with the electromagnetic field (LSPR) generated at a
metallic nanostructure, usually made of gold or silver, upon
excitationwith lightofappropriateenergy.SERScanbecarried
out using theLSPR from individual nanoparticles, for example,
in a colloidal suspension, which is known as average SERS.
However, particle aggregates havebeen found toprovidemuch
higher enhancement due to coupling between the LSPRs of the
different particles within the aggregate, resulting in a
significantly higher electromagnetic field at certain regions
g GmbH & Co. KGaA, Weinheim small 2010, 6, No. 5, 604–610
Figure 1. Schematic view of SERS on both non-interacting nanoparticles
and aggregates. Non-interacting nanoparticles display well-defined LSPR
but these broaden and red-shift in aggregates due to electromagnetic
coupling. Coupling also leads to the formation of hot spots, with large
effects on the intensity of the SERS signal (additional enhancement
factors up to 103), in this case for a protein.
(typically at interstices) within the interacting nanostructures,
which are called ‘‘hot spots’’ (Figure 1). Since the goal of this
Concept article is not to provide a detailed description of SERS
and the mechanisms involved, we direct the interested readers
to excellent reviews that have been recently published.[6–8]
Since bioanalytes are in general highly sensitive to their
environment, changes in parameters such as pH, temperature,
or ionic strength can easily affect them. Thus, the possibility of
acquiring SERS spectra directly from aqueous solution
constitutes a unique advantage over other utrasensitive
techniques, such as mass spectrometry, which typically require
demanding processing conditions (e.g., high vacuum).
Additionally, the biocompatibility of gold nanoparticles and
other complex enhancing substrates, which can be coated or
functionalizedwithappropriateprotecting shells, allows theuse
of SERS spectroscopy for detection even inside living cells and
animals.[9]
This article is not conceived as a thorough review of the
literature but rather a brief introduction and discussion of both
recent advances and limitations of SERS in the context of
diagnosis and biodetection, ranging from direct sensing to the
use of encoded nanoparticles, in particular focusing on
ultradetection of relevant bioanalytes, rapid diagnosis of
diseases, marking of organelles within individual cells, and
tagging of anomalous tissues in living animals.
2. Direct Biomolecule Sensing
The most usual way of using SERS is simply the direct
detection of the target analyte, that is, the identification of the
small 2010, 6, No. 5, 604–610 � 2010 Wiley-VCH Verlag Gmb
specific SERS spectral fingerprint of the analyte through direct
binding onto a metallic, enhancing nanostructure. This can be
carried out in different ways, which we describe here. In typical
experiments using silver or gold colloids, a small volume (a few
mL) of the sample to be analyzed is mixed with the colloidal
dispersion (a fewmL).After some time (a few seconds) needed
for the mixture to reach thermodynamic equilibrium, the
sample is directly analyzed by measuring the SERS spectrum
with a Raman spectrometer. These experiments, known as
average SERS, give rise to well-defined SERS spectra with
reproducible intensity because the signal is not acquired from a
restricted number of particles and analytes but from a dynamic
system where, because of Brownian motion, the particles that
are being sampled continuously change.[10] This largely
attenuates any sample damage induced by the laser (heat is
rapidly released to the solvent) so that more energetic
excitation lines can be used, with higher power densities at
the sample.Unfortunately, average SERS is usually carried out
on an ensemble of dilute (widely spaced) colloidal particles,
therefore with basically no interaction between them. This
means that theprobability of forminghot spots is extremely low
and small intensities are usually registered, as compared to
other operation modes, which leads to lower detection limits.
As an alternative to this method, nanostructured metal films
can be used. Preparation methods include direct casting, spin-
coating, self-assembly, or layer-by-layer assembly of colloidal
particles on a surface, as well as the direct buildup of the
nanostructures by physical evaporation or lithography.[11,12]
Because these substrates contain a dense arrangement of small
metal particles, a general characteristic is the close interaction
between different nanostructured components, which favors
the formation of hot spots, thereby dramatically increasing the
electromagnetic field necessary for SERS and thus improving
the detection limits.[13] These platforms are usually self-
sustained, portable, and stable in time, and the analyte is
usually deposited by casting or by dip-coating of the surface in
the problem solution for subsequent analysis with a Raman
microscope. Although these platforms provide much stronger
signals than those obtained using metal colloids, they also pose
important restrictions. Because the analyte molecules under
study are always the same (the laser beam is focused on a
specific region of the substrate) and the radiation is not
attenuated, there is a restriction in the laser energy and density
at the sample that can be effectively used without inducing
undesired reactions on the sample such as photobleaching,
combustion, sublimation, and even photocatalysis.[14]
Additionally, due to the static nature of these platforms, their
implementation within on-line devices for real-time analyte
monitoring is complicated.
Numerous examples have been reported of the application
of colloidal dispersions for ultrasensitive detection and
characterization of both small and macro-biomolecules such
asDNAandRNA,[15] small proteins,[16] as well as for the study
of kinetics in single enzymes,[17] and evaluation of drug
interactions with their specific receptors.[18] An elegant
application of nanoparticle colloids can be found in label-free
in vivo cell studies. Gold nanoparticles have been proven to
be freely uptaken by living organisms upon suitable surface
functionalization.[19] However, the distribution of nanoparticles
H & Co. KGaA, Weinheim www.small-journal.com 605
concepts R. A. Alvarez-Puebla and L. M. Liz-Marzan
Figure 2. Probing and imaging pH values within individual living cells using a SERS nanosensor. a) Photomicrograph of an NIH/3T3 cell after 4.5 h
incubation with the pMBA gold nanoparticle colloid. Lysosomal accumulations can be observed as black spots at the resolution of the optical
microscope. b) pH map of the cell displayed as false-color plot of the ratios between SERS lines at 1423 and 1076 cm–�1. The values given in the color
scale bar indicate the upper limit of each respective color. Scatteringsignals below a defined signal threshold (i.e., where no SERS signals exist) appear
in dark blue. c) Typical SERS spectra collected in the endosomal compartments with different pH. Reproduced with permission from Reference [20].
Copyright 2007, American Chemical Society.
606
in the cytosol or retained at the different organelle membranes
is stronglyaffectedbypH(Figure 2).[20] Interestingly, SERS is a
suitable technique to study the different cell compartments as a
function of medium and membrane pH.
Regarding the use of silver or gold nanostructured films as
direct sensing elements for SERS, plenty of examples can be
found in the literature. Films have been successfully employed
in the ultradetection and in vivo monitoring of metabolites,[21]
biodetection of pathogens,[22] characterization of pro-
teins,[23,24] and even detection and classification of living
organisms.[25,26] Systems that couple statistics to resolve
complex vibrational patterns deserve special attention. For
example, by coupling principal component analysis (PCA) to
theoutput from theSERS system (i.e., spectra), one candiscern
with no uncertainty the presence of a metabolic analyte in a
complex mixture extracted from a living animal (Figure 3).[27]
This, in the era of genomic doping drugs, is essential in sports as
well as very useful in the prediction of anomalies caused by
structural mutations.[28]
In an effort to combine the specific advantages of colloids
and films as sensor elements for SERS, a new family of
platforms is currently under rapid development. These
materials are characterized by using micrometer- or submic-
rometer-sized particles to support the enhancing plasmonic
nanostructures (see examples in Figure 4).[30-34] The final
hybrid materials are thus sufficiently small to behave as
colloidal suspensionsbutare largeenough tobeobservedunder
a conventional confocal microscope. The deposition of
plasmonic nanostructures on their surface can be carried out
indifferentways, througheitherdirectgrowth[31] orassemblyof
preformed nanoparticles,[32] and usually are aggregated in such
away that they become a dense collection of hot spots. Because
they can be considered as a discrete surface, one single particle
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is in principle enough to carry out the SERS analysis.
These colloidal platforms allow the addition of other
functionalities (such as magnetic response; see Figure 4a and
b), do not aggregate or settle in physiological media, and can
carry bulky macromolecules, which renders them optimal
candidates to be used in Raman flow cytometry[35] and
microfluidics.[36]
Althoughwe have seen that direct sensing can be applied in
a very flexible manner and can lead to ultrasensitive detection,
it poses a major drawback related to the complex nature of
biological fluids, since they contain a wide variety of molecular
moieties. A separation (purification) step is thus usually
required prior to SERS analysis because otherwise the
interpretation of the obtained signal becomes a hard task
and can even be impossible. Therefore, newmethodologies are
being developed that can allowdetection inblood, saliva, urine,
and other biofluids. One attractive option is the exploitation of
specific interactions between a capture agent, chemically
bound to the SERS enhancing substrate, and the analyte
present in the fluid of interest.[37,38] The strategy comprises
contact between the sensing colloidal composite and the fluid
of interest, followed by separation from the fluid by means of a
suitable functionality, such as magnetism or simply centrifuga-
tion, and finally washing and analysis. The SERS spectra
obtained before and after coupling will very likely correspond
to the part of themacromolecule close to the enhancing surface
as antibodies and DNA/RNA are usually very large and
efficient vibrational enhancement is never extended over more
than 2 nm from the surface (Figure 4c–e). Thus, these sensors
work indirectly (as opposed to ‘‘direct sensing’’), registering
the changes induced on the structure of the antibody, in
response to coupling of the antigen. This method offers some
advantages: first, it allows efficient separation of the analyte of
bH & Co. KGaA, Weinheim small 2010, 6, No. 5, 604–610
Figure 3. a) Surface plasmon resonance spectrum and scanning electron microscopy (SEM) image
of a pillared substrate used as optical enhancer. [28] b) SERS spectra of mixtures of three
different corticotrophin releasing factors (CRFs) belonging to human (H-CRF), bovine (B-CRF), and
sheep (S-CRF). c) Classification of human, bovine, and sheep CRFs and mixtures. Score plot
represents the first and second principal components (PC) for the partial least-square (PLS)
regression model of human (�, H-CRF), bovine (&, B-CRF), and sheep (~, S-CRF) CRFs, and
mixtures thereof (&, 0.9:0.1 H-CRF/B-CRF; ^, 0.75:0.25 H-CRF/B-CRF; �D, 0.5:0.5 H-CRF/S-CRF;
*, 0.5:0.5 H-CRF/B-CRF). d) Contour plot showing the angular correlation between the reference
and predicted identity matrices for B-CRF (samples 1–9), H-CRF (samples 10–19), S-CRF (samples
20–29), H-CRF/B-CRF/S-CRF 1:1:1 (samples 30, 31), H-CRF/BCRF 0.9:0.1 (samples 32, 33), H-CRF/
B-CRF 0.75:0.25 (samples 34, 35), H-CRF/S-CRF 0.5:0.5 (samples 36–38), and HCRF/ B-CRF
0.5:0.5 (samples 39–41). Reproduced from Reference [27].
interest from its matrix. Second, it does not require the use of
secondary detection antibodies and thus small analytes can be
detected, which are very unlikely to bind a second macro-
molecule because of sterical hindrance. Finally, and more
importantly, because some SERS peaks arising from parts of
the antibody unaffected by coupling of the antigen remain
constant, they can be used as an internal standard so that the
biomarker can be quantitatively determined.[33] Nevertheless,
this approach also has a key limitation as it is restricted to the
analysis of a small number of analytes so as to avoid the
complication of the vibrational spectra. As a result, the use of
these sensors is restricted to the detection and quantification of
small 2010, 6, No. 5, 604–610 � 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
one or two parameters at a time, not
being useful for multiplex high-
throughput screening.
3. Encoded particles
One of the modern trends in
nanomedicine is the development of
sensors that are capable of drawing a
rapid and accurate diagnostic regard-
ing the health condition of a patient,
or environmental risk in a short time
scale (typically seconds).[39,40] As
discussed above, direct SERS cannot
be used for this task as the interpreta-
tion of the vibrational spectra
recorded from a living system would
become impossible. However, clear
advances have been made toward the
useofencodedmicroparticlesasassay
platforms that can be used for appli-
cationsnotonly restrictedtodiagnosis
and biodetection but also in combi-
natorial chemistry anddrug discovery
or as contrast agents for in vivo
imaging. Such encoded particles
address some of the limitations posed
by conventional substrates. A short
list of advantages offered by these
substrates would include:
a) amenability to high-throughput
screening and multiplexing; b) larger
surface area for receptor conjugation
or solid-phase synthesis; c) better
accessibility of the analytes to the
entire sample volume for interaction
with bead-conjugated receptors, and
d) greater versatility in sample ana-
lysis and data acquisition. Although
the use of encoded nanoparticles as
biolabels is not new,[41] until recently
most of the approaches were mainly
based on fluorescence, particle shape
andsize,andvibrationalpatterns.[42–47]
Interestingly, the incorporation of
SERS labels can provide additional
advantages, such as a) ultrasensitive detection, which may
dramatically decrease the time required for spectral deconvo-
lution and facilitate implementation in real-time applications
such as high-throughput screening in flow-cytometry or
microfluidic systems; b) an unlimited number of barcodes
can be devised, since SERS spectra are essentially vibrational
fingerprints, and thus unique for each specific molecule, which
opens up the possibility of expanding the encoded library
toward infinity through combination of different tags with
comparable SERS cross sections, and c) the same hybrid
systems can be used as optical enhancing platforms for
fluorescence in sandwich key–lock applications such as
www.small-journal.com 607
concepts R. A. Alvarez-Puebla and L. M. Liz-Marzan
Figure 4. a) Transmission electron microscopy (TEM) image of gold nanorods supported on silica-
coated maghemite spindles. b) White-light optical image (left) and SERS mapping (right) of single gold-
coated maghemite spindles after 1-naphthalenethiol adsorption. Reproduced with permission from
Reference [32]. Copyright 2009, American Chemical Society. c) TEM image of a silver-coated carbon
nanotube. d) SERS spectra and e) schematic representation of the detection of the cocaine metabolite
benzoylecgonine (BGC) through conformation changes induced on a selective monoclonal antibody
(Ab) covalently supported on the silver-coated nanotube. Reproduced with permission from Reference
[33]. Copyright 2009, Royal Society of Chemistry.
608
ELISA-like microarrays, which will increase the detection
limits for pathogens or pathogen markers.
In general, the strategies that have been reported for SERS
particle encoding involve coverage of the optical enhancer (i.e.,
silver or gold metal nanoparticles or aggregates thereof) with a
Figure 5. a) TEM image of a SERS-encoded capsule comprising gold nanoparticles and their Raman tags,
coated with a thin layer of silica where a specific antibody has been coupled. Adapted with permission
from Reference [48]. Copyright 2009, American Chemical Society. b) General assay for detection by using
SERS-encoded nanoparticles. Different encoded particles are functionalized, each with a specific
antibody, and a mixture of the different particles is put into contact with the sample so that antigens
present in the sample will bind their specific antibodies. After washing, the beads are immersed in a
solution containing the secondary detection antibodies labeled with a fluorophore. Recognition is
achieved by recording the SERS spectra of the fluorescent beads. Adapted from Reference [53].
www.small-journal.com � 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
suitableprotective shell, usually silica
or poly(ethyleneglycol) (PEG), so
that the Raman code cannot leach
out.Thecoatingshouldbesufficiently
versatile to permit further functiona-
lization with appropriate functional
groups for conjugation of antibodies
or nucleic acids and stabilization in
biological media (Figure 5a).[15,48–50]
Another interesting feature is the
homogeneity in shape and size of the
encoded particles so that they can be
easily arranged as ordered systems
within amicrochip, aswell as to avoid
interference due to different (mor-
phology dependent) Rayleigh scat-
tering signals during read out in
automatic systems such as Raman
flow cytometers.[51,52]
The actual biodetection mech-
anism is based on classical
immunoassays as fluorescence immu-
noanalysis (FIA) or radioimmuno-
analysis (RIA), and thus requires a
secondary antibody, marked with
fluorophores, radioactive, or other
tags for detection of the positive
reaction between the capture antibody and the antigen for each
different barcode (Figure 5b). That is a powerful strategy for
multiplex high-throughput screening in chips but encompasses
several limitations, including the need to use two specific
antibodies for each antigen, which again hinders the detection of
small antigens, as well as the require-
ment of two readout systems: SERS
and fluorescence.
Another stylish application of
SERS-encoded nanoparticles is the
invivo imagingofcells,[54] tissues,and
organs. The high intensity provided
by SERS-encoded particles, together
with the possibility of preparing
extremely bright, biocompatible,
and small capsules, which can be
functionalized and directed against
specific receptors, constitutes a com-
petitive alternative to quantum dots
and magnetic nanoparticles. SERS-
encoded nanoparticles have already
been successfully employed in the
multiplex detection of different
receptors within cells[9,55] and tissues
(Figure6).[54] Inaddition,Nie’sgroup
has recently proven the ability to
irradiate near infrared (NIR) lasers
through tissues and record the SERS
fingerprint of an encoded particle
functionalized with an antibody
selectively binding specific cancer
tumors (Figure 7).[56]
small 2010, 6, No. 5, 604–610
Figure 6. Spectral deconvolution in a multiplex tissue assay where three different encoded particles (BFU, AOH, and YOYO) were conjugated to two
different antibodies: anti-CK18 (BFU–CK18), anti-PSA (AOH–PSA), and a fluorescent-dye-targeted DNA (YOYO). A) Bright-field image of prostate tissue.
Spectra were recorded at each spot in a raster pattern. The raster spans epithelia (E) of two prostate glands, a narrow band of stromal tissue separating
the glands (S), and thegland lumen(L). B) Spectraldeconvolution for a single spot measurement. Upper traces represent the measuredspectrum(gray)
and best-fit spectrum (black). Colored lines represent extracted spectra for BFU–CK18 (red), AOH–PSA (green), and YOYO (blue). C) Fitting of the
obtainedspectra(gray)with thetwoSERStags(BFUandAOH)(black)spectra. D)Purecomponent imagesandco-localization imageof thethreeprobes.
DNA is visualized with the YOYO nucleic acid stain and marked in blue; signals of CK18 are marked in red and PSA in green. The co-localization image
identifies epithelial nuclei (magenta) and co-expression of CK18 and PSA specifically in the epithelium (yellow). Reproduced with permission from
Reference [54]. Copyright 2008, American Chemical Society.
Figure 7. a) In vivocancer targetingand SERSdetectionby usingScFv-antibody conjugated gold
nanoparticles thatrecognizethetumorbiomarkerEGFR.Top:Photographsshowingalaserbeam
focusingonthetumorsiteorontheanatomiclocationof liver.Lower:SERSspectraobtainedfrom
the tumor and the liver locations by using a) targeted and b) non-targeted nanoparticles.
Two nude mice bearing human head and neck squamous cell carcinoma (Tu686) xenograft
tumor (3-mm diameter) received 90mL of ScFv EGFR-conjugated SERS tags or PEGylated SERS
tags (460 pM). The particles were administered via tail-vein single injection. SERS spectra were
taken5 h post injection. InvivoSERSspectrawereobtainedfromthetumorsite(red)andtheliver
site (blue) with 2 s signal integration and at 785-nm excitation. The spectra were background
subtracted and shifted for better visualization. The Raman reporter molecule was malachite
green, with distinct spectral signatures as labeled. Laser power: 20 mW. Reproduced with
permission from Reference [56]. Copyright 2008, Royal Society of Chemistry.
small 2010, 6, No. 5, 604–610 � 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
4. Conclusions and Outlook
To sum up, SERS represents an attrac-
tive technique, which advances hand in
hand with nanoscience and nanotechnol-
ogy, to offer solutions for a diverse array of
biomedical applications. Nanomaterials
can be engineered for individual and multi-
modal applications in SERS, including
biomolecular recognition and characteriza-
tion, ultrasensitive diagnosis and biodetec-
tion, and biomedical imaging. Although
SERS has already been used for a wide
range of applications both in vitro and in
vivo, full realization of its potential requires
addressing a number of open issues, includ-
ing nanoparticle stability in biological
fluids, single-event recognition detection,
label-free analysis and classification, quan-
tification of the analytes of interest, and
acute and long-term health effects of
nanomaterials.
Acknowledgements
R.A.A.-P. acknowledges the RyC (MEC,
Spain) program. This work was funded by
the Spanish Ministerio de Ciencia e Innova-
c i on (Grants MAT2007-62696 and
MAT2008-05755, Consolider Ingenio 2010-
www.small-journal.com 609
concepts R. A. Alvarez-Puebla and L. M. Liz-Marzan
610
CSD2006-12) and the Xunta de Galicia (Grants PGI-
DIT06TMT31402PR, 08TMT008314PR).
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Received: September 26, 2009Revised: November 25, 2009Published online: January 27, 2010
small 2010, 6, No. 5, 604–610