SERS-Based Diagnosis and Biodetection

concepts

604

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: [email protected]; [email protected]

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

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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

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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

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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-

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610

CSD2006-12) and the Xunta de Galicia (Grants PGI-

DIT06TMT31402PR, 08TMT008314PR).

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bH & Co. KGaA, Weinheim

Received: September 26, 2009Revised: November 25, 2009Published online: January 27, 2010

small 2010, 6, No. 5, 604–610

SERS-Based Diagnosis and Biodetection

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