plasmonic materials for ... · plasmonics is an emerging branch of nanophotonics that examines the...

368 MRS BULLETIN • VOLUME 30 • MAY 2005

IntroductionPlasmonics is an emerging branch of

nanophotonics that examines the propertiesof the collective electronic excitations innoble metal films or nanoparticles knowncolloquially as surface plasmons. The excite-ment of plasmonics lies in its potential toachieve highly miniaturized and sensitivephotonic devices by controlling, manipulat-ing, and amplifying light on the nanometerlength scale.1–3

To date, a variety of passive plasmonicdevices have been demonstrated, includingfilters,1 waveguides,1,3 polarizers,4 Braggreflectors,1 and nanoscopic light sources.5On the horizon are active plasmonic de-vices, such as light-output enhancers fororganic light-emitting diodes6,7 as well asswitches and modulators.8 Furthermore,our rapidly improving understanding ofthe interactions between adsorbed mole-cules and plasmonic nanostructures (i.e.,molecular plasmonics)9 is having a signifi-cant impact on a broad spectrum of other

applications, including nanoscale opticalspectroscopy,10 surface-enhanced Ramanspectroscopy,11 surface plasmon resonancesensing,12,13 and nanolithography.14

There are two types of surface plasmonresonance—localized and propagating.This article will mostly be concerned withthe former, which we term localized surfaceplasmon resonance (LSPR). This occurs in silver and gold nanoparticles in the10–200 nm size range and results in am-plification of the electric field E near the particle surfaces such that |E|2 can be100–10,000 times greater in intensity thanthe incident field. The field has a spatialrange on the order of 10–50 nm and isstrongly dependent on nanoparticle size,shape, and local dielectric environment.Propagating plasmons, which are oftencalled surface plasmon polaritons (SPPs),are associated with smooth, thin films ofsilver and gold with thicknesses in the10–200 nm range. Propagating plasmons

lead to smaller field enhancements (10–100times) and a larger spatial range (�1000 nm).

This article will focus on the fabricationand characterization of plasmonic materialsthat show promise in chemical/biologicalsensing and surface-enhanced spectroscopyapplications. In the first part, the simple,massively parallel method of nanospherelithography (NSL) and its use in the fabri-cation of size- and shape-controlled nano-structures is briefly reviewed. Also, theessential physics of LSPR and the theoreti-cal methods used to understand it are de-scribed, and key results concerning theshort- and long-range distance depen-dences of the electromagnetic fields sur-rounding the nanoparticles are summarized.In the second part of this article, we focuson the relationship between LSPR spec-troscopy and surface-enhanced Ramanspectroscopy (SERS), as revealed by surface-enhanced Raman excitation spectroscopy(SERES). SERES provides a systematic, re-producible way to optimize the signal in-tensity in SERS experiments.

Nanosphere LithographyNanosphere lithography (NSL)15 is a sur-

prisingly powerful yet simple approach to the fabrication of nanoparticle arrayswith precisely controlled shape, size, andinterparticle spacing.

Nanosphere lithography (Figure 1) be-gins with the self-assembly of monodis-perse polystyrene or SiO2 nanospheres ofdiameter D to form a single- or double-layer colloidal crystal mask for materialdeposition. A substrate (Figure 1a) is pre-pared so that the nanospheres can freelymove until they reach their lowest energyconfiguration. This is achieved by chemi-cally modifying the nanosphere surfacewith a negative charge that is electrostati-cally repelled by a negatively chargedsubstrate such as mica or chemicallytreated glass. As the solvent (water) evap-orates, capillary forces draw the nano-spheres together, and they crystallize intoan hcp pattern on the substrate. As in allnaturally occurring crystals, nanospheremasks include a variety of defects that ariseas a result of nanosphere polydispersity,site randomness, point defects (vacancies),line defects (slip dislocations), and poly-crystalline domains. Typical defect-free do-main sizes are in the 10–100 �m range.Following self-assembly of the nanospheremask, a metal or other material is then de-posited by physical vapor deposition froma collimated source normal to the substratethrough the nanosphere mask to a con-trolled thickness. The resulting surface isreferred to as a metal (e.g., Ag) “film overnanosphere” (FON) surface. Ag FON sur-faces are robust plasmonic materials for

Plasmonic Materialsfor Surface-EnhancedSensing andSpectroscopy

Amanda J. Haes, Christy L. Haynes, Adam D. McFarland, George C. Schatz, Richard P.Van Duyne, and Shengli Zou

AbstractLocalized surface plasmon resonance (LSPR) excitation in silver and gold nanoparticles

produces strong extinction and scattering spectra that in recent years have been usedfor important sensing and spectroscopy applications.This article describes thefabrication, characterization, and computational electrodynamics of plasmonic materialsthat take advantage of this concept.Two applications of these plasmonic materials arepresented: (1) the development of an ultrasensitive nanoscale optical biosensor basedon LSPR wavelength-shift spectroscopy and (2) the use of plasmon-sampled andwavelength-scanned surface-enhanced Raman excitation spectroscopy (SERES) toprovide new insight into the electromagnetic-field enhancement mechanism.

Keywords: localized surface plasmon resonance spectroscopy, nanosensing,plasmonic materials, surface-enhanced Raman spectroscopy.

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Plasmonic Materials for Surface-Enhanced Sensing and Spectrosopy

MRS BULLETIN • VOLUME 30 • MAY 2005 369

SERS applications.16,17 If the nanospheremask is removed, typically by sonicatingthe entire sample in a solvent, surface-confined nanoparticles are left behind thathave a triangular footprint. In a typicalNSL process, the deposition of 50 nm ofAg over a single-layer mask self-assembledfrom nanospheres with D � 400 nm pro-duces nanotriangles with an in-plane width

a 100 nm, height b 50 nm, and interpar-ticle separation distance dip 230 nm.

Size- and Shape-TunableLocalized Surface PlasmonResonance Spectra

NSL-derived nanoparticles exhibit in-tense UV–visible extinction (i.e., the sum ofabsorption and scattering) bands that are

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not present in the spectrum of the bulkmetal. Figure 1b shows that the LSPRspectra can easily be tuned all the wayfrom the near-UV through the visible spec-trum18 and even into the mid-IR19 by chang-ing the size or shape (triangle or hemisphere)of the nanoparticles. Note that the LSPRbandwidth does not change significantlyas the wavelength at peak maximum, �max,is tuned. Additionally, several other sur-prising LSPR optical properties have beendiscovered for NSL-derived Ag nano-particles: (1) �max shifts by 2–6 nm per1 nm variation in nanoparticle width orheight,18 (2) the molar decadic (tenfold) extinction coefficient is � 3 � 1011

M�1 cm�1,18 (3) the LSPR oscillator strengthper atom is equivalent to that of atomic sil-ver in gas or liquid phases,18 (4) resonantRayleigh scattering20,21 occurs with an effi-ciency equivalent to that of 106 fluo-rophors,22 and (5) local electromagneticfields are amplified by factors of |E|2

104, leading to intense signals in all surface-enhanced spectroscopies.11

Fundamentals of LocalizedSurface Plasmon ResonanceSpectroscopy

The simplest theoretical approach avail-able for modeling the optical properties ofnanoparticles is classical electrodynamics(i.e., solving Maxwell’s equations with themetal dielectric constant taken from bulkmeasurements). For spherical particles, thisleads to the following (Mie theory) expres-sion for the extinction coefficient E(�) in thelong-wavelength limit:23

. (1)

Here, NA is the areal density of the nano-particles, a is the radius of the metallic nano-sphere, m is the dielectric constant of themedium surrounding the nanosphere (as-sumed to be a positive, real number), � is thewavelength, and r and i are the real andimaginary parts of the metal dielectric func-tion. This formula predicts a resonant peakwhen r � �2 m, which for silver and goldoccurs in the visible portion of the spectrum.In addition, any change in the dielectricconstant of the medium (e.g., when mole-cules adsorb on the particle) leads to achange in the resonance wavelength.

When one considers spheroidally shapedparticles, the term r � 2 m in the denomi-nator in Equation 1 is replaced by r � � m ,where � is a parameter that depends onthe shape of the spheroid, increasing from2 for a sphere to 17 for a spheroid with anaspect ratio of 5:1. This leads to strong de-

εεεε

εε

εε

ε

� � εi

�εr � 2εm�2 � εi2�

E�λ� �24πNAa3εm

3�2

λln�10�

�

ε

Figure 1. (a) Schematic representation of the nanosphere lithography (NSL) fabricationprocess.The AFM image in step 3 is 5 �m � 5 �m. (b) Size- and shape-tunable localizedsurface plasmon resonance spectra of various Ag nanoparticles (labeled A–H) fabricatedby NSL.The wavelength of maximum extinction, �max, is changed by varying the in-planewidth a and out-of-plane height b of the nanoparticles.



pendence of �max on particle shape, andthere is also a strong size dependence thatarises from electrodynamic effects that arenot contained in Equation 1. In addition,many of the samples considered in this workcontain an ensemble of nanoparticles thatare supported on a substrate, leading to a dependence on interparticle spacing2 andsubstrate dielectric constant.24

Electrodynamic CalculationsEquation 1 provides useful insight, but it

only applies to spherical particles. In orderto describe particles like those pictured inFigure 1a, it is necessary to use a numericalmethod. Several methods have been devel-oped for solving Maxwell’s equations usingfinite-element-based approaches, and onethat we have found to be particularly use-ful is the discrete dipole approximation(DDA).25–27 In this method, the particle issubdivided into an array of cubical, polar-izable elements. When a plane wave fieldis applied to the particle, the resulting in-duced polarizations in the elements are cal-culated, and from these the extinction andlocal fields are determined. Figure 2a showsextinction cross sections that have beencalculated with this approach for severalparticle shapes, all for silver particles withthe same volume. This shows that a spherehas a plasmon nearer the blue end of thespectrum (Figure 2b), and the particles withpoints (cubes, prisms, pyramids) are nearerthe red end of the spectrum (Figures 2cand 2d).

The electromagnetic (EM) mechanism ofSERS11 predicts an enhancement factor pro-portional to |E(�)|2|E(�’)|2, where � and�’ are the incident and Stokes-shifted fre-quencies, respectively. To estimate the en-hancement factor, in Figures 2b–2d weshow contours of |E|2 around three of theparticles for wavelengths corresponding to�max and for polarizations that lead to thelargest |E|2. These figures show that thepeak field for a sphere is on the order of 102,while that for the tetrahedron is 104. If weapproximate the enhancement factor as|E(�)|4, we see that the highest enhance-ments are on the order of 108, which, asnoted later, is about what is found forNSL-derived particles.

Distance Dependence of theLocalized Surface PlasmonResonance

The electrodynamics results in Figure 2show that the electromagnetic fields sur-rounding Ag nanoparticles excited at �max drop off quickly as one moves a fewnanometers away from the particle sur-face. How can the range of these fields bemeasured experimentally? Scanning near-field optical microscopy is certainly a pos-

sibility; however, the resolution currentlyobtainable (�10 nm) would only providea rough picture.

We have pursued an alternative strategyto experimentally measure the range of theelectromagnetic fields. It is apparent fromEquation 1 that �max of noble metal nano-particles is highly dependent on the dielectric properties of the surroundingenvironment. For NSL-derived Ag nano-particles, it has been demonstrated experi-mentally that �max is a linear function of thesolvent refractive index, nm (where the ex-ternal dielectric constant, m, is equal to nm

2)with a slope of approximately 200 nm perrefractive index unit (RIU, defined as achange of 1 in the refractive index).28 Inaddition, �max is responsive to molecule-induced changes in the local dielectric envi-ronment. For example, the chemisorption ofone monolayer of hexadecanethiol causes�max of a NSL-derived Ag nanotriangle (in-

ε

plane width a � 100 nm, out-of-planeheight b � 50 nm) to shift by �max �40 nm (Figure 3a).29,30

The chemisorption-induced LSPR shiftexperiment provides a method for probingthe short-range (�0.5–3.5 nm) distance dependence of the electromagnetic fields surrounding NSL-derived nanoparticles.Figure 3c demonstrates that the depend-ence of �max on the chain length of analkanethiol monolayer is linear with a re-markably large slope of 3.1 nm per CH2unit.29,30 These results can be interpretedusing a model of the refractive-index re-sponse of propagating SPPs on a planarnoble metal surface:31

, (2)

where �max is the wavelength shift, m isthe refractive-index sensitivity, n is the change in refractive index induced by

�max � mn�1 � exp��2d�ld�

Figure 2. (a) Extinction efficiency (ratio of cross section to effective area) of silver nanoparticles in vacuum having the shapes indicated. Each particle has the same volume, taken to bethat of a sphere with a radius of 50 nm. |E|2 contours (E is electric field) for a (b) sphere,(c) cube, and (d) pyramid, plotted for wavelengths corresponding to the plasmon peak in(a), with peak |E|2 values of 54, 745, and 9770, respectively.



an adsorbate, d is the effective adsorbatelayer thickness, and ld is the characteristicelectromagnetic-field decay length. Thismodel assumes exponential decay of theelectromagnetic field normal to the planarsurface, which is accurate for a SPP, but isundoubtedly an oversimplification for theelectromagnetic fields associated with noblemetal nanoparticles. Nevertheless, Equa-tion 2 does a remarkably good job of ac-counting for the data in Figure 3c if oneassumes a value ld � 5–6 nm. We will returnto a justification of this value in a moment.

Recently, it has become possible to usedark-field resonant Rayleigh scattering toperform the �max measurement for isolatedsingle nanoparticles, and it was found thatfor chemically synthesized Ag triangularprisms (Figure 3e), �max is a linear func-

tion of the refractive index of the externalmedium (Figure 3d) with a slope of�200 nm/RIU.32 In addition, the sameLSPR shift of �40 nm as seen for the NSLparticle array was observed for a mono-layer of hexadecanethiol on a single par-ticle; more important, the dependence of�max on the chain length of the alka-nethiol monolayer (Figure 3f) was found tobe linear, with a large slope of 3.5 nm perCH2 unit.32

The similarity of the adsorbate-inducedLSPR shift behavior for both NSL-derivednanoparticle arrays and single nanoparticlesleads to the following important conclu-sions: (1) the short-range distance depend-ences for both systems can be described byEquation 2, and (2) interparticle coupling ef-fects are minimal at the dip values imposed

by the NSL nanofabrication technique. Inaddition, it should be pointed out that fromthe viewpoint of chemical sensing, thesesingle-nanoparticle LSPR shift results aretruly remarkable. Based on the average sur-face area of the nanoparticles prepared andthe monolayer packing density of moleculeson Ag, this saturation response correspondsto the detection of fewer than 60,000 surface-confined molecules of hexadecanethiol. Ad-ditional experiments (not described here)demonstrate a limit of detection of �2000molecules.32 The real-time kinetics of ad-sorbates binding to single nanoparticles isalso accessible from this experiment.32

LSPR spectroscopy can also be used to probe the long-range (up to 40 nm) dis-tance dependence of the electromagneticfields surrounding NSL-derived Ag nano-particles.33 Figure 4 (see 4a and the solidtriangles in 4c) shows that the dependenceof �max on the thickness of multilayer ad-sorbate shells synthesized through the in-teraction of HOOC(CH2)10SH and Cu2� isnonlinear. The multilayer thickness requiredto saturate the LSPR shift response wasfound to be dependent on the composition(Ag versus Au), shape (triangle versushemisphere), in-plane width, and out-of-plane height of the nanoparticles. Theseresults verify that the linear distance de-pendence found in the case of alkanethiolmonolayers was the thin-shell limit of alonger-range, nonlinear dependence. Theycan be understood on the basis of an ex-ponentially decaying electromagnetic fieldwith ld in the range of 5–6 nm for Ag nanotriangles (a 100 nm, b 50 nm). Amore accurate simulation of the data hasbeen provided by DDA calculations,33 andthe results are in quantitative agreementwith experiment. Comparison of the long-range LSPR spectroscopy results for Ag(Figure 4a and solid circles in 4d) versus Au(Figure 4b and red triangles in 4d) showsthat Ag has a longer spatial electromag-netic range and is more sensitive to adsor-bates than Au.

Nanoscale Optical BiosensorsBased on LSPR Spectroscopy

The LSPR wavelength shift response,�max, has been used to develop a new classof nanoscale optical biosensors.34–38 Twomodel systems, biotin–streptavidin34 andbiotin–anti-biotin,35 were studied to illus-trate the desirable attributes of LSPR-basednanoscale affinity biosensors. Streptavidin, atetrameric protein, can bind up to four biotinylated molecules (i.e., antibodies, in-hibitors, nucleic acids, etc.) with minimalimpact on its biological activity, and there-fore it provides a ready pathway for extend-ing the analyte accessibility of the LSPRnanobiosensor. Anti-biotin, an antibody,

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Figure 3. (top row) Localized surface plasmon resonance (LSPR) spectroscopy of a Agnanoparticle array fabricated by nanosphere lithography in a N2 environment: (a) Extinctionspectrum of the array (curve 1) before chemical modification, wavelength of extinctionmaximum �max � 594.8 nm, and (curve 2) after modification with 1 mM hexadecanethiol,�max � 634.8 nm. (b) Tapping-mode atomic force microscopy (AFM) image of the array in(a); nanosphere diameter D � 390 nm, deposited mass thickness dm � 50 nm; Ag on micasubstrate; scan area, 3.0 �m2. After solvent annealing, nanoparticle in-plane width is100 nm and out-of-plane height is 51 nm. (c) Alkanethiol chain length dependence on theLSPR spectral peak shifts for the array. Even and odd carbon chain lengths are depictedwith different symbols to emphasize the difference in the terminal bond orientation withrespect to the substrate, which leads to different observed trends for the two cases. (bottomrow) LSPR spectroscopy of single Ag nanoparticles produced by chemical synthesis:(d) scattering spectrum of a single Ag nanoparticle (curve 1) before chemical modification,�max � 510.2 nm, and (curve 2) after modification with 1 mM hexadecanethiol, �max � 550.9 nm.The circled nanoparticle in (e) produced the signal for these curves.(e) Dark-field resonant Rayleigh scattering image of a random array of chemicallysynthesized Ag nanoparticles (image dimensions, 130 �m � 130 �m). (f) Alkanethiol chain length dependence on the LSPR spectral peak shifts for a single Ag nanoparticle.The open circles represent an overlay of the array data from (c).The solid circles are singlenanoparticle measurements.



provides a straightforward platform to extend the LSPR nanosensor technology toimmunoassay technology. The �max forbiotin-functionalized Ag NSL nanopar-ticle arrays was measured as a function ofthe concentration of streptavidin (SA) overthe concentration range 10�15 M [SA] 10�6 M. Fitting the data to the theoreticalnormalized response expected for 1:1 binding of a ligand to a multivalent re-ceptor with different sites but invariantaffinities yielded the following values: saturation response Rmax � 26.5 nm,surface-confined thermodynamic binding

constant Ka,surf � 1011 M�1, and limit of detection LOD 1 pM SA.34 Similarly, the LSPR response curve for the bindingof anti-biotin (AB) to biotin-functionalizedAg NSL nanoparticles was measured, and the data analysis yielded Rmax �38.0 nm, Ka,surf � 4.5 � 107 M�1, and LOD 7 � 10�10 M AB.35 As predicted, the LODof the nanobiosensor studied is lower forsystems with higher binding affinities, suchas for the well-studied biotin–streptavidincouple, and higher for systems with lowerbinding affinities, as seen in the anti-biotinsystem.

A comparative analysis of real-time re-sponses of a commercial propagating SPRsensor (planar thin film of gold) and theLSPR sensor (Ag NSL nanoparticle array)was carried out using the binding of Con-canavalin A (ConA), a mannose-specificplant lectin, to mannose-functionalized self-assembled monolayers (SAMs).36 Duringthe association phase in the real-time bind-ing studies, both sensors exhibited qualita-tively similar signal-versus-time curves.However, in the dissociation phase, theSPR sensor showed an approximately fivetimes greater loss of signal than the LSPRsensor. A comprehensive set of nonspecificbinding studies demonstrated that thissignal difference was not the consequenceof greater nonspecific binding to the LSPRsensor, but rather a systematic function of nanoparticle structure. Ag nanoparticleswith larger aspect ratios showed larger dissociation phase responses than thosewith smaller aspect ratios. DDA calcula-tions demonstrated that this response is aconsequence of the similarity in length scalebetween the electromagnetic-field decaylength and the physical size of ConA.36

Recently, the LSPR sensor has been successfully used for the detection of anAlzheimer’s disease biomarker from bothsynthetic37,38 and human patient38 samples(see also the article by Thaxton et al. in thisissue). In this work, the interactions betweenthe biomarker (antigen), amyloid-� deriveddiffusible ligands (ADDLs), and specificanti-ADDL antibodies were studied. Usingthe sandwich assay format, the LSPR sen-sor provided quantitative binding informa-tion for both antigen and second antibodydetection that permits the determinationof ADDL concentration. This unique capa-bility offers the possibility of analyzing theaggregation mechanisms of this putativeAlzheimer’s disease pathogen at physio-logically relevant monomer concentrations.Monitoring the LSPR-induced shifts fromboth ADDLs and a second polyclonal anti-ADDL antibody as a function of ADDL con-centration reveals two ADDL epitopes (theexact binding site on an antigen that bindsto an antibody) that have binding constantsto the specific anti-ADDL antibodies of 7.3 � 1012 M�1 and 9.5 � 108 M�1. Further-more, this study demonstrated for the first time that the LSPR nanosensor was suc-cessful at analyzing human brain extractand cerebrospinal fluid (CSF) samples. Examination of these results from bothAlzheimer’s disease and control patients reveals that the LSPR nanosensor providesnew information relevant to the understand-ing and possible diagnosis of Alzheimer’sdisease. This exciting advance is one of thefirst examples in which nanotechnologyhas been applied to clinical materials forbiomolecular diagnostics.38

Figure 4. Localized surface plasmon resonance (LSPR) spectra for Ag and Au nanoparticlesfabricated by nanosphere lithography. Long-range distance dependence: (a) Ag nanoparticles(in-plane width a � 100 nm, out-of-plane height b � 50.0 nm) for 0–20 layers ofCu2�/HS–(CH2)10COOH; (b) Au nanoparticles (a � 70 nm, b � 50.0 nm) for 0–14 layers ofCu2�/HS–(CH2)10COOH. (c) Shape dependence for Ag nanoparticles: (solid triangles)LSPR shift versus number of layers in the self-assembled monolayer (SAM) thickness forsolvent-annealed Ag nanoparticles; (blue circles) LSPR shift versus number of layers in theSAM thickness for thermally annealed (600�C for 1 h) Ag nanoparticles. (inset, upper left)Atomic force microscopy (AFM) image of solvent-annealed nanoparticles (a � 114 nm, b � 54 nm). (inset, lower right) AFM image of thermally annealed nanoparticles (a � 110 nm, b � 61 nm). (d) Composition dependence: (solid circles) LSPR shift versus layer thicknessfor Ag nanoparticles (a � 70 nm, b � 50.0 nm); (red triangles) LSPR shift versus layerthickness for Au nanoparticles (a � 70 nm, b � 50.0 nm).



with SERS. Consequently, there is renewedexperimental effort in SERES seeking toverify various aspects of the EM theory.

We will now discuss some recent resultsfrom our laboratory obtained using two ap-proaches—plasmon-sampled (PS)42 andwavelength-scanned (WS) SERES. WS-SERES requires both a broadly tunablelaser and detection system. This is not commonly available; however, with thesize- and shape-tunable LSPR spectra ob-tainable with NSL- or EBL-derived plas-monic materials, this problem can becircumvented. NSL and EBL are used toprepare many samples with differentLSPR �max values. Each sample is thor-oughly characterized structurally by atomicforce microscopy or scanning electronmicroscopy. Correlated, spatially resolvedLSPR and SER spectra are measured witha single excitation wavelength at multiplelocations on each sample using a Ramanmicroscope. Figure 5 shows representativeLSPR spectra (Figures 5a–5c) and SERSspectra (Figures 5d–5f) for benzenethioladsorbed on NSL-fabricated Ag nanopar-ticles excited at three common, fixed, exci-tation wavelengths. The SERS enhancementfactors for Figures 5d–5f are 7.6 � 107, 6.3� 107, and 9.0 � 107, respectively,42 whichare results that roughly agree with theoreti-cal estimates (the electrodynamic calcula-tions described earlier). PS-SERES plots(enhancement factor versus LSPR �max) forthe 1575 cm�1 band of benzenethiol ex-cited at three different laser wavelengthseach showed a well-defined maximum

It is important to note that this technol-ogy is in its infancy. There are at least twomain research objectives that must be metbefore it becomes available. First, the sensormust undergo rigorous testing using CSFsamples from many more patients. In orderfor this objective to be met, we must inte-grate this technology with a redesignedchip. Currently, approximately 250 �l of CSFis required for each assay. Because spinaltaps are painful, it is ideal to minimize theamount of sample needed. By incorporat-ing microfluidics and miniaturizing thesample cell, this objective will be met.

The examples just given all involvedNSL-derivatized Ag nanoparticle arrays asthe sensor platform. It has now been demon-strated that all of the outstanding attributesof the propagating SPR sensor, the “goldstandard” in optical biosensing, are retainedor exceeded in a single-nanoparticle sen-sor.32,39 Dark-field LSPR scattering spec-troscopy and microscopy were used todemonstrate zeptomole (10�21) sensitivitycoupled with real-time kinetic analysis.32

Streptavidin biosensing has also beendemonstrated on single Ag nanoparticles.A 12.7 nm redshift in the LSPR �max arisesfrom the detection of 700 streptavidinmolecules.40

Surface-Enhanced RamanSpectroscopy

The local electromagnetic fields that ac-company photon excitation of the LSPR area key factor leading to the intense signalsobserved in all surface-enhanced spectro-

scopies.11 Surface-enhanced Raman spec-troscopy (SERS) is characterized by anensemble-averaged intensity enhancementfactor, EF, of �106, for analytes bound tonoble metal surfaces that possess randomroughness,41 or EF 107–108, for surfaceswith intentionally nanofabricated featuresizes in the �100 nm range.42 Recent reportsof single-molecule detection43,44 using SERSon Ag nanoparticle clusters have rejuve-nated interest in this widely used analyti-cal technique. A clear understanding of themechanism responsible for the enormousenhancement factors (�1014–1015) observedin single-molecule SERS remains elusive.

The electromagnetic (EM) mechanism ofSERS mentioned earlier predicts that thereis a well-defined relationship between theLSPR spectrum and the SERES spectrum ofa SERS-active surface. In particular, very specific quantitative predictions for themagnitude of the enhancement factor onnanoparticle size, shape, and local dielectricenvironment are made. Wavelength-scanned SERES experiments on microfabri-cated surfaces were carried out in the early1980s at Bell Laboratories,45,46 with a viewtoward verifying these predictions of theEM mechanism. Both as a consequence ofexperimental difficulty and the dissolutionof the Bell group, many EM predictions re-main purely in the domain of theory. Theplasmonic materials that are now readilyavailable from NSL and electron-beam li-thography (EBL) provide a new platformfor the detailed study of the electromagnetic-field enhancement mechanism associated

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Figure 5. (a)–(f) Correlated, spatially resolved, localized surface plasmon resonance (LSPR) and surface-enhanced Raman spectroscopy (SERS)results for benzenethiol adsorbed on Ag nanoparticle arrays fabricated by nanosphere lithography. (a), (d) Ag nanoparticles fabricated withnanosphere diameter D � 280 nm and deposited mass thickness dm � 36 nm, probed with an excitation wavelength �ex � 514.5 nm, power � 0.7 mW.A representative atomic force micrograph of the substrate is shown in the inset. (b), (e) Ag nanoparticles fabricated with D � 280 nm, dm � 36 nm,probed with �ex � 532.0 nm, power � 0.7 mW. (c), (f) Ag nanoparticles fabricated with D � 400 nm, dm � 56 nm, probed with �ex � 632.8 nm, power � 1.2 mW. (g)–(i) Plasmon-sampled surface-enhanced Raman excitation spectroscopy (PS-SERES) results for the 1575 cm�1 band ofbenzenethiol with three different excitation wavelengths: (g) �ex � 514.5 nm, (h) �ex � 532.0 nm, and (i) �ex � 632.8 nm. For each �ex, both thewavelength location of the excitation (solid line) and the scattering (dashed line) are marked.The overlaid curves represent the bin-averaged values ofthe LSPR �max and the enhancement factor. Bin widths are (g) 24 nm, (h) 16 nm, and (i) 16 nm.



representing the highest intensity SERSsignal (Figures 5g–5i).42 In each case, thesePS SERES spectra follow the behavior pre-dicted by the EM theory, that is, the largestenhancement factor occurs when the en-ergy corresponding to the LSPR maximumis located near the midpoint between theenergy of laser excitation and the energyof the Raman photons. These results un-ambiguously demonstrate (1) a systematicapproach to the optimization of SERS spec-tra on nanoparticle substrates and (2) thatlarge, ensemble-averaged, SERS enhance-ment factors (�1 � 108) are readily obtain-able from Ag NSL-derived nanoparticlearray surfaces. Recently, these results havebeen corroborated using Au nanoparticlearray surfaces fabricated by EBL.47

Figure 6a shows the SERS spectrum ofbenzenethiol adsorbed on a Ag FON sur-face. The nanostructure of Ag FON surfacescan be quantitatively characterized byatomic force microscopy (Figures 6b and6c). Ag FON surfaces have a nanostruc-ture size distribution that is less broadthan that of a randomly roughened sur-face but not as narrow as a NSL-derivedsurface. Consequently, Ag FON surfaceshave well-defined LSPR spectra (Figures 6dand 6e, solid curves), albeit broader thanthe LSPR spectra for Ag NSL-derivednanoparticle arrays. WS SERES spectra forthe 1081 cm�1 band of benzenethiol areshown in Figures 6d and 6e (data points).Using neat liquid benzenethiol as the nor-mal Raman standard, the peak enhance-ment factor values were calculated to be2.7 � 106 and 1.9 � 106 for Figure 6d and 6e,respectively. Two different behaviors areobserved in the WS-SERES spectra forthese surfaces. In one case, the WS-SERESspectrum peaks to the red of the LSPRspectrum (Figure 6d, data points) and inthe other, it tracks the LSPR spectrum(Figure 6e, data points). This outcome isattributed to the different nanostructuresize distributions for each surface. From apractical perspective, we point out that it isnot yet possible to determine the optimumlaser excitation wavelength for an Ag FONwithout doing SERES.

Figure 6f shows the SERS spectrum of benzenethiol adsorbed on a Ag NSL-derived nanoparticle array surface. A typi-cal atomic force micrograph of a Ag NSLsurface is shown in the inset in Figure 6g.The LSPR spectrum of a Ag NSL array isshown in Figure 6g (solid red line), alongwith the corresponding WS-SERES spec-trum for the 1081 cm�1 band of benzenethioladsorbed on the same Ag NSL array (datapoints). The peak enhancement factor forthe 1081 cm�1 band of benzenethiol ad-sorbed on this surface is 1.9 � 107, againusing neat liquid benzenethiol as the nor-mal Raman standard. Note that the peak

Figure 6. Localized surface plasmon resonance (LSPR), surface-enhanced Ramanspectroscopy (SERS), and wavelength-scanned surface-enhanced Raman excitationspectroscopy (WS-SERES) results for benzenethiol adsorbed on Ag film-over-nanosphere(Ag FON) surfaces and nanoparticle arrays fabricated by nanosphere lithography (NSL). (a) SERS spectrum measured from Ag FON surface with excitation wavelength �ex � 532 nm, power � 3.0 mW, and 100 s data acquisition time. (b) Contact-mode atomicforce microscopy (AFM) image of Ag FON surface (nanosphere diameter D � 410 nm,deposited mass thickness dm � 200 nm) used for SERS in (a). (c) Contact-mode AFM image of Ag FON surface (D � 500 nm, dm � 250 nm) used for WS-SERES in (e). (d) LSPR spectrum (solid line, �max � 562 nm, FWHM � 144 nm) and WS-SERESspectra (data points) for the 1081 cm�1 band of benzenethiol measured from the Ag FONsurface in (b). (e) LSPR spectrum (solid line, �max � 638 nm, FWHM � 131 nm) andWS-SERES spectra (data points) for the 1081 cm�1 band of benzenethiol measured from Ag FON surface in (c). (f) SERS spectrum measured from Ag nanoparticle arraysurface (�ex � 532 nm, power � 3.0 mW, 100 s data acquisition time). (g) LSPR spectrum (solid line, �max � 688 nm, FWHM � 95 nm) and WS-SERES spectra (data points) for the 1081 cm�1 band of benzenethiol measured from a Ag nanoparticle array surface. (inset) Tapping-mode AFM image of a representative array surface.



of the WS-SERES spectrum is blueshiftedby 613 cm�1 (76 meV) with respect to theLSPR �max. The blueshift is an importantprediction of EM theory that has not been previously observed. The results inFigures 5 and 6 are, to the best of ourknowledge, the first correlated LSPR andSERES measurements for a nanofabricated surface.48,49

ConclusionsThe results presented here demonstrate

the strength and versatility of silver andgold nanoparticles for biosensing based onextinction and surface-enhanced Ramanspectroscopy and on single-particle Rayleighscattering. These capabilities are based onthe unique optical properties of localizedsurface plasmon resonances, including theirsensitivity to particle shape and size, andto local changes in the surrounding refrac-tive index due to molecular adsorption. Inaddition, plasmon excitation leads to stronglocal field enhancements that yield 108

enhancements in SERS. For all of theseproperties, theory can be used to provideimportant guidance to the design of these experiments. Recent theoretical workhas suggested that new directions fornanoparticle-based sensing may be devel-oped for interacting nanoparticle struc-tures, including nanoparticle dimers50 andlarge-scale arrays.51 Thus, there is muchyet to do in this field.

AcknowledgmentsThe authors gratefully acknowledge

support from the Air Force Office of Sci-entific Research MURI program (grantF49620-02-1-0381), the National ScienceFoundation (EEC-0118025, DMR-0076097,CHE-0414554), the Institute for Bioengi-neering and Nanoscience in AdvancedMedicine at Northwestern University, andthe National Institutes of Health (1 R21DK066990-01A1).

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