surface-enhanced raman spectroscopy biosensors: excitation

Surface-enhanced Raman spectroscopy biosensors:excitation spectroscopy for optimisation ofsubstrates 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) hasprogressed frommodel system studies of pyridine on a roughened silver electrode to state-of-the-artsurface science studies and real-world sensing applications. Each year, the number of SERSpublications increases as nanoscale material design techniques advance and the importance of traceanalyte detection increases. To achieve the lowest limits of detection, both the relationship betweensurface nanostructure and laser excitation wavelength and the analyte–surface binding chemistrymust be carefully optimised. This work exploits the highly tunable nature of nanoparticle opticalproperties to establish the optimisation conditions. Two methods are used to study the optimisedconditions of the SERS substrate: plasmon-sampled and wavelength-scanned surfaced Ramanexcitation spectroscopy (SERES). The SERS enhancement condition is optimised when the energyof the localised surface plasmon resonance of the nanostructures lies between the energy of theexcitation wavelength and the energy of the vibration band of interest. These optimised conditionsenabled the development of SERS-based sensors for the detection of a Bacillus anthracis biomarkerand glucose in a serum–protein matrix.

1 Introduction

Inelastic photon scattering was first witnessed by C.V.Raman and K.S. Krishnan in 1928 [1], a process later tobear the name Raman scattering and to garner itsdiscoverer a Nobel Prize in 1930. Raman scattering is avery inefficient process, in that approximately one photonis inelastically scattered for every 1010 elastically scatteredphotons. In a simple classical view of the Raman effect,incident light is considered as an electromagnetic wave thatinduces polarisation in a molecule. Then, the induced dipoleemits or scatters light at the optical frequency of the incidentlight wave, plus or minus the energy of a molecularvibration. The magnitude of the induced polarisation, P, isrelated to the molecular polarisability, a, and the magnitudeof the incident electric field, E, by [2]:

P ¼ aE ð1Þ

The molecular polarisability describes the ease with whichthe electron cloud around a molecule can be distorted bythe electric field. The strength of E is dependent on thepower and wavelength of the incident electromagnetic field.In surface-enhanced Raman scattering (SERS), the E field isincreased by a factor of 10–1000 by the nanoscale opticalproperties of the enhancing substrate, as described below.

Several technological developments, such as laser lightsources for excitation, holographic notch filters andgratings, and array-based detectors, led to a renaissance inRaman spectroscopy. Raman spectroscopy finds increasing

application in materials characterisation, biochemistry andeven art conservation, owing to the rich spectral informa-tion and the ability to obtain spectra for non-transparentsolids or aqueous samples. The advent of micro-Ramantechniques permits characterisation of small quantities ofmaterials with high spatial resolutions [2]. Recently, thecombination of laser trapping [3, 4] with Raman micro-scopy has enabled the spatially localised study of chemicalspecies and reactions in single micrometre-sized [5] or evennanometre-sized volumes [6]. Raman spectroscopy is nolonger an under-utilised, but information-rich, spectroscopictechnique; rather, it is a powerful tool for the fundamentalstudy of molecular structure, dynamics and analysis.

As the Raman effect is inherently weak, it is usuallydifficult to use normal Raman scattering either for traceanalysis or for the study of highly fluorescent samples [2].One way to overcome this problem is to use surface-enhanced Raman spectroscopy. SERS is a plasmonicphenomenon whereby molecules at or near a noble metalsurface with nanoscale structural features experience adramatic increase in the incident electromagnetic field,yielding high Raman intensity. SERS can increase theRaman intensity of a molecule in proximity to a nanoscaleroughened surface by 108 [7, 8], and up to 1014 times underspecial circumstances [9, 10]. The mechanism responsible forthis large enhancement in Raman intensity continues to bethe subject of considerable research [11–16]. However, it isgenerally agreed that two mechanisms contribute to theoverall enhancement: a chemical effect (CHEM) and anelectromagnetic enhancement (EM).

The theory of chemical enhancement was invoked toaccount for the discrepancy between the enhancementspredicted in the initial electromagnetic models (B104) andthe empirically determined enhancement (B106). It isthought that ‘active sites’, either crystal defects or adatomson the surface of the metal, undergo a charge-transferprocess enabling the CHEM enhancement. The dependence

The authors are with the Department of Chemistry, Northwestern University,Evanston, IL 60201, USA

Email: [email protected]

r IEE, 2005

IEE Proceedings online no. 20050009

doi:10.1049/ip-nbt:20050009

Paper first received 17th May and in final revised form 4th November 2005

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

of the optimum SERS excitation frequency on electrodepotential suggests that new electronic states may arise uponadsorption of the analyte, which leads to resonantintermediate states for Raman scattering [17]. The CHEMenhancement has been investigated, particularly the effect ofhalide ions on increasing SERS [16, 18]. However, becausethe chemical environment of a given sample may be largelyoutside experimental control (e.g. in biological media),or determined by other factors (e.g. in ultra high vacuumsystems), it is often difficult to exploit the CHEMenhancement mechanism. Furthermore, because electro-magnetic enhancement is the dominant contributor toSERS, and because of the large variety of substratematerials and geometries available, maximising the electro-magnetic enhancement provides the most convenient andeffective handle to optimise SERS in a typical empiricalsetting. Modern advances in both experimental scienceand theoretical modelling allow better understanding of,and control over, SERS than ever before.

The discovery of single-molecule SERS (SMSERS) byEmory and Nie [9] and Kneipp et al. [10], in 1997, hasnecessitated a re-examination of the EM mechanism. TheEM mechanism is based on the significantly amplifiedelectromagnetic fields (E) generated by the localised surfaceplasmon resonance (LSPR) of nanoscale surface roughnessfeatures [12]. It is the intricate relationship between thesurface structure and the structurally dependent opticalcharacteristics that determines the magnitude of the EMportion of SERS enhancement [7, 8]. In the so-called‘lightning rod effect’, nanoscale features such as sharppoints or interparticle junctions can generate intense EMfields that can amplify both the incoming excitation photonsand the exiting Stokes shifted photons by orders of magni-tude [12, 19, 20]. These factors must be well-controlled forSERS to be used effectively [21].

The EM mechanism has several experimental signatures,such as long-range (i.e. a few nanometres) distancedependence [22–24], the weak dependence of the enchant-ment factor (EF) on the chemical identity of the adsorbate[25] and the existence of surface-enhanced second harmonicgeneration (SESHG) [26–28] SMSERS. The improvedunderstanding of the nature of the EM enhancements hasgenerated renewed interest in the use of SERS as ananalytical technique. Many groups continue to generatenew insights as new mathematical models are coupledto more accurate characterisation in the nanoscale regime[29, 30].

SERS possesses many desirable characteristics as a toolfor the chemical analysis of interfacial molecular species,including high specificity attomole to high zeptomole masssensitivity [9, 10], micromolar to picomolar concentrationsensitivity [21] and interfacial generality [31, 32]. Otherexamples include concentration-jump or potential stepstudies of adsorption/desorption kinetics at electrode orcolloid surfaces [33] and the use of ultra fast, pulsed laserexcitation to probe vibrational relaxation, energy transfer orelectron dynamics at surfaces [34, 35]. Recently, SERS hasbeen used in the quantitative detection and analysis ofbio-related molecules [21, 36–38]. The variation in surfaceenhancement is easily corrected for by addition of aninternal standard to the samples [21]. Ideally, the signalintensities from the internal standard should be comparablewith those of the analyte, and one or more bands should bein spectrally silent regions. Internal standards with only afew spectral bands are preferable for easier data evaluation.However, in many instances, the addition of internalstandards is either impossible, or would unduly interferewith the analyte or process under observation. Therefore it

is desirable to be able to produce SERS substrates that notonly provide the optimum EF, but also are stable fromsubstrate to substrate and from experiment to experiment.

The first SERS experiments generated largely randomstructures, and enhancement factors could vary wildly.Modern synthetic techniques allow exquisitely precisecontrol over nanoscale features and thus over the nanoscaleoptical properties upon which SERS intimately depends.The choice of substrates with specific, well-defined andcontrolled LSPR and laser excitation wavelength, lex, is thecritical factor that affects the resulting SERS EF and,accordingly, the analytical limit of detection (LOD). Thisreview will demonstrate how the SERS enhancement factordepends on these characteristics.

The remainder of this review will be organised into threeparts. First, we compare the available methods forfabrication of SERS-active surfaces. Secondly, we discusstechniques designed to optimise the EM enhancement [7, 8].Specifically, we describe both wavelength-scanned andplasmon-sampled SERS excitation spectroscopy. Thirdly,we review the most recent progress in the SERS-basedsensors for two important target molecules: a Bacillusanthracis biomarker [21, 39] and glucose in a serum proteinmixture [36, 38].

2 SERS-active surfaces

SERS is usually performed on Ag [13], Au [40] or Cu [41]surfaces, because these metals have appropriate values ofthe real and imaginary parts of the dielectric constant [42]and can be easily handled in ambient, electrochemicaland ultra-high vacuum environments. With the variety ofSERS-active surfaces available today, e.g. colloids, colloidin sol-gel [43], electrochemically roughened electrodes[11, 13], vapour-deposited metal island films [44] andlithography-produced nanostructures [45], researchers areable to select the SERS substrate architecture that bestmatches their experimental needs.

The preparation of noble metal colloids has been studiedsince ancient times [46]. Colloidal substrates can beprepared in many different shapes [47, 48], such as spheres,rod-shaped [49, 50], cubes, triangles [51, 52] and shells[53, 54]; they can be dispersed throughout the object ofstudy and even inside macrostructures [55], such as cells [56]and tissue samples [57]. However, the distribution of shapesand sizes and the ease of aggregation will influence thesurface-enhancing properties.

The nanoparticle synthesis methods discussed abovecreate suspensions of nanoparticles in solution. Anotherclass of fabrication techniques yields surface-bound nano-structures. The standard approach for making surface-bound nanostructures is electron beam lithography (EBL).In EBL, the desired pattern is serially produced by theexposure of a thin layer of photo resist to high-energyelectrons, followed by chemical development and depositionof the noble metal. Several research groups have focused onthe optical properties of two-dimensional arrays to utilisethese nanoparticle assemblies as surface-enhanced spectro-scopy substrates [30, 58, 59]. Although EBL providesexquisite control over interparticle spacing and can fabricatearbitrary shapes, it is an expensive and slow, serial form oflithography.

An alternative method for the large-scale productionof surface-bound nanoparticle arrays is nanosphere litho-graphy (NSL). NSL is a powerful fabrication technique thatinexpensively produces nanoparticle arrays with controlledshape, size and interparticle spacing [60]. Every NSLstructure 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-dimensional colloidal crystal deposition mask [60–63]. Asthe solvent evaporates, capillary forces draw the nano-spheres together, and the nanospheres crystallise in ahexagonally close-packed pattern on the substrate (Fig. 1).As in all naturally occurring crystals, nanosphere masksinclude a variety of defects that arise as a result ofnanosphere polydispersity, site randomness, point defects,line defects and polycrystalline domains. Typical defect-freedomain sizes are in the 10–100mm range. Following self-assembly of the nanosphere mask, a metal or other materialis then deposited by thermal evaporation, electron beamdeposition or pulsed laser deposition to a target metalthickness, dm. After metal deposition, the nanosphere maskis removed, leaving behind surface-confined nanoparticleswith triangular footprints (Fig. 1a). At mass thicknessesexceeding 100nm, a continuous film of metal is depositedon the nanospheres (Fig. 1b). These metal film overnanosphere (MFON) substrates are some of the mostrobust SERS substrates available in terms of spectralstability, sample-to-sample uniformity and mechanicalruggedness [45]. The diameter of the nanosphere coresand the thickness of the metal film shell determine the sizedistribution of the roughness features and, hence, theoptical response. Even though the nanoscale roughnessfeatures are not homogeneous in size but, instead, aredriven by the larger-scale templating, they are sufficientlyuniform to generate a relatively narrow surface plasmonpeak (FWHM B200nm) [21]. Recent experiments haveconclusively demonstrated that MFON substrates arestable for months [21] (unlike many other nanostructuredsurfaces) and remain SERS-active even when exposed

to large temperature [64] and electrochemical potentialexcursions [39].

3 Optimisation of SERS by surface-enhancedRaman excitation spectroscopy

3.1 Plasmon-scanned surface-enhancedRaman excitation spectroscopyPlasmon-sampled surface-enhanced Raman excitation spec-troscopy (PS SERES) was developed to explore therelationship between nanoparticle optics and the resultingEF [65]. In the PS SERES experiment, a Raman-activemolecule is adsorbed onto the substrate of choice, and thenthe correlated SERS and LSPR spectra of that adsorbateare captured from a microscale domain using a single laserexcitation wavelength. This procedure can be repeated on asingle substrate until all the different LSPR domains havebeen sampled. The use of NSL fabricated arrays permitsagile frequency tuning of the LSPR based on the nano-sphere mask dimensions, metal thickness and metal type.The intensity or enhancement factor of a specific Ramanband is plotted against the LSPR maximum to reveal theoptimised experimental conditions. There are multipleadvantages in performing the PS SERES profiling:

(a) the number of data points in the PS SERES profile isonly limited by the number of microscale sampleregions (and fabricated substrates) with different LSPRcharacteristics

(b) the experiment requires only a single laser excitationwavelength, a single notch filter and a single stagemonochromator

assemblemask

removemask

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depositmetal

depositmetal

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Fig. 1 Nanosphere lithographic fabrication of triangular particle array and metal film over nanosphere (MFON) substratesCarboxylate 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 maska Array of triangular-shaped particles is formed in interstices between spheres, which can be removed by sonicationb If 4100nm of metal is deposited, continuous film covers spheres, forming FON surface


(c) the information gained gives the general guidelines forachieving an optimised EF on any substrate with anarrow LSPR.

NSL-fabricated Ag nanotriangles were used as SERSsubstrates for the PS SERES experiments. The size/shapeof these Ag nanotriangles can be tuned systematically bychanging the nanosphere size in the deposition mask or thedeposition thickness [66, 67]. Slight variations in the size orshape of these nanoparticles yield significant shifts in thenarrow LSPR spectrum.

Even though the structural variations among thenanoparticles on a given NSL sample are small, theygenerate varying LSPR properties between microscaledomains. Although it is possible to eliminate much of thisvariation if necessary, the PS SERES experiment exploitsthe distribution of optical properties to maximise thenumber of data points in the excitation profile. In all casesto date, PS SERES experiments have explored adsorbatesthat bind irreversibly to the silver nanoparticles to simplifydata collection and interpretation.

Below we present three separate PS SERES profilesthat were constructed using benzenethiol adsorbed ontoAg nanoparticles. Each profile was measured with adifferent laser excitation wavelength (514.5, 532.0 or632.8nm). The correlated LSPR and SERS spectra forthe optimised enhancement factor in each case are shownin Fig. 2, and the PS SERES profiles are shown in Fig. 3.The profiles clearly demonstrate that the maximumenhancement factor occurs when the LSPR lmax liesbetween the energy of the excitation wavelength (solidlines) and the vibration mode (broken lines). It is interestingto note that the enhancement factor does not vary by morethan a factor of 10 once the signal is measurable abovebackground.

To ensure that the results observed are not an artifact ofthe molecule adsorbed on the surface, more PS SERESprofiles were constructed with varied molecular adsorbates(3,4-dichlorobenzenethiol, 1,4-benzenedithiol, andFe(bpy)3

2+),and similar results were achieved in all cases. The two non-resonant molecules yielded maximised enhancement factorsof 2.3� 107 and 1.4� 108, and the maximum enhancementfactor for the resonant molecule Fe(bpy)3

2+ was 7.1� 109.To verify that various vibration modes adhere to thedemonstrated pattern, PS SERES profiles were constructedfor five different vibration modes of 3,4-dichlorobenzene-thiol, including in-plane ring deformations, a ring-breathingmode, the C-S stretch and the C-Cl stretch. Again, themaximised enhancement factors occurred when the LSPRlmax was between the excitation wavelength and thewavelength of the vibration mode.

Thus, for practical application, the PS SERES techniquehas revealed a general rule for optimising EF whensubstrates with narrow LSPR spectra and a single laserexcitation source are used. The largest EF is achieved whenthe energy corresponding to the narrow LSPR lmax fallswithin a B120nm window that includes the energy ofthe excitation wavelength and that of the Raman-shiftedscattered photons. The LSPR variation on the NSLsubstrates used in the work presented above can be easilyexploited to find the microscale domains with the bestoptical properties for a given experiment, thus maximisingthe enhancement factor and lowering the detection limit.Although the reported enhancement factors from NSLmicrodomains are significantly smaller than those measuredin single-molecule SERS studies, these ensemble-averagedvalues can be regularly achieved, are temporally stableand are more readily understood.

3.2 Wavelength-scanned surface-enhancedRaman excitation spectroscopyFor all SERS sensing applications, sensitivity is of primaryimportance. To this end, we have undertaken a study of thefundamental mechanism of SERS to ascertain the specificconditions necessary for maximising the enhancement of the

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Fig. 2 Correlated, spatially resolved LSPR extinction and SERSspectra of benzenethiol-dosed NSL substrates with maximisedenhancement factorsa and d Measured from D¼ 280nm, mass thickness of Ag filmdm¼ 36nm, Pex¼ 0.7mW, lex¼ 514.5nmb and e Measured from D¼ 280nm, dm¼ 36nm, Pex¼ 0.7mW,lex¼ 532.0nmc and f Measured from D¼ 400nm, dm¼ 56nm, Pex¼ 0.7mW,lex¼ 632.8nmAll Raman spectra were captured with integration time of 30 s.Reproduced with permission from [7]Copyright 2003, the American Chemical Society


Raman scattering. There are three principle factors thatdetermine the sensitivity of a Raman experiment:

(i) the frequency to the fourth (n4) scattering dependence ofRaman photons

(ii) the wavelength-dependent efficiency of the detectionsystem

(iii) the relationship between the LSPR of the substrate andthe maximum SERS enhancement.

The first factor would indicate that shorter excitationwavelengths are always best, but that is not necessarily thecase. For example, in many biological applications, it isoften desirable to use near IR excitation to avoid damagingthe sample or exciting sample autofluorescence, even at thecost of some signal loss due to n4. The second factor isstraightforward and is simply a function of the configura-tion of the spectrograph and detector. Consultation of theefficiency curves of the dispersive elements and the detectorare readily available from manufacturers and should factorinto purchase planning and experimental design. Forexample, most systems are configured with gratings anddetectors optimised in the visible region of the spectrum,decreasing their efficacy in the UV or NIR region. The thirdfactor, the relationship between the LSPR of the substrateand the maximum SERS enhancement, is more complexand interesting, yet less well understood or utilised. Wehave explored this third factor using a technique calledwavelength-scanned surface-enhanced Raman excitationspectroscopy (WS SERES).

WS SERES involves the measurement of SERS en-hancement for several laser excitation wavelengths, lex. Thistechnique was recognised as a useful tool for exploringthe EM mechanism soon after the discovery of SERS. Anobvious limitation of this technique is that the number ofdata points is determined by the tuneability of the excitationlaser and detection system. These substantial instrumentalrequirements have led to the majority of SERES publica-tions suffering from low data point density and/or limitedspectral coverage [31, 68–70]. These limitations prevent theestablishment of conclusive generalisations from SERESdata. Additionally, most SERES experiments have beenperformed using surface-enhancing substrates with an

unknown or poorly characterised distribution of roughnessfeatures. In the few cases where the surfaces are carefullycharacterised, it has been shown that there is a widedistribution of roughness feature sizes [31, 71]. Other studiesdo not include characterisation of the LSPR of the sub-strate [69, 70], which prevents any direct comparison ofthe excitation profiles with the spectral location of thewavelength of maximum LSPR extinction, lmax.

The most common substrates historically employed inSERES experiments are Ag island films and Ag colloidalsolutions. In these cases, most of the SERS excitationprofiles peak at excitation wavelengths (lex,max) near500–600nm [71–75]. The peaks of the excitation profileshave been shown to shift to longer wavelengths withincreased aggregation [68, 71, 73, 74], which is a qualitativeresult predicted by the EM mechanism. With these types ofsubstrate, it is difficult to make a direct comparison betweenthe LSPR of the substrate and the SERS excitation profile,because the LSPR of the substrate is a superposition ofa wide variety of LSPR wavelengths corresponding to thedifferent roughness features caused by morphological (sizeand shape) polydispersity between islands or colloidalnanoparticles.

Two exceptions to the above statements regardingroughness features are the well-known experiments by Liaoand co-workers on microlithographically prepared Ag posts[76] and recent work by Felidj and co-workers on EBL-produced arrays of gold elongated nanoparticles [77]. Theformer ground-breaking work demonstrated excitationprofiles where lex,max shifted to longer wavelengths withincreased particle aspect ratio and with increased dielectricconstant of the medium surrounding the particles. Theseresults qualitatively agree with the EM mechanism, but theLSPR of these substrates was not measured for a directcomparison. In the latter work, the SERS enhancement wasshown to peak halfway between the excitation wavelengthand the wavelength of the Raman scattered photon. Thisimportant experiment was the first observation of preciselywhat is predicted by the EM mechanism. Unfortunately,this result was only obtained on one sample with a profileconsisting of only three data points.

Herein, we describe the utilisation of a broadly tuneableRaman system to measure excitation profiles with thegreatest number of data points ever achieved in a WSSERES experiment. A broadly tuneable laser system, aversatile detection system and a well-characterised surface-enhancing substrate are all employed to surmount thetraditional shortcomings of WS SERES experiments. Theuse of a CW mode-locked Ti : sapphire and its harmonicsallows for continuous tuneability over the spectral ranges350–500nm and 700–1000nm. The visible region notcovered by the Ti : sapphire system was augmented withthe use of a solid-state laser and a tuneable dye laser. Atriple spectrograph equipped with a CCD camera allowsfor rapid, multichannel spectral acquisition with efficientrejection of Rayleigh-scattered photons. The SERS sub-strates used in this work are triangular nanoparticle arraysfabricated by NSL. These substrates have been wellcharacterised previously [61, 66, 67]. They present asignificant advantage over many of the traditional SERSsubstrates for SERES studies, because NSL-fabricatedtriangular nanoparticles exhibit extremely narrow sizedistributions, making them an indispensable tool forprobing the fundamental characteristics of SERS. Tune-ability of the LSPR lmax of these nanoparticles throughoutthe visible and NIR wavelengths can be achieved by sys-tematic variation of the dimension of the nanoparticles [66].Even though the surface coverage of these nanoparticles is

10

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560 640 600 680cba

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Fig. 3 PS SERES for n8a (1575 cm�1) band of benzenethiol withthree different excitation wavelengthsa lex¼ 514.5nmb lex¼ 532.0nmc lex¼ 632.8nm- - - - - - - - - Wavelength location of excitation- - - - - - - Wavelength location of scatteringOverlaid line represents bin-averaged values of LSPR lmax and EF


B7%, strong SERS intensities are observed from analytesadsorbed to these substrates, owing to the strong enhance-ment NSL-fabricated arrays exhibit [65].

For the work presented below, the intensity of the1575cm�1 band of a benzenethiol monolayer adsorbed tothe Ag nanoparticles was measured at many differentexcitation wavelengths, lex. Benzenethiol is used as a modelmolecule in both this and the following sections because it:

(a) binds via the thiol group to the metal surface

(b) has a high Raman scattering cross-section

(c) lacks electronic transitions in the visible range, renderingit non-resonant at the excitation wavelengths used

(d ) has a well-resolved spectrum.

To account for any variation of the SERS intensity not dueto the enhancement by the substrate, the 1444cm�1 normalRaman scattering band of neat cyclohexane was used as anintensity standard. This standard was used to correct forthe inherent n4 behaviour of Raman scattering, spectraldependence of the detection system and differences in theillumination power.

Figure 4 shows four excitation spectra for the 1575cm�1

peak of benzenethiol, each with an LSPR lmax at adistinctly different location. The SERES spectrum in Fig. 4ais measured over the spectral range 420–500nm. Becausethe formation of a monolayer of benzenethiol on thesenanoparticle arrays results in a large red shift in the positionof the LSPR lmax, it was necessary to anneal this sample at3001C for 1h prior to benzenethiol addition, to achieve afinal LSPR lmax at a wavelength shorter than 500nm. It hasbeen previously shown that annealing NSL-derived samplesresults in a large blue shift of the LSPR owing to the shapeof the nanoparticles being changed [66]. The LSPR lmax ofthis substrate was measured to be 489nm (20450cm�1).The largest SERS enhancement occurs at lex¼ 485nm.Fitting a Gaussian line shape to the data reveals that thepeak of the excitation profile, lex,max, is 480nm(20833cm�1). The peak enhancement factor (EF) valuefor this sample was calculated to be 5.5� 105. This value islow in comparison with the values determined for the othersamples, because the shape of the nanoparticles is mademore ellipsoidal by annealing. In addition to shifting theLSPR lmax to shorter wavelengths, this change decreasesthe intensity of the electromagnetic fields at the nanoparticlesurfaces by removing the sharp corners that are calculatedto possess the greatest field enhancements [12].

The SERES spectrum in Fig. 4b was measured over thespectral range 532–690nm. The LSPR lmax of this substratewas measured to be 663nm (15083cm�1). The largestSERS enhancement occurs for lex¼ 625nm. The maximumof a Gaussian line shape fit to the data is 625nm(16000cm�1). The peak EF value for this sample is1.2� 107. The SERES spectrum in Fig. 4c is measuredover the spectral range 532–740nm. The LSPR lmax ofthis substrate was measured to be 699nm (14306cm�1).The largest SERS enhancement occurs for lex¼ 670nm.The maximum of a Gaussian line shape fit to the data is671nm (14903 cm�1). The peak EF value for this sample is1.4� 107. The SERES spectrum in Fig. 4d is measured overthe spectral range 630–800nm. The LSPR lmax of thissubstrate was measured to be 810nm (12346cm�1).The largest SERS enhancement occurs for lex¼ 770nm.The maximum of a Gaussian line shape fit to the data is765nm (13072 cm�1). The peak EF value for this sample is9.3� 107.

If the peak in the SERS enhancement occurs when theLSPR lmax of the sample is equal to (lex+lvib)/2 (where lvib

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Fig. 4 Surface-enhanced Raman excitation spectra of 1575 cm�1

peak of benzenethiol with cyclohexane as intensity standarda Substrate annealed at 3001C for 1h. LSPR lmax¼ 489nm; profile fitmaximum at lex,max¼ 480nmb LSPR lmax¼ 663nm; profile fit maximum at lex,max¼ 625nmc LSPR lmax¼ 699nm; profile fit maximum at lex,max¼ 671nmd LSPR lmax¼ 810nm; profile fit maximum at lex,max¼ 765nm


denotes the wavelength of the Raman-shifted peak ofinterest), then lex,max should be different for the variousRaman bands of benzenethiol on a single sample. It isexpected that lex,max will have a larger separation from theLSPR lmax for a large Raman shift than for a small shift.Excitation spectra for three benzenethiol peaks on a singlesubstrate are shown in Fig. 5. For this substrate, the LSPRlmax is 729nm. Figure 5a shows the SERS excitation profilefor the 1575cm�1 peak of benzenethiol, normalised tothe 1444cm�1 peak of liquid cyclohexane. The separationin wave numbers between the LSPRs lmax and lex,max is734cm�1. In Fig. 5b, the excitation profile for the 1081cm�1

benzenethiol peak (normalised to the 1028 cm�1 peak ofcyclohexane) is shown. The separation in wave numbersbetween the LSPRs lmax and lex,max is 569cm

�1. Finally, inFig. 5c, the excitation profile for the 1009cm�1 benzenethiolpeak (normalised to the 1028cm�1 peak of cyclohexane) is

shown, and the separation in wave numbers between theLSPR lmax and lex,max is 488 cm�1.

These data demonstrate the qualitative trend whereby thelex,max in the excitation spectra of larger Raman-shiftedbands yield a larger separation from the LSPR lmax thanthose of smaller Raman-shifted bands. These data lendvalidation to the EM mechanism as well as highlightinganother factor that must be kept in mind in the design of anSERS-based sensor. The relationship between the peak inthe SERS enhancement factor and the extinction maximumof the substrate’s LSPR has been shown to be dependentupon the magnitude of the Raman shift.

The work above demonstrates the most thorough WSSERES experiments heretofore performed on optically andtopographically characterised SERS substrates. The experi-mental apparatus utilised has been proven effective for themeasurement of relative SERS enhancements that vary bythree orders of magnitude. This work demonstrates that therelationship between the substrate LSPR and the SERESprofile for size-homogenous nanoparticles is consistentthroughout the visible range. In all cases, the experimentallyobserved results are consistent with those predicted by theEMmechanism. Specifically, the maximum SERS enhance-ment always occurs when the substrate LSPR lmax islocated between the laser excitation wavelength, lex, and thewavelength of the Raman-scattered photon, lvib. Underthese conditions, both the incident and scattered photonsexperience strong enhancement by the LSPR. Eachsubstrate exhibits a WS SERES profile that has a similarline shape to the extinction spectrum of the substrate.The largest EF measured was B108 for the triangularnanoparticle arrays.

Ultimately, refinement of the experimental apparatus andoptimisation of SERS enhancement will allow SERES to beperformed using single nanoparticle substrates. This levelprovides the best possible case in terms of reducing sampleheterogeneity, in that it will remove the effects of ensembleaveraging or particle coupling. These experiments areexpected to provide key information to validate the EMmechanism of SERS and will present an additionaltechnique that can be used to study the SMSERS effect.

Our detailed WS SERES studies unequivocally demon-strate the importance of designing SERS experiments suchthat the LSPR lmax of the substrate is located in betweenthe laser excitation wavelength and the wavelength of theRaman peak of interest. One very attractive feature ofthe NSL-derived nanoparticle substrates is the tuneability ofthe LSPR lmax spectral position. Therefore, by using thesesubstrates for SERS-based sensing, we can optimise thesensor by using an excitation wavelength that utilises the n4

dependence of Raman scattering while taking into accountother factors of the particular application, assembling adetection system that is optimised for highest throughputat the wavelength of interest, and fabricating nanoparticlearrays with an LSPR lmax precisely located in between lex

and lvib.

4 Biological applications of surface-enhancedRaman scattering

4.1 Rapid anthrax biomarker detectionbased on SERSAnthrax is an infectious disease caused by the spore-forming bacterium Bacillus anthracis [78]. Currently, thedemand for better field tests for B. anthracis spores hasincreased as a consequence of contaminated mail and falsealarms. Several techniques have been developed for real-time detection of Bacillus spores [79, 80]. Examples include

wavelength, nm

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c

18 000 20 000

700 600 500

νvib = 1575 cm−1

shift = 734 cm−1

νvib = 1081 cm−1

shift = 569 cm−1

νvib = 1009 cm−1

shift = 488 cm−1

a

b

Fig. 5 Effect of Stokes Raman shifta Profile of 1575cm�1 vibration mode of benzenethiol. Distancebetween LSPR lmax and excitation profile fit line lex,max¼ 734cm�1

b 1081cm�1 vibration mode, shift¼ 569cm�1

c 1009cm�1 vibration mode, shift¼ 488cm�1


fluorescence detection using portable devices, whichis sensitive enough to detect B103 spores in 7min, buthas the potential to give false positive readings [79]. Incontrast, SERS possesses highly specific chemical informa-tion content and therefore is capable of uniquely identifyingtarget analytes, thereby limiting false positives [2]. We havedeveloped a procedure for the rapid extraction of CaDPAfrom B. subtilis spores (simulants for B. anthracis spores),followed by SERS detection on AgFON substrates [21].These substrates were designed and optimised using themethods described in the preceding sections.

Prior work demonstrates that when the localised surfaceplasmon resonance maximum of a AgFON substrateclosely matches the laser excitation wavelength, the max-imum SERS signal intensity results. As AgFONs are notoptically transparent, the reflectivity minimum was used tolocate the LSPR maximum. AgFON surfaces fabricatedusing 600nm spheres show a reflectivity minimum at753nm (Fig. 6a). In fact, further SERS experiments usingAgFON substrates with different polystyrene nanospherediameters demonstrate that the largest SERS enhancementfor 750nm laser excitation was obtained from the AgFONwhere D¼ 600nm [21]. The use of NIR laser light inRaman spectroscopy is advantageous because it causes lessphotochemical damage to the biological samples and resultsin much less fluorescence background in the Ramanspectrum. Therefore this AgFON substrate was chosen asoptimum for the bacillus spore detection experiments thatfollow.

Calcium dipicolinate (CaDPA) was extracted from sporesby sonicating in 0.02M HNO3 solution for 10min. Figure6b shows the SERS spectrum of 3.7� 104 spores in 0.2ml,0.02M HNO3 on a AgFON substrate (D¼ 600nm,dm¼ 200nm), which is dominated by bands associatedwith CaDPA, in agreement with the previous Ramanstudies on CaDPA and bacillus spores [81–84]. The peak at1050cm�1 in Fig. 6b arises from the symmetrical stretchingvibration of NO�3 . This peak is used as an internal standardto reduce the sample-to-sample deviations, because of itshigh intensity and spectral isolation.

We show in Fig. 7a an LOD of 2550 anthrax spores witha data acquisition period of 1min and a laser power of50mW, well below the level believed to trigger an infectionin humans. To put these results into context, previouslypublished SERS studies of bacillus spore detection were 200times less sensitive and required three times more laserpower [83]. Recently, Bell and co-workers reported a SERSLOD of B4 parts per million of dipicolinic acid, i.e.2.4� 10�5M, using laser excitation at 532nm without

indicating the laser power [85]. In contrast, the SERS LODof CaDPA is B3.1� 10�6M using the optimised NSLsubstrate and laser excitation at 750nm [21]. It should benoted that the intensities of Raman spectra with 750nmexcitation are expected to be about a quarter of thosemeasured with 532nm excitation, because the Raman signalintensities are inversely proportional to the fourth power ofthe laser wavelength (see above) [2]. Therefore we calculatean LOD of CaDPA is B30 times more sensitive thanliterature values.

Previous research has demonstrated that bare AgFONsurfaces display extremely stable SERS activity whenchallenged by negative potentials in electrochemical experi-ments [39] and high temperatures in ultrahigh vacuumexperiments [86]. In the present example, the intensity ratiosbetween the strongest CaDPA peak at 1020cm�1 and theNO�3 peak at 1050cm�1 (I1020/I1050) were measured so thatprefabricated AgFON substrates of different ages couldbe compared quantitatively (Fig. 7a, inset). The CaDPAintensity remained constant over the course of 40 days,indicating the temporal spectroscopic stability of AgFONsubstrates over a period of 40 days.

Finally, a portable, handheld SERS device successfullyproduced a SERS spectrum from 104 spores in 5 s usinga one-month-old prefabricated AgFON substrate (Fig. 7a).The SERS peak positions and intensity pattern for thespore sample were similar to those of CaDPA recordedutilising the same device (Fig. 7b). This represents the firstreported use of a compact vibration spectrometer for thedetection of bacillus spores. Coupling the portability andease of use of this type of device to the molecular specificityand spectral sensitivity inherent in SERS, a range ofpossibilities become available in the area of detecting bio-agents and other chemical species, such as environmentalpollutants, toxic industrial chemicals, process reactionstreams, food products etc.

4.2 SERS-based measurement of glucoseAlthough, the CaDPA biomarker in Bacillus subtilis sporesdirectly binds with the silver surface, which is required forSERS detection, many important molecules (e.g. glucose)do not have any natural binding affinity for the silver

refle

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1595

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93

1194

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824

66610

50 (

NO

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1500 1000 500

b

λex = 750 nm

Fig. 6 Reflectance and SERS spectraa UV-vis diffuse reflectance spectrum of AgFON substrate in airb SERS spectrum of 3.1� 10�13M spore suspension (3.7� 104 sporesin 0.2ml, 0.02M HNO3) on AgFON substrate. lex¼ 750nm,Pex¼ 50mW, acquisition time¼ 1min, D¼ 600nm, dm¼ 200nm

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an in

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ity

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Fig. 7 SERS spectraa SERS spectrum of 2.1� 10�14M spore suspension (2.6� 103 sporesin 0.2ml, 0.02M HNO3) on AgFON. Inset shows intensity ratio (I1020/I1050) variation with time. lex¼ 750nm, Pex¼ 50mW, acquisitiontime¼ 1min, D¼ 600nm, and dm¼ 200nmb SERS spectra obtained by portable Raman spectrometer. Upperspectrum is 8.3� 10�14M spore suspension (1.0� 104 spores inB0.2ml, 0.02MHNO3) on 30-day-old prefabricated AgFON. BottomSERS spectrum is 10�4M CaDPA in 0.2ml 0.02M HNO3 on 30-day-old pre-fabricated AgFON substrate. lex¼ 785nm, Pex¼ 35mW,acquisition time¼ 5 s, resolution¼ 15cm�1, D¼ 600nm, anddm¼ 200nm


surface [37]. Although the normal Raman cross-section ofglucose should provide sufficient signal to register on mostdetection systems [2], the inability to observe glucose usingSERS must be attributed to the weak or non-existentinteraction of glucose with bare noble metal surfaces. Thissection demonstrates quantitative glucose detection bytailoring a AgFON substrate similar to that used abovewith a self-assembled monolayer (SAM) to partition glucosewithin the range of the enhanced electromagnetic fields onthe AgFON surface (Fig. 8) [37], in a manner analogousto that used in high-performance liquid chromatography(HPLC) [87–91]. Functionalising the AgFON substratewith a partition layer has three advantages: the SAMstabilises the Ag surface against oxidation; the SAM isexceedingly stable; and pre-concentration functionality isbuilt in and can be tailored by synthetic control of thepartition layer. Such chemical functionalisation of theSERS substrate enables the extension of SERS topreviously inaccessible molecules and provides an additionalparameter that can be controlled in the development ofSERS-based experiments [23].

Straight chain alkanethiols and tri(ethylene glycol)terminated alkanethiol (EG3) were found to be effectivepartition layers. Of the many SAMs tested to determinetheir effectiveness as a partition layer, EG3 was chosen asa partition layer because of its ability to reject non-specificbinding by background proteins [92–95] and its biocompat-ibility [96, 97], with the goal in mind of progression towardsfabrication of an implantable SERS-based glucose sensor.Each EG3-modified AgFON sample was incubated insaline solution with glucose (0–25mM; 0–450mgdl�1) at aphysiological pH of 7.4. Then, the samples were placed inan environmental control flow cell under saline, and SERSspectra were collected. The spectra were normalised usingEG3 peak intensities, followed by partial least squaresanalysis [36, 37]. The resulting cross-validated glucoseconcentration predictions are presented in the Clarke errorgrid (Fig. 10). Clarke and co-workers established the errorgrid as a metric for evaluating glucose sensor efficacy in theclinically relevant concentration range [98].

The EG3-modified AgFON sensor allows quantitativedetection of glucose in the physiological range with acorresponding prediction error of 82mg dl�1 (4.5mM).In Fig. 9, 94% of the predictions fall in zones A and B,whereas a few data points overlap in zone D within thehypoglycaemic area (o70mgdl�1,o3.9mM). The error of82mgdl�1 (4.5mM) can be partially attributed to slightvariation in the EF between different AgFON samples. Thenanostructure on a AgFON substrate varies from point to

point, affecting the localised surface plasmon resonanceand, accordingly, the EF.

Although quantitative detection is an important char-acteristic of a viable biosensor, the glucose sensor must alsobe effective in the presence of interfering proteins. Serumalbumin was used to mimic blood serum protein as achallenge to the glucose sensor. The EG3-functionalisedAgFON substrate was installed in a flow cell under a salineenvironment, and the SERS spectrum was obtained(Fig. 10a). Then, the BSA solution was injected into theflow cell, and the SERS spectrum was collected throughoutthe 240 s incubation (Fig. 10b). Finally, the sample wasexposed to 100mM glucose, and the SERS spectrum wascollected (Fig. 10c). Figure 10d is the difference spectrumbetween the sensor under saline and the same surfaceexposed to the BSA solution, demonstrating that BSA doesnot have a measurable SERS spectrum on the EG3-modified surface. The lack of SERS bands from BSA isattributed to inefficient adsorption of BSA to the EG3partition layer. The data in Fig. 10e confirm that the SERSglucose sensor is still effective after substrate exposure to aninterfering protein and that the peaks correspond to thecrystalline glucose peaks shown for comparative referencein Fig. 10f. This experiment clearly shows that glucosepartitioning into EG3 is not influenced by the presence oflarge molecules such as serum albumin. It is interesting tonote that the peak at 695cm�1 (Fig. 10a) shifts to 710cm�1

(Fig. 10c) in the presence of glucose. The rearrangement ofthe SAM when the glucose molecules partition into EG3may cause this shift. The observed shift in this peak furthercorroborates the hypothesis of glucose penetrating deeplyinto the EG3 monolayer, affecting even the character of theC–S bond.

In addition to the demonstration of quantitative glucosemeasurement in a clinically relevant concentration rangeand detection of glucose in the presence of other interfering

OHOHO

HOH2C

OHOH

partition

departition

AgFON AgFON

Fig. 8 Schematic demonstration of hypothetical glucose concen-tration gradient created by EG3 partition layerAlkanethiol portion of EG3 helps form stable SAM, and glycolportion provides hydrophilic environment amenable to glucosesalvation

actual concentration, mM

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240 320 400

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C

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E

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B

D

E

C

Fig. 9 Clarke error grid of LOO-PLS predicted glucose concen-tration against actual glucose concentration (3 loading vectors)AgFON samples were fabricated (D¼ 390nm, dm¼ 200nm), incu-bated forB16h in 1mM EG3 solution, and dosed in glucose solution(range: 0–450mg/dl, 0–25mM) for 10min. Each SERS measurementwas made in flow cell under saline with pH¼ 7.4, using lex¼ 632.8nm,Pex¼ 1.0mW, t¼ 30 sAll SERS measurements were taken in single spot Reproduced withpermission from [32]Copyright 2003, American Chemical Society


proteins, other characteristics of the EG3-modified AgFONglucose sensor, such as durability and reusability, aredemonstrated in published work [36, 38].

5 Conclusions

SERS is a powerful technique for the sensitive and selectivedetection of low-concentration analytes. To achieve thelowest limits of detection, both the relationship betweensurface nanostructure and laser excitation wavelength,as well as the analyte–surface binding chemistry must becarefully optimised. This work exploits the highly tuneablenature of nanoparticle optical properties to establish thefirst set of optimisation conditions. We conclusivelydemonstrated that the SERS enhancement factor isoptimised when the energy of LSPR lies between the energyof the excitation wavelength and the energy of the vibrationband of interest. With the narrow LSPRs used in this work,it is straightforward to achieve an EF of B108. We alsodetailed the use of the PS SERES technique to aid in thedesign of experiments that are unable to exploit a broadlytuneable laser and detector system, i.e. those most routinelyavailable.

The experience garnered in the development of theSERES work was applied to the analytically challengingtask of detecting biomarkers for bacillus spores. This reviewalso covered the exploitation of chemical modificationof SERS substrates to improve the interaction betweenthe surface and target analytes. Specifically, we used themeasurement of glucose, a molecule with no detectableSERS signal under normal circumstances, to illustrate howeffective surface functionalisation can be for expanding thepotential role of SERS.

6 Acknowledgments

The authors gratefully acknowledge the financial support ofthe Air Force Office of Scientific Research MURI program(F49620-02-1-0381) and the National Science Foundation(DMR-0076097). Any opinions, findings and conclusion orrecommendations expressed in this material are those of theauthors and do not necessarily reflect those of the NationalScience Foundation. This project was also supported by theInstitute for Bioengineering and Nanoscience in AdvancedMedicine at Northwestern University, and the NationalInstitutes of Health (EY13002 and EY13015). Theauthors wish to acknowledge the following individualsfor their experimental assistance, materials or expertise:J.A. Dieringer, L. Chang, M. Glucksberg, C.L. Haynes,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.

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10∗

5∗

inte

nsity

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inte

nsity

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1434

a

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f

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B A

C B

Fig. 10 SERS spectra showing detection of glucose in presence ofserum albumina EG3 monolayer on AgFON substrate, lex¼ 632.8, Pex¼ 0.8mW,t¼ 240 sb 1mgml�1 serum albumin injected into flow cell to challenge EG3-modified AgFON, lex¼ 632.8, Plaser¼ 0.8mW, t¼ 240 sc 100mM glucose injected into flow cell, lex¼ 632.8, Pex¼ 0.8mW,t¼ 240 sd Difference spectrum obtained by subtracting a from b revealing lackof SERS spectrum for adsorbed serum albumine Difference spectrum obtained by subtracting b from c indicatingserum albumin exposure does not interfere with glucose detectionf Normal Raman spectrum of crystalline glucose for comparison,lex¼ 632.8, Pex¼ 5mW, t¼ 30 s*adumW�1 s�1


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surface-enhanced raman spectroscopy biosensors: excitation

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