Surface-Enhanced Raman Scattering Substrates Based on Self-Assembled PEGylated Gold and Gold−Silver Core−Shell NanorodsBoris N. Khlebtsov,†,‡ Vitaly A. Khanadeev,†,‡ Mikhail Yu. Tsvetkov,§ Victor N. Bagratashvili,§

and Nikolai G. Khlebtsov*,†,‡

†Institute of Biochemistry and Physiology of Plants and Microorganisms, Russian Academy of Sciences, 13 Prospekt Entuziastov,Saratov 410049, Russia‡Saratov State University, 83 Ulitsa Astrakhanskaya, Saratov 410012, Russia§Institute of Laser and Information Technologies, Russian Academy of Sciences, 2 Pionerskaya Ulitsa, Moscow, Troitsk, 142190,Russia

*S Supporting Information

ABSTRACT: For routine laboratory practice, surface-enhanced Raman scattering (SERS)spectroscopy must be simple, inexpensive, stable for a long time, and reproducible. In thisstudy, we mixed 1 μL of PEGylated silver-coated gold nanorods (Au−Ag NRs) with 1 μLof Raman reporters to obtain, after deposition on a silicon wafer and drying at roomtemperature for 5 min, a self-assembled NR monolayer with incorporated Ramanmolecules. In addition, PEGylated NR powders were fabricated and used for SERS in twooptions. In one, they were used to prepare SERS colloids with a desired NR concentrationin a matter of seconds; in the other, they were directly dissolved in a drop of analyte andwere then deposited on a silicon wafer. We investigated the SERS spectra of rhodamine 6G(R6G) and the analytical enhancement factor (AEF) as a function of silver shell thicknessand NR concentration at 633 nm laser excitation. A very thin (∼2 nm) silver coating of AuNRs was sufficient to increase the AEF from 103 for Au NRs to 2.5 × 104 for compositeAu−Ag NRs. The increase in the NR concentration from 2 to 14 g/L of Au gave a 3-foldincrease in the SERS intensity but did not affect the AEF. Three-dimensional finite-difference time-domain simulations confirmedthat the electromagnetic mechanism of AEF enhancement for silver-coated NRs was due to the generation of hotspots near theend-to-end and end-to-side nanoparticle contacts. The estimated cost of all reagents for one SERS measurement is about onecent. Coupled with its simplicity, reproducibility, and low cost, the proposed SERS technique can be recommended for routinechemical or biomedical sensing of small analyte amounts with various substrates, such as glass, polymer films, or even Whatmanpaper.

■ INTRODUCTION

Since the first theoretical prediction1 and experimentalobservations in crystals2 and liquids3 (February 21 and 27,1928, respectively4), Raman scattering remained a potentiallyimportant yet unpractical spectroscopic tool for almost 50 yearsuntil the first observations5−7 of the surface-enhanced Ramanscattering (SERS) were published and the electromagnetic6,7

and chemical mechanisms8 of SERS were suggested (for thediscussion of the early SERS history, see, e.g., Haynes et al.9).After these seminal steps,5−7 the annual number of SERS-related papers increased dramatically to about 1500 the pastfew years.10 The principles and applications of SERS have beenextensively discussed in a series of review articles,11−19 bookchapters,20 and monographs.21−23

Owing to the electromagnetic “hotspots” associated withsharp ends and gaps between particles,24−27 the localmagnitudes of the fundamental SERS enhancement28 canreach 1011, but the surface-averaged results are usually about108 in the best recent experiments.29−31 Furthermore, theenormous local enhancements are difficult to distribute evenly

over the whole substrate, and hotspots usually constitute asmall fraction of the substrate area (typically, less than 1%32).Nevertheless, such a negligible (e.g., 0.09%33) percentage ofhotspots can contribute about 50% to the total surface-averagedSERS signal. This explains why numerous attempts have beenmade to fabricate large-scale high-enhancing SERS substrates.Despite the great variety of existing technologies,11,14,15 most

SERS platforms fall into five broad categories: (1) regular metalsubstrates, fabricated by nanosphere lithography34 and otherlithographic techniques (e.g., refs 16 and 31 and referencestherein); (2) substrates fabricated by lithographic patterning35

or multistage transfer printing;36 (3) “film-over-spheres”platforms, obtained with appropriate nanosized tem-plates;30,31,37−43 (4) metal nanoparticles (NPs) assembled onsilicon, glass,44−47 silica nanospheres,48,49 inverse opals,50 orsimply on Whatman filter paper;51,52 and (5) “SERS tags,”

Received: August 21, 2013Revised: October 1, 2013Published: October 2, 2013

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which combine plasmonic NPs with specific Raman reporterorganic molecules.13,18,53

Thanks to the remarkable progress in NP synthesistechnologies,54−56 one of the most simple and cost-effectivestrategies to manufacture SERS substrates is to fabricate self-assembled nanoparticle films. The published examples includeNP assemblies made of gold NRs (Au NRs),44,46,57−65

nanostars,66−69 mesoflowers,70 and silver NRs71−79 and nano-plates.80 In particular, plasmonic nanopowders of PEGylatedparticles81,82 seem to be suitable for the simple and low-costfabrication of SERS platforms based on random NPassembles.46 However, such substrates demonstrated amoderate analytical31 enhancement averaged over the probelaser beam spot (about 103).46 The difference between thefundamental and the analytical enhancement factor (AEF) isdiscussed, for example, in refs 28 and 31. In particular, the AEFis conveniently used to measure the substrate-induced increasein Raman intensity owing to SERS substrate within a relevantrange of analyte concentrations.31

The goal of this work was to increase the AEF of Au NR-based SERS substrates46 while retaining the simplicity offabrication and measurements with self-assembled large-areaSERS substrates based on powders of PEGylated plasmonicNPs.83 Our simple and facile approach includes a thin-layersilver coating of as-prepared Au NRs (Au−Ag NRs), followedby their PEGylation and fabrication of a nanoparticle powder(for long-term use) or of a concentrated suspension forimmediate SERS measurements. In the first case, a solutionwith a desired NP concentration can be obtained in a matter ofseconds by simply adding water to a portion of nanoparticlepowder. Then, a drop of the concentrated suspension is mixedwith an analyte [in our case, rhodamine 6G (R6G)] and placedon a silicon or glass substrate to yield, after drying at roomtemperature for 5 min, a self-assembled NR monolayer withanalyte molecules incorporated into it. Similarly, a portion ofnanorod powder can be dissolved in a drop of an analyte toprepare a self-assembled NR monolayer.83 In agreement withrecent data of Contreras-Caceres et al.,84 we observed anaverage analytical SERS enhancement even for thin (1 to 2 nm)silver coatings of Au NRs. Note that Au−Ag NRs nanorodswith thicker silver coatings have been used recently by Cui andco-workers85,86 as multifunctional composite Raman labels foranalytical purposes and biomedical applications. According toour three-dimensional (3D) finite difference time domain(FDTD) simulations, the electromagnetic enhancement atsubstrate hotspots makes the main contribution to the observedeffects of silver coatings.

■ EXPERIMENTAL METHODSReagents. The following reagents were used: cetyltrime-

thylammonium bromide (CTAB) (98%; Fluka), chloroauricacid (HAuCl4) (99.99%; Alfa Aser), ascorbic acid (Aldrich),sodium borohydride (Aldrich), silver nitrate (AgNO3)(>99.9%; Aldrich), sodium hydroxide (NaOH) (Reachim),hydrochloric acid (pure; Vekton), absolute ethanol (99.99%;Sharlau), potash (Aldrich), Milli-Q water (18 mΩ cm;Millipore), thiolated polyethylene glycol (PEG-SH; Nektar).Fabrication of Au NRs and Au−Ag NRs. Au NRs were

synthesized by a seed-mediated method44 as describedpreviously.87 In brief, gold “seeds” were prepared by theaddition of 0.1 mL of an ice-cold 10 mM sodium borohydridesolution to 1 mL of aqueous CTAB followed by the addition of0.025 mL of HAuCl4. Then, 2 mL of 4 mM AgNO3, 5 mL of 10

M HAuCl4, 1 mL of 100 mM ascorbic acid, 1 mL of 1 M HCl,and 1 mL of the gold “seed” solution were sequentially addedto 90 mL of 0.1 M CTAB. The mixture was kept undisturbedovernight at 30 °C. The mass-volume gold concentration of thesamples was about 100 mg/L.Au−Ag NRs were fabricated by the method of Xiang et al.,88

with minor modifications. Specifically, we used as-prepared AuNRs without additional washing or polymer stabilization.89 Forthe preparation of Au−Ag NRs with a thin silver coating (0.5 to1.6 nm, see below), 0.5 mL of 4 mM AgNO3, 1 mL of 100 MMascorbic acid, 7 mL of water, and 2.5 mL of 200 mM NaOHwere sequentially added to 10 mL of as-prepared Au NRs. Forsynthesis of Au−Ag NRs with a thicker silver shell (1.3 to 2.7nm, see below), 1 mL of 4 mM AgNO3, 2 mL of 100 mMascorbic acid, 5 mL of water, and 3.5 mL of 200 mM NaOHwere sequentially added to 10 mL of as-prepared Au NRs. Inboth cases, the solutions were mixed for 5 min and were thencentrifuged at 14,000g for 1 h and redispersed in 10 mL ofwater. In what follows, the as-prepared Au NRs and NRs withthin and thick silver coatings are designated NR-0, NR-1, andNR-2, respectively. For all three samples, material balanceestimations gave the following gold and silver concentrations,respectively: 100 and 0 mg/L (NR-0), 100 and 21 mg/L (NR-1), and 100 and 42 mg/L (NR-2).

SERS Substrate Fabrication. Self-assembled SERS sub-strates were fabricated by functionalizing Au−Ag NRs withPEG molecules. To this end, 0.1 mL of a 0.2 M solution ofpotassium and 0.1 mL of a 1 mM solution of PEG-SH wereadded to 10 mL of an NP suspension. The mixture was storedat 28 °C for 12 h and was centrifuged at 14,000g for 1 h; thesediment was redispersed in 0.5 mL of water. Accordingly, theNP concentration was increased by 20 times (2 g/L of Au).Alternatively, the PEGylated NRs were used to fabricate water-soluble powders as described in detail elsewhere.81,82 Thesepowders were also used to prepare NR solutions with thedesired concentration (e.g., 2 g/L of Au) or for dissolutiondirectly in a drop of an analyte. All the other manipulationswere identical for both nanoparticle solutions, and no differencebetween the variants of the SERS substrate protocol wasobserved.Silicon wafers were cleaned with H2SO4−H2O2 (70:30 by

volume). A drop of an NR suspension (1 μL) was mixed with 1μL of the R6G dye and was placed on the wafer surface. Fiveminutes after drying, an aureate film was clearly seen, indicatingthe formation of a self-assembled densely packed monolayerwith included R6G molecules.46 The average diameter of theself-assembled films was about 2 mm.

NR Characterization. Extinction spectra of the sampleswere measured with a Specord 250 BU spectrophotometer(Analytik, Jena, Germany). Before measurements, all sampleswere diluted 1:10 and were placed in 2 mm quartz cuvettes.Transmission electron microscopy (TEM) images wereobtained with a Libra-120 transmission electron microscope(Carl Zeiss, Jena, Germany). For analysis of self-assembledlayers on TEM grids, the fabrication protocol was similar tothat used for silicon wafers.

Raman Spectroscopy. SERS and Raman spectra weremeasured with an HR800 spectroscope system (HORIBA JobinYvon), in which a Raman scattering (RS) spectrophotometer iscombined with a confocal microscope (a micro-RS spectrom-eter). The basic measurements were performed with a He−Nelaser (632.8 nm, 7.25 mW); the laser beam was focused into a 5μm spot located near the central area of the substrates. The

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acquisition interval was 10 s, and all SERS spectra wereaveraged over 10 independent runs.FDTD Electrodynamics Simulations. FDTD simulations

were performed with an FDTD Lumerical Solution 7 code. Thesimulation volume was a 200 × 200 × 150 nm parallelepipedwith a uniform mesh of 0.35 nm. The structure of the NRmonolayers was close to the structures observed by TEM.Twenty-four NRs were placed horizontally on a semi-infinitesilicon substrate and were illuminated at 633 nm perpendic-ularly to the silicon plane. In all simulations, the “total field/scattering field” mode was used with the Au and Ag opticalconstants taken from the Drude approximation of Johnson andChristy’s data.90 The FDTD “Field monitor” was placed in themiddle section of the nanoparticle layer. The CPU time of onesimulation run was about 24 h (4 × 2.2 GHz processors, 20 GbRAM).

■ RESULTS AND DISCUSSIONParticle Characterization. Figure 1 shows the extinction

spectra of Au NRs, which demonstrate the well-known blue

shift91 of the longitudinal plasmon resonance with increasingsilver shell thickness. For as-prepared uncoated Au NRs, thelongitudinal and transverse resonances are located near 810 and512 nm, respectively. These resonances are typical of Au NRswith diameters of 10 to 15 nm and an aspect ratio of about 4.87

The ratio of major to minor resonance maximum equals 3.7,indicating that the percentage of impurity particles (spheres,cubes, etc.) is small.92 On the other hand, the full width at half-maximum (fwhm) is about 200 nm, which is typical of themoderate polydispersity of Au NRs over length and width.93

Reduction of silver on the NR-1 rod surface shifts the majorand minor resonances to 700 and 497 nm, respectively. Thisshift is accompanied by an increase in the major resonancequality factor Q, in agreement with the “plasmonic focusing”reported earlier by Becker et al.89 Physically, this effect can beexplained simply by the different spectral properties of gold andsilver dielectric functions and by the corresponding high-qualityresonances of silver colloids as compared to their goldcounterparts. A further increase in silver shell thickness, inaddition to the blue shift of the longitudinal and transverseresonances, gives rise to a new short-wavelength resonance near380 nm. Again, these spectral features are typical of Au−AgNRs with 2−3 nm silver shells, and the new 380 nm maximumis not related to the formation of small silver spheres.94

For composite Au−Ag NRs, the average silver shell thicknesscan be estimated with a calibration curve that correlates themajor resonance shift with the silver shell thickness.94 For NR-1

and NR-2 rods, these estimations give the average silverthicknesses of 1.1 and 1.7 nm, respectively (Figure S1 in theSupporting Information).Although some details of the NR shape can be extracted from

extinction spectra (or, as demonstrated previously,87 incombination with scattering and depolarized light scatteringspectra), a direct comparison with TEM data unambiguouslyconfirms the quality of the optical model. Figure 2a−cdemonstrates TEM images of NR-0, NR-1, and NR-2 rods,respectively. Note that these images do not show the additionalspherical silver particles in samples NR-1 and NR-2. For as-prepared Au NRs, statistical analysis (Figure S2 in theSupporting Information) reveals that they have a “dog bone”shape with an average length of 52.7 ± 6.2 nm, an end-capdiameter of 15.1 ± 3.1 nm, and a central diameter of 11.8 ± 2.7nm. T-matrix simulations of the optical properties of such rodshave been reported,87 and those simulations are in goodagreement with the data of Figure 1. It follows from Figure 2dthat the reduction of silver starts in the thinnest part of theparticle, where the concentration of CTAB molecules ismaximal.95 Accordingly, the NR-1 rods have a cigarlike shape,are longer (53.8 ± 6.7 nm), and have a greater average diameter(16.7 ± 3.4 nm). From TEM data, the average thickness of thesilver layer is 1.05 nm, in excellent agreement with the valuederived from the plasmonic shift (1.1 nm; Figure S1 in theSupporting Information).Similar data were obtained for NR-2 rods, also exhibiting the

anisotropic growth of a silver coating: from 1.6 to 2.7 nm in thecentral part and from 0.5 to 1.3 nm at the ends. Again, theaverage TEM silver thickness of 2 nm (Figure 2d) is in goodagreement with the plasmonic shift estimate of 1.7 nm (see alsoFigure S1 in the Supporting Information). This explains whythe Au−Ag NRs tend to form spheres and prisms while thesilver layer is growing.88 Note that small silver layers cannot beresolved at moderate TEM magnifications (Figure 2 a−c), butthey can be identified in the enlarged and contrasted TEMimage shown in Figure S3 in the Supporting Information.To summarize, our simple and robust protocol allows

controlled deposition of thin silver layers of 1 to 3 nm withoutany temporal change in the optical properties of Au−Ag NRswithin one week. After PEGylation, the Au−Ag NRs are verystable for at least a year of storage in a typical refrigerator.

SERS Substrates. When the concentration of drying PEG-coated NRs on TEM grids is increased, small regions with side-by-side packing appear (Figure 3) in which the nanometer-sized gaps between the NRs are determined by the adsorbedPEG molecules. One can suppose that the analyte moleculesplaced right into the gaps between the NRs would be goodSERS reporters. To achieve this goal, we suggest the the simplescheme depicted in the top panel of Figure 3.In this “immediately before measurement” (IBM) scheme, a

drop of analyte is mixed with a drop of functionalized Au−AgNRs and the mixture is placed on a silicon wafer to dry at roomtemperature for 5−10 min. This yields a quasiregularmonolayer of NRs with nanometer-sized interparticle separa-tions and with a more or less homogeneous distribution ofanalyte molecules. The SERS substrate made of self-assembledAu and Au−Ag nanorods was stable and provided reproducibleenhancements after storage for 1−2 months under ambientconditions.83

The TEM images in Figure 3 show monolayers of NR-0 (a),NR-1 (b), and NR-2 (c) rods formed on Formvar-coated TEMgrids. On the average, for a typical 25 μm2 SERS measurement

Figure 1. Normalized extinction spectra of as-prepared Au NRs (1,NR-0) and silver-coated Au−Ag NRs with Au:Ag weight ratios of 5:1(2, NR-1) and 2.5:1 (3, NR-2).

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area, we observe quasiregular monolayers with some multilayerand void defects caused by Formvar-coating imperfections. Thedeposition of the same NRs on silica wafers looks morehomogeneous (see, e.g., the scanning electron microscopy(SEM) images in Figure S4 of the Supporting Information).Unfortunately, the quality of the SEM images in Figure S4 isworse because of the surface polymer (PEG-SH) layer, andcontrast sputtering could have destroyed the monolayerstructure.For a concentration of the deposited colloid of 2 g/L, the

average particle number density is about 600 particles per 1μm2, corresponding to 50% coverage of the surface area. With a10-fold decrease in the colloid concentration, we observe theformation of islandlike films rather than a homogeneous

decrease in the average covering density (Figure S4 in theSupporting Information). However, regardless of the averagedensity, the particle surface density remains roughly constantwithin each self-assembled island. We believe that the principalrole in the formation of such monolayer islands is played by thePEG molecules on the particle surface. These molecules serveas a self-assembling agent that prevents random particleaggregation, which is typically observed when a drop of non-PEGylated colloidal gold particles is drying.

SERS Spectra of R6G and AEFs. The proposed approachto forming SERS substrates with PEG-coated NRs has severalattractive features. First, the fabrication of a substratesimultaneously with analyte incorporation is very simple andcan be performed routinely with just a 1 μL dispenser. Second,

Figure 2. TEM images of NR-0 (a), NR-1 (b), and NR-2 (c) rods. Panel (d) shows the geometrical model for as-prepared Au NRs and theanisotropic growth of silver shells.

Figure 3. Schematic fabrication of a SERS substrate by the simultaneous deposition of PEG-coated Au−Ag NRs and an analyte on a silicon wafer(top). TEM images of self-assembled monolayers on Formvar-coated grids for NR-0 (a), NR-1 (b), and NR-2 (c) rods.

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the surface distribution of the analyte is stable in time and ishomogeneous over the whole substrate area. In particular, thestandard deviation of the SERS intensity at different points inthe substrate was shown to be less than 10%.46 Finally, suchSERS substrates can be made with various base surfaces,including silicon, glass, polymer films, and Whatman paper.51

For all substrate types, the dense NR layer will strongly shieldthe background and fluorescence signals from the underlyingsurface. The basic disadvantage of such substrates withassembled Au NRs is their small analytical enhancement ascompared with vertical61 or fractal96 particle packing. However,the average analytical SERS enhancement can be increased bycoating Au NRs with silver.84−86

To confirm this approach under our conditions, we usedR6G as a Raman label and measured SERS spectra forsubstrates with NR-0, NR-1, and NR-2 particles by varying theR6G concentration from 0.08 μM to 80 μM (Figure 4). R6G isa typical benchmark molecule with intense Raman bands at614, 766, 1178, 1306, 1361, 1509, and 1647 cm−1,corresponding to the C−H, C−O−C, and C−C vibrations ofan aromatic ring.First of all, we observed no additional Raman bands from

silicon or from the PEG molecules attached to the NR surface.It is well-known that silicon has a characteristic Raman peak at518 cm−1. This peak is clearly seen in the Raman spectra of the

R6G solution deposited on a silicon wafer (Figure S5-I in theSupporting Information). The peak intensity decreases afterdeposition of R6G on a rare NR substrate (Figure S5-II in theSupporting Information), and the peak disappears for a denselypacked NR-0 monolayer on a silicon wafer (Figure S5-III in theSupporting Information). This confirms the silicon backgroundshielding and applicability of PEG coating to SERS measure-ments, as the Raman cross section of PEG molecules is verylow compared to that of R6G.Under our measuring conditions (laser power, wavelength,

and acquisition intervals), the R6G SERS bands could beidentified at 80, 8, and 0.8 μM R6G only for the NR-2substrate. The most intensive band at 614 cm−1 could beidentified for all three substrates within different ranges of dyeconcentrations: 0.08−80 μM for NR-2, 0.8−80 μM for NR-1,and 8−80 μM for NR-0.Figure 4d shows the concentration dependence of the 614

cm−1 Raman band for the three substrates to illustrate theremarkable effects of silver coating: an almost one-orderenhancement for thin silver shells and an almost 30-foldenhancement for thicker silver shells. In general, the analyticalSERS enhancement with NR-2 exceeds our previously reportedvalues for fractal films of Au NRs46 under similar experimentalconditions.

Figure 4. SERS spectra for different R6G concentrations applied simultaneously with NR-0 (a), NR-1 (b), and NR-2 (c) rods. Panel (d) shows theSERS intensity at 614 cm−1 as a function of the R6G concentration. The intensity values were corrected for the baseline.

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One important note is in order here. For elucidation of therole of the silver shell, it seems more reasonable to compare theAEFs of the NR-1 and NR-2 platforms with those of SERSsubstrates made of gold NRs with the same or close plasmonresonance wavelengths. Of course, such a comparison would bequite correct in the case of single-particle measurements or forrare NR monolayers. However, because of the strongelectromagnetic particle coupling in densely packed andfractal-like films, the experimental extinction spectra demon-strate a significant broadening, thus making the plasmonexcitations delocalized within a broad spectral band. Forexample, we have studied three types of SERS platforms (raremonolayers, densely packed monolayers, and fractal-like films)for Au NR-670 and Au NR-810 NRs (plasmon resonances at670 and 810 nm).46 For densely packed and fractal-likesubstrates, the experimental extinction spectra demonstrated adelocalized extinction band from 500 to 1000 nm. This explainswhy we observed no significant differences between Au NR-670and Au NR-810 densely packed and fractal-like substrates at633 nm and 785 nm excitations.46 Therefore, we speculate thatthe difference between the AEFs of Au- and Ag-coatedassembled NRs (Figure 4) may be related to the localelectromagnetic enhancement owing to silver coating.It is instructive to estimate the number of R6G molecules

that contribute to the recorded SERS bands. In the bestenhancement, we introduced 1 μL of an 80 nM solution ofR6G, which contained 4.8 × 109 molecules. Taking intoaccount the ratio between the deposition and the measurementarea, we obtained about 105 contributing molecules. Such amoderate analytical sensitivity should be considered as acompromise between the simplicity, reproducibility, and lowcost of SERS measurements on the one hand and the lowdetection limit on the other. It is known that strongerenhancements can be obtained with aggregated colloids;however, such substrates show poor reproducibility andstability.46

To illustrate the effects of the NR concentration, weperformed similar measurements with more concentrated NRcolloids (14 g/L of Au), which form multilayer SERSsubstrates.46 Figure 5a demonstrates a notable increase inSERS intensities as compared to Figure 4, but the ratiosbetween the detected intensities in the NR-0−NR-1−NR-2series are similar to the ratios in Figure 4 (Figure 5b).The AEF is a figure of merit used to quantify the overall

boost of the SERS intensity provided by a given substrate underspecific experimental conditions.31 From a practical point of

view, this factor is simply the ratio between the analyteconcentrations producing equivalent Raman and SERS signals.By contrast with the fundamental enhancement factor(EF),28,31 the AEF is usually several orders lower than theEF, as the EF parameter is the ratio between SERS and Ramanintensities normalized to the number of molecules excited.Figure 6 demonstrates the determination of AEFs for the

three SERS substrates by comparing SERS spectra at 8 μM

R6G with a Raman spectrum recorded with a much moreconcentrated R6G solution (10 mM). The SERS intensities at614 cm−1 for NR-0, NR-1, and NR-2 were 0.8, 5, and 20 au,respectively. Taking into account the concentration ratio 10mM/8 μM = 1250, we obtained AEFs of 103, 6.25 × 103, and2.5 × 104, respectively. Thus, the maximal AEF for NR-2 rods isan order of magnitude greater than that for fractal films ofdeposited Au NRs.46,49

FDTD Simulations. It is reasonable to assume that theSERS enhancement for silver-coated NRs is due to electro-magnetic rather than to chemical contribution. To confirm thisassumption, we compared the electromagnetic field distribu-tions for monolayers of gold NRs and silver-coated Au NRs.The FDTD simulation parameters were chosen from theexperimental conditions. Specifically, the size and shape of AuNRs were taken from the statistical data for as-prepared AuNRs, whereas the inhomogeneous silver coating was also taken

Figure 5. (a) SERS spectra of R6G (1 μL, 80 μM) deposited with NR-0 (1), NR-1 (2), and NR-3 (3) rods at a Au concentration of 14 g/L. (b)Intensities of the 614 cm−1 band for simultaneous deposition of R6G (1 μL, 80 μM) with NR-0, NR-1, and NR-2 rods at two Au concentrations: 2and 14 g/L. For convenience, the intensity of NR-2 at 14 g/L was multiplied by 0.2.

Figure 6. Determination of AEFs for NR-2 (a), NR-1 (b), and NR-0(c) rods using SERS spectra of 8 μM R6G (a−c) and a Ramanspectrum of 10 mM R6G (d).

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into account from the same TEM data for NR-1 and NR-2. The3D simulation volume had dimensions of 200 × 200 × 150 nm.The particle monolayer was assembled on a silicon wafer, andthe simulation volume included a 20 nm thick layer locatedinside the silicon wafer. According to the TEM data, theaverage number density of the NRs was about 600 μm−2;therefore, the number of randomly distributed rods on the 200× 200 nm simulation area was about 24. The surfacedistribution of the rods was generated randomly, but weselected some configurations with a preferential side-by-sideorientation, in agreement with the experimental TEM images.The light source was a 633 nm plane electromagnetic wave witha vertical incidence direction (Figure 7a). Panels b−d in Figure7 show the electric field distribution (the absolute values of E)in the middle section of the monolayers. For convenience ofcomparison, all distributions are presented with the same colorscale.Nanoscale gaps and sharp points in metal nanostructures are

the primary features needed to produce an electromagneticenhancement in Raman scattering by small molecules. Itfollows from Figure 7 b−d that the field distributions near thecontact points are similar. Furthermore, the parallel packingdoes not produce electromagnetic hotspots and does not

increase the average electromagnetic enhancement, in agree-ment with previous data.64,97 By contrast, strong local fields areobserved near the end-to-end contacts. Our simulations haveconfirmed the remarkable electromagnetic enhancement due tosilver coating: the thicker the silver shell, the larger the numberof hotspots observed in the simulation plots (see also Figure S6in the Supporting Information for the electric field distributionaround individual Au and Au−Ag NRs). For 633 nm, themaximal field enhancement was about 50, which correspondedto a 6 × 106 SERS enhancement. These estimations are close tothe literature data for silver-oversphere substrates.31

■ CONCLUSIONS

SERS substrates have been fabricated by a very simple and low-cost technique. Our approach is based on mixing a drop of Au−Ag PEG-coated NRs with a drop of an analyte to obtain, afterdeposition on a silicon wafer and drying at room temperaturefor 5 min, a self-assembled NR monolayer with analytemolecules incorporated into it. The suggested simplifiedprotocol for synthesizing silver-coated and PEGylatedcomposite NRs produces optically stable concentrated colloidsfor immediate use in SERS measurements. These colloids can

Figure 7. (a) Model for a rod monolayer illuminated by a plane electromagnetic wave. The 200 × 200 × 150 nm gray parallelepiped displays thesimulation region. Panels b−d are 200 × 200 nm and show the electric field distributions in the middle section of NR-0 (b), NR-1 (c), and NR-2 (d)monolayers.

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also serve to fabricate plasmonic nanopowders for long-termSERS purposes, as described earlier.81 In particular, SERSmeasurements can be performed with NR powders directlydissolved in a drop of analyte and then deposited on a siliconwafer. In general, this approach has resulted in AEFs close tothose obtained with the SERS substrates fabricated with a dropof PEG-coated NR solution mixed with a drop of analyte. Evena very thin (1−2 nm) silver coating of Au NRs has broughtabout a 25-fold increase in the AEF at 633 nm excitation: from103 for Au NRs to 2.5 × 104 for Au−Ag NRs. Both as-preparedconcentrated colloidal samples and similar colloids obtainedfrom nanopowders have produced very stable large-scale SERSsubstrates. Increasing the NR concentration from 2 to 14 g/Lof Au has given a 3-fold increase in the SERS intensity but hasnot changed the measured AEFs. 3D FDTD simulations haveconfirmed that the dominant electromagnetic mechanism ofSERS enhancement is due to the formation of hotspots nearthe end-to-end and end-to-side nanoparticle contacts. Theproposed technique can be easily reproduced in routinelaboratory practice, as it is very simple, robust, and inexpensive.Indeed, in a typical synthesis, we fabricate 1 L of NRs for50,000 measurements; as a result, the cost of all reagents forone measurement can be reduced to one cent. Although ourexperiments have been performed mostly with silicon and glass,other types of support (e.g., glass, Whatman filter paper, orpolymer films) seem applicable. For example, we measuredSERS spectra with gold NRs deposited on microscopic coverglass substrates, and in general, the estimated AEFs were closeto those reported here for silicon. In view of its simplicity,reproducibility, and low cost, our technique can be useful in theroutine chemical or biomedical sensing of small amounts ofanalytes with various substrate bases.

■ ASSOCIATED CONTENT*S Supporting InformationCalibration curve for determining the Ag layer thickness fromthe plasmonic shift (Figure S1); TEM histograms of the particlelength and particle width distributions (Figure S2); an enlargedTEM image of Au−Ag NRs (Figure S3); SEM images of self-assembled NR-0 rods for different concentrations (Figure S4);Raman spectra of R6G (8 μM) applied on silicon wafer, rareNR monolayer, and NR-0 monolayer (Figure S5); and FDTDfield distributions calculated for individual NR-0 and NR-2 rods(Figure S6). This material is available free of charge via theInternet at http://pubs.acs.org.

■ AUTHOR INFORMATIONCorresponding Author*E-mail: khlebtsov@ibppm.sgu.ru.NotesThe authors declare no competing financial interest.

■ ACKNOWLEDGMENTSThis research was supported by grants from the RussianFoundation for Basic Research (11-02-00128a, 12-02-00379a,12-02-31056 mol_a, 13-02-01075a, and 13-02-12413), thePrograms of the Presidium of the Russian Academy of Sciences“Basic Sciences for Medicine” and “Basic Technologies forNanostructures and Nanomaterials,” and the Government ofthe Russian Federation (Contracts 14.B25.31.0019 and11.G.34.31.0030 to support scientific research projectsimplemented under the supervision of leading scientists at

Russian institutions and Russian institutions of highereducation). M.Y.T. and V.N.B. were supported by a grantfrom the Ministry of Science and Education of the RussianFederation (Contract 8391). V.A.K. was supported by ascholarship from the President of the Russian Federation anda grant from the OPTEC company. We thank D. N. Tychinin(IBPPM RAS) for his help in preparation of the manuscript.

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SUPPORTING INFORMATION

SERS Substrates Based on Self-Assembled

PEGylated Gold and Gold–Silver Core–Shell

Nanorods

Boris N. Khlebtsova, Vitaly A. Khanadeeva, Mikhail Yu. Tsvetkovb, Victor N. Bagratashvili,b

and Nikolai G. Khlebtsova,c,*

aInstitute of Biochemistry and Physiology of Plants and Microorganisms, Russian Academy of

Sciences,

13 Prospekt Entuziastov, Saratov 410049, Russia, e-mail: khlebtsov@ibppm.sgu.ru

bInstitute of Laser and Information Technologies, Russian Academy of Sciences, 2 Pionerskaya

Ulitsa, Moscow, Troitsk, 142190, Russia

cSaratov State University, 83 Ulitsa Astrakhanskaya, Saratov 410012, Russia

*Corresponding author: khlebtsov@ibppm.sgu.ru (Nikolai Khlebtsov).

Figure S1. Calibration curve for determining the Ag layer thickness through the measured

relative shift of the extinction plasmon resonance.1,2 The red circles show the determination of

Ag thicknesses for NR-1 and NR-2 rods.

Figure S2. Histograms of the particle-length and particle-width distributions for NR-0 (a, d),

NR-1 (b, e), and NR-2 (c, f) rods, as derived from the TEM images. For NR-0, the width is the

arithmetic average between the minimal (central) and the maximal (end-cap) diameter.

Figure S3. Enlarged and contrasted fragment of a TEM image for NR-2 rods. The arrows

indicate the gold core and the silver shell.

Figure S4. Overview (a, c) and enlarged (b, d) SEM images of NR-0 monolayers deposited on a

silicon wafer. The NR concentrations (in terms of Au content) are 2 g/L (a, b) and 0.2 g/L(c, d).

Figure S5. Raman spectra of R6G (8 µM) applied to a silicon wafer (I), a rare NR monolayer (II;

see Fig. S4d), and a densely packed NR-0 monolayer (III).

Figure S6. FDTD field distributions calculated for individual NR-0 (a) and NR-2 (b) rods.

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2 Khlebtsov, B. N.; Khanadeev, V. A.; Khlebtsov, N. G. Attenuation, Scattering, and

Depolarization of Light by Gold Nanorods with Silver Shells. Opt. Spectrosc. 2010, 108 (1), 59–

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