Gold Nanoparticle-Based pH Sensor in Highly Alkaline Region atpH . 11: Surface-Enhanced Raman Scattering Study

JONG KUK LIM and SANG-WOO JOO*Department of Chemistry, Soongsil University, Seoul 156-743, Korea

A surface plasmon resonance spectroscopy study showed that citrate-

reduced gold nanoparticles (;15 nm diameter, ;9 3 10�9 M concentra-

tion, ;2 3 10�2 M ionic strength) were found to be utilized as

a colorimetric sensor by exhibiting a distinct color change at a highly

alkaline pH . 11.5. Surface-enhanced Raman scattering (SERS) of 4-

ethynylpyridine (4-EP) on gold nanoparticle surfaces indicated that the

multiple peaks in the m(C[C) stretching bands should vary significantly in

the highly alkaline region from pH 12 to 14. As the pH value increased,

the m(C[C) stretching band intensity at ;2080 cm�1 became stronger

than that at ;2010 cm�1. The pK1/2 value was determined to be around 13

by the SERS titration of taking intensity ratios of I2080 with respect to

I2010. Using SERS enhancements and conspicuous spectral changes, self-

assembled monolayers (SAMs) of 4-EP on Au nanoparticles holds

potential as a pH sensor for sensitive detection of the hydroxide OH�

concentration at around pH 13 in an aqueous solution. The pH calibration

from SERS titration of 4-EP is expected to have advantages in terms of

higher alkaline detection limit and more precise measurements, if

compared with the indigo carmine, the pK1/2 value of which is 12.2.

Index Headings: Au nanoparticles; Surface plasmon resonance; SPR;

Surface-enhanced Raman scattering; SERS; 4-Ethynylpyridine; pH

sensor.

INTRODUCTION

Gold nanoparticles have attracted much attention in the pastdecade due to their stability, uniformity, and optical proper-ties.1 Optical properties of gold particle aggregates have beenextensively investigated by surface plasma resonance andtransmission electron microscopy (TEM).2 Surface plasmonresonance has been recently used to monitor analyte–surfacebinding interactions for colorimetric sensors.3 For molecularassembly of alipathic thiols on gold nanoparticles, the extent ofaggregation was estimated from a measure of the integratedextinction between 600 and 800 nm.4

Since its discovery, surface-enhanced Raman scattering(SERS) as an ultra-sensitive spectroscopic tool for interfacestudies has been widely used as a chemical sensor in the area ofanalytical chemistry.5–7 It can provide chemically specificinformation on the basis of the unique vibrational modes oftarget adsorbates. Recently we have employed variousexperimental techniques such as SERS, quasi-elastic lightscattering (QELS), and zeta-potential measurement to charac-terize the gold nanoparticle aggregates.8

Self-assembled monolayers (SAMs) have played an impor-tant role in nanoscience and technology due to their potentialapplications in molecular electronic devices and biocompati-bility.1 Interfacial pKa values for SAMs have been evaluated byusing capacitance measurements,9 second-harmonic genera-tion,10 and chemical force microscopy.11 Determination of pHand pKa values from SAMs have been performed using SERStitration.12,13 The pyridine ring mode of 4-mercaptopyridine

appeared to vary at slightly acidic regions of pH 3;6 in theprevious report.14,15 SERS spectra of salicylic acid, pyridine,and 2-naphthalenethiol were found to strongly relate to the pHand zeta potential values.16

Relatively little work has been done to fabricate organicSAMs on metal surfaces without sulfur as the anchoringgroups.17 There have been several reports on amine-terminat-ed18 or carboxylic acid-terminated SAMs19 on metals or metaloxides. Anchoring an aromatic ring via an alkynyl group mayhave an advantage of providing a p-conjugated linkage at thegold surfaces to design novel materials and devices.20–22

Surface-enhanced Raman scattering of ethynylbenzene onsilver and gold was previously reported.23–25 Multiple bandsobserved in the m(C[C) stretching region were interpreted asbeing due to adsorption at different crystals or the existence ofseveral different complexes. A plausible cause of the splittinghas not yet been clearly confirmed, however. The multiplebands in the C[C stretching region for the SERS spectrumwere found to change upon addition of other ions into the solmedium.25

Highly alkaline sensors should be useful in the environmen-tal26 or biological fields.27 To the best of our knowledge, therehas not been a detailed study of gold nanoparticles as a pHsensor in the highly alkaline region of pH . 11 using SERS. Inthis study, we examined the adsorption behaviors of 4-ethynylpyridine on Au nanoparticle surfaces by means ofSERS to aim at the development of a method to monitor highlybasic conditions at pH . 11.

EXPERIMENTAL

Sample Preparation. Citrate-stabilized gold nanoparticleswere synthesized by following the recipes in the literature.28

A 133.5 mg portion of KAuCl4 (from Aldrich) was initiallydissolved in 250 mL of water, and the solution was brought toboiling. A solution of 1% sodium citrate (25 mL) was thenadded to the KAuCl4 solution under vigorous stirring, andboiling was continued for approximately 20 min. Theresulting Au concentration ignoring the evaporated watershould be ;1.28 3 10�3 M and the concentration of the Aunanoparticles was estimated to be ;9.1 3 10�9 M.29 All thechemicals otherwise specified were reagent-grade, and triplydistilled water, of resistivity greater than 18.0 MX�cm, wasused in making aqueous solutions. To compare the Aunanoparticle aggregations induced by SAMs, we chosea simple molecule, 4-ethynylpyridine (4-EP) (.95%), asdrawn in Fig. 1, purchased from Aldrich and used withoutfurther purification.

Characterization of Gold Nanoparticle Aggregates.Transmission electron microscopy images were obtained witha Tecnai F20 Philips or a JEM-2000EXII transmission electronmicroscope after placing a drop of colloidal solution ona carbon-coated copper grid. The statistical analysis revealed

Received 18 February 2006; accepted 22 May 2006.* Author to whom correspondence should be sent. E-mail: sjoo@ssu.ac.kr.

Volume 60, Number 8, 2006 APPLIED SPECTROSCOPY 8470003-7028/06/6008-0847$2.00/0

� 2006 Society for Applied Spectroscopy

a size distribution in diameter for 15 nm particles. Ultraviolet–visible (UV-Vis) absorption spectra of the colloidal solutionswere obtained with a Shimadzu UV-3101PC spectrophotom-eter. The kmax value of the UV-Vis extinction spectrum in thegold nanoparticles was found at ;520 nm. The full-width athalf-maximum (FWHM) for the samples was measured to be;70 nm. After the addition of NaOH or 4-EP, the UV-Visspectra were taken as immediately as possible, within severalminutes. The pH values of Au nanoparticle solutions weremeasured by using a Thermoelectron Orion 3 star benchtop pHmeter.

Instrumentation. The light scattering and zeta potentialmeasurements were performed using an Otsuka ElectronicsFDLS-3000 particle analyzer. Raman spectra were obtainedusing a Renishaw Raman confocal system model 1000spectrometer equipped with an integral microscope (LeicaDM LM).25 Spontaneous Raman scattering was detected with1808 geometry using a peltier cooled (�70 8C) charge-coupleddevice (CCD) camera (400 3 600 pixels). An appropriateholographic supernotch filter was set in the spectrometer for632.8 nm. The holographic grating (1800 grooves/mm) and theslit allowed the spectral resolution to be 1 cm�1. The 632.8 nmirradiation from a 35 mW air-cooled HeNe laser (Melles GriotsModel 25 LHP 928) with a plasma line rejection filter was usedas the excitation source for the Raman experiments. Dataacquisition time used in the Raman measurements wasapproximately 30 s. The Raman band of a silicon wafer at520 cm�1 was used to calibrate the spectrometer.

RESULTS AND DISCUSSION

Colorimetric pH Indicators. There have been variouscolorimetric indicators such as thymolphthalein (pH:9.4;10.6), alizarin yellow (pH: 10.1;12.0), and indigocarmine (pH: 11.4;13.0) available to check the pH changein the alkaline region.31 Using the common color indicatorindigo carmine, the visible absorption spectral changes andresulting pH titration curve by measuring the extinction at;610 nm are shown in Fig. 2. The absorption band at ;610nm was found to decrease drastically as pH increased at pH .12. A titration curve by measuring the intensities is plotted inFig. 2b. The titration curve indicated a rather abrupt change inabsorbance at pH . 12.2.

Surface Plasmon Resonance Spectra of Au Nanopar-ticles. To check the possibility of utilizing gold nanoparticlesas a pH sensor, we monitored UV-Vis spectral changes byvarying pH conditions. As shown in Figs. 3a–3c, the color ofthe gold nanoparticle solution changed upon the addition ofNaOH. At pH , 11, the sol color was red without showingflocculation, in line with the previous report,4,16 whereas itbecame purple and greenish black at pH ;12.5 and ;13.5,respectively. Our results suggest that the replacement of thetrivalent citrate ions adsorbed on the nanoparticle surface withmonovalent hydroxide ions should destabilize the particles,causing aggregation and hence the increase in the size of thenanoparticle aggregates.8 Our light scattering and zeta potentialmeasurements also supported that gold nanoparticles became

aggregated and showed surface potential change when thehydroxide ions were added into the sol medium.

As shown in Fig. 3d, the aggregation of gold particles bya self-assembly process could be checked by a red shift in theUV-Vis absorption spectrum due to a decrease in inter-particledistances. Our results appeared to be in line with the recentreport.16 After the addition of NaOH, the red-shift of thesurface plasmon band may indicate the formation of largeraggregates. Aggregation of Au nanoparticles is checked by theappearance of features above 600 nm in the UV-Visabsorbance spectrum. The extent of aggregation (flocculation)was estimated from a measure of the integrated extinctionbetween 600 and 800 nm.4 Measured flocculation was found toincrease as time elapsed and reached a maximum after a certainpoint. On the basis of the estimated flocculation as measuredfrom Fig. 3d, the titration could be plotted depending on pH asshown in Fig. 3e.

Considering that the gold sol in our experiments wasprepared using a 1.28 3 10�3 M KAuCl4 solution, the ionicstrength of Au nanoparticles was estimated to be around 2.0 310�2 M by summing the products of the concentration by thesquare of the charge. The aggregation of gold dispersions wasfound to vary by changing ionic strengths and pH in the

FIG. 2. (a) Extinction spectra of indigo carmine by varying pH . 12. (b) pHtitration calibration curve of indigo carmine with the standard deviations of fivemeasurements by monitoring the extinction at around 610 nm.

FIG. 1. Structure of 4-ethynylpyridine (4-EP).

848 Volume 60, Number 8, 2006

previous report,4 where the flocculation did not occur for theinitial gold nanoparticle concentrations of ;15 pM at pH 10.5with the ionic strength below 10�1 M.

4-Ethynylpyridine may have several ways to bind metalsurfaces via its pyridine ring or acetylene group that can beaffected by pH condition. Figure 4 shows the surface plasmonresonance shifts and resulting color changes of gold nanoparticlesolutions when 4-EP was added before and after the addition ofNaOH. It is likely that not 4-EP but rather the hydroxide OH�

ions should induce the surface plasmon shifts from our UV-Visdata. Even without 4-EP, gold nanoparticles were found toaggregate at highly alkaline pH as shown in Fig. 3.

Raman Spectra of 4-Ethynylpyridine. To examine theadsorption behaviors of 4-EP in a more careful way, we tooka series of SERS spectra at different pH values. Figure 5 showsthe ordinary Raman (OR) spectrum of 4-EP in the liquid stateand the Au SERS spectra at various pH values. Since theaverage diameter of a colloidal Au particle is estimated to be 15nm, its mean surface area and volume are estimated to be 707nm2 and 1770 nm3, respectively. Assuming that the radius ofa Au atom is 0.14425 nm, the number of atoms per Aunanoparticle should be 1.41 3 105. Assuming that each 4-EP isto occupy an area of 0.217 nm2 with a perpendicularorientation on Au as in the case of thiols, the number of 4-EP molecules covering a single Au nanoparticle should be3260. The concentration required to cover all the colloidal goldparticles is accordingly calculated to be 3.0 3 10�5 M, i.e.,(3260) 3 9.1 3 10�9 M.30 The concentration of 4-EP in Aunanoparticle solution was ;10�4 M, which should be abovemonolayer coverage.

In order to obtain information on the surface mechanism, itis necessary to analyze spectral changes according to theadsorption process. Consulting the earlier vibrational assign-ments,14,25 we analyzed the Raman spectra in Fig. 5. It wasrather straightforward to correlate the OR bands with the Au

FIG. 3. Typical images of gold nanoparticle solutions at values of (a) pH ,;11, (b) ;11 , pH , ;13, and (c) pH . 13.0. (d) UV-Vis spectral changeresulting from aggregation of gold nanoparticle solutions by increasing pH from(i) pH , ;11, (ii) ;11 , pH , ;13, and (iii) pH . 13.0. (Horizontal axisunits, nm.) (e) pH titration calibration curve with the standard deviations of fivemeasurements from the integration of the absorbance between 600 and 800 nm.

FIG. 4. (a) Extinction spectra of Au sol (i) before and after the addition of 4-EP without (ii) base (pH ; 6.5) and (iii) with base (pH ; 13.8). Photographs ofAu sol (b) before the addition of 4-EP and after the addition of (c) 4-EP withoutNaOH (pH ; 6.5) and (d) with NaOH (pH ; 13.8).

APPLIED SPECTROSCOPY 849

SERS bands. Their peak positions are listed in Table I alongwith the appropriate vibrational assignments. Also, thebandwidth (FWHM) of the m(C[C) band of a free statebecame broadened upon adsorption on gold surfaces.25 Theseresults should indicate that 4-EP may bind to Au surfaces viaits C[C group. As shown in Fig. 5, strong enhancements ofthe in-plane ring modes support a rather vertical orientation of4-EP on gold.

To date, only qualitative arguments concerning molecularorientation have been offered for data obtained via SERS, sinceit has been difficult to model real surfaces due to the fact thatboth a long range electromagnetic (EM) effect and a shortrange chemical effect are assumed to simultaneously operatefor the overall enhancement.32–34 On the basis of electromag-netic (EM) surface selection rules,32–34 the interfacial structuresof simple aromatic adsorbates on silver and gold nanoparticlescould be explained in a more quantitative way.34 An additionalcontribution to the SERS phenomenon is the charge transfer(CT) mechanism as a resonance Raman process, although itstrongly depends on the nature of the metal–adsorbate systemwithout general rules.32 The analysis of the simple 4-EPmolecule could be possible for a few selected peaks on thebasis of the prediction of the electromagnetic (EM) selectionrule. From the EM surface selection rule,32,33 the vibrationalmode perpendicular to the surface is more enhanced than theparallel mode. For 4-EP, most ring modes were found tobelong to those of in-plane modes, except for the features at;819 and ;488 cm�1. The relative weakness of the out-of-plane bands supported that the adsorbate should have a rathervertical structure. It was reported that the charge transfermechanism could also significantly contribute to the enhance-ment of the SERS intensities.33 Although the SERS spectralfeature could be roughly described by the EM mechanism, it isadmitted that the charge transfer (CT) mechanism may alsocontribute to the SERS intensities of several vibrational bandsof 4-EP on Au. The vibrational band most enhanced by thecharge transfer mechanism was estimated to be that of the m8a

mode.34 It is intriguing that the m8a mode was greatly enhancedfor the present SERS spectrum of 4-EP on Au. In order to

investigate the adsorption behaviors of the acetylene group ongold, a further examination is needed. Invoking the fact that thespectral features in the C[C stretching region of our SERSspectrum on gold nanoparticle surfaces are found to varysignificantly depending on pH condition, the multiple bandscould be ascribed to either several complexes or adsorption ata different crystal plane. On the other hand, it is noteworthythat other in-plane ring modes have not shown much differenceexcept the two peaks at 1620 and 1051 cm�1 for very low pHof ;0.8. This result suggests that the orientation of the phenylring would remain vertical at several binding schemes of theacetylene group.

As exhibited in Fig. 5, several vibrational features of 4-EPwere found to be affected as pH values varied. The ionizationstructures of pyridine and acetylene groups of 4-EP may havedifferent ionization states depending on pH conditions. As inthe previous literature14 for 4-mercaptopyridine, the ringstretching mode appeared at ;1620 cm�1 at acidic conditionsof pH , 6 due to the protonation of the pyridine ring. Thisspectral change occurred in an acidic medium as previouslyreported in the studies14,15 of 4-mercaptopyridine. It isnoteworthy that the m(C[C) stretching peaks were found tovary significantly under alkaline conditions in particular.

m(C[C) Stretching Bands. The m(C[C) stretching peakson Au showed multiple structures upon adsorption. As shownin Fig. 5, the m(C[C) bands changed quite significantly witha variation of pH under highly alkaline conditions (pH 12;13).A more magnified view for the C[C stretching region isexhibited in Fig. 6. Our recent SERS study of ethynylbenzeneon Au nanoparticle surfaces has shown that the m(C[C) bandsshould change significantly upon addition of NaBH4, KCl, andKBr into the sol medium.25 When the BH4

� ions are present,the band at ;1960 cm�1 became considerably weaker than thatat ;2015 cm�1. The m(C[C) band intensities of 4-EP at;2080 cm�1 were found to increase, whereas the band at;2010 cm�1 became quite weakened as the sol medium turned

FIG. 5. SERS spectra of 4-EP on gold nanoparticle surfaces at pH of (a) 0.8,(b) 1.8, (c) 4.4, (d) 6.7, (e) 13.1, and (f) 13.8. (g) Ordinary Raman spectrum of4-EP.

TABLE I. Spectral data and vibrational assignment of 4-EP.a

Ordinary RamanAu SERS (;10�4 M,

at pH ;6.7) Assignmentb

In-plane

1589 1589 8a (A1)1479 19a (A1)

1200 1194 9a (A1)1120 18a (A1)1010 12 (A1)1006 18b (B2)

991 995 1 (A1)670 665 6b (B2)466 6a (A1)

Out-of-plane

774 819 11 (B1)509 488w 16b (B1)

Anchoring group

2096 2085 m(C[C)2010

547 549w b(C–C[C)469 a(C–C–C)

344 b and c(C–CCH)

a w (weak).b Based on Ref. 14 in Wilson notation with symmetries based on C2m point

group for the ring mode and Ref. 25 for the acetylene mode. The symmetry inthe parentheses corresponds to the C2m point group.

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more basic. 4-EP may have different binding schemes such asvinylidene intermediates or a flat orientation on Au as proposedin the previous report.21 It is not absolutely certain, however,whether the intermediates and orientational change may resultin the m(C[C) band variance at different pH values. Regardingthe reproducibility of the measurements, it has to be admittedthat our spectra appeared to be affected by the conditions of thesol medium. The m(C[C) stretching bands at ;2010 cm�1

were not found to decrease drastically, but weakly even athighly alkaline pH values for several Au sols (initial pH ;5.6)that we have tested. It is evident, however, that the spectralfeatures of the m(C[C) stretching bands are changed in thehighly alkaline region and hold potential as a pH sensor.

Surface-Enhanced Raman Scattering Titration of 4-Ethynylpyridine for pH Sensors. By taking intensity ratios ofI1590 with respect to I1620 and I2080 with respect to I2010,sigmoidal calibration curves can be obtained as shown in Figs.7a and 7b, respectively. The pH calibration from SERS titrationof 4-EP is expected to have advantages in terms of a higheralkaline detection limit and more precise measurements, ifcompared with the indigo carmine as previously shown in Fig. 2.

The pK1/2 value was determined to be around 13 from thecalibration curve. By the Gouy–Chapman–Stern theory,12,13

the local pHbulk and pHsurface can be given by pHsurface¼ pHbulk

þ ew/2.3kT, where e is the electronic charge, k is the Boltzmannconstant, T is the temperature, and w is the potential at the

surface. The surface potential was found at around �50 mVfrom the zeta potential measurements.8 pHsurface is thusestimated to be around 14. The pKa value21 of the acetylenegroup in the neutral phenyl acetylene is known to around 30. Itis not absolutely certain whether such high pKa values could bereduced on metal surfaces.14 Pyridine ring groups as well as theacetylene group should also affect the spectral changes in 4-EPunder alkaline conditions. Our results indicate that Aunanoparticles should act as a pH indicator at around pH12;14 in the alkaline region. Also, a highly basic region ofaround pH 13 could be further checked by means of SERStitration of 4-EP by monitoring the m(C[C) bands. Ourmethods should be potentially useful to check highly alkalineconditions of pH . 11.

CONCLUSION

Gold nanoparticles on the basis of SERS spectroscopic toolscan be potentially utilized as a pH indicator in the range of pH12;14 under highly alkaline conditions. Citrate-reduced goldnanoparticles with ;15 nm diameter, ;9 3 10�9 Mconcentration, and ;0.02 M ionic strength showed a distinct

FIG. 6. C[C stretching region in SERS spectra of 4-EP on gold nanoparticlesurfaces at pH of (a) 6.7, (b) 7.2, (c) 11.7, (d) 12.7, (e) 13.0, (f) 13.1, (g) 13.2,(h) 13.3, (i) 13.6, (j) 13.7, and (k) 13.8.

FIG. 7. pH titration calibration curves with the standard deviations of fivemeasurements for 4-EP by taking a ratio of (a) the two m8a bands at 1590 and1620 cm�1 and (b) the two C[C bands 2080 and 2010 cm�1.

APPLIED SPECTROSCOPY 851

color change at a high alkaline pH . 11.5. The relativeintensities of the m(C[C)bound band at ;2080 cm�1 for SERSspectra of 4-EP on gold nanoparticles were found to vary underhighly alkaline conditions of around pH 13. As the pH valuesincreased, the m(C[C)bound band at ;2080 cm�1 becameprominent over that at ;2010 cm�1 on Au colloidal surfaces.Using SERS enhancements and conspicuous spectral changes,SAMs of 4-EP assembled on Au nanoparticles holds potentialas a pH sensor for highly sensitive detection of the hydroxideOH� concentration at around pH 13 in an aqueous solution.

ACKNOWLEDGMENTS

S.-W.J. would like to thank Bum Keun Yoo and In-Hyun Kim for helpingwith experiments, Prof. Kwan Kim for the introduction to SERS research, andProf. Seong Keun Kim for helpful considerations. This work was supported bythe Soongsil University Research Fund.

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