Synthesis of Silver Nanoparticles with ControllableSurface Charge and Their Application toSurface-Enhanced Raman Scattering

Ramon A. Alvarez-Puebla and Ricardo F. Aroca*

Materials and Surface Science Group, Faculty of Sciences, University of Windsor, Windsor, ON, Canada, N9B 3P4

The objective of the present work was to explore newmethods of synthesis of silver nanocolloids using aminoacids as reducing agents. The goal of the study was tofabricate nanostructures with controllable surface charge(zeta potential) that may allow optimizing the adsorptionof target molecules for ultrasensitive analysis using surface-enhanced Raman scattering (SERS). The average SERSproperties of these colloids are tested with two organicanalytes and compared with those obtained by using themost commonly used citrate Ag sols.

Surface-enhanced Raman scattering (SERS) is a powerfulanalytical technique for ultrasensitive chemical or biochemicalanalysis.1 Since the first reported SERS on silver and gold colloidalsolutions in 1979,2 metal colloids have become one of the mostcommonly used nanostructures for SERS. The localized surfaceplasmon resonances (LSPR)3 in colloidal metal particles haveserved as a testing ground for the most thorough of theoreticalmodeling and, with the achievement of the single moleculedetection (SMD),4-6 they have become central to single moleculeRaman spectroscopy. For analytical applications, it is importantto distinguish two types of SERS signals when using colloidalnanoparticles.7 First, the “average SERS” enhanced spectra,1 i.e.,the SERS spectrum of a given analyte obtained from an ensembleof colloidal particles and aggregates and characterized by a stableintensity pattern, with well defined and reproducible frequenciesand bandwidths. Second, SERS intensities obtained from silveror gold nanostructures sustaining a “hot spot” (large enhancementfactors), which permits the detection of a few molecules withfluctuating spectral charateristics.8,9 Average SERS is nowadays

a quantitative analytical tool,10 and today the work is turning tothe specifics of tuning the experimental conditions for a givenanalyte. For example, enhancement factors (EF) reported fororganic acids and alcohols are not competitive with those achievedwith thiols and amines.11 Of course, for a proper comparison, itis the first order of business to examine the correspondingscattering cross sections of the analytes. However, the adsorptionproperties of the analyte remain as a central issue, and theadsorption is partly controlled by the surface charge of thecolloidal metal particles. Recently, it was demonstrated that withvariation of the pH, one can gain control of the surface charge ofthe colloidal suspensions.12,13 Thereby, it is possible to maximizethe adsorption. Silver and gold colloids used in SERS have surfacecharge (i.e., zeta potential, �) varying between -60 mV at pH )10 and 5 mV at pH ) 2.12,13 In the present work, new methods ofsynthesis of silver nanocolloids are carried out using amino acidsas reducing agents. The colloidal nanostructures show optimaloptical properties for SERS and a controllable � ranging from -60 to 30 mV. Amino acids are also retained at the surface,stabilizing the colloids as predicted by the double diffuse layertheory.14 SERS properties of these colloids are tested with twodifferent analytes at different conditions (average and hot-spotSERS), and results are compared with those obtained by usingthe most commonly used citrate Ag sols.

EXPERIMENTAL METHODSAll the chemicals were purchased from Aldrich and used

without further purification. Reducing agent solutions of 1%(w/v) were prepared with glycine (Gly), D-cysteine, (Cys), D-lysine(Lys), and citrate. These solutions were adjusted at pH 9 to ensurethat they were in their carboxylate forms. Silver colloids were pre-pared by adding 6 mL of the reducing agent (2 mL in the case ofcitrate) to 100 mL of boiling solution of Ag+ 10-3 M undervigorous stirring. The solution was kept boiling during 30 min.After that, aliquots of the colloidal dispersion were adjusted atthe desired pH (1-11) by adding HNO3 or NaOH 1 M(Metrohm Titrino 702SM autoburet). Colloids were then

* Author to whom correspondence should be addressed. E-mail: raroca1@cogeco.ca. Fax: 1-519-973-7098. Phone: 1-519-253-3000 (ext. 3528).

(1) Aroca, R. Surface-Enhanced Vibrational Spectroscopy; John Wiley & Sons:Chichester, U.K., 2006.

(2) Creighton, J. A.; Blatchford, C. G.; Albrecht, M. G. J. Chem. Soc., FaradayTrans. 2 1979, 75, 790–798.

(3) Anker, J. N.; Hall, W. P.; Lyandres, O.; Shah, N. C.; Zhao, J.; Van Duyne,R. P. Nat. Mater. 2008, 7, 442–453.

(4) Kneipp, K.; Wang, Y.; Kneip, H.; Perelman, L. T.; Itzkan, I.; Dasari, R. R.;Feld, M. Phys. Rev. Lett. 1997, 78, 1667–1670.

(5) Nie, S.; Emory, S. R. Science 1997, 275, 1102–1106.(6) Constantino, C. J. L.; Lemma, T.; Antunes, P. A.; Aroca, R. Anal. Chem.

2001, 73, 3674–3678.(7) Pieczonka, N. P. W.; Aroca, R. F. ChemPhysChem 2005, 6, 2473–2484.(8) Le Ru, E. C.; Blackie, E.; Meyer, M.; Etchegoin, P. G. J. Phys. Chem. C

2007, 111, 13794–13803.(9) Etchegoin, P. G.; Meyer, M.; Blackie, E.; Le Ru, E. C. Anal. Chem. 2007,

79, 8411–8415.

(10) Bell, S. E. J.; Sirimuthu, N. M. S. Chem. Soc. Rev. 2008, 37, 1012–1024.(11) Alvarez-Puebla, R. A.; Contreras-Caceres, R.; Pastoriza-Santos, I.; Perez-

Juste, J.; Liz-Marzan, L. M. Angew. Chem., Int. Ed. 2009, 48, 138–143.(12) Alvarez-Puebla, R. A.; Arceo, E.; Goulet, P. J. G.; Garrido, J. J.; Aroca, R. F.

J. Phys. Chem. B 2005, 109, 3787–3792.(13) Aroca, R. F.; Alvarez-Puebla, R. A.; Pieczonka, N.; Sanchez-Cortez, S.; Garcia-

Ramos, J. V. Adv. Colloid Interface Sci. 2005, 116, 45–61.(14) Shaw, D. J. Introduction to Colloid and Surface Chemistry; Butterworths:

London, 1980.

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characterized by UV-vis spectroscopy (Cary 50, Varian), fieldemission scanning electron microscopy (FE-SEM, HitachiS4800), and photon correlation spectroscopy (Zetasizer 3000HS,Malvern Instruments) and tested for SERS (Renishaw Invia)with two different analytes, 2-naphthoic (2-NA) acid and2-naphthalenethiol (2-NT).

For the study of � and size variation as a function of pH,colloidal suspensions were diluted 10 times. Colloidal size wasdetermined by introducing 5 mL of the colloidal suspension intoa 1 cm path length-quartz cell and employing dynamic lightscattering at an angle of 90°. � values were determined by injecting10 mL aliquots of the colloidal solutions into a photon correlationspectrometer with an applied voltage of 100 V and a 5 cm quartzcell. To ensure reproducibility, two subsamples were studied, andthree measurements were coadded for each one.

Sample for “average SERS” were prepared by adding aliquotsof 10 µL of analyte solution (10-4 M) per milliliter of colloidalsuspension. After 2 h, allowing for the thermodynamic equi-librium to be reached, “average SERS” was directly recordedfrom these suspensions. To acquire the “hot-spot SERS”spectra, solid thin films of the colloidal suspensions wereprepared by casting and air-drying 50 µL of them on a glassslide. Then 10 µL of analyte solution (10-4 M) were spread onthem.

The inelastic scattered radiation was collected with a RenishawInvia system, equipped with Peltier charge-coupled device (CCD)detectors and a Leica microscope. The spectrograph has 1800g/mm gratings with additional band-pass filter optics. Sampleswere excited with three different laser lines 514 (Ar ion) 633(He-Ne), and 785 nm (diode). Spectra were collected in Renishawextended mode with accumulation times of 10 s. Power at thesample was varied between 1 µW and 5 mW. For “average SERS”a macrosampling 90° objective adaptor was used. For hot-spotSERS, the laser line was focused onto the sample in backscatteringgeometry using a 50× objective (NA 0.75) providing scatteringareas of ∼1 µm2.

RESULTS AND DISCUSSIONCharacterization of the Colloidal Suspensions. The maxima

of localized surface plasmon absorption resonances for silvernanoparticles (AgNP) prepared with the different reducing agentsas a function of pH were observed at 432, 421 and 422, for citrate,glycine, and lysine, respectively (Figure S1 in the SupportingInformation). AgNP reduced with citrate yields mainly sphericalcolloids of around 30-50 nm (Figure 1A) stable in suspensionsin a range of pH from 3 to 11. Nanoparticles produced using Glyas reducing agents are stable between pH 3-11. Ag-Gly are mainlyspherical-like as prepared (Figure 2), with sizes ranging from 40to 80 nm and with a small presence of nanorods. Notably, as thepH decreases to 7, LSPR of the suspension completely changes,showing a new absorption 546 nm and a blue shift of the mainband at 412 nm. These plasmonic absorptions are consistent withthe formation of nanorods characterized by transversal (412 nm)and longitudinal (546 nm) absorptions,15,16 which is also supportedby SEM (Figure 2) and hydrodynamic size measurements (Figure

3). Mechanism for the formation of rods from spherical NP atneutral and acidic pH is very likely due to self-assembly pro-cesses.17 Gly presents its isoelectric point (pI) at 5.97 (Gly, pKa1

) 2.34, and pKa2 ) 9.60).18 Assuming that retention of Gly onthe Ag colloidal surfaces is random, either through the aminoor the carboxylic groups, the net result is colloidal NPs with amix of positive (amino) and negative (carboxylic) surfacegroups between pH 3 and 9, being the optimal ratio chargesfor electrostatic attraction interaction between pH 5 and 7. Onthe other hand, it has been suggested that atoms on differentcrystallographic facets might have different interaction strengthswith the capping reagent, leading to the anisotropic growth of

(15) Link, S.; Mohamed, M. B.; El-Sayed, M. A. J. Phys. Chem. B 1999, 103,3073–3077.

(16) Sun, Y.; Yin, Y.; Mayers, B. T.; Herricks, T.; Xia, Y. Chem. Mater. 2002,14, 4736–4745.

(17) Xia, Y.; Yang, P.; Sun, Y.; Wu, Y.; Mayers, B.; Gates, B.; Yin, Y.; Kim, F.;Yan, H. Adv. Mater. 2003, 15, 353–389.

(18) Nelson, D. L.; Cox, M. M. Lehninger Principles of Biochemistry; W. H.Freeman and Company: New York, 2008.

Figure 1. SEM images for silver nanoparticles prepared with (A)citrate, (B) lysine, and (C) cysteine at pH 6.

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a solid material.19 For silver, {100} facets interact more stronglywith the capping agent than the {111} facets.20 In other words,preferential capping on the {100} facets leads to a one-directional self-assembly (through the {111} facets) of thespherical nanoparticles yielding nanorods.21

The lysine reduction of silver gives rise to colloidal suspensionsstable in a pH interval from 3 to 12 and partially stable at pH 2.

SEM shows spherical-like nanoparticles with sizes between 40 and80 nm (Figure 1B). In this case, the decrease in the pH valuedoes not lead to other particle shapes probably due to the presenceof an extra amino group in the lateral chain of the amino acid. Inthe case of cysteine, no plasmonic bands were observed at anypH (Figure S1 in the Supporting Information). From pH 12 to 8,the solution remained colorless. At pH 7 and below, a whiteprecipitate was formed. SEM supported the reduction of the silverto metal; however, in this case, particles ranging from severalhundred nanometers to micrometer size were produced (Figure1C), too large to support radiative localized surface plasmons.

The variation of � with pH for Ag-Citrate, Ag-Gly, and Ag-Lysis shown in Figure 4. In all the cases, the � decreases with pH.However, while Ag-citrate colloids have negative � in all the pHrange, those prepared with Gly or Lys give rise to positive valueswith isoelectric points at pH at 2.23 and 4.44 and maximum valuesat 15.4 and 27.7 mV for Gly and Lys, respectively. Colloidsprepared with amino acids have comparable stability to aggrega-tion and flocculation processes at alkaline pHs to citrate Agcolloids. At neutral and acidic pHs, the stability of Ag-Gly andespecially Ag-Lys colloids are even better due to the extrastabilization provided by the amino groups.

(19) Ahmadi, T. S.; Wang, Z. L.; Green, T. C.; Henglein, A.; El-Sayed, M. A.Science 1996, 272, 1924–1925.

(20) Sun, Y.; Mayers, B.; Herricks, T.; Xia, Y. Nano Lett. 2003, 955–960.(21) Wiley, B.; Sun, Y.; Xia, Y. Acc. Chem. Res. 2007, 40, 1067–1076.

Figure 2. Surface plasmon and SEM images for silver nanoparticles prepared with glycine at pH 6 and 8.

Figure 3. Hydrodynamic size of silver colloids prepared with citrate,glycine, and lysine as a function of pH, before and after the additionof the analytes.

Figure 4. � of silver colloids prepared with citrate, glycine, and lysineas a function of pH.

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The applicability of a colloidal suspension as an opticalenhancer for SERS strongly relies on the fact that the reducingagent does not have a large Raman cross-section in order to avoidstrong background signals coming from it. Figure S2 in theSupporting Information) shows the Raman spectra of the 1 Maqueous solution of Gly and Lys and the “average” and “hot-spot”SERS spectra of the Ag-Gly and Ag-Lys colloids and theirassignment. Notably, the background SERS spectrum is veryweak, consistent with the small cross-section expected for a smallaliphatic molecule. However, the SERS background spectrum isclearly seen in the “hot-spot” experiment, very likely because ofthe increase of the enhancement factor due to the coupling ofthe electromagnetic fields of aggregated nanoparticles.22

Average SERS on the Colloidal Suspensions As a Functionof pH. For the study of the optical enhancing properties of theprepared colloids under different conditions, two structurallysimilar analytes, but with different pH properties were chosen,2-naphtahalenethiol (2NT), and 2-naphthoic acid (2NA). Ramanof the powder and SERS spectra for these analytes are presentedin Figure 5. The characteristic SERS spectra of 2NT are dominatedby ring stretching (1621, 1378 cm-1), C-H bending (1080 cm-1),ring deformation, and C-S stretching (364 cm-1) vibrationalmodes.23 For 2NA, SERS spectra are characterized by the ringstretching at 1632 and at 1388 cm-1, CH bending at 1468 cm-1,ring breathing at 1018 cm-1, and C-H deformation at 770 cm-1.Notably, SERS spectra for both analytes fit band to band witheach other independent of the excitation laser line used forthe acquisition (514, 633, or 785 nm), consistent with theposition of their absorption bands, at 242 and 232 nm for 2NTand 2NA, respectively, far away from the excitation laser lines.

Average SERS Measurements. Figure 6 shows the “average”SERS spectra for both analytes on silver citrate, glycine, and lysinereduced colloids, as a function of pH excited with three laser lines(514, 633, or 785 nm). In citrate colloids, both analytes presentsimilar behavior. Both increase their intensity with a maximumat pH 4. Then the SERS intensity decays. To test for possibleaggregation in the colloidal systems as a function of pH, sizemeasurements with DLS were carried out after the addition ofthe analytes. Dynamic light scattering size measurements (Figure

3) show that all the colloids presented similar sizes before andafter the addition of the analyte in between pH 4 and 11. Therefore,no evidence of heavy aggregation in the suspensions between pH4 and 11 was found. That is also consistent with the role ofelectrostatic interactions24 between adsorbent (metal colloids) andadsorbate (analytes) in the measured intensity of the SERSsignal.12 In short, as the � of the colloidal surfaces increases (asthe pH decreases), the adsorption process of the analytes on thecolloids are favored. Thus, an increase in the SERS signal frompH 11 to 4 is likely due to a boost in adsorbed molecules. At pH3 and 2, the size of the remaining colloids noticeably increased(Figure 3). Adsorption of the analytes onto the colloidal surfacesat pH 3 and 2 lead to the aggregation and flocculation of thesystem25 with the consequent decrease in the concentration ofcolloids and the SERS intensity.

For Gly and Lys silver colloids, the behavior of the analytes isdifferent. 2NT shows an extraordinary increase in the SERSintensity from pH 11 to 5 on Ag-Gly and 11 to 4 on Ag-Lys (Figure6). Notably, DLS of the suspensions after the addition of 2NT showevidence of aggregation and flocculation at pH 4 for Ag-Gly and3 for Ag-Lys (Figure 3). The 2NA on Ag-Gly shows a continuousincrease until pH 3 and flocculation at pH 2 (Figure 3). On Ag-Lys, an increase in the SERS signal is extended to pH 3, becomingsmaller from this pH downward. 2NT is characterized by a pKa

of 4.22,26 and conditions around pH 3 with Ag-Gly and 4 withAg-Lys extremely favor the adsorption process due to a smallelectrostatic reduction or even attraction (colloids are slightlynegative or positively charged and acid-base equilibrium isaround the pKa).

The overall conclusion for average SERS experiments is thatsurface charge, controlled through the variation of the pH of the

(22) Etchegoin, P.; Cohen, L. F.; Hartigan, H.; Brown, R. J. C.; Milton, M. J. T.;Gallop, J. C. J. Chem. Phys. 2003, 119, 5281–5289.

(23) Alvarez-Puebla, R. A.; dos Santos, D. S., Jr.; Aroca, R. F. Analyst 2004,129, 1251–1256.

(24) Zhong, Z.; Patskovskyy, S.; Bouvrette, P.; Luong, J. H. T.; Gedanken, A. J.Phys. Chem. B 2004, 108, 4046–4052.

(25) Moskovits, M.; Vlckova, B. J. Phys. Chem. B 2005, 109, 14755–14758.(26) Underberg, W. J. M.; Schulman, S. G. Anal. Chim. Acta 1979, 105, 311–

317.

Figure 5. Raman and SERS (on Ag-Gly colloids suspension) spectrafor 2-naphthalenethiol (pH 5) and 2-naphthoic acid (pH 3).

Figure 6. Comparison of average SERS intensity for 2-naphthale-nethiol (1378 cm-1) and 2-naphthoic acid (1389 cm-1) as a functionof pH, using silver nanoparticles prepared with citrate, glycine, andlysine as optical enhancers.

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surrounding media, plays a key role in the SERS intensity of thestudied molecule. However, this is one of many factors such ascolloidal stability that increases the � (favoring the adsorptionprocess), whereas the adsorption process of a nonpolar analytemay dramatically decrease the stability of the colloids leading toaggregation and flocculation processes. Subsequently SERS signaldecreases because of the decrease in the colloidal concentrationon the suspension.

Hot-Spot SERS on Cast Films. To test for the influence ofsurface charge on “hot-spot SERS”, cast films with the differentcolloidal suspensions were prepared by casting and air-drying 50µL of each suspension at different pH (3-11) on a glass slide.Analytes were then deposited by casting 10 µL of a 10-4 Msolution, air-dried and surfaces studied with 514, 633 and 785nm laser lines (Figure 7). SERS intensity for 2NT shows acontinuous increase as the pH of the cast film decreases. In thecase of 2NA, the citrate colloids present similar behavior. However,silver colloids produced with amino acids still present a decreasein the intensity at pH 3. In this case, a selected volume with agiven concentration of analyte is added to the film. The evapora-tion of the solvent leads to an effective increase of the analyteconcentration promoting the adsorbent-adsorbate surface interac-tions. 2NA at pH 3 is an exception for both Ag colloidalnanoparticles formed with amino acid and is likely due to a highlyrepulsive interaction between the positively charged surface andthe protonated concentrated solution of the analyte which finallyyields to a partial surface nucleation process rather than thecomplete adsorption of the analyte.27

Second, it is known that surface charge of the initial solutionaffects the aggregation of the colloids.24,28,29 For nanoparticleswith high surface charge density, small clusters are preferentially

formed (Figure S3 in the Supporting Information), and as thenanoparticles surface charges decreases larger clusters are found,in full agreement with the diffusion-limited aggregation (DLA)mechanism.30 This, together with the fact that the formation ofcolloidal aggregates is essential in the generation of hot-spots,31-33

suggest that a systems where the aggregation process forformation of large clusters of nanoparticles is favored will verylikely yield a higher density of hot-spots. To explore this empiricalconcept, several clusters with different sizes were studied in oneof the cast films (Figure 8A). Figure 8C shows the dependenceof the of the SERS signal with the size of the cluster. Results showa sigmoidal trend of the intensity versus the size of the cluster.The first part of the curve is characterized by an exponentialincrease until diameters around 25 µm. From this point onward,intensity varies slightly and tends to be stabilized.

CONCLUSIONS

We demonstrate the advantages of the synthesis of silvernanoparticles using two amino acids, Gly and Lys as reducingagents. The silver colloidal nanostructures show a controllable �ranging from -60 to 30 mV. The average SERS obtained for twoorganic analytes was examined at different pH values, which arein close correspondence with variations in the surface charge. Insolution, controlling the surface charge can be correlated withthe degree of adsorption and with the average SERS signal. TheSERS spectra of cast colloids are also discussed. Here, there isevidence of the increase in SERS activity due to nanoparticleaggregation.

(27) Stumm, W. Chemistry of the Solid-Water Interface; Wiley: New York, 1992.(28) Stankiewicz, J.; Cabrerizo-Vilchez, M.; Hidalgo-Alvarez, R. Phys. Rev. E

1993, 47, 2663–2671.(29) Fernandez-Barbero, A.; Cabrerizo-Vılchez, M.; Martınez-Garcıa, R.; Hidalgo-

Alvarez, R. Phys. Rev. E 1996, 53, 4981–4989.

(30) Weitz, D. A.; Lin, M. Y. Phys. Rev. Lett. 1986, 57, 2037–2040.(31) Garcia-Vidal, F. J.; Pendry, J. B. Phys. Rev. Lett. 1996, 77, 1163–1166.(32) Gresillon, S.; Aigouy, L.; Boccara, A. C.; Rivoal, J. C.; Quelin, X.; Desmarest,

C.; Gadenne, P.; Shubin, V. A.; Sarychev, A. K.; Shalaev, V. M. Phys. Rev.Lett. 1999, 82, 4520–4523.

(33) Braun, G.; Lee, S. J.; Dante, M.; Nguyen, T.-Q.; Moskovits, M.; Reich, N.J. Am. Chem. Soc. 2007, 129, 6378–6379.

Figure 7. Comparison of hot-spot SERS intensity for 2-naphthale-nethiol (1378 cm-1) and 2-naphthoic acid (1389 cm-1) as a functionof pH, using silver nanoparticles prepared with citrate, glycine, andlysine as optical enhancers.

Figure 8. (A) Optical micrograph of cast silver colloids cast colloidfilm of silver nanoparticles prepared with lysine at pH 5 with2-naphthalenethiol 10-5 M. (B) SERS spectra of cast colloids. (C)Dependence of the SERS intensity (1378 cm-1) with the size of theaggregates. Excitation laser line: 785 nm.

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ACKNOWLEDGMENTFinancial assistance from the Natural Science and Engineering

Research Council of Canada (NSERC) is acknowledged.

SUPPORTING INFORMATION AVAILABLELocalized surface plasmons of the prepared colloids at

different pH; Raman and SERS spectra of Gly and Lys; andoptical and SEM micrographs of Ag-Lys cast films produced

at different pHs. This material is available free of charge viathe Internet at http://pubs.acs.org.

Received for review November 18, 2008. AcceptedJanuary 23, 2009.

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Fig S1A

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Fig S2

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Average SERS (LL: 633 nm)

SERS of the cast colloids (633 nm)

Raman shift/cm-1

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