ARTICLES

Surface-Enhanced Raman Scattering on Aggregates of Silver Nanoparticles withDefinite Size

Mustafa Culha,* Mehmet Kahraman, Nilgun Tokman, and Guler TurkogluYeditepe UniVersity, Faculty of Engineering and Architecture, Department of Genetics and Bioengineering,34755 Kayisdagi-Istanbul, Turkey

ReceiVed: NoVember 26, 2007; ReVised Manuscript ReceiVed: April 18, 2008

Aggregation behavior of colloidal silver nanoparticles for surface-enhanced Raman scattering (SERS) on ahydrophobic surface is investigated. A regular glass slide is used to deposit dicholoromethylsilane in order toinactivate the free hydroxyl groups and increase the hydrophobicity of the glass surface. A submicron volumeof colloidal suspension is spotted on the hydrophobic surface. During the evaporation of water from thedroplet, the nanoparticles start to form aggregates with a definite size and shape. Increasing colloidal suspensionconcentration by more than 4 times helped to complete the size of silver nanoparticles aggregates to about 1µm diameter. The SERS activity of aggregates was investigated using Rhodamine 6G as a probe. The aggregatesformed from higher colloidal suspension concentrations show a significant improvement in SERS activity.The SERS enhancement on the aggregates formed on hydrophobic surfaces is at least 1 order of magnitudegreater than the disordered aggregates prepared on the regular glass surface using the same colloidal suspension.The SERS enhancement factor for the aggregates and the limit of detection (LOD) for Rhodamine 6G areestimated as about 3 × 105 and 1.0 × 10-5, respectively. The percent coefficient of variance (CV) improvedabout 300% by increasing the colloidal suspension concentration 8-fold on the prepared aggregates. A minimum2-fold increase in SERS enhancement on the aggregates prepared from silver colloidal suspension containingNaCl and SDS is also observed.

Introduction

The self-assembly of nanoparticles into a variety of nano-structured morphologies is an important concept for thedevelopment of novel materials with unique properties, whichcan be applicable in nonlinear optic,1,2 catalysis,3,4 and SERSapplications.5 In preparation of materials using nanoparticlesas building blocks, it is possible to influence the properties ofthe materials by not only changing the properties of nanopar-ticles as building blocks but also tuning the interparticle distanceor the morphological structure of the entire system. There area number of techniques and approaches to construct orderedstructures for a variety of applications. The examples can begiven as photolithography,6,7 electron beam lithography,8 tem-plate assisted assembly9 and convective assembly.10

Because SERS is a vibrational spectroscopic technique andprovides rich chemical information about the molecule ormolecular structure under investigation, it has been used fornumerous applications ranging from detection of biomolecules11,12

and investigation of cellular processes in single living cells13

to explosives14–17 since its discovery.18–20 The simplicity ofsample preparation and competing sensitivity with fluorescenceunder certain experimental conditions are the other importantfactors for its popularity. Due to their easy preparation and highSERS enhancement, the colloidal gold and silver nanoparticlesand their aggregates were popularly used in early SERS

experiments.21–28 However, stability and reproducibility ofcolloidal suspensions as SERS substrate is limited. Therefore,new strategies and approaches such as evaporating metals ontoa support material such as glass or silica,29,30 self-assembly,28,31

deposition,32 and mirror reaction33 were explored. Althoughthese structures have significant advantages such as higherstability and reasonable reproducibility over colloidal suspen-sions, they do not have the advanced structures such as porosityand periodicity to improve the enhancement characteristics ofthe substrate.34,35 The more sophisticated 3D structures usingcolloidal nanoparticles were also reported with superior SERSenhancement.36,37 However, the difficulties generating 3Dstructures with uniform enhancement properties and complexityof preparation methods indicate that still some effort must bedevoted for the complete development of such substrates.

It is now better understood that it is necessary to generateoverlapping localized surface plasmons (LSPs) for optimumSERS enhancement.38,39 Thus, the aggregates of gold or silvernanoparticles are excellent structures for optimal SERS en-hancement, and the first single molecule detection was indeedperformed on aggregates of silver colloidal nanoparticles.40–44

In order for LSPs to overlap for intense enhancement, theinterparticle distance in an aggregate is thought to be smallerthan 2 nm, and it is difficult to construct such periodic structureswith current nanofabrication technology.45

The aggregation properties of nanoparticles are mostlyinfluenced by their surface charge properties, which are definedby the stabilizing agents used in the synthesis procedure or the

* Phone: +90 216 578 15 87. Fax: +90 216 578 00 40. E-mail:mculha@yeditepe.edu.tr.

J. Phys. Chem. C 2008, 112, 10338–1034310338

10.1021/jp711177z CCC: $40.75 2008 American Chemical SocietyPublished on Web 06/21/2008

other additives added after the synthesis of colloidal nanopar-ticles such as halides.46–50 With a simple addition of an additiveinto the colloidal suspension, there is almost no control overthe aggregate size and aggregation degree. Therefore, otherapproaches to construct SERS substrates using colloidal goldor silver nanoparticles as building blocks are also investigatedand reported to have an excellent SERS enhancement asmentioned earlier.36,37

The formation of 3D structures at the air-water interfacewas reported.51 Because air is hydrophobic in nature, thenanoparticles formed well-defined structures due to the Cou-lombic, hydrophobic, and van der Waals interactions at thehydrophobic/hydrophilic, air/suspension interface.51 In thisstudy, we prepared reproducible and well-defined shape and size3D silver nanoparticle aggregates by sandwiching the silvercolloidal suspension between two hydrophobic interfaces, airand a hydrophobic surface prepared by inactivating the freehydroxyl groups on regular glass surface. We also investigatedtheir use as SERS substrate. Rhodamine 6G was used as probemolecule to study the generated structures. The presence of ionicspecies in the colloidal suspension influences the surface chargeproperties of the colloidal nanoparticles and their aggregationproperties. In addition, due to the known effect of additives suchas NaCl and SDS (sodium dodecyl sulfate) on SERS enhance-ment, we also investigated their influence on aggregationproperties of silver colloidal nanoparticles and the SERSperformance. SEM was used to characterize the aggregates.

Materials and Methods

All chemicals were purchased from Sigma-Aldrich (St. Louis,MO) and used without further purification.

Preparation of Silver Nanoparticles. Silver colloid wasprepared using the Lee method.27 Briefly; 90 mg of AgNO3 wasdissolved in 500 mL of water. This solution was heated to boil.A 10 mL aliquot of 1% sodium citrate was added into thesolution and kept boiling until the volume reached half of theinitial volume. The maximum of its absorption was recorded at420 nm. Characterization with SEM revealed that several sizesand shapes of silver nanoparticles with a mean diameter of 50nm had been formed.

Preparation of SERS Substrate. The glass slides were leftovernight in the chromic acid solution and washed with distilledwater by being submerged several times. Silver colloidalsuspension was concentrated to 4, 8, 16, and 32 times bycentrifugation at 5500 rpm for 30 min. The two separate 40mL portions of the silver colloidal suspensions were centrifugedat 5500 rpm for 30 min, and 30 and 35 mL of the supernatantfrom the centrifuged suspensions were discarded to obtain 4×and 8×, respectively. For the concentrations of 16× and 32×,two separate 32 mL colloidal suspension portions were centri-fuged at 5500 rpm for 30 min, and 30 and 31 mL of thesupernatant were discarded. A cleaned glass slide was used toprepare the hydrophobic surface by exposing the glass slide todichloromethylsilane in a closed beaker. The silver colloidalsolutions containing NaCl and SDS were prepared by additionof the proper amount of each additive to the concentrated silvercolloidal suspension. The concentration is confirmed as 0.001M. A 0.5 µL of colloidal suspension was spotted onto the glassslides and dried at ambient temperature.

Raman Instrumentation. All spectra were obtained by usinga Renishaw in Viva Reflex Raman Microscopy System. Thesystem is automatically calibrated against silicon wafer peak at520 cm-1. A diode laser at 830 nm and 50× (NA: 0.75)objective were used for all experiments. All spectra were taken

at the same power of 0.3 mW except the enhancement factordetermination and LOD experiment, which were performed at3 mW.

Scanning Electron Microscopy. The SEM images weretaken using Carl Ziess Evo 40 at high vacuum and EHT ) 10kV.

Results and Discussions

Characterization of Silver Nanoparticle Aggregates. Figure1 shows the SEM image of a spot of colloidal suspension at aconcentration of 32× on plain glass surface.The Lee methodgenerates silver nanoparticles with several sizes ranging from30 to 200 nm with an average size of 50 nm. A few rod-shapedsilver nanoparticles are also present along with spherical ones.The surface charge of silver nanoparticles prepared with citratereduction is negative as synthesized,51 and a double layer (�potential) is present on the surface of each silver nanoparticle.This double charge layer is solvated by water molecules incolloidal suspension. The regular glass surface is relativelyhydrophilic due to the presence of free hydroxyl groups on thesurface. Since water molecules surround the charged nanopar-ticles, the repulsion and attraction forces are diminished. Hence,on a bare glass surface, the interaction tendency of the silvernanoparticles with the surface increases. With the help of thewater molecules, which are on the move toward the edges ofthe droplet due to the capillary effect, the colloidal particleseasily spread on the glass surface forming arbitrary clusters.Therefore, the nanoparticles extend on the glass surface withsome degree of aggregation generating random aggregates withseveral sizes. The inset in Figure 1 clearly shows the looselybound colloidal silver nanoparticles in the aggregates, whichare possible places for “hot spots”.

Formation of Silver Nanoparticle Aggregates on Hydro-phobic Surfaces. In spite of all the limitations, the colloidalnanoparticles are still excellent constructing building blocks toprepare SERS substrates with improved enhancement. Thus, weare motivated to prepare 3D structures using silver colloidalsuspension through self-assembly of colloidal nanoparticles.Since air provides an excellent hydrophobic environment anddepositing dicholoromethylsilane onto the surface of a cleanedglass slide inactivates the free silanol groups, the colloidalsuspension droplet is sandwiched in between two hydrophobicenvironments. Figure 2 compares the general view of a 0.5 µLsilver colloidal suspension spotted on a hydrophobic surface(A) and a regular glass slide surface (B). As seen, the spot sizeis almost perfectly round on the hydrophobic surface. With thisfeature, it allows us to locate the multiple spots on the glasssurface without mixing as an additional advantage for highthroughput applications.

Figure 3 shows the formation of the aggregates of colloidalsilver nanoparticles as the concentration of the colloidal sus-pension is increased. When a small volume of colloidalsuspension is left on a hydrophobic surface, the nanoparticlessolvated with water molecules try to avoid the hydrophobicsurfaces (air and hydrophobic surface) as bulk water evaporatesfrom the suspension. During this process, the nanoparticles areforced to adhere together to form aggregates. However, theaggregate size remains constant after a certain size. At lowercolloidal suspension concentrations, for example, at a concentra-tion as it was synthesized, the aggregation was incomplete dueto the low number of the nanoparticles in the spotted volume.This incomplete aggregation even at 4× is visible as seen inFigure 3A. At concentrations higher than 8×, the aggregationwas complete, and increasing the concentration only increased

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the number of aggregates, which is seen in Figure 3B (8×), C(16×), and D (32×). One important point is that the size of theaggregates is not significantly altered as the concentration isincreased. This could be due to the necessity for the particlesto remain at a certain surface area/volume ratio under the exertedpressure on the entities in the droplet. During the evaporationof water from the colloidal suspension, some of the nanoparticlesare trapped at the air-suspension interface at the circular edgesof the spotted volume of colloidal suspension and accumulatedwith help of the capillary effect as well. Figure 3E is an overallview of such an edge. Increasing the colloidal suspensionconcentration further to 32× improved the surface coverage ofaggregates on the spotted area. The average size of silvernanoparticle aggregates is measured as 0.975 ( 0.122 µm fromthe SEM images (Figure 3F).

Previously, the SERS performance of the aggregates isreported to be independent of the size, and it is satisfactory ifthe size in the range 100 nm-1.0 µm.13 However, a more recentstudy shows that aggregation morphology has a critical respon-sibility on the SERS enhancement as well.52 In order to measureperformance of the aggregates, 1.0 mM Rhodamine 6G wasspotted onto the aggregates. Since the aggregate size is about1.0 µm, this almost corresponds to the size of the laser spotused for SERS experiments in this study (a 50× objective withNA of 0.75). In addition, the aggregates are easily visible undera 50× microscope objective due to their surface plasmons ofsilver nanoparticles, and the laser spot can easily be directedon one isolated aggregate (see Figure 5). This allowed us tostudy the SERS performance of aggregates and whether theSERS performance changes from aggregate to aggregate. It

Figure 1. SEM image of silver nanoparticle aggregates from the concentrated silver colloidal suspension on bare glass substrate.

Figure 2. Comparison of SEM images of spot size and shape prepared on hydrophobic (A) and regular (B) glass slide surfaces.

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should be noted that although the isolated individual aggregatesare targeted and it is theoretically possible to locate the laserlight on one individual aggregate, there could be some contribu-tions from the other aggregates closely located to the targetaggregate and from the intersection of aggregates in theaggregate clusters (see the inset of Figure 5).

Figure 4 shows the improvements in SERS enhancement withincreasing colloidal suspension concentration on the hydrophilicand hydrophobic surfaces based on the area of the peak at 1514cm-1. The interparticle distances are critical and are found tobe in the 1-4 nm range for better SERS enhancement.53–56 Asthe concentration of colloidal suspension was increased on bothsurfaces, the SERS enhancement performance was improved.This improvement was much more significant on the aggregatesformed on the hydrophobic surfaces. A reasonable improvementin the SERS enhancement obtained from the aggregates on aregular glass slide with the increased colloidal suspensionconcentration is seen in Figure 4. This is possibly due to theimproved aggregation and, as a result, diminished interparticle

distances with increased colloidal suspension concentration. Theaggregation of silver colloidal particles on the hydrophobicsurfaces appears to be incomplete at the low colloidal suspensionconcentrations (2×, 4×, and 8×). As the concentration ofcolloidal suspension is increased (16× and 32×), the formationof aggregates seems to be completed. As mentioned earlier, theincreased size of aggregates and the aggregates closely locatedto the target aggregate at higher concentration on the hydro-phobic surfaces may also contribute to the SERS enhancementdepending on the location of the laser spot and its real size.The comparison of the SERS spectra obtained from theaggregates on the hydrophobic surface and regular glass slidesurfaces revealed that the SERS enhancement from the ag-gregates on hydrophobic surface is greater than 1 order ofmagnitude at all colloidal concentrations. This can be explainedagain with the changes in the interparticle distances in theaggregates as the spherical aggregates formed, which is muchgreater in the loosely formed aggregate clusters on the regularglass surfaces while it is much smaller in the aggregates formedon the hydrophobic surfaces. This can easily be observed bythe comparison of the SEM images in Figures 1 and 3F. Inaddition, there could be some contribution from surfacemorphology changes of the aggregates as stated in a more recentstudy.52 Overall, this is an excellent opportunity to improveenhancement performance of silver colloidal nanoparticles bysimply spotting on a hydrophobic surface. The reproducibilityof SERS spectra improved significantly as the concentration ofthe colloidal suspension increased, and the best reproducibilitywas obtained on aggregates prepared with 32× colloidalsuspension. The CV (percent coefficient of variance) wasdropped 23.49 to 7.63 by increasing the concentration from 4×to 32×, which corresponds to more than 300% improvementbased on the calculation using the intensity of the peak at 1365cm-1. Figure 5 shows the reproducibility of Rhodamine 6G onaggregates prepared from 32× colloidal suspension. Each of

Figure 3. SEM images of aggregated silver nanoparticles on hydrophobic surface for colloidal silver concentrations of 4× (A), 8× (B), and 16×(C), 32× (D). Parts E and F show the view of the spot at the edge and the closer view of aggregates, respectively.

Figure 4. The improvement in SERS enhancement on the hydrophilicand hydrophobic surfaces as the concentration of the colloidal suspen-sion increases. The peak area at 1514 cm-1 was chosen for thecalculations.

Aggregates of Ag Nanoparticles with Definite Size J. Phys. Chem. C, Vol. 112, No. 28, 2008 10341

the 10 spectra in Figure 5 was collected from an isolatedaggregate. The white light image of the aggregates is seen onthe inset.

The SERS enhancement factor for the peak at 1514 cm-1 ofRhodamine 6G was estimated to be ∼3 × 105. The enhancementfactor was calculated by using the following formula, ISERS/IR

× CR/CSERS. By using 0.1 M Rhodamine 6G, a bulk Ramanspectrum was obtained and compared to the spectrum obtainedusing 1.0 × 10-5 M Rhodamine 6G. The ratio of the intensityof the peak at 1514 cm-1 was ∼30, and the concentration ratiowas calculated as 1.0 × 104. The diameter of the laser spotimpinging on the sample was assumed to be 1.0 µm.

Influence of Surface Charge Properties on Silver Nano-particle Aggregation. The presence of the charged species inthe colloidal solution can influence the SERS performance byinfluencing the aggregation properties of colloidal nanoparticlesdue to the strong relationship between aggregation and surfacecharge properties of nanoparticles and the absorption of theanalyte onto the surface of the nanoparticles. Anions, especiallyhalides, have an impact on SERS activity of silver or goldcolloidal nanoparticles by stabilizing nanoparticles, increasingactive sites, and enhancing molecular adsorption.46,49,57,58 Theaddition of NaCl and SDS into the colloidal suspension beforespotting on the hydrophobic surface increased the SERSperformance of the aggregates. The effect of NaCl is explainedwith increasing the active sites on the silver nanoparticles. TheSDS molecule has a hydrophobic tail and negative headgroup,which may have repulsive forces with negatively charged silver

nanoparticles while its hydrophobic tail avoids the water. Theseforces may orient the hydrophilic head groups away from thesilver nanoparticle surfaces and the hydrophobic tail toward tothe surfaces. As a result, the interparticle distance in theaggregates becomes smaller than the packing of nanoparticleswithout SDS. Besides, as mentioned earlier, the surfacemorphology of the aggregates may contribute to the enhance-ment in both cases. Mixing the analytes with colloidal suspen-sion may induce the aggregation as it is in the case ofRhodamine 6G and may cause poor SERS performance. Thus,Rhodamine 6G solution was spotted on top of the preparedaggregates not mixed with the colloidal suspension solution.Figure 6 shows comparison of the peak area at 1514 cm-1 ofRhodamine 6G spectra obtained from the spots on the hydro-phobic surface. The intensity of peaks on Rhodamine 6G SERSspectra improved at a minimum 2-fold by addition of SDS andNaCl into the colloidal suspension compared to plain colloidalsuspension at the same concentration. At this point, it is notplausible to make a prediction about the reason for theenhancement, but we believe it could be due to a change ininterparticle distance, enhanced analyte adsorption, and apositive change in the surface morphology of the aggregates.We finally attempted to predict the LOD (limit of detection) ofRhodamine 6G on the aggregates prepared on the hydrophobicsurfaces. As seen in Figure 7, spectral features with 1.0 × 10-5

M are easily visible.

Figure 5. Reproducibility of SERS spectra of Rhodamine 6G on silver nanoparticle aggregates prepared from 32× colloidal suspension on thehydrophobic surface.

Figure 6. Influence of SDS and NaCl on SERS enhancement of silveraggregates. The peak area at 1514 cm-1 was chosen for the calculations.

Figure 7. SERS spectra of several concentrations of Rhodamine 6Gon aggregates prepared on hydrophobic surfaces.

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Conclusions

A simple method for the preparation of silver nanoparticleaggregates with an average of 1.0 µm size was developed. Thismethod uses a concentrated silver colloidal suspension and ahydrophobic surface. The improvement in SERS enhancementwas about 1 order of magnitude compared to the aggregatesprepared on a regular glass surface. The enhancement factorup to 3 × 105 was easily achieved. The addition of NaCl andSDS resulted in an additional minimum 2-fold increase in SERSenhancement. The concentration study with Rhodamine 6Grevealed that the minimum concentration, which can be detectedon these aggregates, was 1.0 × 10-5 M. The existence of “hotspots” on the aggregates was not observed. However, thereliability and reproducibility of the SERS substrates are moreimportant for routine analytical applications.

Acknowledgment. The financial support from Yeditepe Uni-versity Research Fund and TUBITAK is greatly acknowledged.

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