A Facile Deposition of Silver onto the Inner Surface of a GlassCapillary Tube for Micro-Surface-Enhanced Raman ScatteringMeasurements

HYOUNG KUN PARK, HYANG BONG LEE, and KWAN KIM*Laboratory of Intelligent Interfaces, Department of Chemistry, Seoul National University, Seoul 151–742, Korea

Silver can be deposited very efficiently onto glass substrates using only

ethanolic solutions of AgNO3 and butylamine. This paper reports that the

inner surface of a glass capillary can also be coated evenly with silver by

shaking it after soaking in ethanolic solutions of AgNO3 and butylamine;

the silver deposited outside the capillary can be easily wiped off with

cotton wool before drying. The grain size of the silver deposited onto the

inner surface can be readily controlled within the range from 20 to 100 nm

by varying the relative molar ratio of butylamine and AgNO3 used as

reactants. Due to its nanoaggregated structure, the Ag coated capillary is

a very efficient surface-enhanced Raman scattering (SERS) active

substrate, particularly usable in the microanalysis of chemicals; the

detection limit of adenine is as low as 1.0 3 10�7 M based on a signal-to-

noise (S/N) ratio of 3. Since the proposed method is cost-effective and is

suitable for the mass production of Ag coated capillaries, we fully expect it

to play a significant role in the development of SERS based microchip

analyzers and even in the fabrication of Ag coated hollow glass

waveguides.

Index Headings: Surface-enhanced Raman scattering; SERS; Glass

capillary; AgNO3; Butylamine; Microanalysis.

INTRODUCTION

For several decades, the electroless deposition of metal filmshas been a continuous research topic in the scientificcommunity because of its immense application prospects.1 Itis especially challenging to coat thin metal films in a controlledway onto the inner surfaces of glass and plastic capillaries andmicrochannels.2–4 Silver-coated capillaries, for instance, can beincorporated into microchip analyzers, running on the basis ofsurface-enhanced Raman scattering (SERS) spectroscopy.5,6

On the other hand, it should be noted that the energy losses ofhollow-glass waveguides (HGWs),7,8 which are used for thedelivery of high-power laser pulses and optical pulsecompression, can be reduced significantly by coating theinside surface of the hollow waveguide with a highly reflectivemetallic layer. For instance, the transmission of the silver-coated HGW was reported to be 95%, which was considerablyhigher than the transmission of HGWs with no coating.9

In principle, the silver film can be deposited inside thecapillary, as well as the hollow silica waveguide, by a liquid-phase deposition process in which the thickness of the silverfilm may be controlled by varying the reaction time, the flowrate, the temperature, and the concentration of the reactingsolutions.10 On one hand, for the preparation of Ag coatedHGWs, a photosensitive alkaline silver amine solution wasusually mixed with a reducer solution composed of dextroseand a sodium salt of ethylenediaminetetracetic acid, and thenthe resultant solution was pumped into the HGW. The two

primary solutions reacted to yield a film of metallic silver onthe inner surface of the HGW.9 On the other hand, Farquharsonet al. prepared silver-doped sol-gel coated capillaries byinitially drawing a mixture of two precursor solutions, oneconsisting of silver nitrate and hydroxylamine and the otherconsisting of methanol and tetramethyl orthosilicate, into acapillary to form sol-gels, and then reducing the incorporatedsilver ions using dilute sodium borohydride.11–14 Thosecapillaries were demonstrated by them to be very effectivefor the detection of dipicolinic acid as a biochemical signatureof Bacillus cereus spores by SERS.15 A similar SERS basedchemical detection has long been exploited to improve theperformance of gas chromatography, liquid chromatography,capillary electrophoresis, and flow injection analysis.16–20

We have recently reported that very stable and opticallytunable Ag films can be reproducibly fabricated simply bysoaking glass substrates in ethanolic solutions of AgNO3 andbutylamine.21 These Ag films were shown to possess a veryhomogeneous morphology and SERS activity. We havedemonstrated in this work that silver can be deposited in asimilar fashion onto the inside surface of a glass capillary. Asreactants, only AgNO3, butylamine, and absolute ethanol areneeded, and the thickness and the surface roughness of theresulting silver film can be readily controlled by varying thereaction conditions (reaction time, temperature, and concentra-tion). We show in this work that the Ag coated capillary can beused as a miniature SERS sampling device to detect minimalamounts of chemicals. We are sure that this work willcontribute to the development of SERS based microchipanalyzers since silvering is possible not only on glasses butalso on plastic microchannels. The method is also expected tohave an application to the fabrication of Ag coated HGWs.

EXPERIMENTAL

Chemicals. Butylamine (99.5%), silver nitrate (99%),benzenethiol (99%), rhodamine 6G hydrochloride (99%),adenine (99%), L-tyrosine (98%), and (-)-riboflavin werepurchased from Aldrich and used as received. Other chemicals,unless specified, were of reagent grade, and also used asreceived. Highly pure water (Millipore), of resistivity greaterthan 18.0 MX�cm, was used throughout.

Method of Silver Deposition. Silver deposition was carriedout employing the electroless plating method developedrecently in our laboratory.21,22 Glass capillary columns havingdimensions of 1.1 mm inner diameter 3 0.2 mm thickness 3 75mm length were used as the silvering substrates. Initially, theglass capillaries were soaked in a basic cleaning solution (pH¼9.2; 0.5% Hellmanex II, Hellma) for 3 h and sonicated indistilled water for 10 min, followed by rinsing with ethanol,and then dried in an oven at 70 8C for 20 min. The cleaned

Received 16 August 2006; accepted 27 October 2006.* Author to whom correspondence should be sent. E-mail: kwankim@snu.ac.kr.

Volume 61, Number 1, 2007 APPLIED SPECTROSCOPY 190003-7028/07/6101-0019$2.00/0

� 2007 Society for Applied Spectroscopy

glass capillaries were placed inside the polypropylene reactionvessel, and then a reaction mixture composed of ethanolicAgNO3 and butylamine was added to it. The reason for using apolypropylene container as the reaction vessel was to avoidnonspecific silvering of the reaction vessel. The reaction vesselwas incubated for 40 min at 45 6 1 8C with vigorous shakingparallel to the longitudinal axes of the capillaries. As a reactionmixture, the concentration of AgNO3 in absolute ethanol waskept at 0.83 mM for all experiments, while that of butylaminewas subjected to change. The Ag coated glass capillaries werefinally rinsed with ethanol, and then the outer surfaces weregently wiped using cotton wool to maintain exclusively theinner Ag surfaces as SERS substrates after air-drying.

Scanning Electron Microscopy. The morphologies of thesilver nanostructures formed on the inner surfaces of capillarieswere examined by taking field emission scanning electronmicroscope (FE-SEM) images using a JSM-6700F FE-SEMoperated at 5.0 kV; the silvered capillaries were broken intoseveral pieces and then mounted on carbon tapes to take SEMimages.

Raman Spectroscopy. Raman spectra were obtained using aRenishaw Raman system Model 2000 spectrometer equippedwith an integral microscope (Olympus BH2-UMA). The 514.5nm line from a 20 mW Arþ laser (Melles-Griot Model351MA520) or the 632.8 nm line from a 17 mW HeNe laser(Spectra Physics Model 127) were used as the excitationsource. Raman scattering was detected using 1808 geometrywith a Peltier cooled (�70 8C) charge-coupled device (CCD)camera (400 3 600 pixels). The laser beam was focused onto aAg coated spot of the sampling capillary with an objectivemicroscope on the order of 203 magnification. The dataacquisition time was usually 30 s. The holographic grating(1800 grooves/mm) and the slit allowed the spectral resolutionto be 1 cm�1. The Raman band of a silicon wafer at 520 cm�1

was used to calibrate the spectrometer, and the accuracy of thespectral measurement was estimated to be better than 1 cm�1.

RESULTS AND DISCUSSION

The protocol of electroless deposition of silver onto the innersurfaces of a glass capillary and its usage to detect chemicalsby SERS are schematically drawn in Scheme 1. The silveringof a glass substrate, qualitatively sketched in panels a and b, isvery effective, particularly because the hydroxyl groups of theglass surfaces are partially deprotonated in absolute ethanol,leaving behind lots of negatively charged surface oxygenatoms. Upon adding silver ions, the oxygen sites are bound byAgþ ions such that the surface-bound Agþ ions cansubsequently function as seeds for the growth of Ag nano-structures on the glass substrates by the action of a mildreductant, i.e., butylamine.22 Once Ag nanostructures areformed on the inner surface of a glass capillary, an analytesolution can be introduced into the capillary, as sketched inpanel c of Scheme 1, for chemi- or physisorption of the analytemolecules that can subsequently be detected by SERS, assketched in panel d.

In the Experimental section, we described that the silveringof glass capillaries was conducted in a reaction vessel undervigorous shaking. The advantage of this method was thatsilvered capillaries with the same properties could be multiplyfabricated in one batch. The silver films coated on the outersurfaces can be wiped off in the final stages, for the productionof inner-surface-only coated capillaries. Much the same silver-coated capillary can also be fabricated by drawing silveringreactants into the capillary using a syringe pump. Figure 1shows the cross-sectional SEM micrograph of an Ag coatedglass capillary that has been prepared by introducing ethanolicsolutions of AgNO3 (1 mM) and butylamine (1 mM) into acapillary for 30 min at the rate of 1 mL/min using a syringepump at 45 8C. The left side of Fig. 1 shows the Agnanoaggregates formed inside the capillary; the right siderepresents the glass part in contact with Ag. Relativelyhomogeneous nanoparticles with diameters of ;70 nm aredensely packed onto the inner surface of the capillary. Theaverage size of the Ag nanoparticles deposited can be tuned byvarying the relative molar ratios of the butylamine and silvernitrate reactants.21

Figure 2 shows the FE-SEM images of Ag coated capillariesprepared at various silver ion-to-butylamine molar ratios in apolypropylene reaction vessel at 45 8C. As described in theExperimental section, the concentration of AgNO3 in absolute

FIG. 1. Cross-sectional SEM micrograph of an Ag coated glass capillaryprepared by the flow of 1 mM AgNO3 and 1 mM butylamine in ethanol for 30min at the rate of 1 mL/min using a syringe pump at 45 8C.

SCHEME 1. Electroless deposition of silver onto the inner surfaces of a glasscapillary and its usage to detect chemicals by SERS; (a) formation of surface-bound Agþ ions to function as seeds for growth of Ag nanostructure, (b) actualformation of silver nanostructures, (c) chemi- and/or physisorption of analytemolecules onto Ag nanostructures for SERS analysis, and (d) SERSmeasurement taken by focusing laser light through the capillary wall ontoAg nanostructures formed inside the capillary.

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ethanol was kept at 0.83 mM, but that of butylamine wasvaried, from 0.08 mM to 0.16, 0.42, or 0.83 mM, to obtain thesurfaces corresponding to Figs. 2a through 2d, respectively.The mean grain sizes of Ag nanostructures were thendetermined to be 24 6 7, 40 6 11, 63 6 22, and 91 6 40nm, suggesting that larger and more aggregated silver grainsare produced by increasing the molar ratio of butylamine usedas the reductant.21 Considering the fact that SERS usuallyoccurs with aggregated structures of silver particles in the rangeof 20–200 nm, these capillaries would be expected to showhigh SERS activity.23 This is particularly because a greatlyenhanced electric field can be built at the junctions betweenparticles, resulting in the enhancement of Raman signals for themolecules adsorbed at those locations.24–26

The four capillaries whose SEM images are shown in Figs.2a–2d are colored to be yellow, reddish brown, purple, anddeep blue, respectively (see Fig. 3). This indicates that thenanostructured Ag films can be tuned to possess specificplasmon bands ranging from the green to the red region. Inreference to the electromagnetic mechanism of SERS,27 thisimplies that a wavelength-dependent SERS active capillary canbe fabricated simply by varying the amount of butylamineadded initially to the ethanolic solution of silver ions.Consulting the previous report, the absorption maximum ofthe deep blue capillary (Fig. 3d) is presumed to be located at;600 nm, while that of the purple capillary (Fig. 3c) is around;500 nm.21 Hence, at least when 632.8 nm radiation is used asthe excitation source, a greater SERS signal is expected to beobtained using a capillary corresponding to Figs. 2d and 3d,among others. This was confirmed experimentally by takingthe SERS spectra of benzenethiol (BT) adsorbed onto the innersurfaces of the four different Ag coated capillaries in Fig. 2.Before taking the SERS spectra, an aliquot of methanolic BT(2.7 3 10�5 M) was drawn into each Ag coated capillary up to3 mm in columnar length by a capillary force and then left todry under ambient conditions. A laser beam of 632.8 nm

radiation was subsequently focused through the glass wall onto

the inner Ag coated surface. Figures 4a through 4d show the

SERS spectra of BT observed in that way using capillaries

corresponding to those in Figs. 2a–2d or 3a–3d, respectively.

In agreement with expectation, the more intense SERS spectra

were observed from the more aggregated structures of silver

particles. A statistical calculation revealed that the spot-to-spot

relative deviations were at most 8%, 10%, 6%, and 18% for the

four capillaries corresponding to Figs. 4a–4d, respectively (see

also the inset of Fig. 4), warranting the homogeneity of the

surface activity of our Ag coated glass capillary.

The relative peak intensity of the 8a mode of BT at 1573

cm�1, shown in the inset of Fig. 4, clearly illustrates that the Ag

film fabricated from a 1:1 ratio solution of Agþ:butylamine is at

least an order of magnitude more SERS active than that

fabricated from a 1:0.1 ratio solution of Agþ:butylamine. On

these grounds, we have attempted to estimate the SERS

enhancement factor (EF) of the capillary d in Fig. 4 using the

following relationship:

EF ¼ ðISERS=INRÞðNNR=NSERSÞ ð1Þ

FIG. 2. FE-SEM images of Ag coated capillaries prepared in a polypropylenereaction vessel at different molar ratios of AgNO3 and butylamine: (a) 1:0.1,(b) 1:0.2, (c) 1:0.5, and (d) 1:1. The mean particle sizes are 24, 40, 63, and 91nm, respectively; scale bar¼ 100 nm.

FIG. 3. Photographs of Ag coated capillaries made from solutions ofAgþ:butylamine ratios of (a) 1:0.1, (b) 1:0.2, (c) 1:0.5, and (d) 1:1, each ofwhich corresponds to that in Fig. 2.

FIG. 4. SERS spectra of benzenethiol adsorbed on Ag coated capillaries madefrom solutions of Agþ:butylamine ratios of (a) 1:0.1, (b) 1:0.2, (c) 1:0.5, and(d) 1:1; the inset shows the relative peak intensities of the 8a mode at 1573cm�1 measured using the four Ag coated capillaries in Fig. 2; 632.8 nmradiation was used as the excitation source, the laser power at the samplingposition was 2.0 mW, and the signal integration time was 30 s.

APPLIED SPECTROSCOPY 21

where ISERS and INR are the SERS intensity of BT on Ag filmsand the normal Raman (NR) scattering intensity of BT in bulk,respectively, and NSERS and NNR are the number of BTmolecules illuminated by the laser light to obtain thecorresponding SERS and NR spectra, respectively. ISERS andINR were measured at 1573 cm�1, and NSERS and NNR werecalculated on the basis of the estimated concentration of surfaceBT species, density of bulk BT, and the sampling areas. Takingthe sampling area (approximately 1 lm in diameter) and thenumber of moles soaked inside a capillary up to 3 mm incolumnar length into account, NSERS¼ 5.8 3 10�18 mol, whichis comparable to the equilibrated surface concentration of BTon Au and Ag reported in the literature, i.e., ;7.1 3 10�10 molcm�2.28 When taking the NR spectrum of pure BT, thesampling volume will be the product of the laser spot(approximately 1 lm in diameter) and the penetration depth(approximately 2 lm) of the focused beam. As the density ofneat BT is 1.07 g cm�3, NNR is calculated to be 1.4 3 10�14

mol. Since the intensity ratio, ISERS/INR, was measured as ;70,EF could then be as large as ;1.6 3 105 if 632.8 nm radiationis used as the excitation source.

Consulting the previous report, a silver film formed on glassfrom a 1:1 ratio solution of Agþ and butylamine should exhibitan absorption maximum at ;600 nm, accompanying asignificant band broadening. The absorbance at ;500 nm ismerely 10% lower than that at ;600 nm in that case.21 The Agcoated capillary corresponding to that in Fig. 2d (and Fig. 3d)is supposed to have similar absorption characteristics. Thecapillary would then be highly SERS active, not only by aHeNe laser emitting 632.8 nm radiation but also by an Arþ

laser emitting 514.5 nm radiation. To confirm the SERSactivity at 514.5 nm, R6G was chosen as a probing molecule.R6G does not chemisorb on Ag; rather, it can be regarded as aprototype physisorbing organic molecule.29 Therefore, obser-vation of its SERS spectrum is thought to be invaluable for thedevelopment of SERS based microchip analyzers. We alsotook into consideration that R6G is a strongly fluorescent

xanthene dye that can show a molecular resonance Ramaneffect when excited into its visible absorption band centerednear 530 nm. In fact, the fluorescence band of R6G extendsover practically all the visible range. Owing to this fluores-cence, it is not possible to obtain Raman spectra of acceptablequality in either the solid state or the dissolved state at 514.5nm excitation. However, the fluorescence is dramaticallyreduced once adsorbed on silver nanoaggregates, since Agparticles can provide an energy relaxation channel forfluorescence quenching.30

Figure 5 shows a series of Raman spectra taken after R6Gsolutions at 10�4, 10�6, or 10�8 M in methanol were drawn intosame-batch Ag coated capillaries for 10 min, followed bywashing with ethanol for 10 s, and then air-drying thecapillaries. The Ag coated capillaries were the same ones usedin taking the spectrum in Fig. 4d, but all the spectra in Fig. 5were taken using 514.5 nm radiation as the excitation source.When methanol was drawn into the capillary for a blankexperiment, the spectrum recorded was completely featureless,implying that the spectra in Fig. 5 must be the surface-enhanced resonance Raman scattering (SERRS) spectra ofR6G. The spectrum obtained after exposing the Ag coatedcapillary to 10�4 M R6G (Fig. 5a) is very intense, and thepronounced peaks at 1312, 1365, 1510, and 1650 cm�1 can allbe assigned to the aromatic stretching vibrations of thexanthene skeleton.29 When the Ag coated capillary wasexposed to 10�6 M R6G, the Raman signal was dramaticallydiminished, as can be seen in Fig. 5b. The Raman signal wouldthen decrease further if the Ag coated capillary were exposed toan even more diluted R6G solution. In fact, an about five timesweaker SERRS signal was detected when the Ag coatedcapillary was exposed to 10�8 M R6G. Nevertheless, thesignal-to-noise (S/N) ratio was obviously greater than 10 evenafter exposure to 10�8 M R6G as long as the laser power at thesampling position was 0.17 mW and the data acquisition timewas 1 s. Therefore, all the characteristic peaks of R6G can stillbe identified even after exposure to 10�8 M R6G, as can beseen in Fig. 5c. The present observation supports our earlierpresumption that the Ag coated capillary fabricated herein ishighly SERS active, not only due to 632.8 nm excitation butalso due to 514.5 nm excitation. The Ag coated capillary maythen be utilized to detect, via SERS, especially the fluorescingmolecules separated by chromatography columns since fluo-rescence is quenched at metal substrates.31

The Ag coated capillary can also be used to detect very smallvolumes of chemicals and/or those of extremely lowconcentration, which may have to be collected or reused afteranalysis. Sampling can be conducted simply via capillary force,and the volume needed to fill the capillary up to a length of 1mm is as low as ;1 lL. To illustrate its usefulness, we havetaken SERS spectra of a few compounds of biologicalsignificance, i.e., 0.1 mM aqueous solutions of adenine(DNA base), tyrosine (amino acid), and riboflavin (biosyntheticcofactor), after sampling via capillary force. At first, Fig. 6ashows the SERS spectrum of adenine taken using 632.8 nmradiation as the excitation source. In agreement with theprevious reports, one distinct band is observed at 734 cm�1,along with a series of weak bands in the region 1200–1500cm�1; all these bands were assigned previously to the skeletalvibration modes.23 Secondly, Fig. 6b shows the SERSspectrum of tyrosine. Although its spectral feature is somewhatbroad, the overall intensity profile associated with the peaks

FIG. 5. SERRS spectra of R6G on Ag nanostructure taken after methanolicsolutions of R6G at (a) 10�4, (b) 10�6, and (c) 10�8 have been drawn into a Agcoated capillary for 10 min, followed by washing with ethanol for 10 s, andthen air-drying the capillary; 514.5 nm radiation was used as the excitationsource, the laser power at the sampling position was 0.17 mW, and the signalintegration time was 1 s; the Ag coated capillary used here was the same oneused in Fig. 3d.

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around 1163, 1358, and 1585 cm�1, as well as the spectral gaparound 1420 cm�1, is in good agreement with the previousSERS data obtained in Ag colloidal solutions.32 Thirdly, Fig.6c shows the SERS spectrum of riboflavin, known asbiological cofactor (vitamin B2), and the observed spectralpattern is also very similar to the reported data.33

The ready acquisition of Raman spectra for small amounts ofsubstances of biological importance is very promising. Asshown in Fig. 6, sub-millimolar concentration can be readilydetected by SERS using Ag coated capillaries. The concentra-tion of bio-analytes is frequently in the sub-micro molarregime, however, so it is worth examining the detection limit ofthe present methodology. In this light, SERS spectra wereobtained after sampling adenine at various concentrationsranging from 10�4 to 10�8 M into Ag coated capillaries for 10min. Figure 7 shows the intensity of the adenine peak at 734cm�1 drawn versus the concentration of adenine solution. It isseen that the SERS signal attains a plateau value when theadenine concentration is above 53 10�5 M, probably due to thefull coverage of adenine molecules onto silver.34 When theadenine concentration is below 10�5 M, the number ofadsorbed adenine molecules decreases, resulting in weakerSERS signals. Nonetheless, we are able to detect as low as 1.93 10�7 M of adenine with an S/N ratio of 5.5 6 1.0 (see theinset of Fig. 7); the detection limit would then be 1.0 3 10�7 Mbased on an S/N ratio of 3. This clearly suggests that our Agcoated capillaries can be used to detect by SERS bio-analytesdown to a sub-micro molar regime.

CONCLUSION

We have succeeded in depositing Ag onto the inner surfaceof a glass capillary simply by shaking it inside a polypropylenereaction vessel containing only ethanolic AgNO3 and butyl-amine solution. A similar electroless Ag deposition can also beaccomplished by allowing the reactants to flow through thecapillary using a syringe pump. The silver deposited onto theinside surface consists of aggregated granular particles whosemean grain sizes were determined merely by the relative molar

ratio of butylamine and AgNO3 used as reactants, similarly tothose formed on glass slides21 and silica beads.22 Owing to theaggregated structures of Ag in the range of 20–100 nm, the Agcoated capillary was a very efficient SERS active substrate,probably usable in the microanalysis of bio- and hazardouschemicals; the detection limit of adenine is, for instance, as lowas 1.0 3 10�7 M based on an S/N ratio of 3. Since the presentmethod is cost-effective and is suitable for the mass productionof Ag coated capillaries, its application prospects are expectedto be very high in many areas of science and technology.

ACKNOWLEDGMENTS

This work was supported by the Korea Science and Engineering Foundation(KOSEF R01-2006-000-10017-0).

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FIG. 6. SERS spectra of (a) adenine, (b) tyrosine, and (c) riboflavin on Agnanostructures taken after sampling their aqueous solutions (0.1 mM) into anAg coated capillary via capillary force; 632.8 nm radiation was used as theexcitation source, the laser power at the sampling position was 2.0 mW, and thesignal integration time was 30 s; the Ag coated capillary used here was thesame one used in Fig. 3d.

FIG. 7. SERS intensity of the ring breathing band of adenine at 734 cm�1

versus the concentration of adenine sampled into Ag coated capillaries to takeSERS spectra; 632.8 nm radiation was used as the excitation source, the laserpower at the sampling position was 20 mW, and the signal integration time was1 s; the Ag coated capillary used here was the same one used in Fig. 3d. Theinset shows a SERS spectrum taken after sampling at 1.9 3 10�7 M.

APPLIED SPECTROSCOPY 23

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