Surface-Enhanced Raman Scattering: a Powerful Tool for Chemical Identification

ANALYTICAL SCIENCES AUGUST 2011, VOL. 27 775 2011 © The Japan Society for Analytical Chemistry

Reviews Surface-Enhanced Raman Scattering: A Powerful Tool for Chemical Identification

Kwan KIM*† and Kuan Soo SHIN**†

*Department of Chemistry, Seoul National University, Seoul 151-742, Korea **Department of Chemistry, Soongsil University, Seoul 156-743, Korea

The nature of “hot spots” in surface-enhanced Raman scattering (SERS) and the novel fabrication of Ag thin films for efficient SERS measurements are the main focus of this review. By using 4-aminobenzenthiol or 1,4-phenylenediisocyanide molecules we can characterize the nanogap formed by a metal nanoparticle and a flat metal substrate, or the gap between two spherical metal nanoparticles. These nanogaps are indeed SERS-active sites and the apparent size of “hot spots” is found to be very limited. To use SERS for routine chemical identifications, the substrates should be stable, reproducibly prepared, inexpensive, and easy to make. We introduce a method for the facile fabrication of Ag thin films, simply by

soaking glass substrates in ethanolic solutions of AgNO3 and butylamine, and their application as efficient SERS substrates. Ag-coated glass capillary and Ag deposited magnetic nanoparticles can be used for the microanalysis of bio- and hazardous chemicals.

(Received March 16, 2011; Accepted April 26, 2011; Published August 10, 2011)

1 Introduction 775 3·2 Ag-coated glass capillary for minute 2 The Nature of “Hot Spots” in SERS 776 chemical analysis 2·1 Nanogaps formed by nanoparticles and 3·3 Ag-coated magnetic particles for a flat substrate the induction of SERS 2·2 Ag nanoparticle-mediated induction of SERS 3·4 Ag-coated beads for SERS-based 2·3 Surface potential of metal nanoparticles molecular sensors 2·4 Size of “hot spots” 4 Conclusions and Outlook 782 3 Novel Fabrication of Ag Thin Films for 5 Acknowledgements 782 Efficient SERS 778 6 References 782 3·1 Fabrication of SERS-active Ag films on glass

Raman signals for molecules adsorbed on nanostructured 1 Introduction coinage metals.1–3 In recent years, it has been reported that even single molecule detection is possible by surface-enhanced Surface-enhanced Raman scattering (SERS) is an abnormal resonance Raman scattering (SERRS), suggesting that the surface optical phenomenon resulting in strongly increased enhancement factor (EF) can reach as much as 1014 or 1015;

Kwan KIM received the BS degree in Kuan Soo SHIN received the BS and MS chemistry from Seoul National University degrees in chemistry from Seoul National in 1971, and obtained the PhD degree in University, Korea in 1983 and 1985, and chemistry from Brown University, USA, obtained the PhD degree in physical in 1980. After working as a Research chemistry from The University of Texas Associate and a Research Assistant at Austin, USA, in 1990. After working Professor in Brown University for two as a Post-doctoral Research Associate in years, he has been serving as a Professor Argonne National Laboratory, he has been in the Chemistry Department of Seoul serving as a Professor in the Chemistry National University. His current research Department of Soongsil University. His interest is the preparation, characterization, current research interest is the synthesis, and molecular engineering application of spectroscopic characteriza tion, and sensor nano-scaled systems, including the application of metal nanoparticles. surface-enhanced Raman scattering-based chemical analysis.

† To whom correspondence should be addressed. E-mail: [email protected]; [email protected] This is an invited review article selected from Keynote presenters at IUPAC International Conference on Analytical Sciences 2011 (ICAS 2011). 776 ANALYTICAL SCIENCES AUGUST 2011, VOL. 27 the Raman cross sections are thus comparable to the usual cross sections of fluorescence.4,5 In addition, the SERS technique has many advantages, such as the fact that it is nondestructive, and that little or no sample preparation is required to obtain structural information; further, SERS measurements can be performed in air, vacuum, or water as well as solutions. Since its first discovery in the late 1970s,6 SERS has thus been an object of great interest in many areas of science and technology, including chemical analysis, corrosion, lubrication, heterogeneous catalysis, biological sensors, and molecular electronics.7–11 It has long been believed that two enhancement mechanisms, one called a long-range electromagnetic (EM) effect, and the other called a short-range chemical (CHEM) effect, are simultaneously operative for SERS.1,3,12 Theoretically, at least 8 – 10 orders of magnitude can arise from electromagnetic Fig. 1 Schematic diagram of a nanogap, formed by a planar Au (Pt) surface plasmon excitation, while the enhancement factor due to 1 2 5,13 substrate and an Au (Ag) nanoparticle, in which a probe molecule, the chemical effect is 10 – 10 times. It is not yet clear what 4-aminobenzenethiol, is bound via a thiolate sulfur and an amine structural factors are responsible for single-molecule SERS, but group to Au (Pt) and Au (Ag), respectively. electromagnetic “hot spots” are presumed to exist in large fractal Ag aggregates. The junction of two aggregated Ag nanoparticles is also presumed to be a “hot spot” for SERS.4,14–17 22,23 We have recently confirmed that dramatic increases in Raman solutions of AgNO3 and butylamine. These Ag films have scattering can occur even for molecules sandwiched between been shown to possess a very homogeneous morphology and Au (Ag) nanoparticles and macroscopically smooth Au (Pt) high SERS activity. The Ag-coated capillary and Ag deposited substrates.18–21 The gap formed by a metal nanoparticle and a magnetic particles can then be used in the microanalysis of bio- flat metal substrate, or the gap between two metal nanoparticles and hazardous chemicals.23,29–31 is indeed the SERS-active site. Regarding the “hot spot”, it has been well established theoretically that as two particles approach each other, their transition dipoles couple in such a way that the 2 The Nature of “Hot Spots” in SERS enhanced EM fields around each particle create a pattern of coherent interference.4,14–17 This implies that as the distance 2·1 Nanogaps formed by nanoparticles and a flat substrate between the nanoparticles decreases, the coupled plasmon One of the weak points of SERS is that the surface should be resonance shifts to red, the enhanced EM field increases in the rough in order to observe a large enhancement. It is therefore junction between the particles, and destructive interference of not unexpected that there is no Raman signal measurable for the fields occurs at other points in space. In fact, the EF has 4-ABT molecules adsorbed on a smooth Au substrate. However, been estimated to be ~1010 in a gap of 1 nm formed between Raman peaks can be made observable by attaching Ag or Au two 90-nm sized Ag spheres.4 nanoparticles onto the 4-ABT monolayer (Fig. 1). The Raman To use SERS for routine analytical purposes, the substrates spectrum must be a SERS spectrum, derived from should be stable, reproducibly prepared, inexpensive, and easy electromagnetic coupling of the localized surface plasmon of to make. However, many SERS active surfaces currently Ag (Au) nanoparticles with the surface plasmon polariton of the available are lacking in stability and/or reproducibility. We have Au metal underneath.32–34 The gap formed by a metal recently found a very simple method that can repeatedly produce nanoparticle and a flat metal substrate is functioning as a “hot highly SERS-active Ag films on dielectric substrates, such as spot” for SERS.18–21 The EFs derived from the attachment of a glass.22–25 This is based on the fact that aliphatic amines can act single, 55-nm-sized nanoparticle onto 4-ABT on Au were as reductants and stabilizers simultaneously when preparing estimated to be as large as 8.3 × 105 or 5.0 × 105, for Ag or Au 26 Ag and Au nanoparticles by thermal activation. This method nanoparticles, respectively (for the ring 3 band (b2) near only requires the incubation of negatively charged glass 1390 cm–1), in which a factor of ~102 was presumed to be due to substrates in a solution of Ag ions with aliphatic amines, such as a chemical effect, with the remaining EF contributed by the butylamine at moderate temperatures. Ag films are then formed electromagnetic effect.18 exclusively and evenly on glass substrates by the reduction What is intriguing is that a more intense Raman signal is activity of butylamine.22,23 Since butylamine is a very mild obtained with increasing size of the Au nanoparticles, at least reductant, bulk reactions do not take place, although the amines with dimensions of under ~100 nm. Much the same trend has can reduce Ag ions that have been anchored on negatively been confirmed from a finite-difference time-domain (FDTD) charged glass substrates. Hence, the nonspecific adsorption of calculation.19 This is thought to suggest that the electromagnetic Ag particles on the reaction vessel can be readily controlled. coupling of the localized surface plasmon of Au nanoparticles Herein, we review the nature of “hot spots” in SERS with the surface plasmon polariton of the underlying smooth Au phenomena and a novel fabrication method of Ag thin films for substrate occurs more favorably with larger-sized nanoparticles, efficient SERS measurements. By using 4-aminobenzenthiol leading to the molecules trapped in the gap exhibiting strong (4-ABT) or 1,4-phenylenediisocyanide (1,4-PDI) molecules Raman peaks. Orendorff et al. have also investigated the SERS we can characterize the gap formed by a planar Au (Pt) and spectra of 4-mercaptobenzoic acid molecules sandwiched an Au (Ag) nanoparticle, or the gap between two spherical between Au nanoparticles of various shapes and a smooth Au Au nanoparticles.18–21,27,28 These nanogaps are indeed the substrate.35 They found that SERS EFs depend on the SERS-active sites, and the apparent size of the “hot spot” is nanoparticle shape, and vary by a factor of 102. Similar found to be very limited. Silver thin film can be reproducibly sandwich architectures have been used for immunoassays, most fabricated simply by soaking glass substrates in ethanolic notably by Porter and coworkers.36 ANALYTICAL SCIENCES AUGUST 2011, VOL. 27 777

2·2 Ag nanoparticle-mediated induction of SERS The intrinsic weak point of SERS is that only noble metals, such as Ag and Au, can provide large enhancement effects. However, to use SERS in routine, on-line studies for analytical purposes, SERS spectra should be obtainable even for molecules anchored on solid surfaces with negligible SERS activity. One promising approach would be to introduce so-called tip-enhanced Raman spectroscopy (TERS).37,38 By illuminating the gap between a Ag or Au tip of a scanning probe microscope and a metal substrate with a laser of suitable wavelength, localized surface plasmons can be excited inside the gap, producing a large increase of the electromagnetic field, and thus resulting in a significant increase in the Raman intensity in a region having the size of the tip apex. An alternative way to mimic TERS would be to use Ag or Au nanoparticles, such that the gap between the metal nanoparticle and the planar metal substrate could function as a “hot spot” for SERS. As mentioned above, Fig. 2 SERS spectra of 4-ABT on flat (a) Pt and (b) Au taken after very intense SERS spectra could be obtained for 4-ABT soaking in Ag sol using the 632.8, 568, 514.5, and 488 nm radiation assembled on a macroscopically flat Ag or Au substrate simply (from top to bottom) as the excitation sources. All spectra were taken by attaching Ag or Au nanoparticles to them.18,19 Based on while spinning at 3000 rpm so as to minimize the effect of an inhomogeneous distribution of Ag nanoparticles. In addition, all these observations, we investigated whether unaggregated Ag spectral intensities were normalized with respect to those of silicon nanoparticles can also induce SERS for 4-ABT assembled on a wafers used for instrument calibration, and also to the number of Ag 20 macroscopically smooth Pt substrate (Fig. 1). nanoparticles adsorbed on 4-ABT on Pt or Au (Copyright 2010 Platinum is intrinsically a weak SERS substrate, but we have American Chemical Society). confirmed that very intense SERS spectra can be obtained even from adsorbates on Pt by assembling nanosized Ag particles onto them.20 Specifically, we have compared the SERS intensity of 4-ABT sandwiched between Ag nanoparticles and a smooth sandwich-type molecular nanostructure. As before, no Raman Pt substrate (Ag@4-ABT/Pt) with that sandwiched between Ag signal was observed when 1,4-PDI was self-assembled onto a nanoparticles and a smooth Au substrate (Ag@4-ABT/Au), and flame-annealed Au wire, since it is SERS inactive. When found that the more intense spectra are measured in the former, 1,4-PDI-adsorbed Au wire is soaked in Au sol, Au nanoparticles at least under the illumination of a short-wavelength laser, i.e. are bound to the pendent isocyanide group, such that 1,4-PDI is 488 and 514.5 nm (Fig. 2(a)). This spectral enhancement must situated in the gaps between planar Au and nanosized Au be ascribed to a more facile electromagnetic coupling of the particles. Owing to electromagnetic coupling between the Ag particle and the Pt substrate in short-wavelength regions. localized surface plasmon of Au nanoparticles and the surface That is, the localized surface plasmon of the Ag particle must plasmon polaritons of Au wire, a distinct Raman spectrum can interact more favorably with the surface plasmon polariton of then be observed for the 1,4-PDI molecules.42 It is also the Pt substrate in short-wavelength regions. For Ag@4-ABT/Au, noteworthy in this case that the peak due to the NC group is however, a very intense spectrum of 4-ABT was observed using blue-shifted by as much as 48 cm–1, by the adsorption of Au a long-wavelength laser, i.e. 632.8 nm, reflecting the idea that nanoparticles, from 2127 cm–1 in the NR spectrum to 2175 cm–1. the electromagnetic coupling of the Ag particle with Au should This is caused by the donation of antibonding lone-pair electrons be comparatively stronger in the long-wavelength regions of the pendent isocyanide group to the Au nanoparticles.43–45 (Fig. 2(b)). These wavelength-dependent behaviors were found Another advantage of organic isocyanide as a SERS probe is also to agree with FDTD calculations. In addition to EM that the peak position of the NC stretching band is very sensitive enhancement, we also confirmed the contribution of CHEM to the applied potential under electrochemical conditions. This enhancement. In both Ag@4-ABT/Pt and Ag@4-ABT/Au, the would suggest that the peak position of the NC stretching band CHEM was more favorable in the shorter excitation wavelength could be used as an indicator of the surface potential of metal region. In the longer excitation wavelength region, the CHEM nanostructures. As surmised, a substantial peak shift was appeared to be more facile in Ag@4-ABT/Au than in observed when 1,4-PDI trapped in Au nanogaps was exposed to Ag@4-ABT/Pt. These results could be understood in terms of polar organics, like acetone and ammonia.28 For clarity, only the difference in the Fermi energy of Pt and Au.39,40 It is needed the NC stretching region is shown in Fig. 3(a). In the presence to note that significant changes in spectral features can occur of acetone vapor, the NC stretching band is blue-shifted when the CHEM mechanism contributes to SERS. The by up to 8 cm–1 from 2175 to 2183 cm–1. According to the implication of the present observation is enormous, since it potential-dependent SERS, this corresponds to a potential strongly suggests that the inherent obstacles to a more change from +0.2 to +0.7 V vs. a saturated Ag/AgCl electrode. widespread use of SERS can be overcome by the judicious use The peak shift can be understood by recalling the fact that a of SERS-active nanoparticles, either directly or indirectly. charge transfer occurs from the occupied s orbital of the interacting Au atom to the unoccupied p orbital of the oxygen 2·3 Surface potential of metal nanoparticles atom of acetone.46 Due to the charge transfer exclusively Organic diisocyanide is the other type of adsorbate that can be coming from the s part of the orbital, the surface potential of Au used in the characterization of nanogaps. This is particularly nanoparticles moves in the positive direction, resulting in a because 1,4-PDI, for instance, is known to adsorb on Au via blue-shift of the NC stretching band of 1,4-PDI. The situation only one of its two isocyanide groups.41 The other isocyanide is schematically shown in Fig. 3(b). When ammonia vapor is group is pendent with respect to the Au substrate, so that new passed over the Au nanoparticle-adsorbed 1,4-PDI/Au wire, Au or Ag nanoparticles can further bind onto it, forming a the NC stretching peak is red-shifted from 2175 to 2161 cm–1, 778 ANALYTICAL SCIENCES AUGUST 2011, VOL. 27

Fig. 3 (a) Raman spectra taken under the flow of acetone and ammonia vapor over 1,4-PDI sandwiched between smooth Au and Au nanoparticles. (b) Schematic diagrams showing the origin of blue- and red-shift of the NC stretching band of 1,4-PDI on Au (Copyright 2010 Wiley-VCH).

as shown in Fig. 3(a), corresponding to the potential change of molecules residing only within a 10-nm diameter area of the Au nanoparticles from +0.2 to –0.6 V. The red-peak shift can be center of the gap, adsorbed in the first stage, have thus understood by recalling the fact that ammonia is a typical donor contributed most of the measured Raman signal of 1,4-PDI. to metal substrates,47 thus lowering the surface potential of Au This apparently indicates that the size of “hot spot” is very nanoparticles (Fig. 3(b)). All of these observations suggest that limited. the isocyanide-adsorbed noble metal nanostructures can be used as a platform for a sensor of volatile organic chemicals (VOCs) operating via SERS. 3 Novel Fabrication of Ag Thin Films for Efficient SERS 2·4 Size of “hot spots” It is a noble job to elucidate the identity of the SERS 3·1 Fabrication of SERS-active Ag films on glass “hot spot”. As one of such efforts, we have recently estimated To use SERS in routine, on-line studies for analytical purposes, the apparent size of the “hot spot” for SERS located within the the substrates should be stable, reproducibly prepared, gap between two spherical Au nanoparticles (Fig. 4).27 Initially, inexpensive, and easy to make. The fabrication of SERS-active 55-nm sized Au nanoparticles were laid on a thiol substrates has accordingly been a challenge over the decades.54–56 group-terminated silane film, and then 1,4-PDI molecules In reality, the enhancement properties of a SERS-active surface self-assembled onto the Au nanoparticles. 1,4-PDI must have are highly susceptible to its method of preparation, and therefore bonded to Au by forming one Au–CN bond, with another its detailed nanostructure. Lots of SERS-active surfaces isocyanide group being pendent with respect to the Au surface. currently available are thus lacking in stability and/or Up to this point, no Raman scattering was detected at all for reproducibility. We have recently reported that very stable and 1,4-PDI. Upon attaching new Au nanoparticles onto the pendent optically tunable Ag films can be reproducibly fabricated simply isocyanide groups of 1,4-PDI, a Raman signal was distinctly by soaking glass substrates in ethanolic solutions of AgNO3 and observed. This means that electromagnetic “hot spots” had butylamine.22 These Ag films were shown to possess a very formed at the gaps between pairs of Au nanoparticles.48–53 The homogeneous morphology and SERS activity. Figure 5A shows Raman signal did not increase further, however, even after the AFM images of the silver films prepared at various Ag adsorption of additional 1,4-PDI onto the vacant surfaces of the ion-to-butylamine molar ratios. The mean grain sizes were second Au nanoparticles. As a rough estimate, about 400 determined to be 25 ± 4, 46 ± 6, 71 ± 19, and 110 ± 37 nm for ANALYTICAL SCIENCES AUGUST 2011, VOL. 27 779

curved surfaces of glasses, but also onto the inside surfaces of capillaries, and even onto silica beads and flexible fabrics.22–25,57 It is especially noteworthy that the Ag-nanocoated fabric was validated as an effective antiseptic bandage.57 The advantage of the present Ag film formation is that in addition to the simplicity of the reaction, the loading levels of Ag nanoparticles on cotton fabrics can be controlled simply by varying the

concentration of AgNO3 and butylamine.

3·2 Ag-coated glass capillary for minute chemical analysis As mentioned above, Ag can also be deposited onto the 23 inside surface of a glass capillary. Once again, only AgNO3, butylamine, and absolute ethanol are needed, and the thickness and the surface roughness of the resulting Ag film can be controlled by varying the reaction conditions (reaction time, temperature, and concentration). The Ag deposited onto the inside surface consists of aggregated granular particles whose mean grain sizes are determined merely by the relative molar

ratio of butylamine and AgNO3 used as reactants. Owing to the aggregated structures of Ag in the range of 20 – 100 nm, the Ag-coated capillary is a very efficient SERS-active substrate, usable in the microanalysis of bio- and hazardous chemicals. The protocol of electroless deposition of Ag onto the inner surfaces of a glass capillary and its usage to detect chemicals by SERS are schematically drawn in Fig. 5B. The silvering of a glass substrate, qualitatively sketched in (a) and (b), is very effective, particularly because the hydroxyl groups of the glass surfaces are partially deprotonated in absolute ethanol, leaving behind lots of negatively charged surface oxygen atoms. Upon adding Ag ions, the oxygen sites are bound by Ag+ ions, such that the surface-bound Ag+ ions can subsequently function as seeds for the growth of Ag nanostructures by the action of a mild reductant, i.e. butylamine. Once Ag nanostructures are formed on the inner surface of a glass capillary, an analyte solution can be introduced into the capillary, for chemi- or physisorption of the analyte molecules that can subsequently be Fig. 5 (A) AFM images (500 nm × 500 nm) of Ag films prepared by detected by SERS, as sketched in (d). electroless deposition at different molar ratios of AgNO3 and Figure 6A shows a series of Raman spectra taken after R6G butylamine: (a) 1:0.1, (b) 1:0.2, (c) 1:0.5, (d) 1:1. The mean particle solutions at 10–4, 10–6, or 10–8 M in methanol have been drawn sizes are 25, 46, 71, and 110 nm, respectively; the Z scale = 100 nm into an Ag-coated capillary; all of the spectra were taken using (Copyright 2006 American Chemical Society). (B) Electroless 514.5-nm radiation as the excitation source. When methanol deposition of Ag onto inner surfaces of a glass capillary and its usage was drawn into the capillary for a control experiment, the + to detect chemicals by SERS: (a) formation of surface-bound Ag ions spectrum recorded was completely featureless, implying that the to function as seeds for growth of Ag nanostructure, (b) actual spectra shown in Fig. 6A must be the SERS spectra of R6G. formation of Ag nanostructures, (c) chemi- and/or physisorption of analyte molecules onto Ag nanostructures for SERS analysis, and (d) The spectrum obtained after exposing the Ag-coated capillary to –4 SERS measurement taken by focusing laser light through capillary 10 M R6G is very intense, and the pronounced peaks at 1312, walls onto Ag nanostructures formed inside the capillary (Copyright 1365, 1510, and 1650 cm–1 can all be assigned to the aromatic 2007 Society for Applied Spectroscopy). stretching vibrations of the xanthene skeleton.58 The Raman signal would then decrease further if the Ag-coated capillary were exposed to an even more diluted R6G solution. However, all of the characteristic peaks of R6G can still be identified even the surfaces a – d, respectively. The mean values themselves after exposure to 10–8 M R6G. The present observation supports suggest that larger and more aggregated Ag grains are the idea that the Ag-coated capillary fabricated herein is highly obtained by increasing the molar ratio of butylamine used as a SERS-active, not only due to 632.8-nm excitation, but also due reductant. The SERS EF of the 71 nm Ag film was determined to 514.5-nm excitation. The Ag-coated capillary may then be to be ~2 × 105. In addition, the SERS spectrum on these films utilized particularly, via SERS, to detect the fluorescing has proved to be highly reproducible. A statistical calculation molecules separated by chromatography columns. revealed that the batch-to-batch relative deviation is 7.6%, while The Ag-coated capillary can also be used to detect very small the spot-to-spot relative deviation is 5.5%. The fact that the volumes of chemicals and/or those of extremely low relative standard deviation is <10% from five different concentration, which may have to be collected or reused after measurements for each type of film with five different spots per analysis. Sampling is possible simply via capillary force, and film clearly illustrates the reproducible characteristics of our Ag the volume needed to fill the capillary up to a length of 1 mm is films. as low as ~1 μL. To illustrate its usefulness, we have taken As to be described in a subsequent section, robust Ag Raman spectra of a few compounds of biological significance, nanoparticle films can be fabricated not only onto the planar and i.e. 0.1 mM aqueous solutions of adenine (DNA base), tyrosine 780 ANALYTICAL SCIENCES AUGUST 2011, VOL. 27

Fig. 6 (A) SERS spectra of R6G on Ag nanostructure taken after methanolic solutions of R6G at (a) 10–4, (b) 10–6, and (c) 10–8 have been drawn into an Ag-coated capillary for 10 min, followed by washing with ethanol for 10 s and the capillary air-dried; 514.5 nm radiation was used as the excitation source; the laser power at the sampling position was 0.17 mW and the signal integration time was 1 s. (B) SERS spectra of (a) adenine, (b) tyrosine, and (c) riboflavin on Ag nanostructures taken after sampling their aqueous solutions (0.1 mM) into an Ag-coated capillary; 632.8 nm radiation was used as the excitation source; the laser power at the sampling position was 2.0 mW and the signal integration time was 30 s (Copyright 2007 Society for Fig. 7 (A) SERS spectra of RhBITC adsorbed on (a) Fe3O4/SiO2@Ag Applied Spectroscopy). and (b) commercial μAg particles gathered at 632.8 nm excitation. (B) SERS spectra of adenine on Fe3O4/SiO2@Ag gathered by varying its concentration from 10–4 to 10–8 M. (C) Raman spectrum taken after dropping 1.0 μL of Fe3O4/SiO2@Ag colloids onto 1.5-nm thick (amino acid), and riboflavin (biosynthetic cofactor), after RhBITC–PAH film on a glass slide (Copyright 2010 Elsevier). sampling via capillary force. Figure 6B(a) shows the SERS spectrum of adenine taken using 632.8 nm radiation as the excitation source. In agreement with the previous reports, one distinct band was observed at 734 cm–1, along with a series of 3·3 Ag-coated magnetic particles for the induction of SERS weak bands in the region 1200 – 1500 cm–1; all of these bands Since the magnetic particles are readily recovered from the were assigned previously to the skeletal vibration modes.59 solution phase using a magnet without centrifugation and/or Figure 6B(b) shows the SERS spectrum of tyrosine. Although filtering, any SERS-active magnetic particles will be especially its spectral feature is somewhat broad, the overall intensity useful in the Raman spectroscopic analysis of chemical species profile associated with the peaks around 1163, 1358, and dissolved in aqueous solutions. In conformity with this demand, 1585 cm–1, as well as the spectral gap around 1420 cm–1, is in Ag-deposited magnetic particles can be prepared using the good agreement with previous SERS data obtained under Ag same protocol as mentioned above.29–31,62 The thus-prepared 60 colloidal solutions. Finally, Fig. 6B(c) shows the SERS Ag-deposited Fe2O3 particles were efficient SERS substrates for spectrum of riboflavin, known as a biological cofactor (vitamin the vibrational spectroscopic characterization of molecular

B2). The observed spectral pattern is also very similar to the adsorbates prepared in a similar way on Ag surfaces; the EF of 61 reported data. The ready acquisition of Raman spectra for benzenethiol adsorbed on Ag-deposited Fe2O3 was estimated to small amounts of substances of biological importance like be about 3 × 106. Adenine could be detected down to 10 nM riboflavin is very promising, particularly because it takes part in concentrations, and naphthalene was also detectable down to energy production by aiding in the metabolism of fats, 200 nM by using pentachlorobenzenethiol (PCBT)-adsorbed 29 carbohydrates, and proteins, and also because vitamin B2 is Ag-deposited Fe2O3 particles as the adsorbent. The Fe2O3 necessary for red blood cell formation, respiration, and antibody particles used in these works were commercial products with production, as well as for regulating human growth and very irregular shape and size distribution. Accordingly, it was reproduction. difficult to fully enjoy the properties of the superparamagnetism ANALYTICAL SCIENCES AUGUST 2011, VOL. 27 781

Fig. 8 (A) Fabrication of a Ag-coated polystyrene bead usable as a template for a biosensor operating via surface-enhanced Raman scattering. The biotinylated 4-ABT/Ag/PS units are selectively adsorbed on a streptavidin-patterned solid support (Copyright 2009 Elsevier). (B) Illustration of fabrication processes of RhBITC-embedded SiO2@Ag beads modified further with antibodies for multiplex immunoassays (Copyright 2011 American Chemical Society).

–1 of nanometer sized-Fe2O3 particles. We thus synthesized bands in the region of 1200 – 1500 cm ; all of these bands were 23,59 spherical Fe3O4 particles with a mean diameter of 426 ± 40 nm. assigned previously to the skeletal vibration modes. The They were actually composed of ~13 nm superparamagnetic graph of the intensity of the adenine peak at 734 cm–1 plotted Fe3O4 particles, which were then coated with SiO2, onto which against the concentration of adenine solution allowed us to Ag nanoaggregates were finally assembled to endow them with make analyses of solutions as low as 10 nM based on a both superparamagnetism and SERS activity.31 The particles signal-to-noise (S/N) ratio of 3. The detection limit of denoted by Fe3O4/SiO2@Ag could be magnetically concentrated Fe3O4/SiO2@Ag particles is 10-times lower than that of an and picked up using a small magnet. Ag-coated capillary. These Fe3O4/SiO2@Ag particles can be Figure 7A(a) shows the SERS spectrum of rhodamine B recycled by treating with a mild borohydride solution to desorb isothiocyanate (RhBITC) adsorbed on Fe3O4/SiO2@Ag particles any organic species from the Ag surface. prepared in an ethanolic solution of 1:1 molar ratio of butylamine As another example, we illustrate the induction of SERS 62 and AgNO3. For a reference, Fig. 7A(b) shows the SERS for a colored film. For that purpose, poly(allylamine spectrum of RhBITC adsorbed onto μAg powders, which are hydrochloride)-grafted fluorescent RhBITC (RhBITC-PAH) known to be effective substrates for the IR and Raman film was assembled on a glass slide by spin cast.62 Its thickness spectroscopic characterization of molecular adsorbates. It can was determined to be 1.5 nm by ellipsometry, but an absorption be seen that the SERS peaks in Fig. 7A(a) are an order of band could be identified, although weak, at 540 nm in the magnitude stronger than the corresponding ones in Fig. 7A(b). UV/vis spectrum. However, the resonance Raman spectrum of The EF of a typical aromatic thiol, such as benzenethiol the film was not observable due to a fluorescence background. 5 adsorbed on μAg powders was in fact determined to be 3.8 × 10 When a drop of (~1.0 μL) of Fe3O4/SiO2@Ag colloids and (with 632.8 nm excitation). Hence, the EF factor of toluene were placed on the film, Ag-coated magnetite particles 6 Fe3O4/SiO2@Ag particles may well be more than 3 × 10 . were agglomerated, showing the intrinsic peaks of RhBITC, as Figure 7B shows a series of SERS spectra for adenine, shown in Fig. 7C. The appearance of the RhBITC peaks must a compound of biological significance, in concentrations from be due to the enhanced electromagnetic field caused by –4 –8 10 to 10 M. In agreement with previous reports, one distinct clustering of the suspended Fe3O4/SiO2@Ag particles. After band was observed at 734 cm–1, along with a series of weak taking the Raman spectra, we were able to remove the clustered 782 ANALYTICAL SCIENCES AUGUST 2011, VOL. 27

Fe3O4/SiO2@Ag particles from the glass substrate by using a near the junction of the particles should contribute most of the very strong neodymium magnet. Hence, the present total Raman signal. methodology has proved to be a useful tool in probing the To use SERS in routine, on-line studies for analytical purposes, physicochemical characteristics of surface adsorbates on diverse the substrates should be stable, reproducibly prepared, substrates, including historic paints. inexpensive, and easy to make. We have introduced a facile fabrication method of Ag thin films simply by soaking glass 3·4 Ag-coated beads for SERS-based molecular sensors substrates in ethanolic solutions of AgNO3 and butylamine and A simple electroless plating method can also be applied to their application for efficient SERS substrates. These Ag films prepare Ag-coated silica and polystyrene beads (Fig. 8A).23,24,63 are shown to possess a very homogeneous morphology, and to Robust Ag nanostructures are reproducibly fabricated by soaking be highly SERS active; the SERS EF value was estimated to be 5 polystyrene beads in ethanolic solutions of AgNO3 and as large as ~2 × 10 . The Ag-coated capillary can then be used butylamine. When the molar ratio of butylamine to AgNO3 is in the microanalysis of bio- and hazardous chemicals. In far below 1.0, distinct nanosized Ag particles are formed on the addition, naphthalene, a prototype aromatic molecule whose polystyrene beads, but by increasing the amount of butylamine, adsorption strength to Ag is too weak to observe its intrinsic network-like Ag nanostructures are formed that possess very SERS spectrum, can also be detected by SERS in aqueous broad UV/vis absorption characteristics extending from the solutions by using the PCBT-modified Ag deposited magnetic near-UV to near-infrared regions. In conformity with the particles. UV/vis absorption characteristics, the Ag-deposited polystyrene SERS, firstly discovered in the middle of 1970s, was reborn a beads are highly efficient SERS substrates, with an EF estimated, decade ago as the result of a number of simultaneous innovations. using 4-ABT as a model adsorbate, to be larger than 1.1 × 106.25 These include: firstly, the advent of nanoscience and On the basis of the nature of the SERS peaks of 4-ABT, the nanotechnology, which allowed us to solve many long-standing Ag-deposited polystyrene beads were confirmed, after attaching problems in SERS; secondly, the availability of inexpensive, but biotin groups over 4-ABT, to selectively recognize streptavidin powerful computers, which allowed us to routinely calculate the molecules down to concentrations of 10–11 g mL–1 (i.e. ~0.2 pM). electromagnetic field near nanostructures; thirdly, the advent of Since a number of different molecules can be used as micro Raman spectrometers with confocal probe capability that SERS-marker molecules (like 4-ABT), multiple bioassays are allowed us routinely to measure Raman spectra on samples a readily accomplished via SERS after attaching appropriate host fraction of a micrometer in size. The level of understanding or guest molecules onto them. about SERS is very advanced, so that in the very near future When SERS-active Ag is coated onto dye-embedded silica SERS will be used as a general platform for chemical and beads, a dual-tag sensor for immunoassays is possible that can biological analysis with unprecedented routine levels of be operated via both fluorescence and SERS.64 A one-shot sensitivity, specificity, and reproducibility, combining huge fluorescence image over the whole specimen allows us to save enhancement with the inherent molecular spectroscopic nature considerable time because any unnecessary time-consuming of Raman scattering. SERS measurements can be avoided by using the signature of the fluorescence. The fluorescence signal of the embedded dye can thus be used as a fast readout system of molecular 5 Acknowledgements recognition, whereas the SERS signals are subsequently used as the signature of specific molecular interactions. In this way, This work was supported by National Research Foundation of the antibody-grafted particles could recognize antigens down to Korea Grant funded by the Korean Government (2011-0001218, 1 × 10–10 g mL–1 solely by the SERS peaks of the Raman M10703001067-08M0300-06711-Nano2007-02943, KRF-2008- markers (Fig. 8B). 313-C00390, and 2009-0072467).

4 Conclusions and Outlook 6 References

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