- Email: [email protected]
RAMAN SPECTROSCOPY | Surface-Enhanced
110 RAMAN SPECTROSCOPY / Surface-Enhanced
studying cancerous changes in the colon, urinary bladder, breast, and soft tissue sarcomas has also been examined. NIR-Raman has found applications in the pharmaceutical industry. McCreery and colleagues reported the use of the technique for identification of pharmaceuticals inside amber vials. Even with the signal attenuation through the glass, adequate spectra were obtained for determination of vial content with 1–60 s integration times. Using a library of spectra, identification of the pharmaceuticals in the vials was performed and identification was found to display accuracy between 88% and 96%. This work demonstrated the potential of NIR-Raman for online process monitoring. The environmental community has also found uses for NIR-Raman analysis. A continuous method was developed by Weissenbacher and colleagues to detect trace organic pollutants using flow injection analysis and surface enhanced Raman spectroscopy (SERS). This method uses NIR excitation and FT-SERS detection to detect parts per million of pesticides in aqueous solutions. In this study, the authors describe the simultaneous detection of two pesticides, carbenazim and metazachlorine. A variety of industrial processes benefit from the use of NIR-Raman spectroscopy. In a study examining unleaded petroleum gasoline, with excitation at 514 nm from an argon laser, no Raman features were observed above the strong fluorescence. With He/Ne laser excitation at 633 nm, several Raman features appeared above the background. With excitation at 785 or 852 nm, a good quality Raman spectrum was obtained. In the textile industry, NIRRaman was used to examine ready-made textiles for the discrimination of the different raw materials. NIR-Raman has been introduced for use into the food processing industry. In the production of oils and fats, the determination of the amount of unsaturation, such as cis and trans isomers, can be important for food processing and food labeling. NIR-Raman was reported to measure the total unsaturation and the cis and trans isomers online during the production process. Fluorescence interference with visible excitation of many fats was
drastically reduced at NIR wavelengths. In addition to food processing, NIR-Raman has been used for determination of food quality as the main components of food (carbohydrates, proteins, and lipids) all show characteristic Raman lines using NIR detection. The pairing of SERS with NIR detection has proved to be a powerful DNA detection technique. NIR–SERS was reported to provide excellent discrimination against fluorescent interference and was nonresonant with most molecules. This allowed greater excitation intensities without photobleaching or destruction of the analyte. In one study, Kneipp and colleagues used this technique in rapid DNA sequencing to detect single-molecule DNA bases or nucleotides. NIR–SERS provided a method for detection and identification of the single DNA base, adenine, without any additional labeling. This study reported trace detection levels of adenine and AMP with well-resolved spectra. See also: Raman Surface-Enhanced.
Spectroscopy:
Instrumentation;
Further Reading Hanlon EB, Manoharan R, Koo TW, et al. (2000) Prospects for in vivo Raman spectroscopy. Physical and Medical Biology 45: R1–R59. Isola N, Stokes DL, and Vo-Dinh T (1998) Analytical Chemistry 70: 1352–1356. Keller S, Lochte T, Dippel B, and Schrader B (1993) Quality control of food with near-infrared excited Raman spectroscopy. Fresenius Journal of Analytical Chemistry 346: 863–867. Kneipp K, Kneipp H, Itzkan I, Dasari R, and Feld M (1999) Ultrasensitive chemical analysis by Raman spectroscopy. Chemical Reviews 99: 2957–2975. McCreery RL, Horn AJ, Spencer J, and Jefferson E (1998) Journal of Pharmaceutical Science 87: 1–8. Mahadevan-Jansen A, Mitchell MF, Ramanujam N, et al. (1998) Photochemistry and Photobiology 68: 123–132. Mulvaney SP and Keating CD (2000) Raman spectroscopy. Analytical Chemistry 72: 145R–157R. Weissenbacher N, Lendl B, Frank J, et al. (1997) Journal of Molecular Structure 410–411: 539–542.
Surface-Enhanced R E Littleford, D Graham, and W E Smith, University of Strathclyde, Glasgow, UK I Khan, Imperial College, London, UK & 2005, Elsevier Ltd. All Rights Reserved.
Introduction Surface enhanced Raman scattering (SERS) is a sensitive spectroscopic technique for the detection and
RAMAN SPECTROSCOPY / Surface-Enhanced 111
characterization of analytes adsorbed on suitable metal surfaces. The effect was first discovered experimentally in 1974 by Fleischmann et al., who reported very intense Raman bands from pyridine adsorbed onto an anodized silver surface. They attributed this strong signal to the presence of a large number of pyridine molecules present at the roughened electrode surface due to its large surface area. However, in 1977 Jeanmaire and Van Duyne, and Albrecht and Creighton, independently recognized that the increase in intensity could not be accounted for simply by the number of scatterers present. They observed that compared with the equivalent concentration of pyridine in solution, an enhancement of B106 in Raman scattering was obtained. They concluded that an intrinsic surface enhancement effect played a fundamental role in producing the enhanced Raman scattering. In the basic process of SERS, the analyte is adsorbed onto a roughened metal surface of a suitable metal, usually silver or gold. On excitation of this surface with a laser beam, a change in polarizability of the analyte occurs in a direction perpendicular to the surface, leading to the enhanced scattering. The rough surface required for scattering can be provided in a large number of ways. Common methods are to use aggregated colloidal particles, roughened electrodes, or thin cold-deposited metal films.
Mechanisms of Surface Enhancement Since the discovery of SERS there has been much debate regarding the origins of the effect. However, it is generally accepted that there are two main contributions to the enhancement process, namely electromagnetic enhancement and charge transfer or chemical enhancement. Electromagnetic Enhancement
The collective excitation of an electron cloud on the surface of a metal is termed a surface plasmon. Surface roughness or curvature is required for the scattering of light by surface plasmons. The electromagnetic field at the surface is greatly enhanced upon surface plasmon excitation. If an analyte is present on the roughened metal surface, the molecule experiences a large electric field at the surface. The intensity of the Raman scattering is dependent upon the induced polarization caused by the electric field, and consequently Raman scattering is amplified by a factor of 104 or greater. Electromagnetic enhancement does not require a direct metal–analyte bond but becomes weaker the larger the separation between the analyte and the surface. It can occur
with a separation of up to B20 A˚. Surface selection rules have been developed based on the electromagnetic approach. Charge Transfer
Charge transfer assumes a bond is formed between the analyte and the metal surface. The energy levels of the molecule are shifted and broadened so that they overlap with the Fermi level of the metal and new electronic states are created. On absorption of the incident light by the metal these states serve as resonant intermediate states, allowing efficient charge transfer between the metal and the analyte and hence enhanced Raman scattering. The enhancement obtained in this manner is confined to being a first layer effect. In general, the enhancement predicted is B102.
Advantages and Disadvantages of SERS SERS offers several advantages over normal Raman or resonance Raman scattering. The huge increase in signal intensity allows an extended concentration range to be studied, with detection limits considerably lower than those offered by resonance Raman scattering. Fluorescence from the analyte is quenched due to its proximity to the metal surface, providing an alternative, nonradiative route for energy loss. There is however a much broader low-intensity fluorescence from the SERS process. In addition, for studies of processes such as surface–ligand adhesion and corrosion, it allows the study of an adsorbate at less than monolayer coverage on a suitable metal surface in situ, in contact with water and other solvents. SERS does, however, have some limitations. For the SERS effect to occur, the analyte needs to be adsorbed onto or in close proximity to the roughened metal surface, and only a few metals have so far been shown to be efficient at providing surface enhancement. SERS intensities are also dependent on the roughness of the metal surface, and there are significant problems associated with the preparation of reproducible substrates with uniform roughness features. The spectra obtained are also dependent on the orientation of the molecule on the metal surface, and vibrations with little to no intensity in normal Raman scattering can become relatively intense. This can in some cases make it difficult to identify the analyte positively. Further, contamination can also be a problem. Since SERS is very sensitive, it is possible that very small amounts of a contaminant can be enhanced, and if the analyte to be considered does
112 RAMAN SPECTROSCOPY / Surface-Enhanced
not adsorb efficiently or does not give good SERS, the contaminant can easily dominate the SERS. These problems limit the use of SERS as a quantitative analytical method. However, these problems are largely overcome if surface-enhanced resonanceRaman scattering (SERRS) is used.
A
B
Surface Enhanced Resonance Raman Scattering
(A) Absorbance
So far in this article, both colored molecules and colorless molecules have been treated simply as analytes, and the enhancement has been taken as a combination of electromagnetic and chemical enhancement. However, there are crucial differences where a colored adsorbate is adsorbed onto a metallic surface such that the plasmon resonance frequency and the frequency of an absorption band are close to the frequency of the incident light. This combination of surface enhancement and molecular resonance is called SERRS. Stacy and Van Duyne first reported SERRS in 1983. The increase in sensitivity over SERS can be quite large, with total enhancements of up to 1014 reported. When used practically, there are various combinations of laser frequency, plasmon resonance frequency, and molar absorptivity of the analyte that can be employed to obtain a condition that can be deemed to be SERRS rather than SERS. These are shown diagrammatically in Figure 1. If the excitation is matched to the molecular absorption maximum (Figure 1A), the surface selection rules expected for SERS are much less effective. This is due to the polarization of the laser excitation altering on interaction with the chromophore during the scattering process. This results in a degradation of the effective input polarization required for SERS selection rules, making SERRS less sensitive to the orientation of the molecule to the surface. This usually means that the spectrum resembles that of the resonance spectrum in solution, so that positive identification of the analyte is possible. In addition, the extra enhancement of the analyte discriminates against the detection of contamination. Thus, SERRS is more readily applicable for the development of methods for quantitative analysis. A second possible arrangement illustrated in Figure 1A is where the laser excitation is set away from the absorption maximum of the adsorbate and at the maximum of the plasmon resonance. This is described as preresonant scattering, and often SERRS taken in this way is written as SE(R)RS. The advantages of resonance still apply over quite a wide range of frequencies, gradually tailing off to SERS. Thus, SER(R)S makes it simpler to pick out individual
2
1
1
2
A B
(B) Wavelength (nm) Figure 1 The different arrangements for SERRS: the curves represent (A) molecular absorbance and (B) plasmon resonance. In (A) the molecular absorbance maximum and the plasmon absorbance maximum do not coincide: position 1 represents excitation at the absorbance maximum, 2 that at the plasmon maximum. In (B) the molecular absorbance and the plasmon maximum coincide: position 1 represents excitation away from the absorbance and plasmon maximum where the spectrum has a preresonant component, 2 that at the absorbance maximum and plasmon maximum. (Rodger C, Smith WE, Dent G, and Edmondson M (1996) Surface-enhanced resonance-Raman scattering: An informative probe of surfaces. Journal of Chemical Society, Dalton Transaction 791–799; reproduced by permission of The Royal Society of Chemistry.)
resonant molecules in the presence of a matrix of interferents, but the further away the excitation is from molecular resonance, the more the effect will be dependent on the angle of the adsorbate to the surface. For surface studies this is a key point, and consequently this arrangement may be preferred for some surface analyses. Figure 1B illustrates an alternative case in which the molecular chromophore coincides with the surface plasmon maximum. Similar considerations will apply to those discussed for the case represented in Figure 1A, but a further increase in sensitivity is likely with excitation at the molecular and plasmon resonance frequency. The orientation dependence will become more apparent the greater the difference in frequency between the excitation frequency used and the absorbance and plasmon maximum as the conditions become more appropriate for SERS (1 in
RAMAN SPECTROSCOPY / Surface-Enhanced 113
Intensity (arbitrary)
1652
200
1365
1513 1578
1313 1190
621
400
600
781
800 1000 1200 1400 1600 1800 Raman shift (cm−1)
Figure 2 Resonance Raman and SERRS spectra of rhodamine 6G with excitation at 514.5 nm. The high fluorescence background is effectively quenched in the SERRS spectrum due to adsorption of the rhodamine onto the surface of the silver nanoparticles. (Reproduced with permission from Rodger C and Smith WE (2002) SERS. In: Handbook of Vibrational Spectroscopy. New York: Wiley; & John Wiley & Sons Ltd.)
Figure 1B). As a consequence the sensitivity will decrease. Where SERRS can be applied, it offers many advantages over Raman scattering, resonance Raman scattering, and SERS. It provides both vibrational and electronic information on the adsorbate. Very wide concentration ranges can be studied, and single molecule detection is easier. A further major advantage is the quenching of fluorescence by the surface, which allows fluorescing dyes, which are ineffective in visible resonance Raman studies, to give very good SERRS. For example, rhodamine 6G fluoresces strongly in the visible region, preventing the use of normal Raman scattering, but gives good SERRS (Figure 2). In addition, there is less dependence of the signal on orientation. The combination of sensitivity, selectivity, and robustness of signal makes SERRS a very sensitive and selective form of vibrational spectroscopy that can be used semiquantitatively or qualitatively.
SERS Substrates SERS activity requires that very specific substrates be prepared. The metal chosen requires a plasmon in the frequency region close to that of the excitation laser. In addition, the ratio of scattering to adsorption of the substrate is important. Since visible frequency lasers are widely used in this technique, silver, gold, and copper, which have plasmons resonant with visible light, are obvious effective substrates. Silver is particularly useful, having a better ratio of scattering to adsorption in the visible region than gold. Copper tends to be too chemically reactive, and although other metals including lithium, sodium, and some
transition metals are effective, silver is by far the most widely used. It is possible to form reasonably time-stable roughened silver surfaces that can be used under normal laboratory conditions. Many types of SERS-active substrate have been made, each containing many microscopic metal domains. These include the following: electrode surfaces roughened by oxidation–reduction cycles; island films consisting of small metal particles; cold-deposited films prepared by evaporation or sputtering in a vacuum; lithographically produced assemblies; metal gratings; metal colloids prepared by reducing a dissolved metal salt with an appropriate reducing agent; metal colloids encapsulated in sol–gel-derived xerogel layers; silver particle-doped cellulose gel films; and microbeads as a carrier of silver colloid. Metal nanoparticles, usually prepared as colloidal suspensions, are one of the most widely studied SERS substrates. They exhibit unique optical properties due to excitation of the surface plasmons. The frequency of the surface plasmon resonance depends strongly on the size, shape, and dielectric environment of the particles. Additionally, when particles are closely spaced, the surface plasmons of individual particles interact to create new resonances dependent upon the interparticle distance and the incident angle and polarization of the light. Thus, optical properties of individual particles and arrays of interacting particles can be conveniently tuned. Significant progress has been made in controlling the size and shape of metal nanoparticles. Monodisperse single crystal nanocubes, nanorods, and nanoshells have all been prepared using simple methods. The size and shape of gold particles are more easily controlled than those of silver, and consequently silver-clad gold nanoparticles have been prepared and shown to possess distinct optical properties. Silver colloid is popular due to the relative ease of manufacture, low cost, and stability. One widely used method of silver colloid preparation is citrate reduction of silver nitrate (Figure 3). This results in an overall net negative charge on the colloid, which is high compared with many preparations (Figure 4). As a result, the colloid is stable for months and even years, and in some cases, with careful attention to detail, quantitative SERRS can be obtained from this colloid with relative standard deviations of less than 5%. There is, however, a change in enhancement with time, and so a standard needs to be used.
Studies of SERS/SERRS Enhancement The reason for SERS/SERRS enhancement has been studied by a number of groups. It has been shown that aggregated silver colloids produce fractal
114 RAMAN SPECTROSCOPY / Surface-Enhanced
Figure 3 A representative transmission electron microscopic image of citrate-reduced silver colloid. The particles are mainly hexagonal, with a largest face diameter of B36 nm. A few rods are present. (Reprinted with permission from Faulds K, Littleford RE, Graham D, Dent G, and Smith WE (2004) Comparison of surface-enhanced resonance Raman scattering from unaggregated and aggregated nanoparticles. Analytical Chemistry 76: 592–598; & American Chemical Society.)
O
H H
H
O
H
O
H O
O O
Ag
O
Ag
Figure 4 Representation of the surface of a Lee and Meisel colloidal particle. The citrate is believed to be bonded to the silver, which is present as AgI, with negatively charged carboxylate groups. (Rodger C, Smith WE, Dent G, and Edmondson M (1996) Surface-enhanced resonance-Raman scattering: An informative probe of surfaces. Journal of Chemical Society, Dalton Transaction 791–799; reproduced by permission of The Royal Society of Chemistry.)
clusters. Upon excitation, the electromagnetic field over a fractal object is not distributed uniformly but is localized in hot spots much smaller than the wavelength of light (10–30 nm). A large volume of the fractal therefore remains inactive or active at a lower level, while the portions of the fractal within these hot spots carry the full activity. This
localization of optical excitation indicates that very high electromagnetic fields lead to the huge enhancement of Raman scattering. Other experiments have shown an anomalously high enhancement in the first layer, suggesting the need to consider the charge transfer or chemical mechanism of enhancement as well as the electrochemical enhancement. Since only analytes attached to or close to metal surface are enhanced, the need for effective surface attachment is crucial if reproducible results are to be obtained. In many early experiments this was widely ignored. Commercial dyes were simply added to colloid or placed on surfaces and the results, where favourable, have been reported in many studies. The problem with this approach is that the surface chemistry of an element such as silver is complex. Little is understood about the surfaces on which the analyte is adsorbed, particularly in aqueous solution or, as is often done, with particles prepared in aqueous solutions and dried out on a surface for investigation. Further, where colloidal suspensions are used, it is common practice to aggregate the colloid in order to shift the frequency of the plasmon to a value that would place it in resonance with the laser. In fact, it is clear from ultraviolet–visible absorption that a range of clusters are made in most conditions and only a few are likely to be in resonance with the laser. However, this gives a bigger SERS enhancement than for single particles. The aggregating agent usually breaks up a stable colloid by reducing the charge on the surface. This can often happen by chemical action. Under the aerobic conditions used in most experiments, and shown in some to be essential for good SERS, the silver surface in the colloidal suspension is likely to be coated with a silver oxide or related layer containing silver(I) ions. It may also contain other adsorbed molecules from the preparation of the colloid. When a reagent such as sodium chloride is added to this surface, it is likely that a layer of silver chloride will form. This is known to be the case for electrode surfaces. With solid substrates, similar approaches are sometimes taken. For example, using the most commonly used analyte for fundamental studies, rhodamine 6G, the normal process for obtaining the best signals is to activate the surface with the sodium chloride. This particular combination of rhodamine and sodium chloride does give a more effective spectrum than without the addition of sodium chloride. However, studies of other analytes with colloidal suspensions have shown that sodium chloride is not always the most effective aggregating agent and no special activation procedures have been required. Additionally, with higher concentrations of rhodamine, not all the dye adsorbs and large fluorescent
RAMAN SPECTROSCOPY / Surface-Enhanced 115 NH2
OH N
MeO
OMe N
N
N
N (A)
N
NH
N
O2 N
NO2
(B)
Figure 5 Chemical structure of two dyes specifically designed to complex strongly to the silver surface for effective SERS. (A) 4(50 -azobenzotriazyl) 3,5-dimethoxyphenylamine; (B) 5-(2-methyl-3,5-dinitro-phenylazo) quinolin-8-ol.
backgrounds are obtained (see Figure 2). This can limit the use of SERRS and is often a problem where weak signals are obtained, and the temptation is to use higher, not lower, concentrations. Thus, for quantitative work and for obtaining a better understanding of the effect, effective surface adhesion is essential. In addition, the simpler the nature of the surface, the easier it is to understand the nature of the enhancement. For this reason, a set of special dyes has been developed for SERS/SERRS. These contain groups added to the dye specifically to obtain metal complexing with silver ions present on the surface. Two effective groups of this type are shown in Figure 5. One contains the benzotriazole group, known to be an effective method of preventing tarnishing in silver due to its strong attachment to the surface. The other contains an 8-hydroxyquinoline group, which will create a complex with silver ions on the metal surface that is insoluble. Both these reagents have proved to be effective for obtaining SERS/SERRS with sodium chloride and other aggregating agents, including organic aggregating agents such as poly-L-lysine.
Single Molecule Spectroscopy In 1997 two groups independently claimed single molecule detection using SERS/SERRS using different approaches. In some of the single molecule studies, the difference between SERS and SERRS is ignored, but most, not all, of the studies use chromophores. Nie and Emory reported detection of single rhodamine 6G dye molecules adsorbed on immobilized single silver nanoparticles using SERRS, and Kneipp et al. reported detection of a single molecule of crystal violet adsorbed on aggregated clusters of silver particles in a colloidal suspension using near infrared excitation. The large SERS
enhancement was attributed to excitation of the coupled surface plasmons of the colloidal aggregate. The SERS enhancement quoted by both groups for the detection of single molecules is of the order of 1014. Such enhancements are many orders of magnitude greater than predicted ensemble averaged values. This can be attributed to the removal of population averaging effects, whereby all the molecules and particles are assumed to contribute equally to the observed signals. Instead, only the active particle or active site is considered. Other groups have subsequently reported single molecule detection using SERS. Although detection of a single molecule adsorbed onto an immobilized single silver nanoparticle has been reported, studies on the protein hemoglobin adsorbed on immobilized colloidal particles revealed that the minimum state of aggregation necessary for SERS detection of a single hemoglobin molecule was the dimer. Detection of molecules adsorbed to single particles was not observed. Consequently, it is suggested that the surface enhancement is predominantly electromagnetic in nature and dominated by the increased local electric field between the two particles. Calculations suggest the maximum electromagnetic contribution to SERS enhancement for two interacting spheres to be of the order of 1011, and therefore an additional enhancement of 103 is required to explain the enhancement factors necessary for single molecule detection. This extra enhancement may be electromagnetic due to further surface roughness present on individual particles or may be chemical in nature. It is proposed that the additional electromagnetic enhancement is obtained only under special positions such as the interstitial site between two particles and outside sharp surface protrusions.
Applications of SERS Sensors
Due to the molecular specificity of SERS/SERRS, combined with its inherent sensitivity, it has found utility in a wide range of applications, only a few of which can be described here. The development of SERS sensor technology has allowed the detection and identification of seven structurally similar monosaccharides in aqueous solution using a sample volume of only 5 ml with a concentration of 1 10 2 mol dm 3. This has led to the initial development of a glucose-based biosensor with true in vivo, real time, minimally invasive sensing. The development of a volatile organic compound sensor using SERS detection has also been reported. The SERS substrate is chemically modified with a
116 RAMAN SPECTROSCOPY / Surface-Enhanced
thiol coating to prevent oxidation of the roughened silver surface and attracts the analyte of interest to the SERS surface. Detection of chlorinated solvents, aromatic compounds, and methyl t-butyl ether have all been demonstrated. Drugs
There is considerable interest in the development of novel platinum-based anticancer drugs that overcome the disadvantages associated with the widely used drug cisplatin, namely its inactivity against some types of tumors and toxic side effects. SERS has been shown to be suitable for the characterization of platinum complexes at physiological concentrations, allowing the determination of binding strengths of different ligands. The large SERRS intensities from the anticancer drug mitoxantrone have been used to provide a quantitative method of detection. The method uses a flow cell, which causes a dilution of plasma and serum samples, thus overcoming the fluorescent background. Even with these dilution steps the detection limits are superior to the previously published high-performance liquid chromatography (HPLC) method and are sufficiently good to be used for clinical analysis. Since the SERRS technique takes minutes from sample introduction to detection, and the chromatography technique hours, the SERRS technique could be considered the technique of choice for analysis of this drug. Intracellular SERS
SERS from living cells has been reported using several techniques. Etched and silver-coated glass fiber tips have been used as the SERS substrate, allowing the recording of spectra of biological samples, such as plant tissue and microbiological cells, with high spatial resolution. The deposition of colloidal gold particles inside single living cells by fluid-phase uptake has been demonstrated, providing strong SERS signals from the native chemical constituents of the cells. The sensitivity of SERS allowed measurements to be made in relatively short collection times (1 s for one mapping point) using 3–5 mW of near infrared excitation. SERS mapping over a cell monolayer with a 1 mm lateral resolution showed different Raman spectra at almost all places, reflecting the very inhomogeneous chemical constitution of the cells. This has opened up exciting possibilities for the study of cell biology and biomedical studies. Atomic Force Microscopy/Raman
Atomic force microscopy (AFM) uses a fine tip to probe surfaces. It has extremely high resolution. By
adding a roughened silver coating to AFM tips, a technique has been developed whereby when the AFM tip is touched on the surface it is irradiated with a laser, providing good SERS. The advantage of this technique is that the effective resolution of the technique is the dimension of the tip and consequently is much smaller than optical dimensions. The tip position is known from the AFM scan. This type of method has been used for analysis of thin films, which are undetectable with Raman microprobe systems but easily acquired with a suitably gold-coated AFM tip. More recently, a combined scanning electron microscope/Raman scattering detection system has been created. It is possible to detect the Raman scattering during the time of the SEM scan. This provides detailed structural information on the surface in a noncontact manner, which is faster and sometimes more effective than AFM. Since Raman scattering only occurs from the tip, and the tip position is known from the SEM, the dimensions of the tip are essentially the resolution of the instrument. DNA
The single molecule detection capacity of SERRS has been used to develop analytical techniques for DNA. These can use either a substrate or a colloid. In either case, this is most effective when the label is within the monolayer of the surface and hence the largest SERRS enhancement can be obtained. Methods have been developed that enable spatial separation of different events and their detection on a substrate. The use of SERS to monitor DNA hybridization of a fragment of the breast cancer susceptibility gene, BRCA1, on modified silver surfaces has been shown to be effective. A number of SERRS active DNA probes with different dyes have been synthesized that are used with colloids. Detection limits that are better than those obtained with fluorescence can now be obtained with SERRS. In addition, whereas fluorescence gives a very broad signal, SERRS gives a sharp signal and consequently is much more effective for multiplexing. This technique has been improved by the addition of a flow cell and in particular a lab on a chip flow cell, where control of the process of aggregation through the laminar flow region of the chip has shown a great improvement in quantitation as well as an improvement in sensitivity. The sensitivity of this method is known to be down to single molecule level, and it is possible to obtain this using commercial equipment within a maximum of 10 s. This technique is showing considerable promise for more effective multiplexed forms of DNA analysis (Figure 6). Recently, multiplexing of six probes with
RAMAN SPECTROSCOPY / Surface-Enhanced 117 8000
7000
Intensity
1650
1510
6000
1573
1360
Intensity
5000
550
1314 1441
1186
4000
1131
1632
3000
2000
1503 R6G
4000 3500 3000 2500 2000 1500 1000 500 0
1303
1000 HEX Blank 0 1000 1100 1200 1300 1400 1500 1600 1700 1800 Raman shift (cm−1) Figure 6 SERRS from 2,5,10 ,30 ,90 -hexachloro-6-carboxyfluorescein (HEX) and rhodamine 6G labeled oligonucleotides at 2 10 9 mol l 1 (1 s accumulation time). The unique SERRS spectral fingerprints from each dye allow multiplex analysis of SERRS/DNA complexes. (Reproduced with permission from Graham D, Mallinder BJ, and Smith WE (2000) Detection and identification of labeled DNA by surface enhanced resonance Raman scattering. Biopolymers (Biospectroscopy) 57: 85–91; & John Wiley & Sons, Inc.)
gold nanoparticles has been demonstrated, indicating the large multiple detection capability for SERRS/ DNA assays. Distance Detection
The ability to obtain sharp molecularly specific spectra from SERRS prompted the concept that a number of codes could be written simply by changing the combination of analytes and by changing their concentration. In this way, it is believed, hundreds of thousands of codes can be written that can be read using a Raman spectrometer without any spatial separation. The initial demonstration for this for one dye has shown that Raman scattering from suitable polymer films can be detected at up to 10 m (Figure 7) and therefore there is considerable potential for further expansion in this direction. Proteins
One of the first important demonstrations of SERRS was to detect heme groups in proteins. It is a good
1m
560
590 570 580 Wavelength (nm)
2&3 m 4&5 m 10 m 600
Figure 7 Change in spectral intensity as the distance from the sample is increased. (Reproduced with permission from McCabe A, Smith WE, Thomson G, et al. (2002) Remote detection using surface enhanced resonance Raman scattering. Appl Spectrosc 56: 820–826.)
example of the selectivity of the method in that practically no signals are obtained from the protein other than from the heme group. The heme on the other hand can be detected down to below monolayer coverage, although a monolayer is often used since it helps prevent protein degradation. A silver colloid stabilized with citrate layers also helps. The advantage of SERRS is that marker bands are available for the oxidation state, spin state, and coplanarity of the vinyl groups with the heme group. In addition, compared with resonance Raman scattering, it is easier to obtain SERRS from weaker chromophore absorption bands such as the Q band. Bacteria
The treatment of bacteria with sodium borohydride provides a nucleating substrate for the reduction of silver ions, forming a rough silver metal coating around the microorganism. Intense SERS of the coated bacteria showed four different types of bacteria to all produce similar spectra, suggesting that the spectra are selective and sensitive to a specific molecular species that dominates the spectra and that is found in all the bacteria analyzed. See also: Blood and Plasma. Clinical Analysis: Glucose. DNA Sequencing. Fluorescence: Overview. Forensic Sciences: Drug Screening in Sport. Microscopy Techniques: Electron Microscopy; Scanning Electron Microscopy; Atomic Force and Scanning Tunneling Microscopy. Nucleic Acids: Spectroscopic Methods. Raman Spectroscopy: Instrumentation. Sensors: Overview.
Further Reading Albrecht MG and Creighton JA (1977) Anomalously intense Raman spectra of pyridine at a silver electrode. Journal of the American Chemical Society 99: 5215–5217.
118 RAMAN SPECTROSCOPY / Surface-Enhanced Billmann J and Otto A (1982) Electronic surface state contribution to surface enhanced Raman scattering. Solid State Communications 44: 105–107. Creighton JA (1998) The selection rules for surface enhanced Raman spectroscopy. In: Clark RHJ and Hester RE (eds.) Spectroscopy of Surfaces, ch. 2, p. 37. New York: Wiley. Emory SR and Nie S (1997) Probing single molecules and single nanoparticles by surface enhanced Raman scattering. Science 275: 1102–1106. Fleischmann M, Hendra PJ, and McQuillan AJ (1974) Raman spectra of pyridine adsorbed at a silver electrode. Chemical Physics Letters 26: 163–166. Jeanmaire DL and Van Duyne RP (1977) Surface Raman spectroelctrochemistry. Part 1. Heterocyclic, aromatic, and aliphatic amines adsorbed on the anodized silver electrode. Journal of Electroanalytical Chemistry 84: 1–20. Kneipp K, Kneipp H, Irving I, Dasari RR, and Feld MS (2002) Surface enhanced Raman scattering and biophysics. Journal of Physics: Condensed Matter 14: R597– R624.
Kneipp K, Wang Y, Kneipp H, et al. (1997) Single molecule detection using surface enhanced Raman scattering (SERS). Physical Review Letters 78: 1667–1773. Moskovitis M (1978) Surface roughness and the enhanced intensity of Raman scattering by molecules adsorbed on metals. Journal of Chemical Physics 69: 4159–4161. Otto A, Mrozek I, Grabhorn H, and Akemann W (1992) Surface enhanced Raman scattering. Journal of Physics: Condensed Matter 4: 1143–1212. Stacy AM and Van Duyne RP (1983) Surface enhanced Raman and resonance Raman spectroscopy in a nonaqueous electrochemical environment: Tris(2,20 -bipyridine) ruthenium (II) adsorbed on silver from acetonitrile. Chemical Physics Letters. 102: 365–370. Xu HX, Aizpurua J, Kall M, and Apell P (2000) Electromagnetic contributions to a single-molecule sensitivity in surface enhanced Raman scattering. Physical Review E 62: 4318–4324. Xu HX, Bjerneld EJ, Kall M, and Borjesson L (1999) Spectroscopy of single haemoglobin molecules by surface enhanced Raman scattering. Physical Review Letters 83: 4357–4360.
REAGENTS See ANALYTICAL REAGENTS: Specification; Purification. RADIOCHEMICAL METHODS: Radio-Reagent Methods
REDOX INDICATORS See INDICATORS: Redox
REDOX TITRATION See TITRIMETRY: Overview; Potentiometric; Photometric
REFERENCE MATERIALS See QUALITY ASSURANCE: Reference Materials; Production of Reference Materials
REFLECTOMETRY See OPTICAL SPECTROSCOPY: Refractometry and Reflectometry
studying cancerous changes in the colon, urinary bladder, breast, and soft tissue sarcomas has also been examined. NIR-Raman has found applications in the pharmaceutical industry. McCreery and colleagues reported the use of the technique for identification of pharmaceuticals inside amber vials. Even with the signal attenuation through the glass, adequate spectra were obtained for determination of vial content with 1–60 s integration times. Using a library of spectra, identification of the pharmaceuticals in the vials was performed and identification was found to display accuracy between 88% and 96%. This work demonstrated the potential of NIR-Raman for online process monitoring. The environmental community has also found uses for NIR-Raman analysis. A continuous method was developed by Weissenbacher and colleagues to detect trace organic pollutants using flow injection analysis and surface enhanced Raman spectroscopy (SERS). This method uses NIR excitation and FT-SERS detection to detect parts per million of pesticides in aqueous solutions. In this study, the authors describe the simultaneous detection of two pesticides, carbenazim and metazachlorine. A variety of industrial processes benefit from the use of NIR-Raman spectroscopy. In a study examining unleaded petroleum gasoline, with excitation at 514 nm from an argon laser, no Raman features were observed above the strong fluorescence. With He/Ne laser excitation at 633 nm, several Raman features appeared above the background. With excitation at 785 or 852 nm, a good quality Raman spectrum was obtained. In the textile industry, NIRRaman was used to examine ready-made textiles for the discrimination of the different raw materials. NIR-Raman has been introduced for use into the food processing industry. In the production of oils and fats, the determination of the amount of unsaturation, such as cis and trans isomers, can be important for food processing and food labeling. NIR-Raman was reported to measure the total unsaturation and the cis and trans isomers online during the production process. Fluorescence interference with visible excitation of many fats was
drastically reduced at NIR wavelengths. In addition to food processing, NIR-Raman has been used for determination of food quality as the main components of food (carbohydrates, proteins, and lipids) all show characteristic Raman lines using NIR detection. The pairing of SERS with NIR detection has proved to be a powerful DNA detection technique. NIR–SERS was reported to provide excellent discrimination against fluorescent interference and was nonresonant with most molecules. This allowed greater excitation intensities without photobleaching or destruction of the analyte. In one study, Kneipp and colleagues used this technique in rapid DNA sequencing to detect single-molecule DNA bases or nucleotides. NIR–SERS provided a method for detection and identification of the single DNA base, adenine, without any additional labeling. This study reported trace detection levels of adenine and AMP with well-resolved spectra. See also: Raman Surface-Enhanced.
Spectroscopy:
Instrumentation;
Further Reading Hanlon EB, Manoharan R, Koo TW, et al. (2000) Prospects for in vivo Raman spectroscopy. Physical and Medical Biology 45: R1–R59. Isola N, Stokes DL, and Vo-Dinh T (1998) Analytical Chemistry 70: 1352–1356. Keller S, Lochte T, Dippel B, and Schrader B (1993) Quality control of food with near-infrared excited Raman spectroscopy. Fresenius Journal of Analytical Chemistry 346: 863–867. Kneipp K, Kneipp H, Itzkan I, Dasari R, and Feld M (1999) Ultrasensitive chemical analysis by Raman spectroscopy. Chemical Reviews 99: 2957–2975. McCreery RL, Horn AJ, Spencer J, and Jefferson E (1998) Journal of Pharmaceutical Science 87: 1–8. Mahadevan-Jansen A, Mitchell MF, Ramanujam N, et al. (1998) Photochemistry and Photobiology 68: 123–132. Mulvaney SP and Keating CD (2000) Raman spectroscopy. Analytical Chemistry 72: 145R–157R. Weissenbacher N, Lendl B, Frank J, et al. (1997) Journal of Molecular Structure 410–411: 539–542.
Surface-Enhanced R E Littleford, D Graham, and W E Smith, University of Strathclyde, Glasgow, UK I Khan, Imperial College, London, UK & 2005, Elsevier Ltd. All Rights Reserved.
Introduction Surface enhanced Raman scattering (SERS) is a sensitive spectroscopic technique for the detection and
RAMAN SPECTROSCOPY / Surface-Enhanced 111
characterization of analytes adsorbed on suitable metal surfaces. The effect was first discovered experimentally in 1974 by Fleischmann et al., who reported very intense Raman bands from pyridine adsorbed onto an anodized silver surface. They attributed this strong signal to the presence of a large number of pyridine molecules present at the roughened electrode surface due to its large surface area. However, in 1977 Jeanmaire and Van Duyne, and Albrecht and Creighton, independently recognized that the increase in intensity could not be accounted for simply by the number of scatterers present. They observed that compared with the equivalent concentration of pyridine in solution, an enhancement of B106 in Raman scattering was obtained. They concluded that an intrinsic surface enhancement effect played a fundamental role in producing the enhanced Raman scattering. In the basic process of SERS, the analyte is adsorbed onto a roughened metal surface of a suitable metal, usually silver or gold. On excitation of this surface with a laser beam, a change in polarizability of the analyte occurs in a direction perpendicular to the surface, leading to the enhanced scattering. The rough surface required for scattering can be provided in a large number of ways. Common methods are to use aggregated colloidal particles, roughened electrodes, or thin cold-deposited metal films.
Mechanisms of Surface Enhancement Since the discovery of SERS there has been much debate regarding the origins of the effect. However, it is generally accepted that there are two main contributions to the enhancement process, namely electromagnetic enhancement and charge transfer or chemical enhancement. Electromagnetic Enhancement
The collective excitation of an electron cloud on the surface of a metal is termed a surface plasmon. Surface roughness or curvature is required for the scattering of light by surface plasmons. The electromagnetic field at the surface is greatly enhanced upon surface plasmon excitation. If an analyte is present on the roughened metal surface, the molecule experiences a large electric field at the surface. The intensity of the Raman scattering is dependent upon the induced polarization caused by the electric field, and consequently Raman scattering is amplified by a factor of 104 or greater. Electromagnetic enhancement does not require a direct metal–analyte bond but becomes weaker the larger the separation between the analyte and the surface. It can occur
with a separation of up to B20 A˚. Surface selection rules have been developed based on the electromagnetic approach. Charge Transfer
Charge transfer assumes a bond is formed between the analyte and the metal surface. The energy levels of the molecule are shifted and broadened so that they overlap with the Fermi level of the metal and new electronic states are created. On absorption of the incident light by the metal these states serve as resonant intermediate states, allowing efficient charge transfer between the metal and the analyte and hence enhanced Raman scattering. The enhancement obtained in this manner is confined to being a first layer effect. In general, the enhancement predicted is B102.
Advantages and Disadvantages of SERS SERS offers several advantages over normal Raman or resonance Raman scattering. The huge increase in signal intensity allows an extended concentration range to be studied, with detection limits considerably lower than those offered by resonance Raman scattering. Fluorescence from the analyte is quenched due to its proximity to the metal surface, providing an alternative, nonradiative route for energy loss. There is however a much broader low-intensity fluorescence from the SERS process. In addition, for studies of processes such as surface–ligand adhesion and corrosion, it allows the study of an adsorbate at less than monolayer coverage on a suitable metal surface in situ, in contact with water and other solvents. SERS does, however, have some limitations. For the SERS effect to occur, the analyte needs to be adsorbed onto or in close proximity to the roughened metal surface, and only a few metals have so far been shown to be efficient at providing surface enhancement. SERS intensities are also dependent on the roughness of the metal surface, and there are significant problems associated with the preparation of reproducible substrates with uniform roughness features. The spectra obtained are also dependent on the orientation of the molecule on the metal surface, and vibrations with little to no intensity in normal Raman scattering can become relatively intense. This can in some cases make it difficult to identify the analyte positively. Further, contamination can also be a problem. Since SERS is very sensitive, it is possible that very small amounts of a contaminant can be enhanced, and if the analyte to be considered does
112 RAMAN SPECTROSCOPY / Surface-Enhanced
not adsorb efficiently or does not give good SERS, the contaminant can easily dominate the SERS. These problems limit the use of SERS as a quantitative analytical method. However, these problems are largely overcome if surface-enhanced resonanceRaman scattering (SERRS) is used.
A
B
Surface Enhanced Resonance Raman Scattering
(A) Absorbance
So far in this article, both colored molecules and colorless molecules have been treated simply as analytes, and the enhancement has been taken as a combination of electromagnetic and chemical enhancement. However, there are crucial differences where a colored adsorbate is adsorbed onto a metallic surface such that the plasmon resonance frequency and the frequency of an absorption band are close to the frequency of the incident light. This combination of surface enhancement and molecular resonance is called SERRS. Stacy and Van Duyne first reported SERRS in 1983. The increase in sensitivity over SERS can be quite large, with total enhancements of up to 1014 reported. When used practically, there are various combinations of laser frequency, plasmon resonance frequency, and molar absorptivity of the analyte that can be employed to obtain a condition that can be deemed to be SERRS rather than SERS. These are shown diagrammatically in Figure 1. If the excitation is matched to the molecular absorption maximum (Figure 1A), the surface selection rules expected for SERS are much less effective. This is due to the polarization of the laser excitation altering on interaction with the chromophore during the scattering process. This results in a degradation of the effective input polarization required for SERS selection rules, making SERRS less sensitive to the orientation of the molecule to the surface. This usually means that the spectrum resembles that of the resonance spectrum in solution, so that positive identification of the analyte is possible. In addition, the extra enhancement of the analyte discriminates against the detection of contamination. Thus, SERRS is more readily applicable for the development of methods for quantitative analysis. A second possible arrangement illustrated in Figure 1A is where the laser excitation is set away from the absorption maximum of the adsorbate and at the maximum of the plasmon resonance. This is described as preresonant scattering, and often SERRS taken in this way is written as SE(R)RS. The advantages of resonance still apply over quite a wide range of frequencies, gradually tailing off to SERS. Thus, SER(R)S makes it simpler to pick out individual
2
1
1
2
A B
(B) Wavelength (nm) Figure 1 The different arrangements for SERRS: the curves represent (A) molecular absorbance and (B) plasmon resonance. In (A) the molecular absorbance maximum and the plasmon absorbance maximum do not coincide: position 1 represents excitation at the absorbance maximum, 2 that at the plasmon maximum. In (B) the molecular absorbance and the plasmon maximum coincide: position 1 represents excitation away from the absorbance and plasmon maximum where the spectrum has a preresonant component, 2 that at the absorbance maximum and plasmon maximum. (Rodger C, Smith WE, Dent G, and Edmondson M (1996) Surface-enhanced resonance-Raman scattering: An informative probe of surfaces. Journal of Chemical Society, Dalton Transaction 791–799; reproduced by permission of The Royal Society of Chemistry.)
resonant molecules in the presence of a matrix of interferents, but the further away the excitation is from molecular resonance, the more the effect will be dependent on the angle of the adsorbate to the surface. For surface studies this is a key point, and consequently this arrangement may be preferred for some surface analyses. Figure 1B illustrates an alternative case in which the molecular chromophore coincides with the surface plasmon maximum. Similar considerations will apply to those discussed for the case represented in Figure 1A, but a further increase in sensitivity is likely with excitation at the molecular and plasmon resonance frequency. The orientation dependence will become more apparent the greater the difference in frequency between the excitation frequency used and the absorbance and plasmon maximum as the conditions become more appropriate for SERS (1 in
RAMAN SPECTROSCOPY / Surface-Enhanced 113
Intensity (arbitrary)
1652
200
1365
1513 1578
1313 1190
621
400
600
781
800 1000 1200 1400 1600 1800 Raman shift (cm−1)
Figure 2 Resonance Raman and SERRS spectra of rhodamine 6G with excitation at 514.5 nm. The high fluorescence background is effectively quenched in the SERRS spectrum due to adsorption of the rhodamine onto the surface of the silver nanoparticles. (Reproduced with permission from Rodger C and Smith WE (2002) SERS. In: Handbook of Vibrational Spectroscopy. New York: Wiley; & John Wiley & Sons Ltd.)
Figure 1B). As a consequence the sensitivity will decrease. Where SERRS can be applied, it offers many advantages over Raman scattering, resonance Raman scattering, and SERS. It provides both vibrational and electronic information on the adsorbate. Very wide concentration ranges can be studied, and single molecule detection is easier. A further major advantage is the quenching of fluorescence by the surface, which allows fluorescing dyes, which are ineffective in visible resonance Raman studies, to give very good SERRS. For example, rhodamine 6G fluoresces strongly in the visible region, preventing the use of normal Raman scattering, but gives good SERRS (Figure 2). In addition, there is less dependence of the signal on orientation. The combination of sensitivity, selectivity, and robustness of signal makes SERRS a very sensitive and selective form of vibrational spectroscopy that can be used semiquantitatively or qualitatively.
SERS Substrates SERS activity requires that very specific substrates be prepared. The metal chosen requires a plasmon in the frequency region close to that of the excitation laser. In addition, the ratio of scattering to adsorption of the substrate is important. Since visible frequency lasers are widely used in this technique, silver, gold, and copper, which have plasmons resonant with visible light, are obvious effective substrates. Silver is particularly useful, having a better ratio of scattering to adsorption in the visible region than gold. Copper tends to be too chemically reactive, and although other metals including lithium, sodium, and some
transition metals are effective, silver is by far the most widely used. It is possible to form reasonably time-stable roughened silver surfaces that can be used under normal laboratory conditions. Many types of SERS-active substrate have been made, each containing many microscopic metal domains. These include the following: electrode surfaces roughened by oxidation–reduction cycles; island films consisting of small metal particles; cold-deposited films prepared by evaporation or sputtering in a vacuum; lithographically produced assemblies; metal gratings; metal colloids prepared by reducing a dissolved metal salt with an appropriate reducing agent; metal colloids encapsulated in sol–gel-derived xerogel layers; silver particle-doped cellulose gel films; and microbeads as a carrier of silver colloid. Metal nanoparticles, usually prepared as colloidal suspensions, are one of the most widely studied SERS substrates. They exhibit unique optical properties due to excitation of the surface plasmons. The frequency of the surface plasmon resonance depends strongly on the size, shape, and dielectric environment of the particles. Additionally, when particles are closely spaced, the surface plasmons of individual particles interact to create new resonances dependent upon the interparticle distance and the incident angle and polarization of the light. Thus, optical properties of individual particles and arrays of interacting particles can be conveniently tuned. Significant progress has been made in controlling the size and shape of metal nanoparticles. Monodisperse single crystal nanocubes, nanorods, and nanoshells have all been prepared using simple methods. The size and shape of gold particles are more easily controlled than those of silver, and consequently silver-clad gold nanoparticles have been prepared and shown to possess distinct optical properties. Silver colloid is popular due to the relative ease of manufacture, low cost, and stability. One widely used method of silver colloid preparation is citrate reduction of silver nitrate (Figure 3). This results in an overall net negative charge on the colloid, which is high compared with many preparations (Figure 4). As a result, the colloid is stable for months and even years, and in some cases, with careful attention to detail, quantitative SERRS can be obtained from this colloid with relative standard deviations of less than 5%. There is, however, a change in enhancement with time, and so a standard needs to be used.
Studies of SERS/SERRS Enhancement The reason for SERS/SERRS enhancement has been studied by a number of groups. It has been shown that aggregated silver colloids produce fractal
114 RAMAN SPECTROSCOPY / Surface-Enhanced
Figure 3 A representative transmission electron microscopic image of citrate-reduced silver colloid. The particles are mainly hexagonal, with a largest face diameter of B36 nm. A few rods are present. (Reprinted with permission from Faulds K, Littleford RE, Graham D, Dent G, and Smith WE (2004) Comparison of surface-enhanced resonance Raman scattering from unaggregated and aggregated nanoparticles. Analytical Chemistry 76: 592–598; & American Chemical Society.)
O
H H
H
O
H
O
H O
O O
Ag
O
Ag
Figure 4 Representation of the surface of a Lee and Meisel colloidal particle. The citrate is believed to be bonded to the silver, which is present as AgI, with negatively charged carboxylate groups. (Rodger C, Smith WE, Dent G, and Edmondson M (1996) Surface-enhanced resonance-Raman scattering: An informative probe of surfaces. Journal of Chemical Society, Dalton Transaction 791–799; reproduced by permission of The Royal Society of Chemistry.)
clusters. Upon excitation, the electromagnetic field over a fractal object is not distributed uniformly but is localized in hot spots much smaller than the wavelength of light (10–30 nm). A large volume of the fractal therefore remains inactive or active at a lower level, while the portions of the fractal within these hot spots carry the full activity. This
localization of optical excitation indicates that very high electromagnetic fields lead to the huge enhancement of Raman scattering. Other experiments have shown an anomalously high enhancement in the first layer, suggesting the need to consider the charge transfer or chemical mechanism of enhancement as well as the electrochemical enhancement. Since only analytes attached to or close to metal surface are enhanced, the need for effective surface attachment is crucial if reproducible results are to be obtained. In many early experiments this was widely ignored. Commercial dyes were simply added to colloid or placed on surfaces and the results, where favourable, have been reported in many studies. The problem with this approach is that the surface chemistry of an element such as silver is complex. Little is understood about the surfaces on which the analyte is adsorbed, particularly in aqueous solution or, as is often done, with particles prepared in aqueous solutions and dried out on a surface for investigation. Further, where colloidal suspensions are used, it is common practice to aggregate the colloid in order to shift the frequency of the plasmon to a value that would place it in resonance with the laser. In fact, it is clear from ultraviolet–visible absorption that a range of clusters are made in most conditions and only a few are likely to be in resonance with the laser. However, this gives a bigger SERS enhancement than for single particles. The aggregating agent usually breaks up a stable colloid by reducing the charge on the surface. This can often happen by chemical action. Under the aerobic conditions used in most experiments, and shown in some to be essential for good SERS, the silver surface in the colloidal suspension is likely to be coated with a silver oxide or related layer containing silver(I) ions. It may also contain other adsorbed molecules from the preparation of the colloid. When a reagent such as sodium chloride is added to this surface, it is likely that a layer of silver chloride will form. This is known to be the case for electrode surfaces. With solid substrates, similar approaches are sometimes taken. For example, using the most commonly used analyte for fundamental studies, rhodamine 6G, the normal process for obtaining the best signals is to activate the surface with the sodium chloride. This particular combination of rhodamine and sodium chloride does give a more effective spectrum than without the addition of sodium chloride. However, studies of other analytes with colloidal suspensions have shown that sodium chloride is not always the most effective aggregating agent and no special activation procedures have been required. Additionally, with higher concentrations of rhodamine, not all the dye adsorbs and large fluorescent
RAMAN SPECTROSCOPY / Surface-Enhanced 115 NH2
OH N
MeO
OMe N
N
N
N (A)
N
NH
N
O2 N
NO2
(B)
Figure 5 Chemical structure of two dyes specifically designed to complex strongly to the silver surface for effective SERS. (A) 4(50 -azobenzotriazyl) 3,5-dimethoxyphenylamine; (B) 5-(2-methyl-3,5-dinitro-phenylazo) quinolin-8-ol.
backgrounds are obtained (see Figure 2). This can limit the use of SERRS and is often a problem where weak signals are obtained, and the temptation is to use higher, not lower, concentrations. Thus, for quantitative work and for obtaining a better understanding of the effect, effective surface adhesion is essential. In addition, the simpler the nature of the surface, the easier it is to understand the nature of the enhancement. For this reason, a set of special dyes has been developed for SERS/SERRS. These contain groups added to the dye specifically to obtain metal complexing with silver ions present on the surface. Two effective groups of this type are shown in Figure 5. One contains the benzotriazole group, known to be an effective method of preventing tarnishing in silver due to its strong attachment to the surface. The other contains an 8-hydroxyquinoline group, which will create a complex with silver ions on the metal surface that is insoluble. Both these reagents have proved to be effective for obtaining SERS/SERRS with sodium chloride and other aggregating agents, including organic aggregating agents such as poly-L-lysine.
Single Molecule Spectroscopy In 1997 two groups independently claimed single molecule detection using SERS/SERRS using different approaches. In some of the single molecule studies, the difference between SERS and SERRS is ignored, but most, not all, of the studies use chromophores. Nie and Emory reported detection of single rhodamine 6G dye molecules adsorbed on immobilized single silver nanoparticles using SERRS, and Kneipp et al. reported detection of a single molecule of crystal violet adsorbed on aggregated clusters of silver particles in a colloidal suspension using near infrared excitation. The large SERS
enhancement was attributed to excitation of the coupled surface plasmons of the colloidal aggregate. The SERS enhancement quoted by both groups for the detection of single molecules is of the order of 1014. Such enhancements are many orders of magnitude greater than predicted ensemble averaged values. This can be attributed to the removal of population averaging effects, whereby all the molecules and particles are assumed to contribute equally to the observed signals. Instead, only the active particle or active site is considered. Other groups have subsequently reported single molecule detection using SERS. Although detection of a single molecule adsorbed onto an immobilized single silver nanoparticle has been reported, studies on the protein hemoglobin adsorbed on immobilized colloidal particles revealed that the minimum state of aggregation necessary for SERS detection of a single hemoglobin molecule was the dimer. Detection of molecules adsorbed to single particles was not observed. Consequently, it is suggested that the surface enhancement is predominantly electromagnetic in nature and dominated by the increased local electric field between the two particles. Calculations suggest the maximum electromagnetic contribution to SERS enhancement for two interacting spheres to be of the order of 1011, and therefore an additional enhancement of 103 is required to explain the enhancement factors necessary for single molecule detection. This extra enhancement may be electromagnetic due to further surface roughness present on individual particles or may be chemical in nature. It is proposed that the additional electromagnetic enhancement is obtained only under special positions such as the interstitial site between two particles and outside sharp surface protrusions.
Applications of SERS Sensors
Due to the molecular specificity of SERS/SERRS, combined with its inherent sensitivity, it has found utility in a wide range of applications, only a few of which can be described here. The development of SERS sensor technology has allowed the detection and identification of seven structurally similar monosaccharides in aqueous solution using a sample volume of only 5 ml with a concentration of 1 10 2 mol dm 3. This has led to the initial development of a glucose-based biosensor with true in vivo, real time, minimally invasive sensing. The development of a volatile organic compound sensor using SERS detection has also been reported. The SERS substrate is chemically modified with a
116 RAMAN SPECTROSCOPY / Surface-Enhanced
thiol coating to prevent oxidation of the roughened silver surface and attracts the analyte of interest to the SERS surface. Detection of chlorinated solvents, aromatic compounds, and methyl t-butyl ether have all been demonstrated. Drugs
There is considerable interest in the development of novel platinum-based anticancer drugs that overcome the disadvantages associated with the widely used drug cisplatin, namely its inactivity against some types of tumors and toxic side effects. SERS has been shown to be suitable for the characterization of platinum complexes at physiological concentrations, allowing the determination of binding strengths of different ligands. The large SERRS intensities from the anticancer drug mitoxantrone have been used to provide a quantitative method of detection. The method uses a flow cell, which causes a dilution of plasma and serum samples, thus overcoming the fluorescent background. Even with these dilution steps the detection limits are superior to the previously published high-performance liquid chromatography (HPLC) method and are sufficiently good to be used for clinical analysis. Since the SERRS technique takes minutes from sample introduction to detection, and the chromatography technique hours, the SERRS technique could be considered the technique of choice for analysis of this drug. Intracellular SERS
SERS from living cells has been reported using several techniques. Etched and silver-coated glass fiber tips have been used as the SERS substrate, allowing the recording of spectra of biological samples, such as plant tissue and microbiological cells, with high spatial resolution. The deposition of colloidal gold particles inside single living cells by fluid-phase uptake has been demonstrated, providing strong SERS signals from the native chemical constituents of the cells. The sensitivity of SERS allowed measurements to be made in relatively short collection times (1 s for one mapping point) using 3–5 mW of near infrared excitation. SERS mapping over a cell monolayer with a 1 mm lateral resolution showed different Raman spectra at almost all places, reflecting the very inhomogeneous chemical constitution of the cells. This has opened up exciting possibilities for the study of cell biology and biomedical studies. Atomic Force Microscopy/Raman
Atomic force microscopy (AFM) uses a fine tip to probe surfaces. It has extremely high resolution. By
adding a roughened silver coating to AFM tips, a technique has been developed whereby when the AFM tip is touched on the surface it is irradiated with a laser, providing good SERS. The advantage of this technique is that the effective resolution of the technique is the dimension of the tip and consequently is much smaller than optical dimensions. The tip position is known from the AFM scan. This type of method has been used for analysis of thin films, which are undetectable with Raman microprobe systems but easily acquired with a suitably gold-coated AFM tip. More recently, a combined scanning electron microscope/Raman scattering detection system has been created. It is possible to detect the Raman scattering during the time of the SEM scan. This provides detailed structural information on the surface in a noncontact manner, which is faster and sometimes more effective than AFM. Since Raman scattering only occurs from the tip, and the tip position is known from the SEM, the dimensions of the tip are essentially the resolution of the instrument. DNA
The single molecule detection capacity of SERRS has been used to develop analytical techniques for DNA. These can use either a substrate or a colloid. In either case, this is most effective when the label is within the monolayer of the surface and hence the largest SERRS enhancement can be obtained. Methods have been developed that enable spatial separation of different events and their detection on a substrate. The use of SERS to monitor DNA hybridization of a fragment of the breast cancer susceptibility gene, BRCA1, on modified silver surfaces has been shown to be effective. A number of SERRS active DNA probes with different dyes have been synthesized that are used with colloids. Detection limits that are better than those obtained with fluorescence can now be obtained with SERRS. In addition, whereas fluorescence gives a very broad signal, SERRS gives a sharp signal and consequently is much more effective for multiplexing. This technique has been improved by the addition of a flow cell and in particular a lab on a chip flow cell, where control of the process of aggregation through the laminar flow region of the chip has shown a great improvement in quantitation as well as an improvement in sensitivity. The sensitivity of this method is known to be down to single molecule level, and it is possible to obtain this using commercial equipment within a maximum of 10 s. This technique is showing considerable promise for more effective multiplexed forms of DNA analysis (Figure 6). Recently, multiplexing of six probes with
RAMAN SPECTROSCOPY / Surface-Enhanced 117 8000
7000
Intensity
1650
1510
6000
1573
1360
Intensity
5000
550
1314 1441
1186
4000
1131
1632
3000
2000
1503 R6G
4000 3500 3000 2500 2000 1500 1000 500 0
1303
1000 HEX Blank 0 1000 1100 1200 1300 1400 1500 1600 1700 1800 Raman shift (cm−1) Figure 6 SERRS from 2,5,10 ,30 ,90 -hexachloro-6-carboxyfluorescein (HEX) and rhodamine 6G labeled oligonucleotides at 2 10 9 mol l 1 (1 s accumulation time). The unique SERRS spectral fingerprints from each dye allow multiplex analysis of SERRS/DNA complexes. (Reproduced with permission from Graham D, Mallinder BJ, and Smith WE (2000) Detection and identification of labeled DNA by surface enhanced resonance Raman scattering. Biopolymers (Biospectroscopy) 57: 85–91; & John Wiley & Sons, Inc.)
gold nanoparticles has been demonstrated, indicating the large multiple detection capability for SERRS/ DNA assays. Distance Detection
The ability to obtain sharp molecularly specific spectra from SERRS prompted the concept that a number of codes could be written simply by changing the combination of analytes and by changing their concentration. In this way, it is believed, hundreds of thousands of codes can be written that can be read using a Raman spectrometer without any spatial separation. The initial demonstration for this for one dye has shown that Raman scattering from suitable polymer films can be detected at up to 10 m (Figure 7) and therefore there is considerable potential for further expansion in this direction. Proteins
One of the first important demonstrations of SERRS was to detect heme groups in proteins. It is a good
1m
560
590 570 580 Wavelength (nm)
2&3 m 4&5 m 10 m 600
Figure 7 Change in spectral intensity as the distance from the sample is increased. (Reproduced with permission from McCabe A, Smith WE, Thomson G, et al. (2002) Remote detection using surface enhanced resonance Raman scattering. Appl Spectrosc 56: 820–826.)
example of the selectivity of the method in that practically no signals are obtained from the protein other than from the heme group. The heme on the other hand can be detected down to below monolayer coverage, although a monolayer is often used since it helps prevent protein degradation. A silver colloid stabilized with citrate layers also helps. The advantage of SERRS is that marker bands are available for the oxidation state, spin state, and coplanarity of the vinyl groups with the heme group. In addition, compared with resonance Raman scattering, it is easier to obtain SERRS from weaker chromophore absorption bands such as the Q band. Bacteria
The treatment of bacteria with sodium borohydride provides a nucleating substrate for the reduction of silver ions, forming a rough silver metal coating around the microorganism. Intense SERS of the coated bacteria showed four different types of bacteria to all produce similar spectra, suggesting that the spectra are selective and sensitive to a specific molecular species that dominates the spectra and that is found in all the bacteria analyzed. See also: Blood and Plasma. Clinical Analysis: Glucose. DNA Sequencing. Fluorescence: Overview. Forensic Sciences: Drug Screening in Sport. Microscopy Techniques: Electron Microscopy; Scanning Electron Microscopy; Atomic Force and Scanning Tunneling Microscopy. Nucleic Acids: Spectroscopic Methods. Raman Spectroscopy: Instrumentation. Sensors: Overview.
Further Reading Albrecht MG and Creighton JA (1977) Anomalously intense Raman spectra of pyridine at a silver electrode. Journal of the American Chemical Society 99: 5215–5217.
118 RAMAN SPECTROSCOPY / Surface-Enhanced Billmann J and Otto A (1982) Electronic surface state contribution to surface enhanced Raman scattering. Solid State Communications 44: 105–107. Creighton JA (1998) The selection rules for surface enhanced Raman spectroscopy. In: Clark RHJ and Hester RE (eds.) Spectroscopy of Surfaces, ch. 2, p. 37. New York: Wiley. Emory SR and Nie S (1997) Probing single molecules and single nanoparticles by surface enhanced Raman scattering. Science 275: 1102–1106. Fleischmann M, Hendra PJ, and McQuillan AJ (1974) Raman spectra of pyridine adsorbed at a silver electrode. Chemical Physics Letters 26: 163–166. Jeanmaire DL and Van Duyne RP (1977) Surface Raman spectroelctrochemistry. Part 1. Heterocyclic, aromatic, and aliphatic amines adsorbed on the anodized silver electrode. Journal of Electroanalytical Chemistry 84: 1–20. Kneipp K, Kneipp H, Irving I, Dasari RR, and Feld MS (2002) Surface enhanced Raman scattering and biophysics. Journal of Physics: Condensed Matter 14: R597– R624.
Kneipp K, Wang Y, Kneipp H, et al. (1997) Single molecule detection using surface enhanced Raman scattering (SERS). Physical Review Letters 78: 1667–1773. Moskovitis M (1978) Surface roughness and the enhanced intensity of Raman scattering by molecules adsorbed on metals. Journal of Chemical Physics 69: 4159–4161. Otto A, Mrozek I, Grabhorn H, and Akemann W (1992) Surface enhanced Raman scattering. Journal of Physics: Condensed Matter 4: 1143–1212. Stacy AM and Van Duyne RP (1983) Surface enhanced Raman and resonance Raman spectroscopy in a nonaqueous electrochemical environment: Tris(2,20 -bipyridine) ruthenium (II) adsorbed on silver from acetonitrile. Chemical Physics Letters. 102: 365–370. Xu HX, Aizpurua J, Kall M, and Apell P (2000) Electromagnetic contributions to a single-molecule sensitivity in surface enhanced Raman scattering. Physical Review E 62: 4318–4324. Xu HX, Bjerneld EJ, Kall M, and Borjesson L (1999) Spectroscopy of single haemoglobin molecules by surface enhanced Raman scattering. Physical Review Letters 83: 4357–4360.
REAGENTS See ANALYTICAL REAGENTS: Specification; Purification. RADIOCHEMICAL METHODS: Radio-Reagent Methods
REDOX INDICATORS See INDICATORS: Redox
REDOX TITRATION See TITRIMETRY: Overview; Potentiometric; Photometric
REFERENCE MATERIALS See QUALITY ASSURANCE: Reference Materials; Production of Reference Materials
REFLECTOMETRY See OPTICAL SPECTROSCOPY: Refractometry and Reflectometry