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Surface-Enhanced Raman Scattering (SERS), Applications
SURFACE-ENHANCED RAMAN SCATTERING (SERS), APPLICATIONS 2329
Surface-Enhanced Raman Scattering (SERS), Applications WE Smith and C Rodger, University of Strathclyde, Glasgow, UK Copyright © 1999 Academic Press
Surface enhanced Raman scattering (SERS) was first demonstrated by Fleischmann and colleagues in 1974. In a study of the adsorption of pyridine at a silver electrode, they noted that the Raman scattering was considerably stronger when the surface of the electrode was roughened. Jeanmaire and Van Duyne and Albreicht and Creighton reported that the Raman scattering from pyridine adsorbed on a roughened surface was enhanced by a factor of 106 compared to the equivalent concentration of pyridine in solution. This huge increase in signal stimulated a great interest in the technique and it remains one of its main advantages. The technique has been applied in many fields, including surface science, medicinal chemistry and analytical chemistry. Several books and reviews have been written: early developments were surveyed by Furtak and Reyez and Laserna has produced an informative overview indicating the potential to develop a powerful quantitative and qualitative analytical methodology. Chang and Furtak have written a comprehensive book on the subject. Articles directed towards specific applications include one by Vo Dinh targeted at chemical analysis and two by Nabiev and colleagues and Cotton and colleagues targeted at biological and medicinal applications.
The mechanism of the surface enhancement The nature of the mechanism that produces SERS is still the subject of debate. Two main mechanisms of enhancement are now most commonly proposed. These are electromagnetic enhancement and charge transfer or chemical enhancement. Electromagnetic enhancement does not require a chemical bond between the adsorbate and the metal surface. It arises from an interaction between surface plasmons on the metal surface and the adsorbed molecule. Chemical or charge transfer enhancement requires a specific bond between the adsorbate and the metal plus energy transfer between the metal and the adsorbate during the Raman scattering process. There is evidence for both mechanisms. The predominant view appears to be that both may occur.
VIBRATIONAL, ROTATIONAL & RAMAN SPECTROSCOPIES Applications Electromagnetic enhancement
On smooth surfaces, surface plasmons exist as waves of electrons bound and confined to the metal surface. However, on a roughened metal surface, the plasmons become localized and are no longer confined and the resulting electric field can radiate both in a parallel and in a perpendicular direction. When an incident photon falls on the roughened surface, excitation of the plasmon resonance of the metal may occur, causing the electric field to be increased both parallel and perpendicular to the surface. The adsorbate is bathed in this field and the Raman scattering is amplified. This mechanism has been studied and reviewed by Weitz, Moskovits and Creighton. Since SERS has been obtained from molecules spaced off the surface, the existence of enhancement from this type of mechanism is well established. Charge transfer enhancement
The enhancement from the charge transfer mechanism is believed to result from resonance Raman scattering from new resonant intermediate states created by the bonding of the adsorbate to the metal. The adsorbate molecular orbitals are broadened into resonance by interaction with electrons in the conduction band. Resonance states whose energies lie near the Fermi energy are partially filled, while those lying well below are completely filled. Otto has provided much evidence of the existence of this effect. He showed that there was a specific first layer and has extensively reviewed the field. Campion reported direct experimental evidence linking new features in the electronic spectrum of an adsorbate to SERS, under conditions where electromagnetic enhancements were unimportant. He noted that it was difficult to observe charge transfer only because the electromagnetic effects had to be accounted for and removed. This problem was overcome by conducting SERS on an atomically flat, smooth single-crystal surface where the electromagnetic effects were small and well understood. He adsorbed pyromellitic dianhydride (PMDA) on to copper(III) and observed an enhancement of a factor of 30. In addition, a low-
2330 SURFACE-ENHANCED RAMAN SCATTERING (SERS), APPLICATIONS
energy band in the electronic spectrum from the adsorbed PMDA was observed that was absent in the solution PMDA spectrum.
Selection rules Selection rules have been derived for electromagnetic SERS enhancement. The advantage of electromagnetic enhancement is that, since no new chemical species is formed on the surface, the selection rules can be based on the properties of the molecular adsorbate rather than on an ill-understood surface complex. In its simplest form, assuming no specific symmetry rules, the most intense bands are those where a polarization of the adsorbate electron cloud is induced perpendicular to the metal surface. However, more detailed selection rules can be obtained when the molecule has symmetry elements. Creighton and Moskovits have independently reviewed the principles.
Nature of the substrate The active substrates are usually made from a limited number of metals. Silver, gold and copper are the most commonly used SERS-active metals, although the use of lithium is well established. These substrates were chosen because their surface plasmons exist in or close to the visible region. Ideally, the excitation from the laser should coincide with the plasmon resonance frequency of the particular surface created and conditions such that the efficiency of absorption of the light is reduced and the efficiency of scattering increased. Silver is the most commonly used substrate, although gold is often used particularly in the near infrared. The original experiments used electrochemistry and this is a good method of obtaining a suitable surface. The scale of the roughness required is between about 40 and 250 nm for visible excitation with silver. SERS of pyridine obtained using an electrode setup results in certain bands appearing strongly in the pyridine spectra and the relative intensity and absolute intensity is dependent on voltage. The maximum enhancement is believed to be when the Fermi level matches the energy of the π orbital of pyridine. The electrode working surface can be difficult to reproduce and is prone to annealing with time in certain environments. However, sensitive qualitative analysis is feasible. Colloidal suspensions are particularly attractive as they can be prepared in a one-pot process and are inexpensive. Reliable SERS analysis is possible as a fresh surface is available for each analysis. Many different methods of colloid preparation have been re-
ported. Some groups always use freshly prepared colloid for their experiments, but recent emphasis has been on obtaining reproducible, monodisperse colloid that is stable for several months. In particular, colloid prepared by the citrate reduction of silver can be produced in almost monodisperse form and with a lifetime of up to one year or more. The particle size of these colloids varies. In one standard preparation of citrate-reduced silver colloid, a transmission electron microscopy study indicated that the particles were approximately 36 nm in their longest dimension and were small hexagonal units (Figure 1). Photoelectron correlation spectra of the suspension indicated that the average particle size approximated to a sphere was 28 nm. Metal colloidal particles adsorbed upon or incorporated into porous membranes such as filter papers, gels, beads, polymers, etc. have been developed as SERS-active substrates. Although these substrates are claimed to be reproducible, they are not widely used, probably because they involve complicated preparative procedures and are susceptible to contamination and self-aggregation. Ruled gratings can be used to give good reproducibility and abraded surfaces, although not so reproducible, they are attractive because of their simplicity of preparation. Numerous researchers have reported that immobilization of the colloidal particles as ordered arrays on films gives reproducible and sensitive SERS sensors.
Surface enhanced resonance Raman scattering Surface enhanced resonance Raman scattering (SERRS) is obtained by using a molecule with a
Figure 1 Transmission electron microscopy image of silver colloid, × 250.
SURFACE-ENHANCED RAMAN SCATTERING (SERS), APPLICATIONS 2331
chromophore as the adsorbate and tuning the excitation radiation to the frequency of the chromophore. The effect was originally reported by Stacey and Van Dyne in 1983. The enhancement obtained is very much greater than that of either resonance Raman or SERS, enabling very sensitive analysis and low detection limits to be achieved. Although SERRS is best considered as a single process, it arose experimentally from the combination of two previously studied effects, namely resonance scattering and surface enhanced Raman scattering (SERS). It is a unique process and different effects can be obtained depending on the nature of the chromophores used and the choice of laser excitation. Figure 2 illustrates the main choices. In Figure 2A, the molecular chromophore (curve a) is chosen not to coincide with the maximum of the plasmon resonance (curve b). If laser excitation at the molecular absorption maximum is used, the maximum contribution to the overall effect from resonance enhancement would be expected. With the arrangement in Figure 2A and with the excitation at the molecular resonance maximum, it has been reported that for azo dyes there is reduced sensitivity to surface enhancement mechanisms, providing a signal that is less sensitive to the nature of the surface and that has a recognizable molecular fingerprint related to the resonance spectrum, making this arrangement better for quantitative analysis. The second possible arrangement illustrated in Figure 2A is where the laser excitation is set off the frequency of the adsorbate resonance and at the maximum of the plasmon resonance (2). For resonance experiments on the molecule alone, this would be described as a preresonant condition and often SERRS undertaken in this way is written as SE(R)RS. More orientation information is to be expected and additional bands have been observed and assigned as due to mechanisms of surface enhancement. However, in this preresonant condition, the selectivity of resonance still applies. Thus, it is possible to pick out individual molecules in the presence of a matrix of interferents, but the effect will now be more dependent on the angle of the adsorbate to the surface. For many surface studies this is a key point and consequently this experimental process may be preferred for surface analysis. Figure 2B gives an alternative case in which the molecular chromophore (curve a) coincides with the surface plasmon maximum (curve b). Similar considerations will apply, but a greater increase in sensitivity is likely at the resonance and plasmon resonance maximum frequency. Hildebrandt and Stockburger carried out an extensive study on SERRS of Rhodamine 6G in order to explore the enhancement mechanisms involved. They
Figure 2 Illustration of the different arrangements for SERRS: curve a is the molecular absorbance and curve b is the plasmon absorbance. In (A) the molecular and plasmon absorbances do not coincide. Position (1) represents excitation at the molecular maximum and (2) represents excitation at the plasmon maximum. In (B) the molecular and plasmon maxima coincide. Position (1) represents excitation away from molecular and plasmon maxima and (2) represents excitation at the absorbance and plasmon maximum.
reported that two different types of adsorption sites on the colloid surface were responsible for the enhancement experienced: an unspecific adsorption site that had high surface coverage on the colloid surface resulted in an enhancement factor of 3000 and could be explained by a classical electromagnetic mechanism; a specific adsorption site was only activated in the presence of certain anions (Cl−, I−, Br−, F− and ). This specific site had a low surface coverage (approximately 3 per colloidal particle); however, an enhancement of 106 was claimed to result. This enhancement was believed to be due to a charge transfer mechanism. This study was continued into the near-infrared region and extended to include gold colloid and gold and silver colloid supported on filter
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papers. The enhancement experienced from anion activation with silver colloid was stronger by a factor of 47 in the near-infrared region compared with the visible region. The authors concluded that this phenomenon could be accounted for by the charge transfer transition being shifted towards the red for Rhodamine 6G, increasing the resonance effect in the near-infrared region.
Advantages and disadvantages of SERS/SERRS SERS incorporates many of the advantages of Raman spectroscopy in that visible lasers can be used so that flexible sampling is possible, and there is little signal from water so that in situ examination of such surfaces as those of colloidal particles in aqueous suspension or of electrode surfaces under solvent can be carried out. The greatest advantages are the sensitivity that can be obtained and the selectivity of the signals. Since SERS will detect compounds down to a level of about 10−9 M, adsorbates at monolayer coverage or less can be studied easily. Experiments on pyridine are a classic example. At well below monolayer coverage, pyridine is believed to lie with the plane of the ring almost flat on the metal surface. Under these conditions, there is very little intense Raman scattering since the main polarizability changes in the molecule are parallel to the surface. As the surface density of pyridine increases, the molecule is forced into a more vertical position and the signal begins to appear quite rapidly. This forms a good probe of when monolayer coverage occurs. Further, the existence of selection rules means that an indication of the nature of the surface processes can be obtained. There are a number of key limitations on the method. First, to obtain a large effect, SERS can be used only for adsorbates on a limited number of metal surfaces in correctly prepared (roughened) form. Second, the very large surface enhancement coupled to the need for a specific molecule to be adsorbed on the surface makes the technique prone to interference. Contaminants that give strong surface enhancement can be detected in much lower concentration than the adsorbate studied, leading to problems in identification. The additional complexity that the intensity of the bands depends on only partially understood selection rules and can change depending on the angle of the molecule to the surface and the degree of packing makes it difficult to assign bands. Finally, there is a tendency in SERS for photodecomposition to occur on the surface. Characteristic broad signals that have been reported as being due to specific
surface adsorbates are probably pyrolysed species on the surface. Notwithstanding these problems, SERS is unique in providing a fascinating insight into the adsorption mechanisms of molecules on suitable surfaces in situ. The technique of SERRS might be assumed to have some of the same disadvantages as SERS and more limitations, but in fact SERRS is proving to be a much more effective technique for analytical science. The major advantage of SERRS is that, if correctly applied, the chromophore signal dominates. Since related spectra are obtained by resonance from solution, the spectra on the surface can easily be recognized, and since the Raman signal from the chromophore is enhanced more than any other molecule this particular species is very readily identified at the surface. Thus, in contrast to the difficulty in assigning signals in SERS in some cases, the signal assignment in SERRS is often simple and reliable. Further, and rather surprisingly, a fluorescence quenching mechanism occurs on the surface so that both fluorescent and nonfluorescent dyes give good SERRS. Provided the molecule is attached to the surface, there is little fluorescence background. In fact, it is often useful to establish the fluorescence background against the strong SERRS signals in order to measure the degree of adsorption and desorption from the surface. Thus, a wide range of chromophores is available. Further, the technique requires very low laser powers and consequently the photodegradation common in SERS is seldom a problem. The characteristic spectra routinely observed with SERRS permit the identification of mixtures without the need for preseparation. Munro and colleagues have reported the analysis and characterization of 20 similar monoazo dyes, all of which produced unique characteristic spectra that in turn permitted the simultaneous analysis and detection of five dyes presented in a crude mixture. They addressed the problems associated with reproducibility and have focused much attention on improving and standardizing the production of the silver colloid generally used to obtain SERS. They concluded that, with careful attention to detail, a relative standard deviation (RSD) of 5% was routinely obtained.
Applications of SERRS and SERS The advantages reported above have been exploited in numerous research fields, including the following. Biochemistry SERRS methodology can be modified in order to provide a biocompatible environment for biological materials. The identification of watersoluble porphyrins and their photostability and
SURFACE-ENHANCED RAMAN SCATTERING (SERS), APPLICATIONS 2333
interaction with roughened metal surfaces have been reported. For copper chlorophyllin, spectra obtained by using different excitation frequencies permitted a better understanding of a complex system. SERRS enabled the identification of novel chromophores in eye lenses without preseparation of the crude mixture. Finally the oxidation and spin state of proteins such as myoglobin and cytochrome P450 can be probed, and the use of a fluorescent low-concentration protein solution to study labelled tyrosines has been reported. Medicinal chemistry SERRS has been used to detect the antitumour drug mitoxantrone and its interaction with DNA in situ. The adsorption of the drug complex onto a colloidal surface did not destroy or interfere with the native structure. Therefore, bonding information from the complex was extracted from the SERRS spectra. Selective detection on DNA at ultra-low detection by SERRS has been developed. Surface chemistry SERRS has been used to probe electrode surfaces in situ to extract structural information and to provide quantification. The in situ SERRS detection of compounds such as 2,4,6trinitrobenzene sulfonic acid covalently bound to tin oxide was observed when the chemically modified surface was coated with silver. The spectra collected provided rapid and sensitive structural information that was semiquantitative. SERRS has also been used to follow reactions occurring at well below monolayer coverage at roughened metal surfaces. Polymer science The ability to probe surfaces and boundaries using in situ SERS has been exploited extensively in polymer chemistry to characterize the surface of polymers for comparison with the bulk properties, and to determine the molecular geometry, orientation of polymer side groups adjacent to the metal surface and information on bonding, for example of polymermetal composites such as adhesives and coatings. Forensic science Modern Raman spectrometers connected to microscopes enable the examination of small amounts of material such as single fibres. The sensitivity and selectivity of SERRS can be exploited in forensic science by determining the nature of the dye mixture in situ from a single fibre, from an ink or from a lipstick smear. Corrosion science Studies of corrosion inhibitors, particularly for copper using SERS of the inhibitor adsorbed on the roughened metal surface, have been used to selectively identify the species. The limitation of requiring a roughened metal surface of a particular
metal can be overcome by applying colloid to a smooth nonactive surface, but this field has yet to be exploited. Practical uses of SERRS have been developed. It has been used to prepare a robust disulfide pH indicator by coupling pH-sensitive dyes methyl red, cresol violet and 4-pyridinethiol to cystamine, which adsorbs strongly to the roughened metal surface, forming monolayer coverage of the complex with colloidal silver and allowing strong SERRS to be recorded. Changes in the pH result in changes in the chromophores of the dyes that were easily detected by SERRS. As the SERRS spectrum obtained from the complex was pH sensitive, it was possible to obtain quantitative pH determination. Another example involves the exploitation of the sensitivity of the technique to analysis of trace amounts of nitrite in fresh and sea waters: sulfanilamide was added to the water sample and reacted with any nitrite present, forming a diazonium salt that was then coupled with ethylenediamine to produce an azo dye that was then detected by SERRS. An enhancement of a factor of 109, a relative standard deviation of 1015% and a limit of detection of picograms were reported. This method was superior to existing colorimetric and chemiluminescence techniques used to analyse the water samples for nitrite. Ultrasensitive detection of metal ions has been reported. A limit of detection at the nanogram level was claimed. The metal ions nickel or cobalt and a ligand, a mixture of 2-pyridinecarboxyaldehyde and 2-pyridinehydrazone or 1,10-phenolanthroline, form a complex on the roughened metal surface that is then detected by SERRS.
Single molecule detection Rhodamine 6G has been used extensively as a model dye to probe the nature of the SERRS effect. It is an extremely strong fluorophore when excited by visible radiation. Hence normal Raman is not observed except with near-infrared excitation. However, when SERRS is used the dye adsorbs strongly to the roughened metal surface and consequently this strong fluorescence is quenched and an extremely strong, enhanced Raman signal is observed. Figure 3 illustrates the resonance Raman and SERRS spectra collected from Rhodamine 6G. Attomolar levels (10−18 M) of detection have been reported for this system, which is approaching single molecule detection. The fluorescence-quenching properties of surface enhancement coupled with the additional sensitivity obtained from SERRS have been exploited by several researchers. Rhodamine 6G adsorbs very effectively on the roughened silver
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The resulting spectra provide molecular information and are unique to individual molecules. Sample preparation is simple and it is possible to undertake analysis in situ under water or in air or in vacuum. Since there are new selection rules and the effect is dependent on the metal used and the degree of surface roughness, there is a wealth of surface information to be obtained from SERS provided the limitations in terms of contamination and photodecomposition are remembered. The main problems are the limited number of surfaces to which the method can be applied and difficulties in interpreting the spectra. Additional advantages can be obtained from the use of SERRS. It is more sensitive and the resulting spectrum can be related back to the molecular resonance spectrum, making for more confidence in assignments. Fluorescence is quenched, signals from the adsorbate are much more intense than from contaminants and there is less dependence on the exact nature of the surface. This makes for unique applications for SERRS, which include simpler, more sensitive and more selective quantitative analysis and single molecule detection. Figure 3 Curve a: Solution spectrum from a 10−6 M Rhodamine 6G solution using 514.5 nm excitation, demonstrating the predominance of fluorescence over resonance Raman scattering. Curve b: SERRS spectrum taken from a suspension of aggregated silver colloid to which 150 µL of a 10−8 M Rhodamine 6G solution has been added using 514.5 nm excitation.
surface. However, the detection of single adsorbates of dopamine or phthalazine on colloidal clusters, with a limit of detection at picogram levels, illustrates that ultrasensitivity of this technique for other adsorbates is possible. The ability of SERRS to detect one molecule has recently been demonstrated by three groups. Nie has isolated colloidal particles with rhodamine adsorbed onto glass slides and obtained spectra from the individual particles. The particles are preselected for size to ensure that the surface plasmon of the single particle is in the visible region. Kneipp has used near-infrared anti-Stokes scattering and statistical methods to demonstrate that single molecules can be observed in suspension and Graham and colleagues have shown that one molecule of DNA labelled with a covalently attached fluorescein dye can be detected in the interrogation volume of suspended and aggregated colloid.
Conclusion In summary, surface enhancement results in a huge enhancement in Raman scattering and the ability to observe Raman signals at very low concentrations.
See also: Biochemical Applications of Raman Spectroscopy; Dyes and Indicators, Use of UV-Visible Absorption Spectroscopy; FT-Raman Spectroscopy Applications; IR and Raman Spectroscopy of Inorganic, Coordination and Organometallic Compounds; MRI of Oil/Water in Rocks; Polymer Applications of IR and Raman Spectroscopy.
Further reading Campion A, Ivanecky JE, Child CM and Foster M (1995) Journal of the American Chemical Society 117: 11 807. Chang RK and Furtak TE (1982) Surface Enhanced Raman Scattering. New York: Plenum Press. Cotton TM and Chumanov G (1991) Journal of Raman Spectroscopy 22: 729. Creighton JA (1988) In: Clark RIH and Hester RE (eds) Spectroscopy of Surfaces. Chichester: Wiley. Fleischmann M, Hendra PJ and McQuillan AJ (1974) Chemical Physics Letters 26: 163. Furtak TE and Reyez J (1980) Surface Science 93: 351. Laserna JJ (1993) Analytica Chimica Acta 283: 607. Moskovits M (1982) Journal of Chemical Physics 77: 4408. Nabiev I, Chourpa I and Manfait M (1994) Journal of Raman Spectroscopy 25: 13. Vo Dinh T, Alak A and Moody RL (1988) Spectrochimica Acta 43B: 605. Weitz DA, Moskovits M and Creighton JA (1986) In: Hall RB and Ellis AB (eds) Chemistry and Structure at Interfaces, New Laser and Optical Techniques, p 197. Florida: VCH.
Surface-Enhanced Raman Scattering (SERS), Applications WE Smith and C Rodger, University of Strathclyde, Glasgow, UK Copyright © 1999 Academic Press
Surface enhanced Raman scattering (SERS) was first demonstrated by Fleischmann and colleagues in 1974. In a study of the adsorption of pyridine at a silver electrode, they noted that the Raman scattering was considerably stronger when the surface of the electrode was roughened. Jeanmaire and Van Duyne and Albreicht and Creighton reported that the Raman scattering from pyridine adsorbed on a roughened surface was enhanced by a factor of 106 compared to the equivalent concentration of pyridine in solution. This huge increase in signal stimulated a great interest in the technique and it remains one of its main advantages. The technique has been applied in many fields, including surface science, medicinal chemistry and analytical chemistry. Several books and reviews have been written: early developments were surveyed by Furtak and Reyez and Laserna has produced an informative overview indicating the potential to develop a powerful quantitative and qualitative analytical methodology. Chang and Furtak have written a comprehensive book on the subject. Articles directed towards specific applications include one by Vo Dinh targeted at chemical analysis and two by Nabiev and colleagues and Cotton and colleagues targeted at biological and medicinal applications.
The mechanism of the surface enhancement The nature of the mechanism that produces SERS is still the subject of debate. Two main mechanisms of enhancement are now most commonly proposed. These are electromagnetic enhancement and charge transfer or chemical enhancement. Electromagnetic enhancement does not require a chemical bond between the adsorbate and the metal surface. It arises from an interaction between surface plasmons on the metal surface and the adsorbed molecule. Chemical or charge transfer enhancement requires a specific bond between the adsorbate and the metal plus energy transfer between the metal and the adsorbate during the Raman scattering process. There is evidence for both mechanisms. The predominant view appears to be that both may occur.
VIBRATIONAL, ROTATIONAL & RAMAN SPECTROSCOPIES Applications Electromagnetic enhancement
On smooth surfaces, surface plasmons exist as waves of electrons bound and confined to the metal surface. However, on a roughened metal surface, the plasmons become localized and are no longer confined and the resulting electric field can radiate both in a parallel and in a perpendicular direction. When an incident photon falls on the roughened surface, excitation of the plasmon resonance of the metal may occur, causing the electric field to be increased both parallel and perpendicular to the surface. The adsorbate is bathed in this field and the Raman scattering is amplified. This mechanism has been studied and reviewed by Weitz, Moskovits and Creighton. Since SERS has been obtained from molecules spaced off the surface, the existence of enhancement from this type of mechanism is well established. Charge transfer enhancement
The enhancement from the charge transfer mechanism is believed to result from resonance Raman scattering from new resonant intermediate states created by the bonding of the adsorbate to the metal. The adsorbate molecular orbitals are broadened into resonance by interaction with electrons in the conduction band. Resonance states whose energies lie near the Fermi energy are partially filled, while those lying well below are completely filled. Otto has provided much evidence of the existence of this effect. He showed that there was a specific first layer and has extensively reviewed the field. Campion reported direct experimental evidence linking new features in the electronic spectrum of an adsorbate to SERS, under conditions where electromagnetic enhancements were unimportant. He noted that it was difficult to observe charge transfer only because the electromagnetic effects had to be accounted for and removed. This problem was overcome by conducting SERS on an atomically flat, smooth single-crystal surface where the electromagnetic effects were small and well understood. He adsorbed pyromellitic dianhydride (PMDA) on to copper(III) and observed an enhancement of a factor of 30. In addition, a low-
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energy band in the electronic spectrum from the adsorbed PMDA was observed that was absent in the solution PMDA spectrum.
Selection rules Selection rules have been derived for electromagnetic SERS enhancement. The advantage of electromagnetic enhancement is that, since no new chemical species is formed on the surface, the selection rules can be based on the properties of the molecular adsorbate rather than on an ill-understood surface complex. In its simplest form, assuming no specific symmetry rules, the most intense bands are those where a polarization of the adsorbate electron cloud is induced perpendicular to the metal surface. However, more detailed selection rules can be obtained when the molecule has symmetry elements. Creighton and Moskovits have independently reviewed the principles.
Nature of the substrate The active substrates are usually made from a limited number of metals. Silver, gold and copper are the most commonly used SERS-active metals, although the use of lithium is well established. These substrates were chosen because their surface plasmons exist in or close to the visible region. Ideally, the excitation from the laser should coincide with the plasmon resonance frequency of the particular surface created and conditions such that the efficiency of absorption of the light is reduced and the efficiency of scattering increased. Silver is the most commonly used substrate, although gold is often used particularly in the near infrared. The original experiments used electrochemistry and this is a good method of obtaining a suitable surface. The scale of the roughness required is between about 40 and 250 nm for visible excitation with silver. SERS of pyridine obtained using an electrode setup results in certain bands appearing strongly in the pyridine spectra and the relative intensity and absolute intensity is dependent on voltage. The maximum enhancement is believed to be when the Fermi level matches the energy of the π orbital of pyridine. The electrode working surface can be difficult to reproduce and is prone to annealing with time in certain environments. However, sensitive qualitative analysis is feasible. Colloidal suspensions are particularly attractive as they can be prepared in a one-pot process and are inexpensive. Reliable SERS analysis is possible as a fresh surface is available for each analysis. Many different methods of colloid preparation have been re-
ported. Some groups always use freshly prepared colloid for their experiments, but recent emphasis has been on obtaining reproducible, monodisperse colloid that is stable for several months. In particular, colloid prepared by the citrate reduction of silver can be produced in almost monodisperse form and with a lifetime of up to one year or more. The particle size of these colloids varies. In one standard preparation of citrate-reduced silver colloid, a transmission electron microscopy study indicated that the particles were approximately 36 nm in their longest dimension and were small hexagonal units (Figure 1). Photoelectron correlation spectra of the suspension indicated that the average particle size approximated to a sphere was 28 nm. Metal colloidal particles adsorbed upon or incorporated into porous membranes such as filter papers, gels, beads, polymers, etc. have been developed as SERS-active substrates. Although these substrates are claimed to be reproducible, they are not widely used, probably because they involve complicated preparative procedures and are susceptible to contamination and self-aggregation. Ruled gratings can be used to give good reproducibility and abraded surfaces, although not so reproducible, they are attractive because of their simplicity of preparation. Numerous researchers have reported that immobilization of the colloidal particles as ordered arrays on films gives reproducible and sensitive SERS sensors.
Surface enhanced resonance Raman scattering Surface enhanced resonance Raman scattering (SERRS) is obtained by using a molecule with a
Figure 1 Transmission electron microscopy image of silver colloid, × 250.
SURFACE-ENHANCED RAMAN SCATTERING (SERS), APPLICATIONS 2331
chromophore as the adsorbate and tuning the excitation radiation to the frequency of the chromophore. The effect was originally reported by Stacey and Van Dyne in 1983. The enhancement obtained is very much greater than that of either resonance Raman or SERS, enabling very sensitive analysis and low detection limits to be achieved. Although SERRS is best considered as a single process, it arose experimentally from the combination of two previously studied effects, namely resonance scattering and surface enhanced Raman scattering (SERS). It is a unique process and different effects can be obtained depending on the nature of the chromophores used and the choice of laser excitation. Figure 2 illustrates the main choices. In Figure 2A, the molecular chromophore (curve a) is chosen not to coincide with the maximum of the plasmon resonance (curve b). If laser excitation at the molecular absorption maximum is used, the maximum contribution to the overall effect from resonance enhancement would be expected. With the arrangement in Figure 2A and with the excitation at the molecular resonance maximum, it has been reported that for azo dyes there is reduced sensitivity to surface enhancement mechanisms, providing a signal that is less sensitive to the nature of the surface and that has a recognizable molecular fingerprint related to the resonance spectrum, making this arrangement better for quantitative analysis. The second possible arrangement illustrated in Figure 2A is where the laser excitation is set off the frequency of the adsorbate resonance and at the maximum of the plasmon resonance (2). For resonance experiments on the molecule alone, this would be described as a preresonant condition and often SERRS undertaken in this way is written as SE(R)RS. More orientation information is to be expected and additional bands have been observed and assigned as due to mechanisms of surface enhancement. However, in this preresonant condition, the selectivity of resonance still applies. Thus, it is possible to pick out individual molecules in the presence of a matrix of interferents, but the effect will now be more dependent on the angle of the adsorbate to the surface. For many surface studies this is a key point and consequently this experimental process may be preferred for surface analysis. Figure 2B gives an alternative case in which the molecular chromophore (curve a) coincides with the surface plasmon maximum (curve b). Similar considerations will apply, but a greater increase in sensitivity is likely at the resonance and plasmon resonance maximum frequency. Hildebrandt and Stockburger carried out an extensive study on SERRS of Rhodamine 6G in order to explore the enhancement mechanisms involved. They
Figure 2 Illustration of the different arrangements for SERRS: curve a is the molecular absorbance and curve b is the plasmon absorbance. In (A) the molecular and plasmon absorbances do not coincide. Position (1) represents excitation at the molecular maximum and (2) represents excitation at the plasmon maximum. In (B) the molecular and plasmon maxima coincide. Position (1) represents excitation away from molecular and plasmon maxima and (2) represents excitation at the absorbance and plasmon maximum.
reported that two different types of adsorption sites on the colloid surface were responsible for the enhancement experienced: an unspecific adsorption site that had high surface coverage on the colloid surface resulted in an enhancement factor of 3000 and could be explained by a classical electromagnetic mechanism; a specific adsorption site was only activated in the presence of certain anions (Cl−, I−, Br−, F− and ). This specific site had a low surface coverage (approximately 3 per colloidal particle); however, an enhancement of 106 was claimed to result. This enhancement was believed to be due to a charge transfer mechanism. This study was continued into the near-infrared region and extended to include gold colloid and gold and silver colloid supported on filter
2332 SURFACE-ENHANCED RAMAN SCATTERING (SERS), APPLICATIONS
papers. The enhancement experienced from anion activation with silver colloid was stronger by a factor of 47 in the near-infrared region compared with the visible region. The authors concluded that this phenomenon could be accounted for by the charge transfer transition being shifted towards the red for Rhodamine 6G, increasing the resonance effect in the near-infrared region.
Advantages and disadvantages of SERS/SERRS SERS incorporates many of the advantages of Raman spectroscopy in that visible lasers can be used so that flexible sampling is possible, and there is little signal from water so that in situ examination of such surfaces as those of colloidal particles in aqueous suspension or of electrode surfaces under solvent can be carried out. The greatest advantages are the sensitivity that can be obtained and the selectivity of the signals. Since SERS will detect compounds down to a level of about 10−9 M, adsorbates at monolayer coverage or less can be studied easily. Experiments on pyridine are a classic example. At well below monolayer coverage, pyridine is believed to lie with the plane of the ring almost flat on the metal surface. Under these conditions, there is very little intense Raman scattering since the main polarizability changes in the molecule are parallel to the surface. As the surface density of pyridine increases, the molecule is forced into a more vertical position and the signal begins to appear quite rapidly. This forms a good probe of when monolayer coverage occurs. Further, the existence of selection rules means that an indication of the nature of the surface processes can be obtained. There are a number of key limitations on the method. First, to obtain a large effect, SERS can be used only for adsorbates on a limited number of metal surfaces in correctly prepared (roughened) form. Second, the very large surface enhancement coupled to the need for a specific molecule to be adsorbed on the surface makes the technique prone to interference. Contaminants that give strong surface enhancement can be detected in much lower concentration than the adsorbate studied, leading to problems in identification. The additional complexity that the intensity of the bands depends on only partially understood selection rules and can change depending on the angle of the molecule to the surface and the degree of packing makes it difficult to assign bands. Finally, there is a tendency in SERS for photodecomposition to occur on the surface. Characteristic broad signals that have been reported as being due to specific
surface adsorbates are probably pyrolysed species on the surface. Notwithstanding these problems, SERS is unique in providing a fascinating insight into the adsorption mechanisms of molecules on suitable surfaces in situ. The technique of SERRS might be assumed to have some of the same disadvantages as SERS and more limitations, but in fact SERRS is proving to be a much more effective technique for analytical science. The major advantage of SERRS is that, if correctly applied, the chromophore signal dominates. Since related spectra are obtained by resonance from solution, the spectra on the surface can easily be recognized, and since the Raman signal from the chromophore is enhanced more than any other molecule this particular species is very readily identified at the surface. Thus, in contrast to the difficulty in assigning signals in SERS in some cases, the signal assignment in SERRS is often simple and reliable. Further, and rather surprisingly, a fluorescence quenching mechanism occurs on the surface so that both fluorescent and nonfluorescent dyes give good SERRS. Provided the molecule is attached to the surface, there is little fluorescence background. In fact, it is often useful to establish the fluorescence background against the strong SERRS signals in order to measure the degree of adsorption and desorption from the surface. Thus, a wide range of chromophores is available. Further, the technique requires very low laser powers and consequently the photodegradation common in SERS is seldom a problem. The characteristic spectra routinely observed with SERRS permit the identification of mixtures without the need for preseparation. Munro and colleagues have reported the analysis and characterization of 20 similar monoazo dyes, all of which produced unique characteristic spectra that in turn permitted the simultaneous analysis and detection of five dyes presented in a crude mixture. They addressed the problems associated with reproducibility and have focused much attention on improving and standardizing the production of the silver colloid generally used to obtain SERS. They concluded that, with careful attention to detail, a relative standard deviation (RSD) of 5% was routinely obtained.
Applications of SERRS and SERS The advantages reported above have been exploited in numerous research fields, including the following. Biochemistry SERRS methodology can be modified in order to provide a biocompatible environment for biological materials. The identification of watersoluble porphyrins and their photostability and
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interaction with roughened metal surfaces have been reported. For copper chlorophyllin, spectra obtained by using different excitation frequencies permitted a better understanding of a complex system. SERRS enabled the identification of novel chromophores in eye lenses without preseparation of the crude mixture. Finally the oxidation and spin state of proteins such as myoglobin and cytochrome P450 can be probed, and the use of a fluorescent low-concentration protein solution to study labelled tyrosines has been reported. Medicinal chemistry SERRS has been used to detect the antitumour drug mitoxantrone and its interaction with DNA in situ. The adsorption of the drug complex onto a colloidal surface did not destroy or interfere with the native structure. Therefore, bonding information from the complex was extracted from the SERRS spectra. Selective detection on DNA at ultra-low detection by SERRS has been developed. Surface chemistry SERRS has been used to probe electrode surfaces in situ to extract structural information and to provide quantification. The in situ SERRS detection of compounds such as 2,4,6trinitrobenzene sulfonic acid covalently bound to tin oxide was observed when the chemically modified surface was coated with silver. The spectra collected provided rapid and sensitive structural information that was semiquantitative. SERRS has also been used to follow reactions occurring at well below monolayer coverage at roughened metal surfaces. Polymer science The ability to probe surfaces and boundaries using in situ SERS has been exploited extensively in polymer chemistry to characterize the surface of polymers for comparison with the bulk properties, and to determine the molecular geometry, orientation of polymer side groups adjacent to the metal surface and information on bonding, for example of polymermetal composites such as adhesives and coatings. Forensic science Modern Raman spectrometers connected to microscopes enable the examination of small amounts of material such as single fibres. The sensitivity and selectivity of SERRS can be exploited in forensic science by determining the nature of the dye mixture in situ from a single fibre, from an ink or from a lipstick smear. Corrosion science Studies of corrosion inhibitors, particularly for copper using SERS of the inhibitor adsorbed on the roughened metal surface, have been used to selectively identify the species. The limitation of requiring a roughened metal surface of a particular
metal can be overcome by applying colloid to a smooth nonactive surface, but this field has yet to be exploited. Practical uses of SERRS have been developed. It has been used to prepare a robust disulfide pH indicator by coupling pH-sensitive dyes methyl red, cresol violet and 4-pyridinethiol to cystamine, which adsorbs strongly to the roughened metal surface, forming monolayer coverage of the complex with colloidal silver and allowing strong SERRS to be recorded. Changes in the pH result in changes in the chromophores of the dyes that were easily detected by SERRS. As the SERRS spectrum obtained from the complex was pH sensitive, it was possible to obtain quantitative pH determination. Another example involves the exploitation of the sensitivity of the technique to analysis of trace amounts of nitrite in fresh and sea waters: sulfanilamide was added to the water sample and reacted with any nitrite present, forming a diazonium salt that was then coupled with ethylenediamine to produce an azo dye that was then detected by SERRS. An enhancement of a factor of 109, a relative standard deviation of 1015% and a limit of detection of picograms were reported. This method was superior to existing colorimetric and chemiluminescence techniques used to analyse the water samples for nitrite. Ultrasensitive detection of metal ions has been reported. A limit of detection at the nanogram level was claimed. The metal ions nickel or cobalt and a ligand, a mixture of 2-pyridinecarboxyaldehyde and 2-pyridinehydrazone or 1,10-phenolanthroline, form a complex on the roughened metal surface that is then detected by SERRS.
Single molecule detection Rhodamine 6G has been used extensively as a model dye to probe the nature of the SERRS effect. It is an extremely strong fluorophore when excited by visible radiation. Hence normal Raman is not observed except with near-infrared excitation. However, when SERRS is used the dye adsorbs strongly to the roughened metal surface and consequently this strong fluorescence is quenched and an extremely strong, enhanced Raman signal is observed. Figure 3 illustrates the resonance Raman and SERRS spectra collected from Rhodamine 6G. Attomolar levels (10−18 M) of detection have been reported for this system, which is approaching single molecule detection. The fluorescence-quenching properties of surface enhancement coupled with the additional sensitivity obtained from SERRS have been exploited by several researchers. Rhodamine 6G adsorbs very effectively on the roughened silver
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The resulting spectra provide molecular information and are unique to individual molecules. Sample preparation is simple and it is possible to undertake analysis in situ under water or in air or in vacuum. Since there are new selection rules and the effect is dependent on the metal used and the degree of surface roughness, there is a wealth of surface information to be obtained from SERS provided the limitations in terms of contamination and photodecomposition are remembered. The main problems are the limited number of surfaces to which the method can be applied and difficulties in interpreting the spectra. Additional advantages can be obtained from the use of SERRS. It is more sensitive and the resulting spectrum can be related back to the molecular resonance spectrum, making for more confidence in assignments. Fluorescence is quenched, signals from the adsorbate are much more intense than from contaminants and there is less dependence on the exact nature of the surface. This makes for unique applications for SERRS, which include simpler, more sensitive and more selective quantitative analysis and single molecule detection. Figure 3 Curve a: Solution spectrum from a 10−6 M Rhodamine 6G solution using 514.5 nm excitation, demonstrating the predominance of fluorescence over resonance Raman scattering. Curve b: SERRS spectrum taken from a suspension of aggregated silver colloid to which 150 µL of a 10−8 M Rhodamine 6G solution has been added using 514.5 nm excitation.
surface. However, the detection of single adsorbates of dopamine or phthalazine on colloidal clusters, with a limit of detection at picogram levels, illustrates that ultrasensitivity of this technique for other adsorbates is possible. The ability of SERRS to detect one molecule has recently been demonstrated by three groups. Nie has isolated colloidal particles with rhodamine adsorbed onto glass slides and obtained spectra from the individual particles. The particles are preselected for size to ensure that the surface plasmon of the single particle is in the visible region. Kneipp has used near-infrared anti-Stokes scattering and statistical methods to demonstrate that single molecules can be observed in suspension and Graham and colleagues have shown that one molecule of DNA labelled with a covalently attached fluorescein dye can be detected in the interrogation volume of suspended and aggregated colloid.
Conclusion In summary, surface enhancement results in a huge enhancement in Raman scattering and the ability to observe Raman signals at very low concentrations.
See also: Biochemical Applications of Raman Spectroscopy; Dyes and Indicators, Use of UV-Visible Absorption Spectroscopy; FT-Raman Spectroscopy Applications; IR and Raman Spectroscopy of Inorganic, Coordination and Organometallic Compounds; MRI of Oil/Water in Rocks; Polymer Applications of IR and Raman Spectroscopy.
Further reading Campion A, Ivanecky JE, Child CM and Foster M (1995) Journal of the American Chemical Society 117: 11 807. Chang RK and Furtak TE (1982) Surface Enhanced Raman Scattering. New York: Plenum Press. Cotton TM and Chumanov G (1991) Journal of Raman Spectroscopy 22: 729. Creighton JA (1988) In: Clark RIH and Hester RE (eds) Spectroscopy of Surfaces. Chichester: Wiley. Fleischmann M, Hendra PJ and McQuillan AJ (1974) Chemical Physics Letters 26: 163. Furtak TE and Reyez J (1980) Surface Science 93: 351. Laserna JJ (1993) Analytica Chimica Acta 283: 607. Moskovits M (1982) Journal of Chemical Physics 77: 4408. Nabiev I, Chourpa I and Manfait M (1994) Journal of Raman Spectroscopy 25: 13. Vo Dinh T, Alak A and Moody RL (1988) Spectrochimica Acta 43B: 605. Weitz DA, Moskovits M and Creighton JA (1986) In: Hall RB and Ellis AB (eds) Chemistry and Structure at Interfaces, New Laser and Optical Techniques, p 197. Florida: VCH.