Anal. Chem. 2000, 72, 145R-157R
Raman Spectroscopy Shawn P. Mulvaney and Christine D. Keating*
Department of Chemistry, 152 Davey Laboratory, The Pennsylvania State University, University Park, Pennsylvania 16802 Review Contents Books, Periodicals, and Reviews Instrumentation and Techniques Combination Techniques Raman Microscopy Other Advances in Instrumentation and Techniques Surface-Enhanced Raman Spectroscopy SERS Substrates SERS at Transition Metal Surfaces “Sandwich” Geometries in SERS Self-Assembled Monolayers Ultrasensitive SERS Ultrasensitive SERS for DNA Detection Biological Applications of Raman Spectroscopy Biomedical Applications of Raman Spectroscopy Raman for Diagnostics Pharmaceutical Applications General Information Materials Characterization Stationary-Phase Characterization Carbonaceous Materials Environmental Applications Reaction Monitoring New Technique: Two-Dimensional Raman Spectroscopy Prospectus Literature Cited
145R 147R 147R 149R 149R 149R 150R 150R 151R 151R 152R 152R 153R 153R 153R 154R 154R 154R 154R 155R 155R 155R 155R 156R 156R
This review of Raman spectroscopy covers the two-year period from late 1997 through late 1999. The previous two-year period has been comprehensively reviewed (1). There continues to be a great deal of activity in Raman spectroscopy, as this technique takes its place among the established workhorses for characterization and industrial applications. Due to the enormous volume of Raman literature, it has been necessary to limit the scope of this review. We have chosen to focus on the developments of greatest interest to analytical chemists and, therefore, have omitted many excellent works on applications of Raman, particularly in the areas of materials characterization and biochemistry. This review is not meant to be comprehensive, but rather to offer a glimpse of recent progress in Raman spectroscopy and impart a sense of where this technique is headed. A recurrent theme in Raman spectroscopy is the pervasive effect of technological advances in lasers, holographic notch filters, and detectors. Although these advances occurred prior to the twoyear period covered in this review, their effects are still being felt throughout the ever-growing Raman community. Experiments that only recently were thought to be impossible have become almost * Author to whom correspondence should be addressed: (e-mail) keating@ chem.psu.edu. 10.1021/a10000155 CCC: $19.00 Published on Web 05/10/2000
© 2000 American Chemical Society
routine. More accessible instrumentation has also led to adoption of this technique by an increasing number of nonspecialists, with applications to environmental chemistry, archeology, forensics, and even analysis of samples from space. Raman has been enthusiastically adopted by the materials community and is becoming more popular for industrial process monitoring. At the same time, surface-enhanced Raman scattering is poised to challenge fluorescence in ultrasensitivity. Already detection of single dye molecules has been reported by several groups; given the greater information content of vibrational spectra as compared to fluorescence, we expect ultrasensitive SERS to become increasingly important in chemical sensing. This review is organized into sections covering review articles, advances in instrumentation and techniques, progress in surfaceenhanced Raman scattering, and applications of Raman spectroscopy to materials, biological, and biomedical samples. Extensive coverage is given to surface-enhanced Raman scattering (SERS) not only because of the sheer number of SERS publications within the past two years but also because of the increasing relevance of this technique to analytical chemists. The time period covered by this review has seen important advances in SERS, including reports of single-molecule detection and of useful enhancements at surfaces other than noble metals. Other areas in which Raman spectroscopy shows considerable promise are as a detection method for a variety of separations techniques, where Raman offers not only elution profiles but structure-specific spectra of analytes, and in biomedical diagnostics, where it may be possible to generate Raman “fingerprints” for disease states. Finally, we will briefly mention a new direction in Raman spectroscopy, which currently remains in the realm of physicists: multidimensional pulsed Raman. These techniques may one day revolutionize Raman by enabling the use of pulsed techniques to gather information analogous to that now possible with 2-D NMR. Many hurdles remain before multidimensional pulsed Raman becomes analytically useful; however, we thought it worth mentioning at this time because of its potential impact on Raman spectroscopy. A short prospectus is given at the end of the review. BOOKS, PERIODICALS, AND REVIEWS In this section, we briefly describe some of the recent review articles concerning Raman spectroscopy which we expect to be of the greatest interest to the analytical community. In some cases, review articles have been included in later sections in order to maintain the continuity of the discussion. Strommen has written an accessible overview of Raman spectroscopy for the neophyte wishing to evaluate this technique for possible adoption (2). This review is packed with practical details such as instrumentation requirements, sample requirements, and even educational requirements for operators. Brief introductions to many of the special classes of Raman are given Analytical Chemistry, Vol. 72, No. 12, June 15, 2000 145R
(e.g., resonance Raman, Raman depolarization ratios, etc.), and a few specific applications are described. In the final section, entitled “Nuts and Bolts”, Strommen provides estimates of setup costs for various types of instruments, contacts for commercial Raman equipment, and suggested readings. This article is a good starting point for persons new to the technique. For those interested in more detail, an extensive review of instrumentation for Raman and IR spectroscopies has appeared (3). This review covers the historical development of instruments for vibrational spectroscopy, as well as modern instrument design and performance, including detectors, monochromators, and lasers. Callender et al. have reviewed Raman difference spectroscopy (RDS) for investigations of protein structure, binding, and enzymatic activity (4). It is only recently that, as a result of improvements in Raman instrumentation, RDS has become a viable method for protein studies. RDS is useful for zeroing in on small changes in the vibrational spectra of proteins, for which spectral crowding makes data difficult to interpret. The basis of the technique is collection of spectra for two samples identical except for the change under investigation (e.g., presence or absence of ligand, isotopic labeling). Split sample cells are employed and pains taken to ensure that the two “parent” spectra are as error-free as possible, so that small differences are interpretable. This review pays special attention to applications of RDS in protein folding and ligand binding for enzymatic catalysis. A review by Carey reflects on the first 25 years of Raman in enzymology (5). While Raman offers the ability to interrogate bond lengths and electron distribution in the active site of enzymes, few studies have yet made use of these advantages. Carey concludes that the future of Raman spectroscopy in elucidation of enzyme mechanisms is bright, due in large part to recent advances in instrumentation, including longer excitation wavelengths, which have led to better sensitivity and a reduction in fluorescence interference. Near-infrared (NIR) excitation has been particularly important for biological and biomedical applications of Raman spectroscopy. Fluorescence has been a limiting factor for Raman analysis of biological samples, particularly whole-cell or whole-tissue samples. As described in a review by Schrader et al., NIR excitation avoids interference from fluorescence and has enabled researchers to obtain spectra for a wide variety of biomaterials (6). Thomas has reviewed Raman spectroscopy for characterization of supramolecular assemblies of proteins and nucleic acids (7). This review first describes Raman spectroscopy in general, then relates recent developments in instrumentation, and finally discusses applications ranging from analysis of virus capsid assembly to elucidation of enzyme mechanisms. Information such as hydrogen bonding and the time dependence of molecular structure in large macromolecules can be obtained by Raman spectroscopy. The use of labels for Raman and resonance Raman (RR) spectroscopy of biological samples has been reviewed (8). Either type of label works by virtue of yielding a much stronger Raman signal than the surrounding macromolecule, thus enabling the properties of a particular site of interest to be probed (e.g., a chromophoric or highly polarizable substrate mimic). Although resonance Raman labels have been used for decades, recent developments in instrumentation have encouraged the use of 146R
Analytical Chemistry, Vol. 72, No. 12, June 15, 2000
nonresonant labels, which are highly polarizable molecules that are not resonantly excited. This review article discusses the advantages of both RR and normal Raman (NR) labels for investigation of biochemical systems. NR labels are preferred when photochemistry and/or fluorescence are significant problems for RR labels. Resonance Raman intensity analysis of excited-state dynamics has been reviewed by Myers (9). This review describes the theory of RR intensities, focusing on condensed-phase systems and on time-domain approaches. It then describes several applications of the theory to understand experimental data on excited-state dynamics. A second review on RR for the study of excited-state structure and ultrafast dynamics has also appeared (10). The use of experimental spectra and time-dependent wave packet dynamical calculations to elucidate chemical dynamics is discussed. Raman spectroscopy is a valuable method for materials characterization due to the high information content of Raman spectra, the ability to obtain spectra for nontransparent solids, and the advent of micro-Raman techniques, which permit characterization of small quantities of materials and/or small regions within a material. Consequently, interest in Raman spectroscopy for materials analysis continues to grow. Raman for diagnostics of semiconductor fabrication has been reviewed (11); in this article, the focus is on Raman from dry-etched planar surfaces, nanostructured surfaces, and doped quantum wires and quantum dots. Husson has reviewed Raman for the analysis of structural phase transitions, focusing on methods, recent advances, and examples of experimental data (12). Quantitative analysis of vibrational spectra for inorganic glasses (13) and IR and Raman spectra of volatile inorganic hydrides (14) are the subject of other recent reviews. The in situ analysis of aerosol particles using Raman has been reviewed (15), as has the role of Raman in high-pressure research (16). Raman has been popular for characterization of carbonaceous materials for some time. Areas of particular recent interest include the use of this technique for study of carbon nanotubes. A review on this subject has appeared (17), in which the authors compare high-pressure Raman studies of single walled carbon nanotubes with different phases of C60 and C70. A review of several vibrational spectroscopies for analysis of molecules at interfaces has appeared (18). This article covers techniques from IR and Raman to high-energy electron loss and sum-frequency generation. Particular attention is paid to reactions at technologically important surfaces and to vibrational dynamics. Raman is finding increasing application for in situ analysis of catalytic materials and surfaces. Not only analyses at high temperatures but also transient spectroscopy is possible with modern instrumentation. The pros and cons of Raman for such applications are presented in a recent review (19). The application of Raman spectroscopy for in situ studies of catalysts has been reviewed by Wachs (20). He concludes that Raman is gaining in popularity as a result of new sample cells, as well as its ability to provide vibrational information on catalyst structure and intermediates in situ. Several heterogeneous catalytic systems are discussed to illustrate the types of information that can be gained from Raman. Two reviews covering applications of Raman spectroscopy to combinatorial chemistry have been published in the last two years. In the first, the authors discuss Raman for chemistry validation,
library deconvolution, and structure elucidation and go on to describe acquisition of Raman in an agar matrix as well as automation of this technique for combinatorial chemistry (21). While Raman has several advantages for combinatorial analysis, the necessity for functional groups having high Raman cross sections (aromatics, thioethers, multiple bonds, and other highly polarizable groups) is a significant drawback at this time. Nevertheless, Raman has been applied to the validation of library chemistry and has been used to deconvolute both split and mixed libraries. The authors have set up an automated system capable of overnight acquisition of a large number of spectra. The second review is a more general discussion of optical spectroscopy in combichem (22). This article focuses on vibrational methods (i.e., IR and Raman) as the “premier analytical methods” for onsubstrate characterization. Coupling Raman spectrometers to optical microscopes ranging from standard and confocal far-field instruments to near-field scanning optical microscopes (NSOM) has been an active area of research in the last several years. Any of these combinations offers some degree of spatial resolution and structure-dependent contrast. An introduction to Raman microspectroscopy, including instrumentation and experimental considerations as well as advantages and limitations of the technique, has appeared (23). Another review on Raman chemical imaging spectroscopy (not necessarily microspectroscopy) stresses the impact of technological advances including improvements in liquid crystal tunable filters and image-processing capabilities (24). This article compares experimental approaches for Raman imaging microscopy, as well as several applications. The authors foresee continued growth in this technique, in particular for remote monitoring in harsh environments over time, as well as biomedical and forensics applications. Also mentioned are efforts toward development of a hand-held LCTF Raman imaging system for remote use via fiber optics. Applications of confocal Raman and fluorescence microscopies in industrial research have been reviewed (25). Each technique is described, including its use in correlation spectroscopy (both fluorescence correlation spectroscopy and Raman correlation spectroscopy), and a few applications are discussed. Both techniques are valuable for investigation of heterogeneous systems. The authors are particularly enthusiastic about the potential of Raman correlation spectroscopy for investigations of the dynamics of chemical species ranging from pigments to pharmaceutical formulations. Recent progress in confocal Raman microspectroscopy has also been reviewed (26). This article focuses in particular upon the use of this technique for remote analysis (up to several hundred meters away). Application of confocal Raman microscopy to analysis of materials, and in particular to polymeric materials, has been reviewed by Sammon et al. (27). Advances in Raman spectroscopy and microspectroscopy for polymer analysis (28) and uses of Raman for industrial polymer characterization (29) have been reviewed. The second of these articles discusses challenges for Raman analysis of polymers in an industrial setting, which include fluorescence, chromatic aberrations and turbidity of the polymer solutions. A review on the use of in-line fiber-optic Raman spectroscopy for following emulsion polymerizations has also appeared (30).
A collection of short papers (in many cases no more than extended abstracts) from the 1997 European Conference on Spectroscopy of Biological Molecules (ECSBM) have been compiled in Spectroscopy of Biological Molecules: Modern Trends. Raman spectroscopy featured prominently in this conference, and reports of Raman spectroscopy on protein, nucleic acid, polysaccharide, and drug samples are given. In addition, the section on biomedical, biotechnological, and environmental applications contains Raman studies of various healthy and diseased tissues, ranging from skin to liver (31). A short review of the potential for using surface-enhanced resonance Raman scattering (SERRS) in forensic analysis has appeared (32). The authors conclude that the high sensitivity and structure-specific information available for SERRS, combined with its nondestructive nature and the ability to detect several chromophores in a sample without prior separation, make it well-suited for forensic applications. Spectra for a variety of forensically important chromophores are presented (e.g., lipstick, shoe polish, and inks). Several special issues in Journal of Raman Spectroscopy have appeared in the period covered by this review; among the topics covered are Raman of proteins (33), resonance Raman (34), Raman in art, medicine, and archaeology (35), ab initio and density functional methods for Raman (36), and SERS (37). Many interesting articles appear in these issues, each issue providing highlights of active research areas in the field. Extensive reviews of vibrational spectra for compounds of main group elements and of transition metals have appeared (38, 39). In addition, a Handbook of FT Raman and Infrared Spectra of Polymers has been published (40). INSTRUMENTATION AND TECHNIQUES The breadth of instrumental advances impacting Raman spectroscopy was quite extensive in the past two years. For that reason, it is not possible to review all of the papers published. Instead we have opted to highlight the use of Raman spectroscopy in combination with other analytical techniques. Raman spectroscopy offers molecule-specific information, which is a distinct advantage over commonly used detection schemes (e.g., UVvisible). As Raman instrumentation has become more accessible, this technique has been embraced as a detection mechanism in a wide variety of separation methods. Following the review of the hyphenated techniques is a look at novel instrumental designs of interest to the analytical community. Combination Techniques. Michael Morris’ group and collaborators have reported on the combination of isotachophoresis and Raman spectroscopy. Their work included both capillary systems (41, 42) and microfabricated chip devices (43). UVvisible or fluorescence are the standard detection methods for isotachophoresis, however, the use of Raman facilitated simultaneous collection of qualitative and quantitative data. As a proof of concept, the authors demonstrated the separation and detection of adenosine, cytidine, guanosine, and uridine on a capillary system, with reported preconcentration values as high as 1000fold. The detection scheme used sulfur as an internal standard and subtracted the water background before Raman peak identification. However, a background arising from the silica fiber was present and 2 W of excitation was required to collect resolved Analytical Chemistry, Vol. 72, No. 12, June 15, 2000
147R
Raman spectra. These problems were addressed in the authors’ microfabricated chip device. In this construct, the microfabricated channels are covered by a thin glass cover slip. In this geometry, a microscope objective was selected based on collection distance instead of being limited by the need to match the numerical aperture of the silica fiber. Thus, the silica background was eliminated. Furthermore, the 40-µm-wide channels dissipate heat more efficiently than capillaries. The operation of the microchip device was demonstrated for the detection of two pesticides, paraquat and diquat, in the ppb range. A mixture of these pesticides was separated and detected by the combination of isotachophoresis and Raman in just 5 s. The combination of FT-Raman spectroscopy with solid-phase extraction (SPE) was recently explored by Strawn and co-workers (44). The novel aspect of this paper was that the spectroscopic analysis was performed on the intact SPE medium; typical SPE experiments involve the extraction of the analyte and then the subsequent detection after being eluted from the extraction phase. The authors presented data for the unambiguous detection of two photographic dyes on an aminopropyl extraction phase. The aminopropyl phase has minimal spectral activity in the 700-2000cm-1 range and, therefore, results in no background interference. Finally, the detection of an unknown dye was demonstrated by collection of the FT-Raman spectra and deconvolution with a commercially available spectral library. The dye identification was further confirmed with mass spectrometry and IR spectroscopy. While the authors present an elegant combination of analytical techniques, it is important to note that not all combinations of SPE and FT-Raman will have a near-zero background. Overlapping Raman bands are a real possibility for other pairings of analyte and SPE medium. Raman spectroscopy has also been implemented as a detection technique for HPLC analysis. This combination of techniques dates back to the early 1980s and, when coupled with SERS and resonant Raman enhancements, has detection limits as low as 10-15 mol/L. However, there were practical difficulties to the use of SERS films in HPLC and resonant enhancement required the analytes to be photostable. Bettermann and co-workers have proposed a new Raman-HPLC instrument that utilized neither enhancement technique but still has detection levels down to 10-6 mol/L (45). The authors attained their detection levels by using excitation of up to 7 W. A feedback mirror located after the sample cell returns the original 3 W of 488-nm light back through the sample, into the laser, and a final power of up to 8 W was measured at the sample. With this setup, the authors detected micromolar concentrations of p- and o-xylene. The two isomers can be distinguished on the basis of their Raman profile at these trace levels. The authors have built a highly sensitive RamanHPLC instrument, but its practical application will be limited to samples that can withstand high laser intensity. Meanwhile, several researchers were addressing the problems of implementing SERS with LC through new designs for flow injection analysis (FIA)-SERS flow cells. Carron and co-workers have designed a cascade flow cell with an etched Ag foil SERS film (46). The SERS substrate can be removed and replaced with a new substrate with relative ease. The remainder of the flow cell has a dead volume of 2.5 µL and was made out of Perspex, a material that can be polished for optical transparency and is inert 148R
Analytical Chemistry, Vol. 72, No. 12, June 15, 2000
to mild solvents (e.g., methanol). The operation of the flow cell was demonstrated with several aromatic compounds. Good detection limits were obtained for both single component and multicomponent samples. In this work, the SERS substrate was coated with propanethiol in order to facilitate reversible analyte interactions. Adsorption to these modified SERS substrates was described as analogous to that for reversed-phase chromatography. However, the optimal conditions for separations on C8 or C18 columns are not compatible with SERS detection. The modified SERS substrates performed best in 100% water; this may be a benefit in some types of analyses, e.g., water quality monitoring via FIA. Lendl and co-workers have proposed another flow cell made from Teflon and having a dual-injection design (47). The injection ports delivered either analyte solution or rinsing solution to the solid supported Ag film. The SERS film was rinsed with a 0.1 M NaOH or 3 M KCl solution between each sample run. Although these treatments significantly decreased the SERS enhancement of the Ag film, good reproducibility in SERS signal (5%) was obtained after the Ag film had gone through 10 wash cycles. This led to calibration curves with r2 values of 0.97. Another way to achieve enhanced Raman signals in LC without resorting to SERS or resonant excitation was found in the use of liquid core waveguides (LCW). LCWs operate on the principle of total internal reflection. This criterion is met when the index of refraction for the capillary cell material is less than that of the LC eluent. Only recently has a material with a low enough index of refraction (η) to be used with conventional solvents (ηwater 1.33) been made commercially available. The material of interest is a fluropolymer named Teflon AF and it has an index of refraction of 1.29. Teflon AF has been implemented by several groups with up to a 1000-fold improvement in sensitivity (48-50). Furthermore, it has been employed with cell volumes as low as 6 nL and in the detection of 2 ppm of 2-propanol. Nonetheless, larger enhancements will need to be realized for this technique to compete with some of the SERS-LC and resonant Raman-LC combinations already in use. Larger excitation powers and resonance enhancement are potential routes to achieve the desired sensitivity. Nikitin et al. have developed a hyphenated optical technique that combines surface plasmon resonance (SPR) with SERS (51) at a Si-based grating. In so doing, the often complimentary data of SPR and SERS can be simultaneously detected. This sensor was designed for compatibility with existing Si technology, with an eye toward miniaturization. The device was based on a Si grating that was coated with Ag, Au, and then phthalocyanine. In this scheme, the electromagnetic enhancement mechanism dominated the SERS signal and little background scatter was seen for the grating structure. The authors demonstrated ppb detection levels for the gas adsorption of NO2 onto the grating structure. Both the SERS and SPR channels tracked the surface-bound molecules as the gas pressure was cycled. FT-Raman spectroscopy has been used in conjunction with differential scanning calorimetry (DSC) (52). This combination technique allowed for the phase transitions in ammonium nitrate to be studied between room temperature and just below its melting point. A fiber-optic probe head was constructed in order to perform Raman measurements at elevated temperatures. The insertion of the probe head into the calorimeter resulted in only a 1 K change
in operating temperature, presumably due to altered gas flow, and a small broadening in the DSC curves. Furthermore, the two measurements could be made simultaneously, or if better resolution was desired, the FT-Raman spectrum could be gathered in the absence of the DSC measurement. Future application of the technique may involve increased sensitivity with the use of visible light, but that choice results in the loss of fluorescent discrimination associated with near-IR excitation. Raman Microscopy. Combining the lateral resolution of nearfield scanning optical microscopy with Raman spectroscopy has made nanoscale materials characterization possible. NSOM instruments achieve lateral resolution as high as λ/20 by forcing the incident light through an aperture smaller than λ and maintaining a tip-sample separation less than λ. The working distance between a sample and the aperture greatly affects the data collection. It has been shown that an exponential decay occurs with increasing probe to sample distance. Jordan et al. have demonstrated this decay for Raman intensity, reporting that a 50nm separation resulted in a 50% decay of Raman intensity (53). They measured near-field to far-field Raman intensity ratios of 5:1 and 9:1 for transmission and reflection, respectively. This type of information is valuable in assessing the local fields at the probe tip. A lateral resolution of 100 nm has been demonstrated for surface-bound DNA and a scratched silicon surface with SERSNSOM. Deckert et al. have used SERS-NSOM to detect dyelabeled DNA on Ag-coated Teflon microspheres (54). With this experimental setup, the authors were able to compare SERS and topographical information from the same surface sites. Therefore, they were able to probe regions of exceptionally high SERS enhancement (“special sites” or “hot spots”). The data suggested that a variety of “hot spots” exist and that they corresponded to locales on top of particles and between particles. The authors give a thorough description of what artifacts may lead to poor data interpretation. Their system allows them to monitor both reflected and Raman scattered light; this is useful for uncoupling the effects of local reflectivity from the surface-enhanced Raman signal. Meanwhile, Batchelder and co-workers have demonstrated a fully applied use of NSOM and Raman. They have imaged and determined the phases in and around a scratched silicon chip by Raman spectral profiles (55). They not only presented data for the damaged area but also measured the effect of stress on the surrounding area. The stress-damaged area was measured from a high-stress regime, 2-3 µm from the scratch, to an area of zero stress, 10-15 µm from the scratch. This work clearly demonstrates the commercial importance of a Raman-NSOM instrument in the silicon microchip industry. Other Advances in Instrumentation and Techniques. A variety of clever techniques have been implemented to expand or contract the dimensionality of Raman data. Barnett et al. have proposed the use of liquid tunable filters in order to collect Raman spectra at various wavelengths from the UV to the near-IR (56). The impetus for this work was to better differentiate between two materials based on shifts in the Raman spectrum due to changing wavelength. Thus, a fifth dimension has been added to their repertoire of X-dimension, Y-dimension, Raman shift, and Raman intensity. The authors demonstrated the multidimensional approach by examining GaN and diamond-like carbon films. Mean-
while, Ma and Ben-Amotz compressed 3-D (x-y-λ) data into a 2-D array (57). The authors have taken an 18-fiber bundle and aligned one end into a 2-D array. By doing so, all three dimensions of data can be gathered in every scan of the CCD. The operation of the instrument was reported for a mixture of KNO2 and K2SO4 salts where the salts’ lateral position and identity were determined in one scan. A new method for discriminating between fluorescence and Raman signals in luminescent samples has been described by Bell et al. This technique, called subtracted shifted Raman spectroscopy (SSRS), operates by taking several Raman spectra at different, but closely spaced spectrometer positions (58). This results in the removal of the fixed pattern irregularities found in spectra taken with a CCD camera. SSRS is analogous to shifted excitation Raman difference spectroscopy (SERDS) but has the major advantage of not requiring a tunable laser source. Furthermore, the error associated with spectral reconstruction can be determined by comparing shifts of δ and 2δ. A detailed discussion of data processing was presented, using cyclohexane in a solution containing fluorescent dye as the model analyte. SURFACE-ENHANCED RAMAN SPECTROSCOPY The field of SERS has arguably been the most active aspect of Raman research in past several years. Two recent developments in surface-enhanced Raman spectroscopy have had particularly significant impact. First, reports of extraordinarily high enhancement factors and single-molecule detection from several groups have raised the possibility that SERS may one day rival fluorescence as the ultrasensitive detection method of choice. A second development of potentially wide-ranging impact has been the finding that pinhole-free thin films of transition metals atop a SERSactive surface can be used as SERS substrates. This is an important result because a number of new surfaces are amenable to SERS study, among them surfaces important in heterogeneous catalysis. Other noteworthy developments in SERS include several recent reports of SERS “sandwiches”, in which the analyte is located between two metal features and hence experiences a greatly amplified electromagnetic (EM) field. Theory has long predicted large EM fields, and correspondingly large SERS enhancements, for such geometries, and previous experimental results have implicated such geometries. However, it is only within the past few years that “sandwich” structures have been intentionally prepared in a well-controlled fashion. This section also covers progress in SERS microscopy and remote sensing with metal-coated fiber-optic cables, as well as the movement toward harnessing large enhancement factors in DNA detection schemes. Two reviews on surface-enhanced Raman scattering have appeared, one by Campion and Kambhampati (59), and the other by Vo-Dinh, focusing on the use of metal nanostructured solid substrates (60). The review by Campion and Kambhampati is a good introduction for newcomers to this technique and will also be of interest to long-time SERS afficionados. After an introduction to SERS, its historical development, and the current understanding of this phenomenon, the authors focus on single-molecule/singleparticle SERS, fractal SERS substrates, and recent progress in understanding the chemical enhancement mechanism. They go on to predict continued progress in SERS, including a quantitative Analytical Chemistry, Vol. 72, No. 12, June 15, 2000
149R
elucidation of enhancement mechanisms within the next few years. This would indeed be an exciting development. The review by Vo-Dinh is narrower in scope, discussing SERS on metal-coated nanostructures on solid substrates. A wide variety of such substrates is described, and a theoretical background for understanding enhancement factors is given. Detailed information on preparation and characterization for several substrate types is given. The author then goes on to describe analytical applications for these substrates including a SERS-based dosimeter, singlemolecule detection, and several DNA detection schemes, from sequencing to gene probes. SERS Substrates. With the variety of SERS substrates available today, researchers are able to select a SERS substrate architecture to best match their experimental needs. Two recent reports have made this process substantially easier. Rowlen and co-workers made a comparative study of five common SERS substrates (61). They reported limit of detection data for 1,2-bis(4-pyridyl)ethylene (BPE) on electrochemically roughened electrodes, vapor-deposited Ag films, acid-etched Ag foils, Tollensproduced Ag films, and photoreduced Ag films on TiO2. Of these films postannealed, vapor-deposited Ag films were the most sensitive and the Tollens-produced films were the least. Viets and Hill have done a similar study for the comparison of fiber-optic SERS tips (62). In their work they compared tips coated with alumina/Ag, Ag islands, Ag-coated after sandblast treatment, and diamond/Ag film. All four preparations produced tips that were enhancing for the detection of thiophenol. No tip significantly outperformed another in terms of enhancement, so the authors argued that reproducibility, mechanical properties, and recyclability make the sandblasted tip best suited for “real” applications. The Ag film attached in this manner gains structural stability from a Cr underlayer and its nanoscale roughness from the sandblasted topography of the silica fiber. Therefore, they suggested that the fiber is recyclable because the Ag film can be removed and another attached with the same nanostructure. These two reports only scratch the surface of SERS substrate architectures but emphasize the need to compare substrate performance with one instrument and set of experimental parameters. A SERS substrate architecture not tested by Rowlen was the metal-coated dielectric nanoparticles proposed by Halas and coworkers (63). In this work, the authors coated monodisperse silica particles with thin layers of Au. Depending on the Au film thickness, the plasmon frequency could be shifted from the nearIR to the near-UV. Excitation was from a Nd:YAG laser at 1.06 µm. The optimal particle size for this wavelength was a 170-nm silica core with a discontinuous, 20-nm shell of Au. The discontinuous or islandlike Au film resulted in higher enhancement than continuous films, presumably due to the interisland “hot spots”. These particles have advantages in control over size and optical properties as compared to some traditionally prepared SERS substrates. Improvements in SERS substrates have also renewed efforts for quantitative measurements. Harris and co-workers have done a careful study on the dependence of the electromagnetic enhancement with changing analyte concentration (64). By controlling the analyte solution concentration and dipping procedure, Harris was able to apply a known concentration of analyte to an SiO2-overcoated Ag island SERS substrate. Using a fluores150R
Analytical Chemistry, Vol. 72, No. 12, June 15, 2000
cent dye as the analyte allowed the application of optical absorption and fluorescent measurements to follow analyte adsorption. The most significant result reported was that the enhancement drops by as much as 1 order of magnitude for coverage exceeding one monolayer. Meanwhile, the sensitivity remained constant for coverage up to 80% when benzoic acid was used as the analyte. In a similar study, Smith and co-workers quantitatively examined the resonant SERS response for dyes on aggregated colloidal Ag (65). Reproducibility measurements with colloidal aggregates were difficult to make, in part because it is nontrivial to determine the number of molecules adsorbed to the Ag aggregate. The authors acknowledged this point, and the importance of the paper lies in the examination of aggregation methods. They found that preaggregation gave the best reproducibility. From a selection of six aggregation agents, poly(L-lysine) and spermine showed the best reproducibility in SERS response. Quantitation was possible for some dyes, but not all. The authors conclude that the SERS response is effected by surface interactions between the dye and the Ag surface, and for some dyes, quantitation will require careful optimization of these interactions. Internal standards remain the most effective method to collect quantitative SERS and Raman data. Young and co-workers reported a diamond film coating on a silica fiber-optic probe which served as such an internal standard (66). The diamond film reduced optical transmission to ∼14% and resulted in a decreased Raman signal. However, the coated probe was effective for the detection of several solvents under varying experimental conditions. This flexibility was obtained by taking the ratio of the 1332cm-1 diamond band to the main band of each solvent. Linear calibration curves were obtained with r2 values of better than 0.98. While this paper does not utilize a SERS fiber-optic probe, it demonstrates the possibility of quantitative analysis with diamond/ Ag probes such as those examined by Viets and Hill. SERS at Transition Metal Surfaces. There has been a large effort in the Weaver group applying surface-enhanced Raman scattering to in situ monitoring of catalysis. This work is predicated on the finding that deposition of a “pinhole-free”, thin (1-2 nm) layer of non-SERS-active transition metals atop a roughened noble metal surface yields substrates with optical properties (i.e., SERS enhancement) characteristic of the underlying Au substrate but with the surface chemistry of the transition metal (67). Recent advances in electrodeposition of Pd, Rh, Pt, and Ir on SERS-active Au substrates coupled with voltammetric and vibrational tests for pinhole formation have made this technique applicable to a wider range of systems than previously thought possible. Using these surfaces, the Weaver group has obtained SERS spectra for benzene adsorbed at Pd in an aqueous medium (68). The choice of benzene was driven by the inability of competing in situ vibrational methods (i.e., infrared reflection-absorption spectroscopy and sum-frequency generation) to detect vibrations parallel to the surface, as are expected for benzene. Weaver has since gone on to use SERS at transition metal surfaces to investigate a wide variety of catalytic systems. A review appeared in 1998, detailing progress in coupling SERS with mass spectroscopy for real-time studies of heterogeneous catalysis (69). Since then, numerous reports on new systems have appeared. For example, the chemical vapor deposition (CVD) of the high-dielectric material tanatalum oxide onto a Pt surface has been monitored
in situ (70). In another study, the mechanism of oxidation of five metals (Pt, Pd, Ir, Rh, Ru) was studied by SERS (71). That useful enhancements could be obtained at these distances from the underlying SER-active Au film is somewhat counter to the conventional wisdom in the SERS community, where rapid decreases in enhancement have been observed for molecules located tens of angstroms from the metal surface. A recent paper reports the distance dependence of SERS from molecules adsorbed to Rh atop Au, for 5-200 monolayers of Rh (72). For five monolayers of Rh on Au, and enhancement factor of 5 × 104 is reported. Interestingly, it is found that the Rh itself is also SERSactive, with an enhancement factor on the order of 103 and at different wavelength dependence than Au. While this is not impressive as SERS enhancement factors go, when coupled with sensitive detection it is sufficient to allow monitoring of reactions at that surface. Taken together, the recent body of work in SERS of adsorbates at transition metal films is an exciting new direction in Raman scattering. Not only catalysis but any interfacial process at an increasingly large variety of metal films can now be monitored via this technique. “Sandwich” Geometries in SERS. Several authors have probed the SERS response for analytes that reside between two metal nanoparticles. This architecture is often referred to as “sandwich” geometry and the overall SERS enhancement is modulated by the interaction of the analyte with both nanoparticles. Keating et al. have presented an in-depth report on SERRS from sandwiches of the heme protein cytochrome c (Cc) between Au and Ag nanoparticles (73). In these experiments, half of the sandwich was an aggregate of colloidal particles, while the other half was a Cc-coated nanoparticle. Several types of sandwiches (Ag-Cc-Au, Au-Cc-Au, Ag-Cc-Ag) were examined with excitation wavelengths ranging from 488.0 to 647.1 nm. Great care was taken to ensure that results could be interpreted in terms of the effect of sample geometry and interparticle coupling. The wavelength dependence of SERS spectra for Ag-Cc-Au sandwiches evidenced a strong interparticle electromagnetic coupling. In addition, improved signal was obtained for Ag-Cc-Ag sandwiches as compared to Cc directly adsorbed to aggregated Ag sols under identical conditions. Another approach to probing the contribution of each metal nanoparticle’s role in a sandwich geometry was reported by Liu and co-workers. In this experiment, an azobenzene molecule with alkyl spacers of varying lengths was placed between an Au film and thermally deposited Ag islands (74). The data clearly demonstrated an exponential decay in SERS intensity as the azobenzene moiety becomes more distant from either metal layer. This paper marks the authors’ first efforts to probe the sandwich geometry and comparisons with theoretical prediction were preliminary, in part because the Ag islands were not as well characterized as separately prepared colloid nanoparticles. Nevertheless, the authors present an elegant method to examine the distance dependence of sandwich geometry SERS enhancement. A sandwich geometry immunoassay with SERS detection was developed by Ni et al (75). In this experiment, an antibody was immobilized on a gold film and an antigen was allowed to bind. Then a colloidal Au nanoparticle, which was tagged with a SERSactive molecule and the same antibody, was bound to the immobilized antigen. Thus, a sandwich geometry was created
between the gold particle and metal film. By choosing SERS tags with large cross sections, the authors improved the sensitivity of the method. A detection limit of 30 ng/mL was estimated for rabbit IgG. The authors also demonstrated that multiple immunoassays were simultaneously detectable. This was possible because the narrow bandwidth of SERS peaks (20 cm-1) eliminated peak overlapping and the specificity of the antigen-antibody interaction controlled nonspecific binding. Sarychev and co-workers observed similar sandwichlike enhancements for Ag island film SERS substrates (76). By probing their substrate with NSOM, they were able to locate the “hot spots” for SERS enhancement. The hot spots were located between two Ag island features, a geometry that closely resembles a sandwich motif. The nanometer-scale lateral resolution of the NSOM-Raman technique allowed the authors to isolate hot spots and compare their results with theoretical predictions. Self-Assembled Monolayers. Self-assembled monolayers (SAMs) and other surface coatings have served roles as varied as analyte, spacer molecule, or surface functionalization in SERS experiments. Three recent papers demonstrated the variety of use and importance of SAMs in SERS experiments. First, Norrod and Rowlen have shown that SAMs of decanethiol were an effective method to remove carbonaceous contamination from etched Ag foils (77). The thiol moiety’s strong affinity for noble metal surfaces led to the displacement of contaminants. The thiol layer was then removed by ozone treatment, leaving a weakly bound film of oxidized sulfur species (RSOx). This weakly bound film could be simply rinsed away, leaving a substrate with a 6-fold lower background signal and a 3-fold more intense spectrum for BPE. The authors attributed the increased SERS signal to a distance effect; the carbonaceous layer removed is ∼6 Å in thickness, a distance that can be theoretically shown to cause the change in signal intensity. While this treatment provides an effective cleaning method, it is not suitable for all SERS films because it alters the surface properties of some metal films, notably Ag island films (77). Second, Liu and co-workers reported the first determination of surface pKa of SAMS by SERS titration (78). By examining 4-mercaptopyridine on an Ag electrode, differences in spectral profile were interpreted in terms of pKa. The intensity ratio of bands at 1000 and 1100 cm-1 was plotted against pH to give a titration curve. These data were considered in conjunction with the Gouy-Chapman model to differentiate the surface and bulk pKa values. This method compared favorably with contact angle titration. A third example of SAM coatings in SERS experiments involves a thiolated cyclodextrin (CD) film attached to colloidal Ag. In this study, the complexation of azo dyes with free CD and surfacebound CD was probed using SERS (79). The complexation of the two dyes can be monitored by changes in the spectral profile that occur upon the binding event. From the data collected, association constants were calculated and the surface-bound film was shown to have a higher association constant. The authors further demonstrated that SAM layers of CD were stereoselective by comparing the binding of (S)- and (R)-o-methyl red. While the level of stereoselectivity was not impressive, this work lays the groundwork for further studies by SERS and possible optimization for sensor applications. Analytical Chemistry, Vol. 72, No. 12, June 15, 2000
151R
Azo dyes are often used in SERS studies because of their high polarizability and because an additional enhancement can be achieved by exciting with a wavelength that is resonant with a molecular transition. In surface-enhanced resonant Raman scattering, the overall enhancement can rival fluorescence in sensitivity. Graham et al. have synthesized the first dyes specifically intended for SERRS (80). Each of these dyes used benzotriazole as a surface-tethering group. This group not only shows a strong affinity for noble metals, it also binds in a manner that determines the orientation of the molecule. Controlling the surface orientation was important because it determines the position and relative intensity of the spectral bands observed. Four dyes (λmax of 360, 392, 435, and 488 nm) were made by attaching phenylamine analogues to the benzotriazole moiety. The SERRS spectra of each were shown for adsorption to Ag colloid. Ultrasensitive SERS. In light of the high information content of Raman as compared with fluorescence, this technique would seem to be preferable for trace analysis. Historically, however, Raman has been limited by poor sensitivity. Recent reports, by several groups, of single-molecule SERS may change this. There have been a sufficient number of publications claiming singlemolecule or few-molecule Raman detection that two reviews have been published on this topic in the last two years. While large enhancements are expected in SERS experiments at appropriately roughened surfaces, there remains some skepticism in the analytical community concerning claims of single-molecule SERS. Proving that signals result from single molecules is nontrivial. These issues are addressed in a short review consisting of an editorial introduction followed by statements from Kneipp and Nie in support of single-molecule SERS (81). A more comprehensive review on ultrasensitive SERS has also appeared (82). In this second review, Kneipp et al. give an introduction to the SERS effect and then a detailed discussion of SERS enhancement factors and cross sections, including a comparison of SERS cross sections to fluorescence cross sections. They then go on to discuss singlemolecule SERS (which had been observed for five different molecules), including a description of an experimental setup to conduct single-molecule SERS. Concluding with a critical analysis of single-molecule Raman, they summarize the evidence for singlemolecule SERS, which includes simple concentration/interrogated volume considerations, statistical analysis, and the observation of quantized and strongly polarized signals. While it seems likely at this point that single-molecule SERS has in fact been observed, it remains unclear to what extent we will be able to take advantage of ultrasensitive SERS in chemical analysis. To date, the molecules have been carefully chosen and often have benefited from resonant enhancement in addition to SERS. In addition, the highly enhanced molecules have not always been representative of the majority of analyte molecules in solution. An example of this is the work of Nie and co-workers, described below (83-85). The vast majority of colloidal SERS substrates found in the literature report enhancement factors (EF) that reflect the average population of the colloidal sol. Nie and co-workers have initiated fundamental research to determine the contribution of individual particles. In fact, Nie reports EFs up to 1015 for single Ag nanoparticles and 1014 for Au nanoparticles when the size has been appropriately matched with the excitation wavelength (83-85). 152R
Analytical Chemistry, Vol. 72, No. 12, June 15, 2000
By filter fractionation of a heterogeneous Ag colloid solution, narrow size distributions were isolated. The EF of these fractions was then tested with three wavelengths of excitation. This revealed that the optimal Ag particle size was 70, 140, and 190-200 nm for 488-, 568-, and 647-nm excitation, respectively. In a similar study, it was shown that 63-nm Au particles were optimal when exciting with 647-nm red light. Furthermore, TEM and AFM revealed that the “hot particles” have a 1.5:1 ratio for the major and minor axes. The identification of these hot particles was highly significant because they represent less than 1% of the colloidal sols produced. In fact, the truly “hot particles” only comprised ∼15% of the fractionated samples. While the EFs reported by these authors are difficult to rationalize on the basis of electromagnetic or chemical enhancement theories, the examination of individual properties was insightful and an important first step if nanoparticles are to be used as optoelectronic components, as biological detection labels, or in chemical sensors. Regardless of the precise value of the EF, it is clear from this work that the “hot particles” are responsible for the vast majority of signal from the sample and that they comprise but a small fraction of the total population. Ultrasensitive SERS for DNA Detection. Several groups have begun investigating the use of SERS for DNA hybridization assays. Kneipp and co-workers were active in this arena by applying their knowledge of near-IR SERS on Ag colloidal clusters (86). Near-IR SERS provides excellent discrimination against fluorescent interference and is nonresonant with most molecules; this allows greater excitation intensities without photobleaching or destruction of the analyte. Overall EFs on the order of 1014 were reported, and Kneipp used this advantage to detect singlemolecule DNA bases. The data show trace detection levels of adenine and AMP with well-resolved spectra. Indeed, the magnitudes of EFs observed in this study also lend credence to the surprisingly high values reported by Emory and Nie. Meanwhile, Vo Dinh and co-workers were examining DNA hybridization to construct a surface-enhanced Raman gene probe (87). In this work, the dye, cresyl fast violet, was attached to a DNA sequence as a Raman tag. After polymerase chain reaction (PCR), the DNA gene probe was exposed to target DNA and allowed to hybridize. The hybridized product was attached to a polystyrene bead and coated with 10 nm of thermally evaporated Ag; the Ag film had to be applied in the last step because it was adversely affected by PCR and the rinsing procedures. Proof-ofconcept data were given for the HIV gag gene and sensitivity and selectivity of the technique. Multiplexed experiments were a main advantage Vo Dinh offers for the SERS gene probe technique, but no data were presented. Still, this work marks the foundation of a potentially powerful gene identification technique. A SERRS technique that was sensitive enough to eliminate the use of PCR has been presented by Smith and co-workers (88). In this work, the DNA strands analyzed have a Raman active dye attached that is resonantly enhanced. Greater efficiency of dye excitation was achieved by studying the binding of DNA to the aggregated Ag colloid. The negative charge on DNA was limiting the number and conformation of DNA molecules binding to the Ag colloids. The authors circumvented this limitation by using spermine as an aggregant and also substituting several bases with propargylamino-modified analogues, thus facilitating a more favorable interaction with the Ag surface. The end result was that the
attached dye resides in closer proximity to the SERS surface and was therefore better enhanced. A limit of detection of 8 × 10-13 M for a 17-mer oligonucleotide was reported (88). This work did not detect the 17-mer in a sequence-selective fashion, and therefore, is not yet viable for detection of specific oligonucleotides. Nonetheless, the reported detection limit surpasses fluorescence, and approaches similar to this may lead to hybridization assays soon. BIOLOGICAL APPLICATIONS OF RAMAN SPECTROSCOPY Raman spectroscopy is a powerful tool for determining the active site structure of metalloenzymes, monitoring changes in protein structure during reactions or in response to changes in solution conditions, investigating substrate binding, etc. Just about every type of Raman study, from Raman difference spectroscopy to resonance and surface-enhanced Raman, has been employed in studies of biomolecules. Through these studies, Raman has played a critical role in many important discoveries in biomolecular structure and function and continues to do so. However, since this literature is vast and since the articles are primarily focused on the molecule of interest rather than the development of Raman scattering as an analytical technique, these papers are not reviewed here. In this section, we instead discuss several papers wherein Raman was applied to new systems, and which may result in new general applications for this technique. One example of a potentially far-reaching new application of Raman to biological systems is recent work by the Asher group, using nanosecond UVRR in combination with a laser-induced temperature jump, to follow peptide folding and unfolding kinetics (89, 90). UVRR selectively interrogates vibrations in the peptide backbone, making it well-suited to studies of protein secondary structure. This method joins fluorescence and IR as tools for studying the “protein folding problem” and may play an important role in predicting protein three-dimensional structure from amino acid sequence, which has been a longstanding goal of biochemists. Schwartz and Berglund report monitoring of protein concentration in 5-µL hanging drops during crystallization via Raman spectroscopy (91). While the measured standard deviations are rather large, and there is some concern over whether the laser light could cause slight heating of the droplets during measurement, this work is exciting in that it opens a new window into understanding protein crystallization through monitoring supersaturation levels in situ. A SERS-based assay has been developed for detection of membrane-bound prostaglandin H synthases (PGHS) in hepatocyte and hepatocellular carcinoma cells (92). The assay involves immunolabeling with specific primary and enzyme-labeled secondary antibodies. Enzymatic production of azoaniline, a strong Raman scatterer, is detected via SERS at Ag nanoparticles added to the sample. The entire assay is conducted and read out (using Raman microspectroscopy) in microwells. Cancerous cells were easily distinguishable by their elevated levels of PGSH-2. The sensitivity of this method (∼0.1 pg/well) was limited not by signal detection but by background signal from nonspecific binding and the presence of interference from other enzymes in the cells (92). Similar strategies could be adapted for detection of other biomolecules in cells.
SERS has been evaluated as a rapid, noninvasive method for the study of membrane transport processes (93). This work differed from previous studies in which diffusion through thin films onto bulk SERS substrates was measured in that the membranes were preformed rather than prepared atop the SERS substrate, and detection was at SERS-active colloidal Ag aggregates placed on the underside of the membrane. In this study, transport of two easily detectable probe molecules (pyridine and diphenyl disulfide) across a thin (0.02 in.) poly(dimethylsiloxane) membrane was followed as a function of time. The membrane and molecules in this paper were clearly chosen to illustrate the technique in the best possible light; most drug molecules are unlikely to give rise to SERS signals as strong as pyridine. Nevertheless, many drug molecules should be detectable, and this method is superior to standard Franz cell methods in speed and in its ability to take measurements in situ. Applications include analysis of transport across biological (e.g., skin) and nonbiological (e.g., food packaging) barriers (93). BIOMEDICAL APPLICATIONS OF RAMAN SPECTROSCOPY There continues to be interest in both in vitro and in vivo use of Raman spectroscopy of tissues for diagnostics. Autofluorescence from the biological materials (which can be very much larger than the Raman signal, even with NIR excitation) and interference from fiber materials used to deliver and collect light from the sample are the main obstacles to Raman spectroscopy of tissues. One recent study describes optimization of fiber-optic probes for an instrument designed for in vivo Raman scattering (94). These probes, which are designed with “in the tip” filters to suppress fluorescence and Raman from the silica fibers, and to improve collection efficiency, were tested in a turbid solution to simulate in vivo use. Raman for Diagnostics. NIR Raman has been evaluated as a diagnostic for cervical precancers (95). Although spectra could not be collected in vivo due to long integration times, Raman spectroscopy was found to have specificity superior to standard methodologies (i.e., coloscopy and cytology) for differentiating squamous intraepithelial lesions (SIL) from non-SIL. Confocal Raman spectroscopy has been used for in vivo monitoring of concentration and transport of a topically applied drug through the rabbit cornea (96). The effect of the drug on corneal hydration was also monitored. The authors determined that confocal Raman offered sufficient sensitivity, specificity, reproducibility, and spatial resolution to be promising for ocular pharmacokinetic studies. Cancerous and noncancerous cells have been distinguished on the basis of their UV-resonance Raman spectra with 257-nm excitation (resonant for nucleic acid absorbance ∼260 nm) (97). Spectra acquired for suspensions of normal and malignant cultured breast and cervical cells showed features attributable to DNA, RNA, and tyrosine. The ratios of peaks at 1480/1614 and 1480/ 1540 cm-1, which relate nucleic acid and protein concentrations, were elevated in malignant cells. The ratio of 1330/1480 cm-1 intensities also was found to be a marker of cell type and may result from changes in the vibrational structure of the nucleic acids in malignant cells. Two publications on using Raman in the characterization of human coronary artery have appeared, both from the same group. Analytical Chemistry, Vol. 72, No. 12, June 15, 2000
153R
They describe early steps toward development of an intravascular technique for analysis of arterial tissue in vivo based on Raman (98, 99). Spectra would be acquired via fiber-optic probes, either during surgery or by catheterization. Current efforts are focused on developing the scientific basis and technology upon which this technique would be based. In a 1997 study, NIR Raman spectra were acquired for coronary artery segments, and the relative amounts of various chemical components (e.g., cholesterol esters, calcium salts, etc.) were determined via fitting to a model by assuming the tissue spectrum resulted from a linear superposition of individual components (98). Seven components were necessary to obtain good fits. Results from Raman agreed well with standard chemical assays for samples from a variety of disease states. Since in principle Raman could acquire this information nondestructively, it may provide a powerful new means to ascertain the chemical composition of artherosclerotic lesions in vivo. In a 1998 paper, the authors move closer to their goal, by collecting spectra for artherosclerotic plaques in intact sections of human coronary artery (99). The ability to detect plaques was investigated as a function of their depth in the artery wall. This was done in a controlled fashion by placing tissue atop the plaques to increase their “depth”. The results from Raman correlated well with other techniques for determination of cholesterol content. Pharmaceutical Applications. Raman is finding applications in the pharmaceutical industry. Two recent examples are given to highlight uses of this technique. First, McCreery et al. have reported the use of NIR Raman spectroscopy for identification of pharmaceuticals inside amber USP vials (100). Although the amber glass greatly attenuates the signal, adequate spectra were obtained for determination of vial contents with 1-60-s integration. Identification was performed using a library of spectra and ranged in accuracy from 88% to 96%. Most of the errors were due to fluorescent samples. When these samples were omitted, a marked improvement in accuracy of the search algorithm resulted. This work demonstrates the potential of Raman for on-line process monitoring. In a different type of pharmaceutical application, confocal Raman imaging was used to map solid dispersions of drugs in polymeric matrixes (101). This method was found to be a promising tool for monitoring the spatial distribution of drugs in solid dispersions and to detect changes in formulation (e.g., recrystallization). General Information. Hyperspectral Raman imaging of mature and newly generated mammalian bone has been used to map the distribution of PO43- and HPO42-. Newly generated bone shows sharper gradients of these ions than does mature bone tissue (102). Freeman et al. have used Raman microscopy to generate intracellular concentration maps for zinc pthalocyanines, which are potential photodynamic therapy (PDT) agents (103). PDT is used to selectively damage tumor cells by light-activated generation of singlet oxygen; thus, the localization of these compounds is critical to their efficacy. The Raman maps generated in this paper are of lower resolution than can be acquired by fluorescence; however, use of Raman allows data to be collected without activating the PDT agents. This is a significant advantage, since activation can result in their relocalization. Finally, an entire symposium on Biomedical Applications of Raman Spectroscopy was held at the conference of The Interna154R
Analytical Chemistry, Vol. 72, No. 12, June 15, 2000
tional Society for Optical Engineering and The International Biomedical Optics Society, held in 1999 in San Jose, CA (104). The proceedings volume for this series contains a wide variety of reports of work in progress in this area. Sessions were held on histopathology, pharmaceutical analysis, dermatology and opthamology, Raman imaging, instrument development, and surfaceenhanced Raman scattering. MATERIALS CHARACTERIZATION There have been hundreds of references on the use of Raman spectroscopy in materials characterization and applications in the past few years. This volume of literature resoundingly indicates that Raman is fulfilling its potential in industrial circles. In light of this development, extensive coverage was afforded the topic in our last review of Raman spectroscopy. In this installment, we have only selected a few papers to highlight some important advancements since that review was published. Stationary-Phase Characterization. Several authors have looked at the conformation of and bonding to stationary phases with Raman spectroscopy. For example, Ho and Pemberton have studied responses in octadecylsilane packing conformation in response to temperature changes (105). The impetus for this work was that partitioning into stationary phases is not fully understood and knowledge of the alkyl chain conformation will facilitate a fundamental understanding. Raman is a good technique to study alkyl chains because it can probe the C-C and C-H bonding environments in the presence of H2O, a condition not met by IR spectroscopy. Stationary phases at temperatures between -15 and +95 °C were examined and chain ordering was evident in the lower temperature samples. Furthermore, the degree of chain conformity was also affected by the number of silane tethering points to the silica column, with more defects being present for multiply bonded alkyl chains. Pemberton’s work marks a significant step forward in understanding stationary-phase behavior, but a full understanding will require additional studies with more solvents. In another study, Horvath and co-workers have examined bonding of serine, phenylalanine, and mandelic acid enantiomers to a chiral stationary phase (106). The goal of this work was to elucidate the binding interaction and thus create a knowledge base for selecting the stationary phase that best matches a set of analytes. Through a detailed study of amide and ring stretching bands, it was determined that hydrogen bonding and π-stacking were the predominant interaction mechanisms for these three sets of enantiomers and N-3,5-dinitrobenzoyl-L-leucine. Rahman and co-workers have examined organic compounds on single resin beads for combinatorial libraries (107). In this work, IR- and Raman-active groups were attached to the growing organic construct in order to monitor the combinatorial synthesis. Raman tag molecules were selected on the basis of their spectral profile, such that they have a characteristic band between 2000 and 2300 cm-1. By using these two microscopy techniques, the authors have developed an identification method that was both in situ and considerably faster than analysis of the compound after cleaving from the support resin. Moreover, spectroscopic evaluation of libraries has the potential to revolutionize the combinatorial synthesis process; the attachment of a molecular tag is an excellent way to facilitate the use of IR and Raman microscopy in these studies.
Carbonaceous Materials. Raman spectroscopy has become a significant characterization technique for carbon nanotubes. In fact, so many papers have been published that the topic could be reviewed in and of itself. The most intriguing of these papers, reported on the detection of nanotubes of finite size (108). It was demonstrated that the length of single-walled nanotubes can be determined by measuring the Raman intensity of the z-axis breathing modes. For nanotubes of the same diameter, a linear relationship between tube length and the intensity of the z-axis breathing exists. It was also possible to determine which nanotubes were kinked or nonlinear. However, length measurements on nanotubes were nontrivial to make, became less effective with longer nanotubes, and were difficult for samples with a heterogeneous distribution of diameters. Nonetheless, a spectroscopic analysis of nanotube length would be a powerful way to characterize a sample of single-walled carbon nanotubes. Raman microscopy was employed by McCreery and coworkers to study defect sites on and adsorption to carbon electrodes (109). The glassy carbon (GC) or highly ordered pyrolytic graphite (HOPG) electrode was contained in a specially designed Teflon cell. First, the heterogeneity of the carbon surface was probed by comparing the ratio of D band (∼1360 cm-1) to the E2g band (∼1582 cm-1). From these data, seven distinguishable areas were assigned. Next, the physisorption of Rhodamine 6G revealed that adsorption occurred at disordered regions, more particularly at defect sites. In contrast, chemisorption of dinitrophenylhydrazine occurred at edge planes where carboxyl groups were localized. These assignments came from Raman spectra that reveal both the adsorbed analyte and the carbon understructure. These results represent the first spatially resolved look at physand chemisorption on carbon surfaces. Environmental Applications. The environmental community continues to embrace Raman spectroscopy because nature’s preferred solvent, water, interferes minimally. The ability of fulvic and humic acids to bind environmental analytes, specifically transition metal cations, remains a major difficulty, as this renders them unavailable for detection by most methods. Liang et al. have attacked the problem from a new direction by examining the binding sites of organic matter with SERS (110). In this work, humic and fulvic acids adsorbed to a roughened Cu electrode were probed with SERS. The spectra demonstrated that carboxylic groups and pyridine-substituted rings were the major binding domains. The authors took advantage of the distance dependence of SERS enhancement to only probe the binding domains; the bulk of the organic acids reside too far away from the Cu electrode to be SERS enhanced. Instead well-resolved and simple spectra of the binding domain were collected and provide a more fundamental understanding of how organic acids interact with metal species. A continuous method for the detection of trace organic pollutants by flow injection analysis and SERS has been developed (111). This new method used near-IR excitation and FT-SERS detection to look at ppm aqueous pesticide solutions. While the authors chose a pair of pesticides, carbenazim and metazachlorine, with decidedly different binding affinities for the Ag film, they demonstrated the ability to simultaneously detect two analytes with their SERS-FIA system. Introducing NaOH between samples affectively cleaned the SERS film, but this treatment led to a
decrease in EF with each treatment. This problem needs to be resolved before SERS-FIA will be robust enough to be a common detection method. Reaction Monitoring. Raman spectroscopy remains an effective method to track reactants, intermediates, and products during chemical reactions. Langkilde and co-workers have used Raman to monitor the synthesis and hydrolysis of ethyl acetate (112). The relative concentrations of ethanol, acetic acid, and ethyl acetate were determined from their Raman spectra. Continuous monitoring of the reaction and subsequent chemometric analysis revealed that the reaction was of first-order kinetics. Furthermore, the calculated rate constants agreed well with literature values. On a similar note, Oyama and co-workers have monitored the decomposition of ozone on a manganese oxide catalyst (113). The presence of a peroxide intermediate was determined by in situ Raman measurements and a careful study of oxygen isotope ratios. The reaction process determined involves the adsorption of O2 and monotonic O species, reaction of the O species with ozone to form a peroxide, decomposition of the peroxide intermediate, and desorption of the peroxide and molecular oxygen. These two studies reveal the power of Raman spectroscopy for monitoring reactions in progress. NEW TECHNIQUE: TWO-DIMENSIONAL RAMAN SPECTROSCOPY The last two years have seen a precipitous increase in papers concerning two-dimensional Raman scattering. While this technique is not yet close to becoming analytically useful, we consider it briefly here because of its great potential for opening up an entirely new realm of Raman spectroscopy, analogous in many ways to 2-D NMR. While significant advances in both theory and experiment are needed before these methods can become useful to any but a small class of simple molecules, this area is worth keeping an eye on. Tokmakoff et al. have used 2-D Raman spectroscopysin particular time-domain fifth-order Ramansto probe vibrational interactions in liquid CCl4 and CHCl3 and in mixtures of the two liquids (114). The authors observe combination peaks in the 2-D Raman spectrum which give qualitative evidence for vibrational coupling. Cho and co-workers have proposed frequency-domain 2-D Raman scattering based on theoretical analysis (115). Cho has also theoretically described resonant coherent 2-D Raman scattering (116). This technique would be used to investigate vibrational mode couplings in electronic excited states. A significant setback came last year when Blank and coworkers reported that the fifth-order 2-D spectrum of CS2, a favorite molecule in 2-D Raman studies due to its high polarizability, was in fact dominated by third-order (one-dimensional) cascade processes. The actual 2-D signal for CS2 is vanishingly small compared to that from the third-order cascades. While a possible solution to this problem could be envisioned, it will be experimentally difficult (117). Mukamel et al. have written a review on the subject of 2-D Raman echoes (118). This review not only serves as an introduction to multidimensional Raman methods but also compares and contrasts 2-D Raman methods with the more familiar 2-D NMR techniques. Although the two are in many ways analogous, there are important differences, not the least of which lies in the greater Analytical Chemistry, Vol. 72, No. 12, June 15, 2000
155R
theoretical and experimental difficulties inherent in optical methods as compared to their NMR counterparts. PROSPECTUS Raman spectroscopy continues to grow in popularity both among analytical chemists and in other fields. It has become a standard method for materials characterization and biochemistry and is finding greater acceptance in industrial process monitoring. In addition, biomedical diagnostic applications look promising. Raman is being developed as a detection mechanism in many hyphenated techniques, where it can not only detect but also identify eluents. The future of SERS for general ultrasensitive detection remains to be seen. While the enhancement factors observed for a few molecules yield Raman scattering cross sections on the order of fluorescence cross sections, single-molecule SERS is in its infancy and it is not yet known how general these extraordinary enhancements will be. Nonetheless, the much greater information content of Raman as compared with fluorescence engenders much enthusiasm for its use in ultrasensitive detection and analysis. Another question to be answered in the coming years is whether 2-D Raman methods will fulfill their early promise. ACKNOWLEDGMENT
Financial support from NSF, NIH, and the Jet Propulsion Laboratory is gratefully acknowledged. Shawn P. Mulvaney is a graduate student in the Chemistry Department at The Pennsylvania State University. He received his ACS-certified B.S. in chemistry from the College of William and Mary in 1997. His research interests lie in the development and implementation of SERSbased sensors for environmental and pharmaceutical analysis. Notable applications include a SERS-solid-phase microextraction substrate architecture and core-shell constructs made from glass and Raman-tagged metal nanoparticles. Christine D. Keating is currently an Assistant Professor of Chemistry at The Pennsylvania State University. Christine received her B.S. in chemistry and biology from St. Francis College in 1991 and her Ph.D. in chemistry from The Pennsylvania State University in 1997. She then conducted postdoctoral research on membrane biophysics in the laboratories of Professors Paul Weiss and Michael Natan at The Pennsylvania State University. Her research interests include directed self-assembly of nanoparticles, amphiphiles, and/or biomolecules to form functional structures ranging from sensor architectures to models of biological cells. Of particular current interest are nanoparticle assemblies for ultrasensitive surface-enhanced Raman scattering and bioanalytical applications of nanoparticle-amplified surface plasmon resonance. LITERATURE CITED (1) Lyon, L. A.; Keating, C. D.; Fox, A. F.; Baker, B. E.; He, L.; Nicewarner, S. R.; Mulvaney, S. P.; Natan, M. J. Anal. Chem. 1998, 70, 341R-361R. (2) Strommen, D. P. In The Handbook of Instrumental Techniques for Analytical Chemistry; Settle, F. A., Ed.; Prentice Hall, PTR: Upper Saddle River, NJ, 1997; p 994. (3) Coates, J. Appl. Spectrosc. Rev. 1998, 33, 267-425. (4) Callender, R.; Deng, H.; Gilmanshin, R. J. Raman Spectrosc. 1998, 29, 15-21. (5) Carey, P. R. J. Raman Spectrosc. 1998, 29, 7-14. (6) Schrader, B.; Dippel, B.; Erb, I.; Keller, S.; Lochte, T.; Schultz, H.; Tatsch, E.; Wessel, S. J. Mol. Struct. 1999, 480/481, 21-32. (7) Thomas, G. J., Jr. Annu. Rev. Biophys. Biomol. Struct. 1999, 28, 1-27. (8) Carey, P. R. J. Raman Spectrosc. 1998, 29, 861-868. (9) Myers, A. B. Acc. Chem. Res. 1997, 30, 519-527. (10) Biswas, N.; Umapathy, S. Curr. Sci. 1998, 74, 328-340. (11) Torres, C. M. S. NATO ASI Ser., Ser. E 1997, 344, 331-354. (12) Husson, E. Key Eng. Mater. 1999, 155/156, 1-40. (13) Efimov, A. M. J. Non-Cryst. Solids 1997, 253, 95-118. (14) Devyatykh, G. G.; Sennikov, P. G.; Nabiev, Sh. Sh. Russ. Chem. Bull. 1999, 48, 623-639. (15) Schweiger, G. In Analytical Chemistry of Aerosols; Spurney, K. R., Ed.; Lewis: Boca Raton, FL, 1999; pp 319-352. (16) Jayaraman, A.; Sharma, S. K. Curr. Sci. 1998, 74, 308-316. 156R
Analytical Chemistry, Vol. 72, No. 12, June 15, 2000
(17) Sood, A. K.; Teresdesai, P. V.; Muthu, D. V. S.; Sen, R.; Govindaraj, A.; Rao, C. N. R. Phys. Status Solidi B 1999, 215, 393-401. (18) Dumas, P.; Weldon, M. K.; Chabal, Y. J.; Williams, G. P. Surf. Rev. Lett. 1999, 6, 225-255. (19) Knozinger, H.; Mestl, G. Top. Catal. 1999, 8, 45-55. (20) Wachs, I. E. Top. Catal. 1999, 8, 57-63. (21) Hochlowski, J.; Whittern, D.; Pan, J.; Swenson, R. Drugs Future 1999, 24, 539-554. (22) Gremlich, H.-U. Biotechnol. Bioeng. 1999, 61, 179-187. (23) Zhang, S.; Franke, F. S.; Niemczyk, T. M. Mod. Technol. Appl. Spectrosc. 1998, 291-322. (24) Schaeberle, M. D.; Morris, H. R.; Turner, J. F., II; Treado, P. J. Anal. Chem. 1999, 71, 175A-181A. (25) Schrof, W.; Klinger, J.; Heckmann, W.; Horn, D. Colloid Polym. Sci. 1998, 276, 577-588. (26) Huong, P. V.; Cavagnat, R.; Bruneel, E. J. L. Spectra Anal. 1999, 28, 26-30. (27) Sammon, C.; Hajatdoost, S.; Eaton, P.; Mura, C.; Yarwood, J. Proc. Macromol. Symp. 1999, 141, 247-262. (28) Williams, K. P. J.; Wilcock, I. C. Polym. Test. 1997, 3, 4, 1-6. (29) Everall, N.; King, B. Proc. Macromol. Symp. 1999, 141, 103116. (30) Al-Khanbashi, A.; Dhamdhere, M.; Hansen, M. Appl. Spectrosc. Rev. 1998, 33, 115-131. (31) Carmona, P.; Navarro, R.; Hernanz, A. Spectroscopy of Biological Molecules: Modern Trends; Kluwer Academic Publishers: Dordrecht, The Netherlands, 1997; p 639. (32) White, P. C.; Rodgers, C.; Rutherford, V.; Finnon, Y.; Smith, W. E.; Fitzgerald, M. Proc. SPIE-Int. Soc. Opt. Eng. 1999, 3576, 7786. (33) J. Raman Spectrosc. 1998, 29 (1). (34) J. Raman Spectrosc. 1998, 29 (10/11). (35) J. Raman Spectrosc. 1997, 28 (2/3). (36) J. Raman Spectrosc. 1998, 29 (6). (37) J. Raman Spectrosc. 1998, 29 (8). (38) Davidson, G. Spectrosc. Prop. Inorg. Organomet. Compd. 1997, 30, 233-276. (39) Davidson, G. Spectrosc. Prop. Inorg. Organomet. Compd. 1997, 30, 277-318. (40) Kuptsov, A. H.; Zhizhin, G. N. Handbook of FT Raman and Infrared Spectra of Polymers; Elsevier: Amsterdam, NY, 1998; p 566. (41) Walker, P. A., III.; Morris, M. D. J. Chromatogr., A 1998, 805, 269-275. (42) Li, H.; Walker, P. A., III.; Morris, M. D. J. Microcolumn Sep. 1998, 10, 449-453. (43) Walker, P. A., III.; Morris, M. D.; Burns, M. A.; Johnson, B. N. Anal. Chem. 1998, 70, 3766-3769. (44) Bristow, A. W. T.; Strawn, A. W.; Courbariaux, Y.; Sewell, C. Anal. Commun. 1998, 35, 297-299. (45) Steinert, R.; Bettermann, H.; Kleinermanns, K. Appl. Spectrosc. 1997, 51, 1644-1647. (46) Kennedy, B. J.; Milofsky, R.; Carron, K. T. Anal. Chem. 1997, 69, 4708-4715. (47) Weissenbacher, N.; Lendl, B.; Frank, J.; Wazenbock, H. D.; Kellner, R. Analyst 1998, 123, 1057-1060. (48) Holtz, M.; Dasgupta, P. K.; Zhang, G. Anal. Chem. 1999, 71, 2934-2938. (49) Dijkstra, R. J.; Bader, A. N.; Hoornweg, G. P.; Brinkman, U. A. T.; Gooijer, C. Anal. Chem. 1999, 71, 4575-4579. (50) Marquardt, B. J.; Vahey, P. G.; Synovec, R. E.; Burgess, L. W. Anal. Chem. 1999, 71, 4808-4814. (51) Nikitin, P. I.; Beloglazov, A. A.; Valeiko, M. V.; Creighton, J. A.; Wright, J. D. Rev. Sci. Instrum. 1997, 68, 2554-2557. (52) Sprunt, J. C.; Jayasooriya, U. A. Appl. Spectrosc. 1997, 51, 14101414. (53) Jordan, C. E.; Stranick, S. J.; Cavanagh, R. R.; Richter, L. J.; Chase, D. B. Surf. Sci. 1999, 433-435, 48-52. (54) Deckert, V.; Zeisel, D.; Zenobi, R.; Vo-Dinh, T. Anal. Chem. 1998, 70, 2646-2650. (55) Webster, S.; Smith, D. A.; Batchelder, D. N. Vib. Spectrosc. 1998, 18, 51-59. (56) Barnett, S. M.; Bormett, R. W.; Whitley, A. Proc. SPIE-Int. Soc. Opt. Eng. 1999, 3605, 308-316 (Three-Dimensional and Multidimensional Microscopy: Image Acquisition and Processing VI). (57) Ma, J.; Ben-Amotz, D. Appl. Spectrosc. 1997, 51, 1845-1848. (58) Bell, S. E. J.; Bourguignon, E. S. O.; Dennis, A. Analyst 1998, 123, 1729-1734. (59) Campion, A.; Kambhampati, P. Chem. Soc. Rev. 1998, 27, 241250. (60) Vo-Dinh, T. Trends Anal. Chem. 1998, 17, 557-582. (61) Norrod, K. L.; Sudnik, L. M.; Rousell, D.; Rowlen, K. L. Appl. Spectrosc. 1997, 51, 994-1001. (62) Viets, C.; Hill, W. Sens. Actuators, B 1998, B51, 92-99. (63) Oldenburg, S. J.; Westcatt, S. L.; Averitt, R. D.; Halas, N. J. J. Chem. Phys. 1999, 111, 4729-4735. (64) Lacy, W. B.; Olson, L. G.; Harris, J. M. Anal. Chem. 1999, 71, 2564-2570. (65) Jones, J. C.; McLaughlin, C.; Littlejohn, D.; Sadler, D. A.; Graham, D.; Smith, W. E. Anal. Chem. 1999, 71, 596-601. (66) Xiao, H.; Dai, S.; Young, J. P.; Feigerle, C. S.; Edwards, A. G. Appl. Spectrosc. 1998, 52, 626-628. (67) Zou, S.; Weaver, M. J. Anal. Chem. 1998, 70, 2387-2395.
(68) Zou, S.; Williams, C. T.; Chen, E. K.-Y.; Weaver, M. J. J. Am. Chem. Soc. 1998, 120, 3811-3812. (69) Williams, C. T.; Chan, H. Y. H.; Tolia, A. A.; Weaver, M. J.; Takoudis, C. G. Ind. Eng. Chem. Res. 1998, 37, 2307-2315. (70) Chan, H. Y. H.; Takoudis, C. G.; Weaver, M. J. J. Am. Chem. Soc. 1999, 121, 9219-9220. (71) Chan, H. Y. H.; Zou, S.; Weaver, M. J. J. Phys. Chem. B 1999, 103, 11141-11151. (72) Zou, S.; Weaver, M. J.; Li, X. Q.; Ren, B.; Tian, Z. Q. J. Phys. Chem. B 1999, 103, 4218-4222. (73) Keating, C. D.; Kovaleski, K. K.; Natan, M. J. J. Phys. Chem. B 1998, 102, 9414-9425. (74) Yu, H.-Z.; Zhang, J.; Zhang, H.-L.; Liu, Z.-F. Langmuir 1999, 15, 16-19. (75) Ni, J.; Lipert, R. J.; Dawson, G. B.; Porter, M. D. Anal. Chem. 1999, 71, 4903-4908. (76) Gresillon, S.; Rivoal, J.-C.; Gadenne, P.; Quelin, X.; Shalaev, V.; Sarychev, A. Phys. Status Solidi A 1999, 175, 337-343. (77) Norrod, K. L.; Rowlen, K. L. Anal. Chem. 1998, 70, 4218-4221. (78) Yu, H.-Z.; Xia, N.; Liu, Z.-F. Anal. Chem. 1999, 71, 1354-1358. (79) Yamamoto, H.; Maeda, Y.; Kitano, H. J. Phys. Chem. B 1997, 101, 6855-6860. (80) Graham, D.; MaAnally, G.; Jones, J. C.; Smith, W. E. Chem. Commun. 1998, 1187-1188. (81) Zenobi, R. Chimia 1999, 53, 35-37. (82) Kneipp, K.; Kneipp, H.; Itzkan, I.; Dasari, R. R.; Feld, M. S. Chem. Rev. 1999, 99, 2957-2975. (83) Krug, J. T., II.; Wang, G. D.; Emory, S. R.; Nie, S. J. Am. Chem. Soc. 1999, 121, 9208-9214. (84) Emory, S. R.; Nie, S. J. Phys. Chem. B 1998, 102, 493-497. (85) Emory, S. R.; Haskins, W. E.; Nie, S. J. Am. Chem. Soc. 1998, 120, 8009-8010. (86) Kneipp, K.; Kneipp, H.; Kartha, V. B.; Manoharan, R.; Deinum, G.; Itzkan, I.; Dasari, R. R.; Feld, M. S. Phys. Rev. E 1998, 57, R6281-R6284. (87) Isola, N.; Stokes, D. L.; Vo-Dinh, T. Anal. Chem. 1998, 70, 13521356. (88) Graham, D.; Smith, W. E.; Linacre, A. M. T.; Munro, C. H.; Watson, N. D.; White, P. C. Anal. Chem. 1997, 69, 4703-4707. (89) Lednev, I. K.; Karnoup, A. S.; Sparrow, M. C.; Asher, S. A. J. Am. Chem. Soc. 1999, 121, 4076-4077. (90) Lednev, I. K.; Karnoup, A. S.; Sparrow, M. C.; Asher, S. A. J. Am. Chem. Soc. 1999, 121, 8074-8086. (91) Schwartz, A. M.; Berglund, K. A. J. Cryst. Growth 1999, 203, 599-603. (92) Hawi, S. R.; Rochanakij, S.; Adar, F.; Campbell, W. B.; Nithipatikom, K. Anal. Biochem. 1998, 259, 212-217. (93) Wood, E.; Sutton, C.; Beezer, A. E.; Creighton, J. A.; Davis, A. F.; Mitchell, J. C. Int. J. Pharm. 1997, 154, 115-118. (94) Shim, M. G.; Wilson, B. C.; Marple, E.; Wach, M. Appl. Spectrosc. 1999, 53, 619-627. (95) Mahadevan-Jansen, A.; Mitchell, M. F.; Ramanujam, N.; Malpica, A.; Thomsen, S.; Utzinger, U.; Richards-Kortum, R. Photochem. Photobiol. 1998, 68, 123-132.
(96) Bauer, N. J. C.; Motamedi, M.; Wicksted, J. P.; March, W. F.; Webers, C. A. B.; Hendrikse, F. J. Ocul. Pharmacol. Ther. 1999, 15, 123-134. (97) Yazdi, Y.; Ramanujam, N.; Lotan, R.; Mitchell, M. F.; Hittelman, W.; Richards-Kortum, R. Appl. Spectrosc. 1999, 53, 82-85. (98) Brennan, J. F., III.; Romer, T. J.; Lees, R. S.; Tercyak, A. M.; Kramer, J. R., Jr.; Feld, M. S. Circulation 1997, 96, 99-105. (99) Romer, T. J.; Brennan, J. F., III.; Baker Schutt, T. C.; Wolthuis, R.; van den Hoogan, R. C. M.; Emeis, J. J.; van der Laarse, A.; Bruschke, A. V. G.; Puppels, G. J. Atherosclerosis 1998, 141, 117124. (100) McCreery, R. L.; Horn, A. J.; Spencer, J.; Jefferson, E. J. Pharm. Sci. 1998, 87, 1-8. (101) Breitenbach, J.; Schrof, W.; Neumann, J. Pharm. Res. 1999, 16, 1109-1113. (102) Timlin, J. A.; Carden, A.; Morris, M. D.; Bonadio, J. F.; Hoffler, C. E., II.; Kozloff, K. M.; Goldstein, S. A. J. Biomed. Opt. 1999, 4, 28-34. (103) Freeman, T. L.; Cope, S. E.; Stringer, M. R.; Cruse-Sawyer, J. E.; Brown, S. B.; Batchelder, D. N.; Birbeck, K. Appl. Spectrosc. 1998, 52, 1257-1263. (104) Morris, M. D.; Katzir, A. Proc. SPIE-Int. Soc. Opt. Eng. 1999, 3608, 1-250. (105) Ho, M.; Pemberton, J. E. Anal. Chem. 1998, 70, 4915-4920. (106) Horvath, E.; Kocsis, L.; Frost, R. L.; Hren, B.; Szabo, L. P. Anal. Chem. 1998, 70, 2766-2770. (107) Rahman, S. S.; Busby, D. J.; Lee, D. C. J. Org. Chem. 1998, 63, 6196-6199. (108) Saito, R.; Takeya, T.; Kimura, T.; Dresselhaus, G.; Dresselhaus, M. S. Phys. Rev. B: Condens. Matter Mater. Phys 1999, 59, 2388-2392. (109) McCreery, R. L.; Horn, A. J.; Spencer, J.; Jefferson, E. J. Pharm. Sci. 1998, 87, 1-8. (110) Liang, E. J.; Yang, Y.; Kiefer, W. Spectrosc. Lett. 1999, 32, 689701. (111) Weissenbacher, N.; Lendl, B.; Frank, J.; Wanzenbock, H. D.; Mizaikoff, B.; Kellner, R. J. Mol. Struct. 1997, 410-411, 539542. (112) Svensson, O.; Josefson, M.; Lngkilde, F. W. Chemom. Intell. Lab. Syst. 1999, 49, 49-66. (113) Li, W.; Gibbs, G. V.; Oyama, S. T. J. Am. Chem. Soc. 1998, 120, 9041-9046. (114) Tokmakoff, A.; Lang, M. J.; Larsen, D. S.; Fleming, G. R. Phys. Rev. Lett. 1997, 79, 2702-2705. (115) Cho, M.; Okumura, K.; Tanimura, Y. J. Chem. Phys. 1998, 109, 1326-1334. (116) Cho, M. J. Chem. Phys. 1998, 109, 5327-5337. (117) Blank, D. A.; Kaufman, L. J.; Fleming, G. R. J. Chem. Phys. 1999, 111, 3105-3114. (118) Mukamel, S.; Piryatinski, A.; Chernyak, V. Acc. Chem. Res. 1999, 32, 145-154.
A10000155
Analytical Chemistry, Vol. 72, No. 12, June 15, 2000
157R
