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SERS chemical sensors and biosensors: new tools for environmental and biological analysis
Ac'rwoRS
cHEBcAL
ELSEVIER
Sensors and Actuators B 29 (1995) 183 189
SERS chemical sensors and biosensors" new tools for environmental and biological analysis Tuan Vo-Dinh Advaneed Monitoring Development Group, Health Sciences Research Division, Oak Ridge National Laboratory, Oak Ridge, TN 37831-6101, USA
Abstract This paper describes recent advances in chemical sensors and biosensors based on surface-enhanced Raman scattering (SERS) detection. The SERS-active probes involve silver-coated microstructured substrates designed to amplify the Raman signals of adsorbed molecules and to detect trace chemicals in liquid as well as vapor samples. The development and application of several SERS devices such as a fiber-optic remote sensor and a vapor dosimeter are described. The results illustrate the different uses of the SERS method for the identification and quantification of important environmental and biological compounds.
Keywords: SERS sensors; Spectroscopy; Environmental analysis; Biological analysis
1. Introduction The development of optical chemical sensors and biosensors for detection of trace quantities of toxic chemicals and related biological indicators is critical to the achievement of environmentally viable and safe technologies. Problems pertaining to the identification of specific compounds at ultratrace levels, the analysis of complex mixtures and the assessment of biological effects continue to create new analytical challenges. An important problem area in chemical sensing is the sensitive identification of trace compounds in complex environmental and biological samples. In order to detect minute amounts of a compound in a complex 'real-life' sample, sensors must be able not only to differentiate compounds having different molecular sizes but also to identify specific substituents and/or derivative chemical groups attached to the basic structure. In many situations, several techniques are required to provide unambiguous identification and accurate quantification. The various spectrochemical techniques investigated in our laboratories for use in optical sensing and in trace organic analysis include synchronous
÷*Invited paper. The submitted manuscript has been authored by a contractor of the US Government under Contract No. DE-AC0584OR21400. Accordingly, the US Government retains a nonexclusive, royalty-free license to publish or reproduce the published form of the contribution, or allow others to do so, for US Government purposes. 0925-4005/95/$09.50
© 1995
S S D I 0925-4005(95)01681 -K
Elsevier Science S.A. All rights reserved
luminescence [1,2], room temperature phosphorescence [3,4], surface-enhanced R a m a n spectroscopy [5-8] and fiberoptics-based laser antibody sensor technology [9,10]. Heavy atoms and cyclodextrins were used to enhance phosphorescence analysis [4], whereas antibodies were developed to improve the specificity and sensitivity of laser-based biochemical sensors [9,10]. This paper presents an overview of some recent advances in the development of surface-enhanced Raman scattering (SERS) substrates for use in chemical sensors and biosensors. The development of various SERS-active materials for optical probes is discussed. The substrates consisted of glass plates covered with silver-coated microstructured particles. The results of SERS-active substrates for in situ analysis in liquid and vapor samples are presented to illustrate the usefulness of SERS sensors in environmental and biological analysis.
2. Background: the SERS effect Since its discovery in the late 1970s [11,12], the SERS effect has received increased interest only in the last few years. Extensive research efforts have been devoted to the investigation and determination of the sources of enhancement. The experimental observations related to SERS, and the origin of the enormous Raman enhancement are believed to be the result of several mechanisms. There are at least two major types
184
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&,nsors and Actuators B 29 (1995) 183-189
of mechanisms that contribute to the SERS effect: (a) an electromagnetic effect associated with large local fields caused by electromagnetic resonances occurring near metal surface structures; and (b) a chemical effect involving a scattering process associated with chemical interactions between the molecule and the metal surface. Some aspects of SERS, such as the contribution of electromagnetic interactions, have been extensively investigated and are reasonably well understood. Other aspects, such as the contribution of chemical effects, are less known and are currently topics of extensive research. The reader is referred to a number of reviews for further details [8,13-15]. It has been shown that electromagnetic interactions between the molecule and the substrate provide the dominant enhancement in the SERS process. These electromagnetic interactions are divided into two major classes: (a) interactions that occur only in the presence of a radiation field; and (b) interactions that occur even without a radiation field. The first class of interactions between the molecule and the substrate is believed to play a major role in the SERS process. A major contribution to electromagnetic enhancement is due to surface plasmons, which are associated with collective excitations of surface conduction electrons in metal particles. Raman enhancements result from excitation of these surface plasmons by the incident radiation. At the plasmon frequency, the metal becomes highly polarizable, resulting in large field-induced polarizations and thus large local fields on the surface. These local fields increase the Raman emission intensity, which is proportional to the square of the applied field at the molecule. Additional enhancement is due to excitation of surface plasmons by the Raman emission radiation of the molecule. Surface plasmons are not the only sources of enhanced local electromagnetic fields [13,14]. Other types
Fig. 1. SEM photograph of a silver-coatedfused silica microparticle substrate.
of electromagnetic enhancement mechanisms are: (a) concentration of electromagnetic field lines near highcurvature points on the surface, i.e., the 'lightning rod' effect; (b) polarization of the surface by dipole-induced fields in adsorbed molecules, i.e., the image effect; and (c) Fresnel reflection effects. The chemical effect is associated with the overlap of metal and adsorbate electronic wavefunctions, which leads to ground-state and light-induced charge-transfer processes [13,14]. In the charge-transfer model, an electron of the metal, excited by the incident photon, tunnels into a charge-transfer excited state of the adsorbed molecule. The resulting negative ion (adsorbate molecule electron) has a different equilibrium geometry from that of the original neutral adsorbate molecule. Therefore, the charge-transfer process induces a nuclear relaxation in the adsorbate molecule which, after the return of the electron to the metal, leads to a vibrationally excited neutral molecule and to emission of a Raman-shifted photon. The 'adatom model' also suggests additional Raman enhancement for adsorbates at special active sites of atomic-scale roughness, which may facilitate charge-transfer enhancement mechanisms [13-15].
3. Experimental 3.1. Instrumentation and procedures 3.1.1. Raman measurements The Raman spectra were measured using two Raman spectrometers. Recently a portable SERS field spectrometer (Gamma-Metrics, Inc., San Diego, CA) was available for measurements. The first instrument consisted of a Spex model 1403 double monochromator with a Spex Datamate DM1 control and data acquisition system. The detection employed the photon-counting technique, accomplished by using a cooled RCA C31034-02 photomultiplier tube. Excitation was provided by a Spectra Physics model 166 argon laser, a Coherent Radiation model Innova 90K krypton ion laser, or a Liconix model 4240PS helium cadmium laser. The second system was based on a Jobin-Yvon/ISA Ramanor 2000M double-grating monochromator. The data acquisition system was an LSI 11/23 minicomputer purchased from Data Translation Corporation and a DSD/880 Winchester/floppy disk drive. Photon counting was accomplished using a cooled RCA C31034-02 photomultiplier tube. The excitation was provided by a Spectra Physics model 171 argon ion laser. Scanning electron microscope (SEM) photographs were obtained with an ISI DS-130 scanning electron microscope. The substrate preparation involved two steps. The first step was the deposition of microbodies (such as
72 Vo-Dinh / Sensors and Actuators B 29 (1995) 183 189
polystyrene latex spheres, fumed silica, titanium oxide and aluminum oxide particles) on glass plates. This deposition was accomplished by placing a glass slide on a spin-coating device. A few drops of the microparticle/ water solution were placed on the glass slide, which was then immediately spun at 2000 rpm for 20 s. Spinning has been found necessary to preclude clumping of the microparticles on the glass surface. The microparticles adhered to the glass providing a uniform coverage. The second step was the coating of the microparticle-covered glass slide with silver. The glass slide was placed inside a vacuum evaporator. The pressure was less than 5 x 10 -~ Torr. The rate of silver deposition was controlled at approximately 1.5 2.0nm/s. The rate and thickness of silver deposition were measured using a Kronon model QM-311 quartz crystal thickness monitor. D a t a from the quartz crystal thickness monitor exhibited a standard deviation of 10%. Following silver evaporation, 2 - 4 ml of sample solution were spotted on the glass plate substrate. The R a m a n spectrum was then scanned over the region of interest. For solution measurements, 1 ml of the sample solution was pipetted into a standard quartz cell. The SERS substrate was then inserted directly into the cell and the SERS spectrum was recorded. For in situ measurements, the substrates were mounted on a fiberoptic probe and inserted into liquid samples for spectral recording. The SERS substrates were also used in chemical vapor sensors. Following exposure to the analyte vapor, the silver-coated alumina SERS substrate was removed from the dosimeter and inserted directly into the R a m a n spectrometer. The SERS spectrum was then recorded. SERS data were collected in two ways. The most c o m m o n technique used the front side excitation/ collection geometry. In this geometry, the excitation beam was focused onto the front (metal) side of the SERS substrate and scattered radiation was also collected from the front side at 90 ° with respect to the excitation beam. An optical fiber-based auxiliary optical setup was also used for some measurements. With this remote setup, the excitation fiber was placed at the back (glass) side of the SERS substrate, while the collection fiber was placed at the front (metal) side of the substrate at an angle of 180 ° with respect to the excitation fiber.
3.2. Vapor exposure system A simple system was set up for vapor monitoring. Air was passed through a charcoal filter to remove impurities and through a calcium sulfate filter to remove water vapor. The air flow rate was determined by a flowmeter. A humidity source allowed for the testing of humidity effects during vapor-phase studies. Air temperature was controlled by the use of a heating coil placed in the air flow path just prior to the sample
Bandpass
185
Filter
Laser Coupling
j Optics
Optical
Fibers~
Spectrograph
m
Portable
SERS Monitor Controller Probe i
Computer
Fig. 2. Schematic diagram of a fiberoptic remote SERS sensor.
injection point. The sample was injected at a fixed rate using a syringe and a syringe pump. An additional flowmeter monitored the air flow into the sampling chamber.
4. Results and discussion
4.1. The SERS probes The substrate can serve as a probe to collect analyte compounds adsorbed onto its SERS-active surface. In general, microparticles of oxides (alumina, titania, silica) have been used on glass plates, which provide simple and inexpensive practical supports [8]. Fig. 1 shows an SEM photograph of the surface of a SERS probe consisting of a glass plate covered with fused silica microparticles coated with a 75-nm layer of silver. For the present studies we used glass plates as solid planar supports. The size of the surface microstructure can be easily controlled by simply selecting the appropriate microparticle sizes. In most of our studies, silver was used as the coating metal for SERS substrates. Previous research indicates that the type of metal on the surfaces is an important factor affecting the SERS effect [13]. Silver exhibits the strongest enhancement, followed by copper and gold. The development of SERS as an analytical technique is relatively recent and many experimental factors require careful optimization in order to obtain the maximum signal enhancement. One of the major difficulties in the development of the SERS technique for analytical applications is the development of surfaces or media that have an easily controlled protrusion size and reproducible structure. In a previous work [7] we have shown that the SERS effect depends upon several factors including excitation wavelength, microparticle size and silver coating thickness. Using a 364-nm-diameter microsphere, we measured
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the S E R S signal intensity using different excitation frequencies. In the previous study, we investigated this excitation dependence effect for a variety o f sphere sizes and silver-coating thickness combinations [7]. The 1240-cm-~ vibration o f 1-nitropyrene was used as the reference signal. Different excitation frequencies from five argon-ion laser emission lines, three krypton-ion laser lines and one h e l i u m - c a d m i u m laser line were used. Comparative measurements o f the Ram a n enhancement were made by varying the excitation wavelength for six sphere sizes from 38 to 482 nm. F o r our spectrometer/photomultiplier combination the sensitivity drops strongly toward the red spectral region. M a x i m u m SERS signals are obtained with the 514.5-nm excitation. The relative standard deviation o f our measurements is approximately + 15%. 4.2. The f i b e r o p t i c remote S E R S
tures are described here. A single optical fiber was used to transmit the laser excitation into the SERS probe, and a second fiber was used to collect the scattered radiation from the sample. The laser beam transmitted t h r o u g h a bandpass filter was focused into one end o f the excitation fiber with the use o f a microscope objective lens. This end o f the excitation fiber was held by a fiberoptic holder. The terminus end o f the excitation fiber was positioned close to the SERS substrate in order to contain the laser beam to a very small spot on the substrate. The SERS probe was prepared with a glass backing (microscope slide, 1 m m thick) so that the excitation and collection fibers could be positioned either head on, with the fibers positioned on opposite sides o f the SERS substrate, or side-by-side, with the two fibers on the same side o f the substrate. The terminus end o f the collection fiber was positioned next to the
sensor
The development o f SERS-active substrates that allow direct measurements in liquid samples is critical for in situ analysis. SERS has been observed using different solid substrates such as metal electrodes, metal islands, films, glass or cellulose coated with silver-covered microparticles. However, with the exception o f metal electrodes and colloidal solutions, most o f the S E R S studies performed with solid substrates to date have been performed in the dry state. Recently, we have developed the technique o f measuring S E R S in solution using probes covered with silver-coated substrates m o u n t e d in fiberoptic sensors. Fig. 2 shows a schematic diagram o f a prototype fiberoptic remote S E R S monitor. A preliminary version o f the S E R S fiberoptic probe has been developed and described previously [16,17]. Only the salient fea-
m.0.
80000.0
40000.0
~ . 0
Table 1 Some detection limits using SERS probes a Compound
Limit of detection
Comments b
p-Aminobenzoic acid p-Diacetyl benzene Terephthaldehyde Terephthalic acid p-Cresol Benzoic acid m-Nitrobenzoic acid 3-Nitroaniline acid p-Aminobenzoic acid Benzoic acid Benzo(a)pyrene tetraol Formothion Pyrene Terephthalic acid Carbonphenothran Bremophos Methyl chloropyrifos Dichloran Linuron Chlordane l-Hydroxychlorodene Methylparathion Fonofoxon Chlorofenvinhos Cyanax Diazinon Formothion Dimethoate Trichlofon Benzo(a)pyrene Carbazole
0.4 43 1 3 28 50 87 36 17 17 8 11 0.002 15 32 36 32 20 25 40 35 < 26 < 25 < 36 < 25 < 25 < 26 < 23 < 26 0.1 0.2 1.4 0.3
fiberoptic sensor fiberoptic sensor fiberoptic sensor fiberoptic sensor fiberoptic sensor fiberoptic sensor fiberoptic sensor fiberoptic sensor
1-Aminopyrene 0.0 '164 II
14111
112 ~ Ii ItlIIIWl S l l l f t | e l ' - | )
~'.1 §
344
Fig. 3. Schematic SERS monitor for 1-aminopyrene (1 ~tl spot o f 10 - 3 M solution; alumina substrate; excitation 632.8 nm, 3 mW; RE-ICCD system w/20-m optical fiber system, gain 5.86, exp. 5 s, acc. 5.
Benzoic acid
chlorinated pesticide chlorinated pesticide chlorinated pesticide chlorinated pesticide chlorinated pesticide chlorinated pesticide chlorinated pesticide organophosphorus organophosphorus organophosphorus organophosphorus organophosphorus organophosphorus organophosphorus organophosphorus
"Data taken from Refs. [6,7,21-24]. b The limits of detection (in nanograms) are given for a complete sample spot although only about 1% of the spot is illuminated by the analyzing laser. The actual limits of detection are therefore 100 times less.
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cinogenic metabolite involved in binding to D N A [18]. Detection of BPT can provide a measure of genetic damage due to BP exposure [19]. In previous works BPT samples were analyzed in the dry state using the room temperature phosphorescence [18]. In this study we were able to obtain SERS spectra with a very high signal-to-noise ratio with a portable, air-cooled argon ion laser using a laser power of only 10mW. This aspect is important because this would eliminate the need of powerful and expensive lasers that are normally used with Raman spectroscopy and demonstrate the feasibility of a portable SERS system. Fig. 4 shows the solution SERS spectrum of 32 part-per-million (ppm) BPT obtained with a glass coated with silver-coated microspheres and obtained with the portable argon ion laser. The spectrum was recorded using the back-geometry arrangement. The solution SERS spectrum of BPT shown in Fig. 4 is very similar to the SERS spectrum of BPT adsorbed on silver-coated cellulosic substrate. The solution SERS spectrum of BPT (Fig. 4) shows three major bands at 1256, 1384 and 1620 cm -~. Note that the peaks at 1384 and 1620cm ] are superimposed on broad bands (1300-1600cm l) that might be related to the substrate. These broad bands were similar to those observed previously in Raman scattering on silver surface in tunneling structures and on polycrystalline silver treated in K C N solutions. These broad bands were attributed to Raman scattering from contaminated car-
entrance slit of a spectrometer. Since the f / n u m b e r of the fiber and that of the spectrometer were different, it was necessary to focus the input radiation from the collection fiber with lenses. An f/1 lens was used to collect and collimate the output beam from the colection fiber. A second lens with an f / n u m b e r matching that of the spectrometer (f/7) was then used to focus the collected SERS signal into the slit of the spectrometer equipped with a red-enhanced intensified chargecoupled device (ICCD) from Princeton Instruments, Inc. The length of excitation and collection fibers used was 1-20 m, with minor alteration in the SERS signal. Fig. 3 shows a SERS spectrum of 1-aminopyrene obtained in only 5 s using the fiberoptic remote SERS sensor. The results indicate that the combination of the ICCD sensitivity and the SERS probe effectiveness allowed rapid in situ chemical sensing. The usefulness of the SERS technique in chemical sensing is illustrated in Table 1.
4.3. Monitoring biological systems The SERS probes can also be used to detect compounds of biological interest. Benzo(a)pyrene r-7,t8,9,10-tetrahydrotetraol, also referred to as benzopyrene tetraol (BPT), a product of the carcinogenic compound, benzo(a)pyrene (BP), was used in this study. BPT is the product obtained by acid hydrolysis of benzo(a)pyrene-diol-epoxide (BPDE), a major car-
14
12
Benzo[a]pyreneTetraol
I.-p,-
~
10
'
~
~
ot
m
8
6 Z UJ I,Z --
4
W
I
200
I
400
I
600
I
I
800
I
1000
I
1200
I
1400
1600
R A M A N S H I F T ( c m "1)
Fig. 4. SERS biomonitoring of benzo(a)pyrene tetraol, a product of the carcinogen benzopyrene.
1800
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T. Vo-Dinh ,' Sensors and Actuators B 29 (1995) 183 189
bon. The exact nature of this broad emission is under investigation. Weak bands at 1520, 1172, 990 and 815 cm ] are also present in the solution SERS spectrum of BPT. In general, the bands and the spectral shape of the solution SERS spectrum of BPT is comparable to that of BPT adsorbed on cellulosic substrate. Using the maximum SERS peak at 1384 cm ~, the limit of detection (LOD) of BPT was also calculated. Although the experimental parameters were not optimized in our current system, the limit of detection for BPT in solution was 0.73 ppm.
b,-
4.4. The S E R S vapor dosimeter g
The SERS dosimeter is a self-contained, badge-size passive monitor [20]. The device weighs about 30 g and can be conveniently worn by a person or placed at a stationary location. The dosimeter is designed to be compatible with the R a m a n spectrophotometers currently used for measuring the SERS signal. The dosimeter basically consists of a badge-size sample holder, a SERS-active substrate and an interchangeable diffusion tube. The dosimeter body is a 4.0-cm long and 1.0-cm wide pen-size badge made of aluminum. The sample collection area is a circle having 0.5-cm diameter. The screen device serves to prevent air turbulence from affecting the diffusion process within the dosimeter. The type of diffusion chamber used in the version of the dosimeter investigated here consists of a honeycomb tube which is composed of parallel cylindric holes of 0.1 m diameter and 0.04 m length. The use of a honeycomb is effective in preventing artifacts caused by turbulent mass transport in windy environments. The honeycomb decreases the effective surface collection value, and therefore the sampling rate, but increases the reproducibility of the method of sampling. With the honeycomb tube, it is not necessary to cover the open end of the diffusion tube with a windscreen since the honeycomb geometry can effectively prevent disruption of the diffusion process by ambient air movements. Fig. 5 shows the SERS measurements of formaldehyde vapor (saturated vapor condition at room temperature) using a SERS probe. Measurements were also conducted to detect vapors of benzoic acid (BA), which was used as the model compound since the SERS characteristic has been extensively studied previously [20]. The vapor dosimeter spectrum could be detected after 1 h of dosimeter exposure in a chamber containing the BA at an equilibrium vapor concentration of 1 ppm. The SERS spectrum of vapor is similar to that observed for BA from solutions spotted on the SERS substrate after solvent evaporation. The SERS spectrum of BA vapor exhibited the strong peak characteristic of BA SERS signal observed with a spotted sample.
Z Z
I
I
600 WAVENUMBER
I 800
(CM - 1 )
Fig. 5. SERS detection of formaldehyde vapor. Calibration measurements were conducted with BA vapor. The air-vapor concentration of BA was kept constant at 100 ppb and the temperature maintained at 25 °C. The data were linear over the entire time period, with no indication of substrate saturation, even after 8 h. The results demonstrated that the dosimeter can be designed to produce a response linear to the exposure time for over 8 h under saturated equilibrium vapor conditions. This feature is important for quantitative applications where daily occupational exposure of workers for 8 h workday shifts is generally monitored.
5. Conclusions
R a m a n and surface-enhanced R a m a n spectroscopies are spectrochemical techniques that have a number of important advantages to chemical sensing. The examples shown in this work illustrate the different uses of the technique for the detection of important environmental and biological compounds. At room temperature, R a m a n spectroscopy can provide
T. Vo-Dinh / Sensors and Actuators B 29 (1995) 183-189
an analytical tool having analytical figures of merit that complement luminescence. The Raman technique is well known for its high selectivity. With the advances of fiberoptic technology, the SERS technique, which can amplify the Raman signal by several orders of magnitude, can provide a remote sensing technique with the added merit of improved sensitivity due to the surfaceenhanced effect. The development of SERS-active probes will open new horizons for in situ remote sensors and biosensors for environmental and biomedical applications.
Acknowledgements This research is sponsored by the Office of Health and Environmental Research, US Department of Energy under Contract DE-AC05-84OR21400 with Lockheed Martin Energy Systems, Inc. and the Biosensor LDRD Project. The sponsorship of BMDO managed by the Office of Naval Research (NAVY No. N0001491-F-0042) is acknowledged. The author also thanks D.L..Stokes, J.P. Alarie, A.M. Helmenstine, V.A. Narayanan, W.S. Sutherland, R.L. Moody, J.M. Bello and P. Enlow for their assistance in the SERS measurements discussed in this work. References [1] T. Vo-Dinh, Anal. Chem., 50 (1978) 396. [2] T. Vo-Dinh, Appl. Spectrosc., 36 (1982) 576.
189
[3] T. Vo-Dinh, Room Temperature Phosphorimetry, Wiley, New York, 1984. [4] A.M. Alak and T. Vo-Dinh, Anal. Chem., 65(1988) 596. [5] J.P. Goudonnet, G.M. Begun and E.T. Arakawa, Chem. Phys. Lett., 92(1982) 197. [6] T. Vo-Dinh, M.V.K. Hiromoto, G.M. Begun and R.L+ Moody, Anal. Chem., 56(1984) 1667. [7] R.L. Moody, T. Vo-Dinh and W.H. Fletcher, Appl. Spectrosc., 41 (1987) 966. [8] T. Vo-Dinh, Surface-enhanced Raman spectroscopy, in T. VoDinh (ed.), Chemical Analysis of Polyeyclic Aromatic Compounds, Wiley, New York, 1989. [9] B.J. Tromberg, M.J. Sepaniak, T. Vo-Dinh and G.D. Griffin, Anal, Chem., 59 (1987) 1226. [10] T. Vo-Dinh, B+J. Tromberg, G.D. Griffin, K.R. Ambrose, M.J. Sepaniak and E.M. Gardenhire, Appl. Spectrosc., 5 (1987) 735. [11] D.J. Jeanmaire and R.P. Van Duyne, J. Electroanal. Chem., 84 (1977) 1. [12] M.G. Albrecht and J.A. Creighton, J. Am. Chem. Soc., 99 (1977) 5215. [13] R.K. Chang and T.E. Furtak (eds.), Surjace-enhanced Raman Scattering, Plenum Press, New York, 1982. [14] M. Moskovits, Rev. Mod. Phys., 57(1985) 783. [15] G.C. Schatz, Acc. Chem. Res., 17(1984) 376. [16] J.M. Bello, V.S. Narayana, D.L. Stokes and T. Vo-Dinh, Anal. Chem., 62 (1990) 2437. [17] J.P. Alarie, D.L. Stokes, W.S. Sutherland, A.C. Edwards and T. Vo-Dinh, Appl. Spectros~., 46 (1992) 1608. [18] T. Vo-Dinh and M. Uziel, Anal. Chem., 59(1987) 1093. [19] T. Vo-Dinh (ed,), Chemical Analysis of Polycyclic Aromatic Compounds', Wiley, New York, 1989. [20] T. Vo-Dinh and D.L. Stokes, Appl. Speetrosc., 47 (1993) 1728. [21] J.M. Bello and T. Vo-Dinh, Appl. Spectrosc., 44 (1990) 63. [22] J.M. Bello, D.L. Stokes and T. Vo-Dinh, Appl. Spectrosc., 43 (1989) 1325. [23] A.M. Alak and T. Vo-Dinh, Anal. Chim. Acta., 206 (1988) 333. [24] A.M. Alak and T. Vo-Dinh, Anal. Chem., 59 (1987) 2149.
cHEBcAL
ELSEVIER
Sensors and Actuators B 29 (1995) 183 189
SERS chemical sensors and biosensors" new tools for environmental and biological analysis Tuan Vo-Dinh Advaneed Monitoring Development Group, Health Sciences Research Division, Oak Ridge National Laboratory, Oak Ridge, TN 37831-6101, USA
Abstract This paper describes recent advances in chemical sensors and biosensors based on surface-enhanced Raman scattering (SERS) detection. The SERS-active probes involve silver-coated microstructured substrates designed to amplify the Raman signals of adsorbed molecules and to detect trace chemicals in liquid as well as vapor samples. The development and application of several SERS devices such as a fiber-optic remote sensor and a vapor dosimeter are described. The results illustrate the different uses of the SERS method for the identification and quantification of important environmental and biological compounds.
Keywords: SERS sensors; Spectroscopy; Environmental analysis; Biological analysis
1. Introduction The development of optical chemical sensors and biosensors for detection of trace quantities of toxic chemicals and related biological indicators is critical to the achievement of environmentally viable and safe technologies. Problems pertaining to the identification of specific compounds at ultratrace levels, the analysis of complex mixtures and the assessment of biological effects continue to create new analytical challenges. An important problem area in chemical sensing is the sensitive identification of trace compounds in complex environmental and biological samples. In order to detect minute amounts of a compound in a complex 'real-life' sample, sensors must be able not only to differentiate compounds having different molecular sizes but also to identify specific substituents and/or derivative chemical groups attached to the basic structure. In many situations, several techniques are required to provide unambiguous identification and accurate quantification. The various spectrochemical techniques investigated in our laboratories for use in optical sensing and in trace organic analysis include synchronous
÷*Invited paper. The submitted manuscript has been authored by a contractor of the US Government under Contract No. DE-AC0584OR21400. Accordingly, the US Government retains a nonexclusive, royalty-free license to publish or reproduce the published form of the contribution, or allow others to do so, for US Government purposes. 0925-4005/95/$09.50
© 1995
S S D I 0925-4005(95)01681 -K
Elsevier Science S.A. All rights reserved
luminescence [1,2], room temperature phosphorescence [3,4], surface-enhanced R a m a n spectroscopy [5-8] and fiberoptics-based laser antibody sensor technology [9,10]. Heavy atoms and cyclodextrins were used to enhance phosphorescence analysis [4], whereas antibodies were developed to improve the specificity and sensitivity of laser-based biochemical sensors [9,10]. This paper presents an overview of some recent advances in the development of surface-enhanced Raman scattering (SERS) substrates for use in chemical sensors and biosensors. The development of various SERS-active materials for optical probes is discussed. The substrates consisted of glass plates covered with silver-coated microstructured particles. The results of SERS-active substrates for in situ analysis in liquid and vapor samples are presented to illustrate the usefulness of SERS sensors in environmental and biological analysis.
2. Background: the SERS effect Since its discovery in the late 1970s [11,12], the SERS effect has received increased interest only in the last few years. Extensive research efforts have been devoted to the investigation and determination of the sources of enhancement. The experimental observations related to SERS, and the origin of the enormous Raman enhancement are believed to be the result of several mechanisms. There are at least two major types
184
T. Vo-Dinh
&,nsors and Actuators B 29 (1995) 183-189
of mechanisms that contribute to the SERS effect: (a) an electromagnetic effect associated with large local fields caused by electromagnetic resonances occurring near metal surface structures; and (b) a chemical effect involving a scattering process associated with chemical interactions between the molecule and the metal surface. Some aspects of SERS, such as the contribution of electromagnetic interactions, have been extensively investigated and are reasonably well understood. Other aspects, such as the contribution of chemical effects, are less known and are currently topics of extensive research. The reader is referred to a number of reviews for further details [8,13-15]. It has been shown that electromagnetic interactions between the molecule and the substrate provide the dominant enhancement in the SERS process. These electromagnetic interactions are divided into two major classes: (a) interactions that occur only in the presence of a radiation field; and (b) interactions that occur even without a radiation field. The first class of interactions between the molecule and the substrate is believed to play a major role in the SERS process. A major contribution to electromagnetic enhancement is due to surface plasmons, which are associated with collective excitations of surface conduction electrons in metal particles. Raman enhancements result from excitation of these surface plasmons by the incident radiation. At the plasmon frequency, the metal becomes highly polarizable, resulting in large field-induced polarizations and thus large local fields on the surface. These local fields increase the Raman emission intensity, which is proportional to the square of the applied field at the molecule. Additional enhancement is due to excitation of surface plasmons by the Raman emission radiation of the molecule. Surface plasmons are not the only sources of enhanced local electromagnetic fields [13,14]. Other types
Fig. 1. SEM photograph of a silver-coatedfused silica microparticle substrate.
of electromagnetic enhancement mechanisms are: (a) concentration of electromagnetic field lines near highcurvature points on the surface, i.e., the 'lightning rod' effect; (b) polarization of the surface by dipole-induced fields in adsorbed molecules, i.e., the image effect; and (c) Fresnel reflection effects. The chemical effect is associated with the overlap of metal and adsorbate electronic wavefunctions, which leads to ground-state and light-induced charge-transfer processes [13,14]. In the charge-transfer model, an electron of the metal, excited by the incident photon, tunnels into a charge-transfer excited state of the adsorbed molecule. The resulting negative ion (adsorbate molecule electron) has a different equilibrium geometry from that of the original neutral adsorbate molecule. Therefore, the charge-transfer process induces a nuclear relaxation in the adsorbate molecule which, after the return of the electron to the metal, leads to a vibrationally excited neutral molecule and to emission of a Raman-shifted photon. The 'adatom model' also suggests additional Raman enhancement for adsorbates at special active sites of atomic-scale roughness, which may facilitate charge-transfer enhancement mechanisms [13-15].
3. Experimental 3.1. Instrumentation and procedures 3.1.1. Raman measurements The Raman spectra were measured using two Raman spectrometers. Recently a portable SERS field spectrometer (Gamma-Metrics, Inc., San Diego, CA) was available for measurements. The first instrument consisted of a Spex model 1403 double monochromator with a Spex Datamate DM1 control and data acquisition system. The detection employed the photon-counting technique, accomplished by using a cooled RCA C31034-02 photomultiplier tube. Excitation was provided by a Spectra Physics model 166 argon laser, a Coherent Radiation model Innova 90K krypton ion laser, or a Liconix model 4240PS helium cadmium laser. The second system was based on a Jobin-Yvon/ISA Ramanor 2000M double-grating monochromator. The data acquisition system was an LSI 11/23 minicomputer purchased from Data Translation Corporation and a DSD/880 Winchester/floppy disk drive. Photon counting was accomplished using a cooled RCA C31034-02 photomultiplier tube. The excitation was provided by a Spectra Physics model 171 argon ion laser. Scanning electron microscope (SEM) photographs were obtained with an ISI DS-130 scanning electron microscope. The substrate preparation involved two steps. The first step was the deposition of microbodies (such as
72 Vo-Dinh / Sensors and Actuators B 29 (1995) 183 189
polystyrene latex spheres, fumed silica, titanium oxide and aluminum oxide particles) on glass plates. This deposition was accomplished by placing a glass slide on a spin-coating device. A few drops of the microparticle/ water solution were placed on the glass slide, which was then immediately spun at 2000 rpm for 20 s. Spinning has been found necessary to preclude clumping of the microparticles on the glass surface. The microparticles adhered to the glass providing a uniform coverage. The second step was the coating of the microparticle-covered glass slide with silver. The glass slide was placed inside a vacuum evaporator. The pressure was less than 5 x 10 -~ Torr. The rate of silver deposition was controlled at approximately 1.5 2.0nm/s. The rate and thickness of silver deposition were measured using a Kronon model QM-311 quartz crystal thickness monitor. D a t a from the quartz crystal thickness monitor exhibited a standard deviation of 10%. Following silver evaporation, 2 - 4 ml of sample solution were spotted on the glass plate substrate. The R a m a n spectrum was then scanned over the region of interest. For solution measurements, 1 ml of the sample solution was pipetted into a standard quartz cell. The SERS substrate was then inserted directly into the cell and the SERS spectrum was recorded. For in situ measurements, the substrates were mounted on a fiberoptic probe and inserted into liquid samples for spectral recording. The SERS substrates were also used in chemical vapor sensors. Following exposure to the analyte vapor, the silver-coated alumina SERS substrate was removed from the dosimeter and inserted directly into the R a m a n spectrometer. The SERS spectrum was then recorded. SERS data were collected in two ways. The most c o m m o n technique used the front side excitation/ collection geometry. In this geometry, the excitation beam was focused onto the front (metal) side of the SERS substrate and scattered radiation was also collected from the front side at 90 ° with respect to the excitation beam. An optical fiber-based auxiliary optical setup was also used for some measurements. With this remote setup, the excitation fiber was placed at the back (glass) side of the SERS substrate, while the collection fiber was placed at the front (metal) side of the substrate at an angle of 180 ° with respect to the excitation fiber.
3.2. Vapor exposure system A simple system was set up for vapor monitoring. Air was passed through a charcoal filter to remove impurities and through a calcium sulfate filter to remove water vapor. The air flow rate was determined by a flowmeter. A humidity source allowed for the testing of humidity effects during vapor-phase studies. Air temperature was controlled by the use of a heating coil placed in the air flow path just prior to the sample
Bandpass
185
Filter
Laser Coupling
j Optics
Optical
Fibers~
Spectrograph
m
Portable
SERS Monitor Controller Probe i
Computer
Fig. 2. Schematic diagram of a fiberoptic remote SERS sensor.
injection point. The sample was injected at a fixed rate using a syringe and a syringe pump. An additional flowmeter monitored the air flow into the sampling chamber.
4. Results and discussion
4.1. The SERS probes The substrate can serve as a probe to collect analyte compounds adsorbed onto its SERS-active surface. In general, microparticles of oxides (alumina, titania, silica) have been used on glass plates, which provide simple and inexpensive practical supports [8]. Fig. 1 shows an SEM photograph of the surface of a SERS probe consisting of a glass plate covered with fused silica microparticles coated with a 75-nm layer of silver. For the present studies we used glass plates as solid planar supports. The size of the surface microstructure can be easily controlled by simply selecting the appropriate microparticle sizes. In most of our studies, silver was used as the coating metal for SERS substrates. Previous research indicates that the type of metal on the surfaces is an important factor affecting the SERS effect [13]. Silver exhibits the strongest enhancement, followed by copper and gold. The development of SERS as an analytical technique is relatively recent and many experimental factors require careful optimization in order to obtain the maximum signal enhancement. One of the major difficulties in the development of the SERS technique for analytical applications is the development of surfaces or media that have an easily controlled protrusion size and reproducible structure. In a previous work [7] we have shown that the SERS effect depends upon several factors including excitation wavelength, microparticle size and silver coating thickness. Using a 364-nm-diameter microsphere, we measured
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the S E R S signal intensity using different excitation frequencies. In the previous study, we investigated this excitation dependence effect for a variety o f sphere sizes and silver-coating thickness combinations [7]. The 1240-cm-~ vibration o f 1-nitropyrene was used as the reference signal. Different excitation frequencies from five argon-ion laser emission lines, three krypton-ion laser lines and one h e l i u m - c a d m i u m laser line were used. Comparative measurements o f the Ram a n enhancement were made by varying the excitation wavelength for six sphere sizes from 38 to 482 nm. F o r our spectrometer/photomultiplier combination the sensitivity drops strongly toward the red spectral region. M a x i m u m SERS signals are obtained with the 514.5-nm excitation. The relative standard deviation o f our measurements is approximately + 15%. 4.2. The f i b e r o p t i c remote S E R S
tures are described here. A single optical fiber was used to transmit the laser excitation into the SERS probe, and a second fiber was used to collect the scattered radiation from the sample. The laser beam transmitted t h r o u g h a bandpass filter was focused into one end o f the excitation fiber with the use o f a microscope objective lens. This end o f the excitation fiber was held by a fiberoptic holder. The terminus end o f the excitation fiber was positioned close to the SERS substrate in order to contain the laser beam to a very small spot on the substrate. The SERS probe was prepared with a glass backing (microscope slide, 1 m m thick) so that the excitation and collection fibers could be positioned either head on, with the fibers positioned on opposite sides o f the SERS substrate, or side-by-side, with the two fibers on the same side o f the substrate. The terminus end o f the collection fiber was positioned next to the
sensor
The development o f SERS-active substrates that allow direct measurements in liquid samples is critical for in situ analysis. SERS has been observed using different solid substrates such as metal electrodes, metal islands, films, glass or cellulose coated with silver-covered microparticles. However, with the exception o f metal electrodes and colloidal solutions, most o f the S E R S studies performed with solid substrates to date have been performed in the dry state. Recently, we have developed the technique o f measuring S E R S in solution using probes covered with silver-coated substrates m o u n t e d in fiberoptic sensors. Fig. 2 shows a schematic diagram o f a prototype fiberoptic remote S E R S monitor. A preliminary version o f the S E R S fiberoptic probe has been developed and described previously [16,17]. Only the salient fea-
m.0.
80000.0
40000.0
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Table 1 Some detection limits using SERS probes a Compound
Limit of detection
Comments b
p-Aminobenzoic acid p-Diacetyl benzene Terephthaldehyde Terephthalic acid p-Cresol Benzoic acid m-Nitrobenzoic acid 3-Nitroaniline acid p-Aminobenzoic acid Benzoic acid Benzo(a)pyrene tetraol Formothion Pyrene Terephthalic acid Carbonphenothran Bremophos Methyl chloropyrifos Dichloran Linuron Chlordane l-Hydroxychlorodene Methylparathion Fonofoxon Chlorofenvinhos Cyanax Diazinon Formothion Dimethoate Trichlofon Benzo(a)pyrene Carbazole
0.4 43 1 3 28 50 87 36 17 17 8 11 0.002 15 32 36 32 20 25 40 35 < 26 < 25 < 36 < 25 < 25 < 26 < 23 < 26 0.1 0.2 1.4 0.3
fiberoptic sensor fiberoptic sensor fiberoptic sensor fiberoptic sensor fiberoptic sensor fiberoptic sensor fiberoptic sensor fiberoptic sensor
1-Aminopyrene 0.0 '164 II
14111
112 ~ Ii ItlIIIWl S l l l f t | e l ' - | )
~'.1 §
344
Fig. 3. Schematic SERS monitor for 1-aminopyrene (1 ~tl spot o f 10 - 3 M solution; alumina substrate; excitation 632.8 nm, 3 mW; RE-ICCD system w/20-m optical fiber system, gain 5.86, exp. 5 s, acc. 5.
Benzoic acid
chlorinated pesticide chlorinated pesticide chlorinated pesticide chlorinated pesticide chlorinated pesticide chlorinated pesticide chlorinated pesticide organophosphorus organophosphorus organophosphorus organophosphorus organophosphorus organophosphorus organophosphorus organophosphorus
"Data taken from Refs. [6,7,21-24]. b The limits of detection (in nanograms) are given for a complete sample spot although only about 1% of the spot is illuminated by the analyzing laser. The actual limits of detection are therefore 100 times less.
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T. Vo-Dinh / Sensors and Actuators B 29 (1995) 183 189
cinogenic metabolite involved in binding to D N A [18]. Detection of BPT can provide a measure of genetic damage due to BP exposure [19]. In previous works BPT samples were analyzed in the dry state using the room temperature phosphorescence [18]. In this study we were able to obtain SERS spectra with a very high signal-to-noise ratio with a portable, air-cooled argon ion laser using a laser power of only 10mW. This aspect is important because this would eliminate the need of powerful and expensive lasers that are normally used with Raman spectroscopy and demonstrate the feasibility of a portable SERS system. Fig. 4 shows the solution SERS spectrum of 32 part-per-million (ppm) BPT obtained with a glass coated with silver-coated microspheres and obtained with the portable argon ion laser. The spectrum was recorded using the back-geometry arrangement. The solution SERS spectrum of BPT shown in Fig. 4 is very similar to the SERS spectrum of BPT adsorbed on silver-coated cellulosic substrate. The solution SERS spectrum of BPT (Fig. 4) shows three major bands at 1256, 1384 and 1620 cm -~. Note that the peaks at 1384 and 1620cm ] are superimposed on broad bands (1300-1600cm l) that might be related to the substrate. These broad bands were similar to those observed previously in Raman scattering on silver surface in tunneling structures and on polycrystalline silver treated in K C N solutions. These broad bands were attributed to Raman scattering from contaminated car-
entrance slit of a spectrometer. Since the f / n u m b e r of the fiber and that of the spectrometer were different, it was necessary to focus the input radiation from the collection fiber with lenses. An f/1 lens was used to collect and collimate the output beam from the colection fiber. A second lens with an f / n u m b e r matching that of the spectrometer (f/7) was then used to focus the collected SERS signal into the slit of the spectrometer equipped with a red-enhanced intensified chargecoupled device (ICCD) from Princeton Instruments, Inc. The length of excitation and collection fibers used was 1-20 m, with minor alteration in the SERS signal. Fig. 3 shows a SERS spectrum of 1-aminopyrene obtained in only 5 s using the fiberoptic remote SERS sensor. The results indicate that the combination of the ICCD sensitivity and the SERS probe effectiveness allowed rapid in situ chemical sensing. The usefulness of the SERS technique in chemical sensing is illustrated in Table 1.
4.3. Monitoring biological systems The SERS probes can also be used to detect compounds of biological interest. Benzo(a)pyrene r-7,t8,9,10-tetrahydrotetraol, also referred to as benzopyrene tetraol (BPT), a product of the carcinogenic compound, benzo(a)pyrene (BP), was used in this study. BPT is the product obtained by acid hydrolysis of benzo(a)pyrene-diol-epoxide (BPDE), a major car-
14
12
Benzo[a]pyreneTetraol
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~
10
'
~
~
ot
m
8
6 Z UJ I,Z --
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I
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I
400
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600
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800
I
1000
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1200
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1400
1600
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Fig. 4. SERS biomonitoring of benzo(a)pyrene tetraol, a product of the carcinogen benzopyrene.
1800
188
T. Vo-Dinh ,' Sensors and Actuators B 29 (1995) 183 189
bon. The exact nature of this broad emission is under investigation. Weak bands at 1520, 1172, 990 and 815 cm ] are also present in the solution SERS spectrum of BPT. In general, the bands and the spectral shape of the solution SERS spectrum of BPT is comparable to that of BPT adsorbed on cellulosic substrate. Using the maximum SERS peak at 1384 cm ~, the limit of detection (LOD) of BPT was also calculated. Although the experimental parameters were not optimized in our current system, the limit of detection for BPT in solution was 0.73 ppm.
b,-
4.4. The S E R S vapor dosimeter g
The SERS dosimeter is a self-contained, badge-size passive monitor [20]. The device weighs about 30 g and can be conveniently worn by a person or placed at a stationary location. The dosimeter is designed to be compatible with the R a m a n spectrophotometers currently used for measuring the SERS signal. The dosimeter basically consists of a badge-size sample holder, a SERS-active substrate and an interchangeable diffusion tube. The dosimeter body is a 4.0-cm long and 1.0-cm wide pen-size badge made of aluminum. The sample collection area is a circle having 0.5-cm diameter. The screen device serves to prevent air turbulence from affecting the diffusion process within the dosimeter. The type of diffusion chamber used in the version of the dosimeter investigated here consists of a honeycomb tube which is composed of parallel cylindric holes of 0.1 m diameter and 0.04 m length. The use of a honeycomb is effective in preventing artifacts caused by turbulent mass transport in windy environments. The honeycomb decreases the effective surface collection value, and therefore the sampling rate, but increases the reproducibility of the method of sampling. With the honeycomb tube, it is not necessary to cover the open end of the diffusion tube with a windscreen since the honeycomb geometry can effectively prevent disruption of the diffusion process by ambient air movements. Fig. 5 shows the SERS measurements of formaldehyde vapor (saturated vapor condition at room temperature) using a SERS probe. Measurements were also conducted to detect vapors of benzoic acid (BA), which was used as the model compound since the SERS characteristic has been extensively studied previously [20]. The vapor dosimeter spectrum could be detected after 1 h of dosimeter exposure in a chamber containing the BA at an equilibrium vapor concentration of 1 ppm. The SERS spectrum of vapor is similar to that observed for BA from solutions spotted on the SERS substrate after solvent evaporation. The SERS spectrum of BA vapor exhibited the strong peak characteristic of BA SERS signal observed with a spotted sample.
Z Z
I
I
600 WAVENUMBER
I 800
(CM - 1 )
Fig. 5. SERS detection of formaldehyde vapor. Calibration measurements were conducted with BA vapor. The air-vapor concentration of BA was kept constant at 100 ppb and the temperature maintained at 25 °C. The data were linear over the entire time period, with no indication of substrate saturation, even after 8 h. The results demonstrated that the dosimeter can be designed to produce a response linear to the exposure time for over 8 h under saturated equilibrium vapor conditions. This feature is important for quantitative applications where daily occupational exposure of workers for 8 h workday shifts is generally monitored.
5. Conclusions
R a m a n and surface-enhanced R a m a n spectroscopies are spectrochemical techniques that have a number of important advantages to chemical sensing. The examples shown in this work illustrate the different uses of the technique for the detection of important environmental and biological compounds. At room temperature, R a m a n spectroscopy can provide
T. Vo-Dinh / Sensors and Actuators B 29 (1995) 183-189
an analytical tool having analytical figures of merit that complement luminescence. The Raman technique is well known for its high selectivity. With the advances of fiberoptic technology, the SERS technique, which can amplify the Raman signal by several orders of magnitude, can provide a remote sensing technique with the added merit of improved sensitivity due to the surfaceenhanced effect. The development of SERS-active probes will open new horizons for in situ remote sensors and biosensors for environmental and biomedical applications.
Acknowledgements This research is sponsored by the Office of Health and Environmental Research, US Department of Energy under Contract DE-AC05-84OR21400 with Lockheed Martin Energy Systems, Inc. and the Biosensor LDRD Project. The sponsorship of BMDO managed by the Office of Naval Research (NAVY No. N0001491-F-0042) is acknowledged. The author also thanks D.L..Stokes, J.P. Alarie, A.M. Helmenstine, V.A. Narayanan, W.S. Sutherland, R.L. Moody, J.M. Bello and P. Enlow for their assistance in the SERS measurements discussed in this work. References [1] T. Vo-Dinh, Anal. Chem., 50 (1978) 396. [2] T. Vo-Dinh, Appl. Spectrosc., 36 (1982) 576.
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[3] T. Vo-Dinh, Room Temperature Phosphorimetry, Wiley, New York, 1984. [4] A.M. Alak and T. Vo-Dinh, Anal. Chem., 65(1988) 596. [5] J.P. Goudonnet, G.M. Begun and E.T. Arakawa, Chem. Phys. Lett., 92(1982) 197. [6] T. Vo-Dinh, M.V.K. Hiromoto, G.M. Begun and R.L+ Moody, Anal. Chem., 56(1984) 1667. [7] R.L. Moody, T. Vo-Dinh and W.H. Fletcher, Appl. Spectrosc., 41 (1987) 966. [8] T. Vo-Dinh, Surface-enhanced Raman spectroscopy, in T. VoDinh (ed.), Chemical Analysis of Polyeyclic Aromatic Compounds, Wiley, New York, 1989. [9] B.J. Tromberg, M.J. Sepaniak, T. Vo-Dinh and G.D. Griffin, Anal, Chem., 59 (1987) 1226. [10] T. Vo-Dinh, B+J. Tromberg, G.D. Griffin, K.R. Ambrose, M.J. Sepaniak and E.M. Gardenhire, Appl. Spectrosc., 5 (1987) 735. [11] D.J. Jeanmaire and R.P. Van Duyne, J. Electroanal. Chem., 84 (1977) 1. [12] M.G. Albrecht and J.A. Creighton, J. Am. Chem. Soc., 99 (1977) 5215. [13] R.K. Chang and T.E. Furtak (eds.), Surjace-enhanced Raman Scattering, Plenum Press, New York, 1982. [14] M. Moskovits, Rev. Mod. Phys., 57(1985) 783. [15] G.C. Schatz, Acc. Chem. Res., 17(1984) 376. [16] J.M. Bello, V.S. Narayana, D.L. Stokes and T. Vo-Dinh, Anal. Chem., 62 (1990) 2437. [17] J.P. Alarie, D.L. Stokes, W.S. Sutherland, A.C. Edwards and T. Vo-Dinh, Appl. Spectros~., 46 (1992) 1608. [18] T. Vo-Dinh and M. Uziel, Anal. Chem., 59(1987) 1093. [19] T. Vo-Dinh (ed,), Chemical Analysis of Polycyclic Aromatic Compounds', Wiley, New York, 1989. [20] T. Vo-Dinh and D.L. Stokes, Appl. Speetrosc., 47 (1993) 1728. [21] J.M. Bello and T. Vo-Dinh, Appl. Spectrosc., 44 (1990) 63. [22] J.M. Bello, D.L. Stokes and T. Vo-Dinh, Appl. Spectrosc., 43 (1989) 1325. [23] A.M. Alak and T. Vo-Dinh, Anal. Chim. Acta., 206 (1988) 333. [24] A.M. Alak and T. Vo-Dinh, Anal. Chem., 59 (1987) 2149.