Anal. Chem. 1998, 70, 2991-2996
Immunoassays Based on Surface-Enhanced Infrared Absorption Spectroscopy Chris W. Brown,*,† Yue Li,† John A. Seelenbinder,† Phillip Pivarnik,‡ Arthur G. Rand,‡ Stephen V. Letcher,§ Otto J. Gregory,⊥ and Michael J. Platek|
Partnership for Sensors and Surface Technology, Departments of Chemistry, Food Science and Nutrition, Physics, Chemical Engineering, and Electrical Engineering, University of Rhode Island, Kingston, Rhode Island 02881
A new type of biosensor for pathogens has been developed. The sensor produces spectral fingerprints of biological systems by using surface-enhanced infrared absorption (SEIRA) spectroscopy. Antibodies were immobilized onto a 10-nm-thick film of gold which had been previously deposited on a Si wafer. SEIRA spectra of the antibodies measured in the external reflection mode exhibited two new bands at 1085 and 990 cm-1. These new bands were observed with p-polarized radiation but were absent with s-polarized radiation. The spectrum of water on the surface of the sensor was observed under both directions of polarization. The sensor was first tested with a model system consisting of glucose oxidase (GOX) and the antibodies for glucose oxidase (anti-GOX). In addition to the bands due to the anti-GOX at 1085 and 990 cm-1, new bands were observed at 1397, 1275, and 930 cm-1 when the GOX antigens were present. The same type of sensor was prepared for Salmonella (SAL) by immobilizing antibodies for Salmonella (anti-SAL) on a gold-surfaced Si wafer. The SEIRA spectra for anti-SAL antibodies were very similar to those for anti-GOX, with bands at 1085 and 990 cm-1; however, a sharp new band was observed at 1045 cm-1 after the sensor was exposed to the SAL antigens. In addition to specific new bands due to antigens, both GOX and SAL sensors exhibited changes in the regions of water absorptions at ∼3500 and 850 cm-1 when the antigens were present. Currently, there is considerable interest in the development of sensors for detecting chemicals and biological materials. Biosensors are a special class of sensors which associate a biological component with some type of transducer to provide a mechanism for detecting changes in the biological component. Most biosensors consist of enzymes or antibodies which interact with substrates or antigens. Generally, the enzymes or antibodies are immobilized on a platform, and the interaction between an the enzyme and substrate (or the antibody and antigen) is monitored by their attenuation of electrical, magnetic, optical, or acoustical signals. In some cases, the platform used for im†
Department of Chemistry. Department of Food Science and Nutrition. § Department of Physics. ⊥ Department of Chemical Engineering. | Department of Electrical Engineering. ‡
S0003-2700(98)00058-4 CCC: $15.00 Published on Web 06/12/1998
© 1998 American Chemical Society
mobilizing the biological material may be the transducer that is used for detecting the change. In other cases, an external signal is used to monitor the change or attenuation at the surface of the platform. A number of types of biosensors are under development or at the testing stage.1-5 However, to date, biosensor technology has been faced with several obstacles, even though antibody-based immunoassays can be very sensitive and, potentially, very selective. For example, fluorescence and/or luminescence techniques can provide very low levels of detection.6-8 However, the immunoassays based on fluorescence generally require that a sandwich be formed by combining the antibody-antigen with a labeled antibody; thus, this is not a single-step process. Moreover, fluorescence interferences are often encountered in the UVvisible region due to naturally occurring or other contaminating fluorophores;9 this has led to major efforts to develop near-IR fluorophores for labeling the antibodies,10,11 since naturally occurring fluorescence is mostly confined to the UV-visible regions. Biosensors based on surface plasmon resonance (SPR) have received considerable attention in recent years.4,5,12-21 In this method, antibodies are immobilized on the surface of a thin film of a precious metal such as Ag or Au, which was previously deposited on the base of an optically transparent prism. Visible/ (1) Van der Lelie, D.; Corbisier, P.; Baeyens, W.; Wuertz, S.; Diels, L.; Mergeay, M, Res. Microbiol. 1994, 145, 67-74. (2) Hartman, N. F. In Food Microbiological Analysis; Tortorello, M. L., Gendel, S. M., Eds.; Marcel Dekker: New York, 1997. (3) Deshpande, S. S.; Rocco, R.M. Food Technol. 1994, 48, 146-150. (4) Griffths, D.; Hall, G. Trends Biothechnol. 1993, 11, 122-130. (5) Byfield, M. P.; Abuknesha, R. A. Biosens. Bioelectron. 1994, 9, 373-400. (6) Herron, J. N.; Caldwell, K. D.; Christensen, D. A.; Dyer, S.; Hlady, V.; Huang, P.; Janatova, V. Advances in Fluorescence Sensing Technology. Proc. SPIEInt. Soc. Opt. Eng. 1995, 1885, 28-38. (7) Wyatt, G. M.; Lee, H. A.; Morgan, M. R. A. Immunoassays for Food-Poisoning Bacteria and Bacterial Toxins; Chapman & Hall: London, 1992; pp 29-67. (8) Rogers, K. R.; Gerlach, C. L. Environ. Sci. Technol. 1996, 30, 486A-491A. (9) Rahavendran, S. V.; Karnes, H. T. Pharm. Res. 1993, 10, 328-334. (10) Soper, S. A.; Mattingly, Q. L.; Vegunta, P. Anal. Chem. 1993, 65, 740-747. (11) Patonay, G.; Antoine, M. D. Anal. Chem. 1991, 63, 321A-327A. (12) Jorgenson, R. C.; Yee, S. S. Sens. Actuators B 1993, 12, 213-220. (13) De Maria, L.; Martinelli, M.; Vegetti, G. Sens. Actuators B 1993, 12, 221223. (14) Hodgson, J. Bio/Technology 1994, 12, 32-35. (15) Mouvet, C.; Maciag, C.; Wilkson, J. S. Anal. Chim. Acta 1997, 338, 109. (16) Simpson, T. R. E.; Cook, M. J.; Russell, D. A. Analyst 1996, 121, 1501. (17) Kondo, M.; Taya, T.; Yasukawa, K. Biotech. Technol. 1996, 10, 547. (18) Kruchinin, A. A.; Vlasov, Y. G. Sens. Actuators B 1996, 30, 77. (19) Demeule, M.; Vachon, V.; Beliveau, R. Anal. Biochem. 1995, 230, 239. (20) Drake, P. A.; Bohn, P. W. Anal. Chem. 1995, 67, 1766. (21) Brink, G.; Sigl, H.; Sackmann, E. Sens. Actuators B 1995, 25, 756.
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near-IR radiation is passed through the prism at such an angle as to cause an internal reflection at the base face of the prism. At a certain wavelength in the red or near-IR region, the light interacts with the electrons in metal to form a surface plasmon wave, and this resonance results in a strong absorption. The exact wavelength of this absorption depends on the angle of incidence, the metal (size, amount and type), and the surrounding material. The wavelength of the maximum absorbance is shifted by immobilizing antibodies onto the precious metal.4,5,12 The presence of antigens coupling with these antibodies causes an additional shift in the resonance wavelength, and the amount of the shift can be related to the concentration of antigens present in the surrounding medium. Antigens can also be detected by the SPR method using the change in the incident angle which produces the maximum resonance.20 If monochromatic radiation having a wavelength near the resonance is passed through the prism, the presence of antigens changes the resonance angle; thus, both wavelength and angle shifts can be used to monitor the presence and concentration of antigens. SPR is a very sensitive technique; however, it suffers from the fact that nonspecific adsorptions can occur on the immobilized surfaces, e.g., proteins can cling to the surface and cause a change in wavelength or angle of maximum resonance, and this would be falsely interpreted as the antigen. Efforts have been made to minimize nonselective adsorptions by protecting the surface with materials that limit this type of adsorption;22-24 however, there is no guarantee that it is eliminated completely. Interferences due to fluorescence9-11 and nonspecific adsorptions22-24 have been two of the drawbacks in the development of biosensors for toxic chemicals and pathogens. These difficulties decrease the ability of the biosensor to be selective and limit the minimum level of detection. Thus, it has been our goal to find a biosensor-based method which is both selective and sensitive but excludes potential interferences. Our laboratory has been exploring the feasibility of using surface-enhanced infrared absorption (SEIRA) spectroscopy as a means of providing selective and sensitive detection of antigens having biomedical and environmental importance. The SEIRA effect was first reported by Hartstein et al.25 in 1980. Since that time, the effect has been studied in a number of laboratories.26-37 At the present time, the exact cause of this phenomenon is still (22) Davis, K. A.; Leary, T. R. Anal. Chem. 1989, 61, 1227-1230. (23) Muramatsu, H.; Kajiwara, K.; Tamiya, E.; Karube, I. Anal. Chim. Acta 1986, 188, 257-261. (24) Prusak-Sochaczewski, E.; Luong, J. H. T. Enzyme Microb. Technol. 1990, 12, 173-177. (25) Hartstein, A.; Kirtley, J. R.; Tsang, T. C. Phys. Rev. Lett. 1980, 45, 201204. (26) Hatta, A.; Suzuki, Y.; Suetaka, W. Appl. Phys. A 1984, 35, 135-140. (27) Kamata, T.; Kato, A.; Umemura, T.; Takenaka, T. Langmuir 1987, 3, 11501154. (28) Osawa, M.; Ikeda, M. J. Phys. Chem. 1991, 95, 9914-9919. (29) Nishikawa, Y.; Fujiwara, K.; Shima, T. Appl. Spectrosc. 1990, 44, 691-694; 1991, 45, 747-751. (30) Osawa, M.; Ataka, K.; Ikeda, M.; Uchihara, H.; Nanba, R. Anal. Sci. 1991, 7, 503-506. (31) Nishikawa, Y.; Fujiwara, K.; Ataka, K.; Osawa, M. Anal. Chem. 1993, 65, 556-562. (32) Matsuda, N.; Yoshii, K.; Ataka, K.; Osawa, M.; Matsue, T.;. Uchida, I. Chem. Lett. 1992, 1385. (33) Osawa, M.; Ataka, K. Surf. Sci. 1992, 262, L118-L122. (34) Osawa, M.; Ataka, K.; Yoshii, K.; Nishikawa, Y. Appl. Spectrosc. 1993, 47, 1497-1501.
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not completely understood, but it is known that multiple, overlapping mechanisms are most likely involved in the enhancement. The general model used to explain the enhancement is related to the SPR effect. In the case of SEIRA effect, a very thin film of a precious metal such as Ag or Au is deposited on the surface of a substrate. On certain substrates, such thin films (on the order of 10 nm thick) consist of very small islands which appear as oblate ellipsoids whose long axes are parallel to the substrate surface. In the infrared region, the incident radiation has a wavelength much larger than that of the metal islands. It has been proposed that the incident radiation induces an oscillating dipole in the metal islands which, in turn, causes a strong electromagnetic field around the metal islands.34 This latter field is perpendicular to the ellipsoidal surface of the metal islands, as shown in Figure 1. Molecules either physically or chemically absorbed on the surface of the metal can modulate this electromagnetic field. It has been shown that the modulations due to the vibrations can cause either an increase or a decrease in radiation reflected from the surface, depending upon the angle of incidence.31,35 Moreover, it has been shown that chemically adsorbed molecules lying perpendicular to the metal surface cause the greatest modulation.34 Samples being tested for the SEIRA effect are prepared by methods similar to those used for SPR. A thin film of Au (or other precious metal) is deposited onto a substrate, such as a germanium or silicon plate. The organic chemical of interest is then dispersed onto the thin metal film. Much of the early investigations of the infrared enhancement were performed with the model compound, p-nitrobenzoic acid (PNBA).26,28,30,33,36,37 Most of the studies have shown that the SEIRA phenomenon has a very short range and affects only the first layer (monolayer) of the organic chemical. Moreover, the enhancements are observed only in a limited number of normal vibrations of the molecules. In the case of PNBA, the enhancements are observed in the symmetric COOand symmetric NO2 vibrations, in which the dipole moment changes are perpendicular to the metal surface. The absorptions due to these two vibrations are strong, whereas other absorptions are very weak or not observed. The intensification of the two “resonant” vibrations for a monolayer of PNBA molecules is very dramatic and suggests that the SEIRA effect could lead to a very sensitive method for detecting small amounts of organic chemicals or changes in chemicals. The models for the SEIRA effect indicate that a molecule such as PNBA becomes chemically bonded to the metal substrate.26,36 Evidence has been presented to show that the acid proton is removed from the molecule and that the COO- group bonds the acid anion, PNBA-, to the surface in such a way that the molecule is perpendicular to the surface, with the COO- headgroup attached to the metal island and the phenyl NO2 extending away from the metal island. We might picture the metal island as a threedimensional oblate ellipsoid covered with PNBA- ions lined up parallel to each other and perpendicular to the metal in such a way that all of the NO2 groups form the outer surface. Basically, the metal serves as a highly efficient material for coupling the (35) Merkin, G. I.; Griffiths, P. R. J. Phys. Chem. 1997, 101, 5810; 1997, 101, 7408. (36) Merkin, G. I.; Griffiths, P. R. Langmuir 1997, 13, 6159. (37) Wanzenbock, H. D.; Mizakoff, B.; Weissenbacher, N.; Kellner, R. J. Mol. Struct. 1997, 410-411, 535-538.
Figure 1. Cartoon of a single gold metal island coated with antibodies (Y) on the surface of the substrate. The dipole moment induced in the metal island produces a electromagetic field which is perpendicular to the island and parallel to the common or contant region (Fc) of the antibodies.
radiation to the attached molecules so that particular vibrational absorptions are greatly enhanced. The above picture of metal ellipsoids covered with the PNBAanions is similar to the one we might picture for a monolayer of antibodies immobilized on the metal ellipsoids. The nitro groups on the surface mimic the active or Y sections of antibodies. Other than this vague geometrical similarity of the two configurations, there was no previous evidence to suggest that infrared absorptions due to vibrational frequencies of antibodies could be enhanced by the SEIRA effect. However, since antibodies had been shown to work in the SPR effect, we decided to explore the feasibility of obtaining the SEIRA effect with immobilized antibodies to develop a sensitive and selective biosensor. EXPERIMENTAL SECTION Chemicals. Polyclonal anti-glucose oxidase, anti-salmonella, mouse IgG, goat IgG, and Salmonella typhimurim ATCC strain 13311 were purchased from Biodesign International (Kennebunk, ME). Glucose oxidase and all other chemicals were obtained from Sigma Chemical Co. (St. Louis, MO). Preparation of Sensors. In the present investigation, polished Si plates were used as sensor platforms. The plates were cleaned with consecutive 40-min applications of acetone, methanol, and deionized water in an ultrasonic bath. The clean plates were then sputter-coated with gold using a MRC 8667 multitarget sputtering system (Material Research Corp., Orangeburg, NY) to form a 10-nm-think film. The plates were then washed with phosphate buffer solution (PBS at pH of 7.4) and deionized water. Background spectra were measured at this point. Antibodies were immobilized on the surface of the sensor by immersing the plates in a solution of the appropriate antibodies (5 µg/mL of solution) and glutaraldehyde (2.5%) for 1 h; the glutaraldehyde was used for cross-linking the antibodies. After the immersion, the plates were washed again with phosphate buffer solution and immersed in a solution containing bovine serum albumin (BSA at 1%) and glutaraldehyde (2.5%) at pH of
7.4 for 1 h; the BSA was used as a blocking agent to reduce nonselective adsorptions. The plates were then washed with phosphate buffer solution and spectra of the antibodies measured. Finally, the sensor plates were immersed in a solution of the antigens, removed, and washed with buffer and the spectra measured. Spectra. Spectra were measured on a Bio-Rad FTS40 FT-IR from 4000 to 400 cm-1 with 64 scans at 4-cm-1 resolution. Reflection infrared spectra were measured with the Si substrate mounted at an angle of ∼75° in a Foxboro-Wilks model 9D reflection attachment (Foxboro, MA). Prior to each sensor investigation, a background spectrum of the Au film was measured. Polarized spectra were measured with a wire grid ZnSe polarizer. RESULTS AND DISCUSSION Model SystemsSensor for Glucose Oxidase. Our first investigation was on the innocuous system of glucose oxidase (GOX) and antibodies of glucose oxidase (anti-GOX). In the initial experiments, anti-GOX was immobilized onto a gold surface which had been deposited on a 2- × 3-cm wafer of Si. The wafer was placed in an external reflection attachment of a FT-IR, and the reflection spectra were measured at an angle of 75°. Polarized infrared spectra of the anti-GOX are shown in Figure 2. In the s-polarized spectrum, where the electric vector of the incident radiation is perpendicular to the plane formed by the direction of the incident radiation and the normal to the reflecting surface (this vector is parallel to the reflecting surface of the substrate), only bands due to water at ∼3400, 1600, and 800 cm-1 are observed. In the p-polarized spectrum, where the electric vector of the incident radiation is in the plane formed by the direction of the incident and the normal, new bands due to anti-GOX are observed at 1085 and 990 cm-1. The 1085-cm-1 band is assigned to a C-O stretching vibration of the protein,38 which is enhanced by the attachment to the gold particles. The sensor (i.e., the silicon wafer with the immobilized antibodies on the Au film) was removed from Analytical Chemistry, Vol. 70, No. 14, July 15, 1998
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a
Figure 2. SEIRA spectra of the antibodies for glucose oxidase (antiGOX) immobilized on a Au thin film which was deposited on a silicon wafer. In the s-polarization spectrum, the electric vector of the radiation is polarized parallel to the wafer, and, in the p-polarization, the electric vector of the radiation is polarized perpendicular to the wafer. Water bands appear at both polarizations, but the anti-GOX bands at 990 and 1085 cm-1 appear only with the p-polarization.
the reflection attachment and placed in a solution containing glucose oxidase (GOX) for a few minutes. The wafer was then removed from the solution, washed with distilled water, and returned to the reflection attachment and the reflection spectrum measured. The complete spectra with and without GOX are shown in Figure 3a. There are distinctive shape changes in the water bands at 3400 and 800 cm-1, and new bands appear in the region from 1400 to 900 cm-1. An expanded spectrum of the lowfrequency region is shown in Figure 3b. New bands due to the GOX-antibody interaction are observed at 1397, 1275, and 930 cm-1. The band at 1397 cm-1 has been assigned the C-N stretching vibration of a primary amide, whereas the band at 1275 cm-1 has been assigned to the amide III band, which is a mixed vibration involving OCN and N-H modes.39 The weak shoulder at 930 cm-1 is assigned as an NH or NH2 wagging mode. This experiment was performed two additional times with identical results. In the present configuration, the sensor is being used in a “dipstick” configuration. The sensor was prepared by depositing a thin film of Au on a silicon plate, followed by the immobilization of antibodies on the Au surface. The reflection spectrum of this sample was measured, and this was used as the background for future measurements. The sensor was then dipped into a solution containing antigens, removed, washed with distilled water, and placed in the measurement device (in this case, the FT-IR). Changes in the reflection spectra are indicative of the antigen. To determine the source of changes in the SEIRA spectra after the addition of antigens, a “reverse” sensor experiment was performed. GOX was first immobilized on a fresh Au film and its SEIRA spectrum measured. This sensor was then dipped into a solution containing anti-GOX for several minutes and the SEIRA spectrum remeasured. The resulting spectra, shown in Figure (38) Colthup, N. B.; Daly, L. H.; Wiberley, S. E. Introduction to Infrared and Raman Spectroscopy, 3rd ed.; Academic Press: Boston, MA, 1990. (39) Parker, F. S. Applications of Infrared Spectroscopy in Biochemistry, Biology, and Medicine; Plenum Press: NewYork, 1971.
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b
Figure 3. (a) SEIRA spectra of anti-GOX with and without GOX (glucose oxidase) antigen interactions. (b) Expansion of the spectra. In addition to the anti-GOX bands at 990 and 1085 cm-1, new bands due to the GOX-antibody interaction are observed at 930, 1275, and 1397 cm-1.
Figure 4. Spectra of “reversed” sensor with GOX imobilized on the gold surface and anti-GOX collected from solution onto the GOX antigen.
4, are very similar to those shown in Figure 3a. The spectrum of GOX exhibits only very weak bands at 1085 and 990 cm-1, which are weaker but similar to the bands in the spectrum of anti-GOX only shown in Figure 3. The spectrum with anti-GOX interacting
a
b
Figure 5. (a) SEIRA spectra of anti-SAL with and without SAL (Salmonella) antigen interactions. (b) Expansion of the spectra showing the anti-SAL bands at 990 and 1085 cm-1 with a new band at 1045 cm-1 due to the SAL-antibody interaction.
with GOX in Figure 4 is virtually identical to the spectrum of GOX interacting with anti-GOX in Figure 3. Thus, it appears that the immobilized antibodies or antigens have very similar spectra, which are probably due to enhanced bands of the protein vibrations. The new bands (at 1397, 1275, and 930 cm-1) in the spectra after the addition of the antigens to the antibodies or the antibodies to the antigens are due to changes in either the antibody or the antigen caused by interaction. Apparently, the antibody-antigen interaction causes selected vibrations to be perpendicular to the gold surface, and these are enhanced by the SEIRA phenomenon. SEIRA Sensor for Salmonella. A second sensor was next prepared for the pathogen, Salmonella (SAL). The procedure for preparing the sensor was identical to that described above. The antibodies for Salmonella (anti-SAL) were immobilized onto a thin film of Au which had been deposited onto a silicon wafer. A spectrum of the immobilized antibodies was measured, and the sensor was then placed in a solution containing SAL for a few minutes, removed, washed, and inserted into the reflection attachment. The spectra of the anti-SAL sensor with and without SAL are compared in Figure 5. The spectrum of anti-SAL without SAL is almost identical to that of the anti-GOX, with bands due to the antibody at 1085 and 990 cm-1. However, in the presence
Figure 6. SEIRA spectra of mouse IgG and goat IgG showing enhanced bands at 990 and 1085 cm-1.
of SAL, the anti-SAL spectrum exhibits a sharp new band at 1045 cm-1. This single band, which is assigned to the P-O stretching vibration of the phospholipid in the cell wall, is a clear indication of the presence of SAL in the solution. In addition to the new bands due to SAL, there is also a dramatic contour change in the OH stretching region (3700-3000 cm-1) and an increase in the absorption at ∼850 cm-1 after the antigens interact with the antibodies. The first of these changes is certainly related to changes in the OH and/or NH stretches. The increase in the intensity at 3600 cm-1 suggests that either the amount of H2O is reduced (indicated by the intensity decrease at 3400 cm-1) or the amount of free -OH is increased, or both. There also appears to be a less pronounce band at ∼ 3100 cm-1, possibly due to NH or NH2 stretching vibrations. The spectra of all samples discussed herein were measured in an external reflection mode with the samples damp, i.e., they were measured immediately after removal from the aqueous solutions. (This explains the presence of water vapor bands in some of the spectra, since water could not be effectively removed from the spectrometer.) Presently, we are investigating the changes in the OH regions due to the antibody-antigen interaction by measuring spectra of dried samples and samples measured with attenuated total reflection (ATM). These results will be reported soon. Similarity of Antibody Spectra. At the present time, our investigations have focused on the ability of the SEIRA sensors to detect the desired antigen. It appears that IgG antibodies may have identical SEIRA background spectra, since the reflection spectra of the antibodies without the antigens are the very similar. We have tested this concept on two additional antibodies, mouse IgG and Goat IgG. The spectra, shown in Figure 6, are identical to each other in the 900-1200-cm-1 region and are identical to those for the polyclonal antibodies for GOX and SAL. CONCLUSIONS Herein, we demonstrated that the SEIRA effect can greatly enhance spectra of biological materials such as antibodies. More importantly, additional (enhanced) bands appear during an antibody-antigen interaction; these changes can be used to signal the presence of antigens and also for identification purposes, since the two antigens under investigation produce different infrared Analytical Chemistry, Vol. 70, No. 14, July 15, 1998
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bands. Potentially, other antibody-antigen interactions could produce identifying fingerprints, and we plan to explore this possibility. External reflection was used to measure the spectra in the present investigation rather than either attenuated total reflection (ATR) or direct transmission. A large number of sensor plates were needed to develop the gold coating and antibody immobilization procedures in addition to performing the initial tests; thus, the use of ATR prisms would have been impractical for this investigation. Moreover, transmission spectra of the Si plates exhibited bands due to the native oxide in the 600-1200-cm-1 region which could interfere with bands due to the antibodies and antigens; therefore, we did not use transmission spectra for the present study. Currently, we are investigating the limits of detection for the IR biosensors. Our initial investigations were performed on concentrations of antigens in the 104-106 colonies/mL range. We have not determined the capability to quantitate or the limits of detection. Since it appears that new IR bands due to antibody-
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antigen interactions are antigen specific, we are currently working on a sensor that will contain antibodies for several pathogens. It is possible that single sensor could be developed for a larger number of pathogens or chemicals. Potentially, antibodies can be grown for any chemical or pathogen, and each antibodyantigen interaction would have a specific infrared fingerprint. ACKNOWLEDGMENT This work was supported, in part, by the Cooperative State Research, Education and Extension Service, U.S. Department of Agriculture (NRICGP Grant No. 93-37201-9197); by the U.S. Army Natick Research & Development Center (Contract No. DAAK6095C2035); and by the URI Partnership for Sensors and Surface Technology. This is contribution no. 3598 of the Rhode Island Agricultural Experiment Station. Received for review January 22, 1998. Accepted April 29, 1998. AC980058K
