Monoclonal Antibody−Gold Biosensor Chips for ... - ACS Publications

Jul 19, 2000 - University of Nebraska Medical Center, 600 South 42nd Street, ... Eppley Institute for Research in Cancer and Allied Diseases, Universi...
0 downloads 0 Views 251KB Size
Download PDF
Anal. Chem. 2000, 72, 3709-3716

Monoclonal Antibody-Gold Biosensor Chips for Detection of Depurinating Carcinogen-DNA Adducts by Fluorescence Line-Narrowing Spectroscopy Scott D. Duhachek,† Jeremy R. Kenseth,†,‡ George P. Casale,§ Gerald J. Small,† Marc D. Porter,†,‡ and Ryszard Jankowiak†,*

Ames Laboratory-USDOE and Department of Chemistry, Microanalytical Instrumentation Center, Iowa State University, Ames, Iowa 50011, Eppley Institute for Research in Cancer and Allied Diseases, University of Nebraska Medical Center, 600 South 42nd Street, Omaha, Nebraska 68198

A new direct readout methodology for detection and quantitation of fluorescent carcinogen-DNA adducts is described. It combines the binding specificity of an immobilized monoclonal antibody (MAb) with highresolution, low-temperature fluorescence spectroscopy. The MAb, which is covalently bound to a gold surface via a chemisorbed disulfide coupling agent, binds the adduct of interest in an aqueous sample. Laser-induced fluorescence under nonline narrowing (FNLN) and line-narrowing (FLN) conditions was used to detect (benzo[a]pyren6-yl)guanine (BP-6-N7Gua) bound to immobilized MAb. At room temperature, the BP-6-N7Gua fluorescence was not detected, most likely because of quenching by the gold surface and/or efficient dynamical quenching. However, fluorescence was observed at room temperature when the surface was covered with a thin layer of glycerol, and possible reasons for the fluorescence enhancement are considered. Lowering of the temperature to 77 K led to nearly an order of magnitude increase in fluorescence intensity. Highly structured FLN spectra obtained at 4.2 K allowed for definitive adduct identification. The potential of this methodology for risk assessments of individuals exposed to polycyclic aromatic hydrocarbons is discussed. Benzo[a]pyrene (BP) is considered a reliable indicator of exposure to carcinogenic polycyclic aromatic hydrocarbons (PAHs).1 A high level of exposure to BP and other PAHs occurs among workers directly participating in numerous industrial processes, such as aluminum production, processes occurring in iron and steel foundries, coke production, and coal gasification.1,2 For aluminum workers in an anode factory, ambient BP levels as high * To whom correspondence should be addressed (E-mail: jankowiak@ ameslab.gov). † Ames Laboratory-USDOE and Department of Chemistry, Iowa State University. ‡ Microanalytical Instrumentation Center, Iowa State University. § Eppley Institute for Research in Cancer and Allied Diseases, University of Nebraska Medical Center. (1) International Agency for Research in Cancer. In IARC Monographs on the Evaluation of Carcinogenic Risk of Chemicals to Humans, Volume 34, Polynuclear Aromatic Hydrocarbons. Part 3; Lyon, France, 1984. 10.1021/ac000472w CCC: $19.00 Published on Web 07/19/2000

© 2000 American Chemical Society

as 11 600 ng/m3 were reported.3 As the result of burning coal in their dwellings, residents in Xuan Wei County (China) were exposed to indoor BP levels of up to 2 485 ng/m3.4 Cigarette smoking continues to be a significant source of BP exposure, i.e., mainstream smoke produced by a single cigarette contains 2040 ng of BP.5 BP can be activated metabolically by one-electron oxidation to yield reactive radical cations,6 by monooxygenation to produce bay-region diol epoxides,6,7 or by the formation of the reactive and redox active o-quinones catalyzed by dihydrodiol dehydrogenases.8 The active intermediates formed bind to DNA and form “stable” and/or “depurinating” DNA adducts.6-8 Since depurinating adducts are released spontaneously from DNA by hydrolysis of the N-glycosidic bond,6,7 their presence reports directly on DNA damage. Therefore, the development of new methodologies for rapid, low-level detection of depurinating DNA adducts in human fluids is of considerable importance. Recently, the BP-derived 7-(benzo[a]pyren-6-yl)guanine (BP6-N7Gua), a depurinating one-electron oxidation adduct, has been detected in urine extracts of humans exposed to coal smoke.9 This adduct (Figure 1) was identified on-line with capillary electrophoresis-fluorescence line-narrowing spectroscopy (CE-FLNS) at 4.2 K9,10 as well as by mass spectrometry.10 The daily excretion of BP-6-N7Gua in human urine of individuals exposed to coal (2) Van Schooten, F. J.; Van Leeuwen, F. E.; Hillebrand, M. J. X.; Rijke, M. E. D.; Hart, A. A. M.; Van Veen, H. G.; Oosternik, S.; Kriek, E. J. Natl. Cancer Inst. 1990, 82, 927-933. (3) Van Schooten, F. J.; Jongeneelen, F. J.; Hillebrand, M. J. X.; Van Leeuwen, F. E.; Looff, A. J. A. D.; Dijkmans, A. P. G.; Van Rooij, J. G. M.; Englese, L. D.; Kriek, E. Cancer Epidemiol. Biomarkers Prev. 1995, 4, 69-77. (4) Mumford, J. L.; Li, X.; Hu, F.; Lu, X. B.; Chuang, J. C. Carcinogenesis 1995, 16, 3031-3036. (5) Surgeon General. In The Health Consequences of Smoking: Cancer. A report of the Surgeon General; United States Department of Health and Human Services; United States Government Printing Office: Washington, DC, 1986. (6) Cavalieri, E.; Rogan, E. Pharmacol. Ther. 1992, 55, 1083-1099. (7) Cavalieri, E.; Rogan, E. In The Handbook of Environmental Chemistry; Neilson, A. H., Ed.; Springer-Verlag: Heidelberg, Germany, 1998; Vol. 3, pp 81117. (8) Penning, T. M.; Burczynski, M. E.; Hung, C.-F.; McCoull, K. D.; Palackal, N. T.; Tsuruda, L. S. Chem. Res. Toxicol. 1999, 12, 1-18. (9) Roberts, K.; Lin, C.-H.; Singhal, M.; Casale, G.; Small, G. J.; Jankowiak, R. Electrophoresis 2000, 21, 799-806.

Analytical Chemistry, Vol. 72, No. 16, August 15, 2000 3709

Figure 1. Molecular structure of the BP-6-N7Gua adduct.

smoke was 150-300 fmol/mg creatinine equivalence of urine.9,10 In addition, the 7-(Benzo[a]pyren-6-yl)adenine depurinating adduct (BP-6-N7Ade) was detected at the level of 10-20 fmol/mg creatinine equivalence of urine.10 BP-6-N7Ade was also present in urine from cigarette smokers, though at a significantly lower level, i.e., 0.5-1.0 fmol/mg creatinine equivalence of urine.10 Because of their high levels of excretion, it was proposed that the BP-6-N7Gua and BP-6-N7Ade adducts found in urine could serve as effective biomarkers for risk-assessment studies of BP exposure. Techniques, such as CE-FLNS,9-13 however, would require extensive development to function effectively in routine risk assessments. It is important, therefore, to develop simpler methods that possess adequate sensitivity and selectivity. In this regard, we believe that an FLNS-based approach that utilizes a monoclonal antibody (MAb)-based heterogeneous direct immunoassay (IA) on a gold biosensor chip has considerable potential. This concept potentially simplifies the analysis, in that the chip selectively extracts the analyte from the sample, reducing the complexity in sample workup and the needed instrumentation. The feasibility of this approach is demonstrated herein using the BP-6-N7Gua adduct, for which a monoclonal antibody (MAb CB53) with high affinity has been developed.14,15 Immunoassays play a vital role in clinical and environmental chemistry.16-18 Most traditional forms of IA employ absorbance, fluorescence, amperometric, or radiochemical transduction mechanisms, requiring labeled receptors to report indirectly the (10) Casale, G. P.; Singhal, M.; Bhattacharya, S.; RamaNathan, R.; Roberts, K. P.; Zhao, J.; Jankowiak, R.; Gross, M. L.; Cavalieri, E. L.; Small, G. J.; Rennard, S. I.; Mumford, J. L.; Chen, M. Chem. Res. Toxicol. 1999, submitted for publication. (11) Roberts, K.; Lin, C.-H.; Jankowiak, R.; Small, G. J. J. Chromatogr., A 1999, 853, 159-170. (12) Jankowiak, R.; Zamzow, D.; Ding, W.; Small, G. J. Anal. Chem. 1996, 68, 2549-2553. (13) Zamzow, D.; Lin, C.-H.; Small, G. J.; Jankowiak, R. J. Chromatogr., A 1997, 781, 73-80. (14) Casale, G. P.; Rogan, E. G.; Stack, D.; Devanesan, P.; Cavalieri, E. L. Chem. Res. Toxicol. 1996, 9, 1037-1043. (15) Bhattacharya, S.; Shen, M.; Duhachek, S.; Roberts, K. P.; Jankowiak, R.; Cavalieri, E. C.; Casale, G. P., manuscript in preparation. (16) Diamandis, E. P.; Christopoulos, T. K. In Immunoassay; Academic Press: San Diego, CA, 1996. (17) Aga, D. S.; Thurman, E. M. In ImmunochemicaI Technology for Environmental Applications; ACS Symposium Series 657; American Chemical Society: Washington, DC, 1996. (18) Jones, V. W.; Kenseth, J. R.; Mosher, C. L.; Henderson, E.; Porter, M. D. Anal. Chem. 1998, 70, 1233-1241.

3710

Analytical Chemistry, Vol. 72, No. 16, August 15, 2000

presence of an analyte.19 The use of labeled receptors, however, generally results in additional preparative steps and higher overall costs. Thus, the development of IA strategies which provide direct detection of an analyte without the need for additional labeling steps is of clear importance. Several techniques have been developed which allow for the direct detection of analytes in IA. Examples include capacitance,20-22 surface plasmon resonance,23 piezoelectric,24-26 mass spectrometry,27 and optical diffraction.28-30 Recently, it was demonstrated that atomic force microscopy (AFM) could be used for the direct detection of antibody-antigen binding.18 Previous studies had demonstrated that AFM possesses sufficient sensitivity to detect the height increase that occurs as a result of a single antibodyantigen binding event.31,32 Since this approach relies on the change in height that results from ligand-receptor binding, the use of labeled receptors is not necessary. Of the direct readout approaches, only mass spectrometry allows for definitive identification of the bound analyte.27 Specific binding of analyte and nonspecific binding to an immunosensor surface must be differentiated through the use of additional controls. In addition, when employing direct-detection IA methods, the smaller the molecules of interest, the more difficult they are to detect.33 For example, direct detection of bound DNA adducts by AFM using a height-based scheme is difficult, since the height changes upon adduct binding are close to the noise level of the measurement. Therefore, examining the potential of alternative forms of IA for the direct chemical identification of small analytes is of both fundamental and technological interest. Fluorescence spectroscopy is one of the most widely used readout methods for detection of various antigens, primarily because of its high sensitivity. In this regard, FLNS is noteworthy because it is capable of distinguishing, for example, between a given PAH metabolite covalently bound to different DNA bases and that covalently bound to different nucleophilic centers of a given base.34-39 The high level of selectivity and sensitivity of the FLNS technique can, therefore, be exploited for characterization and determination of fluorescent analytes. In this paper, we (19) Gosling, J. P. Clin. Chem. 1990, 36, 1408-1427. (20) Bataillard, P.; Gardies, F.; Jaffrezic-Renault, N.; Martelet, C. Anal. Chem. 1988, 60, 2374-2379. (21) Berggren, C.; Johansson, G. Anal. Chem. 1997, 69, 3651-3657. (22) Gebbert, A.; Alvarez-Icaza, M.; Stocklein, W.; Schmid, R. D. Anal. Chem. 1992, 64, 997-1003. (23) Frutos, A.; Corn, R. Anal. Chem. 1998, 70, 449A-455A. (24) Roederer, J. E.; Bastiaans, G. J. Anal. Chem. 1983, 55, 2333-2336. (25) Welsch, W.; Klein, C.; Von Schickfus, M.; Hunklinger, S. Anal. Chem. 1996, 68, 2000-2004. (26) Nakanishi, K.; Muguruma, H.; Karube, I. Anal. Chem. 1996, 68, 16951700. (27) Nelson, R. W.; Krone, J. R.; Bieber, A. L.; Williams, P. Anal. Chem. 1995, 67, 1153-1158. (28) Tsay, Y. G.; Lin, C. I.; Lee, J.; Gustafson, E. K.; Appleqvist, R.; Magginetti, P.; Norton, R.; Teng, N.; Charlton, D. Clin. Chem. 1991, 37, 1502-1505. (29) John, P. S.; Davis, R.; Cady, N.; Czajka, J.; Batt, C. A.; Craighead, H. G. Anal. Chem. 1998, 70, 1108-1111. (30) Bernard, A.; Bosshard, H. R. Eur. J. Biochem. 1995, 230, 416-423. (31) Browning-Kelley, M. E.; Wadu-Mesthrige, K.; Hari, V.; Liu, G. Y. Langmuir 1997, 13, 343-350. (32) Quist, A. P.; Bergman, A. A.; Reimann, C. T.; Oscarsson, S. O.; Sundqvist, B. U. R. Scanning Microsc. 1995, 9, 395-400. (33) Karlsson, R. Anal. Biochem. 1994, 221, 142-151. (34) Jankowiak, R.; Small, G. J. Anal. Chem. 1989, 61, 1023A-1032A. (35) Devanesan, P. D.; RamaKrishna, N. V. S.; Todorovic, R.; Rogan, E. G.; Cavalieri, E. L.; Jeong, H.; Jankowiak, R.; Small, G. J. Chem. Res. Toxicol. 1992, 5, 302-312.

demonstrate, for the first time, that low-temperature laser-induced fluorescence (LIF), under nonline narrowing (FNLN) and linenarrowing (FLN) conditions, can be used for direct detection of DNA adducts on a gold biosensor chip. MATERIALS AND METHODS Cautions: BP is a hazardous chemical and should be handled carefully in accordance with NIH guidelines. Piranha solution is highly oxidizing and should be handled with extreme care. Preparation of Gold Substrates. Substrates were prepared on 10 mm × 10 mm silicon wafers (100 single crystal, Montco Silicon) or glass slides (Fisher) by the vapor deposition of 15 nm of chromium at 0.1 nm/s followed by 300 nm of gold (99.9% purity) at 0.3 nm/s. The metal depositions were carried out in an Edwards 306A cryo-pumped evaporator at a pressure of 4 × 10-6 Torr. The chromium layer was used to enhance the adherence of the gold film to the substrate. Prior to metal deposition, the silicon substrates were cleaned in an ultrasonic bath for 20 min in deionized water and 20 min in methanol and then dried using high-purity argon. The glass slides were sonicated in Contrad 70 detergent (Fisher) for 20 min and then in neat methanol for 20 min, and finally, dried under a stream of purified argon. Before monolayer deposition, the gold surfaces were cleaned with Piranha solution (3:1 H2SO4 and H2O2) and quickly dried under a stream of argon. Chemicals. Dithiobis(succinimidyl undecanoate) (DSU) was synthesized from a combination of literature procedures.40,41 Octadecanethiol (Aldrich) was recrystallized from ethanol. All other reagents were used as received. Immobilization of Monoclonal Antibody and Adlayer Characterization by Infrared Reflection Spectroscopy (IRS). Infrared reflection spectra were acquired using a Nicolet 750 FTIR spectrometer purged with boil-off from liquid N2. Spectra were obtained in an external reflection mode using p-polarized light incident at 82° with respect to the surface normal. A liquid-N2cooled HgCdTe detector was used for all measurements. The spectra were recorded as -log(R/R0) where R is the reflectance of the sample and R0 is the reflectance of an octadecanethiolated37 monolayer-coated Au(111) reference. The spectra are an average of 512 scans and were taken at a resolution of 2 cm-1 with Happ-Genzel apodization. For IRS characterizations of the effectiveness of the immobilization process, gold-coated glass slides were immersed in a dilute (0.1 mM) ethanol solution of dithiobis(succinimidyl undecanoate) (DSU) for 14 h, rinsed with ethanol, and dried under a stream of argon. Following acquisition of the IR spectrum of the DSU monolayer, the sample was exposed to a solution of MAb CB53 in borate buffer (100 mM H3BO3, 100 mM KCl, pH 8.5, 1% (36) Rogan, E. G.; Devanesan, P. D.; RamaKrishna, N. V. S.; Higginbotham, S.; Padmavathi, N. S.; Chapman, K.; Cavalieri, E. L.; Jeong, H.; Jankowiak, R.; Small, G. J. Chem. Res. Toxicol. 1993, 6, 356-376. (37) Rogan, E. G.; RamaKrishna, N. V. S.; Higginbotham, S.; Padmavanthi, N. S.; Chapman, K.; Cavalieri, E. L.; Jeong, H.; Jankowiak, R.; Small, G. J. Chem. Res. Toxicol. 1990, 3, 441-444. (38) Jankowiak, R.; Small, G. J. Chem. Res. Toxicol. 1991, 4, 256-269. (39) Jankowiak, R.; Small, G. J. In The Handbook of Environmental Chemistry; Neilson, A. H., Ed.; Springer-Verlag: Heidelberg, Germany, 1998; Vol. 3J pp 81-97. (40) Wagner, P.; Hegner, M.; Kernen, P.; Zaugg, F.; Semenza, G. Biophys. J. 1996, 70, 2052-2066. (41) Nakano, K.; Taira, H.; Maeda, M.; Takagi, M. Anal. Sci. 1993, 9, 133-136.

Tween 80) for 21 h. The MAb CB53 concentration was 40 µg/ mL, and the total solution volume translated to about 10 times the MAb CB53 level required for saturation coverage. The sample was again rinsed with buffer and distilled, deionized water (Millipore); dried under a stream of argon; and characterized by IRS. Immobilization of Monoclonal Antibody and Characterization by Atomic Force Microscopy (AFM). To verify further the MAb immobilization process, a compositionally patterned glycolterminated (GLY)/DSU monolayer was prepared on the goldcoated silicon substrates, noting that the incorporation of regions within the patterned surface which resist nonspecific binding of MAb (i.e., the glycol-terminated areas) provides a topographical “reference plane” for the characterization of the immobilized MAb layer.18,42 The first step of the preparation consisted of immersing 10 mm × 10 mm gold-coated silicon chips in an ethanolic solution containing 0.1 mM DSU and a 1000-fold molar excess of 2-(2aminoethoxy)ethanol (2-AE) (Aldrich) for 14-18 h. The DSU and 2-AE were allowed to react in solution for 1 h prior to the addition of the gold substrates. This process resulted in the chemisorption of a glycol-terminated disulfide formed from the solution reaction between DSU and 2-AE. The substrates were then rinsed thoroughly with ethanol and dried under a stream of argon. We note that characterizations using IRS and X-ray photoelectron spectroscopy of the monolayer formed (data not shown) confirm the presence of a chemisorbed glycol-terminated amide species on the gold surface, as expected, with no detectable amounts of unreacted DSU on such surfaces. The second step involved an established photopatterning process.18,43-46 The pattern was formed by sandwiching a copper transmission electron microscopy (TEM) grid (2000 mesh, Electron Microscopy Sciences) between the GLY-coated substrate and a quartz plate. The sandwich was then irradiated with UV light (220-260 nm region) from a mercury lamp (Oriel) for 20 min. The light power density at the sample was 550 mW/cm2. This process converted the exposed gold-bound GLY to various forms of oxygenated sulfur, which could be readily removed from the surface by rinsing with most organic solvents.18,43-46 The last step consisted of thoroughly rinsing the substrate with distilled, deionized water and ethanol to remove the sulfonated portion of the monolayer and, following that, drying under a stream of argon. The three steps resulted in a pattern of 7.5 µm × 7.5 µm squares of uncoated gold surrounded by a grid-like pattern of the GLY monolayer. The grids separating the squares were 5-µm-wide. The gold squares were then modified by immersing the sample in an ethanolic solution of DSU (0.1 mM) for 12-14 h. This process resulted in a patterned monolayer surface of DSU squares separated by GLY grids, with each region possessing similar heights. The effectiveness of the patterning process was verified by AFM operating in the friction force mode (data not shown). (42) O’Brien, J. C.; Jones, V. W.; Mosher, C. L.; Henderson, E.; Porter, M. D. Anal. Chem. 2000, 72, 703-710. (43) Tarlov, M. J.; Donald, R. F.; Burgess, J.; Gillen, G. J. Am. Chem. Soc. 1993, 115, 5305-5306. (44) Huang, J.; Hemminger, J. C. J. Am. Chem. Soc. 1993, 115, 3342-3343. (45) Lewis, M.; Tarlov, M.; Carron, K. J. Am. Chem. Soc. 1995, 117, 95749575. (46) Wang, J.; Kenseth, J. R.; Jones, V. W.; Green, J.-B. D.; McDermott, M. T.; Porter, M. D. J. Am. Chem. Soc. 1997, 119, 12796-12799.

Analytical Chemistry, Vol. 72, No. 16, August 15, 2000

3711

Figure 2. (A) Construction of the MAb-gold biosensor chip. (1) A gold substrate is exposed to an octadecanethiol (ODT)-soaked PDMS stamp with a 0.5-cm hole cut in its center, “inking” the outer portions of the chip with a chemisorbed monolayer of ODT. (2) DSU is chemisorbed from solution to the unmodified central region of the chip. (3) MAb CB53 is covalently bound to the central DSU region of the chip. (B) On-chip direct immunoassay using FLNS. (1) BP-6N7Gua is specifically bound to the MAb-gold biosensor chip surface. (2) Bound BP-6-N7Gua is detected via high-resolution, low-temperature fluorescence spectroscopy.

The covalent immobilization of MAb CB53, a monoclonal antibody specific for BP-6-N7Gua, was accomplished by exposure of the compositionally patterned GLY/DSU monolayer surface to a 0.5 mg/mL MAb CB53 solution in borate buffer (pH 8.5) with 1% Tween 80 surfactant for 18 h. This resulted in the covalent attachment of MAb CB53 to the DSU-coated regions (i.e., the squares) of the surface. The effectiveness of the attachment was verified by topographic imaging with AFM (see below). The AFM images were acquired using a Digital Instruments Multimode Nanoscope IIIa equipped with a 150-µm tube scanner operating in the contact mode under deionized water. Oxidesharpened 200-µm Si3N4 cantilevers with normal bending and torsional force constants of ∼0.06 and ∼80 N/m, respectively, were utilized for all measurements. Binding of BP-6-N7Gua. Binding confirmation studies of depurinating DNA adducts were performed using biosensor chips constructed differently than the GLY/DSU patterned monolayer used in the AFM assessments. As shown in Figure 2A, gold-coated silicon surfaces (10 mm × 10 mm) were exposed for ∼30 s to an octadecanethiol (ODT)-soaked polydimethyl siloxane (PDMS) stamp,47 which had a 0.5-cm-diameter hole cut in its center. After exposure to the stamp, the surface was rinsed with ethanol and dried under a stream of argon. The substrate was then immersed in a solution of 0.1 mM DSU in ethanol for 12-14 h. After removal from the DSU solution, the substrate was rinsed with ethanol and dried under a stream of argon. This facile process reproducibly formed a well-defined DSU-modified region in the center of the chip surrounded by a hydrophobic ODT region, providing a localized region for confinement of the added MAb CB53 solution that improved the quantitative repeatability of the measurements. In the final step of chip construction, the center DSU-modified (47) Kumar, A.; Whitesides, G. M. Appl. Phys. Lett. 1993, 63, 2002-2004.

3712

Analytical Chemistry, Vol. 72, No. 16, August 15, 2000

area was exposed to 40 µL of a solution consisting of MAb CB53 at 0.5 mg/mL in borate buffer (pH 8.5) for a period of 24 h. For on-chip binding studies, the chips were incubated with solutions of BP-6-N7Gua (5 × 10-6 - 5 × 10-8 M) in 50 mM Tris(hydroxymethyl)-aminomethane (TRIS) buffer (150 mM NaCl, pH 7.5) with dimethyl sulfoxide (DMSO) added at 1% for 18 h. After removal, the samples were rinsed once with TRIS buffer and rinsed three times with Nanopure (18 MΩ) water. The surface was then dried under helium. Next, several microliters of glycerol (Aldrich spectroscopic grade) was pipetted onto the circular sample area of the chip which resulted in a film ∼500 µm thick before freezing. The immunoassay procedure is depicted in Figure 2B. Fluorescence Measurements. Fluorescence spectra were obtained in a glass cryostat with quartz optical windows using a Lambda Physik FL 2002 pulsed dye laser (10 Hz) pumped by a Lambda Physik Lextra 100 XeCl excimer laser as the excitation source. A 1-m McPherson Monochromator (model 2601) and a Princeton Instruments photodiode array were used for dispersion and detection of sample fluorescence, respectively. FNLN spectra at 77 K were obtained using excitation at 381 nm. FLN spectra at 4.2 K were generated using several excitation wavelengths, each revealing a portion of the S1 excited-state vibrational frequencies of the DNA adduct. The resolution employed for FLN and FNLN spectra was 0.1 and 0.8 nm, respectively. A Princeton Instruments FG-100 pulse generator was used for time-resolved spectroscopy; detector delay times from 0 to 80 ns and a gate width of 200 ns were used. The collected spectra are the average of 10 one-second acquisitions. The chips, covered with a thin layer of spectroscopicgrade glycerol, were held above the cryoliquid surface until the glycerol was frozen. After freezing, the sample was immersed in liquid nitrogen or helium for low-temperature fluorescence measurements. A grazing angle of ∼30°, of the laser beam relative to the sample surface, was used to minimize the amount of laser light entering the collection optics. RESULTS AND DISCUSSION Chip Characterization. To verify the effectiveness of the MAb immobilization strategy employed (Figure 2A), characterizations of the adlayer were carried out using infrared reflection spectroscopy (IRS) and atomic force microscopy (AFM). Figure 3A shows the IR spectrum of a DSU-derived monolayer chemisorbed on a gold surface, with an idealized depiction of the adlayer structure shown in the figure inset. The DSU monolayer is designed to serve as a coupling agent for the covalent binding of MAb CB53 to the chip surface.40,41 On the basis of earlier assignments,48 the three bands observed between 1820 and 1750 cm-1 are attributed to the CdO stretch of the ester (1817 cm-1) and the in-phase (1788 cm-1) and out-of-phase (1751 cm-1) CdO stretches of the succinimidyl endgroup. The bands at 1218 cm-1 (C-N-C stretch) and 1077 cm-1 (N-C-O stretch) also arise from the succinimidyl endgroup. These data confirm the formation of a DSU adlayer on the gold surface. IRS was also used to confirm the covalent binding of MAb CB53 to the DSU adlayer. The immobilization was accomplished by exposing the DSU-coated sample to a solution containing MAb CB53 (40 µg/mL in borate buffer (pH 8.5) and 1% Tween 80) for (48) Frey, B. L.; Corn, R. M. Anal. Chem. 1996, 68, 3187-3193.

Figure 3. Infrared reflection spectra for a monolayer of DSU on gold (shown in inset) before (spectrum A) and after (spectrum B) exposure to MAb CB53. The major bands observed in spectrum A (at 1751, 1218, and 1077 cm-1) represent vibrations that belong to the leaving succinimidyl endgroup of the DSU molecule. Appearance of bands at 3295, 1666, and 1544 cm-1 in spectrum B along with an appreciable decrease in the intensity of the DSU endgroup bands arise from the binding of the antibody.

21 h. The spectroscopic result, using the same sample as in Figure 3A, is shown in Figure 3B. As expected, exposure to MAb CB53 induces an appreciable decrease in the magnitude of the bands corresponding to the succinimidyl and ester functionalities (e.g., the magnitude of the band at 1751 cm-1 has decreased ∼90% relative to that observed for DSU). In addition, three new bands have appeared. These bands are located at 3295, 1666, and 1544 cm-1, and correspond to a N-H stretch and the amide I and amide II modes, respectively.49 The bands are diagnostic of the presence of amide linkages and are attributed to either the formation of a covalent secondary amide bond upon reaction of the N-hydroxysuccinimidyl (NHS) ester with the primary amines of the MAb CB53 lysine residues and/or the amides present in the native antibody. The disappearance of the NHS ester bands and the appearance of bands corresponding to the protein argue that MAb CB53 is covalently bound to the DSU surface. This conclusion is supported by the fact that the hydrolysis of DSU over the course of the antibody incubation period proceeds to a notably lesser extent (e.g., the band at 1751 cm-1 decreases by less than 20%), as determined by exposure of samples to buffer solutions devoid of antibody for similar lengths of time. Additional proof that MAb CB53 binds to DSU was provided by AFM. Figure 4A shows an AFM height image of a compositionally patterned GLY/DSU surface following the coupling procedure for MAb CB53. This image was obtained in contact (49) Lin-Vien, D.; Colthup, N. B.; Fateley, W. G.; Grasselli, J. G. In The Handbook of Infrared and Raman Characteristic Frequencies of Organic Molecules; Academic Press: San Diego, CA, 1991.

Figure 4. (A) 45 µm × 45 µm AFM height image of a MAb CB53 antibody array obtained in the contact mode under deionized water at an imaging force of 2 nN and a scan rate of 6 Hz. (B) Crosssectional image of the rectangular area marked in (A), indicating an approximate 4.5-nm height increase in the square-shaped regions where MAb CB53 is bound.

mode under deionized water at an imaging force of 2 nN. As expected from the patterning process, the image consists of a periodic array of topographically higher, square-shaped regions separated by lower grid-shaped regions. Figure 4B shows the average vertical cross-section of the rectangular area marked on the image in Figure 4A, and indicates a ∼4.5 nm higher topography for the DSU-modified square regions in which the antibody is immobilized. We conclude that the 4.5 ( 1 nm height of the antibody-modified regions relative to the unmodified grid regions is consistent with the presence of a monolayer of MAb CB53 on the DSU regions of the surface. This result additionally demonstrates that the glycol-terminated regions of the surface effectively resist any nonspecific adsorption of the protein in the presence of small amounts of Tween surfactant in the buffer solution.50 As a result, an array of proteinmodified squares separated by glycol-terminated grids of lower height is observed, with lateral dimensions on the order of the mesh size of the TEM grid mask used for patterning. We also (50) We note that, on some patterned GLY/DSU surfaces, proteins were nonspecifically adsorbed on the glycol-terminated regions when surfactant was not added to the buffer solution. Recent work has shown that the ability of oligo(ethylene glycol) (OEG)-terminated monolayers to resist protein adsorption was strongly dependent on the monolayer molecular conformation.64-66 Thus, the presence of hydrogen bonding from the amide linkages incorporated into our OEG-terminated monolayers versus alkylOEG monolayers67 may result in molecular conformations less resistant to protein adsorption. A more detailed investigation of this effect is in progress.

Analytical Chemistry, Vol. 72, No. 16, August 15, 2000

3713

Figure 5. NLN fluorescence spectra of BP-6-N7Gua standard (5 × 10-6 M) on a gold biosensor chip. Spectrum a was obtained without a glycerol overlayer at T ) 294 K. Spectra b and c were generated with a glycerol overlayer at T ) 294 and 77 K, respectively. λex ) 381 nm.

note that the AFM tip did not displace the protein on the DSU regions, even after imaging under relatively high forces (50 nN). This finding, based on the ability to “scrape” nonspecifically adsorbed antibodies off similar surfaces with an AFM tip,18 further supports the covalent attachment of MAb CB53 to the DSUmodified surface. On-Chip Immunoassays of BP-6-N7Gua via Fluorescence Measurements. As demonstrated by the IRS and AFM results, a monolayer of MAb CB53 with a high affinity for BP-6-N7Gua was successfully immobilized on our gold chip. This section assesses the utility of FLNS as a rapid approach for chip readout. Samples were prepared by exposure of prepared MAb-gold chips to BP-6-N7Gua adduct solutions of differing concentrations and then by thoroughly rinsing them with buffer and triply rinsing them with distilled, deionized water. At room temperature, there was no detectable fluorescence from BP-6-N7Gua bound to the chip, independent of adduct concentration. This result is most likely due to quenching by the gold surface and/or dynamical quenching (vide infra).51,52 However, when the surface of the chip was covered with a thin layer of glycerol, which upon cooling forms a glassy matrix conducive to FLN spectroscopy,38,53 the fluorescence spectrum expected for BP-6-N7Gua was clearly observed. Representative findings are illustrated in Figure 5 where spectra a and b correspond to FNLN spectra obtained at room temperature without (spectrum a) and with (spectrum b) glycerol pretreatment. These spectra were obtained with an excitation wavelength (λex) of 381 nm and a 40ns detection delay time. Importantly, the fluorescence intensity with the glycerol layer increased by a factor of 10 upon lowering the temperature to 77 K (spectrum c). This increase is partly due to an observed increase in fluorescence lifetime (data not shown). There are two additional pieces of evidence that are diagnostic of the presence of the bound adduct. First, the fluorescence origin band for the immobilized antigen obtained with a 0-ns delay time (51) Hellen, E. H.; Axelrod, D. J. Opt. Soc. Am. B 1987, 4, 337-350. (52) Pockrand, I.; Brillante, A.; Mobius, D. Chem. Phys. Lett. 1980, 69, 499504. (53) Meyer, B. In Low-Temperature Spectroscopy; Optical Properties of Molecules in Matrixes, Mixed Crystals, and Frozen Solutions; American Elsevier: New York, 1971.

3714 Analytical Chemistry, Vol. 72, No. 16, August 15, 2000

Figure 6. Spectrum A: NLN fluorescence spectrum of BP-6-N7Gua standard on a gold biosensor chip at T ) 4.2 K coated with a thin layer of glycerol; λex ) 381 nm. The spectrum was taken in continuous-wave mode, and the origin band was located at 406.7 nm. The brackets encompass the spectral region shown in B. Spectrum B: Vibronically excited FLN spectrum obtained for BP-6N7Gua on the same chip surface under the same conditions; λex ) 386.5 nm. The numbers correspond to the excited-state vibrational frequencies in centimeters-1.

at 77 K was located near 406 nm, as expected for BP-6-N7Gua.9 The NLN fluorescence spectrum was similar to that of BP-6-N7Gua in a standard glycerol/water glass except for a matrix-induced red shift for the origin band of ∼1 nm. In addition, the fluorescence origin band underwent a blue shift with increasing delay times (i.e., 406.7, 406.5, 406.0, and 405.7 nm (data not shown) with detection delay times of 0, 20, 40, and 60 ns, respectively). This type of blue shift with delay time has been previously observed for BP-6-N7Gua in standard glasses and was attributed to a distribution of adduct conformations.9,54 Further evidence for the definitive identification of the BP-6N7Gua adduct on the chip was provided by FLN spectra obtained at 4.2 K using several different excitation wavelengths. The spectra were essentially identical to those of the adduct in a glycerol/ water glass except for small variations in the relative intensities of the vibronic bands that are caused by a matrix-induced red shift of the origin band. The high-resolution FLN spectrum for a λex of 386.5 nm (0 ns delay time) shown in Figure 6B represents the bracketed region of the FNLN spectrum shown in Figure 6A. The FLN spectrum corresponds to the BP-6-N7Gua adduct as revealed by the characteristic vibrational frequencies of the S1 excited state at 1164, 1218, 1250, 1316, and 1397 cm-1. Other FLN spectra (obtained at various λex), also diagnostic of the presence of the adduct through revealed characteristic low-frequency vibrational modes, were obtained and will be described in detail elsewhere.55 The Glycerol-Induced Fluorescence Enhancement. The mechanism that gives rise to the fluorescence enhancement in the presence of glycerol is under investigation. However, some insights have been gained through a comparison of the immobilized MAb CB53 structure in the absence or presence of glycerol. Figure 7A shows an AFM height image of the MAb CB53 (54) Lin, C.-H.; Zamzow, D.; Small, G. J.; Jankowiak, R. Polycyclic Aromat. Compd. 1999, 14-15, 43-52. (55) Duhachek, S.; Kenseth, J.; Casale, G.; Small, G.; Porter, M.; Jankowiak, R., manuscript in preparation.

Figure 7. (A) 45 µm × 45 µm AFM contact mode height image of the same MAb CB53 antibody array as in Figure 3 after exposure to a 10% glycerol solution. The image was collected under an imaging force of 2 nN at a scan rate of 10 Hz. (B) Cross-sectional image of the rectangular area marked in (A), indicating an approximate 10nm height increase of the MAb CB53-bound square-shaped regions versus the glycol-terminated grid regions (see text for details).

array shown in Figure 4 after exposure to deionized water containing 10% glycerol (imaging of surfaces exposed to higher concentrations of glycerol proved unsuccessful due to the relatively high viscosity of glycerol). Some nonspecific adsorption of glycerol was initially observed when imaging in the glycolterminated grid regions. However, this material was removed after several scans at increased imaging force (from 2 to 25 nN). Interestingly, Figure 7B reveals a marked increase in the AFM measured cross-sectional height of the MAb CB53 immobilized regions of the surface when compared with the height evident in Figure 4B. The average height of the MAb CB53 adlayer versus the glycol-terminated grid regions has increased to 10 ( 1 nm in the presence of 10% glycerol, a factor of 2 greater than that in the absence of glycerol. This effect appears to be reversible, as subsequent rinsing and exposure to only deionized water resulted in a decrease in height of the MAb CB53 regions of the surface back to the original height of 4-5 nm. It is worthwhile to compare the observed changes in the average height of the immobilized MAb CB53 with the dimensions of an IgG antibody as determined by X-ray crystallography (14 nm × 10 nm × 5 nm).56 The 4-5 nm average height of the MAb CB53 layer observed in Figure 4 is consistent with earlier studies (56) Sarma, V. R.; Silverton, E. W.; Davies, D. R.; Terry, W. D. J. Biol. Chem. 1971, 246, 3753-3759.

of antibody layers by AFM.31,32,57,58 Taking into account the “Y” structure of the antibody, this would imply, on average, that upon immobilization the antibody is lying flat, thereby maximizing contact with the surface.58 However, the presence of glycerol induces an increase in the average height to what one might expect for a film of IgG adopting a more upright orientation on the surface. We attribute the above increase in topography to a glycerolinduced structural reorganization of the adsorbed MAb CB53. From the topographic observations of the antibody surfaces in the absence of glycerol, it appears that interactions between hydrophobic regions of the protein and the surface lead to a partial unfolding or denaturation of the native antibody structure. Since glycerol strongly repels nonpolar substances, its addition to the solution may induce a reordering of the protein structure such that the nonpolar groups at the outermost portions of the protein surface avoid contact with the glycerol/water solvent,59 thereby restabilizing the native structure of the protein.60 This explanation, albeit highly speculative, is supported by the fact that glycerol is widely used as a protein stabilization agent.61 The resultant doubling in the topography of the MAb CB53 layer upon the addition of glycerol therefore reflects the decrease in interaction between the protein and surface. As a consequence, the distance, on average, between the bound BP-6-N7Gua fluorophore and the metal surface increases, accounting for the increase in fluorescence due to a decrease in surface quenching by the underlying gold. The dependence of quenching on separation distance has been previously reported in other systems.51,52 The addition of glycerol may also increase fluorescence through a decrease in dynamical quenching, that is, the presence of glycerol reduces large-amplitude motions of BP and protein residues that result in structures conducive for efficient radiationless decay of the S1 state. In addition, glycerol may disrupt MAb-antigen interactions. In this regard, our studies62,63 of BPdiolepoxide (BPDE)-DNA adducts are relevant. These studies have shown that the addition of glycerol to a buffer solution leads to a significant reduction of π-π interactions between the pyrene fluorophore and the DNA bases. We presently believe that, while both processes play important roles in the fluorescence quenching, the increased separation between the fluorophore and metal substrate is more dominant. This assertion is based primarily on the lack of a detectable fluorescence signal at low temperatures for samples devoid of glycerol. (57) Droz, E.; Taborelli, M.; Descouts, P.; Wells, T. N. C.; Werlen, R. C. J. Vac. Sci. Technol., B 1996, 14, 1422-1426. (58) Delamarche, E.; Sundarababu, G.; Biebuyck, H.; Michel, B.; Gerber, C.; Sigrist, H.; Wolf, H.; Ringsdorf, H.; Xanthopoulos, N.; Mathieu, H. J. Langmuir 1996, 12, 1997-2006. (59) Sinanoglu, O.; Abdulnur, S. FASEB J. 1965, 24, 12-23. (60) Gekko, K.; Timasheff, S. N. Biochemistry 1981, 20, 4667-4676. (61) Sousa, R. Acta Crystallogr., Sect. D 1995, D51, 271-277. (62) Suh, M.; Ariese, F.; Small, G. J.; Jankowiak, R.; Liu, T.-M.; Geacintov, N. E. Biophys. Chem. 1995, 56, 281-296. (63) Suh, M.; Ariese, F.; Small, G. J.; Jankowiak, R.; Hewer, A.; Phillips, D. H. Carcinogenesis 1995, 16, 2561-2569. (64) Harder, P.; Grunze, M.; Dahint, R.; Whitesides, G. M.; Laibinis, P. E. J. Phys. Chem. B 1998, 102, 426-431. (65) Pertsin, A. J.; Grunze, M.; Garbuzova, I. A. J. Phys. Chem. B 1998, 102, 4918-4926. (66) Valiokas, R.; Svedhem, S.; Svensson, S. C. T.; Liedberg, B. Langmuir 1999, 15, 3390-3394. (67) Pale-Grosdemange, C.; Simon, E. S.; Prime, K. L.; Whitesides, G. M. J. Am. Chem. Soc. 1991, 113, 12-20.

Analytical Chemistry, Vol. 72, No. 16, August 15, 2000

3715

Practical Aspects of Biosensor Chip Use. One of our general operational concerns with this concept was that the chip would be physically damaged by the low-temperature cooling process (e.g., cracking of the substrate surface or glycerol overlayer) which would prohibit its reuse. Of the potential mechanical problems, the only notable difficulty was encountered when using excess amounts of glycerol. While thin layers of glycerol consistently formed a clear glassy matrix, thicker layers (>500 µm) would often crack severely and flake off of the surface. The durability of the antibody/antigen interface under repeated temperature cycling was also tested. The test procedure included: warming the chip to room temperature after analysis; rinsing with buffer; triple rinsing with deionized, distilled water; recoating with glycerol; and then reanalyzing at 77 K. This protocol was repeated three times, and the generated spectra did not reveal any noticeable degradation. Moreover, it was found that samples could be refrigerated (at 4 °C) for a period of several months and still function effectively. Preliminary experiments also indicated that the chips could be regenerated using a 30% DMSO and 70% TRIS buffer solution. These attributes should facilitate reuse, making this methodology more cost-effective. Binding Specificity and Estimated Detection Limits. The efficient binding observed above was anticipated considering that MAb CB53 binds with an affinity to BP-6-N7Gua (affinity constant (Ka) ) 1.4 × 108 M-1) that is more than 1000 times that for the parent compound BP.14 In contrast, samples modified with a generic mouse IgG1 antibody, as a test for nonspecific adsorption, showed no detectable binding of BP-6-N7Gua. However, MAb CB53 displays significant cross-reactivity with BP-6-N7Ade, another depurinating adduct of BP. Although distinguishable by CEFLNS,9 these closely related adducts could not be differentiated on the biosensor chip if present at comparable concentrations. Fortunately, in the systems studied thus far,9,10 only one major BP-derived adduct is detected with its identity dependent on the source of BP exposure. For example, in the urine of humans exposed to coal smoke, BP-6-N7Gua is the major adduct present, while in cigarette smokers BP-6-N7Ade is predominant.9,10 In cases where both adducts are present at comparable concentrations, the total amount of -N7Gua and -N7Ade adducts can still be assessed by low-temperature fluorescence spectra obtained under NLN conditions. We note that the weak cross-reactivity of MAb CB53 with various BP metabolites (e.g., BP-trans-7,8-dihydrodiol)14 does not pose a problem, since LIF spectroscopy performed at low temperatures with selective excitation can readily distinguish between BP-DNA adducts and corresponding metabolites. For example,

3716

Analytical Chemistry, Vol. 72, No. 16, August 15, 2000

the origin bands of BP, BP-trans-7,8-dihydrodiol, and BP-6-N7Gua are at 403, 395, and ∼407 nm, respectively,9,36,39,54 and are easily distinguishable by FLNS. As a preliminary assessment of detection capability, we exposed our chip to a 5 × 10-8 M solution of BP-6-N7Gua. The resulting fluorescence signal at 4.2 K yielded a spectrum with a signal-to-noise ratio (SNR) of ∼24 at the emission maximum of 407 nm, which translates to a detection limit of 6 × 10-9 M at a SNR of three. This level of detection is comparable to that of the recently developed competitive ELISA mentioned earlier.14 Importantly, the FLNS-biochip technique has the advantage of additional discrimination between cross-reacted metabolites. Furthermore, we believe that increasing the fluorescence integration time, optimizing the experimental setup (e.g., angle of incidence and optical collection efficiency), and adjusting assay conditions will lower the detection limit to less than 1 × 10-9 M. CONCLUSIONS AND FINAL REMARKS The results presented in this manuscript establish that the MAb-gold biosensor chip approach for detection and characterization of DNA adducts has considerable potential for risk assessment studies of human exposure to various carcinogens (e.g., PAHs). The method is very sensitive and selective due to high-resolution low-temperature fluorescence detection. At present, the MAb-gold biosensor chip methodology is under evaluation for use in the molecular dosimetry of depurinating PAH- and catechol estrogenderived DNA adducts in samples of human and rat urine. It is anticipated that this approach may potentially become an alternative monitoring method for cancer risk assessment. ACKNOWLEDGMENT Ames Laboratory is operated for the U.S. Department of Energy by Iowa State University under contract no. W-7405-Eng82. The work was supported in part by the National Cancer Institute (Grant PO1 CA49210), by the National Science Foundation (Grant BIR-9601789), and by the Office of Health and Environmental Research, Office of Energy Research. J.R.K. gratefully acknowledges the support of a Phillips Petroleum Corp. graduate research fellowship. The authors thank Dr. E. L. Cavalieri (Eppley Institute for Research in Cancer, University of Nebraska Medical Center, Omaha, NE) for providing the BP-6-N7Gua adduct standards. Received for review April 25, 2000. Accepted June 7, 2000. AC000472W