Anal. Chem. 1983, 65, 3713-3716
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Acoustic Network Analysis as a Novel Technique for Studying Protein Adsorption and Denaturation at Surfaces Mengsu Yang: Foun Ling Chung, and Michael Thompson* Department of Chemistry, University of Toronto, 80 St. George Street, Toronto, Ontario, M5S 1Al Canada
INTRODUCTION The understanding of the interaction of proteins with solid is important in the development of implantable medical devices, biotechnology, and biosensors. Some of the more common techniquesfor studyingproteins at an interface include radiolabeling and fluorescent tagging; spectroscopic methods such as ellipsometry, total internal reflectance fluorescence (TIRF),and attenuated total reflectance Fouriertransform infrared spectroscopy (ATR-FTLR); and surface characterization methods such as X-ray photoelectron spectroscopy (XPS) and scanning electron microscopy (SEM). These techniques have been thoroughly reviewed.’ They provide information either on the amount and rate of adsorptionof proteins or on the conformation of the adsorbate. However, protein adsorption and the orientation of the adsorbed molecules involves a large number of time-dependent dynamic phenomena where conformationalchanges can occur during the adsorption process. Few of the above techniques are capable of monitoring in situ adsorption and providing evidence of conformationaleffects in real time. In this preliminary report, we demonstrate that the response of the thickness-shear mode (TSM) acoustic wave sensor can be correlated to interfacial processes involvingprotein adsorption and denaturation at surfaces. A number of studies have employed the so-called quartz crystal microbalance technique to determine the amount of adsorbed protein.&‘ The detection scheme is based on the changes of the oscillating frequency of piezoelectric devices upon mass loading.8 The mass-frequency relationship is describe by the Sauerbrey equation Af=-
2f:Am A(P~M~)’’~
where frequency shift Af is a function of the fundamental frequencyfo, the mass loading Am, the piezoelectrically active area A, the density pg (2.648 g/cm3),and the shear modulus p~ (2.947 X 10’’ g/(cm s2)) of the quartz, respectively. The most common device is an AT-cut quartz crystal with electrodes deposited on opposite sides of the plate. Applying an ac voltage across the quartz plate will generate an acoustic standing wave in the thickness-shear mode. For a 9-MHz AT-cut TSM device, Af(Hz) = -0.91Am (ng). In the liquid phase, the shear coupling of the standing wave and a propagating acoustic wave into liquid results in a velocity shiftand attenuation of the TSM device.”’ The performance t Present address: Department of Molecular Biology, The Scripps Research Institute, La Jolla, CA 92037. (1)Andrade, J. D.,Ed. Surface and Interfacial Aspects of Biomedical Polymers; Plenum Press: New York, 1985;Vola. 1 and 2. (2)Norde, W.Adv. Colloid Interface Sci. 1986,25,267. (3)Brash, J. L., Horbett, T. A., Eds. Proteins at Interfaces; ACS Symposium Series 343;American Chemical Society: Washington,DC, 1987. (4)Grabbe, E. S.;Buck, R. P.; Melroy, 0.R. J. Electroanal. Chem. 1987,223,67. (5)Ebersole, R.; Ward, M. D. J. Am. Chem. SOC. 1988,110,8623. (6) . . Laatikainen.. M.:. Lindstrom, M. J. Colloid Interface Sci. 1988,125, . . 610. (7)Lacour, F.; Torresi, R.; Gabrielli, C.; Caprani, A. J.Electrochem. Soe. 1992,136,1619. (8) Sauerbrey, G. 2.Phys. 1950, 155,206.
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of the device is influenced by both the bulk properties of the contactingliquid and the interfacial properties at the sensorliquid interface. Previous studies have employed the oscillator methodl”’6 to measure the resonant frequency of the quartz crystal. This method only provides limited information on the behavior of the sensor in the liquid phase, and the measurement is restricted by a number of factor^.'^-^^ More recently, the network analysis method has been developed to characterize completely the TSM device in In thismethod, the impedance characteristics of the TSM sensor are determined for a wide range of frequencies covering the resonant region. The electrical properties of the quartz crystal can be described by an equivalent circuit (Figure l).23 Multidimensional information can be obtained from the network analysis, including the resonant frequencies, the magnitude and phase of the impedance, and the equivalent circuit elements. These parameters can be related to the viscosity, density, dielectric constant, and conductive properties of the contacting l i q ~ i d s . 2 ~ ~ ~Furthermore, 9 ~ 4 ~ ~ 6 it has been demonstrated that the sensor response is also affected by the interfacial properties of the sensor-liquid interface, such as the surface free energy (wettability),*u the morphology of the electrode surface,ml and the double-layer s t r u ~ t u r e . The ~ ~ ,multiple ~~ chemical information capability has been successfully applied to the detection of nucleic acid hybridization at metal surfaces.% (9)Kanazawa, K. K.; Gordon, J. G. Anal. Chim. Acta 1986,175,99. (10)Hager, H. E. Chem. Eng. Commun. 1986,43,25. (11)Shana, 2.A.; Radtke, D. E.; Kelkar, U. R.; J w e , F.; Harworth, H. E. Anal. Chim. Acta 1990,231,317. (12)Nomura, T.; Okuhara, M. Anal. Chim. Acta 1982,142,281. (13)Bruckenstein, S.;Shay, M. Electrochim. Acta 1985,30,1295. (14)Thompson, M.; Dhaliwal, G. K.; Arthur, C. L.; Calabrese, G. 5. IEEE Trans. Ultrason.Ferroelectr. Freq. Control 1987,UFFC-34,127. (15)Barnes, C. Sens. Actuators 1991,A29,59. (16)Hayward, G. Anal. Chim. Acta 1992,264,23. (17)Thornpeon, M.; Kipling, A. L.; Duncan-Hewitt, W. C.; RajakoviE, Lj. V.; CaviE-Vlaeak, B. A. Analyst 1991,116,881. (18)Rajakovi6,Lj.V.;CaviC-vlasak,B. A.; Ghaemmaghami,V.;Kallury, K. M. R.; Kipling, A. L.; Thompson, M. Anal. Chem. 1991,63,615. (19)Barnes, C. Sens. Actuators 1992,A30, 197. (20)Beck, R.; Pittermann, U.; Weil, K. G. Ber. Bunsen-Ces. Phys. Chem. 1988,92,1363. (21)Kipling, A. L.; Thompson, M. Anal. Chem. 1990,62, 1514. (22)Martin, S.J.; Granstaff, V. E.; Frye, G. C. Anal. Chem. 1991,63, 2272. (23)Cady, W. G. Piezoelectricity; Dover: New York, 1964. (24)Muramatau, H.;Tamiya, E.; Karube, 1. Anal. Chem. 1988,60, 2142. (25)Yang, M.; Thompson, M. Anal. Chem. 1993,65,1158. (26)Thompson, M.; Dhaliwal, G. K.; Arthur, C. L. Anal. Chem. 1986, 58,1206. (27)Duncan-Hewitt, W. C.; Thompson, M. Anal. Chem. 1992,64,94. (28)Yang, M.; Thompson, M.; Duncan-Hewitt, W. C. Langmuir 1993, 9,802. (29)Schumacher, R.;Borges, G.; Kanazawa, K. K. Surf. Sei. 1985,163, L621. (30)Beck, R.;Pittermann, U.; Weil, K. G. J. Electrochem. SOC. 1992, 139,453. (31)Yang, M.; Thompson, M. Langmuir 1993,9,1990. (32)Niemczyk, T.M.; Martin, S. J.; Frye, G. C.; Ricco, A. J. J. Appl. Phys. 1988,64,5002. (33)Yang, M.; Thompson, M., Anal. Chem., companion paper in this issue. (34)Su,H.; Yang, M.; Kallury, K. M. R.; Thompson, M. Analyst 1993, 118,309. @ 1993 American Chemlcal Society
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ANALYTICAL CHEMISTRY, VOL. 65, NO. 24, DECEMBER 15, 1993
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B
'T-
CO
1
-j
OCO Flgwo 1. Equivalent electrical circuit of the TSM sensor with the clrcuit elements and their impedances. The subscript m is used to denote the fact that the RLC series Is associated with the motion of the quartz plate. The static capacitanceCO representsa slmpk parallec plate capacitor of the quartz at frequencies away from the resonance.
EXPERIMENTAL SECTION Reagents. a-Chymotrypsinogen A (Type I1 from bovinepancreas; chromatographically pure) was obtained from Sigma (St.Louis,MO) and used without further purification. Phosphate buffer (0.1 M NazHPOd in 0.15 M NaCl, pH 6.4) was used to prepare the protein solutions. Apparatus. Quartz crystals 9-MHz AT-cut with gold electrodes were obtained from International Crystal Manufacturer (Oklahoma City, OK). The gold electrodes (ca. 1000-1500 A) were vacuum-deposited onto polished quartz plates cf x 0.2) with a chromiumunderlayer (ca. 100A). Scanning microscopies (SEMand STM) show that the surface features of the electrode are less than 0.02 pm in depth. The gold surface was plasmacleaned under a Nz environment to afford a hydrophilic surface (advancing contact angle of water
