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Energy Dissipation Kinetics for Protein and Antibody-Antigen Adsorption under Shear Oscillation on a Quartz Crystal Microbalance F. Ho¨o¨k,*,†,‡ M. Rodahl,† P. Brzezinski,‡ and B. Kasemo† Department of Applied Physics, Chalmers University of Technology and Go¨ teborg University, S-412 96 Go¨ teborg, Sweden, and Department of Biochemistry and Biophysics, Lundberg Laboratory, Medicinareg 9C, Chalmers University of Technology and Go¨ teborg University, S-41390 Go¨ teborg, Sweden Received July 21, 1997. In Final Form: November 11, 1997 A new quartz crystal microbalance instrument, allowing simultaneous frequency (f) and dissipation factor (D) measurements, has been used to study protein adsorption kinetics by measuring time-resolved data of both the D-factor, measuring the energy dissipation due to the added overlayer, and the f-shift, measuring the effective mass load on the sensor. Four model proteins (myoglobin, hemoglobin, human serum albumin (HSA), ferritin) and one antibody-antigen reaction (antibody against HSA) were studied on a hydrophobic, methyl-terminated (-CH3) gold surface. In all five cases system-specific, positive D-shifts and negative f-shifts were observed, revealing different adsorption phases. The D-factor measurements provide new information about protein adsorption and improve the interpretation of the frequency shift in terms of mass uptake. Possible mechanisms for the adlayer-induced dissipation are discussed.
Introduction Protein adsorption, and generally polymer adsorption, at solid-liquid interfaces is of rapidly increasing scientific and practical importance.1,2 Important scientific issues are the thermodynamics, bonding, conformational changes, phase transitions, and kinetics of macromolecular systems at phase boundaries.3,4 Technologically, protein adsorption is central, e.g., in medical diagnostics (sensors) employing antibody-antigen reactions,5 in the biocompatibility of medical implant materials,6,7 and in biofouling.8 We have used a new version of the quartz crystal microbalance (QCM),9 allowing precise, time-resolved measurements of the energy dissipation factor, together with the more commonly measured frequency shift. A QCM sensor consists of a disk-shaped, AT-cut piezoelectric quartz crystal, usually coated with thin metal film electrodes deposited on its two faces. The crystal is excited to oscillation in the thickness shear mode at its fundamental (or an overtone) resonant frequency, f, by applying a suitable rf voltage across the electrodes. A mass, ∆m, added to the electrodes, induces a decrease in the resonant frequency, which is proportional to ∆m if the mass is small compared to the mass of the crystal, does not slip on the * Corresponding author (email,
[email protected]). † Department of Applied Physics. ‡ Department of Biochemistry and Biophysics. (1) Interfacial Phenomina and Bioproducts; Brash, J. L., Wojciechowski, P. W., Eds.; Marcel Dekker, Inc.: New York, Basel, Hong Kong, 1996. (2) Cho, K. C.; Leung, W. P.; Mok, H. Y.; Choy, C. L. Biochim. Biophys. Acta 1985, 830, 36. Proteins at interfaces II; Horbett, T. A., Brash, J. L., Eds.; ACS Symposium Series 602; American Chemical Society: Washington, DC, 1995. (3) Norde, W. Cells Mater. 1995, 5, 97. (4) Lundstro¨m, I. Prog. Colloid Pollym. Sci. 1985, 70, 76-82. (5) Marco, M. P.; Barcelo, D. Meas. Sci. Technol. 1996, 7, 1547. (6) Andrade, J. D.; Hlady, V. In Advances in Polymer Science; Dusek, K., Ed.; Springer: New York, 1986; Vol. 79. (7) Kasemo, B.; Lausma, J. CRC Crit. Rev. Biocompat. 1986, 2, 335380. (8) Fouling and Cleaning in Food Processing; Kessler, G. H., Lund, D. B., Eds.; Federal Republic of Germany, 1989. (9) Rodahl, M.; Ho¨o¨k, F.; Krozer, A.; Brzezinski, P.; Kasemo, B. Rev. Sci. Instrum. 1995, 66, 3924-3930.
electrode(s), and is sufficiently rigid and/or thin to have negligible internal friction. In this case the Sauerbrey relation10 holds
∆f ) -∆m/nC
(1)
where C is the mass sensitivity constant (C ) 17.7 ng‚cm-2‚Hz-1 at 5 MHz) and n is the overtone number (n ) 1, 3, ...). The QCM technique has by several investigators been shown to be a sensitive tool to study protein adsorption kinetics in aqueous solutions, with a sensitivity in the ng/cm2 (submonolayer) regime.9,11-15 However, the applicability of the technique for such studies has been questioned,16,17 primarily due to the uncertainty about how to translate ∆f into mass uptake, since protein overlayers (i) are not rigid,11,17,18 (ii) may trap liquid,12,14 and/or (iii) may slip on the moving electrode surface.11 There has also been uncertainty about the influence of the salt concentration of the solvent on the measured f-shifts,19 but this question has, in our opinion, been resolved recently.20 When independent methods have been used in parallel with the QCM technique, the latter has often yielded larger apparent mass uptakes,12,14 which we in this and parallel studies attribute to trapped water.21 Due to (i)-(iii) above, we emphasize that whenever the (10) Saurbrey, Z. Z. Phys. 1959, 155, 206-222. (11) Thompson, M.; Dhaliwal, G. K.; Arthur, C. L. Anal. Chem. 1986, 58, 1206. (12) Muratsugu, M.; Ohta, F.; Miya, Y.; Hosokawa, T.; Kurosawa, S.; Kamo, N.; Ikeda, H. Anal. Chem. 1993, 65, 2933. (13) Janshoff, A.; Steinem, C.; Sieber, M.; Galla, H. J. Eur. Biophys. J. Biophys. Lett. 1996; 25 (2), 105-113. (14) Caruso, F.; Furlong, D. N.; Kingshott, P. J. Colloid Interface Sci. 1997, 186, 129. (15) Aberl, F.; Wolf, H.; Koesslinger, C.; Drost, S.; Woias, P.; Koch, S. Sens. Actuators, B 1994, 18-19, 271. (16) Ramsden, J. J. J. Statistical Phys. 1993, 73, 853. (17) Ward, M. D.; Buttry, D. A. Science 1990, 249, 1000. (18) Su, H.; Chong, S.; Thompson, M. Lanmuir 1996, 12, 2247-2255. (19) Josse Proc. IEEE Ultrasonics Symp. 1 1993, 425-430. (20) Rodahl, M.; Ho¨o¨k, F.; Kasemo, B. Anal. Chem. 1996, 68, 22192227. (21) Rodahl, M.; Ho¨o¨k, F.; Fredriksson, C.; Keller, C.; Krozer, A.; Brzezinski, P.; Voinova, M.; Kasemo, B. Faraday Discuss., in press.
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expression “mass” is used below in connection with measured f-shifts that do not obviously obey eq 1, we really mean the “effective mass load”. In most previous studies of protein adsorption with the QCM technique only the change in the resonant frequency of the crystal has been measured. However, the QCM can also provide information about changes in dissipative energy losses in the oscillating system, as demonstrated by Krim et al.22 for adsorbed noble gas monolayers in vacuum, by Su et al.18 for RNA-hybridization on gold in the liquid phase, by Janshoff et al.,13,23 Keller et al.,24 and Fredriksson et al.25 for vesicle and cell adsorption on gold. Dissipation effects are directly related to the validity of the Saurbrey relation and to the phenomena (i)-(iii) above and are not unlikely to occur also for adsorbed proteins. The latter has indeed been shown by Young et al.26 for the adsorption of R-chymotrypsinogen on gold. In the few studies referred to above including D-shift measurements, the adsorption-induced dissipation shifts were obtained from a full impedance analysis of the QCM. This is a time-consuming measurement which to our knowledge hitherto has restricted the time resolution for high-precision measurements of adsorption kinetics to about e0.05 Hz.27,28 We have developed a technique to simultaneously and sensitively measure changes in both the frequency (
