Langmuir 2002, 18, 5283-5286
5283
Measurement of the Rate of Adsorption of Electroactive Cytochrome c to Modified Gold Electrodes by Electrochemical Impedance Spectroscopy Tal M. Nahir* Department of Chemistry, California State University, Chico, California 95929-0210
Edmond F. Bowden* Department of Chemistry, North Carolina State University, Raleigh, North Carolina 27695-8204 Received February 11, 2002. In Final Form: April 16, 2002 The electrochemistry of a system containing a surface-confined electroactive species is characterized by electrochemical impedance spectroscopy, and the results are displayed on a Cole-Cole plot. The rate of adsorption of electroactive horse-heart cytochrome c to a carboxythiol-modified gold electrode is experimentally monitored by continuously measuring the change in the magnitude of the pseudocapacitance, which is directly proportional to the amount of adsorbed protein. The time dependence of the adsorption of cytochrome c to the thiol-modified gold electrode shows a significant period of diffusion-controlled behavior. Limitations on the frequency of measurement are outlined in relationship to the magnitude of the standard rate constant of the electron-transfer reaction.
Introduction The adsorption of biomolecules at the liquid-solid interface is an important topic of current interest.1 A number of practical applications, such as biosensor fabrication,2 chromatography,3 and the maintenance of biocompatibility of materials under conditions of unwanted protein adsorption,4 have already been addressed. In addition, recent theoretical studies have considered kinetic5 and thermodynamic6 aspects of adsorption of proteins. In many instances, biomolecules adsorb to surfaces at (sub) monolayer levels. This process is often irreversible, in the sense that the adsorbed state is highly favored over the solution state. Consequently, there are numerous situations in which the analysis of the kinetics of adsorption can be more important than the determination of thermodynamic, or equilibrium, parameters. The experimental measurement of the rate of adsorption of proteins and enzymes to various substrates has been vigorously pursued using a variety of techniques, including quartz crystal microgravimetry,7 total internal reflection fluorescence,8 attenuated total reflection,9 ellipsometry,10 surface plasmon resonance,11 and Brewster-angle microscopy.12 This work outlines a new method to measure the rate of adsorption of redox proteins by exploiting their electrontransfer capabilities. Following a previous suggestion for (1) Malmsten, M. Biopolymers at Interfaces; Marcel Dekker: New York, 1998. (2) Wang, J.; Rivas, G.; Luo, D.; Cai, X.; Valera, F. S.; Dontha, N. Anal. Chem. 1996, 68, 4365. (3) Lin, S.; Drake, L. R.; Rayson, G. D. Anal. Chem. 1996, 68, 4087. (4) Ishihara, K.; Nakabayashi, N.; Nishida, K.; Sakakida, M.; Shichiri, M. ACS Symp. Ser. 1994, 556, 194. (5) Zhdanov, V. P.; Kasemo, B. Proteins 1998, 30, 177. (6) Zhdanov, V. P.; Kasemo, B. Langmuir 2001, 17, 5407. (7) (a) Calvo, E. J.; Etchenique, R.; Pietrasanta, L.; Wolosiuk, A.; Danilowicz, C. Anal. Chem. 2001, 73, 1161. (b) Tanaka, M.; Mochizuki, A.; Motomura, T.; Shimura, K.; Onishi, M.; Okahata, Y. Colloids Surf., A 2001, 193, 145. (c) Facci, P.; Cavatorta, P.; Cristofolini, L.; Fontana, M. P.; Riccio, P. Chem. Phys. Lett. 1999, 304, 197.
the monitoring of the adsorption process by observing changes in electrochemical activity during cyclic voltammetry,13 we measure the faradaic capacity,14 or the pseudocapacitance, of the system by electrochemical impedance spectroscopy. The reproducible and highly stable model system of cytochrome c and a carboxythiolmodified gold electrode is used here to illustrate the procedure. The irreversible adsorption and sequential electrochemistry of this positively charged protein have already been documented at negatively charged carboxythiol-modified gold electrodes (and tin oxide) at approximately neutral pH.15 Experimental Section The instruments, reagents, cells, and procedure for preparation of thiol-modified gold electrodes were described earlier.16 Complete impedance analysis was conducted at the formal potential of adsorbed cytochrome c with frequency sweeps of 10 steps per decade. To observe the rate of adsorption, a sequence of steps was followed using a gold electrode modified with mercaptohexanoic acid (0.32 cm2 electrode area). Initially, the modified electrode was equilibrated inside the cell with an inert electrolyte (
