Superoxide sensor based on cytochrome c immobilized on mixed-thiol SAM with a new calibration method

Cytochrome c was immobilized on a mixed-thiol (mercaptoundecanoic acid/mercaptoundecanol) modified gold electrode (MUA:MU/cyt c electrode). Characterization of the cyt c electrode showed a quasi-reversible, electrochemical redox behavior with a formal potential of - 13 +/- 5 mV (versus Ag/AgCl) for the surface adsorbed protein and 3 +/- 5 mV for covalently immobilized cyt c. The heterogeneous electron transfer rate constants were determined to be about 70 and 40 s(-1) for both states of the protein, respectively. They were found to be significantly higher than those of pure MUA-modified cyt c electrodes (MUA/cyt c electrodes). The interaction of superoxide radicals (O-2(.-)) with the (MUA:MU)/cyt c electrode was characterized and used for an amperometric O-2(.-) detection. The influence of H2O2 and uric acid on the sensor signal was investigated. The sensitivity of the (MUA:MU)/cyt c electrode to O-2(.-) was significantly improved compared with that of the MUA/cyt c electrode. Based on a kinetic model for the superoxide detection system, a new calibration method was established. This simple and fast method used the spontaneous dismutation of KO2 and was compared with the enzymatic superoxide generation system using xanthine oxidase. (C) 2002 Elsevier Science B.V. All rights reserved.