To measure the direct electron transfer (DET) reaction of cytochrome c (Cytc) immobilized on a bare ITO electrode after removing the adsorbed molecules, automated solution exchange (ASE) processes were performed to induce their desorption. By fitting the absorbance decay curve observed at the Soret band peak position of Cytc at around 408 nm during the ASE processes with a double exponential equation, the final immobilized fraction was estimated to be 58.6% of the Cytc adsorbed on bare ITO electrodes under the experimental conditions. Cyclic voltammograms (CVs) of Cytc adsorbed on the bare ITO electrodes were measured for 60 min to elucidate the DET activity of immobilized Cytc. After repeated CV measurements, approximately 90% of immobilized Cytc was found to remain from the evaluation based on the coulombic amount of reduction and oxidation peaks. The scan rate dependent peak separation data from the immobilized Cytc between reduction and oxidation peaks in CVs produced 2.7 times larger DET reaction rate constant than that previously reported for the Cytc adsorbed on the bare ITO electrode.
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