Inhibition of direct-electron-transfer-type bioelectrocatalysis of bilirubin oxidase by silver ions.

In enzyme-based biosensors, Ag+ eluted from the reference electrode inhibits the enzyme activity. Herein, to suppress the inhibition of bilirubin oxidase (BOD) by Ag+, kinetic analysis was used to examine the effect of Ag+ on the activity of BOD. It was confirmed that the addition of Ag+ decreased the bioelectrocatalytic activity of BOD. Atomic absorption spectroscopy (AAS) suggested that Ag+ was attached to BOD. Moreover, the changes in the visible absorption spectra after Ag+ addition showed that Ag+ was bound to the type I Cu sites in BOD. During oxygen reduction by BOD, the direct-electron-transfer-type bioelectrocatalytic current decreased after Ag+ was added. The decay of the catalytic current was evaluated using kinetic analysis (assuming a pseudo-first-order reaction). Based on the analysis, the inhibition of BOD was suppressed when the Ag+ concentration was below 0.1 µM. Referring to the solubility product of AgCl, Cl- at a concentration of 1 mM suppressed the inhibition of the enzymatic activity by 95%.