Diffusion-limited biosensing of dissolved oxygen by direct electron transfer-type bioelectrocatalysis of multi-copper oxidases immobilized on porous gold microelectrodes

Diffusion-controlled amperometric biosensors for dissolved oxygen (O 2 ) were constructed by immobilization of multi-copper oxidases (copper efflux oxidase and bilirubin oxidase) on porous gold microdisk electrodes fabricated by anodization in a glucose solution. The immobilized enzymes rapidly consumed O 2 near the electrode at potentials more negative than 0.2 V vs. Ag|AgCl|sat. KCl via direct electron transfer-type bioelectrocatalysis and the reduction current reached the steady state limiting value under static conditions. The fabricated biosensor exhibited a linear response to dissolved O 2 concentration and was almost identical to the theoretical sensor, based on nonlinear diffusion of O 2 around the microdisk electrode. The biosensor response was fast enough to monitor the catalytic consumption of dissolved O 2 by glucose oxidase and exhibited storage stability for more than six days.