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 (O2) 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 O2 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 O2 concentration and was almost identical to the theoretical sensor, based on nonlinear diffusion of O2 around the microdisk electrode. The biosensor response was fast enough to monitor the catalytic consumption of dissolved O2 by glucose oxidase and exhibited storage stability for more than six days. •Porous structures on a gold surface enhance the bioelectrocatalytic activity of multi-copper oxidases.•Diffusion-controlled biocatalytic oxygen reduction occurs at microelectrodes.•Oxygen biosensors operate without calibration.•Biosensors function under neutral and weakly acidic conditions.•Response of the biosensor is fast enough to monitor oxidase reactions.