Mechanistic characterization of an oxygen reduction reaction-driven, fully enzymatic and self-calibrating pH biosensor based on wired bilirubin oxidase

Abstract

The oxygen reduction reaction (ORR) is important due to high electrochemical potential. Multicopper oxidases (e.g., bilirubin oxidase (BOx)) belong to a class of enzymes that catalyze ORR with low overpotential, and have attracted significant attention in designing bioelectronic devices, viz., biosensors and enzymatic cathodes of biofuel cells. In this work, we present a new concept – an enzyme driven pH electrode system composed of two electrodes: a pH-sensitive BOx-based working electrode and pH-insensitive, a BOx-based reference electrode. ORR-driven pH monitoring was conducted by measuring the potential difference between the two electrodes. Moreover, the electrode system was self-supported and self-calibrating, and no additional electrodes (Pt counter or Ag/AgCl reference) were needed for measurements. The BOx-based pH sensor was tested in continuous and real-time pH monitoring with high accuracy. Also, we present an in-depth mechanistic study explaining the electrode potential-pH dependence, which brings new insights into the operational principle of bilirubin oxidase at different pH.