Bilirubin oxidase as a single enzymatic oxygen scavenger for the development of reductase-based biosensors in the open air and its application on a nitrite biosensor

The commercialization of amperometric or voltammetric biosensors that operate at potentials lower than −0.2 V vs SHE has been hindered by the need for anoxic working conditions due to the interference of molecular oxygen, whose electrochemical reduction can potentially mask other redox processes and generate reactive oxygen species (ROS). A deoxygenation step must be thus integrated into the analytical process. To this end, several (bio)chemical oxygen scavenging systems have been proposed, such as the bi-enzyme system, glucose oxidase/catalase. Still, a few issues persist owing to enzyme impurities and the formation of oxygen reactive species. Here in, we propose a new mono-enzymatic oxygen scavenging system composed of a multicopper oxidase as a single biocatalytic oxygen reducer. As a model, we used bilirubin oxidase (BOD), which catalyzes the direct reduction of oxygen to water in the presence of an electron donor substrate, without releasing hydrogen peroxide. Both the direct electron transfer and mediated electrochemical approach using different co-substrates were screened for the ability to promote the enzymatic reduction of oxygen. An optimal combination of BOD with sodium ascorbate proved to be quick (5 min) and effective. It was subsequently employed, as a proof-of-concept, in a voltammetric biosensor based on a multiheme cytochrome c nitrite reductase, which performs the reduction of nitrite to ammonia at potentials below −0.3 V vs SHE. The nitrite biosensor performed well under ambient air, with no need for a second enzyme to account for the build-up of oxygen reactive intermediaries.