Bienzymatic mediatorless sensing of total hydrogen peroxide with catalase and multi-copper enzyme co-adsorbed at carbon nanotube-modified electrodes

The rapid, sensitive, and accurate determination of hydrogen peroxide (H2O2) is of practical importance in physiological, pathological, and environmental fields. In this work, we propose a highly sensitive and selective amperometric biosensor for the detection of hydrogen peroxide. The biosensor consists of a glassy carbon electrode (GCE) covered with multi-walled carbon nanotubes (MWCNTs) and adsorbed enzymes: catalase (Cat) and either laccase (Lac) or bilirubin oxidase (BOX). The stability and durability of the electrode was improved by using glutaraldehyde (GAD). The determination of H2O2 by cyclic voltammetry and chronoamperometry experiments proved the synergy of the laccase and catalase co-adsorbed on the carbon nanotubes. Catalase from bovine liver catalyzed the transformation of H2O2 into water and oxygen, which was further transformed into water by multi-copper enzymes (MCO), either laccase from Trametes versicolor or bilirubin oxidase from Myrothecium verrucaria. The unique property of such a bienzymatic sensing layer is the ability to detect oxygen originating both from catalase activity and from the self-decomposition of H2O2. This makes it possible to evaluate the initial concentration of H2O2 in the analyzed sample.