Potential pulse-assisted immobilization of Myrothecium verrucaria bilirubin oxidase at planar and nanoporous gold electrodes

A potential pulse-assisted approach was used to immobilize Myrothecium verrucaria bilirubin oxidase at planar and nanoporous gold electrodes (NPG) containing pores of ca. 20 nm and ca. 40 nm in diameter. An increase in the current due to the bioelectrocatalytic reduction of oxygen by MvBOD-modified gold electrodes obtained from a 20 μL drop by the proposed pulse-assisted approach was observed when compared to the response obtained with electrodes modified by drop-casting. This increase likely arises from a preferential orientation of MvBOD molecules at the planar gold surface obtained by fast switching of the potential pulses between opposite charges. The concomitant ion stirring effect induces the attraction of the enzymes to the charged gold surface and forces access to the internal pore volume of the NPG. Immobilization of MvBOD using the potential pulse-assisted approach significantly increases current densities by facilitating the electron transfer between the enzyme and the electrode surface.