Nitrogen-doped carbon nanotubes supported by macroporous carbon as an efficient enzymatic biosensing platform for glucose Anal. Chem. 88 (2016) 1371-7-0.384 V vs.

Stable, site-specific immobilization of redox proteins and enzymes is of interest for the development of biosensors and biofuel cells, where the long-term stability of enzymatic electrodes as well as the possibility of controlling the orientation of the biomolecules at the electrode surface have a great importance. Ideally, it would be desirable to immobilize redox proteins and enzymes in a specific orientation, but still with some flexibility to optimize reaction kinetics. In this work, we establish such an approach by using site-directed mutagenesis to introduce cysteine residues at specific locations on the protein surface and the reaction between the free thiol group and maleimide groups attached to the electrode surface to immobilize the mutated enzymes. Using cellobiose dehydrogen-ase (CDH) as a model system, carbon nanotube electrodes were first covalently modified with maleimide groups following a modular approach based on electrografting of primary amines at the carbon surface and solid-phase synthesis methodology to elaborate the surface-modified electrode. The CDH-modified electrodes were tested for direct electron transfer (DET), showing high catalytic currents as well as excellent long-term storage stability. The key advantage of this method is its great flexibility, as the main components of the modification can be independently varied to change the local environment at the electrode surface and a wide range of redox proteins or enzymes can be specifically engineered to present cysteine residues at their surface for oriented immobilization.

In this work, we extended the generic approach for the sitedirected immobilization of enzymes based on maleimide\thiol coupling of engineered enzymes to the oriented immobilization of variants of bilirubin oxidase from Magnaporthe oryzae (MoBOD) to electrodes. We show that this approach leads to the stable attachment of the enzyme to the electrode surface and that the immobilized MoBOD variants are active for bioelectrocatalytic reduction of dioxygen through direct (unmediated) electron transfer (DET) from the electrode. For the three MoBOD variants studied, significant differences are observed in the kinetics of DET that relate to the orientation of the enzyme and the distance of the T1 site from the electrode surface. The stability of the immobilized enzymes allows us to compare the DET and mediated electron-transfer (MET) pathways and to investigate the effects of pH and Cl − . Our studies show a change in the slope of pH dependence at pH 6.0 and highlight the effect of Cl − on the direct oxygen reduction by MoBOD as a function of pH for the immobilized enzyme and the interconversion of the resting oxidized (RO) form of the immobilized enzyme and the alternative resting (AR) state formed in the presence of Cl − .