A modular approach for a controlled immobilization of enzymes

Stable, site-specific immobilization of redox proteins and enzymes is of great 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. For such applications, it would be desirable to immobilise redox proteins and enzymes in a specific orientation on the electrode in order to improve direct electron transfer.In this work, we describe such an approach using site directed mutagenesis to introduce cysteine residues at specific locations on the enzyme surface, and the reaction between the free thiol of the cysteine and maleimide groups attached on the electrode surface to immobilise the mutated enzymes. Using cellobiose dehydrogenase (CDH) as a model system, different types of electrodes (carbon and gold-based, flat and nanostructured) were firstly modified with maleimide groups using a modular approach based on electrografting and solid-phase synthesis. Therefore, the electrodes were used to covalently immobilise CDH variants bearing the free cysteine in different locations at their surface. The key point of this method is that the main elements of the modification can be independently varied to tune the architecture of the electrode surface as required, by simply changing the “bricks” of the structure.The CDH-modified electrodes were tested for direct and mediated electron transfer, showing excellent long-term storage stability as well as good catalytic responses. The mechanisms of the direct and mediated electron transfer were fully investigated, as well as the kinetics of the CDH electrodic reactions, also employing computer simulations