Immobilization of Nicotinamide Adenine Dinucleotide Cofactors Using Aryl Diazonium Electrografting

The development of electrochemical biosensors and enzymatic fuel cells is based on the design of bioelectrodes working with oxidoreductases. Among them, NAD-dependent dehydrogenases represent one of the largest groups of redox enzymes using adenine dinucleotide cofactors (NAD), such as the redox couples NAD⁺/NADH and NADP⁺/NADPH. The electrochemical redox reactions with these cofactors leading to their regeneration have been explored intensively during the last 30 years. The regeneration of NAD(P)⁺ from NAD(P)H would allow the enzymatic oxidation of a wide range of biochemical substrates with high chemo-, regio-, and stereospecificity. However, the direct electrochemical oxidation of NADH to NAD⁺ involves single-electron reaction mechanisms, which generate radicals leading to the dimerization of the cofactor (NAD)₂ and its biological inactivation. This can be prevented using redox mediators capable of both operating at lower potentials and chemically oxidizing or reducing NAD through a two-electron reaction process. The formation of (NAD)₂ dimer through the direct electrochemistry of NAD can also be limited by immobilizing the cofactor on the electrode surface. In addition, the cofactor immobilization facilitates and enhances the electron transfers at the electrode surface. This chapter describes the different steps for the covalent tethering of NAD onto phenyl boronic acid (PBA)-modified electrode obtained by electrografting of diazonium.