Amperometric detection of pyridine nucleotides via immobilized viologen-accepting pyridine nucleotide oxidoreductase or immobilized diaphorase

The immobilization and electrical connection of a viologen-accepting pyridine nucleotide oxidoreductase (VAPOR) on an electrode surface by coadsorption with an amphiphilic pyrrole viologen and electropolymerization of this pyrrole monomer are described. The immobilized VAPOR catalyzes the reduction of NAD(P)(+) to NAD(P)H by the viologen redox couple (v2(+/.+)). The sensitivity of this biosensor is 1.4 and 3.5 mA M(-1) cm(-2) for NAD(+) and NADP(+) respectively. The immobilization of diaphorase within a laponite gel adsorbed on an electrode surface is described. The incorporation and electropolymerization of Methylene Blue in the biolayer allows an electron transfer communication between diaphorase molecules and the electrode surface. The diaphorase electrode thus obtained responds to NADH at 0 V. The sensitivity and detection limit of this biosensor are 11.2? mA M(-1) cm(-1) and 1 mu M respectively.