This review deals with the use of pyridine-nucleotide dependent dehydrogenase enzymes in amperometric biosensor design. The electrochemistry of the nicotinamide coenzymes, their mechanism and the challenges associated with their direct oxidation on solid electrodes are discussed. In addition, a survey of the different solutions proposed in the literature for addressing these problems and some of their applications are presented.This article provides an overview of the challenges associated with the direct oxidation of the pyridine-coenzymes on solid electrodes, the different solutions proposed in the literature and the application of these enzymes to the design of amperometric biosensors.

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An amperometric ethanol electrode is described based on carbon paste modified with alcohol dehydrogenase (ADH) and a mediator. The possibility of coupling this enzymatic system with some commercially available phenoxazines [Meldola Blue (MB), Nile Blue (NB) and Brilliant Cresyl Blue (BCB)] and phenothiazines [Toluidine Blue 0 (TBO)], that allows the electrocatalytic oxidation of NADH, was investigated. Different strategies of mediator immobilization in the carbon paste were compared. The most suitable immobilization technique was the coverage of the modified carbon paste surface with a dialysis membrane and the best electrocatalytic structure was the phenothiazine TBO. The TBO(lO%)-ADH(S%)-modified carbon paste electrode shows steady state signals for ethanol within 30 s. The influence of various experimental conditions was explored for optimum analytical performance. Amperometric detection of ethanol at an applied potential of 50mV (Ag/AgCl) gives linear responses over the concentration range of 10-lOOpM, with a detection limit of 5 x 10-6M. The modified electrode was applied to the determination of ethanol in wine.

An amperometric ethanol sensor based on carbon paste, chemically modified with alcohol dehydrogenase, nicotinamide adenine dinucleotide and Toluidine Blue 0 as a mediator, is described. The surface of the sensor is protected by covering with a dialysis membrane. Dialysis membranes with different molecular weight cut-off were tested for preventing leaching of soluble species from the electrode. The influence of various experimental conditions was investigated for optimum analytical performance. Linear responses for ethanol in the range 2 x M at 0.05 V (Ag/AgCl) and a response time of 60 s were obtained. The sensor was also used in flow-injection analysis and tested on wine samples.

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