A generic amperometric bioassay based on the enzymatic oxidation catalysed by the stable NADH oxidase (NAox) from Thermus thermophilus has been developed for NADH measurements. The NAox uses O-2 as its natural electron acceptor and produces H2O2 in a two-electron process. Electrochemical and spectrophotometric experiments showed that the NAox used in this work, presents a very good activity towards its substrate and, in contrary to previously mentioned NADH oxidases, does not require the addition of any exogenous flavin cofactor neither to promote nor to maintain its activity. In addition, the NAox used also works with artificial electron acceptors like ferrocene derivatives. O-2 was successfully replaced by redox mediators such as hydroxymethyl ferrocene (FcCH(2)OH) for the regeneration of the active enzyme. Combining the NAox with the mediator and the horseradish peroxidase we developed an original, high sensitive "redox-flexible" NADH amperometric bioassay working in a large window of applied potentials in both oxidation and reduction modes. The biosensor has a continuous and complementary linearity range permitting to measure NADH concentrations starting from 5 x 10(-6) M in reduction until 2 x 10(3) M in oxidation. This redox-flexibility allows choosing the applied potential in order to avoid electrochemical interferences. The association of the "redox-flexible" concept with NADH dependent enzymes opens a novel strategy for dehydrogenases based bioassays and biosensors. The great number of dehydrogenases available makes the concept applicable for numerous substrates to analyse. Moreover it allows the development of a wide range of biosensors on the basis of a generic platform. This gives several advantages over the previous manufacturing techniques and offers a general and flexible scheme for the fabrication of biosensors presenting high sensitivities, wide calibration ranges and less affected by electrochemical interferences. (c) 2006 Elsevier Ltd. All rights reserved.