The performance of the enzyme based biosensors depends on many parameters, in particular on the enzyme activity. Our approach consists in layer-by-layer building of a dehydrogenase biosensor using electrostatic interactions in order to prevent damaging of the enzyme by direct interactions with the electrode. In order to increase the sensitivity and stability of the sensors, organised porous metal structures has been used as electrode substrates [1].
Our attention is focused on one particular mediator family based on a skeleton of nitrofluorenone, i.e., (2,4,7-trinitro-9-fluorenylidene)-malonitrile, (TNFM), which adsorbs on the surface of gold electrodes and exhibits appreciable electrocatalytic activity towards NADH (nicotinamide adenine dinucleotide) oxidation [2, 3]. The adsorption of this molecule has been studied by cyclic voltammetry and surface infrared spectroscopy in order to elucidate the detailed mechanism of the molecule-surface and mediator-NADH interactions. These studies suggest that the monolayer of redox mediator is irreversibly bound to the gold surface via the CN groups when the electrode is exposed to negative potentials in order to transform one ore more nitro groups into the catalytically active NO/NHOH couple. Electrochemical results show that macroporous electrodes can be modified with the redox mediator on their entire inner surface. Therefore, the amount of the adsorbed mediator directly depends on the surface area of the electrode and the total inner surface is also accessible for the catalytic NADH oxidation. The currents can be further enhanced by adding Ca²⁺ ions to the solution and the produced NAD⁺ seems to be enzymatically active [4].

References
[1] S.A.G. Evans, J.M.Elliott, L. M. Andrews, P.N. Bartlett, P. J. Doyle, and G. Denault, Anal. Chem. 74, 1322-1326, (2002).
[2] N. Mano and A. Kuhn, Electroanal. Chem., 477, 79-88, (1999).
[3] N. Mano and A. Kuhn, Biosensors & Bioelectronics, 16, 653-660, (2001).
[4] S. Ben Ali, D.A. Cook, A. Thienpont, P.N. Bartlett and A. Kuhn. Electrochem. Commun., 5, 747-751, (2003).

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1. Formation and properties of donor-acceptor dyads of Zn porphyrins and C₆₀-imidazole adduct in the Langmuir and Langmuir-Blodgett Films