Studies of the electrocatalytic oxidation of β-nicotinamide adenine dinucleotide (NADH) at glassy carbon rotated disk electrodes modified with electrodeposited films derived from 3,4-dihydroxybenzaldehyde (3,4-DHB) indicate that the mechanism of such electrooxidation proceeds via the formation of an intermediate complex. The reaction also appears to be strongly influenced by the presence of Ca(2+) and Mg(2+) ions as well as by pH. Ascorbate can also be electrocatalytically oxidized at these modified electrodes, giving rise to an electrochemical response very similar to that obtained for NADH. Due to this similarity, the presence of ascorbate in NADH determinations presents a severe interference that cannot be mitigated on the basis of electrochemical responses alone. However, this interference effect can be virtually suppressed by the presence of ascorbate oxidase in solution or immobilized on a nylon mesh which, in turn, is in contact with the electrode modified with the film of 3,4-DHB. Using this approach, we describe the construction of an alcohol biosensor based on alcohol dehydrogenase and which is, furthermore, free from interference effects due to ascorbate.

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Exposure of gold surfaces to solutions of dithiobis N-succinimidyl propionate (DTSP) gives rise to the modification of the surface with N-succinimidyl-3-thiopropionate (NSTP) which can, in turn, react with amino groups allowing for the covalent immobilization of enzymes such as horseradish peroxidase (HRP). The coverage of NSTP has been estimated to be of the order of 1.3 x 10(-10) from the charge consumed during its reductive desorption. The binding reaction of HRP with NSTP modified gold surfaces has been studied with the quartz crystal microbalance, and the results suggest that the immobilization process involves two steps in which the first (faster) appears to correspond to the rapid incorporation of the enzyme whereas the second is likely due to the slow incorporation of additional enzyme and/or reorganization of the immobilized layer. Spectrophotometric and electrochemical assays indicate that the immobilized HRP retains its enzymatic activity after immobilization onto the DTSP modified gold surface. The amount of immobilized (and active) HRP was estimated from QCM and spectrophotometric measurements to be of the order of 1.5 x 10(-11) mol/cm2. A peroxide biosensor was developed making use of a gold surface modified with DTSP and HRP employing Os and Ru complexes of 1,10-phenanthroline 5,6-dione (phen-dione) of the type [M(phendione)x(L)3-x]+2 (where L = 1,10-phenanthroline or 2,2'-bipyridine, x = 1-3) as mediators with the quinone moieties being the active component. The efficiency of the mediators increased with increasing number of phendione ligands.