Synergistic substrates determination with biosensors.

High sensitive biosensors for heterocyclic compounds determination were built using oxidases-catalyzed hexacyanoferrate(III) reduction in the presence of these compounds. As oxidases Aspergillus niger glucose oxidase and recombinant Microdochium nivale carbohydrate oxidase were used. The biosensors were build using graphite electrodes and entrapped solution of the oxidases. The sensitivity of the biosensors achieves 5.2-14.5 microA microM-1 cm-2. The detection limit of some heterocyclic compounds was 0.2 microM. The sensitivity of biosensors was 300-10,000 times larger in comparison to hexacyanoferrate(III). To background the scheme of biosensors action kinetics of synergistic substrates oxidation was investigated in homogenous solution. The measurements showed that the rate of the reduction of low reactive substrate (hexacyanoferrate(III)) increased due to synergistic action of high reactive substrates (oxidized heterocyclic compounds). The modeling revealed the limiting step of the process. The increase of hexacyanoferrate(III) reduction rate is determined by the rate of reduced enzymes interaction with oxidized heterocyclic compound. The oxidation of heterocyclic compounds (mediators) with hexacyanoferrate(III) does not limit the process. The analysis of macrokinetics of biosensors action showed that synergistic effect may be realized and high biosensors sensitivity may be achieved if diffusion module of the enzyme reaction with the oxidized mediator and of a cross reaction is larger than 0.5. The calculated relative sensitivity is about three times higher in comparison to experimentally determined that may be caused by the limited stability of oxidized heterocyclic compounds and/or some external diffusion limitation of substrates.