So-called 'second generation' biosensors are based on redox mediators aimed to deliver electrons between the enzyme active site and the electrode.

Among the advantages of the second generation oxidase-based biosensors are: operation in oxygen free media and a possibility for coulometric detection. However, because the redox mediators are small, in vivo leaching of the mediator is a serious concern. To eliminate this disadvantage we need to find a new method for mediator immobilization without covalent linking either to membrane forming polymer or to the enzyme.

We investigated glucose oxidase immobilization in gel of siloxanes using novel protocol based on water-organic mixtures with the high content of organic solvent reported for lactate oxidase. Obviously, to provide the mediator immobilization in such membrane the water insoluble azines were chosen. We tested unsubstituted phenothiazine, phenoxazine and their oligomers. Among them the only phenothiazine displayed electroactivity when cycled in neutral solutions.

Siloxane gels with immobilized glucose oxidase and the mediator were optimized considering concentrations of all components to provide both the highest stability and the highest response. The optimal membranes were investigated both in flow-injection system with amperometric detection and by cyclic voltammetry.

Upon  the adding of glucose in solution the usual catalytic electrooxidation wave was observed. Such well-defined wave normally observed for both the enzyme and the mediator in solution, indicates diffusion ability of phenothiazine in the membrane. We noted that being water-insoluble phenothiazine was unknown as the mediator for glucose oxidase. Indeed, when immobilized a dip-coating from its organic solution phenothiazine did not show mediation ability even in the presence of both glucose and glucose oxidase in solution.

Analytical performances of the elaborated second generation biosensor based on glucose oxidase and phenothiazine immobilized into siloxane gel were investigated in flow-injection system. Sensitivity of the biosensor in FIA mode is 2 mA M^-1cm^-2 The lower detection limit for glucose (0.015 mM) is among the lowest ones for second generation biosensors. The linear range is from 0.05 to 50 mM glucose.

The crucial point for reagentless second generation biosensors is their operational stability. Accordingly, the latter for the elaborated biosensor was investigated. The wall-jet detector equipped with the biosensor remains its initial response (precisely, 98±1%) after 50 injections. Hence, the elaborated second generation biosensor with immobilized both the enzyme and the mediator is stable enough for multiple measurements and even can be used for continuous monitoring.

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