ABSTRACT
Amperometric biosensors based on flavin-containing oxidases undergo several steps which produce a measurable current that is related to the concentration of substrate. In the initial step, the substrate converts the oxidized flavin adenine dinucleotide (FAD) or flavin mononucleotide (FMN) into the reduced form (FADH2 or FMNH2). Because these cofactors are located well within the enzyme molecule, direct electron transfer to the surface of a conventional electrode does not occur to a measurable degree. A common method of facilitating this electron transfer is to introduce oxygen into the system because it is the natural acceptor for the oxidases; the oxygen is reduced by the FADH2 or FMNH2 to hydrogen peroxide, which can then be detected electrochemically. The major drawback to this approach is the fact that oxidation of hydrogen peroxide requires a large overpotential thus making these sensors susceptible to interference from electroactive species. In order to lower the necessary applied potential, several non-physiological redox couples have been employed to shuttle electrons between the flavin moieties and the electrode. For example, sensors based on the ferrocene/ferricinium redox couple [1,2] and on electrodes consisting of conducting salts such as TTF-TCNQ (tetrathiafulvalene- 196tetracyanoquinodimethane) [3,4] have previously been reported. Electron relays have also been attached directly to the enzyme molecule in order to facilitate electron transfer [5,6]. More recently, these studies have been extended to include systems where the mediating redox species are covalently attached to polymers such as poly(pyrrole) [7], poly(vinylpyridine) [8], and poly(siloxane) [9]. The present paper describes the development of amperometric biosensors based on flavin-containing enzymes and this latter family of polymeric mediators.
