Peroxidase based amperometric biosensors for the determination of gamma-aminobutyric acid

This work presents the realization of enzymatic bioelectrodes, suitable for the determination of gamma-aminobutyric acid (GABA). The biosensors are based on the catalytic activity of the enzymes gamma-aminobutyric glutamic transaminase (GABA-T), succinic semialdehyde dehydrogenase (SSDH) and horseradish peroxidase (HPO). The first two enzymes are usually indicated by the general term ''GABASE''. All the biosensors presented in this work are realized by immobilizing the enzyme HPO on the tip of an amperometric oxygen selective electrode: the resulting NADPH-sensitive biosensor is used in combination with GABASE to determine the concentration of GABA in aqueous samples. Since SSDH depends on the NADP(+)/NADPH equilibrium, it follows that, in the presence of HPO, the NADPH formed is oxidized to NADP, and the decrease in the concentration of dissolved oxygen is proportional to the concentration of NADPH and, in turn, to that of GABA. The experiments were performed either with GABASE free in solution or co-immobilized with HPO on the surface of the oxygen electrode. In the latter case, the immobilization of the three enzymes has been performed either on a single membrane or on two separated membranes. In both cases there is an almost perfect linearity between the electrode signal and GABA concentration in the range 5.0x10(-5)-1.2x10(-3) M, with a lower detection limit of 2.0x10(-5) M; but the single-membrane biosensor showed a better overall performance, especially in terms of repeatability of measurements and lifetime of operation.