ANUYTICA CHIMICA ACTA Analytica Chimica Acta 328 (1996) 41-46 Peroxidase based amperometric biosensors for the determination of y-aminobutyric acid Franc0 Mazzeia, Francesco Botrhb9*, Giampiero Lorentia, Fernando Porcelli” “Cattedra di Chimica Fisica, Dipartimento di Studi di Chimica e Tecnologia delle Sostanze Biologicamente Attive. Universiti2 “La Sapienza”, I?le Aldo Moro 5, Rome 00185, Italy blstituto di Merceologia, Universitci “LA Sapienza”, via de1 Cast, Laurenziano 9, Rome 00161, Italy ‘Cattedra di Chimica Fisica, Dipartimento di Scienze Ambientali, Universitk della Tuscia, Via San Camille de L&is, viterbo 01100, Italy Received 14 September 1995; revised 29 January 1996; accepted 3 February 1996 zyxwvutsrqponmlkjihgfedcbaZ Abstract This work presents the realization of enzymatic bioelectrodes, suitable for the determination of y-aminobutyric acid (GABA). The biosensors are based on the catalytic activity of the enzymes y-aminobutyric glutamic transaminase (GABA-T), succinic semialdehyde dehydrogenase (SSDH) and horseradish peroxidase (IWO). 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 HP0 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 HP0 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 5.0x 10-5-1.2x10-3M, with a lower detection limit of 2.0x10-‘M; but the single-membrane biosensor showed a better overall performance, especially in terms of repeatability of measurements and lifetime of operation. Keywords: Arnperometric biosensors; GABA, GABA-T; GABASE; BPO; SSDH 1. Introduction The problem of the rapid and direct determination of r-aminobutyric acid (GABA) with no significant perturbation in the system under investigation, as it is * Corresponding author. Tel.: +39/6/49766217; fax: +39/6/ 4941621; e-mail: botre@axrma.uniromal.it. 0003-2670/96/$15.00 0 1996 Elsevier Science B.V. All rights reserved PII SOOO3-2670(96)00089-X required in the study of biological systems, is of great relevance owing to the fundamental role that GABA plays in the central nervous system (CNS) under both physiological and pathological conditions [ 1,2]. The steady state conditions for the concentration of GABA in the CNS are determined by the continuous balancing between the production of GABA, formed by the rw-decarboxylation reaction of r_-glutamic acid