Amperometric polymer/enzyme composite (PEC) biosensors, incorporating a poly(o-phenylenediamine) ultra-thin permselective barrier, possess a variety of characteristics that make them suitable for monitoring brain energy and neurotransmitter dynamics in vivo. This review highlights PEC sensitivity and selectivity parameters, which allow development of the basic design in a systematic way in order to improve their performance and to diversify the analyte range of these novel probes of brain function. ª

Abstract:Reports that globular proteins could enhance the interference blocking ability of the PPD (poly(o-phenylenediamine) layer used as a permselective barrier in biosensor design, prompted this study where a variety of modifying agents were incorporated into PPD during its electrosynthesis on Pt-Ir electrodes. Trapped molecules, including fibrous proteins and β-cyclodextrin, altered the polymer/modifier composite selectivity by affecting the sensitivity to both H 2 O 2 (signal molecule in many enzyme-based biosensors) and the archetypal interference species, ascorbic acid. A comparison of electrochemical properties of Pt and a Pt-Ir alloy suggests that the benefits of the latter, more rigid, metal can be exploited in PPD-based biosensor design without significant loss of backward compatibility with studies involving pure Pt.

Biosensors for glutamate (Glu) were fabricated from Teflon-coated Pt wire (cylinders and disks), modified with the enzyme glutamate oxidase (GluOx) and electrosynthesized polymer PPD, poly(o-phenylenediamine). The polymer/enzyme layer was deposited in two configurations: enzyme before polymer (GluOx/PPD) and enzyme after polymer (PPD/GluOx). These four biosensor designs were characterized in terms of response time, limit of detection, Michaelis-Menten parameters for Glu (J max and K M (Glu)), sensitivity to Glu in the linear response region, and dependence on oxygen concentration, K M (O 2 ). Analysis showed that the two polymer/enzyme configurations behaved similarly on both cylinders and disks. Although the two geometries showed different behaviors, these differences could be explained in terms of higher enzyme loading density on the disks; in many analyses, the four designs behaved like a single population with a range of GluOx loading. Enzyme loading was the key to controlling the K M (O 2 ) values of these first generation biosensors. The counterintuitive, and beneficial, behavior that biosensors with higher GluOx loading displayed a lower oxygen dependence was explained in terms of the effects of enzyme loading on the affinity of GluOx for its anionic substrate. Some differences between the properties of surface immobilized GluOx and glucose oxidase are highlighted.L-Glutamate (Glu) is the most widespread excitatory neurotransmitter in the mammalian central nervous system, 1 plays a major role in a broad range of brain functions, and has been implicated in a number of neurological disorders. 2 Indeed, the development of devices for Glu detection has become an important research area due to the value of monitoring this key amino acid in a number of complex matrixes, including food processing, 3,4 cell cultures, 5-7 tissue slices ex vivo, 8,9 and intact brain in vivo. [10][11][12][13][14][15] A number of designs of biosensors for Glu monitoring have been reported and, although Glu receptors 16 Brain Res. Bull. 2002, 58, 401-404. (10) Burmeister, J. J.; Gerhardt, G. A. Trends Anal. Chem. 2003, 22, 498-502. (11)

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The apparent Michaelis constant, K M , for glutamate oxidase (GluOx) immobilised on Pt electrodes increased systematically with enzyme loading. The effect was due, at least in part, to electrostatic repulsion between neighbouring oxidase molecules and the anionic substrate, glutamate (Glu). This understanding has allowed us to increase the Glu sensitivity of GluOxbased amperometric biosensors in the linear response region (100 ¡ 11 nA cm 22 mM 21 at pH 7.4; SD, n = 23) by incorporating a polycation (polyethyleneimine, PEI) to counterbalance the polyanionic protein. Differences in the behaviour of glucose biosensors of a similar configuration highlight a limitation of using glucose oxidase as a model enzyme in biosensor design.