An amperometric biosensor for l-glutamic acid (Glu) was constructed by the adsorption and dip coating of l-glutamate oxidase (GluOx, 200 U ml 21 phosphate buffer, pH 7.4) onto 60-mm radius Teflon-coated Pt wire (1 mm exposed length). The enzyme was then trapped on the surface by electropolymerisation of o-phenylenediamine that also served to block electroactive interference. This procedure afforded electrodes with similar substrate sensitivity compared with the classical approach of immobilising enzyme from a solution of monomer, and represents an approximately 10 000-fold increase in the yield of biosensors from a batch of enzyme. A number of strategies were examined to enhance the sensitivity and selectivity of the Pt/PPD/GluOx sensors operating at 0.7 V versus SCE. Pre-coating the Pt with lipid and incorporation of the protein bovine serum albumin into the polymer matrix were found to improve the performance of the electrode. The sensors had a fast response time, high sensitivity to Glu, with an LOD of about 0.3 mmol l 21 , and possessed selectivity characteristics suggesting that monitoring Glu in biological tissues in vivo may be feasible.

The response of glucose oxidase (GOx) modified poly(& phenylenediamine) coated P t disk electrodes to glucose was well-behaved with a rapid response time and displaying Michaelis-Menten kinetics. However, the glucose response was lowered in a concentration-dependent manner by ascorbic acid when the glucose calibrations were carried out in solutions containing this reducing agent. The possibility of a homogeneous redox reaction in which the H202 generated by the enzymatic oxidation of glucose at the GOx/polymer surface is consumed by ascorbate was investigated. Similar "negative" interference at GOx-modified carbon powder electrodes not involving membranes and for H202 calibrations at bare Pt electrodes supported the hypothesis. The observation that this interference could be blocked by the chelating agent EDTA suggests that the homogeneous reaction is catalyzed by trace metal ion impurities in solution. A model for the homogeneous reaction based on these experimental findings is proposed and tested by comparing quiescent and stirred solutions. No homogeneous interference by uric acid was observed. The electrodes were found to be free from lipid fouling in vitro, and experiments monitoring brain glucose levels in vivo indicate the absence of the homogeneous reaction in this environment. The results highlight the need to test each individual assay procedure involving HzO2 under relevant conditions for both positive and negative interference by ascorbic acid.The search for the ideal glucose sensor continues to be one of the main focuses of biosensor research despite 3 decades of intense investigation since the development of the first electrochemical glucose sensor by Clark and Lyons in 1962.' This is primarily due to the important role of such a glucose monitoring sensor in industrial2t3 and clinicalw applications, ranging from the analysis of fermentation media, to the development of the artificial @-cell or pancreas for the treatment of the metabolic disease diabetes mellitus.'J'Because of the complexity and serious specificity problems associated with direct electrooxidation of glucose9J0 the (11) where FAD is the oxidized form of the prosthetic group, flavin adenine dinucleotide. The development over the last 30 years of enzyme-based amperometric devices for glucose determination can essentially be divided into three categories. The first involves "classical" devices which monitored either the consumption of oxygen' or the formation of hydrogen peroxide.12 Such devices were originally affected by the ambient concentration of oxygen in the sample and required a large overpotential. In order to avoid these problems "second generation" systems were developed in which the natural dioxygen in reaction I1 is replaced by a mediat0r3-13.1~ resulting in electrodes which are relatively insensitive to changes in dioxygen tension and which, depending on the choice of mediator, can be operated at lower applied potentials. The successful application of these mediated systems relies on the appropriate choice of mediator, b...

ABSTaCTGlucose oxidase (GOx) modified poly-phenylenediamine (PPD) coated 250 p m Pt-disk electrodes were prepared by electropolymerization in phenylenediamine solutions containing GOx. Both ortbo and meta monomers were tested, and the om30 isomer was found to give better glucose responses. The response of Pt/o-PPD/GOx electrodes to glucose was well behaved with a rapid response time and displaying Michaelis-Menten kinetics. The behavior of these electrodes in vitro to ascorbic acid (a principal interferant in brain tissue) was complex in that the sensitivity was concentration dependent. At low ascorbate concentrations (<50 pM), the sensitivity was similar to that of bare Pt (72 ? 13 nA/ mM, n = 10) whereas at higher levels it decreased sharply to 0.2 ? 0.1 nA/mM ( n = 3) at 1 mmol/L ascorbate. At still higher concentrations (1 to 10 mrnol/L), the sensitiviv was concentration independent, 0.14 k 0.02 nA/mM (n = 3). Calibrations for another potential interferant, uric acid, were linear in the range studied (0 to 100 pM) with a slope of 0.41 2 0.03 nA/mM (n = 3), indicating negligible interference by this substrate for concentrations present in brain extracellular fluid. Surprisingly, the permeability of both substrates was greater in PPD films containing no GOx. The results are discussed in terms of the suitability of these electrodes for monitoring brain glucose in uiuo .

Very rough guide; depends on electrode material and its state of 9 Very rough guide of non-stimulated values; depends on brain Paper 31'046426

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

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The oxygen dependence of a first generation amperometric biosensor was investigated in vitro and in vivo by monitoring its glucose response as a function of solution pO 2 . The biosensor was a glucose oxidase (GOx) modified poly(o -phenylenediamine) coated Pt cylinder electrode (Pt/PPD/GOx) that has been designed for neurochemical analysis in vivo. Two types of oxygen probes were used: a self-calibrating commercial macroelectrode in vitro; and a carbon paste microelectrode in vivo. Calibrations in vitro showed that oxygen interference in the operation of Pt/PPD/GOx electrodes was minimal for concentrations of glucose ( Â/0.5 mM) and oxygen ( Â/50 mM) found in brain ECF. This observation was confirmed by simultaneous monitoring in vivo of brain glucose and oxygen in the awake rat. However, at levels of glucose normally found in peripheral tissues ( Â/5 mM), the oxygen dependence was severe. We conclude that the oxygen sensitivity of Pt/PPD/GOx biosensors does not preclude their reliable use in media containing low glucose levels, such as brain ECF. #

A range of simple aromatic amines were used to modify Pt microcylinders with insulating electrosynthesised polymers generated amperometrically in the presence or absence of a typical globular protein, bovine serum albumin (BSA), at neutral pH. Sensitivity to a typical interference species, ascorbic acid (AA), and the most common electrochemical signal molecule for oxidase enzymes, H 2 O 2 , was used to compare the suitability of the resulting polymer-protein modified electrodes for biosensor applications. Pt modified with poly(aminobenzene), PANI, gave the lowest sensitivity to AA ( ~3000 times lower than bare Pt); incorporation of BSA into the polymer during synthesis had a detrimental effect on its AA-rejecting capability. The H 2 O 2 sensitivity of the PANI-based electrodes was poor ( ~7 times lower) compared to bare Pt. The behaviour of poly(1,2-diaminobenzene), PoPD, was different in many respects: its AA-blocking ability was enhanced 3-fold by the presence of BSA and its H 2 O 2 sensitivity was similar to bare Pt. EQCM data recorded in PBS indicated that PANI films electrosynthesised at pH 7.4 were two orders of magnitude thicker than for PoPD. These differences may be due, at least in part, to the 'ladder' structure with phenazine rings proposed for PoPD, since they were not duplicated by other mono-substituted derivatives of aniline, such as 2-methylaniline or 1,3-diaminobenzene.