Characterization of Glucose Oxidase-Modified Poly(phenylenediamine)-Coated Electrodes in vitro and in vivo: Homogeneous Interference by Ascorbic Acid in Hydrogen Peroxide Detection

Abstract

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...

An amperometric glucose-oxidase/poly(o-phenylenediamine) biosensor for monitoring brain extracellular glucose: in vivo characterisation in the striatum of freely-moving rats

“…Pt/ PPD/GOx disk electrodes were prepared from Tefloncoated platinum/iridium (Pt/Ir 90%/10%) wire (125 vm bare diameter, 160 vm coated diameter (5T), Advent Research Materials, Suffolk, UK) following a previously reported procedure (Lowry et al, 1994a). Pt/ PPD/GOx cylinder electrodes were made from 5 cm lengths of the same 5T wire.…”

“…In order to ensure good GOx adsorption the exposed 4 mm of wire was then scored repeatedly with a scalpel blade. GOx was immobilised in PPD by potentiostatic electropolymenzation of the o-phenylenediamine monomer (o-PD, 300 mM) following a modified form of a previously reported procedure (Lowry et al, 1994a). Briefly, a deoxygenated solution of the monomer was prepared in PBS, pH 7.4.…”

“…The current was then measured when the solution was quiescent. The electrodes were held at a constant potential of +700 mV, which is the value generally used for H 2 O 2 detection (Lowry et al, 1994a). All calibrations were carried out at room temperature with the exception of a series of experimental measurements done at 37°C.…”

“…This is because of the importance of glucose monitoring in the disease diabetes mellitus and the fact that glucose determination in various body fluids, such as blood, plasma and urine, remains one of the most common analysis carried out in clinical laboratories. However, only a few such devices have been used in vivo in neurochemical studies.With the exception of a report by Boutelle et al (1986) these have all been carried out in anaesthetized animals and have all involved amperometric enzyme-modified electrodes, predominantly for glucose (Lowry et al, 1994a;Silver and Ereciñ ska, 1994;Netchiporouk et al, 1996;, with some recent reports for glutamate (Albery et al, 1992;Hu et al, 1994;Asai et al, 1996) and choline (Garguilo and Michael, 1993;Garguilo and Michael 1994). The small number of such reports reflects the numerous difficulties associated with performing direct neurochemical measurements in such a hostile and complex environment as brain extracellular fluid (ECF).…”

“…interferencefree, high enzyme activity and low response time) indicating ideal suitability for detecting substrates in biological fluids. Several groups, including ourselves, have now used this technology to develop and characterise biosensors for a variety of substrates (Bartlett and Birkin, 1994;Dong, 1994;Lowry et al, 1994a;Friedemann et al, 1996). We have focused on the development of a glucose sensor for applications in the mammalian brain, as glucose is the primary energy source for the brain and a constant supply is essential to maintain normal cerebral function.…”