Oxygen tolerance of an implantable polymer/enzyme composite glutamate biosensor displaying polycation-enhanced substrate sensitivity.

Biosensors were fabricated at neutral pH by sequentially depositing the polycation polyethyleneimine (PEI), the stereoselective enzyme L-glutamate oxidase (GluOx) and the permselective barrier poly-ortho-phenylenediamine (PPD) onto 125-microm diameter Pt wire electrodes (Pt/PEI/GluOx/PPD). These devices were calibrated amperometrically at 0.7 V versus SCE to determine the Michaelis-Menten parameters for enzyme substrate, l-glutamate (Glu) and co-substrate, dioxygen.

Control of the oxygen dependence of an implantable polymer/enzyme composite biosensor for glutamate.

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)(O2).

The efficiency of immobilised glutamate oxidase decreases with surface enzyme loading: an electrostatic effect, and reversal by a polycation significantly enhances biosensor sensitivity.

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 GluOx-based amperometric biosensors in the linear response region (100+/-11 nA cm(-2)microM(-1) at pH 7.

The selectivity of electrosynthesised polymer membranes depends on the electrode dimensions: implications for biosensor applications.

A biosensor selectivity coefficient defined for poly(o-phenylenediamine) electrosynthesised onto Pt microdisks and cylinders was unexpectedly found to change as the scale of the electrodes decreased, mainly due to enhanced permeability of a ubiquitous interference species in biological systems, ascorbic acid.