On-line measurement of brain glutamate with an enzyme/polymer-coated tubular electrode

Abstract

An on-line tubular electrode system is described which provides real time measurements of glutamate in brain microdialysate. It is based on the enzyme glutamate oxidase (EC 1.4.3.11) and the detection of H2O2 on a platinum electrode at 600 mV vs Ag/AgCl. The enzyme is immobilized in a layer of o-phenylenediamine electropolymerized at 750 mV in a phosphate buffer, pH 7.0. The layer is 10-15 nm thick and enables good enzyme loading and fast response time. The ability of the polymer to block out electroactive interferents like ascorbate and uric acid combined with a preoxidation system run at 600 mV resulted in a virtually interference free glutamate assay with a lower detection limit of 0.3 microM in the presence of physiological levels of interferents, a sensitivity of 4 nA/microM, and a linear region up to 30 microM. The system is optimized for use with a microdialysis probe implanted in the brain and perfused at 2 microL/min. It provides a time resolution of < 1 min and has been tested in vivo.