The determination of the extracellular concentration of brain glutamate using quantitative microdialysis.

Quantitative microdialysis with two enzyme-based assays was used to determine the extracellular concentration of glutamate in the striatum of freely moving rats. From the difference between infused and dialysate glutamate a value of 3.0 +/- 0.6 microM for the extracellular glutamate concentration was computed by regression analysis. The in vivo recovery, derived from the slope of the regression line, was 50%.

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

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.

New technologies for amperometric biosensors.

Amperomeric-based detectors have successfully been used as personal monitors for blood glucose levels. However, there is a desire to increase the number of compounds measured in a small blood sample, the speed of detection and enhance the reliability of the measurement. Furthermore, with the increasing use of microdialysis as a clinical sampling method in metabolic medicine, paediatric medicine and neurointensive care, there is a need for rapid on-line detection of analytes such as lactate, glucose and glutamate in low microlitre volume samples. Two approaches to these problems are described. The first uses enzymes immobilized in a packed bed with electrochemical detection of a ferrocene mediator as a flow-injection assay for use with microdialysis. Results from microdialysis of the brain of freely moving rats are described. In the second approach, thin-film techniques are used to fabricate arrays of microdisk and micro line electrodes. The properties of these arrays in free solution and in a flow cell are presented together with an example using multiple arrays to identify an analyte by oxidation potential. Finally, different enzymes are entrapped onto the surface of two arrays by electrochemical polymerization of o-phenylenediamine. The resulting device detects glucose and lactate in real-time.