An online sensor with a low detection limit for L-glutamate was developed in order to monitor the change in the extracellular L-glutamate concentration as a result of stimulated release from cultured nerve cells. The sensor consisted of a microdialysis (MD) probe fixed at the manipulator, a small-volume L-glutamate oxidase enzymatic reactor (0.75 mm i.d. and 2.5 cm long), and an electrochemical detector in a thin-layer radial flow cell with an active volume of 70-340 nL. Glassy carbon bulk or carbon film ring-disk electrodes were used as detectors by modifying them with Os poly(vinylpyridine) mediator containing horseradish peroxidase. The overall efficiency of L-glutamate detection with the sensor is 94% under optimum conditions, due to an efficient enzymatic reaction in the reactor and a high conversion efficiency in the radial flow cell. As a result, we achieved a sensitivity of 24.3 nA/muM and a detection limit of 7.2 nM (S/N = 3). The effect of interferents such as L-ascorbic acid can be minimized effectively by applying a low potential to the electrode for hydrogen peroxide detection (O mV) and via the ring-disk electrode geometry by using the disk for preoxidation. In the in vitro experiment, an MD probe for sampling was connected to a manipulator that controls distance between the probe and the stimulated cells. The cells were stimulated by KCl in a glass capillary or electrically with microarray film electrodes fabricated on a substrate. By using the sensor, we can monitor L-glutamate concentration changes at the submicromolar level caused by KCl stimulation of a single nerve cell and micromolar L-glutamate concentration increases caused by electrical stimulation of a brain slice. An increase in L-glutamate concentration can also be measured by positioning the probe near the cell that is connected synaptically to the stimulated cell.

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An amperometric method suitable for the continuous on-line measurement of cerebral hydrogen peroxide from a microdialysate has been successfully performed for the first time by using an enzyme-modified ring-disk plastic carbon film electrode (PCFE) in a thin-layer radial flow cell. PCFE consists of a ring electrode modified with horseradish peroxidase to detect H2O2 at 0.0 V (vs Ag/ AgCl) and a disk electrode coated with ascorbate oxidase (AOx) to preoxidize ascorbic acid (AA) and thus suppress interference via direct oxidation. Analytes in solution (brain dialysates or standards) are mixed on-line with a phosphate-buffered solution containing dissolved oxygen and chelating agent, EDTA. The buffered solution is used to provide the O2 necessary for the AOx catalytic reaction, stabilize the changes in dialysate pH that are associated with the in vivo formation of H2O2, and remove heavy metal ion impurities and thus suppress reactions between AA and H2O2. This procedure enables trace levels of H2O2 to be readily monitored, virtually interference-free from physiological levels of AA, uric acid, electroactive neurotransmitters and their principle metabolites, in a continuous-flow system.