CONCENTRATION OF EXTRACELLULAR L-GLUTAMATE RELEASED FROM CULTURED NERVE-CELLS MEASURED WITH A SMALL-VOLUME ONLINE SENSOR

An online sensor with a low detection limit for L-glutamate was develo ped in order to monitor the change in the extracellular L-glutamate co ncentration as a result of stimulated release from cultured nerve cell s, The sensor consisted of a microdialysis (MD) probe fixed at the man ipulator, a small-volume L-glutamate oxidase enzymatic reactor (0.75 m m i.d, and 2.5 cm long), and an electrochemical detector in a thin-lay er radial now cell with an active volume of 70-340 nL, Glassy carbon b ulk or carbon film ring-disk electrodes were used as detectors by modi fying 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 now cell. As a result, we achieved a sensitivity of 24.3 nA/mu M 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 pot ential 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 stimula ted cells. The cells were stimulated by KCI in a glass capillary or el ectrically 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 KCI stimulation of a single nerve cell and micromolar L-glutamate concentration increases caused by ele ctrical stimulation of a brain slice, An increase in L-glutamate conce ntration can also be measured by positioning the probe near the cell t hat is connected synaptically to the stimulated cell.