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 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/μM 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 (0 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.