Continuous Monitoring of L-Glutamate Released from Cultured Rat Nerve Cells with a Microfabricated On-Line Sensor at a Slow Flow Rate

A microfabricated online L-glutamate sensor was developed to monitor changes in the local concentration of L-glutamate released from cultured nerve cells. The sensor consists of two glass plates and a fused silica sampling capillary. One of the glass plates has rectanglar flow channels cut into it with a dicing saw and the other has three carbon film based electrodes. One of the electrodes was modified with an Ospolyvinylpyrridine-based polymer layer containing horseradish peroxidase. A GluOx enzyme layer was immobilized upstream of the working electrode. A syringe pump in the suction mode was used to sample extracellular fluid continuously via a glass capillary with a pointed end. The sensor sensitivity did not decrease with decreasing flow rate when the flow rate was reduced to about 200 nL/min. This is because the increase in the conversion efficiency compensated for the decrease in the analyte flow into the sensor. As a result, a low detection limit (6.4 nM) was obtained at a slow flow rate. In an in vitro experiment, the extracellular fluid near a particular nerve cell was sampled through a capillary and continuously introduced into the sensor via the suction provided by a syringe pump. The nerve cells were stimulated by KCl and a transient L-glutamate release with a sharp peak was monitored with a slow flow rate of 500 nL/min to 2 μL/min.