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 change s in the local concentration of L-glutamate released from cultured nerve ce lls. The sensor consists of two glass plates and a fused silica sampling ca pillary. One of the glass plates has rectanglar flow channels cut into it w ith a dicing saw and the other has three carbon film based electrodes. One of the electrodes was modified with an Os-polyvinylpyrridine-based polymer layer containing horseradish peroxidase. A GluOx enzyme layer was immobiliz ed upstream of the working electrode. A syringe pump in the suction mode wa s used to sample extracellular fluid continuously via a glass capillary wit h a pointed end. The sensor sensitivity did not decrease with decreasing fl ow rate when the Bow rate was reduced to about 200 nL/min. This is because the increase in the conversion efficiency compensated for the decrease in t he 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 extracel lular fluid near a particular nerve cell was sampled through a capillary an d continuously introduced into the sensor via the suction provided by a syr inge pump. The nerve cells were stimulated by KCl and a transient L-glutama te release with a sharp peak was monitored with a slow Bow rate of 500 nL/m in to 2 mu L/min.