We used a micromachining technique to fabricate a very reliable amperometric glutamate sensor consisting of dual channel thin layer flow cells for the highly selective measurement of glutamate released from cultured nerve cells. Our microfabricated sensor has two carbon film electrodes separated by a separator in the thin layer channel flow cells. We modified one carbon electrode with a bilayer of Os-gel-HRP/BSA film containing GluOx, and the other with a bilayer of Os-gel-HRP/BSA film without GluOx. Therefore, any difference between the currents at the dual electrodes should be due to the oxidation of glutamate by GluOx. This dual mode measurement technique can eliminate current changes caused by the change in the double layer capacitance when KCl solution is added to stimulate the cells or by baseline fluctuations resulting from pumping noise. As a result, we were able to monitor the transient peak caused by the glutamate release from cultured rat cortex cells with high selectivity by stimulating the cells with 100 mM of KCl solution. The specificity of the GluOx for glutamate was improved when the sensor was pre-treated with 6-diazo-5-oxo-L-norleucine (DON), which is known to be a glutaminase inhibitor. The signal we obtained at the DON-treated sensor when we measured the extracellular solution was lower than that at an untreated sensor. This result shows that the current caused by extracellular glutamine was eliminated by the DON treatment. Moreover, the extracellular glutamate concentration measured with the DON pre-treated sensor was consistent with that obtained by HPLC.
- Dual electrode