Improvement in signal reliability when measuring L-glutamate released fromcultured cells using multi-channel microfabricated sensors

We used a micromachining technique to fabricate a very reliable amperometri c glutamate sensor consisting of dual channel thin layer flow cells for the highly selective measurement of glutamate released from cultured nerve cel ls. Our microfabricated sensor has two carbon film electrodes separated by a separator in the thin layer channel flow cells. We modified one carbon el ectrode with a bilayer of Os-gel-HRP/BSA film containing GluOx, and the oth er with a bilayer of Os-gel-HRP/BSA film without GluOx. Therefore, any diff erence between the currents at the dual electrodes should be due to the oxi dation of glutamate by GluOx. This dual mode measurement technique can elim inate current changes caused by the change in the double layer capacitance when KCI solution is added to stimulate the cells or by baseline fluctuatio ns resulting from pumping noise. As a result, we were able to monitor the t ransient peak caused by the glutamate release from cultured rat cortex cell s with high selectivity by stimulating the cells with 100 mM of KCI solutio n. 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 whe n we measured the extracellular solution was lower than that at an untreate d sensor. This result shows that the current caused by extracellular glutam ine was eliminated by the DON treatment. Moreover, the extracellular glutam ate concentration measured with the DON pre-treated sensor was consistent w ith that obtained by HPLC. (C) 2001 Elsevier Science B.V. All rights reserv ed.