Redox hydrogel-based bienzyme microelectrodes for amperometric monitoring of L-glutamate

Fabrication and characterization of amperometric bienzyme L-glutamate sensitive microelectrodes are the prerequisite for monitoring changes Of L-glutamate concentration at glutamate-secreting cell cultures. The design of the glutamate microelectrodes is based on incorporating L-glutamate oxidase and horseradish peroxidase into a redox-hydrogel containing PVI19-dmeOs as the redox mediator and immobilizing this system onto the surface of platinum microdisk electrodes using, a dip-coating procedure. For amperometric measurements Of L-glutamate, these redox hydrogel-based bienzyme microelectrodes can be operated at low working potentials (-50 mV vs. Ag/AgCl) decreasing the influence of electroactive interferants possibly present in biological samples. The L-glutamate microsensors are characterized by a good operation stability and sensitivity (0.038+/-0.005 mAM(-1)), a low detection limit (0.5 muM in a conventional amperometric set-up and 0.03 muM in a Faraday cage, defined as three times the signal-to-noise ratio), a linear range up to 50 muM and a response time of about 35 s. The glutamate biosensors have been applied for the direct measurement Of L-glutamate release (upon chemical stimulation) from a population of immortalized hippocampal neurons (HN10 cells) demonstrating the possibility to amperometrically monitor in-Situ L-glutamate secretion from these cells.