Redox Hydrogel-Based Bienzyme Microelectrodes for Amperometric Monitoring ofL-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 mA M−1), a low detection limit (0.5 μM in a conventional amperometric set-up and 0.03 μM in a Faraday cage, defined as three times the signal-to-noise ratio), a linear range up to 50 μM 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.