Indirect Voltammetry Detection of Non-Electroactive Neurotransmitters Using Glassy Carbon Microelectrodes: The Case of Glutamate

Glassy carbon (GC) microelectrodes have been successfully used for the detection of electroactive neurotransmitters such as dopamine and serotonin through voltammetry. However, non-electroactive neurotransmitters such as glutamate, lactate, and gamma-aminobutyric acid (GABA) are inherently unsuitable for detection through voltammetry techniques without functionalizing the surface of the microelectrodes. To this end, we present here the immobilization of the L-glutamate oxidase (GluOx) enzyme on the surface of GC microelectrodes to enable the catalysis of a chemical reaction between L-glutamate, oxygen, and water to produce H2O2, an electroactive byproduct that is readily detectable through voltammetry. This immobilization of GluOx on the surface of bare GC microelectrodes and the subsequent catalytic reduction in H2O2 through fast-scan cyclic voltammetry (FSCV) helped demonstrate the indirect in vitro detection of glutamate, a non-electroactive molecule, at concentrations as low as 10 nM. The functionalized microelectrodes formed part of a four-channel array of microelectrodes (30 mu m x 60 mu m) on a 1.6 cm long neural probe that was supported on a flexible polymer, with potential for in vivo applications. The types and strengths of the bond between the GC microelectrode surface and its functional groups, on one hand, and glutamate and the immobilized functionalization matrix, on the other hand, were investigated through molecular dynamic (MD) modeling and Fourier transform infrared spectroscopy (FTIR). Both MD modeling and FTIR demonstrated the presence of several covalent bonds in the form of C-O (carbon-oxygen polar covalent bond), C=O (carbonyl), C-H (alkenyl), N-H (hydrogen bond), C-N (carbon-nitrogen single bond), and C equivalent to N (triple carbon-nitrogen bond). Further, penetration tests on an agarose hydrogel model confirmed that the probes are mechanically robust, with their penetrating forces being much lower than the fracture force of the probe material.