Glutaraldehyde Cross-Linked Glutamate Oxidase CoatedMicroelectrode Arrays: Selectivity and Resting Levels of Glutamatein the CNS

摘要

Glutaraldehyde is widely used as a cross-linking agent for enzyme immobilization onto microelectrodes. Recent studies and prior reports indicate changes in enzyme activity and selectivity with certain glutaraldehyde cross-linking procedures that may jeopardize the performance of microelectrode recordings and lead to falsely elevated responses in biological systems. In this study, the sensitivity of glutaraldehyde cross-linked glutamate oxidase-based microelectrode arrays to 22 amino acids was tested and compared to glutamate. As expected, responses to electroactive amino acids (Cys, Tyr, Trp) were detected at both nonenzyme-coated and enzyme-coated microelectrodes sites, while the remaining amino acids yielded no detectable responses. Electroactive amino acids were effectively blocked with a m-phenylene diamine (mPD) layer and, subsequently, no responses were detected. Preliminary results on the use of poly(ethylene glycol) diglycidyl ether (PEGDE) as a potentially more reliable cross-linking agent for the immobilization of glutamate oxidase onto ceramic-based microelectrode arrays are reported and show no significant advantagesover glutaraldehyde as we observe comparable selectivities and responses.These results support that glutaraldehyde-cross-linked glutamate oxidaseretains sufficient enzyme specificity for accurate in vivo brain measuresof tonic and phasic glutamate levels when immobilized using specific“wet” coating procedures.