L-Glutamate Biosensor for In Vitro Investigations: Application in Brain Extracts

Abstract

Investigations of L-glutamate release in living organisms can help to identify novel L-glutamate-related pathophysiological pathways, since abnormal transmission of L-glutamate can cause many neurological diseases. For the first time, a nitrogen-modified graphene oxide (GO) sample (RGO) is prepared through a simple and facile one-pot hydrothermal reduction of GO in the presence of 20 wt.% of the dye malachite green and is used for amperometric biosensing. The biosensor demonstrates adequate stability and is easy to prepare and calibrate. The biosensor detects the current generated during the electrooxidation of hydrogen peroxide released in the L-glutamate that is converted to the alpha-ketoglutarate catalyzed by L-glutamate oxidase. The biosensor consists of a semipermeable membrane, with L-glutamate oxidase (EC 1.4.3.11) immobilized in albumin and RGO and the working Pt electrode. First, the basic version of the L-glutamate biosensor is examined in PBS to investigate its sensitivity, reliability, and stability. To demonstrate the applicability of the L-glutamate biosensor in the analysis of complex real samples, quantification of L-glutamate in bovine brain extract is performed and the accuracy of the biosensor is confirmed by alternative methods. The enhanced version of the L-glutamate biosensor is applied for L-glutamate release investigations in a newly developed strain of rats (DAT-knockout, DAT-KO).