OPTIMIZATION OF AMPEROMETRIC BIOSENSOR FOR EVALUATION OF RATE OF GLUTAMATE UPTAKE BY ISOLATED BRAIN NERVE TERMINALS

Abstract

An amperometric glutamate biosensor was developed and adapted for analysis of velocity of glutamate uptake by isolated nerve terminals of rat’s brain. Glutamate oxidase was used for creation of biomembrane of the biosensor; the enzyme was co-immobilized with bovine serum albumin via glutaraldehyde on the surface of platinum disc electrode. Additional semi-permeable membrane based on phenylenediamine was placed onto the surface of the electrode for improvement of selectivity of the biosensor. Conditions for the enzyme immobilization onto the surface of working electrode were optimized (dependence of the biosensor work on concentrations of the enzyme and glutaraldehyde, as well as immobilization time were studied). The influence of parameters of working buffer (ionic strength, buffer capacity, pH) on the biosensor work was investigated. Linear range of the biosensor was 2-600 µM of glutamate, limit of glutamate detection was 0.5-2 µM, sensitivity was 250-300 nA/ mM. The biosensor was characterized by good reproducibility of responses and operational stability. The presence of endogenous glutamate in preparations of isolated nerve terminals of brain was shown by using the biosensor. It was demonstrated that the initial rate of Na-dependent glutamate uptake and accumulation by the nerve terminals measured by the biosensor were not significantly different from the results obtained by using radioactive-labeled L-[14C] glutamate. The described work will allow wide usage of the developed biosensor in medicine, as well as in biotechnological and biochemical studies.