Glutamate (Glu) is the most abundant excitatory neurotransmitter in the human central nervous system (CNS) which plays a major role in metabolic signalling pathways involved in learning and memory. Whereas excess Glu concentrations in synapses can excite neurons to the point of cell death in a process referred to as excitotoxicity. Powerful uptake systems (glutamate transporters) prevent excessive activation of Glu receptors present on the surface of brain cells by continuously removing Glu from the extracellular brain fluid. Neurodegenerative diseases known to be associated with expressing excess glutamate exciting nerve cells include Parkinson's disease, Alzheimer's disease and Huntington's disease [1]. Whereas insufficient brain glutamate concentration is thought to result in trouble concentrating, mental exhaustion, insomnia, and low energy. Therefor it is essential our CNS maintains moderate extracellular glutamate concentrations. Extracellular glutamate concentration estimates conducted in vitro from tissue slices vary around 0.02–0.1 μM [2].
An option to facilitate real-time, continuous monitoring of analytes such as glutamate is by use of enzymatic electrochemical sensors. Enzymatic sensors provide potential for obtaining high measurement sensitivity. However, they are often expensive and decrease in stability over time, resulting in a signal reduction causing inaccuracies in measurement. Biosensors are the biological manipulation of electrochemical sensors, where a biological analyte is detected on a bioreceptor. In order to obtain plausible sensitivity and prove biosensor reliability it is essential to assess its technical suitability by accurately optimizing its detection conditions, such as electrolyte solution parameters and provide reassurance that the measured analyte is undeniably glutamate.
Therefor the approach to detect extracellular glutamate levels was using first generation biosensors to utilize the reaction enzyme - glutamate oxidase (GluOx). When the analyte (glutamate) is detected a reduction–oxidation (redox) reaction takes place, indirectly producing an electron through the production of hydrogen peroxide [3]. The following reactions occur, Eq (1):
$$Glutamate + H_2O + O_2 \xrightarrow{GluOx} H_2O_2 + \alpha - ketoglutarate + NH_3 \tag{1}$$$$H_2O_2 \rightarrow H_2 + O_2 + 2e^-$$The used three-electrode potentiometric sensor consists of a platinum (Pt) working electrode on which the redox reaction occurs; a silver chloride (AgCl) reference electrode as an electrode which has a stable and well-known electrode potential (acts as a dynamic baseline to compare the measured potentials of the other electrodes); a titanium (Ti) counter electrode which provides a means of applying input potential to the working electrode. The electrodes were submerged in different concentration acid/base/salt electrolyte solutions to optimize which buffer pH would ensure electrical neutrality and allow ions to migrate with minimal resistance. Electrodes were connected to a data processor to relay the results
As a result, we present a first generation GluOx Pt electrode biosensor with an adjusted membrane which specifically measures glutamate levels in mouse brain tissue. Biosensor results assure reliability by providing high sensitivity measurements and a low limit of detection (LOD) for hydrogen peroxide (up to 0.05 μM) while submerging electrodes in a 10 mM, pH 5.5 electrolyte solution.
Considering Glu is linked to various neurodegenerative diseases and mental health disorders its significance as a biological marker and fast methods of sensing [Glu] in biofluids remains a perspective and important matter to elaborate in future research.