Poster V19 - Development of a Continuous, Real-Time Electrochemical L-Glutamate Sensor using Quasi-Direct Electron Transfer Type L-Glutamate oxidase

Introduction: L-Glutamate (L-Glu) is an excitatory neurotransmitter, responsible for memory and cognition. L-Glu is recognized as the important biomarker associated to the development of several diseases such as anxiety, depression, Parkinson’s disease, Alzheimer’s disease, and Huntington’s disease. Moreover, to understand brain in situ L-Glu monitoring is important to understand the neurotransmission under physiological and pathological conditions. Therefore, the development of a miniaturized, real-time, continuous sensor for L-Glu is expected. L-Glu oxidase (EOx) have been mainly used as the biorecognition molecule for the L-Glu detection, and several electrochemical sensors are reported using EOx. While most of EOx based electrochemical sensors are based on the first-generation principle, where the hydrogen peroxide produced by enzymatic reaction is measured amperometrically. To realize in vivo L-Glu electrochemical sensors employing amperometric principle, the challenge is to downsize electrochemical sensors, which is required for spatial resolution. Here, we propose an alternative principle to amperometric L-Glu sensor, the open circuit potential (OCP) based sensor employing engineered EOx, which is capable of direct electron transfer (DET) with electrode. OCP based sensor signal is dependent on the ratio of oxidized to reduced enzyme states or charged molecules on electrode surface, therefore the sensor signal is independent of the size of electrode.

Materials and

Methods: Mutant EOx, which was engineered to eliminate its reactivity toward oxygen while maintaining its dye-mediated activity was developed in this study by introducing mutations. Engineered EOx was produced recombinantly using Escherichia coli as the host organism and purified. The oxidase activity and the dye-mediated dehydrogenase activity of prepared enzyme were evaluated. Amine-reactive phenazine ethosulfate (arPES) was used to modify enzyme. Prepared engineered EOx was immobilized on the gold disk electrode via self-assembled monolayer, followed by the modification by the arPES. The chronoamperometry measurement and OCP measurement were performed to measure L-Glu.

Results, Conclusions, and Discussions: Engineered EOx was produced as the soluble protein, and purified enzyme showed dye-mediated dehydrogenase activity, while no oxidase activity was observed. Cyclic voltammetry measurement with PES modified engineered EOx immobilized electrode revealed the successful modification of PES on the surface of enzyme and its reactivity with the electrode. The L-Glu concentration dependent signal was successfully observed with OCP measurement from 1 mM to 40 mM L-Glu. This result indicated that the PES modified engineered EOx showed quasi-DET ability successfully. In this study, we have successfully developed an OCP based L-Glu sensor using EOx with quasi-DET ability. Further engineering of EOx to improve the quasi-DET ability via arPES will be expected to develop more sensitive electrochemical sensor suitable for the real-time continuous monitoring of L-Glu.

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