Microfabrication and Characterization of Microwire Microbiosensors for Simultaneous Detection of Glutamate and GABA

Abstract

Glutamate (Glu) and γ-Aminobutyric acid (GABA) are the two most prominent neurotransmitters in mammalian brains. GABA, a major inhibitory neurotransmitter in the cerebral cortex, maintains the inhibitory tone of inhibition of neuronal excitation. Glu is a major excitatory neurotransmitter and plays an important role in neuronal excitation. High levels of Glu and GABA cause calcium overload and dysfunction of mitochondria and oxidative stress, which results in a variety of neuronal diseases such as Alzheimer's disease, stroke and epilepsy. The current detection methods (e.g. HPLC followed by electrochemical or spectroscopy detection) is bulky, time consuming and not suitable for real time continuous monitoring. In contrast, microbiosensors are easy to miniaturize and are well suited for in vivo studies. They selectively oxidize Glu and GABA into a secondary electroactive product (usually hydrogen peroxide, H2O2) in the presence of enzymes, which is then detected by amperometry. Unfortunately, this method is cumbersome because it uses a pre-reactor and relies on externally applied reagents. Here we report the design and implementation of a platinum (Pt) microwire-enabled GLU-GABA probe that consists of four enzyme-modified microwires. The microwires are uniquely modified with the enzymes and by simultaneously measuring and subtracting the H2O2 oxidation currents generated from these microbiosensors, GABA and Glu can be detected continuously in real-time without the addition of any externally applied reagents. We report the sensor metrics such as sensitivity, selectivity and long-term stability of the probe in in vitro and ex vivo (rat brain slice) microenvironments.