Gamma-aminobutyric acid (GABA) is a major inhibitory neurotransmitter that is essential for normal brain function. It is involved in multiple neuronal activities, including plasticity, information processing, and network synchronization. Abnormal GABA levels result in severe brain disorders and therefore GABA has been the target of a wide range of drug therapeutics. GABA being non-electroactive is challenging to detect in real-time. To date, GABA is detected mainly via microdialysis with a high-performance liquid chromatography (HPLC) system that employs electrochemical (EC) and spectroscopic methodology. However, these systems are bulky and unsuitable for real-time continuous monitoring. As opposed to microdialysis, biosensors are easy to miniaturize and are highly suitable for in vivo studies; they selectively oxidize GABA into a secondary electroactive product (usually hydrogen peroxide, H₂O₂) in the presence of enzymes, which is then detected by amperometry. Unfortunately, this method requires a rather cumbersome process with prereactors and relies on externally applied reagents. Here, we report the design and implementation of a GABA microarray probe that operates on a newly conceived principle. It consists of two microbiosensors, one for glutamate (Glu) and one for GABA detection, modified with glutamate oxidase and GABASE enzymes, respectively. By simultaneously measuring and subtracting the H₂O₂ oxidation currents generated from these microbiosensors, GABA and Glu can be detected continuously in real-time in vitro and ex vivo and without the addition of any externally applied reagents. The detection of GABA by this probe is based upon the in-situ generation of α-ketoglutarate from the Glu oxidation that takes place at the Glu microbiosensor. A GABA sensitivity of 36 ± 2.5 pA μM^-1cm^-2, which is 26-fold higher than reported in the literature, and a limit of detection of 2 ± 0.12 μM were achieved in an in vitro setting. The GABA probe was successfully tested in an adult rat brain slice preparation. These results demonstrate that the developed GABA probe constitutes a novel and powerful neuroscientific tool that could be employed in the future for in vivo longitudinal studies of the combined role of GABA and Glu (a major excitatory neurotransmitter) signaling in brain disorders, such as epilepsy and traumatic brain injury, as well as in preclinical trials of potential therapeutic agents for the treatment of these disorders.

Glutamate (GLU) and γ-aminobutyric acid (GABA) are the major excitatory (E) and inhibitory (I) neurotransmitters in the brain, respectively. Dysregulation of the E/I ratio is associated with numerous neurological disorders. Enzyme-based microelectrode array biosensors present the potential for improved biocompatibility, localized sample volumes, and much faster sampling rates over existing measurement methods. However, enzymes degrade over time. To overcome the time limitation of permanently implanted microbiosensors, we created a microwire-based biosensor that can be periodically inserted into a permanently implanted cannula. Biosensor coatings were based on our previously developed GLU and reagent-free GABA shank-type biosensor. In addition, the microwire biosensors were in the same geometric plane for the improved acquisition of signals in planar tissue including rodent brain slices, cultured cells, and brain regions with laminar structure. We measured real-time dynamics of GLU and GABA in rat hippocampal slices and observed a significant, nonlinear shift in the E/I ratio from excitatory to inhibitory dominance as electrical stimulation frequency increased from 10 to 140 Hz, suggesting that GABA release is a component of a homeostatic mechanism in the hippocampus to prevent excitotoxic damage. Additionally, we recorded from a freely moving rat over fourteen weeks, inserting fresh biosensors each time, thus demonstrating that the microwire biosensor overcomes the time limitation of permanently implanted biosensors and that the biosensors detect relevant changes in GLU and GABA levels that are consistent with various behaviors.