A Novel Microbiosensor Microarray for Continuous ex Vivo Monitoring of Gamma-Aminobutyric Acid in Real-Time

Abstract

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.

Approaches to Monitor Circuit Disruption after Traumatic Brain Injury: Frontiers in Preclinical Research

“…However, changes in GABA neurotransmission acutely and chronically offset the balance of excitation and inhibition that underlies several neurological diseases. Recently, electrochemical methods to record near real-time neurotransmission of GABA have been developed and open a wealth of opportunity to understand the role of GABA neurotransmission in the contribution to PCSs [73,74].…”

Novel microwire-based biosensor probe for simultaneous real-time measurement of glutamate and GABA dynamics in vitro and in vivo

“…One of the most common methods for the acquisition of longitudinal measurements is microdialysis, but analyte measurement is limited to large cell populations. In addition, its low sampling rate, on the order of minutes, prevents its use in measuring faster, real-time transients 2,3,[31][32][33][34][35] , introducing a low-pass filtering effect into recorded data 36 . Precision, micromachined dialysis probes have increased the sampling rate to several seconds, but this resolution is still slow relative to the expected dynamics of GLU and GABA in the extracellular space.…”

“…Alternatively, enzyme-based biosensors based on microelectrode arrays (MEAs) may be able to improve spatial and temporal resolution, as well as biocompatibility 10,32 . For these types of biosensors, an analyte-specific enzyme is coated onto an electrode to selectively convert non-electrically-active molecules into substituent molecules, which can be oxidized or reduced further at the electrode to produce a measurable electric current that is proportional to the amount of analyte 32,34 .…”

Clinical applications of neurochemical and electrophysiological measurements for closed-loop neurostimulation

“…Recently, a dual microbiosensor for measurement of GABA and glutamate has been described, with a limit of detection of 2 ± 0.12 μM for GABA. 24 More research is required to further develop techniques with higher sensitivity for the detection of GABA at physiological concentrations. In addition, access to the cerebellum for GABA monitoring may prove challenging.…”