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.
Highlights
The development of multiplexed neural probes for real-time sensing of neurochemicals is a critical step in the study and effective treatment of brain disorders
We report a novel Gammaaminobutyric acid (GABA) microarray probe that can detect GABA without the addition of any external reagents such as α-ketoglutarate and nicotinamide adenine dinucleotide phosphate (NADPH) in vitro
The GABA probe consists of two microbiosensors that were modified with glutamate oxidase (GOx) and GOx+GABASE enzymes
Summary
The development of multiplexed neural probes for real-time sensing of neurochemicals is a critical step in the study and effective treatment of brain disorders. Biosensors selectively oxidize GABA into a secondary electroactive product or reporter molecule in the presence of enzymes, similar to the detection method used for Glu (Hascup et al, 2007) or acetylcholine (Garguilo and Michael, 1994) Electroactive reporter molecules such as β-nicotinamide adenine dinucleotide phosphate (NADPH) or hydrogen peroxide (H2O2) are usually generated through a series of enzymatic reactions by adding nicotinamide adenine dinucleotide phosphate (NADP) as a co-factor, or α-ketoglutarate reagents externally, and electrochemically detecting them on a modified GCE. Able to detect GABA with adequate sensitivity and selectivity in the presence of DA, HT-5 and ascorbic acid (AA) Both approaches are incapable of continuously monitoring the changes in GABA levels in real-time since they require additions of reagents such as NADP and α-ketoglutarate. The other salient features of the GABA probe are: (1) eight individually electrically addressable Pt microelectrodes that can be multiplexed to simultaneously measure other important neurochemicals, such as Glu, DA, adenosine and HT-5, through suitable surface modifications, which is not possible with other commonly available electrodes for chemical sensing, e.g., carbon fiber microelectrodes; (2) GABA and Glu microbiosensors can be placed in close proximity to provide precise measurements of local GABA level changes; (3) an ability to detect GABA realtime without adding reagents (i.e., truly self-contained); (4) the location of MEAs along the long shank allows GABA sensing at multiple depths in the brain; and (5) allows simultaneous sensing of neurochemicals and field potentials for multimodal recordings, which is not possible with the current neurochemical technologies
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