Prabhu U. Arumugam

A Novel Electrochemical Microbiosensor Microarray for Real-Time Continuous In Vitro Monitoring of Gamma-Aminobutyric Acid

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.

Surface Fouling of Ultrananocrystalline Diamond Microelectrodes During Dopamine Detection: Improving Lifetime Via Electrochemical Cycling

In this work, we report the electrochemical response of a boron-doped ultrananocrystalline diamond (BDUNCD) microelectrode during long-term dopamine (DA) detection. Specifically, changes to its electrochemical activity and electroactive area due to DA byproducts and surface oxidation are studied via scanning electron microscopy, energy dispersive spectroscopy, electrochemical impedance spectroscopy, and silver deposition imaging (SDI). The fouling studies with amperometry (AM) and fast scan cyclic voltammetry (FSCV) methods suggest that the microelectrodes are heavily fouled due to poor DA-dopamine- o-quinone cyclization rates followed by a combination of polymer formation and major changes in their surface chemistry. SDI data confirms the presence of the insulating polymer with sparsely distributed tiny electroactive regions. This resulted in severely distorted DA signals and a 90% loss in signal starting as early as 3 h for AM and a 56% loss at 6.5 h for FSCV. This underscores the need for cleaning of the fouled microelectrodes if they have to be used long-term. Out of the three in vivo suitable electrochemical cycling cleaning waveforms investigated, the standard waveform (-0.4 V to +1.0 V) provides the best cleaned surface with a fully retained voltammogram shape, no hysteresis, no DA signal loss (a 90 ± 0.72 nA increase), and the smallest charge transfer resistance value of 0.4 ± 0.02 MΩ even after 6.5 h of monitoring. Most importantly, this is the same waveform that is widely used for in vivo detection with carbon fiber microelectrodes. Future work to test these microelectrodes for more than 24 h of DA detection is anticipated.

Nanocrystalline Diamond Electrodes: Enabling electrochemical microsensing applications with high reliability and stability

The Diamond (D) is one of the most precious materials in the world with unmatched physical and chemical properties, such as hardness, extreme chemical stability, high thermal conductivity, the highest acoustic velocity of any material, an extremely low friction coefficient when smooth, and nearly unmatched biocompatibility. The carbon (C) atoms in Ds are tetrahedrally coordinated, i.e., each C atom is bonded to four others in the D lattice. This bonding is referred to as sp3 bonding, and the strength and configuration of these bonds provide Ds with these unmatched fundamental properties and characteristics. Realizing these properties of the D in a C-based film that can easily be integrated into functional engineering systems and deployed in many applications has been a challenge for several decades. This is of primary concern in microelectronics, sensing, and hard-coating applications.

Electrochemical Assessment of Carbon Nanomaterial-Enabled Microelectrodes for Dopamine Sensing

Chronic neurochemical monitoring is critical for identifying the neural basis of human behavior and treating brain disorders. Studies have already shown that any abnormal neurochemical signaling cause brain disorders such as epilepsy, Parkinsons disease, traumatic brain injury and drug addiction. To treat such disorders, it is important to understand neurochemical dynamics over long-term, preferably in all areas of the brain. Currently, the preferred detection method is fast-scan cyclic voltammetry (FSCV) and the preferred electrode material is carbon fiber microelectrode (CFM). Unfortunately, CFMs increased sensitivity (sub-micromolar levels) is at the expense of increased surface fouling and chemical etching, which limits electrode lifetime to few days. Emerging carbon nanomaterials have spurred renewed interest in investigating new electrode material technology. We report the use of boron-doped ultrananocrystalline diamond (UNCD) and carbon nanotubes (CNTs) as advanced electrode materials for reliably detecting dopamine, a model neurochemical that plays a crucial role in various brain disorders. We present the electrochemical behavior and performance of these emerging materials in detecting dopamine long-term in standard buffer solutions and in biological fluids. Custom microfluidics was developed to study the electrode fouling behavior and the subsequent effect of in situ cleaning methods developed in our laboratory. Finally, development of electrochemical models to explain the progression of surface fouling using impedance techniques will be presented.