Advancing Microelectrode Technology and Voltammetric Methodology for Improved Molecular Monitoring in Live Tissue

SCHMIDT, ANDREAS CARLO. Advancing Microelectrode Technology and Voltammetric Methodology for Improved Molecular Monitoring in Live Tissue. (Under the direction of Dr. Leslie A. Sombers.) The development of real-time electrochemical sensing technologies has significantly impacted our understanding of the dynamic molecular mechanisms underlying basic brain function, as well as a variety of neuropathologies and disease states. However, carbon-fiber microelectrodes (CFME) developed almost 30 years ago are still in use, and electrochemical waveforms have changed very little since fast-scan cyclic voltammetry (FSCV) was first introduced as a promising methodology for monitoring neurotransmission. The research presented here seeks to broaden the chemical sensing capabilities of FSCV and carbon-based microelectrodes through the usage of advanced materials and the development of novel approaches to molecular monitoring using electrochemistry. Cyclic voltammetry typically involves scanning the applied potential at a constant rate using a triangular waveform. Although this works well for monitoring catecholamines, such as dopamine, it is limited in its ability to detect many other classes of molecules. For instance, the opioid peptide methionine-enkephalin (M-ENK) is important because it has been implicated in models of addiction and reward processing; however, it is difficult to monitor. To address this, a waveform was developed that utilizes multiple scan rates in a single voltammetric scan. This novel approach enables the selective detection of M-ENK by increasing sensitivity to the molecule of interest, while decreasing sensitivity to interfering molecules, such as dopamine. Additionally, this innovative waveform incorporates a brief hold at +1.2 V, minimizing electrode fouling and enabling more reproducible measurements. This approach was used to make the first measurements of rapid M-ENK dynamics in tissue. Opioid peptides and dopamine are both highly implicated in reward processing, but the interaction between M-ENK and DA is not well understood. To address this, the effects of MENK on catecholamine dynamics were studied. In brain tissues slices, bath application of MENK increases electrically-evoked dopamine release. In the adrenal gland, acute application of M-ENK evokes immediate catecholamine release, highlighting the critical need for subsecond temporal resolution to study these interactions. In order to detect endogenous M-ENK release in the brain, more sensitive microelectrodes are needed. Previous approaches to improving electrode sensitivity have focused on modifying the carbon-fiber surface itself. In this work, a newly developed electrode material is described. A carbon-based sensing surface fabricated from individual carbon nanotubes spun into yarns was incorporated into disk-shaped microelectrodes. This provides improved sensitivity, selectivity, and spatial resolution as compared to traditional CFME. Electrochemical approaches are also commonly used to study neurotransmitter release from individual cells in culture. In these experiments, a CFME is placed directly onto the cell surface to monitor exocytosis release events. In order to advance such studies, a costand timeeffective plasma etching methodology was developed to create a CFME with a sensing surface that is slightly recessed from the end of the insulation. This ‘cavity’ electrode geometry is beneficial because it hinders mass transport away from the electrode surface, resulting in reliable, reproducible, and sensitive measurements. A key analyte of interest in the brain is hydrogen peroxide (H2O2). Although this molecule is readily detected using FSCV, its electrochemical signature is indistinguishable from some pharmacological agents meant to increase H2O2 concentrations in the brain, convoluting data analysis. Therefore, a size-exclusion polymer, m-phenylenediamine, was electrodeposited onto CFMEs to form a membrane which successfully excluded the contributions of other molecules to the electrochemical signal, thus enabling more chemically selective measurements of H2O2 in tissue. Overall, the work presented herein lays the foundation for exciting studies of target analytes in neurochemical applications. These innovations were accomplished through improving the sensors themselves, and by rethinking how the applied potentials can be used to enhance electrochemical detection.