MEASURING IMPEDANCE OF TISSUES USING A MICROFABRICATED MICROELECTRODE ARRAY By Ashwini Bhat This thesis looks at the possibility of using impedance spectroscopy for differentiating tissue, using a microelectrode array (MEA). The thesis first discusses the background and the motivation for this thesis. It covers the certain basic concepts of the human skin starting from the top epidermis layer all the way to the deep dermis layers of the skin. Then it discusses different types of skin cancer and how they occur, in humans. It also discusses various microfabrication techniques such as oxidation, wet etching, sputtering and photolithography for the creation of a MEA in order to test the tissue. The microfabricated MEA is then used to measure impedance across cooked and raw chicken at different frequencies in order to see if the two types of tissues can be differentiated using their respective impedances. The data shows that the MEA was not able to successfully differentiate the two types of the tissues. It does however list multiple improvements in the fabrication of the MEA and improvements that could be made to the testing procedures which could possible give greater difference in impedance between the two tissues

In translational research, disease models in preclinical studies are used as media for discovery of drugs or novel therapeutics. Development of in vitro models for various neurological diseases that enable efficient pharmacological or toxicological screening has been ongoing but challenging. Recognizing the potential benefit of in vitro disease models, dysfunctions in the cortical neuronal networks were induced to mimic the functional pathology of neurological symptoms using microelectrode arrays. Two different disease states – tinnitusand excitotoxicity – were investigated and discussed. In this model, pentylenetetrazol-induced increase in spontaneous firing rate and synchrony in the auditory cortical networks was used as correlate of tinnitus. Potential tinnitus treatment drugs from several different classes – including the novel class of potassium channel openers – were screened and quantified. The potentialtherapeutic values of these drugs were also discussed as the basis for drug repurposing. Functional excitotoxicity was induced by cisplatin (a cancer drug that causes neurological sideeffects) and glutamate (the major excitatory neurotransmitter). As proof-of-principle that the model may contribute to expediting the development of therapeutics, cisplatin excitotoxicity wasprevented by the antioxidant D-methionine, while glutamate excitotoxicity was prevented by ceftriaxone (a modulator of a glutamate reuptake transporter). In the latter part of the study, with results linking two of the screened drugs L-carnitine and D-methionine to GABAA receptor activation, it was demonstrated that this model not only served as an efficient drug-screening platform, but can be utilized to functionally investigate the underlying mechanism of drugs. Inaddition, several practical or conceptual directions for future studies to improve on this in vitro disease model are suggested.

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Mitochondria provide the main energy required for cardiac excitation-contraction coupling via aerobic oxidative phosphorylation (OXPHOS) process. Accumulation of reactive oxygen species (ROS), by-products of mitochondrial respiration, within dysfunctional mitochondria results in the activation of cardiac cell death pathways and has been associated with heart failure development. Therefore, maintaining mitochondrial homeostasis as a balance between mitochondrial biogenesis and degradation is of great importance toward cardiac proper functioning. In addition to the importance of mitochondrial energy supply, gap junctions, intercellular channels which connect plasma membrane of adjacent cardiomyocytes, by propagating action potential throughout the myocardium maintain cardiac synchronous beating and rhythm. Gap junctions have a rapid turnover and impair of gap junction quality control impacts cell-to-cell communication; resulting in electrical conduction abnormalities and arrhythmogenesis. Therefore, understanding the underlying mechanism the quality control of mitochondria and gap junctions profoundly contributes toward understating the genesis of cardiomyopathy. Furthermore, cardiovascular problems in HIV (Human immunodeficiency virus) positive patients whose viral load is controlled via antiretroviral therapy remains a problem while the underlying mechanism remains elusive. The current study has used an in vitro model of primary neonatal rat ventricular cardiomyocytes (NRVCs) to discover the molecular mechanisms of mitochondrial as well as gap junction quality control under normal and stress conditions. Furthermore, electrical activities of the primary cardiomyocytes were recorded using microelectrode array (MEA) system and important electrophysiological components such as impulse propagation pattern and conduction velocity were extracted from the complex signal recordings. Overall, we have pursued four main aims; Aim 1. Dysregulation of mitochondrial quality control machinery leads to cardiac death; Aim 2. HIV-1 Tat (transcriptional transactivator) dysregulates cardiac homeostasis via mitochondrial pathway; Aim 3. Impairment of protein quality control impacts the quality of gap junction; Aim 4. Inhibition of gap junction quality dysregulates electrical signal propagation within the culture. / Bioengineering

The main objective of the research in this dissertation is to take advantage of unique materials, innovative designs, novel microfabrication techniques, and specialized characterization tools to develop a set of BioMEMS devices and systems further validated with electrical, interface, geometric, and multiphysics models to address unique biological problems emanating from ethical treatment of animals in drug discovery, biological translation, decentralization and personalization of healthcare. This set of devices is designed to interface with multi-sized biological constructs such as 3D cellular networks, viruses, and proteins. The first objective explored a 3D printing-based microfabrication technology to create 2.5D/3D microelectrodes to interface with cellular constructs such as tissues and organoids. Investigations were carried out on how surface roughness and printing parameters play a critical role in the electrical response of the system for in-vitro applications. Three different metallization strategies were investigated and modeled in order to define novel self-insulated 2.5 and 3D microelectrodes. The second objective centered around virus and microparticle detection using a novel combination of microfluidics and Wi-Fi optical detection. Microfluidics were created designing a multilayered system and processing various polymeric materials. The optical system was able to detect and wirelessly transmit information about the presence of viruses including COVID-19 Delta strain and microparticles in the 5 to 10 microns size. The last objective of the dissertation presented the microfabrication of a BioMEMS platform for electrophysiological characterization of Actin protein (smallest entity within the size spectrum). This platform combined interdigitated electrodes, PDMS soft lithography, and impedance and interface modeling to better understand Actin protein dynamics in bundles. This dissertation proposes innovative ideas to the current state of the art for emerging paradigms in the medical technology field involving rapid sensing and manipulating biological entities at various size scales: (proteins, DNA/RNA), (pathogens, virus), and (organoids, spheroids, assembloids).

Thanks to pioneering scientists and clinicians, prosthetic devices that are controlled by intra-cortical electrodes recording one's 'thoughts' are a reality today, and no longer merely in the realm of science fiction. However, widespread clinical use of implanted electrodes is hampered by a lack of reliability in chronic recordings, independent of the type of electrodes used. The dominant hypothesis has been that astroglial scar electrically impedes the electrodes. However, recent studies suggest that the impedance changes associated with the astroglial scar are not high enough to interfere significantly impair neural recordings. Furthermore, there is a time delay between when scar electrically stabilizes and when neural recordings fail (typically >1 month lag), suggesting that scar, per se, does not cause chronic recording unreliability. In this study, an alternative hypothesis was tested in a rat model, namely, that chronic inflammation surrounding microelectrodes causes a local neurodegenerative state. Chronic inflammation was varied in three ways: 1) stab wound control, 2) age-matched control, and 3) inter-shank spacing of a multishank electrode. The results of this study suggest that chronic inflammation, as indicated by activated microglia and reactive astrocytes, is correlated with local neurodegeneration, marked by neuron cell death and dendritic loss. Surprisingly, axonal pathology in the form of hyperphosphorylation of the protein Tau (the hallmark of many tauopathies, including Alzheimer's Disease) was also observed in the immediate vicinity of microelectrodes implanted for 16 weeks. Additionally, work is presented on a fast, non-invasive method to monitor the astrocytic response to intra-cortical electrodes using electrical impedance spectroscopy. This work provides a non-invasive monitoring tool for inflammation, albeit an indirect one, and fills a gap which has slowed the development of strategies to control the inflammatory tissue response surrounding microelectrodes and thereby improve the reliability of chronic neural recordings. The results of these experiments have significance for the field of neuroengineering, because a more accurate understanding of why recordings fail is integral to engineering reliable solutions for integrating brain tissue with microelectrode arrays.

This dissertation presents an investigation of micromachined three-dimensional microelectrode arrays (3-D MEAs) targeted toward in-vitro and in-vivo biomedical applications. Current 3-D MEAs are predominantly silicon-based, fabricated in a planar fashion, and are assembled to achieve a true 3-D form: a technique that cannot be extended to micro-manufacturing. The integrated 3-D MEAs developed in this work are polymer-based and thus offer potential for large-scale, high volume manufacturing. Two different techniques are developed for microfabrication of these MEAs - laser micromachining of a conformally deposited polymer on a non-planar surface to create 3-D molds for metal electrodeposition; and metal transfer micromolding, where functional metal layers are transferred from one polymer to another during the process of micromolding thus eliminating the need for complex and non-repeatable 3-D lithography processes. In-vitro and in-vivo 3-D MEAs are microfabricated using these techniques and are packaged utilizing Printed Circuit Boards (PCB) or other low-cost manufacturing techniques. To demonstrate in-vitro applications, growth of 3-D co-cultures of neurons/astrocytes and tissue-slice electrophysiology with brain tissue of rat pups were implemented. To demonstrate in-vivo application, measurements of nerve conduction were implemented. Microelectrode impedance models, noise models and various process models were evaluated. The results confirmed biocompatibility of the polymers involved, acceptable impedance range and noise of the microelectrodes, and potential to improve upon an archaic clinical diagnostic application utilizing these 3-D MEAs.