Microelectrodes have been found to be a valuable tool in a variety of analytical studies. Their advantages over macro-sized electrodes are well known, including their enhanced mass transport properties (due to their ubiquitous hemispherical diffusion) which lead to steady state responses without external convection. They also exhibit high signal-to-noise ratios (greater sensitivities), furthering their analytical application. Microelectrode arrays are analytical devices with multiple electrodes. There are suitable for practical sensing with all the benefits of microelectrodes but with greater currents, leading to greater ease of measurement. To produce a reliable electroanalytical device the microelectrode response must be reproducible, a fundamental property based on the quality control of their production. Square microelectrode and array fabrication techniques have been developed for this purpose. This research discusses the fabrication and development of closely spaced arrays of square microelectrodes. Simulated and measured responses are compared and used to characterize electrode and array responses by cyclic voltammetry, electrical impedance spectroscopy and current-time transients. Measurements on variably spaced arrays allow insight into overlap of hemispherical diffusion from individual electrodes and the subsequent effect including peak current output on the array device. By studying these devices key insights into the mass transport properties of single square microelectrodes and microelectrode arrays were gained. This study also prepares and develops microelectrodes from materials appropriate for use in the extreme environments of molten salts and concentrated nitric acid solutions. These robust electrodes were developed for use in hydro- and pyro-chemical techniques for nuclear fuel reprocessing. These results demonstrate the practical uses for microelectrode systems across a wide range of chemical systems and in extreme conditions.

Neuronal networks, derived from mouse embryonic frontal cortex (FC) tissue grown on microelectrode arrays, were used to investigate effects of gentamicin pretreatment on pharmacological response to the L-type calcium channel blocker, verapamil. Gentamicin is a broad spectrum antibiotic used to control bacterial contamination in cell culture. The addition of gentamicin directly to medium affects the pharmacological and morphological properties of the cells in culture. A reproducible dose response curve to verapamil from untreated cultures was established and the mean EC50 was calculated to be 1.5 ± 0.5 μM (n=10). 40 μM bicuculline was added to some cell cultures to stabilize activity and verapamil dose response curves were performed in presence of bicuculline, EC50 1.4 ± 0.1 μM (n=9). Statistical analysis showed no significant difference in verapamil EC50s values obtained in presence of bicuculline and hence the data was combined and a standard verapamil EC50 was calculated as 1.4 ± 0.13 μM (n=19). This EC50 was then used to compare verapamil EC50s obtained from neuronal cell cultures with chronic and acute exposures to gentamicin. FC cultures (21- 38 days old) were found to be stable in presence of 2300 μM gentamicin. The recommended concentration of gentamicin for contamination control is 5uL /1 ml medium (108 μM). At this concentration, the verapamil EC50 shifted from 1.4 ± 0.13 μM to 0.9 ± 0.2 μM. Given the limited data points and only two complete CRCs, statistical comparison was not feasible. However, there is a definite trend that shows sensitization of cells to verapamil in presence of gentamicin. The cultures exposed to 108 μM gentamicin for 5 days after seeding showed loss of adhesion and no data could be collected for pharmacological analysis. To conclude, acute gentamicin exposure of neuronal cell cultures causes increased sensitivity to verapamil and chronic or long term exposure to gentamicin may cause loss of adhesion of the cell culture by affecting the glial growth. The effect of chronic exposure to gentamicin on pharmacological responses to verapamil remains inconclusive.

Epilepsy is a disease affecting millions of people worldwide. Despite over 50 years of research, the mechanisms that generate and sustain ictal discharges, a key neural hallmark of seizures, remain unknown. While once thought to be caused by hypersynchronous neuronal firing, we now recognize that the activity underlying ictal discharges is much more complex. With the development of microelectrode arrays (MEAs) suitable for use in humans, it is possible to observe neural activity at fine spatiotemporal scales in human patients with epilepsy. However, the diversity of seizure characteristics and limited patient population has led to a number of conflicting observations and theories. The purpose of this work is to elucidate mechanisms underlying ictal discharges in humans by applying statistical analyses and computational modeling to MEA recordings from human patients with epilepsy. We approach this aim in two projects. In the first project, we unify two seemingly conflicting theories surrounding cortical sources of ictal discharges. According to the ictal wavefront theory, ictal discharges are seeded at an expanding narrow front of high neuronal firing that delineates the boundary between regions of cortex with compromised functionality, and surrounding territory where the seizure is observable in electrical recordings, but cortical function remains intact. A second theory posits that discharges are predominantly seeded from a stationary localized cortical source. The two theories are based on observations from MEA recordings of seizures in two different small cohorts of patients. In this project, we analyze and model the discharge propagation patterns in a combined dataset from both cohorts. We show that discharges are seeded at the ictal wavefront in addition to other–possibly stationary–locations. In the second project, we characterize spatiotemporal patterns in the secondary transients of complex ictal discharges. Electrographic recordings of ictal discharges often have complex waveforms. Existing analyses focus on the spatiotemporal dynamics of the first, high-amplitude transient. In this project, we establish that ictal discharges often comprise multiple transients separated by ≈60 ms. Surprisingly, and contrary to our initial hypothesis, we find that individual transients within a complex discharge may propagate with different speeds, suggesting that different mechanisms are involved in the propagation of different transients.

The advancement of technologies that interface with electrically excitable tissues, such as the cortex and muscle, has the potential to lend greater mobility to the disabled, and facilitate the study of the central and peripheral nervous systems. Myoelectric interfaces are currently limited in their signal fidelity, spatial resolution, and interfacial area. Such interfaces are either implanted in muscle or applied to the surface of the muscle or skin. Thus far, the former technology has been limited in its applications due to the stiffness (several orders of magnitude greater than muscle) of its substrates, such as silicon and polyimide, whereas the latter technology suffers from poor spatial resolution and signal quality due to the physical separation between the electrodes and the signal source. We have developed a stretchable microneedle electrode array (sMEA) that can function while stretching and flexing with muscle tissue, thereby enabling multi-site muscle stimulation and electromyography (EMG) measurement across a large interfacial area. The scope of this research encompassed: (i) the development of a stretchable and flexible array of penetrating electrodes for the purposes of stimulating and measuring the electrical activity of excitable tissue, (ii) the characterization of the electrical, mechanical, and biocompatibility properties of this electrode array, (iii) the measurement of regional electrical activity of muscle via the electrode array, (iv) the study of the effect of spatially distributed stimulation of muscle on the fatigue and ripple of muscle contractions, and (v) the assessment of the extent to which the stretch response of electrically stimulated muscle behaves in a physiological manner.

Embryonic stem cells (ESCs) are being considered as a cell source for cardiac regeneration because of their potency and availability. We studied the electrophysiological implications using co-cultures of ESCs and neonatal rat ventricular myocytes (NRVM) grown on a multi-electrode array (MEA). To mimic expected engraftment rates 5% mouse ESCs were co-cultured with NRVMs. Comparing cultures without and with 5% ESCs at 4 days, the mean bipolar field potential duration (FPD) of NRVMs increased from 26.3 ± 2.2 ms (n=10) to 44.3 ± 6.2 ms (n=9; p < 0.05), the interspike interval (ISI) increased from 358.3 ± 62.8 ms (n=10) to 947.8 ± 214.6 ms (n=7; p < 0.01), and conduction velocity (CV) decreased from 14.2 ± 1.3 cm/s (n=8) to 4.6 ± 1.2 cm/s (n=5; p < 0.01). To evaluate whether ESC were having direct or paracrine effects on NRVMs, media conditioned by 3x106 ESCs for 24 hr was diluted 1:1 with fresh media and then introduced to NRVM cultures on the day of plating. Conditioned media was changed daily and altered mean FPD, ISI, and CV to 46.1 ± 7.8 ms, ISI to 682.0 ± 128.5 ms, and 4.2 ± 0.4 cm/s (n=8; p < 0.01 for each measure), respectively at 4 days. However, changes were not seen in media that was incubated for 24hrs and diluted 1:1 with fresh media and introduced to NRVM cultures in a similar fashion (n=7; p > 0.05). Slowed CV is associated with increased arrhythmic risk and reports demonstrate an inverse relationship between CV and nonphosphorylated Cx43(NP-Cx43). Western blots for total Cx43 expression revealed a decrease in ratio of P-Cx43/NP-Cx43 in the 5% mouse ESCs and ESC conditioned media cultures as compared to controls (n=8; p < 0.01 for each). There was not significant increase in the total Cx43 expression (n=6; p > 0.05). Culturing ESCs with NRVMs resulted in a decreased ISI, prolonged FPD, and slowed CV of the co-cultures as compared to controls leading to pro-arrhythmic conditions. Similar effects on NRVMs were observed when applying media conditioned by ESCs, suggesting that the electrophysiological changes were mediated by soluble factors. The increase in NP-Cx43 leads to gap junction uncoupling being a potential mechanism for these arrhythmogenic substrates. Further research into preventing NP-Cx43 in cultures is currently underway.

No description available.

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).

Les cellules β des îlots de Langerhans jouent un rôle central dans l’homéostasie glucidique car elles seules sécrètent l’insuline, unique hormone hypoglycémiante de l’organisme. La cellule β est un détecteur du glucose qui couple sa réponse sécrétoire et son expression génique aux niveaux ambiants de glucose. Le couplage entre le métabolisme du glucose et l’exocytose des granules d’insuline implique la génération d’une activité électrique. Son étude est importante pour déchiffrer la façon dont la cellule β encode la demande en insuline de l’organisme. Afin de contourner les limites des approches électrophysiologiques classiques incompatibles avec les études à long-terme, les enregistrements extracellulaires par matrice de microélectrodes (MEA) ont été mis en place.L’objectif de ma thèse était de mieux comprendre les signaux complexes enregistrés par MEAs. Cette étude a révélé l’existence d’une nouvelle signature électrique des cellules des îlots, les slow potentials (SP), qui reflète la fonction de couplage des cellules β. Les SP jouent un rôle important dans l’homéostasie du glucose et représentent un biomarqueur de la fonction normale des îlots. La réponse en hystérèse des îlots au glucose suggère l’existence d’un algorithme d’encodage de la demande en insuline intégrée au niveau du micro-organe. De plus, ce nouveau signal a été exploité pour le phénotypage d’îlots de souris invalidées pour le gène GluK2, que nous avons utilisées comme modèle d’interaction entre les cellules α et β. La caractérisation de ce nouveau type de signal constitue aussi une avancée importante pour le développement d’un biocapteur destiné à être intégré dans le futur à un pancréas artificiel. / Pancreatic β cells are central to glucose homeostasis because they are the only cell that secretes insulin, the sole hypoglycemic hormone in the organism. The β cell is a glucose sensor that regulates its secretory response and gene expression according to ambient glucose levels. The coupling between glucose metabolism and insulin granule exocytosis involves the generation of electrical activity. An investigation of this activity is important to decipher how β cells encode the organism’s insulin demand. In order to overcome the limits of classically used electrophysiological approaches that are not compatible with long-term studies, extracellular recordings using multielectrode arrays (MEA) have been set-up.My thesis aim was to better understand the complex signals recorded with MEA. This study revealed the existence of a new electrical signature of islet cells: slow potentials (SP) that reflect the coupling function of β cells. SP play an important role in glucose homeostasis and represent a biomarker of normal functioning of islets. The observed hysteretic response of islets to glucose suggests the existence of an algorithm encoding the insulin demand embedded at the microorgan level. Moreover, this new signal was used for the phenotyping of GluK2 deficient mouse islets that were employed as an α-to-β cell interaction model. The characterization of this new signal is an important progress in the development of a biosensor intended to be integrated in an artificial pancreas in the future.

Brain Machine Interfaces (BMI) provide a communication pathway between the electrical conducting units of the brain (neurons) and external devices. BMI technology may provide improved neurological and physiological functions to patients suffering from disabilities due to damaged nervous systems. Unfortunately, microelectrodes used in Intracortical Neural Interfaces (INI), a subset of the BMI device family, have yet to demonstrate long-term in vivo performance due to material, mechanical and electrical failures. Many state-of-the-art INI devices are constructed using stacks of multiple materials, such as silicon (Si), titanium (Ti), platinum (Pt), parylene C, and polyimide. Not only must each material tolerate the biological environment without exacerbating the inflammatory response, each of the materials used must physically withstand the environment as well as interact well with each other. One approach to address abiotic mechanisms has been optimizing the materials required to fabricate the INI devices. Silicon Carbide (SiC) is a physically robust, hemo and biocompatible, and chemically inert semiconductor. An ‘all-SiC’, or monolithic SiC, device may be the disruptive technology needed in the BMI field to finally achieve long-term and wide-spread use of this technology in humans. The all-SiC device concept is where SiC serves as all device layers: the base (substrate), the conducting traces (electrodes), and the surface conformal insulating layer. The monolithic SiC neural probe is realized by forming high-quality pn junctions of heavily doped SiC on a layer of the opposite polarity. Heavily doped semiconductors display semi-metallic electrical performance, which allow for efficient electrical conduction in the electrode without the deleterious effects of metal ions interacting with the neural environment. The conformal insulator is realized using amorphous-SiC (a-SiC) which can be patterned to open windows to allow electrical conduction to occur between the electrode tips and the brain. Several generations of monolithic SiC devices have been fabricated, tested and are reported in this dissertation. The devices were fabricated utilizing two polytypes of SiC (4H-SiC and 3C-SiC). The monolithic SiC microelectrodes were fabricated utilizing techniques used in the fabrication of Si based microelectrodes. Monolithic SiC devices fabricated include planar single-ended MEAs (with varying lengths and varying active recording area), 60-channel MEAs for in-vitro studies, and 16-electrode Michigan style neural probes for in-vivo studies. Electrical testing of the pn junction demonstrated that the 4H-SiC device can block a forward bias voltage of up to 2.3V and displays reverse bias leakage below 1 nArms well past -20V. Current leakage between adjacent electrodes was ~7.5 nArms over a voltage range of -50V to +50V. Furthermore, electrochemical results show that the 4H-SiC microelectrodes interact with an electrochemical environment primarily through capacitive mechanisms and has an impedance comparable to gold electrodes. Electrode impedance ranged from 675±130 kΩ (GSA = 496 µm2) to 46.5±4.80 kΩ (GSA = 500K µm2). However, the 4H-SiC devices cannot deliver charge as efficiently as other conventionally used microelectrode materials, such as iridium oxide (IrOx), but a larger water window compensates for this since larger stimulation voltages are supported compared to IrOx. All studies and data collected thus far indicate that the monolithic SiC neural device can aid in the advancement of chronic INI use in clinical settings. The all-SiC devices rely on the integration of only robust and highly compatible SiC material, they may offer a promising solution to probe delamination and biological rejection associated with the use of multiple materials used in many current INI devices. Follow-on work is planned to prove this assertion via in vivo studies.

Numerous applications in neuroscience research and neural prosthetics, such as retinal prostheses, spinal-cord surface stimulation for prosthetics, electrocorticogram (ECoG) recording for epilepsy detection, etc., involve electrical interaction with soft excitable tissues using a surface stimulation and/or recording approach. These applications require an interface that is able to set up electrical communications with a high throughput between electronics and the excitable tissue and that can dynamically conform to the shape of the soft tissue. Being a compliant and biocompatible material with mechanical impedance close to that of soft tissues, polydimethylsiloxane (PDMS) offers excellent potential as the substrate material for such neural interfaces. However, fabrication of electrical functionalities on PDMS has long been very challenging. This thesis work has successfully overcome many challenges associated with PDMS-based microfabrication and achieved an integrated technology platform for PDMS-based stretchable microelectrode arrays (sMEAs). This platform features a set of technological advances: (1) we have fabricated uniform current density profile microelectrodes as small as 10 microns in diameter; (2) we have patterned high-resolution (feature as small as 10 microns), high-density (pitch as small as 20 microns) thin-film gold interconnects on PDMS substrate; (3) we have developed a multilayer wiring interconnect technology within the PDMS substrate to further boost the achievable integration density of such sMEA; and (4) we have invented a bonding technology---via-bonding---to facilitate high-resolution, high-density integration of the sMEA with integrated circuits (ICs) to form a compact implant. Taken together, this platform provides a high-resolution, high-density integrated system solution for neural and muscular surface interfacing. sMEAs of example designs are evaluated through in vitro and in vivo experimentations on their biocompatibility, surface conformability, and surface recording/stimulation capabilities, with a focus on epimysial (i.e. on the surface of muscle) applications. Finally, as an example medical application, we investigate a prosthesis for unilateral vocal cord paralysis (UVCP) based on simultaneous multichannel epimysial recording and stimulation.