Development and modelling of a versatile active micro-electrode array for high density in-vivo and in-vitro neural signal investigation

The electrophysiological observation of neurological cells has allowed much knowledge to be gathered regarding how living organisms are believed to acquire and process sensation. Although much has been learned about neurons in isolation, there is much more to be discovered in how these neurons communicate within large networks. The challenges of measuring neurological networks at the scale, density and chronic level of non invasiveness required to observe neurological processing and decision making are manifold, however methods have been suggested that have allowed small scale networks to be observed using arrays of micro-fabricated electrodes. These arrays transduce ionic perturbations local to the cell membrane in the extracellular fluid into small electrical signals within the metal that may be measured. A device was designed for optimal electrical matching to the electrode interface and maximal signal preservation of the received extracellular neural signals. Design parameters were developed from electrophysiological computer simulations and experimentally obtained empirical models of the electrode-electrolyte interface. From this information, a novel interface based signal filtering method was developed that enabled high density amplifier interface circuitry to be realised. A novel prototype monolithic active electrode was developed using CMOS microfabrication technology. The device uses the top metallization of a selected process to form the electrode substrate and compact amplification circuitry fabricated directly beneath the electrode to amplify and separate the neural signal from the baseline offsets and noise of the electrode interface. The signal is then buffered for high speed sampling and switched signal routing. Prototype 16 and 256 active electrode array with custom support circuitry is presented at the layout stage for a 20 μm diameter 100 μm pitch electrode array. Each device consumes 26.4 μW of power and contributes 4.509 μV (rms) of noise to the received signal over a controlled bandwidth of 10 Hz - 5 kHz. The research has provided a fundamental insight into the challenges of high density neural network observation, both in the passive and the active manner. The thesis concludes that power consumption is the fundamental limiting factor of high density integrated MEA circuitry; low power dissipation being crucial for the existence of the surface adhered cells under measurement. With transistor sizing, noise and signal slewing each being inversely proportional to the dc supply current and the large power requirements of desirable ancillary circuitry such as analogue-to-digital converters, a situation of compromise is approached that must be carefully considered for specific application design

Development Of Carbon Based Neural Interface For Neural Stimulation/recording And Neurotransmitter Detection

Electrical stimulation and recording of neural cells have been widely used in basic neuroscience studies, neural prostheses, and clinical therapies. Stable neural interfaces that effectively communicate with the nervous system via electrodes are of great significance. Recently, flexible neural interfaces that combine carbon nanotubes (CNTs) and soft polymer substrates have generated tremendous interests. CNT based microelectrode arrays (MEAs) have shown enhanced electrochemical properties compared to commonly used electrode materials such as tungsten, platinum or titanium nitride. On the other hand, the soft polymer substrate can overcome the mechanical mismatch between the traditional rigid electrodes (or silicon shank) and the soft tissues for chronic use. However, most fabrication techniques suffer from low CNT yield, bad adhesion, and limited controllability. In addition, the electrodes were covered by randomly distributed CNTs in most cases. In this study, a novel fabrication method combining XeF2 etching and parylene deposition was presented to integrate the high quality vertical CNTs grown at high temperature with the heat sensitive parylene substrate in a highly controllable manner. Lower stimulation threshold voltage and higher signal to noise ratio have been demonstrated using vertical CNTs bundles compared to a Pt electrode and other randomly distributed CNT films. Adhesion has also been greatly improved. The work has also been extended to develop cuff shaped electrode for peripheral nerve stimulation. Fast scan cyclic voltammetry is an electrochemical detection technique suitable for in-vivo neurotransmitter detection because of the miniaturization, fast time response, good sensitivity and selectivity. Traditional single carbon fiber microelectrode has been limited to single detection for in-vivo application. Alternatively, pyrolyzed photoresist film (PPF) is a good candidate for this application as they are readily compatible with the microfabrication process for precise fabrication of microelectrode arrays. By the oxygen plasma treatment of photoresist prior to pyrolysis, we obtained carbon fiber arrays. Good sensitivity in dopamine detection by this carbon fiber arrays and improved adhesion have been demonstrated

Graphene Microelectrode Arrays to Combine Electrophysiology with Fluorescence Imaging of Amyloid Proteins

Alzheimer's disease (AD) and Parkinson's diseases (PD) are neurodegenerative diseases that affect $\sim$60\,million people worldwide. Both diseases are linked to the misfolding of proteins from their native conformational state into $\beta$-sheeted amyloid fibrils. In AD the implicated proteins are amyloid-$\beta$ and tau, and for PD the implicated protein is $\alpha$-synuclein (aSyn). The motivation for this work is to develop and use physical techniques to better understand the role of amyloid proteins in neurodegenerative diseases. Two techniques used in amyloid research are fluorescence microscopy, to map the protein location and aggregation state, and electrophysiology, to examine the effect of the proteins on neurons. To enable these techniques to be combined, a transparent graphene microelectrode array (MEA) was designed, fabricated and characterised. The active electrode site was graphene since it is electrically conductive, optically transparent and biocompatible. The graphene MEA was characterised using Raman spectroscopy to check the graphene quality, and electrochemical impedance spectroscopy (EIS) to probe the electrode-electrolyte interface. The graphene MEAs enabled voltage trace recordings from cultured neurons to be combined with widefield, confocal fluorescence and fluorescence lifetime imaging microscopy (FLIM). Combining fluorescence imaging and electrophysiology will allow amyloid aggregation to be correlated with neuronal firing patterns. Another physical technique used was Fourier transform infrared spectroscopy (FTIR). A script was written to estimate the protein secondary structure content, and used to investigate polymorphism in the monomeric amyloid protein aSyn.Engineering and Physical Sciences Research Council, Wellcome Trust, Medical Research Counci

A neural probe with up to 966 electrodes and up to 384 configurable channels in 0.13 μm SOI CMOS

In vivo recording of neural action-potential and local-field-potential signals requires the use of high-resolution penetrating probes. Several international initiatives to better understand the brain are driving technology efforts towards maximizing the number of recording sites while minimizing the neural probe dimensions. We designed and fabricated (0.13-μm SOI Al CMOS) a 384-channel configurable neural probe for large-scale in vivo recording of neural signals. Up to 966 selectable active electrodes were integrated along an implantable shank (70 μm wide, 10 mm long, 20 μm thick), achieving a crosstalk of −64.4 dB. The probe base (5 × 9 mm2) implements dual-band recording and a 1

Biosensor system with an integrated CMOS microelectrode array for high spatio-temporal electrochemical imaging, A

2019 Fall.Includes bibliographical references.The ability to view biological events in real time has contributed significantly to research in life sciences. While optical microscopy is important to observe anatomical and morphological changes, it is equally important to capture real-time two-dimensional (2D) chemical activities that drive the bio-sample behaviors. The existing chemical sensing methods (i.e. optical photoluminescence, magnetic resonance, and scanning electrochemical), are well-established and optimized for existing ex vivo or in vitro analyses. However, such methods also present various limitations in resolution, real-time performance, and costs. Electrochemical method has been advantageous to life sciences by supporting studies and discoveries in neurotransmitter signaling and metabolic activities in biological samples. In the meantime, the integration of Microelectrode Array (MEA) and Complementary-Metal-Oxide-Semiconductor (CMOS) technology to the electrochemical method provides biosensing capabilities with high spatial and temporal resolutions. This work discusses three related subtopics in this specific order: improvements to an electrochemical imaging system with 8,192 sensing points for neurotransmitter sensing; comprehensive design processes of an electrochemical imaging system with 16,064 sensing points based on the previous system; and the application of the system for imaging oxygen concentration gradients in metabolizing bovine oocytes. The first attempt of high spatial electrochemical imaging was based on an integrated CMOS microchip with 8,192 configurable Pt surface electrodes, on-chip potentiostat, on-chip control logic, and a microfluidic device designed to support ex vivo tissue experimentation. Using norepinephrine as a target analyte for proof of concept, the system is capable of differentiating concentrations of norepinephrine as low as 8µM and up to 1,024 µM with a linear response and a spatial resolution of 25.5×30.4μm. Electrochemical imaging was performed using murine adrenal tissue as a biological model and successfully showed caffeine-stimulated release of catecholamines from live slices of adrenal tissue with desired spatial and temporal resolutions. This system demonstrates the capability of an electrochemical imaging system capable of capturing changes in chemical gradients in live tissue slices. An enhanced system was designed and implemented in a CMOS microchip based on the previous generation. The enhanced CMOS microchip has an expanded sensing area of 3.6×3.6mm containing 16,064 Pt electrodes and the associated 16,064 integrated read channels. The novel three-electrode electrochemical sensor system designed at 27.5×27.5µm pitch enables spatially dense cellular level chemical gradient imaging. The noise level of the on-chip read channels allow amperometric linear detection of neurotransmitter (norepinephrine) concentrations from 4µM to 512µM with 4.7pA/µM sensitivity (R=0.98). Electrochemical response to dissolved oxygen concentration or oxygen partial pressure (pO2) was also characterized with deoxygenated deionized water containing 10µM to 165 µM pO2 with 8.21pA/µM sensitivity (R=0.89). The enhanced biosensor system also demonstrates selectivity to different target analytes using cyclic voltammetry to simultaneously detect NE and uric acid. In addition, a custom-designed indium tin oxide and Au glass electrode is integrated into the microfluidic support system to enable pH measurement, ensuring viability of bio-samples in ex vivo experiments. Electrochemical images confirm the spatiotemporal performance at four frames per second while maintaining the sensitivity to target analytes. The overall system is controlled and continuously monitored by a custom-designed user interface, which is optimized for real-time high spatiotemporal resolution chemical bioimaging. It is well known that physiological events related to oxygen concentration gradients provide valuable information to determine the state of metabolizing biological cells. Utilizing the CMOS microchip with 16,064 Pt MEA and an improved three-electrode system configuration, the system is capable of imaging low oxygen concentration with limit of detection of 18.3µM, 0.58mg/L, or 13.8mmHg. A modified microfluidic support system allows convenient bio-sample handling and delivery to the MEA surface for sensing. In vitro oxygen imaging experiments were performed using bovine cumulus-oocytes-complexes cells with custom software algorithms to analyze its flux density and oxygen consumption rate. The imaging results are processed and presented as 2D heatmaps, representing the dissolved oxygen concentration in the immediate proximity of the cell. The 2D images and analysis of oxygen consumption provide a unique insight into the spatial and temporal dynamics of cell metabolism

Monitoring single heart cell biology using lab-on-a- chip technologies

Abstract There has been considerable interest in developing microsensors integrated within lab-on-a-chip structures for the analysis of single cells; however, substantially less work has focused on developing "active" assays, where the cell‘s metabolic and physiological function is itself controlled on-chip. The heart attack is considered the largest cause of mortality and morbidity in the western world. Dynamic information during metabolism from a single heart cell is difficult to obtain. There is a demand for the development of a robust and sensitive analytical system that will enable us to study dynamic metabolism at single-cell level to provide intracellular information on a single-cell scale in different metabolic conditions (such as healthy or simulated unhealthy conditions). The system would also provide medics and clinicians with a better understanding of heart disease, and even help to find new therapeutic compounds. Towards this objective, we have developed a novel platform based on five individually addressable microelectrodes, fully integrated within a microfluidic system, where the cell is electrically stimulated at pre-determined rates and real-time ionic and metabolic fluxes from active, beating single heart cells are measured. The device is comprised of one pair of pacing microelectrodes, used for field-stimulation of the cell, and three other microelectrodes, configured as an enzyme-modified lactate microbiosensor, used to measure the amounts of lactate produced by the heart cell. The device also enables simultaneous in-situ microscopy, allowing optical measurements of single-cell contractility and fluorescence measurements of extracellular pH and cellular Ca2+ from the single beating heart cell at the same time, providing details of its electrical and metabolic state. Further, we have developed a robust microfluidic array, wherein a sensor array is integrated within an array of polydimethylsiloxane (PDMS) chambers, enabling the efficient manipulation of single heart cells and real-time analysis without the need to regenerate either working electrodes or reference electrodes fouled by any extracellular constituents. This sensor array also enables simultaneous electrochemical and optical measurements of single heart cells by integrating an enzyme-immobilized microsensor. Using this device, the fluorescence measurements of intracellular pH were obtained from a single beating heart cell whose electrical and metabolic states were controlled. The mechanism of released intracellular [H+] was investigated to examine extracellular pH change during contraction. In an attempt to measure lactate released from the electrically stimulated contracting cell, the cause of intracellular pH change is discussed. The preliminary investigation was made on the underlying relationship between intracellular pH and lactate from single heart cells in controlled metabolic states

Electrodeposited Poly (3,4-Ethylenedioxythiophene) (PEDOT) for Invasive Recording and Stimulating Neural Electrodes

Les électrodes neuronales sont un des outils médicaux utilisé pour soulager les symptômes des maladies neurodégénératives et pour étudier notre cerveau. Les électrodes neuronales conventionnelles souffrent de quelques inconvénients : leurs faibles dimensions leurs confèrent une haute impédance, et leur nature rigide et métallique couplée au traumatisme créé par la procédure d’implantation entraîne une réaction inflammatoire qui augmente encore plus l’impédance. Les polymères conducteurs sont des matériaux souples et organiques possédant une conductivité ionique-électronique mixte, et sont de candidats idéaux pour les interfaces biotique-abiotique. Ils sont régulièrement utilisés en tant que revêtement d’électrodes neuronales en raison de leur amélioration des propriétés électrochimiques et de leur supposée biocompatibilité. Une technique nommée électropolymérisation est généralement utilisée pour déposer les polymères conducteurs sur les microélectrodes neuronales. Un travail important a déjà été réalisé sur l’optimisation des paramètres de cette méthode de dépositionb. Cependant, les polymères conducteurs électropolymérisés souffrent d’une faible adhésion sur la plupart de leurs substrats. En plus de ce problème, il y a un manque d’études in vivo à long-terme confrontant les revêtements de polymères conducteurs aux conditions de stimulations électriques employés dans le domaine médical. Dans notre étude, nous avons observé l’influence du solvant utilisé lors de la déposition sur l’électropolymérisation, la stabilité électrochimique, et l’adhésion des revêtements en polymères conducteurs. Après avoir défini une procédure précise de déposition nous permettant de produire des revêtements stables, nous avons exploré l’utilité de ces revêtements pour des stimulations cérébrales profondes in vivo. Du poly(3,4-éthylènedioxithipohène) (PEDOT) fut électropolymérisé dans trois solvants différents : acétonitrile, propylène carbonate et eau, sur des microélectrodes de platinium-iridium. Les microélectrodes enrobées furent soumises à différents tests de stabilité : sonication, vieillissement passif, stérilisation à la vapeur et stimulations électriques in vitro. Nous avons découvert que l’acétonitrile et le propylène carbonate nous fournissaient les revêtements les plus résistants. Tous les revêtements de PEDOT produits dans les différents solvants étaient suffisamment stable pour être utilisés dans un contexte médical. Ainsi, nous avons implanté des microélectrodes enrobées de PEDOT dans des rats et avons appliqué des stimulations électriques quotidiennes tout en mesurant l’impédance des microélectrodes. Nous avons observé que les stimulations électriques entraînaient une diminution de l’impédance pour les microélectrodes enrobées de PEDOT et les microélectrodes de contrôle. La chute d’impédance était plus importante pour les microélectrodes de contrôle que pour les microélectrodes enrobées de PEDOT, ce qui remet en cause la pertinence de revêtements en PEDOT pour les stimulations cérébrales profondes et indique qu’un travail conséquent sera nécessaire pour optimiser les revêtements en polymère conducteur pour les électrodes neuronales de stimulation.----------ABSTRACT Neural electrodes are one of the medical tools to improve the symptoms of neurodegenerative diseases and/or to study the brain. Conventional neural electrodes suffer from some disadvantages such as: their smaller dimensions lead to high impedance, and their rigid and metallic nature coupled with the destructive insertion procedure leads to inflammatory response in the body that can further increase the impedance. Conducting polymers are soft and organic materials that possess a mixed electronic-ionic conductivity, and are ideal candidates for biotic-abiotic interfaces. They are regularly used for coating neural electrodes due to their enhanced electrochemical properties and biocompatibility. A technique called electropolymerization is generally used to deposit conducting polymers on neural microelectrodes. Extensive work has been done on the optimization of the parameters in this deposition method. However, electrodeposited conducting polymers coatings suffer from poor adhesion on most of their substrates. Besides this issue, there is a lack of long-term in vivo studies subjecting conducting polymer coatings to electrical stimulation conditions used in medical studies. In this work, we investigated the influence of the processing solvent on the electropolymerization, the electrochemical stability, and the adhesion of conducting polymer coatings. After having defined a precise deposition procedure to produce stable coatings, we investigated the role of these coatings for in vivo deep brain stimulations. Poly(3,4-ethylenedioxithiophene) (PEDOT) was electropolymerized in three different solvent: acetonitrile, propylene carbonate and water, on platinum-iridium microelectrodes. The coated microelectrodes were subjected to different stability tests: sonication, passive aging, steam sterilization, and electrical stimulations in vitro. We found out that acetonitrile and propylene carbonate provided the most resistant PEDOT coatings. All the PEDOT coatings processed in different solvents were stable enough to be used in a medical context. We therefore implanted PEDOT-coated stimulating microelectrodes in rats and applied daily stimulation all the while monitoring the impedance of the microelectrodes. We observed that electrical stimulations decreased the impedance of both the PEDOT-coated microelectrodes and the uncoated control microelectrodes. The decrease in impedance was more prominent for control microelectrodes than for PEDOT-coated ones, which questions the relevance of PEDOT coatings for deep brain stimulation purposes and indicates that more work is required to optimize conducting polymer coatings on neural electrodes for electrical stimulation studies

Parylene Based Flexible Multifunctional Biomedical Probes And Their Applications

MEMS (Micro Electro Mechanical System) based flexible devices have been studied for decades, and they are rapidly being incorporated into modern society in various forms such as flexible electronics and wearable devices. Especially in neuroscience, flexible interfaces provide tremendous possibilities and opportunities to produce reliable, scalable and biocompatible instruments for better exploring neurotransmission and neurological disorders. Of all the types of biomedical instruments such as electroencephalography (EEG) and electrocorticography (ECoG), MEMS-based needle-shape probes have been actively studied in recent years due to their better spatial resolution, selectivity, and sensitivity in chronical invasive physiology monitoring. In order to address the inherent issue of invasiveness that causes tissue damage, research has been made on biocompatible materials, implanting methods and probe structural design. In this dissertation, different types of microfabricated probes for various applications are reviewed. General methods for some key fabrication steps include photolithography patterning, chemical vapor deposition, metal deposition and dry etching are covered in detail. Likewise, three major achievements, which aim to the tagets of flexibility, functionality and mechanical property are introduced and described in detail from chapter 3 to 5. The essential fabrication processes based on XeF2 isotropic silicon etching and parylene conformal deposition are covered in detail, and a set of characterization is summarized

Parylene Based Flexible Multifunctional Biomedical Probes And Their Applications

MEMS (Micro Electro Mechanical System) based flexible devices have been studied for decades, and they are rapidly being incorporated into modern society in various forms such as flexible electronics and wearable devices. Especially in neuroscience, flexible interfaces provide tremendous possibilities and opportunities to produce reliable, scalable and biocompatible instruments for better exploring neurotransmission and neurological disorders. Of all the types of biomedical instruments such as electroencephalography (EEG) and electrocorticography (ECoG), MEMS-based needle-shape probes have been actively studied in recent years due to their better spatial resolution, selectivity, and sensitivity in chronical invasive physiology monitoring. In order to address the inherent issue of invasiveness that causes tissue damage, research has been made on biocompatible materials, implanting methods and probe structural design. In this dissertation, different types of microfabricated probes for various applications are reviewed. General methods for some key fabrication steps include photolithography patterning, chemical vapor deposition, metal deposition and dry etching are covered in detail. Likewise, three major achievements, which aim to the tagets of flexibility, functionality and mechanical property are introduced and described in detail from chapter 3 to 5. The essential fabrication processes based on XeF2 isotropic silicon etching and parylene conformal deposition are covered in detail, and a set of characterization is summarized

Design, characterization and testing of a thin-film microelectrode array and signal conditioning microchip for high spatial resolution surface laplacian measurement.

Cardiac mapping has become an important area of research for understanding the mechanisms responsible for cardiac arrhythmias and the associated diseases. Current technologies for measuring electrical potentials on the surface of the heart are limited due to poor spatial resolution, localization issues, signal distortion due to noise, tissue damage, etc. Therefore, the purpose of this study is to design, develop, characterize and investigate a custom-made microfabricated, polyimide-based, flexible Thin-Film MicroElectrode Array (TFMEA) that is directly interfaced to an integrated Signal Conditioning Microchip (SCM) to record cardiac surface potentials on the cellular level to obtain high spatial resolution Surface Laplacian (SL) measurement. TFMEAs consisting of five fingers (Cover area = 4 mm2 and 16 mm2), which contained five individual microelectrodes placed in orthogonal directions (25-µm in diameter, 75-µm interelectrode spacing) to one another, were fabricated within a flexible polyimide substrate and capable of recording electrical activities of the heart on the order of individual cardiomyocytes. A custom designed SCM consisting of 25 channels of preamplification stages and second order band-pass filters was interfaced directly with the TFMEA in order to improve the signal-to-noise ratio (SNR) characteristics of the high spatial resolution recording data. Metrology characterization using surface profilometry and high resolution Scanning Electron Microscope (SEM) indicated the geometry of fabricated TFMEAs closely matched the design parameters \u3c 0.4%). The DC resistances of the 25 individual micro electrodes were consistent (1.050 ± 0.026 kO). The simulation and testing results of the SCM verified the pre-amplification and filter stages met the designed gain and frequency parameters within 2.96%. The functionality of the TFMEA-SCM system was further characterized on a TX 151 conductive gel. The characterization results revealed that the system functionality was sufficient for high spatial cardiac mapping. In vivo testing results clearly demonstrated feasibility of using the TFMEA-SCM system to obtain cellular level SL measurements with significantly improved the SNRs during normal sinus rhythm and Ventricular Fibrillation (VF). Local activation times were detected via evaluating the zero crossing of the SL electro grams, which coincided with the gold standard (dV/dt)min of unipolar electro grams within ± 1%. The in vivo transmembrane current densities calculated from the high spatial resolution SLs were found to be significantly higher than the transmembrane current densities computed using electrodes with higher interelectrode spacings. In conclusion, the custom-made TFMEASCM systems demonstrated feasibility as a tool for measuring cardiac potentials and to perform high resolution cardiac mapping experiments