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

Technological challenges in the development of optogenetic closed-loop therapy approaches in epilepsy and related network disorders of the brain

Epilepsy is a chronic, neurological disorder affecting millions of people every year. The current available pharmacological and surgical treatments are lacking in overall efficacy and cause side-effects like cognitive impairment, depression, tremor, abnormal liver and kidney function. In recent years, the application of optogenetic implants have shown promise to target aberrant neuronal circuits in epilepsy with the advantage of both high spatial and temporal resolution and high cell-specificity, a feature that could tackle both the efficacy and side-effect problems in epilepsy treatment. Optrodes consist of electrodes to record local field potentials and an optical component to modulate neurons via activation of opsin expressed by these neurons. The goal of optogenetics in epilepsy is to interrupt seizure activity in its earliest state, providing a so-called closed-loop therapeutic intervention. The chronic implantation in vivo poses specific demands for the engineering of therapeutic optrodes. Enzymatic degradation and glial encapsulation of implants may compromise long-term recording and sufficient illumination of the opsin-expressing neural tissue. Engineering efforts for optimal optrode design have to be directed towards limitation of the foreign body reaction by reducing the implant’s elastic modulus and overall size, while still providing stable long-term recording and large-area illumination, and guaranteeing successful intracerebral implantation. This paper presents an overview of the challenges and recent advances in the field of electrode design, neural-tissue illumination, and neural-probe implantation, with the goal of identifying a suitable candidate to be incorporated in a therapeutic approach for long-term treatment of epilepsy patients

Micro- and nano-electrode arrays for electroanalytical sensing

A systematic investigation of the electrochemical behaviour of two sets of microelectrode arrays, fabricated by standard photolithographic and reactive-ion etching techniques, is presented. The first set of microelectrode arrays had a constant relative centre-centre spacing of 10r (where r is the electrode radius). As a value of r was decreased, the cyclic voltammograms recorded from the array became increasingly peak-shaped, due to merging of the diffusion fields of the individual electrodes. Furthermore, it was shown that the peak current densities obtained were largest for the arrays with the smallest individual electrodes, as were the signal-to-noise ratios (SNRs). Electroplating the individuals electrodes with platinum black was also shown to increase the peak currents and the SNRs, due to an increase in the effective surface area. Sigmoidal voltammograms, which are indicative of radial diffusion, were obtained for an individual electrode radius of 25 mm but not for arrays with smaller electrodes. To obtain radial diffusion for an array of 2.5 mm electrodes, it was shown (using a second set of microelectrode arrays) that a minimum relative centre-centre spacing of 40r is required. Further enhancement of the peak current densities were obtained by decreasing the size of the individual electrodes. A series of nanoelectrode arrays were fabricated using electron-beam lithography (EBL). The voltammograms obtained from these arrays exhibited a continual increase in the recorded peak current as the individual electrodes radius was decreased to a value of 110 nm. Since EBL is a slow and costly technique, nanoimprint lithography (NIL) was investigated as an alternative method of fabricating nanoelectrode arrays and comparable results were obtained from arrays produced by EBL and NIL. A dissolved oxygen and temperature sensor incorporating a working microelectrode array was also designed and fabricated. The sector comprised a densely packed array of 2.5 mm radius electrodes and a micro-reference electrode, both of which were covered with an agarose electrolyte gel enclosed in an SU8 chamber. A thermal resistor was included for temperature compensation of the dissolved oxygen measurements. The Ag|AgCl micro-reference electrode was found to be stable for approximately 80 hours in 0.1 M KCl, with 100 nA of current passing through it. Linear calibration curves were obtained from both temperature and dissolved oxygen measurement

DEVELOPMENT OF PIEZORESISTIVE SILICON NANOWIRES (SINWS) AND THE APPLICATIONS IN NEMS TECHNOLOGY.

Ph.DDOCTOR OF PHILOSOPH

Three-dimensional, multifunctional neural interfaces for cortical spheroids and engineered assembloids.

Three-dimensional (3D), submillimeter-scale constructs of neural cells, known as cortical spheroids, are of rapidly growing importance in biological research because these systems reproduce complex features of the brain in vitro. Despite their great potential for studies of neurodevelopment and neurological disease modeling, 3D living objects cannot be studied easily using conventional approaches to neuromodulation, sensing, and manipulation. Here, we introduce classes of microfabricated 3D frameworks as compliant, multifunctional neural interfaces to spheroids and to assembloids. Electrical, optical, chemical, and thermal interfaces to cortical spheroids demonstrate some of the capabilities. Complex architectures and high-resolution features highlight the design versatility. Detailed studies of the spreading of coordinated bursting events across the surface of an isolated cortical spheroid and of the cascade of processes associated with formation and regrowth of bridging tissues across a pair of such spheroids represent two of the many opportunities in basic neuroscience research enabled by these platforms

High-aspect-ratio tridimensional electrode arrays for neural applications

Tese de Doutoramento em Engenharia Biomédica.Neural electrodes are a valuable tool that allows neuroscientists to monitor the neural activity of the brain with great spatial and temporal resolution. Micro and nanotechnology has allowed the development of high-density neural electrode arrays. In the present work, two different materials were used to perform neural arrays, namely silicon and aluminum. The main objective was the ability to reach areas of the brain between 2 mm and 4 mm deep due to its importance in small animal models such as the rat. In such animals, important brain structures such as the hippocampus are situated in this range of depth. Another key objective was the fabrication of probes with high-aspect-ratio in order to minimize tissue displacement and consequent adverse reactions caused by implantation. Four different prototypes each using a specific fabrication approach, were performed and described in detail. Two of these prototypes were performed with aluminum while the other two were performed with silicon. Standard microfabrication processes such as dicing, wet-etching, physical vapor deposition, and non-standard processes such as thermomigration, aluminum anodizing, polymer casting, and sanding were used. The combination of these standard and nonstandard processes allowed a simpler and cheaper fabrication approach when compared with commercially available arrays. The first aluminum prototype was composed by 100 micropillars with a gold electrode at each tip. The aluminum micropillars were encapsulated by aluminum oxide and were 3 mm long with an aspect-ratio of 12:1. The second version was composed by 25 micropillars encapsulated with medical grade epoxy each with a platinum electrode at the tip. Each micropillar was 3 mm long with an aspect-ratio of 19:1. The first silicon prototype was composed by 100 silicon micropillars, each 3 mm long with an aspect-ratio of 5:1. The second version was composed by 36 silicon micropillars encapsulated with medical grade epoxy, each with a platinum electrode at the tip. Each micropillar was 4 mm long with an aspect-ratio of 22:1. A slanted version of the second prototype was also fabricated, allowing progressively increasing penetration depths. Mechanical characterization was performed by implantation in agar gel and porcine cadaver brain while electrical characterization was performed by electrochemical impedance tests as well as cyclic voltammetry. Overall, aluminum showed to be a suitable alternative to silicon in terms of structural material. Also, a dicing based approach proved to be a cost-effective method able to perform high-aspect-ratio neural arrays.Os elétrodos neuronais são uma ferramenta que proporciona aos neurocientistas a capacidade de monitorizar a atividade neuronal do cérebro com uma grande resolução temporal e espacial. As micro e nanotecnologias proporcionaram o desenvolvimento de matrizes de elétrodos neuronais com alta densidade. No presente trabalho dois materiais foram usados para fabricar matrizes de elétrodos neuronais, nomeadamente o silício e o alumínio. O objetivo principal foi a capacidade de chegar a zonas do cérebro entre os 2 mm e 4 mm de profundidade devido a sua importância em modelos animais de pequeno porte como o rato. Nestes animais, estruturas cerebrais importantes como o hipocampo estão situadas nesta gama de profundidades. Outro objetivo chave foi o fabrico de elétrodos com alta razão de aspeto, de forma a minimizar a compressão de tecido neuronal e consequentes reações adversas causadas pela implantação. Quatro protótipos, cada um utilizando um tipo de fabrico específico foram desenvolvidos e descritos em detalhe. Dois desses protótipos foram fabricados com alumínio, enquanto os outros dois foram feitos em silício. Foram usados processos de microfabrico standard como corte com disco, corrosão química, deposição de filmes finos, e também processos não standard como termomigração, anodização do alumínio, molde de polímeros e lixamento. A combinação de processos standard e não standard permitiram uma abordagem de fabrico mais simples e barata quando comparada com a abordagem utilizada em elétrodos comercialmente disponíveis. O primeiro protótipo de alumínio foi composto por 100 micropilares com um elétrodo de ouro na ponta. Os micropilares de alumínio foram encapsulados com óxido de alumínio e tinham 3 mm de comprimento resultando numa razão de aspeto de 12:1. A segunda versão foi composta por 25 micropilares encapsulados com epóxi biocompatível, cada com um elétrodo de platina na ponta. Cada micropillar tinha 3 mm de comprimento com uma razão de aspeto de 5:1. O primeiro protótipo de silício foi composto por 100 micropilares de silício, cada com 3 mm de comprimento e com razão de aspeto de 5:1. A segunda versão foi composta por 36 micropilares de silício encapsulados com epóxi biocompatível e cada com um elétrodo de platina na ponta. Cada micropillar tinha 4 mm de comprimento resultando numa razão de aspeto de 22:1. Uma versão inclinada do segundo protótipo também foi fabricada, permitindo profundidades de penetração progressivas. A caracterização mecânica foi feita através de implantações em gelatina de agar e em cérebro de porco, enquanto a caracterização elétrica foi feita através de testes de impedância eletroquímica assim como testes de voltametria cíclica. No geral, o alumínio demonstrou ser uma alternativa viável ao silício em termos de material estrutural. A abordagem baseada no corte com disco provou ser um método económico capaz de realizar matrizes de elétrodos neuronais de grande razão de aspeto.Portuguese Foundation for Science and Technology (FCT) (SFRH/BD/89509/2012)

Implantable microelectrodes on soft substrate with nanostructured active surface for stimulation and recording of brain activities

Les prothèses neuronales implantables offrent de nos jours une réelle opportunité pour restaurer des fonctions perdues par des patients atteints de lésions cérébrales ou de la moelle épinière, en associant un canal non-musculaire au cerveau ce qui permet la connexion de machines au système nerveux. La fiabilité sur le long terme de ces dispositifs, se présentant sous la forme d'électrodes implantables, est un facteur crucial pour envisager des applications dans le domaine des interfaces cerveau-machine. Cependant, les électrodes actuelles pour l'enregistrement et la stimulation se détériorent en quelques mois voire quelques semaines. Ce défaut de fiabilité sur le long terme, principalement lié à une réaction chronique contre un corps étranger, est induit au départ par le traumatisme consécutif à l'insertion du dispositif et s'aggrave ensuite, durant les mouvements du cerveau, à cause des propriétés mécaniques inadaptées de l'électrode par rapport à celles du tissu. Au cours du temps, l'ensemble de ces facteurs inflammatoires conduit à l'encapsulation de l'électrode par une couche isolante de cellules réactives détériorant ainsi la qualité de l'interface entre le dispositif implanté et le tissu cérébral. Pour s'affranchir de ce phénomène, la biocompatibilité des matériaux et des procédés, ainsi que les propriétés mécaniques de l'électrode doivent être pris en considération. Durant cette thèse, nous avons abordé la question en développant un procédé de fabrication simple pour réaliser des dispositifs implantables souples en parylène. Les électrodes flexibles ainsi obtenues sont totalement biocompatibles et leur compliance est adaptée à celle du tissu cérébral ce qui limite fortement la réaction inflammatoire occasionnée par les mouvements du cerveau. Après avoir optimisé le procédé de fabrication, nous avons focalisé notre étude sur les performances du dispositif et sa stabilité. L'utilisation d'une grande densité d'électrodes micrométriques, avec un diamètre de 10 à 50 µm, permet de localiser les zones d'enregistrement en rendant possible, par exemple, la conversion d'un ensemble de signaux électrophysiologiques en une commande de mouvement. En contrepartie, la réduction de la taille des électrodes conduit à une augmentation de l'impédance ce qui dégrade la qualité d'enregistrement des signaux. Ici, un polymère conducteur organique, le poly(3,4-ethylenedioxythiophene), PEDOT, a été utilisé pour améliorer les caractéristiques électriques d'enregistrement d'électrodes de petites dimensions. Le PEDOT a été déposé sur la surface des électrodes par électrochimie avec une grande reproductibilité. Des dépôts homogènes avec des conductivités électriques très élevées ont été obtenus en utilisant différents procédés électrochimiques. Grâce à l'augmentation du rapport surface/volume induit par la présence de la couche de PEDOT, une diminution significative de l'impédance de l'électrode (jusqu'à 3 ordres de grandeur) a été obtenue sur une large plage de fréquences. De tests de vieillissement thermique accéléré ont également été effectués sans influence notable sur les propriétés électriques démontrant ainsi la stabilité de la couche de PEDOT durant plusieurs mois. Les dispositifs ainsi obtenus, fabriqués en parylène avec un dépôt de PEDOT sur la surface active des électrodes, ont été testés in vitro et in vivo sur des cerveaux de souris. Un meilleur rapport signal sur bruit a été mesuré durant des enregistrements neuronaux en comparaison avec des résultats obtenus avec des électrodes commerciales. En conclusion, la technologie décrite ici, associant stabilité sur le long terme et faible impédance, a permis d'obtenir des électrodes implantables parfaitement adaptées pour le développement d'interfaces neuronales chroniques.Implantable neural prosthetics devices offer, nowadays, a promising opportunity for the restoration of lost functions in patients affected by brain or spinal cord injury, by providing the brain with a non-muscular channel able to link machines to the nervous system. The long term reliability of these devices constituted by implantable electrodes has emerged as a crucial factor in view of the application in the "brain-machine interface" domain. However, current electrodes for recording or stimulation still fail within months or even weeks. This lack of long-term reliability, mainly related to the chronic foreign body reaction, is induced, at the beginning, by insertion trauma, and then exacerbated as a result of mechanical mismatch between the electrode and the tissue during brain motion. All these inflammatory factors lead, over the time, to the encapsulation of the electrode by an insulating layer of reactive cells thus impacting the quality of the interface between the implanted device and the brain tissue. To overcome this phenomenon, both the biocompatibility of materials and processes, and the mechanical properties of the electrodes have to be considered. During this PhD, we have addressed both issues by developing a simple process to fabricate soft implantable devices fully made of parylene. The resulting flexible electrodes are fully biocompatible and more compliant with the brain tissue thus limiting the inflammatory reaction during brain motions. Once the fabrication process has been completed, our study has been focused on the device performances and stability. The use of high density micrometer electrodes with a diameter ranging from 10 to 50 µm, on one hand, provides more localized recordings and allows converting a series of electrophysiological signals into, for instance, a movement command. On the other hand, as the electrode dimensions decrease, the impedance increases affecting the quality of signal recordings. Here, an organic conductive polymer, the poly(3,4-ethylenedioxythiophene), PEDOT, has been used to improve the recording characteristics of small electrodes. PEDOT was deposited on electrode surfaces by electrochemical deposition with a high reproducibility. Homogeneous coatings with a high electrical conductivity were obtained using various electrochemical routes. Thanks to the increase of the surface to volume ratio provided by the PEDOT coating, a significant lowering of the electrode impedance (up to 3 orders of magnitude) has been obtained over a wide range of frequencies. Thermal accelerated ageing tests were also performed without any significant impact on the electrical properties demonstrating the stability of the PEDOT coatings over several months. The resulting devices, made of parylene with a PEDOT coating on the active surface of electrodes, have been tested in vitro and in vivo in mice brain. An improved signal to noise ratio during neural recording has been measured in comparison to results obtained with commercially available electrodes. In conclusion, the technology described here, combining long-term stability and low impedance, make these implantable electrodes suitable candidates for the development of chronic neural interfaces

Group 14 and 15 elements as building blocks for low dimensional functional nanostructures

Carbon nanotubes (CNTs) are an interesting allotrope of carbon which can have a wide range of applications as they have extraordinary mechanical, electrical and thermal properties. All this makes CNTs an interesting nanomaterial for different applications ranging from mechanical sensors to electrical microelectrodes and even biological applications due to their biological compatibility. Beside the excellent material properties, the use of special structuring of these materials is of great importance. The random orientation of CNTs cannot be controlled which usually leads to irreproducible material which is not suitable for real world applications. Therefore, the controlled growth of vertically aligned carbon nanotubes (VACNTs) is considered in this work. VACNTs have been grown on Si/SiO2 substrates using a water-assisted chemical vapor deposition technique. By this technique highly crystalline, pure, low-layer multiwalled CNTs with a vertical orientation to the substrate are obtained. The parameters for the growth are optimized and even structuring of the VACNTs is possible obtaining VACNTs with different heights in one synthesis step. This structuring is the used to construct a nano-microstructured artificial-hair-cell-type sensor as an example for a mechanical sensor which can measure three-dimensional forces by the changing contact resistance between neighboring CNT bundles of different heights. Because to the excellent electrical properties together with the highly-increased surface area due to the vertical alignment of VACNTs, they are compared to randomly oriented CNTs for microelectrode applications. In this, the advantage of vertical alignment becomes clear in the dramatic decrease in impedance and enormous increase in capacity. These microelectrodes are then tested for biological applications for which the compatibility and growth pattern of cortical neurons on VACNTs is studied. While CNTs represent one-dimensional systems, also two-dimensional materials related to carbon such as graphene oxide and reduced graphene oxide are studied and used for gas-adsorption as well as liquids absorbents in combination with bacterial cellulose in the form of aerogels. Finally, phosphorene as an example of a two-dimensional material closely related to graphene is synthesized. Phosphorene, although structurally similar to graphene, it has a band gap in contrast to graphene which makes it an interesting material field-effect transistor devices as shown in this work

Lab-on-chip microsystems for ex vivo network of neurons studies: A review

ABSTRACT: Increasing population is suffering from neurological disorders nowadays, with no effective therapy available to treat them. Explicit knowledge of network of neurons (NoN) in the human brain is key to understanding the pathology of neurological diseases. Research in NoN developed slower than expected due to the complexity of the human brain and the ethical considerations for in vivo studies. However, advances in nanomaterials and micro-/nano-microfabrication have opened up the chances for a deeper understanding of NoN ex vivo, one step closer to in vivo studies. This review therefore summarizes the latest advances in lab-on-chip microsystems for ex vivo NoN studies by focusing on the advanced materials, techniques, and models for ex vivo NoN studies. The essential methods for constructing lab-on-chip models are microfluidics and microelectrode arrays. Through combination with functional biomaterials and biocompatible materials, the microfluidics and microelectrode arrays enable the development of various models for ex vivo NoN studies. This review also includes the state-of-the-art brain slide and organoid-on-chip models. The end of this review discusses the previous issues and future perspectives for NoN studies

Resorbable conductive materials for optimally interfacing medical devices with the living

Implantable and wearable bioelectronic systems are arising growing interest in the medical field. Linking the microelectronic (electronic conductivity) and biological (ionic conductivity) worlds, the biocompatible conductive materials at the electrode/tissue interface are key components in these systems. We herein focus more particularly on resorbable bioelectronic systems, which can safely degrade in the biological environment once they have completed their purpose, namely, stimulating or sensing biological activity in the tissues. Resorbable conductive materials are also explored in the fields of tissue engineering and 3D cell culture. After a short description of polymer-based substrates and scaffolds, and resorbable electrical conductors, we review how they can be combined to design resorbable conductive materials. Although these materials are still emerging, various medical and biomedical applications are already taking shape that can profoundly modify post-operative and wound healing follow-up. Future challenges and perspectives in the field are proposed