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.

Made available in DSpace on 2016-06-02T19:04:00Z (GMT). No. of bitstreams: 1 6814.pdf: 27157124 bytes, checksum: ccc98f69d1fc4cdc487ac2e9917edfc0 (MD5) Previous issue date: 2015-03-09 / Microelectrode arrays (MEA) are devices that allow chemical and electrical stimulation and recording of the extracellular electrical activity from entire neuronal cultures over long periods of time, such as several weeks. Some MEA models have transparent substrate, which enables the imaging of culture using optical microscopy. The images are taken from two channels: fluorescence light and transmitted light channels. In the first one, it is possible to visualize the neurons, while in the other one, it is possible to observe the microelectrodes. The objective of this work is to develop methods that enable performing quantitative analysis of the dissociated culture of rat dorsal root ganglion (DRG) neurons plated on MEA by means of the processing of the images, obtained from confocal fluorescence microscopy. We proposed and developed the following methods in order to achieve this objective: (A) A method to automatically identify the microelectrodes in the transmitted light channel using circular Hough Transform and error correction based on the Delaunay triangulation; (B) the registration of a number of images taken at different parts of the MEA in order to generate a unique and high-resolution representation of the whole culture; (C) the segmentation of the neuron in 2D images taken from the fluorescence channel, composed by the steps: preprocessing, thresholding, morphological filtering, neurons occlusion correction, watershed transform and object classification; (D) 2D quantitative analysis based on the identified microelectrodes and on the segmented neurons; (E) a method for generating 3D polygonal models of the neurons from the volumetric images, to be used for visualizing the culture on the MEA by different points of view and zoom levels; and (F) 3D quantitative analysis performed by the processing of the polygonal surfaces in conjunction with the information about the microelectrodes positioning. The results show that the methods are capable to identify the neurons and microelectrodes on the 2D images efficiently. In the 3D images, the preprocessing step which uses information from the 2D segmentation method, showed to be capable to generate correct polygonal models efficiently. Most of the studies involving the analysis of neuron cultures on MEAs consider only qualitative analysis or simple quantitative measures. However, the methods proposed in this thesis enables to obtain important measures related to the neuron culture, such as: the density and morphology of the neurons, and the spatial and topological distribution of the neurons and microelectrodes. The information about neuron morphology is important because they are related to the behavior of this kind of neuron. The spatial and topological distribution of neurons and microelectrodes are used for providing models of the interface between these elements, for supporting the analysis of the electrophysiological signal recorded by the microelectrodes, as well as in the computational simulations of the neuron culture behavior. / Matrizes de Microeletrodos (MEAs) são dispositivos que permitem estimular quimicamente ou eletricamente e registrar a atividade elétrica extracelular de culturas de neurônios durante um longo período de tempo, da ordem de várias semanas. Modelos de MEAs com o substrato transparente permitem imagear a cultura por meio de microscopia óptica. As imagens são obtidas em dois canais: um de luz de fluorescência e outro de luz de transmissão. O primeiro permite visualizar os neurônios, enquanto o segundo os microeletrodos. O objetivo deste trabalho é desenvolver métodos que permitam realizar análises quantitativas de culturas dissociadas de neurônios de gânglio da raiz dorsal (Dorsal Root Ganglion DRG) de ratos em MEAs por meio do processamento de imagens obtidas por microscopia confocal de fluorescência. Os seguintes métodos foram propostos e desenvolvidos para atingir este objetivo: (A) Identificação automática dos microeletrodos nas imagens do canal de luz de transmissão utilizando a transformada de Hough circular e correção de erros baseado na triangulação de Delaunay; (B) Registro de várias imagens tomadas de diferentes regiões da MEA para gerar uma única imagem em alta resolução que contemple a cultura toda; (C) Segmentação dos neurônios em imagens 2D obtidas a partir do canal de fluorescência, composto por etapas de pré-processamento, segmentação, filtragem morfológica, correção da oclusão de neurônios, transformada watershed e classificação de objetos; (D) Análise quantitativa 2D baseada nos microeletrodos identificados e nos neurônios segmentados; (E) Método para geração de modelos poligonais 3D dos neurônios a partir de imagens volumétricas, modelos os quais são utilizados para visualização da cultura na MEA por diferentes pontos de vista e níveis de zoom; e (F) Análise quantitativa 3D realizada por meio do processamento das superfícies poligonais juntamente com as informações sobre a posição dos microeletrodos. Os resultados mostram que os métodos são capazes de identificar com eficiência os neurônios e microeletrodos presentes nas imagens 2D. Nas imagens 3D, a etapa de pré-processamento utilizando informações resultantes do método de segmentação 2D se mostrou eficiente na geração dos modelos poligonais corretos. Enquanto a maioria das análises de imagens de culturas de neurônios em MEA consideram apenas análises quantitativas simples, os métodos aqui propostos permitem obter importantes medidas quantitativas relacionadas às culturas, tais como: a densidade e morfologia dos neurônios, assim como a distribuição espacial e topológica dos neurônios em relação aos microeletrodos. As informações sobre a morfologia são importantes, pois estão relacionadas com o comportamento desse tipo de neurônio. A distribuição espacial e topológica dos neurônios e microeletrodos permitem modelar a interface entre neurônios e microeletrodos e auxiliar nos estudos dos sinais eletrofisiológicos capturados pelos microeletrodos, assim como em simulações computacionais do comportamento dessas culturas.

Neste trabalho foram realizados estudos eletroquímicos com complexos de rutênio do tipo trans-[Ru(NH3)4L1L2]n+, aos quais se coordenam ligantes apropriados visando à modelagem de estruturas capazes de atuar como \"captadoras\" de NO. A síntese e caracterização desses complexos foram feitas pelo grupo de pesquisa do Professor Dr. Douglas Wagner Franco, IQSC-USP. Estes complexos foram adequadamente imobilizados em eletrodos e microeletrodos de ouro com filme de óxido de molibdênio. Com isso, foi desenvolvido um sensor eletroquímico para NO em solução aquosa de tampão fosfato, simulando o pH fisiológico (pH ˜7,4). Os eletrodos modificados foram empregados na quantificação de NO em amostras de albumina bovina com peroxidonitrito na presença de tempo, verificando-se a produção de NO nesse meio. Essas amostras foram fornecidas pela Professora Dra Ohara Augusto, IQ-USP São Paulo. Os estudos referentes os complexos de rutênio visaram principalmente à determinação da velocidade de liberação do NO desses complexos, através do mecanismo EC. Para isso, utilizaram-se duas técnicas eletroquímicas, cronoamperometria de duplo degrau de potencial e voltametria com eletrodo rotativo de disco-anel. Além dos complexos coordenados com o ligante NO foram estudados complexos coordenados com o ligante sulfato. Os resultados obtidos pelas duas técnicas eletroquímicas foram concordantes, e estavam de acordo com o esperado, mediante aos estudos prévios realizados pelo grupo do Professor Dr. Douglas Wagner Franco. As constantes de velocidades encontradas situaram-se - na faixa de 10 a 10-3 s-1. Essas variações estão relacionadas com as propriedades químicas de cada ligante. Outro estudo eletroquímico realizado com a molécula de óxido nítrico em solução de tampão fosfato (pH ˜7,4) foi feito no intuito de determinar a concentração exata de uma solução saturada de NO. Para isso, utilizou-se a técnica de cronamperometria, com eletrodo de trabalho um microeletrodo de platina de raio igual a 25 µm. A concentração da solução saturada de NO, foi de (2,1 ± 0,3) mmolL-1. Os resultados da concentração foram confirmados pelo método clássico titulação volumétrica, no qual obteve-se o valor da concentração de (1,95 ± 0,02) mmolL-1. Esses resultados estão de acordo com resultados descritos na literatura. / The trans-[Ru(NH3)4NOL]3+ (L = 4-NH2py, Him, L-hist, 4-pic, py, 4-Clpy, nia, isn, 4-CNpy and pz) complexes were eletrochemically investigated by using double potencial step chronoamperometry and rotating ring-disc electrode voltammetry. Values for the rate of NO substitution by water molecule ranged from 0.02 s-1 (4-pic) to 0.34 s-1 (pz) at 25ºC. Gold surfaces were modified with an electrochemically deposited layer of non-stoichiometric molybdenum oxides. At these surfaces, trans-[Ru(III)(NH3)4(4pic)SO4]+ complex was incorporated in a controlled way by cycling consecutively the potential in the range +0.50 to -0.25 V at pH ˜ 3. Very reproducible voltammetric curves corresponding to the electrochemical process of the ruthenium complex were obtained, confirming the immobilisation of the material into the molybdenum oxide film. The anodic oxidation of nitric oxide at physiological pH ˜7.4 in phosphate buffer was investigated at the modified electrode containing the molybdenum oxide + trans-[Ru(III)(NH3)4(4pic)SO4]+ complex and an enhancement in the current response was observed compared to the signal at a bare electrode. The rate for NO electrochemical oxidation was dependent on the amount of catalytic ruthenium sites dispersed into the molybdenum oxide film, suggesting the participation of the metallic ion in an out-sphere mechanism. A linear relationship between current signals measured by square wave voltammetry and NO concentration was obtained in the 0 to 10 µM range. The applicability of the modified electrode as a sensor for real-time NO monitoring was also demonstrated.

De nombreux systèmes électrochimiques sont composés d'un grand nombre d'éléments électroactifs en interaction. Si la réaction électrochimique possède une cinétique non linéaire, des comportements coopératifs complexes peuvent émerger suivant la nature et l’intensité des interactions entre les éléments du système. L'objectif de cette thèse est de comprendre l'influence de la taille finie de l’électrode et des interactions entre les microélectrodes sur le comportement coopératif d'un groupe de microélectrodes de platine soumis à un couplage global. Les réactions choisies pour cette étude sont l’électrooxydation du monoxyde de carbone (CO), une réaction avec une cinétique bistable et l’électrooxydation du formaldéhyde (HCHO), qui présente des oscillations de potentiel sous contrôle galvanostatique. Au cours de l’électrooxydation galvanodynamiques du CO sur une seule microélectrode de Pt, la branche S-NDR a pu être mise en évidence contrairement au comportement observé sur une macroélectrode de Pt. En outre, les nouveaux comportements coopératifs comme l'activation séquentielle des microélectrodes, des oscillations de courant et de potentiel spontanées et un régime de commutation dynamique entre les électrodes ont été découverts pour cette réaction lorsque quatre électrodes ont été couplées globalement. Pendant l’électrooxydation de HCHO, l'introduction du couplage global à deux électrodes conduit à des oscillations de courant en opposition de phase. / Many electrochemical systems are composed of a large number of interacting electroactive elements. If the reaction taking place on them has nonlinear kinetics and their interactions allow them to exchange information, complex cooperative behaviors can emerge. The objective of this thesis is to understand the influence of finite-size effects and cooperative phenomena on the global behavior of a group of coupled Pt microelectrodes. The reactions chosen for this study were CO electrooxidation, a reaction with current bistability, and HCHO electrooxidation, which exhibits oscillations under galvanostatic control. During the galvanodynamic electrooxidation of CO on a single microelectrode the S-NDR branch could be evidenced, on macroelectrodes this is not possible due to the formations of stationary domains. Additionally, novel cooperative behaviors (i.e., sequential activation, oscillations and complex switching) were discovered for this reaction when four electrodes were globally coupled. During HCHO electrooxidation the introduction of global coupling to two electrodes led to anti-phase current oscillations.

The production of reactive oxygen species (ROS) and reactive nitrogen species (RNS) is a metabolic situation, observed in different physiological conditions. When their production is exacerbated, there is an efficient antioxidant system able to control and re-install the homeostasis. Oxidative stress results from an acute and chronic imbalance between the production of ROS and antioxidant capacities of living cells and organisms. In the present case, the release of bursts of ROS and RNS by individual macrophages RAW 264.7 was investigated, in real time, in absence and presence of quinones (alpha- and beta-lapachones) and ascorbic acid. A selective electrochemical detection of each ROS or RNS was conducted at platinized carbon fiber microelectrodes positioned at micrometric distances from single cells, always compared to controls. The results show that the presence of beta-lapachone with 0.1 to 100 µM, within one hour of incubation, leads to a decrease of RONS release by macrophages. Conversely, when the incubation time increases (4 hours and more), for concentrations of 1 µM of ortho-quinone, the quantity of the released species increases. Finally, the increasing of the concentration up to 10 µM with four hours of incubation and more is associated to cell death. The exact nature of the released electroactive species was characterized and the original flux could be reconstructed in terms of O2• and NO•. In the first hour, with 10 µM, the decrease of the oxidative burst concerns mainly the RNS species. The amount of ROS, quantified in terms of H2O2 was shown to be higher than in control. At concentration of 1 µM and after longer time incubation, i. e., 4 hours, the amount of ROS, quantified in terms of H2O2 and O2• and NO2- was shown to be higher than in control. It was observed which ONOO- was decreased in both situations. On the other hand, alpha-lapachone was unable to significantly increase ROS and RNS production in macrophage cells, even after 24 hours of incubation. The cytotoxic concentration observed for alpha-lapachone was ten times higher when compared with beta-lapachone. To overcome solubility and bioavailability problems with beta-lapachone, beta-cyclodextrin was used. UV/vis spectroscopy has been used to monitor the inclusion phenomena of beta-lapachone within the cavity of beta-cyclodextrin, together with studies concerning the competitive effects of ethanol concentration on this behavior. A value of 15 ± 5 M-1 was found for the association constant between beta-lapachone and beta-cyclodextrin in phosphate buffer. This clearly indicates that the equilibrium between the free and the complexed substrate is decreased in the presence of EtOH. Amperometry at platinized carbon fiber ultramicroelectrodes allowed quantitative and kinetic investigations of the overall effect of ascorbic acid on oxidative stress responses produced by single cells pertaining to two different cell lines derived from immune cells: macrophages RAW 264.7 and phagocytes PLB-985. These fine and minute amperometric measurements performed on single cells confirmed that in contradiction with its alleged universal anti-oxidant properties, ascorbic acid may affect positively or negatively the RNS content of oxidative bursts produced by each cell type, through affecting the primary production of NO• by the cells. So, vitamin C acts exclusively as an anti-oxidant (viz., reduced the primary NO• flux) onto PMA-treated PLB 985 cells, thus decreasing the quantity of RNS released by these granulocyte-type cells. Conversely, it gives rise to a prooxidant activity (viz., increased the primary NO• flux) onto RAW267.4 macrophage cells, increasing their production of RNS species. These studies demonstrate the advantage of electrochemical methods for analyzing in real-time and quantitatively the effect of pharmacologically active compounds in cells, in case of oxidative burst. / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Conselho Nacional de Desenvolvimento Científico e Tecnológico / A produção de espécies reativas de oxigênio (ERO), de nitrogênio (ERN), entre outras espécies reativas, é parte integrante do metabolismo e é observada em diversas condições fisiológicas. Quando sua produção é exacerbada, o organismo dispõe de um eficiente sistema antioxidante que consegue controlar e restabelecer o equilíbrio. O estresse oxidativo resulta do desequilíbrio entre o sistema pró e antioxidante, com predomínio dos oxidantes. A liberação de ERO e ERN em macrófagos individuais da linhagem RAW 264.7 foi estudada em tempo real, em ausência e presença de quinonas (alfa- e beta-lapachonas) e de ácido ascórbico. A detecção eletroquímica seletiva de ERO e ERN, em conjunto e individualmente foi conduzida, utilizando-se microeletrodos de fibra de carbono platinizada, posicionados a distâncias micrométricas de uma célula, sempre em relação a controles. Os resultados do tratamento com beta-lapachona (0,1 a 100 µM), com 1 hora de incubação, mostram que ocorre a diminuição de ERON liberados pelos macrófagos. Contrariamente, quando o tempo de tratamento aumenta (4 e 6 horas) para a concentração de 1 µM, ocorre o aumento da quantidade de espécies reativas liberadas. Em concentração de 10 µM e com tempo de tratamento acima de 4 horas ocorre morte celular. A natureza exata das espécies eletroativas liberadas foi caracterizada e o fluxo original foi determinado em termo de O2• e NO•. No tratamento de 1 hora com 10 µM de beta-lapachona, houve diminuição do surto oxidativo relacionado principalmente às ERNs. A quantidade de ERO, em termos de H2O2 foi muito maior do que o controle. Em diferentes condições, com 1 µM com 4 horas de tratamento, a quantidade de ERO, em termos de H2O2, O2• e NO2-, foi maior que o controle. Nas duas condições, obteve-se a diminuição acentuada de ONOO-. Por outro lado, a incubação com alfa-lapachona não foi capaz de alterar significantemente a liberação de ERON pelos macrófagos, mesmo após 24 horas de tratamento. A concentração citotóxica observada para a alfa-lapachona foi dez vezes maior quando comparada com a beta-lapachona. Para contornar problemas relacionados à solubilidade e biodisponibilidade da beta-lapachona, fez-se uso da ciclodextrina. A espectroscopia no UV/Vis foi utilizada para monitorar o fenômeno de inclusão de beta-lapachona na cavidade da beta-ciclodextrina, com análise do efeito competitivo do etanol. O valor da constante de associação entre a beta-lapachona e a beta-ciclodextrina, em tampão fosfato, foi de 15 ± 5 M-1, mostrando que a presença de etanol afeta a formação do complexo. A amperometria utilizando ultramicroeletrodos de fibra de carbono platinizada permitiu investigar quantitativa e cineticamente o efeito total do ácido ascórbico no surto oxidativo produzido por células individuais pertencentes a duas diferentes linhagens derivadas de células imunes: macrófagos (RAW 264.7) e fagócitos PLB-985. Medidas amperométricas finas realizadas em células individuais confirmaram que o ácido ascórbico pode estimular ou atenuar o surto oxidativo produzido por cada linhagem celular, afetando a produção primária de NO• das células. Assim, a vitamina C reduz o fluxo primário de NO• nas células PLB-985 pré-tratadas com PMA, enquanto aumenta o fluxo primário de NO• em macrófagos RAW 264.7. Esses estudos demonstram a vantagem dos métodos eletroquímicos para analisar em tempo real e quantitativamente o efeito farmacológico de compostos ativos em células, no caso de surto oxidativo.

Development of microsized batteries plays an important role in the design of in-situ electrochemical investigation systems and portable/wearable electronics. This emerging field intimately correlates with the topics of rechargeable batteries, nanomaterials, on-chip microfabrication, flexibility with reliable mechanical properties etc. Among the various energy materials, conversion-type materials have been proposed as high-energy-density alternatives to traditional intercalation-based materials. However, these materials usually show complex reaction processes accompanied by multi-reaction intermediates, which poses a great challenge to understand the chemical mechanisms. Benefiting from the merits of microsized battery devices, we develop a novel strategy to investigate and then optimize the electrochemical performance of a specific conversion-type material: nickel-iron oxide (NFO). Subsequently, this kind of materials are employed for flexible minimized energy storage systems. Unlike traditional characterization methods based on slurry-coated electrodes, micro-platforms directly probe the intrinsic electrochemical properties of a single active material in real-time due to the elimination of other additives. In this thesis, we firstly design a micro-lithium batteries (MLBs), based on a single “Swiss-roll” microtubular nanomembrane electrode. This platform enables us to investigate the electrochemical mechanisms of electrode materials in lithium batteries by in-situ Raman spectroscopy, electrical conductivity measurements, and electrochemistry characterization. With this designed MLBs, we systematically studied NFO nanomembranes. Using in-situ Raman spectroscopy during the delithiation/lithiation process, we monitored the transition of the chemical component directly. Guided by our investigations of micro-batteries, composite NFO nanomembrane electrodes were fabricated and tested in coin cells, which showed an excellent rate performance: 440 mAh g-1 at a high rate of 20 A g-1 and a long-term stable cycling performance over 1600 cycles. One step further, a flexible energy storage micro-device is achieved using such optimized materials. We demonstrate a thin, lightweight, and flexible micro-full lithium-ion battery based on nickel-iron oxide with a high-rate performance and energy density that can be repeatedly bent to 180° without structural failure and performance loss. It delivers a stable output capacity of 140 mAh g-1 over 1000 charge/discharge cycles. Meanwhile, the excellent rate performance guarantees high energy output up to 255 W h kg-1 at a high power density of 12000 W kg-1 at the microscale.

Reliable chronic neural recording from focal deep brain structures is impeded by insertion injury and foreign body response, the magnitude of which is correlated with the mechanical mismatch between the electrode and tissue. Thin and flexible neural electrodes cause less glial scarring and record longer than stiff electrodes. However, the insertion of flexible microelectrodes in the brain has been a challenge. A novel insertion method is proposed, and demonstrated, for precise targeting deep brain structures using flexible micro-wire electrodes. A novel electrode guiding system is designed based on the principles governing the buckling strength of electrodes. The proposed guide significantly increases the critical buckling force of the microelectrode. The electrode insertion mechanism involves spinning of the electrode during insertion. The spinning electrode is slowly inserted in the brain through the electrode guide. The electrode guide does not penetrate into cortex. The electrode is inserted in the brain without stiffening it by coating with foreign material or by attaching a rigid support and hence the method is less invasive. Based on two new mechanisms, namely spinning and guided insertion, it is possible to insert ultra-thin micro-wire flexible electrodes in rodent brains without buckling. I have demonstrated successful insertion of 25 µm platinum micro-wire electrodes about 10 mm deep in rat brain. A novel micro-motion compensated ultra-thin flexible platinum microelectrode has been presented for chronic single unit recording. Since manual insertion of the proposed microelectrode is not possible, I have developed a microelectrode insertion device based on the proposed method. A low power low noise 16 channel programmable neural amplifier ASIC has been designed and used to record the neural spikes. The ability to record neural activity during insertion is a unique feature of the developed inserter. In vivo implantation process of the microelectrode has been demonstrated. Microelectrodes were inserted in the Botzinger complex of rat and long term respiratory related neural activity was recorded from live rats. The developed microelectrode has also been used to study brain activity during seizures. In-vivo experimental results show that the proposed method and the prototype insertion system can be used to implant flexible microelectrode in deep brain structures of rodent for brain studies.

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