Um dos principais problemas no emprego do biodiesel como combustível de automóveis é a corrosão que ele pode provocar nos motores, devido à facilidade com que pode degradar. A natureza corrosiva do biodiesel é relacionada com a presença de água, ácidos graxos livres, ions metálicos, impurezas, a incidência da luz, a temperatura e a presença de insaturações na cadeia de alquil ésteres. Na atualidade existem poucos trabalhos na literatura sobre a corrosão dos metais em contato com biodiesel e a maioria deles têm um enfoque mais qualitativo que quantitativo. O maior desafio para avaliar a corrosividade do biodiesel mediante técnicas eletroquímicas é sua baixa condutividade. A espectroscopia de impedância eletroquímica (EIE) é uma técnica sensível que tem sido empregada para estudar meios resistivos como o etanol, por muitos anos. No entanto, os experimentos são difíceis assim como a análise dos resultados, principalmente pelo desenvolvimento da queda ôhmica no meio, devido à alta resistividade do meio ao fluxo da corrente elétrica. Os microeletrodos têm sido empregados em várias pesquisas em eletroquímica e têm apresentado algumas vantagens com respeito aos eletrodos de tamanhos convencionais. O principal benefício é a redução da queda ôhmica em meios resistivos, devido às baixas correntes registradas. No presente trabalho foi avaliada a corrosão do cobre e do aço inoxidável, os quais se encontram presentes no circuito de combustível dos motores, quando entram em contato com o biodiesel obtido a partir do óleo de soja (B100) e etanol. A avaliação foi feita mediante técnicas eletroquímicas com auxilio dos microeletrodos clássicos e na forma de arranjos (de 25 m de diâmetro), sem adição de eletrólito suporte. Adicionalmente, os resultados foram correlacionados com as mudanças nas propriedades fisicoquímicas do biodiesel (viscosidade, teor de água, teor de acidez e estabilidade oxidativa) e com ensaios gravimétricos segundo a norma ASTM G31. Os ensaios de impedância eletroquímica com os microeletrodos clássicos e os arranjos de microeletrodos mostraram duas constantes de tempo e foi possível demostrar que a constante em altas frequências está relacionada com a qualidade do biodiesel, enquanto que a constante nas baixas frequências é uma resposta da interface metal-biodiesel. A viscosidade e o teor de umidade aumentaram com a degradação do biodiesel e a estabilidade oxidativa diminuiu na mesma proporção independentemente do metal que estava em contato com o biocombustível. A presença de defeitos nos arranjos de microeletrodos obtidos por fotolitografia é um problema importante e é preciso estar sempre atento para evitar resultados equivocados. A voltametria cíclica se mostrou como técnica quantitativa para caracterizar e controlar a área exposta dos microeletrodos simples e em arranjos. Finalmente se corroborou com os ensaios eletroquímicos, a maior velocidade de corrosão para o cobre obtida nos ensaios gravimétricos, comparada à do aço inoxidável. / One of the most relevant aspects related with the use of biodiesel in automobile motors is its corrosivity, due to its susceptibility to oxidation reactions. The corrosive nature of biodiesel is related with the presence of water, free fatty acids, metallic ions, impurities, incidence of light, temperature, as well as the insaturation degree of the main chain of the alkyl esters. Currently there are few works in literature about corrosion of metals in contact with biodiesel, and most of them have more a qualitative than a quantitative focus. The main issue for electrochemical studies in biodiesel is its low conductivity. Electrochemistry impedance spectroscopy (EIS) is a sensitive technique that has been used for studying resistive media like ethanol for many years. However the experiments are difficult and the results are complex to understand, mainly because of the ohmic drop developed in this media due to the inherent solution resistance to the flow of electrical current. Microelectrodes have been used in many researches in electrochemistry showing some advantages with respect to conventional electrodes. The principal benefit is the reduction of the ohmic drop in resistive media, due to its negligible value when small currents are registered. In this work corrosiveness of copper and stainless steel which are present in fuel circuit of an automobile motor was evaluated when they come in contact with biodiesel, obtained from soybean oil and ethanol (B100). The evaluation was made using classic microelectrodes and arrays of microelectrodes (25m diameter) without addition of supporting electrolyte. Aditionally the results were correlated with the changes in biodiesel physicochemical properties (viscosity, water content, acid number and oxidation stability) and weight loss tests according to ASTM G31. The electrochemical results with classic microeletrodes and arrays of microelectrodes showed two time constants and it was demonstrated that the time constant at high frequencies is related with biodiesel quality, while the time constant at low frequencies is due to the response of metal-biodiesel interface. The viscosity and water content increased with biodiesel degradation and oxidation stability decrease in the same proportion independently of the metal in contact with biofuel. The presence of defects in the microelectrodes arrays obtained by photolitography is an important issue and the researcher must be atempt to them to avoid equivocated results. Cyclic voltammetry has shown to be a useful technique to characterize and control the exposed are in microlectrodes and their arrays. Finally, the electrochemical measurements corroborate the higher corrosion rate for copper obtained from weight loss tests compaired to corrosion rate for stainless steel.

This thesis describes the development of an optimized method for culturing dissociated, monolayer neuronal networks from murine frontal cortex and midbrain. It is presented as a guidebook for use by cell culture specialists and laboratory personnel who require updated and complete procedures for use with microelectrode array (MEA) recording technology. Specific cell culture protocols, contamination prevention and control, as well common problems encountered within the cell culture facility, are discussed. This volume offers value and utility to the rapidly expanding fields of MEA recording and neuronal cell culture. Due to increasing interest in determining the mechanisms underlying Parkinson's disease, the newly developed procedures for mesencephalon isolation and culture on MEAs are an important research contribution.

La détection quantitative de cellule est généralement réalisée par cytométrie en flux en raison de sa haute sensibilité, cependant cette technique est difficile à mettre en oeuvre pour des analyses de routine ou des analyses au chevet du patient. Les méthodes électrochimiques et en particulier la spectroscopie d'impédance électrochimique ont gagné en popularité en raison de la possibilité de réaliser des analyses sans marquage et de miniaturiser les systèmes d'analyse pour une intégration sur puce. De plus, les avancées récentes dans le domaine des technologies de microfabrication ont permis de développer des électrodes micrométriques ayant de nombreux avantages tels que des hautes impédances dues à des courants très faibles ainsi que la possibilité de les intégrer dans des systèmes microfluidiques. L'objectif de ce travail de thèse se concentre sur la réalisation et l'optimisation de dispositifs microfluidiques contenant les systèmes d'électrodes pour le piégeage immunologique et le comptage impédimétrique de monocytes pro-inflammatoires, marqueurs d'une infection. Compte tenu de l'influence du taux de recouvrement de la surface sur la sensibilité, plusieurs géométries d'électrodes ont été testées. Les meilleures sensibilités et reproductibilités ont été obtenues dans le cas de microélectrodes interdigitées ayant de faibles espaces inter-électrodes (50 µm). D'autre part les études ont également permis de montrer dans ce cas, que la gamme de concentration cellulaire pour laquelle la sensibilité était maximale dépendait de la surface de l'électrode. Les électrodes de plus petites surfaces ont permis d'atteindre une limite de détection inférieure à 10 cellules/mL. De plus, compte tenu de la grande sensibilité des dispositifs ainsi réalisés, ces systèmes ont également été testés pour la caractérisation d'interaction récepteurs-ligands à partir de cellules entières. Ces études ont permis de mettre en évidence l'interaction de cellules CHO exprimant le récepteur A2a à des ligands c-di-AMP pour de très faibles concentrations cellulaires. / The quantitative detection of specific cells is usually carried out by flow cytometry due to its high sensitivity and reliability, however, this technique is not suited for routine screening and point-of-care diagnostics. Electrochemical methods, as electrochemical impedance spectroscopy have gained interest mainly due to a label-free detection and their miniaturization capability required for integration on chip. Furthermore, recent advances in microfabrication based technologies have allowed to develop micron-sized electrodes whose main advantages over conventional electrodes are higher impedances due to smaller currents and the possibility of being integrated inside microfluidic channels. The aim of the present work was the realization and the optimization of microfluidic devices with improved sensitivity targeting the immuno-trapping and counting of pro-inflammatory monocytes as infection markers. Taking into account the influence of the surface coverage on the sensitivity, different geometries were tested. The best sensitivities and reproducibility were recorded in the case of interdigitated micro-electrodes with weak inter-electrodes gap (50 µm). Moreover, experiments carried out with different surfaces demonstrated that there was a threshold beyond which a surface is exploitable for a given slice of concentration. Such microfluidic devices allowed to reach a detection limit around 10 cells/mL. Furthermore, due to the high sensitivity recorded, the devices were also tested to detect ligand binding by cell receptors. These studies have allowed to demonstrate the interaction of CHO-A2a with c-di-AMP for low cell concentrations.

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No description available.

Fast Scan Cyclic Voltammetry (FSCV) is an electrochemical technique, based on ramping a voltage through a microelectrode and measuring the resulting redox current to obtain information about an electroactive molecule. FSCV can be used for the detection of dopamine, which is a vital neurotransmitter. Dopamine is central to conditions such as Parkinson’s disease. The purpose of this thesis was to evaluate if a new type of highly biocompatible microelectrode, called tube electrode, can be used to detect dopamine using FSCV and determine how they compare to standard carbon fibre microelectrodes (CFMEs). In order to achieve this, three main tasks were set and fulfilled. First, a station for in-vitro FSCV was set up and CFMEs of varying sizes were used to detect dopamine at different concentrations. Secondly, the same trials were done with tube electrodes. Finally, the results of the tubes were compared to the CFMEs. In total, four CFMEs and four tube electrodes were investigated. The dopamine concentrations ranged from 20 nM to 40 μM. In short, the results indicate that the tubes generally have lower sensitivity than the CFMEs but better linearity between the increasing dopamine concentration and the resulting current. The tubes also had a marginally higher concentration threshold for dopamine detection. The main challenge encountered was a decrease in sensitivity over time. Here, further investigations are necessary to map the causes responsible. In conclusion, the tube electrodes are able to detect dopamine in-vitro, with concentrations relevant for in-vivo sensing, with a performance comparable to CFMEs. However, further studies are necessary before the tube electrodes can be used for dopamine detection in-vivo. / Fast Scan Cyclic Voltammerey (FSCV) är en elektrokemisk teknik, baserad på att ändra en spänning genom en mikroelektrod och sedan mäta den resulterande redox-strömmen för att få information om en elektroaktiv molekyl. FSCV kan användas för detektering av dopamin, som är en livsviktig signalsubstans. Dopamin har en central roll vid tillstånd så som Parkinsons sjukdom. Syftet med detta examensarbete var att utvärdera om en ny typ av mycket biokompatibel mikroelektrod, kallad tubelektrod, kan användas för att detektera dopamin genom FSCV och fastställa hur de jämför sig med vanliga kolfibermikroelektroder (KFME). För att uppnå detta sattes och uppfylldes tre huvuduppgifter. Först upprättades en station för in-vitro FSCV där KFME med varierande storlek användes för att detektera olika koncentrationer dopamin. Sedan gjordes samma försök med tubelektroder. Slutligen jämfördes resultaten med de från KFME. Totalt testades fyra KFME och fyra tubelektroder. Dopaminkoncentrationerna sträckte sig från 20 nM till 40 μM. Kort sagt indikerar resultaten att tuberna generellt hade lägre känslighet än KFME men bättre linjäritet mellan den ökande dopaminkoncentrationen och den resulterande strömmen. Tuberna hade även något högre koncentrationströskel för detektionen av dopamin. Den största utmaningen som påträffades var en minskning i känslighet över tid. Här krävs vidare undersökningar för att helt förstå de bakomliggande anledningarna. Slutsatsen är att tuberna kan detektera dopamin in-vitro, med koncentrationer som är relevanta för mätningar in-vivo, och med en prestation jämförbar med den för KFME. Dock krävs mer studier innan tubelektroderna kan användas för att detektera dopamin in-vivo.

Esta tese apresenta os achados e avanços obtidos na fabricação, caracterização e aplicação de pontas de prova eletroquímicas multifuncionais para a obtenção de informações eletroquímicas resolvidas no espaço em diversas superfícies/interfaces por meio de técnicas de microscopia de varredura de ponta de prova eletroquímica (SEPM, em inglês). Diferentes designs de pontas de prova multifuncionais foram investigados e, devido a natureza não convencional destas, novos métodos para fabricação e caracterização foram desenvolvidos. Os benefícios da utilização de pontas de prova multifuncionais para a obtenção de informações eletroquímicas resolvidas no espaço ficaram evidente durante a realização de experimentos \"prova de conceito\", onde a maior densidade de informação obtida permitiu o estudo de sistemas mais complexos e a aquisição de informações eletroquímicas livre de interferência topográfica mesmo em superfícies não planas. A hibridização de diferentes técnicas de microscopia de varredura de ponta de prova eletroquímica em uma única ponta de prova também foi investigada o que se provou extremamente útil para a aquisição de imagens eletroquímicas de alta resolução, livres de influências topográficas, quando utilizada a técnica de microscopia de condutividade iônica (SICM, em inglês) como sensor de topografia do substrato investigado. Por ultimo, uma nova técnica, baseada na microscopia de condutividade iônica, que se utiliza de pontas de prova eletroquímicas multifuncionais fabricadas a partir de uma nanopipeta de um único canal, foi desenvolvida. Esta nova técnica se mostrou extremamente ponderosa, capaz de obter informações a respeito da topografia e mapear sítios ativos sobre um substrato utilizando uma nanopipeta de um único canal com alta resolução especial e temporal a uma taxa de aproximadamente 4000 pixels por Segundo. / This thesis presents the findings and advances made on fabrication, characterization and application of multifunctional electrochemical probes to acquire space resolved electrochemical information on diverse surfaces/interfaces employing Scanning Electrochemical Probe Microscopy (SEPM) techniques. Different multifunctional probes designs were investigated and new and innovative methods for fabrication and characterization of those probes were developed, which was necessary due to the unconventional nature of most of the probes studied. The benefits of using multifunctional probes for space resolved electrochemical measurements was clear during \"proof-of-concept\" experiments, where the increased density of information allowed the study of complex systems and the acquisition of topography-free electrochemical information of rough surfaces. The hybridization of different SEPM techniques in a single probe tip was also investigated, and this was found to be extremely beneficial, especially for acquiring high-resolution, topography-free, electrochemical images employing Scanning Ion Conductance Microscopy as a topography feedback. Finally, a new SICM technique, based on the use of a multifunctional probe tip fabricated from a single barrel nanopipette, was developed. This new technique was found to be extremely powerful, capable of acquiring information on topography and map active sites over substrates using a single barrel pipette with high spatial and temporal resolution at a rate of approx. 4000 pixels per second.

Cette thèse porte sur la conception et la réalisation de micros capteurs à électrodes interdigitées pour la caractérisation des milieux biologiques dans la gamme de fréquences : 100 kHz - 10 MHz. L'objectif principal de ce travail est l’optimisation géométrique de la structure d’un capteur à électrodes interdigitées afin d’élargir la plage fréquentielle de mesure en réduisant les effets de polarisation. Le premier chapitre synthétise les données fondamentales relatives au comportement électrique des tissus biologiques ainsi que leurs propriétés électriques notamment en basses fréquences. Le deuxième chapitre concerne une approche théorique pour l’optimisation du capteur pour élargir la bande de fréquence utile de mesure ; ce chapitre recommande également une nouvelle méthode pour déterminer les paramètres de la double couche à la surface en contact entre les électrodes et le milieu biologique. Dans le troisième chapitre nous proposons une modélisation tridimensionnelle du système d’électrodes chargé par un modèle du milieu biologique sous le logiciel ConventorWare®. Les résultats de simulation sont discutés. Les résultats obtenus nous permettent d’évaluer l'influence des paramètres géométriques de la structure interdigitée du capteur ainsi que les propriétés diélectriques du milieu sur l’impédance bioélectrique. Les facteurs d’influence en fonction de la fréquence sont ainsi maîtrisés lors de la conception d’u capteur interdigité destiné à la mesure de bioimpédance. Dans le quatrième chapitre, les dispositifs ainsi que la conception et la fabrication des composants développés au cours de cette thèse sont décrits. Dans le dernier chapitre, les mesures expérimentales effectuées avec de très faibles volumes de différentes solutions (solutions étalons, sang humain) par cinq micros capteurs à électrodes interdigitées. Les mesures sur les échantillons ainsi que la validation des théories d’optimisation ont été élaborées et discutées. Enfin nous avons comparé nos résultats à des valeurs publiées dans la littérature et nous avons justifié expérimentalement la théorie d’optimisation développée. / This thesis focuses on the design and realization of interdigitated sensors for the electrical characterization of the biological medium within the frequency range: 100 kHz - 10 MHz. The main objective of this thesis is to optimize the geometry of the sensor structure according to the specificities of the interdigitated electrodes structure. This optimization leads to widen the measurement frequency range by reducing the effects of polarization at low frequency. The first chapter synthesizes the basics and fundamentals relative to the electric behavior of biological tissues as well as to their electric properties. The second chapter concerns a theoretical approach for the optimization of the sensor to widen the useful frequency band of measurement; this chapter also presents a new method to determine the parameters of the double layer at the contact interface between the electrodes and the biological medium. A three-dimensional modelling of the system, the electrodes being loaded with a biological medium, is implemented using ConventorWare® software and the results discussed in the third chapter. The results obtained allow us to evaluate the influence of the geometrical parameters of the interdigitated structure of the sensor as well as the dielectric properties of the medium on the bioelectric impedance. In the fourth chapter, the devices developed during this thesis are described. The design and the manufacturing of components are presented. The last chapter deals with the experimental measurements obtained with very small volumes of the different solutions (standard solutions, human blood) using five micro sensors that we built in the laboratory. The measurements as well as the validation of the theoretical approach are discussed. Finally we have compared our results with published values in the literature and validated our experimental and theoretical approaches developed in this work.

Esta tese apresenta os achados e avanços obtidos na fabricação, caracterização e aplicação de pontas de prova eletroquímicas multifuncionais para a obtenção de informações eletroquímicas resolvidas no espaço em diversas superfícies/interfaces por meio de técnicas de microscopia de varredura de ponta de prova eletroquímica (SEPM, em inglês). Diferentes designs de pontas de prova multifuncionais foram investigados e, devido a natureza não convencional destas, novos métodos para fabricação e caracterização foram desenvolvidos. Os benefícios da utilização de pontas de prova multifuncionais para a obtenção de informações eletroquímicas resolvidas no espaço ficaram evidente durante a realização de experimentos \"prova de conceito\", onde a maior densidade de informação obtida permitiu o estudo de sistemas mais complexos e a aquisição de informações eletroquímicas livre de interferência topográfica mesmo em superfícies não planas. A hibridização de diferentes técnicas de microscopia de varredura de ponta de prova eletroquímica em uma única ponta de prova também foi investigada o que se provou extremamente útil para a aquisição de imagens eletroquímicas de alta resolução, livres de influências topográficas, quando utilizada a técnica de microscopia de condutividade iônica (SICM, em inglês) como sensor de topografia do substrato investigado. Por ultimo, uma nova técnica, baseada na microscopia de condutividade iônica, que se utiliza de pontas de prova eletroquímicas multifuncionais fabricadas a partir de uma nanopipeta de um único canal, foi desenvolvida. Esta nova técnica se mostrou extremamente ponderosa, capaz de obter informações a respeito da topografia e mapear sítios ativos sobre um substrato utilizando uma nanopipeta de um único canal com alta resolução especial e temporal a uma taxa de aproximadamente 4000 pixels por Segundo. / This thesis presents the findings and advances made on fabrication, characterization and application of multifunctional electrochemical probes to acquire space resolved electrochemical information on diverse surfaces/interfaces employing Scanning Electrochemical Probe Microscopy (SEPM) techniques. Different multifunctional probes designs were investigated and new and innovative methods for fabrication and characterization of those probes were developed, which was necessary due to the unconventional nature of most of the probes studied. The benefits of using multifunctional probes for space resolved electrochemical measurements was clear during \"proof-of-concept\" experiments, where the increased density of information allowed the study of complex systems and the acquisition of topography-free electrochemical information of rough surfaces. The hybridization of different SEPM techniques in a single probe tip was also investigated, and this was found to be extremely beneficial, especially for acquiring high-resolution, topography-free, electrochemical images employing Scanning Ion Conductance Microscopy as a topography feedback. Finally, a new SICM technique, based on the use of a multifunctional probe tip fabricated from a single barrel nanopipette, was developed. This new technique was found to be extremely powerful, capable of acquiring information on topography and map active sites over substrates using a single barrel pipette with high spatial and temporal resolution at a rate of approx. 4000 pixels per second.

An Active Microscaffold System with Fluid Delivery and Stimulation/Recording Functionalities for Culturing 3-D Neuronal Networks Laura Elizabeth Rowe 215 Pages Directed by Dr. A. Bruno Frazier An active microscaffold system with fluid delivery and electrical stimulation/recording functionalities for 3-D neuronal culture studies is presented. The microscaffolds presented in this dissertation consist of an array of microfabricated towers with integrated microfluidic channels, fluid ports, and electrodes. The microfluidic channels and ports allow for perfusion of nutrients, gas exchange, and biochemical control of the extracellular environment throughout the 3-D culture, while the electrodes allow for active stimulation/recording of the 3-D neuronal network. In essence, the microscaffold serves as an artificial circulatory system to enable 3-D in vitro growth and proliferation of re-aggregate neuronal cell cultures. Increased cell survival on microscaffolds with nutrient perfusion at 14 and 21 days in vitro (DIV) is presented. Additionally, the microtower scaffold is built upon a substrate that is compatible with the Multi Channel Systems preamplifier setup to enable electrical stimulation/recording of the cultured network in a 3-D mutilelectrode array (MEA) environment. Impedance measurements on the functioning microtower electrodes were obtained. The overall goal of this research was to develop a BioMEMS technology to provide neuroscientists with a better investigative tool for studying 3-D in vitro neuronal networks than is currently available.