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Les microcapteurs implantables sont des outils de choix pour l’étude du système nerveux central. Ils permettent d’analyser en temps réel la composition du milieu interstitiel du cerveau et les variations de concentration de neurotransmetteurs et de substrats métaboliques dans l’espace extracellulaire. La procédure d’immobilisation de l’enzyme sur l’électrode est une étape cruciale déterminant les performances du biocapteur. Nous avons développé une méthode d’immobilisation simple, non-toxique et peu chère en utilisant une molécule de poly(ethyleneglycol) diglycidyl éther (PEGDE) qui répond bien aux critères des applications cliniques. La méthode a été étudiée et optimisée sur trois enzyme: la Glucose oxydase, la D-amino acide oxydase et la Glutamate oxydase. Les capteurs développés se caractérisent par une forte sensibilité et un temps de réponse suffisamment court pour la détection des événements biologiques en temps réel. Les capteurs à base de PEGDE ont démontrés une bonne stabilité dans le temps et leur capacité de suivre en temps réel la variation de concentration de glucose dans le SNC du rat suite à l’injection d’insuline ou de glucose. Nous avons également adapté les méthodes d’immobilisation d’enzyme les plus utilisées dans le domaine des neurosciences: immobilisation par réticulation dans des vapeurs de Glutaraldéhyde ou par PEGDE, piégeage dans une matrice de sol-gel ou de polypyrrole dérivé, ou immobilisation dans une matrice d’hydrogel. Nous avons comparé les biocapteurs ainsi obtenus en termes de sensibilité, de stabilité in vivo, de temps de réponse et aussi de toxicité. Cette étude comparative nous a permis de conclure que le PEGDE représente un procédé d’immobilisation optimal car il ne demande pas de synthèse organique, contrairement à l’hydrogel, il n’est pas toxique contrairement au glutaraldehyde et il assure une immobilisation covalente plus stable que le piégeage dans des sol-gel ou polypyrrole. Cette étude comparative a mis également en évidence l’effet de la procédure de fixation de l’enzyme sur la spécificité du biocapteur. Nous avons montré que l’immobilisation par glutaraldehyde provoque une importante perte de sélectivité de l’enzyme. Quant au PEGDE, son immobilisation est assez douce pour préserver la spécificité naturelle de l’enzyme. Nous avons montré que la procédure d’immobilisation a un impact important sur la quantification des molécules dans les échantillons biologiques et in vivo. La validité des mesures sur nos capteurs a été contrôlée par HPLC ou électrophorèse capillaire. Nous avons également développé des sondes multisensibles en utilisant les techniques de microfabrication sur silicium. Le dispositif comporte une aiguille de 6mm en longueur, 100µm en largeur et 50 µm en épaisseur. Elle porte trois électrodes de taille 40x200µm. Ces dispositifs, optimisés pour réduire les effets d’interférence entre les électrodes, ont été pour le suivi simultané de glucose et lactate dans le SNC de rats anesthésiés. / Identification, monitoring and quantification of biomolecules in the CNS is a field of growing interest for identifying biomarkers of neurological diseases. In this thesis, silicon needle-shaped multi-molecules sensing microprobes were developed. Our microelectrode array design comprises a needle length of 6mm with 100x50 µm2 cross-section bearing three platinum electrodes with a size of 40x200 µm and 200µm spacing between them. We have used these microprobes for simultaneous glucose and lactate monitoring, using the third electrode for control of non-specific current variations. Local microdroplet protein deposition on the electrode surface was achieved using a pneumatic picopump injection system. Enzyme immobilization on the electrode surface is a key step in microelectrode biosensor fabrication. We have developed a simple, low cost, non-toxic enzyme immobilization method employing poly(ethyleneglycol) diglycidyl ether (PEGDE). Successful biosensor fabrication was demonstrated with glucose oxidase, D-amino acid oxidase, and glutamate oxidase. We found that these biosensors exhibited high sensitivity and short response time sufficient for observing biological events in vivo on a second-by-second timescale. PEGDE-based biosensors demonstrated an excellent long-term stability and reliably monitored changes in brain glucose levels induced by sequential administration of insulin and glucose solution. We then carried out a comparative study of five enzyme immobilization procedures commonly used in Neuroscience: covalent immobilization by cross-linking using glutaraldehyde, PEGDE, or a hydrogel matrix and enzyme entrapment in a sol-gel or polypyrrole-derived matrices. Enzymatic microelectrodes prepared using these different procedures were compared in terms of sensitivity, response time, linear range, apparent Michaelis-Menten constant, stability and selectivity. We conclude that PEGDE and sol-gel techniques are potentially promising procedures for in vivo laboratory studies. The comparative study also revealed that glutaraldehyde significantly decreased enzyme selectivity while PEGDE preserved it. The effects that immobilization can have on enzyme substrate specificity, produce dramatic consequences on glutamate detection in complex biological samples and in the CNS. Our biosensor’s results were systematically controlled by HPLC or capillary electrophoresis. The highly selective PEGDE-based biosensors allowed accurate measurements glutamate concentrations in the anesthetized and awaked rats at physiological conditions and under pharmacological and electrical stimulations. The microfabricated multielectrodes based on silicon needles coupled to the simple, non-toxic and mild immobilization method based on PEGDE, open new possibilities for specific neurotransmitter detection in the central nervous system and the study of cell-cell communication in vivo.

The market for polyamide desalination membranes is expected to continue to grow during the coming decades. Purification of alternative water sources will also be necessary to meet growing water demands. Purification of produced water, a byproduct of oil and gas production, is of interest due to its dual potential to provide water for beneficial use as well as to reduce wastewater disposal costs. However, current polyamide membranes are prone to fouling, which decreases water flux and shortens membrane lifetime. This research explored surface modification using poly(ethylene glycol) diglycidyl ether (PEGDE) to improve the fouling resistance of commercial polyamide membranes. Characterization of commercial polyamide membrane performance was a necessary first step before undertaking surface modification studies. Membrane performance was found to be sensitive to crossflow testing conditions. Concentration polarization and feed pH strongly influenced NaCl rejection, and the use of continuous feed filtration led to higher water flux and lower NaCl rejection than was observed for similar tests performed using unfiltered feed. Two commercial polyamide membranes, including one reverse osmosis and one nanofiltration membrane, were modified by grafting PEGDE to their surfaces. Two different PEG molecular weights (200 and 1000) and treatment concentrations (1% (w/w) and 15% (w/w)) were studied. Water flux decreased and NaCl rejection increased with PEGDE graft density ([microgram]/cm2), although the largest changes were observed for low PEGDE graft densities. Surface properties including hydrophilicity, roughness and charge were minimally affected by surface modification. The fouling resistance of modified and unmodified membranes was compared in crossflow filtration studies using model foulant solutions consisting of either a charged surfactant or an oil in water emulsion containing n-decane and a charged surfactant. Several PEGDE-modified membranes demonstrated improved fouling resistance compared to unmodified membranes of similar initial water flux, possibly due to steric hindrance imparted by the PEG chains. Fouling resistance was higher for membranes modified with higher molecular weight PEG. Fouling was more extensive for feeds containing the cationic surfactant, potentially due to electrostatic attraction with the negatively charged membranes. However, fouling was also observed in the presence of the anionic surfactant, indicating hydrodynamic forces are also responsible for fouling. / text

Identification, monitoring and quantification of biomolecules in the CNS is a field of growing interest for identifying biomarkers of neurological diseases. In this thesis, silicon needle-shaped multi-molecules sensing microprobes were developed. Our microelectrode array design comprises a needle length of 6mm with 100x50 µm2 cross-section bearing three platinum electrodes with a size of 40x200 µm and 200µm spacing between them. We have used these microprobes for simultaneous glucose and lactate monitoring, using the third electrode for control of non-specific current variations. Local microdroplet protein deposition on the electrode surface was achieved using a pneumatic picopump injection system. Enzyme immobilization on the electrode surface is a key step in microelectrode biosensor fabrication. We have developed a simple, low cost, non-toxic enzyme immobilization method employing poly(ethyleneglycol) diglycidyl ether (PEGDE). Successful biosensor fabrication was demonstrated with glucose oxidase, D-amino acid oxidase, and glutamate oxidase. We found that these biosensors exhibited high sensitivity and short response time sufficient for observing biological events in vivo on a second-by-second timescale. PEGDE-based biosensors demonstrated an excellent long-term stability and reliably monitored changes in brain glucose levels induced by sequential administration of insulin and glucose solution. We then carried out a comparative study of five enzyme immobilization procedures commonly used in Neuroscience: covalent immobilization by cross-linking using glutaraldehyde, PEGDE, or a hydrogel matrix and enzyme entrapment in a sol-gel or polypyrrole-derived matrices. Enzymatic microelectrodes prepared using these different procedures were compared in terms of sensitivity, response time, linear range, apparent Michaelis-Menten constant, stability and selectivity. We conclude that PEGDE and sol-gel techniques are potentially promising procedures for in vivo laboratory studies. The comparative study also revealed that glutaraldehyde significantly decreased enzyme selectivity while PEGDE preserved it. The effects that immobilization can have on enzyme substrate specificity, produce dramatic consequences on glutamate detection in complex biological samples and in the CNS. Our biosensor's results were systematically controlled by HPLC or capillary electrophoresis. The highly selective PEGDE-based biosensors allowed accurate measurements glutamate concentrations in the anesthetized and awaked rats at physiological conditions and under pharmacological and electrical stimulations. The microfabricated multielectrodes based on silicon needles coupled to the simple, non-toxic and mild immobilization method based on PEGDE, open new possibilities for specific neurotransmitter detection in the central nervous system and the study of cell-cell communication in vivo.

Submitted by Renata Lopes (renatasil82@gmail.com) on 2017-03-21T18:21:21Z No. of bitstreams: 1 marcosrodriguesfacchinicerqueira.pdf: 6161892 bytes, checksum: a58d0d5ce0d0b333fd8a3b50155105e4 (MD5) / Approved for entry into archive by Adriana Oliveira (adriana.oliveira@ufjf.edu.br) on 2017-03-22T12:39:49Z (GMT) No. of bitstreams: 1 marcosrodriguesfacchinicerqueira.pdf: 6161892 bytes, checksum: a58d0d5ce0d0b333fd8a3b50155105e4 (MD5) / Made available in DSpace on 2017-03-22T12:39:49Z (GMT). No. of bitstreams: 1 marcosrodriguesfacchinicerqueira.pdf: 6161892 bytes, checksum: a58d0d5ce0d0b333fd8a3b50155105e4 (MD5) Previous issue date: 2016-09-09 / CAPES - Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / O foco deste trabalho foi o desenvolvimento e aplicação de microreatores enzimáticos, visando sua aplicação em sistemas de análise por injeção em fluxo. Em cima disso, dois substratos poliméricos foram utilizados para a avaliação de imobilização enzimática: um à base de poli (metil metacrilato) (PMMA) e outro em uma resina do éter diglicídico do bisfenol-A (BADGE). Uma impressora à laser de CO2 foi utilizada para confeccionar os dispositivos nas dimensões desejadas. Para o sucesso da imobilização, os sistemas foram previamente tratados com polietilenoimina (PEI) visando a introdução de grupamentos funcionais reativos na superfície dos materiais de partida. Num primeiro estudo, baseado na modificação de PMMA, a ativação do material foi conseguida após tratamento dos microcanais com PEI em meio de dimetilsulfóxido (DMSO). Já no segundo caso o tratamento com PEI envolveu a simples mistura mecânica dos materiais, objetivando a cura da resina empregada. Após a ativação dos materiais com PEI, as enzimas foram imobilizadas após passagem de uma mistura de glutaraldeído (um agente espaçador) e as enzimas. Dentre as enzimas estudadas estão a glicose oxidase (GOx), a ascorbato-oxidase (AAO), a catalase (CAT), a glutamato dehidrogenase (GDH), além de um sistema híbrido baseado na imobilização simultânea das enzimas glicose oxidase (GOx) e horseradish peroxidase (HPR). A caracterização dos sistemas desenvolvidos foi feita primordialmente por meio da espectroscopia Raman. Além disso, a aplicação de alguns dos sistemas frente a amostras reais e o cálculo de parâmetros cinéticos e operacionais dos microreatores confeccionados foram reralizados. Essas avaliações foram feitas baseadas em sistemas de detecção desenvolvidos no laboratório por técnicas eletroquímicas e por espectroscopia no visível. Como grande benefício dos sistemas desenvolvidos, podem ser destacados a velocidade e a simplicidade de implementação do processo de imobilização e operação. / The focus of this work is the development and application of enzymatic microreactors aiming their application through flow injection analysis systems. On top of that, two polymeric substrates were used for the evaluation of enzyme immobilization: one based on poly (methyl methacrylate) (PMMA) and another baed on a bisphenol-A diglycidyl ether resin (BADGE). A CO2 laser printer was used to fabricate the devices at the desired dimensions. For the success of the immobilization systems have been pretreated with polyethyleneimine (PEI) in order to introduce reactive functional groups on the surface of the starting materials. In a first study, based on PMMA modification, the activation of the material was achieved after treating microchannels with PEI in dimethylsulfoxide (DMSO). In the second case, treatment with PEI involved simply a mechanical mixture of the two materials, in order to cure the resin. After activation of materials with PEI, the enzymes were immobilized after passage of a mixture of glutaraldehyde (a spacer agent) and enzymes. Among the enzymes studied are glucose oxidase (GOx), ascorbate oxidase (AAO), catalase (CAT), dehydrogenase glutamate (GDH), and a hybrid system based on the simultaneous immobilization of the enzymes glucose oxidase (GOx) and horseradish peroxidase (HPR). The characterization of the developed systems was primarily done by Raman spectroscopy. Moreover, application of some of the proposed systems to real samples and calculation of kinetic and operational parameters are presented during the study. These evaluations were made with detection systems based on electrochemical and visible spectroscopy techniques, all developed at the laboratory. One great benefit of the developed systems, are the speed and simplicity of implementation the immobilization process and operation of the devices.

Hyaluronan (HA) is a substance that is commonly used in biomedical applications in the form of hydrogels. One of these biomedical applications is dermatological fillers where HA is cross-linked with 1,4-Butanediol diglycidyl ether (BDDE) to reduce its rapid turnover within tissue. The filler gives a volumetric effect that can fill out wrinkles. This literature study was conducted in collaboration with Galderma to determine if there is research that explores additives to HA hydrogels that give both volumetric and biological effects when applied as filler. Biological effects that improve the skin's appearance and complexion such as a rejuvenation of the skin was preferable. Both polynucleotides and mannitol show great potential to act as additives in injectable hyaluronan hydrogels.  The main effect of polynucleotides (PN) added in hydrogels is that it is collagen stimulating and provides a more natural tissue regeneration. Rheological properties of the filler change with the addition of PN, where elasticity, viscosity and viscoelasticity have been shown to increase. PNs show no toxicity and are considered safe to inject. The study of mannitol has shown that it does not give a volumetric effect after the injected hydrogel has been broken down. What mannitol can help with, is to prolong the life of the hydrogel and reduce the swelling that is a common side effect after an injection. In addition to this, mannitol is a safe substance to inject.