Neste trabalho foram desenvolvidas tecnologias e novos processos de fabricação de microdispositivos analíticos visando o acoplamento desta plataforma microfluídica com a detecção condutométrica sem contato (C4D). Uma segunda etapa do trabalho concentrou esforços em melhorar os níveis de detectabilidade da C4D. Para tanto foi proposto um processo rápido e robusto para selagem de canais de vidro para produção de microdispositivos analíticos para eletroforese. Os canais de vidro foram fabricados por processo fotolitográfico e corrosão química em via úmida. Os microcanais obtidos foram selados contra outra lâmina de vidro previamente recoberta por uma membrana polimérica (PDMS) de 50 µm de espessura. Esta mesma membrana, além de promover a junção das placas de vidro, promoveu também o isolamento elétrico entre a solução no interior do canal microfluídico (de uma lâmina de vidro) e os eletrodos metálicos presentes no substrato de vidro oposto. Assim, foi possível acoplar a detecção condutométrica sem contato (C4D) com a plataforma eletroforética proposta. O desempenho analítico desta foi avaliado usando detecção por fluorescência induzida a laser (LIF) e C4D. Eficiência de aproximadamente 47000 pratos/m foi alcançada com boa repetibilidade chip-a-chip. Fluxo eletrosmótico (EOF) estável foi observado apesar da presença do material polimérico compondo parte da parede interna do canal. Com a metodologia proposta, um chip pode ser fabricado em menos de 120 min, já incluindo as etapas de gravação por fotolitografia, corrosão e selagem. Quando comparada à selagem térmica, além do ganho de tempo e facilidade no manuseio dos substratos, o método proposto não necessita de altas temperaturas e os dispositivos obtidos apresentam repetibilidade satisfatória para análises em diferentes dias e em diferentes microchips. A plataforma analítica desenvolvida foi utilizada em um estudo cinético no qual foi possível determinar os parâmetros cinéticos (Vmax = 12,64 mmol L-1 min-1 e KM = 23,8 mmol L-1) da reação de decomposição de ureia catalisada pela enzima urease. Na segunda etapa do trabalho, foi proposta a alteração de um parâmetro físico, a constante dielétrica, da membrana de PDMS usada como isolante de modo a obter um acoplamento capacitivo mais eficiente e como conseqüência uma melhor resposta no detector. Uma discussão teórica fez-se necessária a respeito do princípio de funcionamento da C4D. Os resultados obtidos com experimentos, nos quais a membrana de PDMS foi dopada com dióxido de titânio (TiO2), mostraram que a discussão sobre o funcionamento deste detector está de acordo com as considerações teóricas apresentadas neste trabalho, onde o sinal analítico é proporcional à capacitância e esta é proporcional à constante dielétrica na cela de detecção. Com esta alternativa foi possível reduzir os limites de detecção em experimentos de análise em fluxo de 385,5 para 14,7 µmol L-1 após adição de 50% em massa de TiO2 na membrana de PDMS. / In this thesis were presented technologies developed aiming new manufacturing processes for analytical microdevices by coupling of this microfluidic platform with capacitively coupled contactless conductivity detection (C4D). In a second stage of the work, we focused on improving the levels of detectability of C4D. We proposed a fast and robust process for sealing glass channels to produce analytical microdevices for electrophoresis. The glass channels were fabricated by photolithographic process and chemical wet etching. The obtained microchannels were sealed against another glass plate, which was previously coated with a 50-µm-thick membrane of poly(dimethylsiloxane) (PDMS). The purpose of this membrane, besides promoting the bonding of the two glass plates, was to act as an electrical insulator between the solution within the microfluidic channel on the top glass plate and the metal electrode present on the bottom glass chip. Thus it was possible to couple the contactless conductivity detection (C4D) with the electrophoretic platform proposed. The analytical performance was evaluated using both laser induced fluorescence (LIF) detection and C4D. Efficiency of about 47,000 plates/m was achieved with good chip-to-chip repeatability. Electroosmotic flow (EOF) was observed and stable despite the presence of polymer composing part of the inner wall of the channel. With the proposed methodology, a chip can be manufactured at less than 120 min, including the patterning step by photolithography, chemical etching, and sealing (bonding) step. When compared to the heat sealing procedure, in addition to time savings, and ease of handling of the substrates, the method does not require high temperatures, and the devices obtained show satisfactory repeatability analysis on different days and different microchips. The proposed analytical platform was used in a kinetic study in which it was possible to determine the kinetic parameters (Vmax = 12.64 mmol L-1 min-1 and KM = 23.8 mmol L-1) for the decomposition of urea catalyzed by the enzyme urease. In the second part of this thesis, it was proposed to change a physical parameter, the dielectric constant of the PDMS membrane used as an insulator, to achieve a more efficient capacitive coupling and consequently a better response in the detector. A theoretical discussion was required regarding the operating principle of C4D. The results obtained from experiments in which the PDMS membrane was doped with titanium dioxide (TiO2) showed that the discussion on the functioning of this detector is in agreement with the theoretical considerations presented in this work. The analytical signal was proportional to the capacitance and this was proportional to the dielectric constant in the detection cell. With this alternative we could reduce the detection limits in flow analysis system experiments from 385.5 to 14.7 µmol L-1 after addition of 50% wt of TiO2 in the PDMS membrane.

Neste trabalho foram desenvolvidas tecnologias e novos processos de fabricação de microdispositivos analíticos visando o acoplamento desta plataforma microfluídica com a detecção condutométrica sem contato (C4D). Uma segunda etapa do trabalho concentrou esforços em melhorar os níveis de detectabilidade da C4D. Para tanto foi proposto um processo rápido e robusto para selagem de canais de vidro para produção de microdispositivos analíticos para eletroforese. Os canais de vidro foram fabricados por processo fotolitográfico e corrosão química em via úmida. Os microcanais obtidos foram selados contra outra lâmina de vidro previamente recoberta por uma membrana polimérica (PDMS) de 50 µm de espessura. Esta mesma membrana, além de promover a junção das placas de vidro, promoveu também o isolamento elétrico entre a solução no interior do canal microfluídico (de uma lâmina de vidro) e os eletrodos metálicos presentes no substrato de vidro oposto. Assim, foi possível acoplar a detecção condutométrica sem contato (C4D) com a plataforma eletroforética proposta. O desempenho analítico desta foi avaliado usando detecção por fluorescência induzida a laser (LIF) e C4D. Eficiência de aproximadamente 47000 pratos/m foi alcançada com boa repetibilidade chip-a-chip. Fluxo eletrosmótico (EOF) estável foi observado apesar da presença do material polimérico compondo parte da parede interna do canal. Com a metodologia proposta, um chip pode ser fabricado em menos de 120 min, já incluindo as etapas de gravação por fotolitografia, corrosão e selagem. Quando comparada à selagem térmica, além do ganho de tempo e facilidade no manuseio dos substratos, o método proposto não necessita de altas temperaturas e os dispositivos obtidos apresentam repetibilidade satisfatória para análises em diferentes dias e em diferentes microchips. A plataforma analítica desenvolvida foi utilizada em um estudo cinético no qual foi possível determinar os parâmetros cinéticos (Vmax = 12,64 mmol L-1 min-1 e KM = 23,8 mmol L-1) da reação de decomposição de ureia catalisada pela enzima urease. Na segunda etapa do trabalho, foi proposta a alteração de um parâmetro físico, a constante dielétrica, da membrana de PDMS usada como isolante de modo a obter um acoplamento capacitivo mais eficiente e como conseqüência uma melhor resposta no detector. Uma discussão teórica fez-se necessária a respeito do princípio de funcionamento da C4D. Os resultados obtidos com experimentos, nos quais a membrana de PDMS foi dopada com dióxido de titânio (TiO2), mostraram que a discussão sobre o funcionamento deste detector está de acordo com as considerações teóricas apresentadas neste trabalho, onde o sinal analítico é proporcional à capacitância e esta é proporcional à constante dielétrica na cela de detecção. Com esta alternativa foi possível reduzir os limites de detecção em experimentos de análise em fluxo de 385,5 para 14,7 µmol L-1 após adição de 50% em massa de TiO2 na membrana de PDMS. / In this thesis were presented technologies developed aiming new manufacturing processes for analytical microdevices by coupling of this microfluidic platform with capacitively coupled contactless conductivity detection (C4D). In a second stage of the work, we focused on improving the levels of detectability of C4D. We proposed a fast and robust process for sealing glass channels to produce analytical microdevices for electrophoresis. The glass channels were fabricated by photolithographic process and chemical wet etching. The obtained microchannels were sealed against another glass plate, which was previously coated with a 50-µm-thick membrane of poly(dimethylsiloxane) (PDMS). The purpose of this membrane, besides promoting the bonding of the two glass plates, was to act as an electrical insulator between the solution within the microfluidic channel on the top glass plate and the metal electrode present on the bottom glass chip. Thus it was possible to couple the contactless conductivity detection (C4D) with the electrophoretic platform proposed. The analytical performance was evaluated using both laser induced fluorescence (LIF) detection and C4D. Efficiency of about 47,000 plates/m was achieved with good chip-to-chip repeatability. Electroosmotic flow (EOF) was observed and stable despite the presence of polymer composing part of the inner wall of the channel. With the proposed methodology, a chip can be manufactured at less than 120 min, including the patterning step by photolithography, chemical etching, and sealing (bonding) step. When compared to the heat sealing procedure, in addition to time savings, and ease of handling of the substrates, the method does not require high temperatures, and the devices obtained show satisfactory repeatability analysis on different days and different microchips. The proposed analytical platform was used in a kinetic study in which it was possible to determine the kinetic parameters (Vmax = 12.64 mmol L-1 min-1 and KM = 23.8 mmol L-1) for the decomposition of urea catalyzed by the enzyme urease. In the second part of this thesis, it was proposed to change a physical parameter, the dielectric constant of the PDMS membrane used as an insulator, to achieve a more efficient capacitive coupling and consequently a better response in the detector. A theoretical discussion was required regarding the operating principle of C4D. The results obtained from experiments in which the PDMS membrane was doped with titanium dioxide (TiO2) showed that the discussion on the functioning of this detector is in agreement with the theoretical considerations presented in this work. The analytical signal was proportional to the capacitance and this was proportional to the dielectric constant in the detection cell. With this alternative we could reduce the detection limits in flow analysis system experiments from 385.5 to 14.7 µmol L-1 after addition of 50% wt of TiO2 in the PDMS membrane.

Doctor of Philosophy (PhD) / Proteomics is rapidly achieving recognition as a complimentary and perhaps superior approach to examine global changes in protein abundance in complex biological systems and the value of these techniques in neuropsychiatry is beginning to be acknowledged. Characterizing the brain’s regional proteomes provides a foundation for the detection of proteins that may be involved in disease-related processes. Firstly, optimal conditions were achieved for the application of two dimensional-gel electrophoresis (2D-GE)-based proteomics with postmortem human brain tissue. These optimized techniques were then applied to soluble fractions of adjacent grey and white matter of a single cytoarchitecturally defined area (Brodmann area 9; BA9) and of two adjacent regions of frontal white matter (BA9 and CC body) from healthy individuals. These normative proteomic comparisons highlighted the importance of correct tissue sampling, i.e. proper separation of regional white matter, as heterogeneity in the respective proteomes was demonstrated. Furthermore, they stressed the necessity for future molecular brain mapping studies. The main focus of this thesis however, was to examine the proteomes of brain regions specifically vulnerable to alcohol-induced damage underlying cognitive dysfunction. Alcoholic patients commonly experience mild to severe cognitive decline. It is postulated that cognitive dysfunction is caused by an alcohol-induced region selective brain damage, particularly to the prefrontal cortex. The cerebellum is increasingly recognized for its role in various aspects of cognition and alcohol–induced damage to the cerebellar vermis could indirectly affect neurocognitive functions attributed to the frontal lobe. We used a 2D-GE-based proteomics approach to compare protein abundance profiles of BA9 grey and white matter and the cerebellar vermis from human alcoholics (neurologically uncomplicated and alcoholics complicated with liver cirrhosis) and healthy control brains. Among the protein level changes observed are disturbances in the levels of a number of thiamine-dependent enzymes. A derangement in energy metabolism perhaps related to thiamine deficiency seems to be important in all regions analysed, even where there are no clinical or pathological findings of Wernicke-Korsakoff Syndrome. Evidence of oxidative changes was also seen in all regions and effects of liver dysfunction in the vermis found. However, overall, these results highlight the complexity of this disease process in that a number of different proteins from different cellular pathways appear to be affected. By identifying changes in protein abundance levels in the prefrontal grey and white matter and the cerebellar vermis, hypotheses may draw upon more mechanistic explanations as to how chronic ethanol consumption causes the structural and functional alterations associated with alcohol-related brain damage. Furthermore, by comparing these results, we may be able to isolate disturbances in molecular pathways specific to the brain damage caused by alcohol, severe liver dysfunction and thiamine deficiency.

Doctor of Philosophy (PhD) / Proteomics is rapidly achieving recognition as a complimentary and perhaps superior approach to examine global changes in protein abundance in complex biological systems and the value of these techniques in neuropsychiatry is beginning to be acknowledged. Characterizing the brain’s regional proteomes provides a foundation for the detection of proteins that may be involved in disease-related processes. Firstly, optimal conditions were achieved for the application of two dimensional-gel electrophoresis (2D-GE)-based proteomics with postmortem human brain tissue. These optimized techniques were then applied to soluble fractions of adjacent grey and white matter of a single cytoarchitecturally defined area (Brodmann area 9; BA9) and of two adjacent regions of frontal white matter (BA9 and CC body) from healthy individuals. These normative proteomic comparisons highlighted the importance of correct tissue sampling, i.e. proper separation of regional white matter, as heterogeneity in the respective proteomes was demonstrated. Furthermore, they stressed the necessity for future molecular brain mapping studies. The main focus of this thesis however, was to examine the proteomes of brain regions specifically vulnerable to alcohol-induced damage underlying cognitive dysfunction. Alcoholic patients commonly experience mild to severe cognitive decline. It is postulated that cognitive dysfunction is caused by an alcohol-induced region selective brain damage, particularly to the prefrontal cortex. The cerebellum is increasingly recognized for its role in various aspects of cognition and alcohol–induced damage to the cerebellar vermis could indirectly affect neurocognitive functions attributed to the frontal lobe. We used a 2D-GE-based proteomics approach to compare protein abundance profiles of BA9 grey and white matter and the cerebellar vermis from human alcoholics (neurologically uncomplicated and alcoholics complicated with liver cirrhosis) and healthy control brains. Among the protein level changes observed are disturbances in the levels of a number of thiamine-dependent enzymes. A derangement in energy metabolism perhaps related to thiamine deficiency seems to be important in all regions analysed, even where there are no clinical or pathological findings of Wernicke-Korsakoff Syndrome. Evidence of oxidative changes was also seen in all regions and effects of liver dysfunction in the vermis found. However, overall, these results highlight the complexity of this disease process in that a number of different proteins from different cellular pathways appear to be affected. By identifying changes in protein abundance levels in the prefrontal grey and white matter and the cerebellar vermis, hypotheses may draw upon more mechanistic explanations as to how chronic ethanol consumption causes the structural and functional alterations associated with alcohol-related brain damage. Furthermore, by comparing these results, we may be able to isolate disturbances in molecular pathways specific to the brain damage caused by alcohol, severe liver dysfunction and thiamine deficiency.

Thesis (M.S.)--Rochester Institute of Technology, 1989. / Includes bibliographical references (leaves 81-83).