Este trabalho descreve o desenvolvimento de um processo de fabricação de mini e microdispositivos fluídicos baseado na utilização de um equipamento de usinagem a laser de CO2 para criação de relevos sobre base de poli(metacrilato de metila) e na selagem térmica contra igual material. Inicialmente, o equipamento laser foi detalhadamente caracterizado, o que possibilitou elaborar métodos para a construção de microcanais de forma mais eficiente e com menores chances de defeitos. Tipicamente, os canais apresentaram seção transversal triangular em torno de 200 µm de largura e 100 µm de profundidade, sendo possível, no entanto, criar canais com outras características. A etapa de selagem entre a tampa e a base que apresentou melhores resultados consiste em pressurização acima de 6 kgf·cm-2 e aquecimento a 110 ºC durante 45 minutos, seguido de resfriamento por 1 h. Os microcanais selados por esta técnica, resistiram a pressões superiores a 3,5·kgf·cm-2. O processo desenvolvido se mostrou adequado para a criação de protótipos, sendo também suas principais características: (1) a facilidade de incorporação de regiões de grandes dimensões (como reservatórios) em conjunto com os microcanais, (2) número reduzido de etapas de produção e (3) boa uniformidade química da parede interna dos canais, o que é particularmente interessante para microdispositivos aplicados à Química Analítica / A microfabrication process based machining using CO2 laser on poly(methyl methacrylate) and thermal sealing is described. Initially, the laser equipment was characterized in detail, which allowed developing strategies for the construction of microchannels more efficiently and less failure-prone. Typically, the channels had a triangular cross section around 200 µm in width and 100 µm in depth. It is possible, however, create channels with other features. The sealing step that showed better results consists in to pressurize at 6 kgf·cm-2 and heating at 110 °C during 45 minutes, followed by natural cooling for 1 h. The microchannels sealed by using this procedure resisted pressures above 3.5 kgf·cm-2. The process proved to be adequate for prototyping and also has other main features: (1) easiness of incorporation of large regions (such as reservoirs) together with the microchannel; (2) reduced number of manufacturing steps and (3) good chemical uniformity of the inner wall of the channel, which is particularly interesting for microdevices applied to Analytical Chemistry.

Este trabalho descreve o desenvolvimento de um processo de fabricação de mini e microdispositivos fluídicos baseado na utilização de um equipamento de usinagem a laser de CO2 para criação de relevos sobre base de poli(metacrilato de metila) e na selagem térmica contra igual material. Inicialmente, o equipamento laser foi detalhadamente caracterizado, o que possibilitou elaborar métodos para a construção de microcanais de forma mais eficiente e com menores chances de defeitos. Tipicamente, os canais apresentaram seção transversal triangular em torno de 200 µm de largura e 100 µm de profundidade, sendo possível, no entanto, criar canais com outras características. A etapa de selagem entre a tampa e a base que apresentou melhores resultados consiste em pressurização acima de 6 kgf·cm-2 e aquecimento a 110 ºC durante 45 minutos, seguido de resfriamento por 1 h. Os microcanais selados por esta técnica, resistiram a pressões superiores a 3,5·kgf·cm-2. O processo desenvolvido se mostrou adequado para a criação de protótipos, sendo também suas principais características: (1) a facilidade de incorporação de regiões de grandes dimensões (como reservatórios) em conjunto com os microcanais, (2) número reduzido de etapas de produção e (3) boa uniformidade química da parede interna dos canais, o que é particularmente interessante para microdispositivos aplicados à Química Analítica / A microfabrication process based machining using CO2 laser on poly(methyl methacrylate) and thermal sealing is described. Initially, the laser equipment was characterized in detail, which allowed developing strategies for the construction of microchannels more efficiently and less failure-prone. Typically, the channels had a triangular cross section around 200 µm in width and 100 µm in depth. It is possible, however, create channels with other features. The sealing step that showed better results consists in to pressurize at 6 kgf·cm-2 and heating at 110 °C during 45 minutes, followed by natural cooling for 1 h. The microchannels sealed by using this procedure resisted pressures above 3.5 kgf·cm-2. The process proved to be adequate for prototyping and also has other main features: (1) easiness of incorporation of large regions (such as reservoirs) together with the microchannel; (2) reduced number of manufacturing steps and (3) good chemical uniformity of the inner wall of the channel, which is particularly interesting for microdevices applied to Analytical Chemistry.

This research utilizes Electrical Impedance Spectroscopy, a technique classically used for electrochemical analysis and material characterization, as the basis for a non-destructive, label-free assay platform for three dimensional (3D) cellular spheroids. In this work, a linear array of microelectrodes is optimized to rapidly respond to changes located within a 3D multicellular model. In addition, this technique is coupled with an on chip micro-pH sensor for monitoring the environment around the cells. Finally, the responses of both impedance and pH are correlated with physical changes within the cellular model. The impedance analysis system realized through this work provides a foundation for the development of high-throughput drug screening systems that utilize multiple parallel sensing modalities including pH and impedance sensing in order to quickly assess the efficacy of specific drug candidates. The slow development of new drugs is mainly attributed to poor predictability of current chemosensitivity and resistivity assays, as well as genetic differences between the animal models used for tests and humans. In addition, monolayer cultures used in early experimentation are fundamentally different from the complex structure of organs in vivo. This requires the study of smaller 3D models (spheroids) that more efficiently replicate the conditions within the body. The main objective of this research was to develop a microfluidic system on a chip that is capable of deducing viability and morphology of 3D tumor spheroids by monitoring both the impedance of the cellular model and the pH of their local environment. This would provide a fast and reliable method for screening pharmaceutical compounds in a high-throughput system.

Digital microfluidics is a new technology that permits manipulation of liquid droplets on an array of electrodes. Using this technology, nanoliter to microliter size droplets of different samples and reagents can be dispensed from reservoirs, moved, split, and merged together. Digital microfluidics is poised to become an important and useful tool for biomedical applications because of its capacity to precisely and automatically carry out sequential chemical reactions. In this thesis, a set of tools is presented to accelerate the integration of digital microfluidics into Lab-on-a-Chip platforms for a wide range of applications. An important contribution in this thesis is the development of three rapid prototyping techniques, including the use of laser printing to pattern flexible printed circuit board (PCB) substrates, to make the technology accessible and less expensive. Using these techniques, both digital and channel microfluidic devices can be produced in less than 30 minutes at a minimal cost. These rapid prototyping techniques led to a new method for manipulating liquid droplets on non-planar surfaces. The method, called All Terrain Droplet Actuation (ATDA), was used for several applications, including DNA enrichment by liquid-liquid extraction. ATDA has great potential for the integration of different physico-chemical environments on Lab-on-a-Chip devices. A second important contribution described herein is the development of a new microfluidic format, hybrid microfluidics, which combines digital and channel microfluidics on the same platform. The new hybrid device architecture was used to perform biological sample processing (e.g. enzymatic digestion and fluorescent labeling) followed by electrophoretic separation of the analytes. This new format will facilitate complete automation of Lab-on-a-Chip devices and will eliminate the need for extensive manual sample processing (e.g. pipetting) or expensive robotic stations. Finally, numerical modeling of droplet actuation on single-plate digital microfluidic devices, using electrodynamics, was used to evaluate the droplet actuation forces. Modeling results were verified experimentally using an innovative technique that estimates actuation forces based on resistive forces against droplet motion. The results suggested a list of design tips to produce better devices. It is hoped that the work presented in this thesis will help introduce digital microfluidics to many of the existing Lab-on-a-Chip applications and inspire the development of new ones.

Digital microfluidics is a new technology that permits manipulation of liquid droplets on an array of electrodes. Using this technology, nanoliter to microliter size droplets of different samples and reagents can be dispensed from reservoirs, moved, split, and merged together. Digital microfluidics is poised to become an important and useful tool for biomedical applications because of its capacity to precisely and automatically carry out sequential chemical reactions. In this thesis, a set of tools is presented to accelerate the integration of digital microfluidics into Lab-on-a-Chip platforms for a wide range of applications. An important contribution in this thesis is the development of three rapid prototyping techniques, including the use of laser printing to pattern flexible printed circuit board (PCB) substrates, to make the technology accessible and less expensive. Using these techniques, both digital and channel microfluidic devices can be produced in less than 30 minutes at a minimal cost. These rapid prototyping techniques led to a new method for manipulating liquid droplets on non-planar surfaces. The method, called All Terrain Droplet Actuation (ATDA), was used for several applications, including DNA enrichment by liquid-liquid extraction. ATDA has great potential for the integration of different physico-chemical environments on Lab-on-a-Chip devices. A second important contribution described herein is the development of a new microfluidic format, hybrid microfluidics, which combines digital and channel microfluidics on the same platform. The new hybrid device architecture was used to perform biological sample processing (e.g. enzymatic digestion and fluorescent labeling) followed by electrophoretic separation of the analytes. This new format will facilitate complete automation of Lab-on-a-Chip devices and will eliminate the need for extensive manual sample processing (e.g. pipetting) or expensive robotic stations. Finally, numerical modeling of droplet actuation on single-plate digital microfluidic devices, using electrodynamics, was used to evaluate the droplet actuation forces. Modeling results were verified experimentally using an innovative technique that estimates actuation forces based on resistive forces against droplet motion. The results suggested a list of design tips to produce better devices. It is hoped that the work presented in this thesis will help introduce digital microfluidics to many of the existing Lab-on-a-Chip applications and inspire the development of new ones.

Electrophoresis separation corresponds to the motion and separation of dispersed particles under the influence of a constant electric field. In molecular biology, electrophoresis separation plays a major role in identifying, quantifying and studying different biological samples such as proteins, peptides, RNA acids, and DNA. In electrophoresis separation, different characteristics of particles, such as charge to mass ratio, size, and pI, can be used to separate and isolate those particles. For very complex samples, two or more characteristics can be combined to form a multi-dimensional electrophoresis separation system, significantly improving separation efficiency. Much effort has been devoted in recent years to performing electrophoresis separations in microfluidic format. Employing microfluidic technology for this purpose provides several benefits, such as improved transport control, reduced sample volumes, simplicity of operation, portability, greater accessibility, and reduced cost. The aim of this study is to develop microfluidic systems for high-performance separation of biochemical samples using electrophoresis methods. The first part of the thesis concerns the development of a fully integrated microfluidic chip for isoelectric focusing separation of proteins with whole-channel imaging detection. All the challenges posed in fabricating and integrating the chip were addressed. The chip was tested by performing protein and pI marker separations, and the separation results obtained from the chip were compared with those obtained from commercial cartridges. Side-by-side comparison of the results validated the developed chip and fabrication techniques. The research also focuses on improving the peak capacity and separation resolution of two counter-flow gradient electrofocusing methods: Temperature Gradient Focusing (TGF) and Micellar Affinity Gradient Focusing (MAGF). In these techniques, a temperature gradient across a microchannel or capillary is used to separate analytes. With an appropriate buffer, the temperature gradient creates a gradient in the electrophoretic velocity (TGF) or affinity (MAGF) of analytes and, if combined with a bulk counter-flow, ionic species concentrate at unique points where their total velocity is zero, and separate from each other. A bilinear temperature gradient is used along the separation channel to improve both peak capacity and separation resolution simultaneously. The temperature profile along the channel consists of a very sharp gradient used to pre-concentrate the sample, followed by a shallow gradient that increases separation resolution. A simple numerical model was applied to predict the improvement in resolution when using a bilinear gradient. A hybrid PDMS/glass chip integrated with planar micro-heaters for generating bilinear temperature gradients was fabricated using conventional sputtering and soft lithography techniques. A specialized design was developed for the heaters to achieve the desired bilinear profiles using both analytical and numerical modeling. To confirm the temperature profile along the channel, a two-color thermometry technique was also developed for measuring the temperature inside the chip. Separation performance was characterized by separating several different dyes, amino acids and peptides. Experiments showed a dramatic improvement in peak capacity and resolution of both techniques over the standard linear temperature gradients. Next, an analytical model was developed to investigate the effect of bilinear gradients in counter-flow gradient electrofocusing methods. The model provides a general equation for calculating the resolution for different gradients, diffusion coefficients and bulk flow scan rates. The results indicate that a bilinear gradient provides up to 100% improvement in separation resolution over the linear case. Additionally, for some scanning rates, an optimum bilinear gradient can be found that maximizes separation resolution. Numerical modeling was also developed to validate some of the results. The final part of the thesis describes the development of a two-dimensional separation system for protein separation, combining temperature gradient focusing (TGF) and sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis (PAGE) in a PDMS/glass microfluidic chip. An experimental study was performed on separating a mixture of proteins using two characteristics: charge to mass ratio, and size. Experimental results showed a dramatic improvement in peak capacity over each of the one-dimensional separation techniques.

This thesis focuses on the design of a versatile, portable, and cost-effective fluorescence detection system for LOC devices. Components that are widely available are used, such as LEDs for excitation and a microcontroller for processing. In addition, a photoresistor is tested for the feasibility of being used as a fluorescence detector, instead of the more commonly used photomultiplier tubes. The device also focuses on upgradeability and versatility, meaning that most of the major components can be replaced as long as power requirements remain unaffected. This allows for future additions to the device once they are available, as well as giving the user the power to choose which add-ons are needed since not all users may have the same requirements. The performance of the device after testing with fluorescein dyes and stained yeast cells indicate that it is capable of executing simple tasks, such as determining the presence and concentration of an analyte if given a sufficient amount. It also provided similar readings to commercial fluorescence analysers, which proves its ability to function as a fluorescence detector device. The thesis also proposes a MEMS diffraction grating that can be used for wavelength tuning. By being able to selectively measure across a range of wavelengths, the capability of the device is increased. Examples include being able to detect multiple fluorescent emissions, which will complement the multicoloured excitation LED nicely. In addition, the device will not be limited to a predetermined set of filters. This effectively allows more fluorescent dyes to be used with the device since any wavelength in the visible range can be selectively filtered for. Simulations of the proposed diffraction grating were performed in ANSYS to confirm the validity of the calculated values. In addition, tests were performed on a slide fabricated with diffraction gratings using values as close to the calculated values as possible. All of the results indicate that there is great promise in the proposed diffraction grating design and that it should be further investigated.

Biological sample analysis is a costly and time-consuming process. It involves highly trained technicians operating large and expensive instruments in a temperature and dust controlled environment. In the world of rising healthcare cost, the drive towards a more cost-effective solution calls for a point-of-care device that performs accurate analyses of human blood samples. To achieve this goal, today's bulky laboratory instruments need to be scaled down and integrated on a single microchip of only a few square centimeters or millimeters in size. Dielectrophoresis (DEP), a phenomenon where small particles such as human blood cells are manipulated by non-uniform electric fields, stands to feature prominently in the point-of-care device. An original device that enhances DEP effect through novel geometry of the electrodes is presented. When activated with two inverting sinusoidal waveforms, the novel-shaped electrodes generate horizontal bands of increasing electric fields on the surface of the microchip. With these bands of electric fields, particles can be manipulated to form a straight horizontal line at a predictable location. Experimental results showing the collection, separation, and transportation of mammalian cells are presented. A strategy for simultaneous processing of two or more types of particles is also demonstrated. With capabilities for an accurate position control and an increased throughput by parallel processing, the novel microchip device delivers substantial improvements over the existing DEP designs. The research presented here explores the effects of novel electrode geometries in cell manipulations and contributes to the overall progress of an automated blood analysis system.

This thesis focuses on the design of a versatile, portable, and cost-effective fluorescence detection system for LOC devices. Components that are widely available are used, such as LEDs for excitation and a microcontroller for processing. In addition, a photoresistor is tested for the feasibility of being used as a fluorescence detector, instead of the more commonly used photomultiplier tubes. The device also focuses on upgradeability and versatility, meaning that most of the major components can be replaced as long as power requirements remain unaffected. This allows for future additions to the device once they are available, as well as giving the user the power to choose which add-ons are needed since not all users may have the same requirements. The performance of the device after testing with fluorescein dyes and stained yeast cells indicate that it is capable of executing simple tasks, such as determining the presence and concentration of an analyte if given a sufficient amount. It also provided similar readings to commercial fluorescence analysers, which proves its ability to function as a fluorescence detector device. The thesis also proposes a MEMS diffraction grating that can be used for wavelength tuning. By being able to selectively measure across a range of wavelengths, the capability of the device is increased. Examples include being able to detect multiple fluorescent emissions, which will complement the multicoloured excitation LED nicely. In addition, the device will not be limited to a predetermined set of filters. This effectively allows more fluorescent dyes to be used with the device since any wavelength in the visible range can be selectively filtered for. Simulations of the proposed diffraction grating were performed in ANSYS to confirm the validity of the calculated values. In addition, tests were performed on a slide fabricated with diffraction gratings using values as close to the calculated values as possible. All of the results indicate that there is great promise in the proposed diffraction grating design and that it should be further investigated.

Biological sample analysis is a costly and time-consuming process. It involves highly trained technicians operating large and expensive instruments in a temperature and dust controlled environment. In the world of rising healthcare cost, the drive towards a more cost-effective solution calls for a point-of-care device that performs accurate analyses of human blood samples. To achieve this goal, today's bulky laboratory instruments need to be scaled down and integrated on a single microchip of only a few square centimeters or millimeters in size. Dielectrophoresis (DEP), a phenomenon where small particles such as human blood cells are manipulated by non-uniform electric fields, stands to feature prominently in the point-of-care device. An original device that enhances DEP effect through novel geometry of the electrodes is presented. When activated with two inverting sinusoidal waveforms, the novel-shaped electrodes generate horizontal bands of increasing electric fields on the surface of the microchip. With these bands of electric fields, particles can be manipulated to form a straight horizontal line at a predictable location. Experimental results showing the collection, separation, and transportation of mammalian cells are presented. A strategy for simultaneous processing of two or more types of particles is also demonstrated. With capabilities for an accurate position control and an increased throughput by parallel processing, the novel microchip device delivers substantial improvements over the existing DEP designs. The research presented here explores the effects of novel electrode geometries in cell manipulations and contributes to the overall progress of an automated blood analysis system.