The master's thesis is about a communication element - a hardware module, its design and implementation. The communication is to be maintained between a Linux embedded device and the sensors elements, using the MiWi protocol. The task is part of the IoT project, developed at FIT BUT. Furthermore, the paper describes design of a driver for the module, its likely solution, as well as the very implementation. Finally, the obtained experience is discussed in a summary, along the next step options on how to proceed further with the driver development.

Laboratory test results are important in making decisions regarding a patient's diagnosis and response to treatment. These tests often measure the biomarkers found in biological fluids such blood, urine, and saliva. Immunoassay is one type of laboratory test used to measure the level of biomarkers using specific antibodies. Microfluidics offer several advantages such as speed, small sample volume requirement, portability, integration, and automation. These advantages are motivating to develop microfluidic platforms of conventional laboratory tests. I have fabricated polymer microfluidic devices and developed immunoassays on-chip for potential cancer markers. Silicon template devices were fabricated using standard photolithographic techniques. The template design was transferred to a poly(methyl methacrylate) (PMMA) piece by hot embossing and subsequently bonded to another PMMA piece with holes for reservoirs. I used these devices to perform microchip immunoaffinity electrophoresis to detect purified recombinant thymidine kinase 1 (TK1). Buffer with 1% methylcellulose acted as a dynamic coating that minimized nonspecific adsorption of protein and as sieving matrix that enabled separation of free antibody from antibody-TK1 complexes. Using this technique, I was able to detect TK1 concentration >80 nM and obtained separation results within 1 minute using a 5 mm effective separation length. Detection of endogenous TK1 in serum is difficult because TK1 is present at the pM range. I compared three different depletion methods to eliminate high abundance immunoglobulin and human serum albumin. Cibacron blue columns depleted abundant protein but also nonspecifically bound TK1. I found that ammonium sulfate precipitation and IgG/albumin immunoaffinity columns effectively depleted high abundance proteins. TK1 was salted out of the serum with saturated ammonium sulfate and still maintained activity. To integrate affinity columns in microfluidic devices, I have developed a fast and easy strategy for initial optimization of monolith affinity columns using bulk polymerization of multiple monolith solutions. The morphology, surface area, and porosity, were qualitatively assessed using scanning electron microscopy. This method decreased the time, effort, and resources compared to in situ optimization of monoliths in microfluidic devices. This strategy could be used when designing novel formulations of monolith columns. I have also integrated poly(ethylene glycol dimethacrylate-glycidyl methacrylate) monolith affinity columns in polymer microfluidic devices to demonstrate the feasibility of extracting human interleukin 8 (IL8), a cancer biomarker, from saliva. Initial results have shown that the affinity column (~3 mm) was successfully integrated into the devices without prior surface modification. Furthermore, anti-IL8 was immobilized on the surface of the monolith. Electrochromatograms showed that 1 ng/mL of IL8 can be detected when in buffer while 10 ng/mL was detected when IL8 was spiked in saliva. Overall, these findings can be used to further develop immunoassays in microfluidic platforms, especially for analyzing biological fluids.

The need for miniaturized, portable devices to separate and detect unknown compounds has greatly multiplied, leading to an increased interest in microfluidics. Total integration of the detector and pump are necessary to decrease the overall size of the microfluidic device. Using previously developed thin film technologies, an electroosmotic flow (EOF) pump was incorporated in a microfluidic liquid chromatography device. An EOF pump was chosen because of its simple design and small size. EOF pumps fabricated on silicon and glass substrates were evaluated. The experimental flow rates were 0.19-2.30 microliters/minute for 40-400 V. The theoretical pump efficiency was calculated along with the generated mechanical power by various pump shapes to elucidate more efficient pump designs. To better understand the EOF on plasma enhanced chemical vapor deposition (PECVD) silicon dioxide, the zeta potential was investigated. PECVD oxide is amorphous and less dense than thermal silicon dioxide, which slightly changes the zeta potential. Zeta potentials were found for pH values from 2.6 to 8.3. Also, surface defects that affect the zeta potential were observed, and procedures to detect and prevent such defects were proposed. Finally, surface modifications to the microfluidic device were attempted to demonstrate that thin film EOF pumps can be used in the liquid chromatographic separation of mixtures. The microfluidic separation channel was coated with chlorodimethyloctadecylsilane, however, due to problems with channel filling and reservoir adhesives, separation was not achieved. The use of new adhesives and external pumps were proposed to resolve these problems for future testing. Also new methods to combine EOF pumps with microfluidic channels and on-chip detectors were suggested.

No description available.

No description available.

La microscopie STED (stimulated emission depletion ou déplétion par émission stimulée) est une des méthodes les plus répandues de microscopie de super-résolution. Dans un microscope STED, un faisceau en anneau se superpose avec le faisceau d'excitation et éteint les fluorophores en périphérie du faisceau d'excitation par émission stimulée. Au centre de l'anneau, où le faisceau STED a une intensité nulle, la fluorescence reste intacte. Cette technique nécessite un montage complexe dans lequel deux faisceaux laser, en général issus de deux sources différentes, doivent être parfaitement alignés et superposés. Dans ce travail de thèse, nous proposons deux configurations STED qui ont pour but de simplifier le montage et de réduire le coût total d'un tel système. L'idée de base dans les deux cas est d'utiliser la même source laser à la fois pour l'excitation et la déplétion par émission stimulée. Dans la première configuration, nous avons développé une source bicolore originale basée sur un laser Nd-YAG microchip. Ce laser microchip délivre simultanément des impulsions sub- ns à deux longueurs d'onde, 355 nm (excitation) et 532 nm (déplétion), qui sont générés par conversion harmonique à partir d'une émission laser Nd-YAG et offrent l'avantage d'être intrinsèquement alignées et synchronisées. Afin de trouver des colorants appropriés pour cette source particulière, nous avons développé une méthode de caractérisation et testé différents colorants Nous avons construit un microscope à partir de cette source laser et obtenu des images avec une résolution améliorée. La réduction du volume d'excitation a été confirmée par spectroscopie de corrélation de fluorescence (FCS). Cependant, les aberrations chromatiques des optiques utilisées limitent les performances du montage actuel. Une perspective prometteuse serait de combiner le STED à la microscopie à feuille de lumière (SPIM), plus tolérante des défauts d'achromatisme, et nous montrons les premiers résultats de cette approche. Dans la seconde configuration, les aberrations chromatiques ne sont plus un problème puisqu' une seule longueur d'onde est utilisée pour l'excitation (par absorption à deux photons) et la déplétion. En jouant sur la durée de l'impulsion (et donc la valeur de l'intensité crête), un de ces deux procédés peut être favorisé. La fluorescence est excitée à deux photons par une impulsion femtoseconde, puis est éteinte par émission stimulée à un photon avec une impulsion étirée. Nous avons utilisé une technique résolue en temps (Time-Correlated Single Photon Counting - TCSPC) pour étudier l'efficacité de déplétion du colorant DCM en solution. Les simulations numériques montrent que cette méthode peut être appliquée à la microscopie de super résolution. En fin de cette partie, nous présentons les premières images obtenues avec un microscope développé au laboratoire qui permet l'excitation à deux photons et la déplétion à un photon avec une seule longueur d'onde, ainsi que l' amélioration de la résolution observée. Dans ce travail, nous avons donc mis en place expérimentalement, pour la première fois, deux concepts destinés à simplifier en utilisant deux sources laser originales. / Stimulated emission depletion (STED) is a well-known super-resolution method. In a STED microscope, a doughnut-shaped beam is superimposed with the excitation beam and keeps the fluorophores in the periphery of the excitation spot in a dark state by stimulated emission, thus effectively improving the spatial resolution in a scanning configuration. This technique requires a complex setup since two laser beams, generally from different sources need to be perfectly aligned. In this work we propose two STED configurations that will simplify the setup and reduce the total cost of such a system. The basic idea in both cases is to use the same laser source for both excitation and stimulated emission depletion. In the first setup we have developed an original two-color source based on a microchip Nd-YAG laser. This microchip laser simultaneously delivers sub-ns pulses at two wavelengths, 355 nm (excitation) and 532 nm (depletion), which are generated by harmonic conversion from an Nd-YAG laser emission and offer the advantage of being intrinsically aligned and synchronized. Further work consisted in determining suitable dyes for this particular source. We have built a microscope setup based on this laser source and obtained images with an improved resolution. The confirmation of the reduction of the excitation volume is showed by Fluorescence Correlation Spectroscopy (FCS) measurements. However, the performance of this system is limited by chromatic aberrations. The combination of Selective Plane Illumination Microscopy (SPIM) with STED is considered. In the second setup the chromatic aberrations are no longer a problem since the same wavelength is used for two photon excitation and one photon depletion. By playing on the duration of the pulse (thus the instantaneous intensity), one of these two processes can be favored. Fluorescence was excited by two photon absorption with a femtosecond pulse, then depleted by one photon stimulated emission with a stretched pulse. We used the Time Correlated Single Photon Counting (TCSPC) method to study the depletion efficiency of DCM dye in solution and numerical simulations show that this method can be applied to super-resolved microscopy. In the end we present the preliminary images obtained with a home-built Two-photon Single wavelength STED microscope and the resolution improvement obtained. Further improvements are to be made to the custom microscope. In this work we have experimentally implemented, for the first time, two concepts meant to simplify the STED setups by using original sources.

Neste trabalho são apresentados processos de microfabricação de estruturas contendo microcanais e sistemas de manipulação hidrodinâmica e eletroosmótica de fluídos. Foram desenvolvidos processos de microfabricação utilizando toner sobre poliéster, toner sobre vidro, toner como resiste, além de métodos alternativos de perfuração de lâminas e selagem de microestruturas em vidro, desenvolvimento de microestruturas para eletroforese capilar e espectrometria de massas com ionização por eletronebulização. A caracterização dos materiais e processos permitiu uma ampla visão das potencialidades e alternativas dos processos de microfabricação, tendo sido demonstrado que os dispositivos produzidos em toner-poliéster são quimicamente resistentes às substâncias tipicamente utilizadas em eletroforese capilar. Neste trabalho, um detector condutométrico sem contato foi implementado em microestruturas de toner-poliéster e a separação eletroforética de alguns metais alcalinos é demonstrada. A microestrutura foi projetada no formato padrão em cruz, tendo o canal de separação 22 mm de comprimento, 12 µm de profundidade e largura típica. A cela condutométrica foi construída sobre o canal de separação utilizando-se fita adesiva de cobre (1 mm de largura) como eletrodos. O sinal aplicado na cela foi de 530 kHz e 10 Vpp . A separação de K+, Na+ e Li+ na concentração de 100 µmol L-1 foi efetuada em torno de 0,8 min, utilizando-se 1 kV como potencial de separação. Foram desenvolvidos microchips para análise por espectrometria de massas com introdução de amostra por eletronebulização, sendo determinado cluster do íon cloreto em concentração de 1 mmol L+. Também solução com 1 mmol/L de glucosamina em água/metanol 1: 1 (v/v), sob corrente de 100 nA gerou sinal estável e livre de descarga corona. Utilizando detecção amperométrica, obteve-se eletroferogramas mostrando a separação de iodeto (10 mmol L-1) e ascorbato (40 mmol L-1) em potencial de separação de 4,0 kV (800 V cm-1 potencial de detecção de 0,9 V (vs. Ag/AgCI), injeção com 1,0 kV/1°s, tampão borato de sódio 10 mmol L+ com CTAH 0,2 mmol L-1, pH 9,2. Obteve-se eficiência de 1,6.104 pratos/m e foi possível obter limites de detecção de 500 nmol L-1 (135 amol) e 1,8 µmol L-1 (486 amol) para iodeto e ascorbato, respectivamente. O processo de fabricação utilizando toner como material estrutural para microchips em vidro foi bem estabelecido, assim como os modos de detecção fotométrico e condutométrico foram demonstrados. Foram obtidos eletroferogramas par detecção condutométrica sem contato de solução 200 µmol L-1 de K+, Na+ e U+, em tampão histidina/ácido lático 30 mmol L-1 9:1 (v/v) água:metanol, injeção eletrocinética de 2,0 kV/5,0 s, potencial de separação de 1 kV, 530 kHz de frequência e tensão de 2,0 Vpp. Também foi implementado um sistema de detecção fotométrico para microchip operando em 660 nm, tendo sido utilizado para a detecção de azul de metileno 1,0 mmol L-1 em tampão de corrida de barato de sódio 20 mmol L-1 (pH 9,2), com o detector posicionado a 40 mm do ponto de injeção e com injeção eletrocinética a 2,0 kV por 12 s com picos bem resolvidos em menos de 1 min. / Microfabrication processes and devices for hydrodynamic and electroosmotic manipulation were developed based on toner-polyester, toner-glass and toner-as-resist techniques. Additionally, techniques to perforate glass slides and sealing of glass devices were introduced. Microdevices for capillary electrophoresis and electrospray for mass spectrometry were developed using these techniques. The characterization of the materiais and the processes demonstrated that the devices obtained by the toner-polyester process are compatible with the media used for capillary electrophoresis. The detection of alkaline ions with capillary electrophoresis with contactless conductivity detection was demonstrated. The typical cross shape microstructure was designed with a 22-mm long and 12-µm deep separation channel. The conductivity cell was implemented with 1-mm wide adhesive copper stripes. The applied signal was 530kHz and 10Vpp . The separation of 100µmo1L-1 K+, Na+, and Li+ was accomplished in 0.8 min under a voltage of 1 kV. Another toner-polyester microchip was developed to demonstrate its usefulness for electrospray/mass spectrometry. Solutions of 1 mmol L-1 potassium chloride and 1 mmol L-1 glucosamine in water/methanol 1:1 (v/v) were introduced with stable current of 100 nA without corona discharge. Capillary electrophoresis with amperometric detection was also demonstrated. The separation of iodide (10 mmol L-1) and ascorbate (40 mmol L-1) was carried out at 4.0 kV (800 V cm-1) with detection potential of 0.9 V (vs. Ag/AgCl), electrokinetic injection at 1.0 kV/10 s, running buffer of sodium borate 10 mmol L-1 with CTAH 0.2 mmol L-1 , pH 9.2. The efficiency was 1.6.104 plates/m and the limits of detection were 500 nmol L-1 (135 9mol) and 1.8 µmol L-1 (486 amol) for iodide and ascorbate, respectively. The toner-glass process was proposed and conductivity and photometric detections were demonstrated for the devices generated by this new technique. The separation of 200 pmol L-1 K+, Na+, and Li+ was achieved in buffer histidine/lactic acid 30 mmol L-1 water/methanol 9: 1 (v/v), electrokinetic injection at 2.0 kV/5.0 s, separation potential of 1 kV, and contactless conductivity detection at 530 kHz and 2.0 Vpp. The photometric detection of methylene blue at 660 nm was carried out in sodium borate 20 mmol L-1 (pH 9.2).

Neste trabalho são apresentados processos de microfabricação de estruturas contendo microcanais e sistemas de manipulação hidrodinâmica e eletroosmótica de fluídos. Foram desenvolvidos processos de microfabricação utilizando toner sobre poliéster, toner sobre vidro, toner como resiste, além de métodos alternativos de perfuração de lâminas e selagem de microestruturas em vidro, desenvolvimento de microestruturas para eletroforese capilar e espectrometria de massas com ionização por eletronebulização. A caracterização dos materiais e processos permitiu uma ampla visão das potencialidades e alternativas dos processos de microfabricação, tendo sido demonstrado que os dispositivos produzidos em toner-poliéster são quimicamente resistentes às substâncias tipicamente utilizadas em eletroforese capilar. Neste trabalho, um detector condutométrico sem contato foi implementado em microestruturas de toner-poliéster e a separação eletroforética de alguns metais alcalinos é demonstrada. A microestrutura foi projetada no formato padrão em cruz, tendo o canal de separação 22 mm de comprimento, 12 µm de profundidade e largura típica. A cela condutométrica foi construída sobre o canal de separação utilizando-se fita adesiva de cobre (1 mm de largura) como eletrodos. O sinal aplicado na cela foi de 530 kHz e 10 Vpp . A separação de K+, Na+ e Li+ na concentração de 100 µmol L-1 foi efetuada em torno de 0,8 min, utilizando-se 1 kV como potencial de separação. Foram desenvolvidos microchips para análise por espectrometria de massas com introdução de amostra por eletronebulização, sendo determinado cluster do íon cloreto em concentração de 1 mmol L+. Também solução com 1 mmol/L de glucosamina em água/metanol 1: 1 (v/v), sob corrente de 100 nA gerou sinal estável e livre de descarga corona. Utilizando detecção amperométrica, obteve-se eletroferogramas mostrando a separação de iodeto (10 mmol L-1) e ascorbato (40 mmol L-1) em potencial de separação de 4,0 kV (800 V cm-1 potencial de detecção de 0,9 V (vs. Ag/AgCI), injeção com 1,0 kV/1°s, tampão borato de sódio 10 mmol L+ com CTAH 0,2 mmol L-1, pH 9,2. Obteve-se eficiência de 1,6.104 pratos/m e foi possível obter limites de detecção de 500 nmol L-1 (135 amol) e 1,8 µmol L-1 (486 amol) para iodeto e ascorbato, respectivamente. O processo de fabricação utilizando toner como material estrutural para microchips em vidro foi bem estabelecido, assim como os modos de detecção fotométrico e condutométrico foram demonstrados. Foram obtidos eletroferogramas par detecção condutométrica sem contato de solução 200 µmol L-1 de K+, Na+ e U+, em tampão histidina/ácido lático 30 mmol L-1 9:1 (v/v) água:metanol, injeção eletrocinética de 2,0 kV/5,0 s, potencial de separação de 1 kV, 530 kHz de frequência e tensão de 2,0 Vpp. Também foi implementado um sistema de detecção fotométrico para microchip operando em 660 nm, tendo sido utilizado para a detecção de azul de metileno 1,0 mmol L-1 em tampão de corrida de barato de sódio 20 mmol L-1 (pH 9,2), com o detector posicionado a 40 mm do ponto de injeção e com injeção eletrocinética a 2,0 kV por 12 s com picos bem resolvidos em menos de 1 min. / Microfabrication processes and devices for hydrodynamic and electroosmotic manipulation were developed based on toner-polyester, toner-glass and toner-as-resist techniques. Additionally, techniques to perforate glass slides and sealing of glass devices were introduced. Microdevices for capillary electrophoresis and electrospray for mass spectrometry were developed using these techniques. The characterization of the materiais and the processes demonstrated that the devices obtained by the toner-polyester process are compatible with the media used for capillary electrophoresis. The detection of alkaline ions with capillary electrophoresis with contactless conductivity detection was demonstrated. The typical cross shape microstructure was designed with a 22-mm long and 12-µm deep separation channel. The conductivity cell was implemented with 1-mm wide adhesive copper stripes. The applied signal was 530kHz and 10Vpp . The separation of 100µmo1L-1 K+, Na+, and Li+ was accomplished in 0.8 min under a voltage of 1 kV. Another toner-polyester microchip was developed to demonstrate its usefulness for electrospray/mass spectrometry. Solutions of 1 mmol L-1 potassium chloride and 1 mmol L-1 glucosamine in water/methanol 1:1 (v/v) were introduced with stable current of 100 nA without corona discharge. Capillary electrophoresis with amperometric detection was also demonstrated. The separation of iodide (10 mmol L-1) and ascorbate (40 mmol L-1) was carried out at 4.0 kV (800 V cm-1) with detection potential of 0.9 V (vs. Ag/AgCl), electrokinetic injection at 1.0 kV/10 s, running buffer of sodium borate 10 mmol L-1 with CTAH 0.2 mmol L-1 , pH 9.2. The efficiency was 1.6.104 plates/m and the limits of detection were 500 nmol L-1 (135 9mol) and 1.8 µmol L-1 (486 amol) for iodide and ascorbate, respectively. The toner-glass process was proposed and conductivity and photometric detections were demonstrated for the devices generated by this new technique. The separation of 200 pmol L-1 K+, Na+, and Li+ was achieved in buffer histidine/lactic acid 30 mmol L-1 water/methanol 9: 1 (v/v), electrokinetic injection at 2.0 kV/5.0 s, separation potential of 1 kV, and contactless conductivity detection at 530 kHz and 2.0 Vpp. The photometric detection of methylene blue at 660 nm was carried out in sodium borate 20 mmol L-1 (pH 9.2).

Doctor of Philosophy / Department of Chemistry / Christopher T. Culbertson / High-performance liquid chromatography (HPLC) and capillary electrophoresis (CE) are two well-established analytical separation techniques that are continuously being adapted for performing distinctive separations and analyses of multitudes of complex and/or unique samples. Since their introduction, these techniques have been pivotal in the discovery, analysis, and understanding of a variety of samples and still prove to be key analytical tools for biological investigation. Using these techniques, one can obtain a wide-range of valuable sample information from the hydrophobicity and molecular weights to size and charge distributions. Furthermore, these techniques allow for sample analysis, purification, and collection for additional sample analysis, such as mass spectrometry analysis. My doctoral dissertation encompasses the full scope of these two techniques and novel approaches for the investigation of distinct, relevant samples. Described herein is the fabrication of glass microfluidic devices used for CE and their diversity for numerous investigations. Chapter 2 shows that the resolution of the photomasks used in microchip fabrication does not alter the separation efficiency of the devices, as the separations remain diffusion-limited. Using an in-house built capillary electrophoresis system, wheat proteins were separated more than 25% faster than previously reported in literature, and the electropherograms used for sample varietal identification. The fabrication of a robust, portable CE system capable of performing biological analysis in microgravity and hypergravity environments is also discussed. The need for and features necessary to achieve a reliable, robust, automated system is further described in Chapter 4. Isolation and analysis of the pea aphid (Acyrthosiphon pisum) salivary secretions was completed for the first time using HPLC. By altering the aphid environment and the sample treatment parameters, sample concentrations were increased above the limit of detection. Coupled with mass spectrometry, identification of pea aphid salivary proteins such as exopeptidase, angiotensin converting enzyme, and Buchnera proteins has been achieved. Finally, a simplified contact conductivity detection system for the detection of jurkat cells was developed that surpasses current, complex optical systems. The experiments described in this dissertation demonstrate novel approaches for the preparation, separation, analysis, and identification of a wide variety of common, and uncommon, samples.

Master of Science / Department of Chemistry / Christopher T. Culbertson / Chemical separation involves selective movement of a component out of a region shared by multiple components into a region where it is the major occupant. The history of the field of chemical separations as a concept can be dated back to ancient times when people started improving the quality of life by separation of good materials from bad ones. Since then the field of chemical separation has become one of the most continually evolving branches of chemical science and encompasses numerous different techniques and principles. An analytical chemist’s quest for a better way of selective identification and quantification of a component by separating it from its mixture is the cause for these ever evolving techniques. As a result, today there are numerous varieties of analytical techniques for the separation of complex mixtures. High Performance Liquid Chromatography (HPLC), Gas Chromatography (GC), Capillary Electrophoresis (CE) and Gel Electrophoresis are a few out of a long list. Each these techniques manipulates the different physical and chemical properties of an analyte to achieve a useful separation and thus certain techniques will be suited for certain molecules. This work primarily focuses on the use of Capillary Electrophoresis as a separation technique. The mechanism of separation in Capillary Zone Electrophoresis and principles of UV detection will discussed in chapter one. Chapter two contains a discussion about the application of Capillary Electrophoresis (CE) on microfluidc devices. This will include sections on: microfabrication techniques of PDMS and photosensitized PDMS (photoPDMS), a UV detector for microfluidic devices and its application for the detection of wheat proteins. In Chapter three we report the experimental part of this project which includes; investigations on the effect of UV exposure time and thermal curing time on feature dimensions of photoPDMS microfluidic device, investigations on the injection and separation performances of the device, characterization of a UV detector set up and its application for the separation and detection of wheat gliadin proteins. The results of these investigations are presented in chapter four.