Different sample preparation steps prior to a detection method are often essential in analytical chemistry. In this thesis, both static extractions and on-line coupled solid-phase extractions have been studied in combination with different detection techniques. Aspects of performing sample preparations in miniaturized analytical devices and the development of poly(dimethylsiloxane) (PDMS) microchips are discussed. Polypyrrole was also evaluated as an electrochemically controllable stationary phase for solid-phase microextraction (SPME) and solid-phase extraction (SPE). The first part of this thesis describes the extraction of an organic compound from a very complex solid matrix utilizing the pressurized-fluid extraction (PFE) technique. The presented results show that PFE is easily optimized and enables rapid extractions and extracts relatively free from interferences. An integrated three-electrode device, which enabled electrochemical (EC) SPME under potential control, was developed. With this device, both anions and cations could be extracted employing two types of polypyrrole films. Planar micro band electrodes positioned at the end of a capillary were also used to electrochemically extract and detect anions in a miniaturized flow system. Different analyte concentrations and preconcentration times were examined, and good linear correlations were found between the extraction time and the detection response. The on-line coupling of a thin layer EC cell, with a polypyrrole coated working electrode, to different mass spectrometric (MS) techniques is also described and evaluated. The results show that EC-SPE, employing polypyrrole as stationary phase, can be used as a preconcentration step prior to detection. In addition, this thesis describes the development and on-line coupling of a microelectrode array equipped PDMS microchip with an integrated graphite electrospray emitter to electrospray ionization (ESI) MS. The system enabled short transfer times and an EC conversion efficiency of 30% at a flow rate of 0.5 μL/min. The on-line EC/ESI-MS experiments were significantly simplified using a wireless Bluetooth battery-powered EC instrument.

A produção de microssistemas de análises totais (µTAS) tem sido objeto de esforços intensos pela comunidade científica. A necessidade de produção de uma plataforma que realize extração, amplificação e separação de DNA--um verdadeiro \"lab on a chip\"--é impulsionada pelas vantagens associadas com as análises em plataformas miniaturizadas. Esta Tese foca no desenvolvimento de métodos para análises de DNA em dispositivos microfluídicos que podem ser associados em µTAS. Inicialmente, foi feito o desenvolvimento de um novo método de extração em fase sólida em que a eficiência de extração depende da manipulação magnética das partículas e não do fluxo da solução através da fase sólida. A utilidade desta técnica em isolar DNA puro de alta qualidade (amplificável) a partir de uma amostra biológica complexa foi demonstrada através da purificação de DNA a partir de sangue total e a subsequente amplificação do fragmento do gene β-globina. A técnica descrita é rápida, simples e eficiente, permitindo uma recuperação de mais de 60% de DNA a partir de 600 nL de sangue em concentração suficiente para amplificação via reação em cadeia da polimerase (PCR). Após o desenvolvimento da extração dinâmica de DNA em fase sólida (dSPE) em microchip de vidro, o método foi adaptado para o uso em microchips de poliéster-toner (PT). Além da extração, a amplificação e separação de DNA também foram realizadas em microchips de PT. O processo convencional de fabricação dos dispositivos de PT produz canais com 12 µm de profundidade. Este trabalho descreve um novo processo de fabricação dos microchips de PT com canais mais profundos. Uma cortadora a laser de CO2 é usada para definir a estrutura desejada no filme de poliéster recoberto com toner. Estes filmes de poliéster recobertos com toner e os canais recortados são utilizados com partes intermediárias no microchip. A tampa e a base (filmes de poliéster) são laminadas juntamente com as partes intermediárias. Desta forma microchips com canais mais profundos podem ser criados. Microchips com 4 filmes de poliéster (base, tampa, e dois filmes centrais) foram utilizados para realizar dSPE. Estes microchips possuem canais com ~270 µm de profundidade. A dSPE adaptada para os microchips de PT demonstrou ser capaz de extrair eficientemente DNA (~65%), e o DNA purificado apresentou qualidade suficiente para PCR. A PCR realizada em microchips de PT demonstrou que os dispositivos de PT são compatíveis com os reagentes da PCR e o sucesso da reação de PCR foi demonstrado através da amplificação do fragmento de 520 pares de bases do λ-DNA. A possibilidade de manipular diferentes soluções que são necessárias para realizar a extração e a PCR demonstra o grande potencial desta plataforma para realizar análises genéticas. Além da extração e amplificação, a separação também foi demonstrada nos dispositivos de PT. Duas integrações foram feitas nos microchips de PT, dSPE-PCR e PCR-separação. Na primeira integração a dSPE e PCR foram realizadas em uma única câmara, e a amplificação do fragmento de 520 pb do λ-DNA foi demonstrada. Na segunda integração, o dispositivo foi fabricado com espessuras diferentes para os diferentes domínios. No domínio da PCR as câmaras possuem profundidade de ~270 µm de profundidade, e para o domínio da eletroforese os canais apresentam 12 µm de profundidade. A integração realizada sem válvulas foi demonstrada através da amplificação e detecção do fragmento de 520 pb do λ-DNA em um mesmo microchip. Este trabalho demonstra o grande potencial dos microchips de PT para produzir dispositivos descartáveis totalmente integrados para análise genética. / Efforts to develop a microfluidic-based total analysis system (µTAS) have been intense in the scientific community. The goal of achieving a device comprising DNA extraction, amplification, and detection in a single device, a true \"lab on a chip,\" is driven by the substantial advantages associated with such a device. This Thesis focus on development of methods for DNA analysis on microdevices, that can be associated with µTAS. Sequentially, the first step was the development of a novel solid-phase extraction technique in which DNA is bound and eluted from magnetic silica beads in a manner that efficiency is dependent on the magnetic manipulation of the beads and not on the flow of solution through a packed bed. The utility of this technique in the isolation of reasonably pure, PCR-amplifiable DNA from complex samples is shown by isolating DNA from whole human blood, and subsequently amplifying a fragment of the β-globin gene. The technique described here is rapid, simple, and efficient, allowing for recovery of more than 60% of DNA from 600 nL of blood at a concentration which is suitable for PCR amplification. The second step was the use of polyester-toner (PT) microchips for DNA analysis (extraction, PCR and separation). The laser-printing of toner onto polyester films has been shown to be effective for generating PT microfluidic devices with channel depths on the order of 12 µm. We describe a novel and innovative process that allows for the production of multilayer PT microdevices with substantially larger channel depths. Utilizing a CO2 laser to create the microchannel in polyester sheets containing a uniform layer of printed toner, multilayer devices can easily be constructed by sandwiching the channel layer between uncoated cover sheets of polyester containing precut access holes. The process allows for the fabrication of channels several hundred microns in depth, with ~270 µm deep microchannels utilized here to demonstrate the effectiveness of multilayer PT microchips for dynamic solid phase extraction (dSPE) and PCR amplification. Dynamic SPE adapted for PT microchip was able to recover more than 65% of DNA from 600 nL of blood and the DNA was compatible with downstream microchip-based PCR amplification. The compatibility of PT microchips was demonstrated by successful amplification of a 520 bp fragment of λ-phage DNA. The ability to handle the diverse chemistries associated with DNA purtification and extraction is a testimony to potential utility of PT microchips beyond separations, and presents a promising new platform for genetic analysis that is low cost and easy to fabricate. Two integrations were carrying out on PT microchip, dSPE - PCR and PCR-ME. The first integration was made in a single chamber and the amplification of 520 bp fragment of λ-phage was demonstrated. The second integration describes a process that allows the production of a multidomain microchip with different channel depths for the different domains for genetic analysis. The final device was made by the conventional sandwiching of the four polyester films of the PCR domain with the two polyester films for the electrophoresis domain. The successful valveless integration of PCR and separation was demonstrated by amplification and detection of a 520 bp fragment of λ-phage DNA. This work shows the enormous potential of PT microchips to be used for total genetic analysis.

Dans cette thèse, nous nous intéressons à la problématique de l’intégration de capteurs plasmoniques performants et bas coût sur des dispositifs de type smartphone, en vue d’applications de diagnostic biomédical. A cette fin, nous proposons deux biocapteurs « smart ». Premièrement, un système de détection colorimétrique à base de nanoparticules d’or est mis en œuvre pour détecter de l’ADN. Le système intègre une détection synchrone logicielle mise en œuvre au sein du smartphone, où les signaux physiques transitent par la voie audio. Le processus de diagnostic prend moins de 15 minutes pour une limite de détection de 0.77 nM, approximativement 6 fois meilleure que la sensibilité usuelle d’un spectromètre UV-Vis conventionnel, à temps de mesure identique. Dans une seconde partie, un capteur à résonance plasmon de surface en configuration de Kretschmann, se distinguant par une sensibilité à la phase optique, est développé. Le design monolithique et compact repose sur un interféromètre à dédoublement latéral et une modulation de phase. Le contrôle et la lecture du prototype s’effectue également par smartphone. La modulation de phase est de type sinusoïdale et une sensibilité importante est obtenue, autour de 2,3 10-6 RIU avec une dynamique de 7 10-3 RIU, chiffres obtenus pour une puce optique standard et un temps d’intégration de 100 ms. Ce second dispositif est ensuite testé pour la détection de protéines (Troponine I cardiaque), en fonctionnalisant la surface par ADN Tro4 / In this thesis, we investigate the possibility and potential for integration of portable optical biosensor for diagnostic purposes. To this end, we propose two “smart” biosensor systems. In the first part of this thesis, a DNA biosensor combining single-wavelength colorimetry and digital Lock-in Amplifier within a smartphone is proposed. Utilizing full advantage of audio channel and digital signal processing capacity of a smartphone, we have built a handheld DNA AuNp colorimetry biosensor. Based on the results, the diagnostic process takes only 15 minutes of reaction time while offering a limit of detection around 0.77 nM which is 6 times better than a desktop UV-Vis spectrometer.In second part of the thesis, a Shearing interferometer based Surface Plasmon Resonance (SiSPR) biosensor is proposed. SiSPR allows for phase sensitive detection on conventional Kretschmann configuration. Its monolithic design reduces optical parts, costs and allows portable application. The essence of SiSPR is a reflective layer in addition to plasmonic layer. To extract phase information from SiSPR, a sinusoidal phase modulation is achieved by modulation of the laser injection current. For a 100 ms measurement and a standard optical chip, the sensitivity of the SiSPR is around 2.3x10-6 RIU with a dynamic range of 7.0x10-3 RIU, which is better than amplitude SPR devices. Finally, Tro4 DNA surface modification on the SiSPR chip is demonstrated for future cardiac Troponin I diagnostic

This thesis deals with design and implementation of flexible modular electronic stopwatch system for use in resport. One of the advantages of the system is possibility of using wireless communication with sensors and displays. Developed system consist of main control unit, external display, wireless modules for communication with the sensors and desktop application that collects data and controls the system. Solution of system components, communication between them and method of time synchronization of wireless devices is also described in this thesis.

This Diploma thesis deals with creation and realisation of extensions for medical dental unit Vizio. Specifically, it deals with electrotechnical solution and software for capacitive keyboard and with proposal of principle of control and regulation of speed for integrated brushless motor. The Diploma thesis contains both the description of used hardware components, and software solution implemented in assembly language. This device is already in commercial use.

The aim of master thesis is implementation of USB host in a microcontroller PIC. The thesis contains description of ways of peripherals to computer connections with detailed description of USB bus. There are also discussed several approaches in implementations USB host for embedded systems. A concept of layered USB host system is designed on the basis of analysis of the state of the art. Finally, the designed system is implemented in microcontroller. Resulting firmware of development kit is tested on real USB device.

This dissertation work deals with the subject of a company in the area of e-commerce, which represents short-term or long-term plans in controlling the financial relationship toward the outer environment but also inside the company. The problem of possible abuse of credit cards is briefly analysed as well. The impulse for choosing this topic was increasing number of falsification and abusing of credit cards. When this subject is analysed in detail, the problem connected with lack of information on this particular area of crime inevitably appears. Clients of individual banking institutions often do not have even the basic knowledge about the ways their cards could be abused or misused. This is why the question of certain, at least minimal, basic information campaign done by the banks, which are supposed to protect the financial means of their clients. This dissertation work sets its goal in creating the methods of investigation of credit cards abuse not only in the Czech Republic but in the growing system of countries of Schengen Treaty and European Union. The benefit of the work can be seen in the level of theory as well of practice. The theoretical part of the work contains the analysis of the current situation of scientific knowledge in the area of information and communication technology of companies, which is implemented into information systems. Then the advantages and disadvantages connected with this kind of e-commerce are assessed and the specific opportunities for abuse of these media of payment are characterised. The problem sees mainly in writing a specialised publication, which would deal with the problem of abuse and falsification of credit cards and its possible use would be at police academies. Its main part should be the characteristics of particular forms of cards forging and also possible identification of these forgeries. This book could also help in prevention and inform clients of financial institutions about the ways of credit cards abuse. In the practical part, which concentrates on the Czech and foreign market, the author sets the basic premises for realisation of safe business of companies in the Czech Republic and its procedures. Furthermore the reasons of e-commerce abuse in the Czech Republic are analysed and results of quantification research presented. This was concentrated on ascertainment of reason of low interest in information among the public. In the conclusive part of the work the whole problem is summarised and there are also outlined perspectives and possibilities of further development

Electrophoretic microsystems (EM) are powerful tools for the separation of species of microsystems analyzes which can easily be combined with electrochemical detection (ECD) and therefore making it ideal for a method of detection. However, the influence of high voltage at the working electrode used for the separation is a problem to be overcome due to the increased signal/noise ratio and possible damage of the electrode and/or the potentiostat. Thus, it was proposed in this thesis one EM hybrid PDMS / glass configuration with dual-channel potentiostat coupled to an electrically isolated in order to minimize the influence of high potential in the separation channel and improve the separation efficiency of the species and subsequently, improve detection limits. The EM contains two separate parallel channels 200 microns and a channel separation and another reference, and each containing a platinum electrode 15 or 50 μm placed about 1 to 4 μm in the channel. An electrode served as the working electrode, positioned in the separation channel, and another electrode as reference electrode, placed in the reference channel. This configuration associated with the electrically isolated potentiostat allowed the amperometric signals were measured without any change or potential interference arising from the high voltage applied separation. Aiming to evaluate the effectiveness of the methodology proposed in this thesis, samples nitrite, tyrosine and peroxynitrite (reactive nitrogen species – RNS), hydrogen peroxide (reactive oxygen species – ROS), ascorbic acid, glutathione and cysteine were injected into the channel containing the working electrode, while simultaneously boric acid buffer pH 11 containing TTAB was injected into the reference channel containing the reference electrode. From this configuration, we obtained a significant reduction in noise level (about 0.94 pA) and a relative improvement in the resolution ratified by electropherograms, compared with using single channel configuration. The limits of detection (LOD) for the chemical species mentioned above were 0.58 μM, 0.14 μM, 0.75 μM, 0.21 μM, 0.82 μM, was not obtained for cysteine and 1.63 μM, respectively. The efficiency can also be seen by analyzing nitrite performed on samples of perfusate blood of sheeps and rats, where have been detected a concentration of 68.05 μM and 22.04 μM, respectively, by the proposed method. It was also proposed in this thesis, microfabrication and evaluation of a PMMA electrophoretic microsystem with single channel configuration coupled to a base made of the same material to fix the microchip with electrochemical detection using a carbon paste electrode. The purpose of the construction of the base was to obtain, by fixing, reproducibility of events. And the microfabrication of PMMA EM aimed the viability of its use in analysis perspective as having the lowest cost per unit made due to the use of CO2 laser for microfabrication, which has a value considerably lower, compared with photolithographic processes. The evaluation of this system was performed through the analysis standards of serotonin and acetaminophen, which proved that the microfabrication of this system showed good reproducibility and repeatability of events, making it viable processing. / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Os microssistemas eletroforéticos (MSE) são ferramentas poderosas para a separação de espécies em microssistemas de análises, onde pode ser facilmente combinada com detecção eletroquímica (DEQ) e tornando-se, portanto, um método de detecção ideal. No entanto, a influência da alta tensão no eletrodo de trabalho utilizada para a separação é um problema a ser contornado devido o aumento da relação sinal/ruído e possíveis danificações do eletrodo e/ou do potenciostato. Assim, foi proposto nesta tese um MSE híbrido de PDMS/vidro com configuração de duplo-canal acoplado a um potenciostato eletricamente isolado com objetivo de minimizar a influência do elevado potencial no canal de separação e melhorar a eficiência de separação das espécies e, subsequentemente, melhorar os limites de detecção. O MSE contém dois canais paralelos separados 200 μm, sendo um canal de separação e outro de referência, e cada um deles contendo um eletrodo de platina de 15 ou 50 μm colocados cerca de 1 a 4 μm dentro do canal. Um eletrodo serviu como eletrodo de trabalho, posicionado no canal de separação, e o outro eletrodo como eletrodo de referência, posicionado no canal de referência. Essa configuração associado ao potenciostato eletricamente isolado permitiu que os sinais amperométricos fossem medidos sem qualquer mudança de potencial ou de interferência oriunda da alta tensão de separação aplicada. Objetivando avaliar a eficiência da metodologia proposta nessa tese, amostras de nitrito e peroxinitrito (espécies reativas de nitrogênio – ERN), tirosina, peróxido de hidrogênio (espécie reativa de oxigênio – ERO), ácido ascórbico, glutationa e cisteína foram injetadas no canal contendo o eletrodo de trabalho, enquanto que simultaneamente o tampão de ácido bórico contendo TTAB pH 11 foi injetado no canal de referência contendo o eletrodo de referência. A partir desta configuração, obteve-se uma significativa diminuição no nível de ruído (cerca de 0,94 pA) e uma relativa melhora na resolução ratificadas pelos eletroferogramas, se comparado com a configuração que utiliza canal único. Os limites de detecção (LOD) para as espécies químicas supracitados foram de 0,58 μM, 0,14 μM, 0,75 μM, 0,21 μM, 0,82 μM, não foi obtida para a cisteína, e 1,63 μM, respectivamente. A eficiência também pode ser vista através das análises de nitrito realizadas em amostras de perfusato de sangue de ovelhas e ratos, onde foram detectados uma concentração de 68,05 μM e 22,04 μM, respectivamente, através da metodologia proposta. Foi proposto também nessa tese, a microfabricação e avaliação de um microssistema eletroforético de PMMA com configuração de canal único acoplado a uma base feita do mesmo material para fixar o microchip, com detecção eletroquímica usando eletrodo de pasta de carbono. O objetivo da construção da base foi obter, através da fixação, reprodutibilidade de eventos. E a microfabricação do MSE de PMMA objetivou a viabilidade do seu uso em análises tendo como perspectiva o baixo custo por unidade confeccionada devido ao uso de laser de CO2 para a microfabricação, o qual possui um valor agregado consideravelmente menor, se comparado com os processos fotolitográficos. A avaliação desse sistema foi feita através das análises de padrões de serotonina e acetaminofeno, onde comprovou-se que a microfabricação desse sistema apresentou boa reprodutibilidade e repetitividade de eventos, tornando-se viável o seu processamento.

The aim of the thesis is the design and realization of the oscilloscope module with wireless data transfer. One part of the thesis is a short overview of the different types of AD converters. The main part is focused on the hardware configuration that allows modification of the wireless module of the oscilloscope, and the possibility of using in the industrial zone. The design takes into account reducing of consumption and EMC compatibility.

Les études génétiques, telles que les études de liaison ou d’association, ont permis d’acquérir une plus grande connaissance sur l’étiologie de plusieurs maladies affectant les populations humaines. Même si une dizaine de milliers d’études génétiques ont été réalisées sur des centaines de maladies ou autres traits, une grande partie de leur héritabilité reste inexpliquée. Depuis une dizaine d’années, plusieurs percées dans le domaine de la génomique ont été réalisées. Par exemple, l’utilisation des micropuces d’hybridation génomique comparative à haute densité a permis de démontrer l’existence à grande échelle des variations et des polymorphismes en nombre de copies. Ces derniers sont maintenant détectables à l’aide de micropuce d’ADN ou du séquençage à haut débit. De plus, des études récentes utilisant le séquençage à haut débit ont permis de démontrer que la majorité des variations présentes dans l’exome d’un individu étaient rares ou même propres à cet individu. Ceci a permis la conception d’une nouvelle micropuce d’ADN permettant de déterminer rapidement et à faible coût le génotype de plusieurs milliers de variations rares pour un grand ensemble d’individus à la fois. Dans ce contexte, l’objectif général de cette thèse vise le développement de nouvelles méthodologies et de nouveaux outils bio-informatiques de haute performance permettant la détection, à de hauts critères de qualité, des variations en nombre de copies et des variations nucléotidiques rares dans le cadre d’études génétiques. Ces avancées permettront, à long terme, d’expliquer une plus grande partie de l’héritabilité manquante des traits complexes, poussant ainsi l’avancement des connaissances sur l’étiologie de ces derniers. Un algorithme permettant le partitionnement des polymorphismes en nombre de copies a donc été conçu, rendant possible l’utilisation de ces variations structurales dans le cadre d’étude de liaison génétique sur données familiales. Ensuite, une étude exploratoire a permis de caractériser les différents problèmes associés aux études génétiques utilisant des variations en nombre de copies rares sur des individus non reliés. Cette étude a été réalisée avec la collaboration du Wellcome Trust Centre for Human Genetics de l’University of Oxford. Par la suite, une comparaison de la performance des algorithmes de génotypage lors de leur utilisation avec une nouvelle micropuce d’ADN contenant une majorité de marqueurs rares a été réalisée. Finalement, un outil bio-informatique permettant de filtrer de façon efficace et rapide des données génétiques a été implémenté. Cet outil permet de générer des données de meilleure qualité, avec une meilleure reproductibilité des résultats, tout en diminuant les chances d’obtenir une fausse association. / Genetic studies, such as linkage and association studies, have contributed greatly to a better understanding of the etiology of several diseases. Nonetheless, despite the tens of thousands of genetic studies performed to date, a large part of the heritability of diseases and traits remains unexplained. The last decade experienced unprecedented progress in genomics. For example, the use of microarrays for high-density comparative genomic hybridization has demonstrated the existence of large-scale copy number variations and polymorphisms. These are now detectable using DNA microarray or high-throughput sequencing. In addition, high-throughput sequencing has shown that the majority of variations in the exome are rare or unique to the individual. This has led to the design of a new type of DNA microarray that is enriched for rare variants that can be quickly and inexpensively genotyped in high throughput capacity. In this context, the general objective of this thesis is the development of methodological approaches and bioinformatics tools for the detection at the highest quality standards of copy number polymorphisms and rare single nucleotide variations. It is expected that by doing so, more of the missing heritability of complex traits can then be accounted for, contributing to the advancement of knowledge of the etiology of diseases. We have developed an algorithm for the partition of copy number polymorphisms, making it feasible to use these structural changes in genetic linkage studies with family data. We have also conducted an extensive study in collaboration with the Wellcome Trust Centre for Human Genetics of the University of Oxford to characterize rare copy number definition metrics and their impact on study results with unrelated individuals. We have conducted a thorough comparison of the performance of genotyping algorithms when used with a new DNA microarray composed of a majority of very rare genetic variants. Finally, we have developed a bioinformatics tool for the fast and efficient processing of genetic data to increase quality, reproducibility of results and to reduce spurious associations.