Bacteria are the oldest, structurally simplest, and most abundant forms of life on earth. Its detection has always been a serious question since the emerging of modern science and technology. There has been a phenomenal growth in the field of real-time bacteria detection in recent years with emerging applications in a wide range of disciplines, including medical analysis, food, environment and many more. Two important analytical functions involved in bacteria detection are cell lysis and polymerase chain reaction (PCR). Cell lysis is required to break cells open to release DNA for use in PCR. PCR is required to reproduce millions of copies of the target genes to reach detection limit from a low DNA concentration. Conventionally, cell lysis and PCR are performed separately using specialized equipments. Those bulky machines consume much more than needed chemical reagents and are very time consuming. An efficient, cost-effective and portable solution involving Nanotechnology and Lab-on-a-Chip (LOC) technology was proposed. The idea was to utilize the excellent antibacterial property of surface-functionalized nanoparticles to perform cell lysis and then to perform PCR on the same LOC system without having to remove them from the solution for rapid detection of bacteria. Nanoparticles possess outstanding properties that are not seen in their bulk form due to their extremely small size. They were introduced to provide two novel methods for LOC cell lysis to overcome problems of current LOC cell lysis methods such as low efficiency, high cost and complicated fabrication process. The first method involved using poly(quaternary ammonium) functionalized gold and titanium dioxide nanoparticles which were demonstrated to be able to lyse E. coli completely in 10 minutes. The idea originated from the excellent antibacterial property of quaternary ammonium salts that people have been using for a long time. The second method involved using titanium dioxide nanoparticles and a miniaturized UV LED array. Titanium dioxide bears photocatalytic effect which generates highly reactive radicals to compromise cell membranes upon absorbing UV light in an aqueous environment. A considerable reduction of live E. coli was observed in 60 minutes. The thesis then evaluates the effect of nanoparticles on PCR to understand the roles nanoparticles play in PCR. It was found that gold and titanium dioxide nanoparticles induce PCR inhibition. How size of gold nanoparticles affected PCR was studied as well. Effective methods were discovered to suppress PCR inhibition caused by gold and titanium dioxide nanoparticles. The pioneering work paves a way for the integration of nanoparticle cell lysis and LOC PCR for rapid detection of bacteria. In the end, an integrated system involving nanoparticle cell lysis and microchip PCR was demonstrated. The prototyped system consisted of a physical microchip for both cell lysis and PCR, a temperature control system and necessary interface connections between the physical device and the temperature control system. The research explored solutions to improve PCR specificity in a microchip environment with gold nanoparticles in PCR. The system was capable of providing the same performance while reducing PCR cycling time by up to 50%. It was inexpensive and easy to be constructed without any complicated clean room fabrication processes. It can find enormous applications in water, food, environment and many more.

Optimization theory is a key technology for inverse problems of reconstruction in science, engineering and economy. Discrete tomography is a modern research field dealing with the reconstruction of finite objects in, e.g., VLSI chip design, where this thesis will focus on. In this work, a framework with its supplementary algorithms and a new problem reformulation are introduced to approximately resolve this NP-hard problem. The framework is modular, so that other reconstruction methods, optimization techniques, optimal experimental design methods can be incorporated within. The problem is being revisited with a new optimization formulation, and interpretations of known methods in accordance with the framework are also given. Supplementary algorithms are combined or incorporated to improve the solution or to reduce the cost in terms of time and space from the computational point of view.

This paper presents a theoretical model of electronic technology applied to Precision Farming and becomes a grant to further increase the use of Precision Farming, setting that is transforming the way you get the results from the works in the field. The design of a radio frequency identification system, currently known as RFID, to be used in the identification and traceability of cattle is the goal to be achieved. It seeks initially emphasize the definition and the use of the use of Precision Farming as a routine in the activities of the field in Brazil. How is the cattle industry and the Precision Farming definition are important items to achieve the proposed goal. The SISBOV system and traceability are presented, showing its importance, but not the obligation deployment along to landowners who work with the creation and marketing of cattle and their meat products. Shows the definition of microprocessors and microcontrollers, their internal architectures and all the associated peripherals to them and is also shown as the definition of embedded systems has made a revolution in the design of these. Plot a marketing scenario and use of electronic products in the Brazilian commercial sphere, indicating how the delay in using technology more turns in the electronics consumers than in technology trainers. Emphasizes in the principle of operation of RFID systems and which the communication protocols that are used. The reader RFID design and miniaturization of electronics has enabled the creation of a device with surface mount technology decreasing the dimensions of the printed circuit board and allowing the reduction of cost in the design of this system. We present all the peripherals that make it up and the way they connect with the master processor, including the slave processor which performs the digital input and output functions and the keyboard. Communication protocols and flowcharts show the use of known commercial channels in using software. The use of solar energy is presented as an alternative source of electric power supply in the operation of the reader equipment. The high cost of commercial RFID modules prevented the practical implementation of the system. / Este trabalho apresenta um modelo teórico da tecnologia eletrônica aplicada à Pecuária de Precisão e transforma-se em um subsídio a mais para incrementar o uso da Agricultura de Precisão, definição que está transformando a maneira como se obtém os resultados advindos do trabalho no campo. O projeto de um sistema de identificação por radiofrequência, conhecido atualmente como RFID, a ser utilizado na identificação e rastreabilidade de gado bovino é o objetivo a ser alcançado. Procura-se inicialmente enfatizar a definição e a utilização do uso da Agricultura de Precisão como uma rotina nas atividades do campo no Brasil. Como está à atividade pecuária e a definição de Pecuária de Precisão são itens importantes para alcançar o objetivo proposto. O sistema SISBOV e a rastreabilidade são apresentados, mostrando a sua importância, mas não a sua obrigação de implantação junto aos proprietários rurais que trabalham com a criação e comercialização de gado e seus produtos cárneos. Mostra-se a definição de microprocessadores e microcontroladores, suas arquiteturas internas e todos os periféricos associados a eles e também é mostrado como a definição de sistemas embarcados tem realizado uma revolução no projeto destes. Traça-se um cenário da comercialização e uso de produtos eletrônicos na esfera comercial brasileira, indicando como o atraso no uso da tecnologia nos transforma muito mais em consumidores de produtos eletrônicos do que em formadores de tecnologia. Enfatiza-se o princípio de funcionamento de sistemas RFID e quais os protocolos de comunicação que são utilizados. O projeto do leitor RFID e a miniaturização da eletrônica possibilitou a criação de um equipamento com tecnologia de montagem em superfície diminuindo as dimensões da placa de circuito impresso e possibilitando a diminuição do custo no projeto deste sistema. Apresentam-se todos os periféricos que o compõem e a maneira como se interligam com o processador mestre, inclusive o processador escravo o qual executa as funções de entrada e saída digitais e o teclado. Protocolos de comunicação e fluxogramas evidenciam a utilização de canais conhecidos comercialmente na utilização de software. O uso da energia solar apresenta-se como uma alternativa de fornecimento de alimentação elétrica no funcionamento do equipamento leitor. O alto custo dos módulos RFID comerciais inviabilizou a parte prática do sistema.

Charles University Faculty of Pharmacy in Hradec Králové Department of Biological and Medical Sciences Author: Bc. Petra Porubová Supervisor: prof. RNDr. Ctirad Andrýs, Ph.D. Title of diploma thesis: Allergic phenotypes in patients with respiratory allergy evaluated by microchip ISAC technique Key words: allergic inflammation, allergen specific IgE, component resolved diagnostics, microchip, ISAC Background: The aim of this diploma thesis was to evaluate the sensitization profile in the group of pacients with allergic rhinitis and/or bronhial asthma and to identify possible differences between patients with different disease severity. Methods: ImmunoCAP ISAC was used to determine specific IgE antibodies against 112 allergen components. The evaluated group included 152 patients (57.9 % women) from the allergology outpatient office of the Department of Clinical Immunology and Allergology, University Hospital Hradec Králové. Results: The most common allergens in this group of patients were the components Phl p 1 (68,4 %) and Bet v 1 (65,1 %). Differences in the number of positive and negative reactions were demonstrated between patients with different severity of allergic rhinitis. Correlations were found between the number of positive/negative reactions and the severity of bronchial asthma. And the...

Although the airbath oven is a reliable heating method for gas chromatography (GC), resistive heating is needed for higher analytical throughput and on-site chemical analysis because of size, heating rate and power requirements. In the last thirty years, a variety of resistive heating methods were developed and implemented for both benchtop and portable GC systems. Although fast heating rates and low power consumption have been achieved, losses in column efficiency and resolution, complex construction processes and difficulties experienced in recovering damaged columns have also become problematic for routine use of resistively heated columns. To solve these problems, a new resistively heated column technique, which uses metal columns and self-insulated heating wires, was developed for capillary gas chromatography. With this method, the total thermal mass was significantly less than in commercial column assemblies. Temperature-programming using resistive heating was at least 10 times faster than with a conventional oven, while only consuming 1—5% of the power that an oven would use. Cooling a column from 350 °C to 25 °C with an air fan only required 1.5 min. Losses in column efficiency and peak capacity were negligible when compared to oven heating. The major trade-off was slightly worse run-to-run retention time deviations, which were still acceptable for most GC analyses. The resistively heated column bundle is highly suitable for fast GC separations and portable GC instruments. Fabrication technologies for microelectromechanical systems (MEMS) allow miniaturization of conventional benchtop GC to portable, microfabricated GC (µGC) devices, which have great potential for on-site chemical analysis and remote sensing. The separation performance of µGC systems, however, has not been on par with conventional GC. Column efficiency, peak symmetry and resolution are often compromised by column defects and non-ideal injections. The relatively low performance of µGC devices has impeded their further commercialization and broader application. This problem can be resolved by incorporating thermal gradient GC (TGGC) into microcolumns. Negative thermal gradients reduce the on-column peak width when compared to temperature-programmed GC (TPGC) separations. This unique focusing effect can overcome many of the shortcomings inherent in µGC analyses. In this dissertation research, the separation performance of µGC columns was improved by using thermal gradient heating with simple set-ups. The analysis time was ~20% shorter for TGGC separations than for TPGC when wide injections were performed. Up to 50% reduction in peak tailing was observed for polar analytes, which significantly improved their resolution. The signal-to-noise ratios (S/N) of late-eluting peaks were increased by 3 to 4 fold. These results indicate that TGGC is a useful tool for bridging the performance gap between µGC and benchtop GC.

Preterm birth is a global health challenge and the leading cause of neonatal mortality. Each year, about 15 million babies are born preterm globally. Traditional tools that have been exploited for the detection of preterm birth biomarkers are expensive, time consuming, or lack multiplexing capabilities. The work described in this dissertation highlights techniques developed to detect preterm birth biomarkers rapidly and accurately in the effort to mitigate preterm birth risk. In this dissertation, I first demonstrated the use of stereolithography digital light processing-based 3D printing and microfluidics for the development of microfluidic devices that had microvalves for fluid control. I then used these devices for microchip electrophoresis and fluorescence detection of five preterm birth biomarkers from a published panel. Next, I presented developments in 3D printed microchip electrophoresis device design. I separated amino acids and preterm birth biomarkers in a serpentine device design, obtaining good resolution, separation efficiency, and improved preterm birth biomarker peak capacity. Finally, I demonstrated the integration of solid-phase extraction with microchip electrophoresis in 3D printed microfluidic devices. These integrated devices enabled a seamless transition from preterm birth biomarker enrichment and labeling to microchip electrophoresis separation and fluorescence detection. The work described in this dissertation shows promise in advancing key tools needed to address preterm birth risk rapidly and effectively.

No description available.

A systemic mock circulatory loop plays a pivotal role as the in vitro assessment tool for left heart medical devices. The standard design employed by many research groups dates to the early 1970's, and lacks the acuity needed for the advanced device designs currently being explored. The necessity to update the architecture of this in vitro tool has become apparent as the historical design fails to deliver the performance needed to simulate conditions and events that have been clinically identified as challenges for future device designs. In order to appropriately deliver the testing solution needed, a comprehensive evaluation of the functionality demanded must be understood. The resulting system is a fully automated systemic mock circulatory loop, inclusive of anatomical geometries at critical flow sections, and accompanying software tools to execute precise investigations of cardiac device performance. Delivering this complete testing solution will be achieved through three research aims: (1) Utilization of advanced physical modeling tools to develop a high fidelity computational model of the in vitro system. This model will enable control design of the logic that will govern the in vitro actuators, allow experimental settings to be evaluated prior to execution in the mock circulatory loop, and determination of system settings that replicate clinical patient data. (2) Deployment of a fully automated mock circulatory loop that allows for runtime control of all the settings needed to appropriately construct the conditions of interest. It is essential that the system is able to change set point on the fly; simulation of cardiovascular dynamics and event sequences require this functionality. The robustness of an automated system with incorporated closed loop control logic yields a mock circulatory loop with excellent reproducibility, which is essential for effective device evaluation. (3) Incorporating anatomical geometry at the critical device interfaces; ascending aorta and left atrium. These anatomies represent complex shapes; the flows present in these sections are complex and greatly affect device performance. Increasing the fidelity of the local flow fields at these interfaces delivers a more accurate representation of the device performance in vivo.

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.

<p>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).</p><p>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.</p><p>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.</p><p>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.</p>