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Novel technologies for cell culture and tissue engineeringGe, Cheng January 2016 (has links)
Cell culture has been a fundamental tool for the study of cell biology, tissue engineering, stem cell technology and biotechnology in general. It becomes more and more important to have a well-defined physiochemical microenvironment during cell culture. Conventional cell cultures employ expensive, manually controlled incubation equipment, making it difficult to maximize a cultures yield. Furthermore, previous studies use qualitative methods to assess cell culture proliferation that are inherently inaccurate and labour intensive, thereby increasing the cost of production. In addition, three dimensional cell culture, in scaffold, has been shown to provide more physiological relevant information as it mimic more accurate conditions that are similar to the physiological conditions of the human body compared with two dimension, which has special interest to regenerative medicine. Therefore, a portable and automated total-analysis-system (μTAS) was proposed with microenvironment control and quantitative analysis techniques to monitor cell proliferation and metabolic activity. The automated portable heating system was validated to be capable to maintain a stable physiochemical microenvironment, with little margin of error, for cellular substrate outside of conventional incubation. A standalone platform system was designed and fabricated with accurate temperature control by employing an optically transparent ITO-film with a large heating area. The transparency of the film is critical for continuous in-situ microscopic observation over long-term cell culture process. Previous studies have attempted to use ITO-film as a heating element, but were unable to distribute the heat evenly onto the microbioreactor platform. This nagging problem in the literature was improved through a novel film design. As a result, the ITO-film based heating system was evaluated and constructed successfully to serve as a heating element for long-term static cell culture with facilitated proliferation rate in gas-permeable PDMS microbioreactor outside of conventional incubation. In addition to maintaining a stable microenvironment, a non-invasive in-situ technology for monitoring cell viability and proliferation rate was constructed and developed based on bioimpedance spectroscopy (BIS). It was primarily focused on making decisions for structure and specification of proposed system-on a chip BIS measurement. The miniaturization of BIS system on microbioreactor platform was achieved by utilizing and integrating switching matrix array, impedance analyzer chip with reliable analogue-front-end circuitry. The realized system was verified with the DLD-1 cells and its monitored data were validated with conventional bioassays. Three dimensional cell cultures with scaffold is a key to the success of tissue engineering. Engineered cornea collagen scaffold may be feasible using re-seeding proper human cells onto a decellularized corneal scaffold. The quality of the scaffold and the interaction of the cells are critical to the key function (i.e transparency, haze and total transmittance) of final products. An integrated corneal collagen scaffold quality assessment system, via optical property inspection unit, was innovatively designed and realized with non-invasive and non-destructive characteristics. The H1299 cells were seeded onto inspected corneal scaffold and BIS system, which were realized in the previous chapter, were used to validate its applicability for 3D cell culture. The cell adhesion as an outcome at different scaffolds with different optical properties has revealed the importance of the microstructure of scaffold on the cell functions. The results showed the developed technologies can be used for the quality control of corneal scaffold and the fabricated μTAS not only enabled environmental control but, with BIS-based in-situ assay, it also facilitate the function (i.e adhesion) and viability monitoring with quantitative and qualitative analysis in 3D-alike cell culture. Additionally, by considering its low decontamination and cost-effective nature with compatibility for high-throughput screening applications, the fabricated and integrated systems has significant applications in tissue engineering.
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Análises genéticas em sistemas microfabricados / Genetic analysis in microfabricated systemsDuarte, Gabriela Rodrigues Mendes 30 July 2010 (has links)
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.
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Análises genéticas em sistemas microfabricados / Genetic analysis in microfabricated systemsGabriela Rodrigues Mendes Duarte 30 July 2010 (has links)
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.
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Desenvolvimento de métodos para coleta e determinação de poluentes em fase gasosa da atmosfera utilizando amostragem por difusão com membranas capilares microporosas / Development of methods for sampling and determination of gaseous pollutants in the atmosphere using diffusional microporous capillary membrane scrubberCoelho, Lúcia Helena Gomes 23 November 2009 (has links)
A tese enfatiza o desenvolvimento e aplicação de amostradores difusionais para pré-concentração de traços de poluentes gasosos e sua conjugação com técnicas de separação e determinação, especialmente, a eletroforese capilar. Foram implementadas novas aplicações para um amostrador com múltiplos filamentos microporosos de polipropileno, previamente desenvolvido pelo grupo de pesquisa, e para o novo dispositivo de coleta de dimensões reduzidas, composto por monofilamento microporoso de polipropileno (CMDS) com volume interno de 30 µL. Para o estabelecimento da vazão estável em 1,0 µL min-1 de fase aceptora pelo capilar poroso, recorreu-se à pressurização do reservatório com uma bomba pneumática de aquário e regulagem de vazão por um capilar de sílica fundida e uma válvula de agulha. Outra bomba pneumática foi utilizada para aspirar o fluxo de ar amostrado. As amostras foram coletadas seqüencialmente em frascos de 200 µL, mantidos sob temperatura reduzida em unidade de refrigeração do tipo Peltier acoplado ao amostrador. O CMDS monofilamentar, mais compacto e robusto, apresentou alta eficiência de coleta para a pré-concentração de formaldeído (CH2O), ácidos fórmico e acético e amônia da fase gasosa da atmosfera. Para as espécies com elevada constante de Henry, água deionizada serviu como fase aceptora, enquanto que para amônia, recorreu-se ao deslocamento do equilíbrio por um aceptor ácido, de modo a reter o analito na forma de NH4 +. A concentração das espécies em fase líquida foi determinada posteriormente por eletroforese capilar com detecção condutométrica sem contato (CE-C4D). Os limites de detecção, em fase líquida, para formiato, acetato, formaldeído (determinado na forma do aduto hidroximetanosulfonato HMS) e amônio foram estimados em 1,0, 1,5, 1,2 e 1,2 µmol L-1, respectivamente (equivalentes à 0,9, 3,0, 1,0 e 0,7 µg m-3 dessas espécies na atmosfera, respectivamente). Para a determinação de CH2O por injeção em fluxo (FIA) acoplada à detecção amperométrica em eletrodo de ouro platinizado, fez-se uso do coletor multifilamentar, por atender melhor à necessidade de volume de amostra. A interferência de espécies comumente presentes na atmosfera, como H2O2 e SO2, pôde ser contornada realizando coletas em presença de peróxido de hidrogênio para promover a oxidação do S(IV) à S(VI) e, posteriormente, destruindo o oxidante em reator contendo enzima catalase imobilizada. A determinação de CH2O foi implementada com sucesso por FIA com detecção por amperometria em eletrodo de ouro platinizado. Um CMDS portátil que funciona à pilha, próprio para amostragens em campo, foi desenvolvido utilizando, para o deslocamento do ar amostrado, uma bomba de pistão retirada de aparelho automático para medida de pressão arterial. O amostrador foi empregado na coleta de H2S, SO2 e alquil-mercaptanas em fase gasosa da atmosfera, utilizando fase aceptora alcalina para promover a desprotonação e conseqüente fixação das espécies em meio líquido. Os analitos foram determinados por CE-C4D ou por microextração em fase sólida (SPME) seguida de determinação por GC. O uso conjunto do antioxidante ascorbato para conservação das amostras e de etanol para fixação dos compostos voláteis em meio aquoso permitiu o estabelecimento de um protocolo completo para coleta e detecção dessas espécies reduzidas de enxofre, com sensibilidade suficiente para monitorar emissões de origem biogênica, o que foi exemplificado na prática coletando amostras próximo a um córrego contaminado com esgoto. A perfeita combinação do diminuto volume de fase aceptora do CMDS com a demanda de nanolitros de amostra da CE-C4D culminou com o desenvolvimento de um sistema de análise total (TAS) com gerenciamento de fluidos baseado em propulsão por bombas de aquário (de baixo custo e alta durabilidade) e válvulas solenóide de estrangulamento, controladas por computador. Como exemplo de aplicação inovador e bem sucedido do TAS desenvolveu-se a análise concomitante e em tempo quase real de ácidos fórmico e acético no ar, com freqüência de 10 pares de dados por hora. As vantagens do sistema CMDS-CE-C4D incluem simplicidade, versatilidade, consumo de reagentes e amostra e geração de resíduos minimizada, robustez e rapidez enquanto uma amostra é coletada, o eletroferograma da anterior é adquirido sem necessidade de bombas de alta pressão ou colunas dispendiosas como as requeridas para HPLC / The development and application of porous membrane diffusion samplers for fast and efficient pre-concentration of an array of trace air pollutants was emphasized in this thesis, in conjunction with compatible separation and determination techniques, especially capillary electrophoresis. New applications were found for a formerly developed diffusion scrubber based on a bundle of microporous hollow polypropylene capillary membranes and for a scaled down version with a single core capillary membrane diffusion scrubber (CMDS) comprising an internal volume of 30 µL, were used for sampling of the trace level pollutants to an adequate liquid acceptor. The low-flow of acceptor solution, 1.0 µL min-1, required by the CMDS was satisfied by pressurization of the reservoir with an aquarium pump combined with flow regulation by a silica capillary as hydrodynamic resistor and a needle valve. Another aquarium pump was used for the aspiration of the sampled air through the sampler. The low volumes collected in the CMDS were stored in 200 µL vials inserted in a cooling plate of a Peltier device. The robust and compact system was used for sampling of formaldehyde (CH2O), formic acid, acetic acid and ammonia in the gaseous phase of the atmosphere. For chemical species with high Henry´s constant, deionized water suffices as acceptor phase. Otherwise, equilibria displacement to a non-volatile ion, like NH4 + for NH3 sampling, promoted quantitative retention in the acceptor phase. The concentrations of the analytes in the liquid phase were determined by capillary electrophoresis with capacitively coupled contactless conductometric detection (CEC4D). The detection limits obtained in the liquid phase for formate, acetate, formaldehyde (in the form of the adduct hydroxymethanesulfonate HMS) and ammonium were 1,0, 1,5, 1,2 e 1,2 µmol L-1 respectively (what corresponds to 0,9, 3,0, 1,0 e 0,7 µg m-3 of the respective gaseous species in the air). The higher volume of acceptor phase provided by the sampler with a bundle of microporous membrane capillaries (~600 µL) are in tune with the needs of flow injection analysis (FIA), as demonstrated for amperometric detection of CH2O on a platinized gold electrode. The interferences from SO2 and H2O2 were overcome by adding H2O2 to the acceptor solution to promote the oxidation of S(IV) to S(VI) and destruction of the oxidant afterwards in a column with immobilized catalase enzyme. The aquarium pump used for gas aspiration was substituted to a piston pump, taken from arterial blood pressure meter fed with 8 V D.C., to turn possible field collections with the CMDS device. The system was used for the sampling of H2S, SO2 e alkyl-mercaptans in the gaseous phase of the atmosphere. To succeed on that, the samplings were performed in alkaline media to promote deprotonation of the species (and stabilization in non-volatile forms). The collected analytes were determined by CE-C4D or by solid phase microextraction (SPME) followed by GC analysis. The joint use of the antioxidant ascorbate for sample preservation and ethanol for fixation of the volatile compounds allowed the establishment of a complete protocol for sampling, storage and detection of the sulfur reduced species with enough sensitivity for monitoring biogenic emissions from waste discharge. Perfect matching of the low-volume characteristics of the CMDS device and the CE-C4D equipment led to the conception of a low-cost automatic CMDS-CE-C4D total analysis system (TAS) and its innovative and successful application to near-realtime simultaneous analysis of formic acid and acetic acid in air. During the evaluation of one sample, the TAS collects a new one, with valves, pumps and high voltage delivery under software control. Advantages include rapidity (10 data points per hour for each analyte), high preconcentration efficiency, simplicity and versatility, minimum sample and reagent consumption and residue generation (green analytical method), no need of costly high pressure pumps and separation columns like those used in HPLC
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Microscale Tools for Sample Preparation, Separation and Detection of Neuropeptides / Mikroskaliga verktyg för provpreparering, separation och detektion av neuropeptiderDahlin, Andreas January 2005 (has links)
<p>The analysis of low abundant biological molecules is often challenging due to their chemical properties, low concentration and limited sample volumes. Neuropeptides are one group of molecules that fits these criteria. Neuropeptides also play an important role in biological functions, which makes them extra interesting to analyze. A classic chemical analysis involves sampling, sample preparation, separation and detection. In this thesis, an enhanced solid supported microdialysis method was developed and used as a combined sampling- and preparation technique. In general, significantly increased extraction efficiency was obtained for all studied peptides. To be able to control the small sample volumes and to minimize the loss of neuropeptides because of unwanted adsorption onto surfaces, the subsequent analysis steps were miniaturized to a micro total analysis system (µ-TAS), which allowed sample pre-treatment, injection, separation, manipulation and detection. </p><p>In order to incorporate these analysis functions to a microchip, a novel microfabrication protocol was developed. This method facilitated three-dimensional structures to be fabricated without the need of clean room facilities. </p><p>The sample pre-treatment step was carried out by solid phase extraction from beads packed in the microchip. Femtomole levels of neuropeptides were detected from samples possessing the same properties as microdialysates. The developed injection system made it possible to conduct injections from a liquid chromatographic separation into a capillary electrophoresis channel, which facilitated for advanced multidimensional separations. An electrochemical sample manipulation system was also developed. In the last part, different electrospray emitter tip designs made directly from the edge of the microchip substrate were developed and evaluated. The emitters were proven to be comparable with conventional, capillary based emitters in stability, durability and dynamic flow range. Although additional developments remain, the analysis steps described in this thesis open a door to an integrated, on-line µ-TAS for neuropeptides analysis in complex biological samples.</p>
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Microfluidics in Surface Modified PDMS : Towards Miniaturized Diagnostic ToolsThorslund, Sara January 2006 (has links)
<p>There is a strong trend in fabricating <i>miniaturized total analytical systems</i>, µTAS, for various biochemical and cell biology applications. These miniaturized systems could e.g. gain better separation performances, be faster, consume less expensive reagents and be used for studies that are difficult to access in the macro world. Disposable µTAS eliminate the risk of carry-over and can be fabricated to a low cost.</p><p>This work focused on the development of µTAS modules with the intentional use for miniaturized diagnostics. Modules for blood separation, desalting, enrichment, separation and ESI-MS detection were successfully fabricated. Surface coatings were additionally developed and evaluated for applications in µTAS with complex biological samples. The first heparin coating could be easily immobilized in a one-step-process, whereas the second heparin coating was aimed to form a hydrophilic surface that was able to draw blood or plasma samples into a microfluidic system by capillary forces. </p><p>The last mentioned heparin surface was further utilized when developing a chip-based sensor for performing CD4-count in human blood, an important marker to determine the stage of an HIV-infection.</p><p>All devices in this work were fabricated in PDMS, an elastomeric polymer with the advantage of rapid and less expensive prototyping of the microfabricated master. It was shown that PDMS could be considered as the material of choice for future commercial µTAS. The devices were intentionally produced using a low grade of fabrication complexity. It was however demonstrated that even with low complexity, it is possible to integrate several functional chip modules into a single microfluidic device.</p>
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Microfluidics in Surface Modified PDMS : Towards Miniaturized Diagnostic ToolsThorslund, Sara January 2006 (has links)
There is a strong trend in fabricating miniaturized total analytical systems, µTAS, for various biochemical and cell biology applications. These miniaturized systems could e.g. gain better separation performances, be faster, consume less expensive reagents and be used for studies that are difficult to access in the macro world. Disposable µTAS eliminate the risk of carry-over and can be fabricated to a low cost. This work focused on the development of µTAS modules with the intentional use for miniaturized diagnostics. Modules for blood separation, desalting, enrichment, separation and ESI-MS detection were successfully fabricated. Surface coatings were additionally developed and evaluated for applications in µTAS with complex biological samples. The first heparin coating could be easily immobilized in a one-step-process, whereas the second heparin coating was aimed to form a hydrophilic surface that was able to draw blood or plasma samples into a microfluidic system by capillary forces. The last mentioned heparin surface was further utilized when developing a chip-based sensor for performing CD4-count in human blood, an important marker to determine the stage of an HIV-infection. All devices in this work were fabricated in PDMS, an elastomeric polymer with the advantage of rapid and less expensive prototyping of the microfabricated master. It was shown that PDMS could be considered as the material of choice for future commercial µTAS. The devices were intentionally produced using a low grade of fabrication complexity. It was however demonstrated that even with low complexity, it is possible to integrate several functional chip modules into a single microfluidic device.
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Novel Microfluidic Devices Based on a Thermally Responsive PDMS CompositeSamel, Björn January 2007 (has links)
The field of micro total analysis systems (μTAS) aims at developments toward miniaturized and fully integrated lab-on-a-chip systems for applications, such as drug screening, drug delivery, cellular assays, protein analysis, genomic analysis and handheld point-of-care diagnostics. Such systems offer to dramatically reduce liquid sample and reagent quantities, increase sensitivity as well as speed of analysis and facilitate portable systems via the integration of components such as pumps, valves, mixers, separation units, reactors and detectors. Precise microfluidic control for such systems has long been considered one of the most difficult technical barriers due to integration of on-chip fluidic handling components and complicated off-chip liquid control as well as fluidic interconnections. Actuation principles and materials with the advantages of low cost, easy fabrication, easy integration, high reliability, and compact size are required to promote the development of such systems. Within this thesis, liquid displacement in microfluidic applications, by means of expandable microspheres, is presented as an innovative approach addressing some of the previously mentioned issues. Furthermore, these expandable microspheres are embedded into a PDMS matrix, which composes a novel thermally responsive silicone elastomer composite actuator for liquid handling. Due to the merits of PDMS and expandable microspheres, the composite actuator's main characteristic to expand irreversibly upon generated heat makes it possible to locally alter its surface topography. The composite actuator concept, along with a novel adhesive PDMS bonding technique, is used to design and fabricate liquid handling components such as pumps and valves, which operate at work-ranges from nanoliters to microliters. The integration of several such microfluidic components promotes the development of disposable lab-on-a-chip platforms for precise sample volume control addressing, e.g. active dosing, transportation, merging and mixing of nanoliter liquid volumes. Moreover, microfluidic pumps based on the composite actuator have been incorporated with sharp and hollow microneedles to realize a microneedle-based transdermal patch which exhibits on-board liquid storage and active dispensing functionality. Such a system represents a first step toward painless, minimally invasive and transdermal administration of macromolecular drugs such as insulin or vaccines. The presented on-chip liquid handling concept does not require external actuators for pumping and valving, uses low-cost materials and wafer-level processes only, is highly integrable and potentially enables controlled and cost-effective transdermal microfluidic applications, as well as large-scale integrated fluidic networks for point-of care diagnostics, disposable biochips or lab-on-a-chip applications. This thesis discusses several design concepts for a large variety of microfluidic components, which are promoted by the use of the novel composite actuator. Results on the successful fabrication and evaluation of prototype devices are reported herein along with comprehensive process parameters on a novel full-wafer adhesive bonding technique for the fabrication of PDMS based microfluidic devices. / QC 20100817
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Microscale Tools for Sample Preparation, Separation and Detection of Neuropeptides / Mikroskaliga verktyg för provpreparering, separation och detektion av neuropeptiderDahlin, Andreas January 2005 (has links)
The analysis of low abundant biological molecules is often challenging due to their chemical properties, low concentration and limited sample volumes. Neuropeptides are one group of molecules that fits these criteria. Neuropeptides also play an important role in biological functions, which makes them extra interesting to analyze. A classic chemical analysis involves sampling, sample preparation, separation and detection. In this thesis, an enhanced solid supported microdialysis method was developed and used as a combined sampling- and preparation technique. In general, significantly increased extraction efficiency was obtained for all studied peptides. To be able to control the small sample volumes and to minimize the loss of neuropeptides because of unwanted adsorption onto surfaces, the subsequent analysis steps were miniaturized to a micro total analysis system (µ-TAS), which allowed sample pre-treatment, injection, separation, manipulation and detection. In order to incorporate these analysis functions to a microchip, a novel microfabrication protocol was developed. This method facilitated three-dimensional structures to be fabricated without the need of clean room facilities. The sample pre-treatment step was carried out by solid phase extraction from beads packed in the microchip. Femtomole levels of neuropeptides were detected from samples possessing the same properties as microdialysates. The developed injection system made it possible to conduct injections from a liquid chromatographic separation into a capillary electrophoresis channel, which facilitated for advanced multidimensional separations. An electrochemical sample manipulation system was also developed. In the last part, different electrospray emitter tip designs made directly from the edge of the microchip substrate were developed and evaluated. The emitters were proven to be comparable with conventional, capillary based emitters in stability, durability and dynamic flow range. Although additional developments remain, the analysis steps described in this thesis open a door to an integrated, on-line µ-TAS for neuropeptides analysis in complex biological samples.
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Microfluidic Devices for Manipulation and Detection of Beads and BiomoleculesJönsson, Mats January 2006 (has links)
This thesis summarises work towards a Lab-on-Chip (LOC). The request for faster and more efficient chemical and biological analysis is the motivation behind the development of the LOC-concept. Microfluidic devices tend to become increasingly complex in order to include, e.g. sample delivery, manipulation, and detection, in one chip. The urge for smart and simple design of robust and low-cost microdevices is addressed and discussed. Design, fabrication and characterization of such microdevices have been demonstrated using low-cost polymer and glass microfabrication methods. The manufacturing is feasible, to a large extent, to perform outside the clean-room, and has subsequently been the chosen technique for most of the work. Issues of bonding reliability are solved by using polymer adhesive tapes. A planar electrocapture device with LOC-compatibility is demonstrated where beads are immobilised and released in a flowing stream. Retention of nanoparticles by means of electric field-flow fractionation using transparent indium tin oxide electrodes is presented. Moreover, a cast PDMS 4-way crossing is enabling a combination of liquid chromatography and capillary electrophoresis to enhance separation efficiency. Sample transport issues and a new flow-cell design in a quartz crystal microbalance bioanalyzer are also investigated. Fast bacteria counting by impedance measurements, much requested by the pharmaceutical industry for biomass monitoring, is carried out successfully. In conclusion, knowledge in micro system technology to build microdevices have been utilised to manipulate and characterise beads and cells, taking one step further towards viable Lab-on-Chip instruments.
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