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.

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>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>

<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>

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.

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

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.

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.