Este estudo tem por objetivo o desenvolvimento de uma metodologia de fabricação de sistemas Lab On Chip, úteis no estudo de processos celulares, a partir da adaptação de tecnologias próprias da microeletrônica. Foram exploradas todas as etapas envolvidas na fabricação de sistemas Lab On Chip em Poli-Di-Metil-Siloxano e desenvolvidos protocolos de fabricação de moldes, técnicas de moldagem e processos de ativação de PDMS com plasma de oxigênio para sua solda química sobre diferentes materiais, obtendo uniões irreversíveis que permitem a integração com outras tecnologias como a microeletrônica em silício e o encapsulamento com cerâmica verde, completando uma metodologia que permite a prototipagem de dispositivos micro-fluídicos de multicamadas com um nível de sofisticação comparável ao estado da arte. Foi desenvolvido o protótipo de um equipamento ótico para litografia por projeção que permite a fabricação de máscaras óticas com resolução de 5 m e oferece a possibilidade de litografia em escala de cinzas para gerar canais e estruturas com relevos arbitrários. Foram adicionalmente abordados três problemas de biofísica celular, para os quais foram propostos novos dispositivos para separação de células móveis de acordo às suas velocidades lineares, dispositivos para crescimento confinado de bactérias e dispositivos para manipulação da curvatura de membranas celulares. / The objective of this study is the development of a methodology for the fabrication of Lab On Chip systems, useful for the analysis of cellular processes, through the adaptation of technologies from microelectronics. All the steps involved with the fabrication of Lab on Chip system in Poly-Di-Methil-Siloxane (PDMS) were explored, developing protocols for mold fabrication, molding techniques and processes for oxygen plasma activation of PDMS for its bonding to different materials, achieving irreversible bonds that enable the integration with other technologies such as silicon microelectronics and green tape packaging. All this techniques constitute a methodology that allows the prototyping of multilayer microfluidic devices comparable with state of the art devices. It was developed the prototype of optical equipment for projection lithography capable of mask fabrication with 5 m resolution, and which offers also the capability of gray scale lithography for the generation of free form microchannels. Additionally three different problems in cellular biophysics where boarded, proposing new devices for the separation of motile cells according to their linear speeds in liquids, new devices for constrained bacterial growth and for curvature manipulation of cell membranes.

Abstract The subject of this thesis is the characterization of chemical reactions and adsorption by means of remote detection (RD) method of nuclear magnetic resonance (NMR). The thesis consists of three related topics: In the first one, novel RD NMR based methods for characterizing chemical reactions were presented. In the second topic RD NMR methods were used to study the performance of new kind of microfluidic reactors. The third project concentrated on the development of a novel way to quantify the adsorption of flowing gas mixtures in porous materials. Even though all the topics cover quite different areas of research, they have few common nominators: remote detection NMR, microfluidics and method development. Microfluidic devices are of interest for many areas of science (such as molecular biology, disease diagnosis, chemistry) as they offer great promises for future technologies. Small dimensions enable, among many other things, the benefits of small sample volumes, large surface to volume ratio, efficient heat exchange and precise control of flow features and chemical reactions. The efficient evolution of microfluidic processes requires also the development of new innovative ways to characterize the performance of microfluidic devices. In this work, remote detection NMR is utilized for the purpose. RD is a method where the encoding and detection of information are separated physically. In many cases, the encoding and detection are performed with two separate RF coils while a fluid is passing through the studied system. In the first part of the thesis work, we introduced the concept of remote detection exchange (RD-EXSY) NMR spectroscopy. We demonstrated that the RD-EXSY method can provide unique chemical information. Furthermore, the time-of-flight (TOF) information, which is a natural side product of the experimental setup used, can be converted into indirect spatial information, showing the active reaction regions in a microfluidic device. Additionally, we demonstrated that by applying the principles of Hadamard spectroscopy in the encoding of the indirect spectral dimension we were able to produce with high efficiency RD-EXSY TOF images with direct spatial information. This allows even more accurate characterization of the active regions. The second topic concentrates on the development of microfluidc hydrogenation reactors. In the project atomic layer depositon (ALD) method was used for the first time to deposit both catalyst nanoparticles and support material on the surface of wall-coated microreactors. As a model reaction continuous flow propene hydrogenation into propane was studied by means of remote detection NMR. Reaction yield, mass transport phenomena and the activity of the catalyst surface were determined from the RD NMR data. Thirdly we presented a novel method for gas adsorption measurements in porous materials using RD TOF NMR. Traditional adsorption measurements are carried out at static conditions for a single gas component, as multi-component adsorption measurements are challenging and time-consuming. We investigated adsorption of continuously flowing propane and propene gases as well as their mixture in packed beds of mesoporous materials. The unique time-of-flight information obtained using the RD NMR method was utilized in the determination of flow velocity, which was then converted into amount of adsorbed gas.

No description available.

This master thesis deals with the use of microfluidics for the purpose of microfluidic enzymatic reactor for drug screening. At first it considers the issue from a theoretical point of view – describes microfluidics as a newly developing and promising field of production of microfluidic devices, materials, biomedical applications and advantages and disadvantages of microfluidics overall. Furthermore, it focuses on an area of analytical utilization of enzymes within enzyme reactors. In the first part of the experimental section, conditions for the testing of enzymes of xenobiotics metabolism in the liver were optimized, namely the model of coumarin metabolism via the spectrofluorimetry method. The second part of the experimental work dealt with optimization of the fabrication conditions of microfluidic chips from OSTE (off-stoichiometry Thiol Ene) via the soft lithography method. Subsequently, the functionality of the produced chips was tested. Based on the results of both parts of the experimental work, an evaluation was carried out to assess the suitability of their interconnection for future research – screening of microsomal enzyme activity and model biotransformation of drugs within the channels of the fabricated devices.

Antibiotic resistance is now a recognized problem in global health. In attempts to find solutions to detect bacteria causing antibiotic resistance we turn to technological solutions that are miniaturized, portable and cheap. The current diagnostic procedures cannot provide correct information outside laboratory settings, at the point-of-care, within necessary time. This has led to ineffective treatment of urinary tract infections causing recurrent infections and multi-drug resistant bacteria to spread. The bacteria genes show which antibiotic that is required to eliminate disease and spread of resistance. Hence, the solution would be to perform nucleic acid testing at the point-of-care. By using new DNA amplification methods it is possible to miniaturize the diagnostic test to a so-called Lab-on-a-chip. These solutions would enable sample-in-results-out capability of the system at the point-of-care. For this to work one of the most important factors is fluorescent signal read-out from DNA amplification products. In this project the design parameters of such a read-out device was investigated with focus on image sensor sensitivity and device integration. During the project it was found that a low-cost commercial image sensor could be used to record images of a (3.76 x 2.74 mm2) micro well array of nanoliter sized PCR chambers. Different imaging artifacts appearing during sample partitioning were observed, distance dependency between sensor surface well array was investigate, and finally the image sensor function was compared to a fluorescent microscope.

I characterize the behavior of microcantilever arrays which utilize the in-plane photonic transduction that I've previously developed and evaluate the performance of the microcantilever arrays in simple sensing scenarios with integrated microfluidics. First the thermal responses of microcantilevers with a variety of patterns of deposited gold films are compared. Using a scanning electron microscope, I observe the deflection thermal sensitivities of 300 µm long microcantilevers to be -170.82 nm/K for a full gold coating and -1.93 nm/K for no gold coating. Using the photonic transduction method I measure a thermal sensitivity of -1.46 nm/K for a microcantilever array with no gold. A microcantilever array integrated with microfluidics is exposed to a solution of bovine serum albumin (BSA) followed by solutions of various pH's. In all cases I observe a previously unreported transient deflection response. We find that the transient response is due to temporary nonuniform concentration distributions. In response to nonspecific binding of BSA, I observe a transient surface stress of -0.23 mN/m that agrees well with the -0.225 mN/m predicted by simulations. We hypothesize that the deflection response to pH changes is due to stress generated by conformational changes of bound BSA.The deflection response of an integrated microcantilever array to different types of flow and different flow rates is observed. Simulations of the deflection response match well with experimental results but disagree at higher flow rates. For flow rates greater than 200 µL/min, the limitation of the differential signal's dynamic range becomes apparent. We then investigate flow driven by an on-chip reciprocating reservoir pump. We demonstrate that it is possible to use the reciprocating pump to achieve high flow rates while making deflection measurements in-between reservoir actuations. Investigations of the microcantilever array noise show that flicker noise dominates below 10 Hz, while above 10 Hz, readout noise dominates. A minimum deflection noise density of 15 pW/√Hz is achieved. To improve the signal-to-noise ratio I develop algorithms for a digital lock-in amplifier with a digital phase-lock loop. In simulation the lock-in amplifier is able to improve the SNR by up to a factor of 6000, and self-lock to a noisy carrier signal without an external reference signal.

Optofluidic flow-through biosensor devices have been in development for fast bio-target detection. Utilizing the fabrication processes developed by the microelectronics industry, these biosensors can be fabricated into lab-on-a-chip devices with a degree of platform portability. This biosensor technology can be used to detect a variety of targets, and is particularly useful for the detection single molecules and nucleic acid strands. Microfabrication also offers the possibility of production at scale, and this will offer a fast detection method for a range of applications with promising economic viability. The development of this technology has advanced to now warrant a descriptive model that will aid in the design of future iterations. The biosensor consists of multiple integrated waveguides and a microfluidic channel. This platform therefore incorporates multiple fields of study: fluorescence, optical waveguiding, microfluidics, and signal counting. This dissertation presents a model theory that integrates all these factors and predicts a biosensor design's sensitivity. The model is validated by comparing simulated tests with physical tests done with fabricated devices. Additionally, the model is used to investigate and comment on designs that have not yet been allocated time and resources to fabricate. Tangentially, an improvement to the fabrication process is investigated and implemented.

Understanding cell migration is a key step in unraveling many physiological phenomena and predicting several pathologies, such as cancer metastasis. In particular, confinement has been proven to be a key factor in the cellular migration strategy choice. As our insight in the field improves, new tools are needed in order to empower biologists’ analysis capabilities. In this framework, microfluidic devices have been used to engineer the mechanical and spatial stimuli and to investigate cellular migration response in a more controlled way. In this work, we will review the existing technologies employed in the realization of microfluidic cellular migration assays, namely the soft lithography of PDMS and hydrogels and femtosecond laser micromachining. We will give an overview of the state of the art of these devices, focusing on the different geometrical configurations that have been exploited to study specific aspects of cellular migration. Our scope is to highlight the advantages and possibilities given by each approach and to envisage the future developments in in vitro migration studies under spatial confinement in microfluidic device

The study of cellular migration dynamics and strategies plays a relevant role in the understanding of both physiological and pathological processes. An important example could be the link between cancer cell motility and tumor evolution into metastatic stage. These strategies can be strongly influenced by the extracellular environment and the consequent mechanical constrains. In this framework, the possibility to study the behavior of single cells when subject to specific topological constraints could be an important tool in the hands of biologists. Two-photon polymerization is a sub-micrometric additive manufacturing technique that allows the fabrication of 3D structures in biocompatible resins, enabling the realization of ad hoc biochips for cell motility analyses, providing different types of mechanical stimuli. In our work, we present a new strategy for the realization of multilayer microfluidic lab-on-a-chip constructs for the study of cell motility which guarantees complete optical accessibility and the possibility to freely shape the migration area, to tailor it to the requirements of the specific cell type or experiment. The device includes a series of micro-constrictions that induce different types of mechanical stress on the cells during their migration. We show the realization of different possible geometries, in order to prove the versatility of the technique. As a proof of concept, we present the use of one of these devices for the study of the motility of murine neuronal cancer cells under high physical confinement, highlighting their peculiar migration mechanisms.

No description available.