Optofluidics is a relatively new and exciting field that includes the integration of optical waveguides into microfluidic platforms. The purpose of this field of study is to miniaturize previously developed optical systems used for biological and chemical analysis with the end goal of placing bench-top optics into microscopic packages. Mundane optical alignment and sample manipulation procedures would then be intrinsic to the platform and allow measurements to be completed quickly and with reduced human interaction. Biosensors based on AntiResonant Reflecting Optical Waveguides (ARROWs) consist of hollow-core waveguides used for fluid sample manipulation and analysis, as well as solid-core waveguides used in interfacing external components located at the chip edges. Hollow-core ARROWs are particularly useful for their ability to provide specifically tailored analyte volumes that are easily configurable depending upon the target experiment. Adaptations of standard planar microfabrication methods allow for complex integrated ARROW designs. Integrated spectral filtering with high rejection can be implemented on-chip, removing the need for additional off-chip components and increasing device sensitivity. Additional techniques to increase device sensitivity and utility, such as hybrid ARROW platforms and optical manipulation of samples, are also explored.

Microcantilevers have been investigated as high sensitivity, label free biosensors for approximately 15 years. In nearly all cases, a thin gold film deposited on the microcantilevers is used as an intermediate attachment layer because of the convenience of thiol-gold chemistry. Unfortunately, this attachment chemistry can be unstable when used with complex sample media such as blood plasma. The Nordin group at BYU has recently developed an all-silicon in-plane photonic microcantilever (PMCL) technology to serve as a platform for label-free biosensing. It has the advantage of being readily scalable to simultaneous readout of many PMCLs in array format, and allows integration with polymer microfluidics to facilitate the introduction of biological samples and reagents. An essential processing step for the transformation of the PMCL into a practical biosensor is the ability to effectively immobilize active biological receptors directly on silicon PMCL surfaces such that ligand binding generates sufficient surface stress to cause measureable PMCL deflection. This dissertation presents the development of a method to functionalize the sensor surface of all-silicon in-plane photonic microcantilever (PMCL) arrays. This method employs a materials inkjet printer for non-contact jetting and a fluid that is custom designed for ink-jetting and biological applications with approximately 1 pL droplet size. The method facilitates the application of different receptors on select PMCLs with drop placement accuracy in the +/- 7.5 μm range. The functionalization fluid facilitates further processing using humidity control to achieve full coverage of only the PMCL's top surface and removal of dissolved salts to improve uniformity of receptor coverage and to prevent fouling of the sensor surface. Once a functionalization method was successfully developed, a series of experiments were performed to investigate the amount of surface stress that can be generated when receptors are immobilized directly to the silicon surface. In one series of experiments, a 4.8 μM streptavidin solution was used with biotin immobilized on multiple PMCLs to demonstrate adsorption-induced surface stress and concomitant deflection of the PMCL. The group observed ~ 15 nm PMCL deflection on average, with a corresponding surface stress of approximately 4 mN/m. These experiments yield the sensor response in real-time and employ a combination of multiple PMCLs functionalized as either sensors or unfunctionalized to serve as references. Investigation of various attachment chemistries is included, as well as a comparison with and without passivation of non-sensor surfaces. Investigated passivation strategies prevented ligand binding from generating a differential surface stress. Failure modes and physical mechanisms for adsorption-induced surface stress are discussed. Immobilization and passivation strategies for antibody-based biosensing are demonstrated with fluorescence microscopy and a corresponding PMCL sensing experiment using rabbit anti-goat F(ab') fragments as the receptors and Alex Fluor 488 labeled goat anti-rabbit IgGs as the ligand. While the results of these experiments remain inconclusive, suggestions for future research involving the PMCL sensor array are recommended.

[pt] A espuma é amplamente usada em operações de recuperação de óleo para melhorar a eficiência de varrido, em operações de armazenamento de gás e acidificação, e para resolver problemas causados por zonas ladras ou segregação gravitacional. A espuma, que pode ser pré-formada e injetada no reservatório ou produzida in situ através da geometria do meio poroso, escoa nas regiões de alta permeabilidade e desvia o fluido de deslocamento na direção do óleo aprisionado, reduzindo a permeabilidade relativa ao gás e levando a uma frente de deslocamento mais estável. A eficiência desses processos depende muito da geração e estabilidade dos filmes de espuma (lamelas) que residem nos poros. A mobilidade do gás injetado é reduzida quando a espuma é formada; esta redução é atribuída ao aumento da viscosidade efetiva do gás e à redução da permeabilidade relativa ao gás. As lamelas formadas criam resistência ao fluxo do gás, impedindo seu movimento livre dentro do meio poroso. A população de lamelas que compõe a espuma está diretamente relacionada com a concentração de surfactante, e seu fluxo e mobilidade são funções da geometria dos poros e das propriedades da espuma. No entanto, a dinâmica da formação de espuma em meios porosos não é totalmente compreendida devido à sua complexidade O objetivo da primeira parte desta pesquisa é compreender o impacto do aumento da concentração de surfactante na formação de espuma durante a injeção de gás em um modelo bidimensional de meio poroso de vidro saturado com uma solução de surfactante. A segunda parte foca na formação de espuma e sua implicação no deslocamento de óleo durante o processo de injeção SAG (injeção alternada de solução de surfactante e gás) considerando diferentes concentrações de surfactante. Uma configuração microfluídica composta por micromodelo de vidro, bomba de seringa, transdutor de pressão e microscópio foi usada para visualizar o deslocamento da escala dos poros e correlacionar a evolução da formação das lamelas durante o processo de injeção com a diferença de pressão para diferentes condições de fluxo através do processamento de imagem. A dinâmica de formação das lamelas é relatada e relacionada ao comportamento do fluxo macroscópico. / [en] Foam is widely used in oil recovery operations to improve sweep efficiency, in gas storage and acidization operations, and to solve problems caused by either a thief zone or gravity override. Foam, which can be preformed and injected into the reservoir or produced in situ through the pore space, fills the high permeability areas known as thief zones and diverts the displacing fluid into the direction of trapped oil, reducing the relative permeability of gas and leading to a more stable displacement front. The efficiency of these processes largely depends on the generation and stability of the foam films (lamellae) residing in the pores. The mobility of the injected gas is reduced when foam is formed; this reduction is attributed to the reduction of the gas phase relative permeability. The lamellae formed create resistance against the gas flow, impeding its free motion inside the porous media. The lamellae population that composes the foam is directly related to surfactant concentration, and their flow and mobility are functions of the pore geometry and foam properties. However, the dynamics of foam formation in porous media is not fully understood due to its complexity. The goal of the first part of this research is to understand the impact of increasing surfactant concentration on foam formation during gas injection in a two-dimensional porous media glass model occupied by a surfactant solution. The second part focuses on foam formation and its implications for oil displacement during the SAG (surfactant-alternating-gas) injection, considering different surfactant concentrations. A microfluidic setup composed of a glass micromodel, syringe pump, pressure transducer and microscope, was used to visualize the pore-scale displacement and correlate the evolution of lamellae formation during the injection process with pressure difference for different flow conditions through image processing. The dynamics of lamellae formation is reported and related to macroscopic flow behavior.

[pt] Microcápsulas são utilizadas em diversos setores da indústria para isolar o material interno do ambiente externo. Elas protegem o conteúdo interno e permitem uma liberação controlada. Neste trabalho, apresenta-se um método de produção de microcápsulas de goma gelana monodispersas a partir da formação de modelos de emulsão dupla óleo-em-água-em-óleo por microfluídica. A extração do óleo externo, após a gelificação ionotrópica, permite a dispersão das microcápsulas em meios aquosos. Assim, o método proposto permite encapsular ativos hidrofóbicos e dispersar as microcápsulas em uma fase aquosa, tendo diversas aplicações. Foram definidas janelas de operação para produção de microcápsulas de gelana monodispersas em função da vazão volumétrica de cada fluido que forma as microcápsulas e das dimensões do dispositivo microfluídico. Produziu-se microcápsulas com diâmetros variando de 95 a 260 um e um coeficiente de variação máximo de 5 per cent. Os resultados mostram que é possível controlar o diâmetro das cápsulas e a espessura da membrana através das vazões da fase externa e intermediária, respectivamente. Além disso, estudamos o escoamento de cápsulas de gelana com diferentes diâmetros e espessuras de membrana por um capilar com constrição através de imagens microscópicas e medidas de diferença de pressão. Mapeamos as condições nas quais a membrana é rompida devido à constrição e o conteúdo interno é liberado durante o escoamento. A gastroresistência das cápsulas de gelana é verificada através de testes in vitro que simulam as fases gástrica e intestinal da digestão. Mostramos, através de imagens fluorescentes, que as cápsulas são capazes de liberar o conteúdo interno apenas no intestino devido ao seu pH. Finalmente, demonstramos ser possível, não só produzir microcápsulas magnéticas, mas controlar a resposta magnética delas regulando a quantidade de ferrofluido que é adicionada à fase interna ou à membrana polimérica. As microcápsulas produzidas neste estudo têm grande potencial de aplicação em diversos setores, como alimentício, biomédico, farmacêutico e de óleo e gás. / [en] Microcapsules are applied in several sectors of industry when a physical barrier between the core material and the external environment is required. They protect their cargo and ultimately release it in a controlled way. In the present work, microcapsules with hydrogel-based shells are produced. Monodispersed microcapsules are formed by ionotropic gelation of gellan gum from monodispersed oil-in-water-in-oil (O/W/O) double emulsion templates obtained using glasscapillary microfluidic devices. An oil extraction step was added after the shell gelation process to enable the dispersion of the microcapsules in an aqueous medium. We report the operability window for the production of monodispersed microcapsules as a function of the flow rate of each fluid phase and the dimensions of the device. Microcapsules with mean diameters ranging from 95 to 260 um and a maximum coefficient of variation of 5 per cent were formed. The results show how to independently control the capsule diameter and shell thickness by varying the outer and middle phase flow rates. After that, we experimentally investigate the flow of monodispersed gellan gum microcapsules through a constricted capillary tube by measuring the evolution of the pressure difference and flow visualization. The maximum pressure difference and capsule deformation is obtained for capsules with different diameter and shell thickness. We map the conditions at which the capsule membrane ruptures during the flow, releasing its internal phase. Then, the gastro-resistance of gellan microcapsules is verified through an in vitro test that mimics the gastric and intestinal phases of digestion. Confocal fluorescence microscopy is used to track microcapsules integrity and we show that microcapsules cargo is released in the intestine mostly due to its pH. Finally, we demonstrate that it is possible to produce magnetic microcapsules with well controlled magnetic response by adding different amounts of ferrofluid to their core or shell. The microcapsules produced have great potential for different applications in food, biomedical, pharmaceutical and oil and gas industries.

Stimuli-sensitive hydrogels have an outstanding potential for miniaturized, integrated sensor, and actuator systems and especially for lab-on-chip technology, but the application is still in its infancy. One major reason may be that design and realization of hydrogel-based systems are exceptionally complex and demanding. Here, the design parameters of a key component, the hydrogel-based valve, are discussed in their entirety. Key developments in the fields of stimuli-sensitive hydrogels are highlighted and the necessary know-how in material behavior, microstructuring technologies, modeling and name five essential design guidelines as well as scaling laws for hydrogelbased components, including microfluidic one-directional valves, microelectromechanical systems valves, self-regulating, chemomechanical valves, and chemofluidic transistors, is provided.

Immunoassays are analytical methods that use the highly specific binding of antibodies in order to detect and quantify an analyte. The technique has become a staple in modern biopharmaceutical research and diagnostics, however the measurement of biomarkers like dysregulated cytokines require ultra-sensitive immunoassays that can detect molecules at sub pg/mL concentrations. One such method is the Exazym® signal amplification. Based on a method called Binding Oligo Ladder Detection (BOLD), it is a set of add-on reagents where a primer is conjugated to a detection antibody which is then combined with a template, polymerase and modified DNA nucleotides to generate a oligonucleotide ladder that is detected with a secondary detection antibody; this amplifies the signal by a factor of 10-100 in an existing immunoassay.  By applying this method to the Gyrolab® microfluidic immunoassay system, a sensitivity increase of 880x-1800x was achieved between a pre-synthesised BOLD product and the polymerised BOLD product. Several key factors for successful polymerisation in the microfluidic system were identified: adding the template separately before the polymerase and using a buffer with low ionic strength for the secondary detection antibody. Applying the BOLD amplification to an existing Gyrolab TNF-α assay only resulted in similar sensitivity as previous methods however. This report demonstrates that BOLD amplification can be successfully performed in a flow-through format on miniaturized affinity columns in the Gyrolab system to increase the sensitivity by orders of magnitude, where both the immunoassay and the amplification steps are automated in the system. However, further optimisation is needed for application in biomarker assays.

Microfluidics is a versatile tool with many applications in biology. Its ability to manipulate small volumes of liquid precisely has led to the development of many microfluidic assay platforms. They could handle small amounts of samples and carry out analysis with high sensitivity and throughput. Microfluidic assays have provided new insights into scarce biological samples at higher resolution. In this thesis, we developed microfluidic tools to conduct low input ChIP-seq and ChIRP-seq. We applied them to a variety of samples profiling different targets. The native MOWChIP-seq platform was developed to map RNA polymerase II, transcription factors and histone deacetylase binding in 1,000-50,000 cells. We examined mouse prefrontal cortex and cerebellum using this technology. We found extensive differences that correlated with distinct neurological functions of the brain regions. The same platform and workflow were used to profile five key histone modifications in human lung tumor and normal tissue samples. Integrative analysis with gene expression data revealed extensive chromatin remodeling in lung tumor. Spatial histone modification mapping was conducted in mouse neocortex in a similar fashion. We generated an epigenomic tomography that demonstrated the molecular state of the brain in 3D. Lastly, we developed a microfluidic version of the ChIRP-seq process which successfully conducted the assay using only 500K cells. This improvement makes ChIRP-seq in tissue samples feasible. / Doctor of Philosophy / Microfluidics is a type of technology that can control small volumes of liquid in a miniature system. It can carry out reactions on very small scales with higher precision and sensitivity than conventional methods. Microfluidics has found many uses in the field of biology, especially dealing with samples available in limited quantities. These low input microfluidic platforms have helped researchers gain new knowledge on many complex questions. In this thesis, we developed microfluidic tools to carry out low input ChIP-seq and ChIRP-seq. These are two established techniques used to map where certain targets are located on the genome of an organism. These targets include specific chemical modifications to the wrapper protein of DNA (histone modification), proteins that take part in transcription and expression of genes (RNA polymerase II, transcription factors) and other molecules. Our nMOWChIP-seq system removed the need for fixation by chemicals. It was able to examine RNA polymerase II, transcription factors and other enzymes using 1,000-50,000 cells. Traditional ChIP-seq requires more than 10 million cells and time-consuming chemical treatment steps. Our technology greatly improved sensitivity and ease of use. We also used this platform to test five important histone modifications in human lung tumors and healthy tissues. We constructed a spatial map of histone modification in mouse brain by analyzing slices of the cortex. Finally, we developed a microfluidic version of ChIRP-seq process to map locations of long non-coding RNAs in cultured human cells. The cells needed for a successful test were reduced to 500K from 20 million of the original workflow.

Abstract  Lack of clinical relevance is assumed to be the main reason behind the high failure rate of medical drugs in the very initial phases of clinical trials. Clinical relevance is difficult to achieve with current tools as they lack the biological and physiological cues found in vivo. Microfluidics, the knowledge of fluid manipulation in small channels, has proven to be a promising science to bridge the gap between the current in vitro and the real in vivo features. In this thesis, a scaffold for the growth of spheroids inside a microfluidic device for potential drug screening was developed. Firstly, the surface of a microfluidic device was coated with the polymers cellulose nanofibrils, polyallylamine hydrochloride, and polyethyleneimine using the Layer-by-Layer technique to achieve an even surface coverage. Here, different chip designs, polymer concentrations, and pressure directions were tested. It was decided that using a negative pressure direction with a polymer concentration of 50 mg/L in a chip design with micropillars was optimal and these conditions were then used for testing the spheroid formation. Secondly, spheroids were grown inside the microfluidic channels using different coatings: the previously mentioned polymer buildup, one non-coated channel, and one coated with attachment factor proteins. These three surface conditions were compared and it was shown that the polymer-based surface cover was indeed superior as a scaffold as it encouraged and promoted cell growth in the spheroid formation of liver cancer cells from the HepG2 cell line. Further development of this cellulose nanofibrils-coated microfluidic device displays a promising future for functioning as an in vitro 3D cell culture model that better mimics the close-to-cell microenvironments by imitating cell proliferation, cell-to-cell, and cell-to-extracellular matrix interactions. / Sammanfattning Den främsta orsaken bakom den höga antal misslyckade kliniska läkemedelsprövningar i de initiala faserna antas bero på brist på klinisk relevans. Klinisk relevans är mycket svår att uppnå med dagens verktyg då de saknar de biologiska och fysiologiska förhållandena som återfinns in vivo. Mikrofluidik, kunskapen om vätskemanipulation i små kanaler har visat sig vara lovande vetenskap för att överbrygga klyftan mellan de nuvarande in vitro och de faktiska in vivo funktionerna. I detta arbete utvecklades en matris för sfäroider att växa på inuti en mikrofluidisk kanal för att potentiellt användas till läkemedelsscreening. Först användes Layer-by-Layer teknologi för att jämnt betäckta ytan inuti en mikrofluidisk kanal med polymererna cellulosananofibriller, polyallylamin hydroklorid samt polyetylenimin. Här testades olika designer på mikrofluidiska chip, polymerkoncentrationer samt tryckriktningar. Utifrån detta gick det att fastställa att negativt tryck med en polymerkoncentration på 50 mg/L i en chippdesign med mikropelare var optimal för en jämn ytbetäckning och dessa förhållanden användes sedan för att pröva sfäroidernas tillväxt. Härnäst testades därmed sfäroidernas tillväxt inuti mikrofluidiska kanaler under tre olika förhållanden: ett med polymerbetäckningen, ett utan betäckning och ett då ytan var täckt med proteiner med fästfaktorer. Dessa tre förhållanden jämfördes sedan med varandra och således gick det att konstatera att den polymerbaseradebetäckningen fungerade överlägset som matris för tillväxt av HepG2 lever cancer cell sfäroider eftersom den tycks främja dess tillväxt och bildning. Det pekar mot att ytterligare utveckling av denna cellulostäckta yta skulle innebära en lovande modell för in vitro 3D cellodling som bättre efterliknar den cellulära mikromiljön genom att imitera cellproliferation, interaktioner celler emellan samt mellan cell och extracellulär matrisen.

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