Aging is a complex process by which a combination of environmental, genetic and stochastic factors generate whole-system changes that modify organ and tissue function and alter physiological processes. Over the last few decades, many genetic and environmental modulators of aging have been found to be highly conserved between humans and a diverse group of model organisms. Yet, an integrative understanding of how these environmental and genetic variables interact over time in a whole organism to modulate the systemic changes involved in aging is lacking. The goal of this thesis project is to advance a systems perspective of aging by providing the experimental tools and conceptual framework for dissecting the regulatory connection between environmental inputs, molecular outputs and long term aging phenotypes in Caenorhabditis elegans, an experimentally tractable multi-cellular model for aging. Specifically, this work advances the quantitative imaging toolsets available to biologists by developing and refining microfluidic, hardware, computer vision, and software integration tools for high-throughput, high-content imaging of C. elegans. As a result of these technological advances, new roles for the TGF-beta and serotonin signaling pathways in encoding environmental food signals to influence longevity were uncovered and quantitatively characterized. Moreover, this work develops and integrates new microfluidic technologies with off-chip support systems to establish a platform for long-term tracking of the health and longevity trajectories of large numbers of individual C. elegans. The capabilities of this platform have the potential to address many important questions in aging including addressing environmental determinants of aging, the sources of inter-individual variability, the time course of aging-related declines and the effects of interventional strategies to improve health outcomes. Together, the toolsets for quantitative imaging and the long-term culture platform permit the large-scale investigation of both the internal state and long-term behavioral and health outputs of an important multicellular model organism for aging.

THz spectroscopy is an emerging technique for studying the dynamics and interactions of cells and biomolecules, but many practical challenges still remain in experimental studies. We present a prototype of simple and inexpensive cell-trapping microfluidic chip for THz spectroscopic study of live cells. Cells are transported, trapped and concentrated into the THz exposure region by applying an AC bias signal while the chip maintains a steady temperature at 37 degrees C by resistive heating. We conduct some preliminary experiments on E. coli and T-cell solution and compare the transmission spectra of empty channels, channels filled with aqueous media only, and channels filled with aqueous media with un-concentrated and concentrated cells.

As electronics reach nanometer size scales, new avenues of integrating biology and electronics become available. For example, nanoscale field-effect transistors have been integrated with single neurons to detect neural activity. Researchers have also used nanoscale materials to build electronic ears and noses. Another exciting development is the use of nanoscale biosensors for the point-of-care detection of disease biomarkers. This thesis addresses many issues that are relevant for electrical sensing applications in biological environments. As an experimental platform we have used carbon nanotube field-effect transistors for the detection of biological proteins. Using this experimental platform we have probed many of properties that control sensor function, such as surface potentials, the response of field effect transistors to absorbed material, and the mass transport of proteins. Field effect transistor biosensors are a topic of active research, and were first demonstrated in 1962. Despite decades of research, the mass transport of proteins onto a sensor surface has not been quantified experimentally, and theoretical modeling has not been reconciled with some notable experiments. Protein transport is an important issue because signals from low analyte concentrations can take hours to develop. Guided by mass transport modeling we modified our sensors to demonstrate a 2.5 fold improvement in sensor response time. It is easy to imagine a 25 fold improvement in sensor response time using more advanced existing fabrication techniques. This improvement would allow for the detection of low concentrations of analyte on the order of minutes instead of hours, and will open the door point-of-care biosensors. / Graduation date: 2011

This thesis includes three studies on related aspects of structure and function control for drug delivery block copolymer nanoparticles manufactured in segmented gas-liquid microfluidic reactors. First, the self-assembly of a series of photoresponsive poly(o-nitrobenzyl acrylate)-b-polydimethylacrylamide copolymers is conducted in the gas-liquid segmented microfluidic reactor at various flow rates. The resulting morphologies are found to be flow-variable and distinct from nanoparticles prepared off-chip by dropwise water addition. Photocleaving of the nanoparticles formed at different flow rates reveal flow-variable photodissociation kinetics. Next, we conduct a direct comparison between a commercially-available single-phase microfluidic mixer and the two-phase, gas-liquid segmented microfluidic reactor used in our group, with respect to nanoparticle formation from a typical block copolymer identified for drug delivery applications, polycaprolactone-b-poly(ethylene oxide). The two-phase chip yields morphologies and core crystallinities that vary with flow rate; however, the same parameters are found to be flow-independent using the single-phase mixer. This study provides the first direct evidence that flow-variable structure control is a unique feature of the two-phase chip design. Finally, we investigate structure and function control for paclitaxel (PAX)-loaded nanoparticles prepared from a series of poly(6-methyl caprolactone-co-ε-caprolactone)-block-poly(ethylene oxide) copolymers with variable 6-methyl caprolactone (MCL) content. For all MCL-containing copolymers, off-chip preparations form nanoparticles with no measurable crystallinity, although PAX loading levels are higher and release rates are slower compared to the copolymer without MCL. Both off-chip and on-chip preparations yield amorphous spheres of similar size from MCL-containing copolymers, although on-chip nanoparticles showed slower release rates, attributed to more homogeneous PAX distribution due to faster mixing. / Graduate

Microfluidic H-cells and surface enhanced Raman spectroscopy are capable of analysing the asphaltene content of petroleum. An H-cell is a microfluidic device that exploits the non-turbulent flow of fluids within a micrometre-dimensioned channel. Diffusive separation in an H-cell permits a liquid that is miscible with the sample matrix to be used as an extractant. It was demonstrated that n-hexane can be used as extractant to obtain an asphaltene-free fraction of oil. The difference between the UV-Vis adsorption spectra of the asphaltene-free oil and the oil sample can then be used to estimate its asphaltene content. This has been demonstrated for a range of oils with asphaltene content between 1-30% and API gravity values between 40-10°, thus liquid petroleum and bitumen can be rapidly assayed by an H-cell; similarly, asphaltene and carboxylic acid content of oil can be determined simultaneously when methanol is used as extractant. The results were shown to be comparable to assays achieved via the ASTM D4124 and ASTM D974 methods respectively. For the first time it was demonstrated that surface enhanced Raman spectroscopy, using a gold substrate and illumination at 514 nm, can detect extremely low concentrations of asphaltene. This was shown to be achievable for asphaltene and related materials at concentrations of 0.001 ppm. In addition, data also demonstrated that the core of the Raman-responsive units within asphaltene have crystallite sizes equivalent to the Raman-responsive units in kerogen (~3 nm). Both methods provide technological advances because they make it possible to detect asphaltene in small sample volumes, using smaller footprint instrumentation. The H-cell method would be extremely useful for appraising oilfield potential, record the attenuation of oil-spills and provide frequent geochemical data that can monitor these at point of need. Similarly, the SERS technique widens the field of application into areas previously inaccessible to current techniques such as the effect of low concentrations of asphaltene-like materials in ecological and living systems.

Master of Science / Department of Chemistry / Christopher T. Culbertson / Chemists have been trying to relate the structure and composition of different cereal proteins to their physical properties to better inform their product use for more than 250 years now. Among these cereals, wheat is considered the most important due to its unique ability to form viscoelastic dough and retain gas during fermentation, the latter being important for bread making. This property is due to the endosperm part of wheat that contains proteins mostly gliadins and glutens. It is known that the composition and relative ratio of these proteins is determined by both the growing environment and genetics. Manipulation of the genetics allows one for control of only about 50% of the end use quality of the wheat and the rest is controlled by environment. Currently, the bread making quality of wheat is determined by baking test loaves of bread. This process is time consuming and wasteful. The main goal of this project was to create fingerprints of gliadin proteins for different wheat cultivars as a function of environmental conditions. This would then allow wheat kernels to be analyzed and assessed right after harvest to determine their appropriateness for making the various wheat products. Researchers have tried to create a catalogue of information for individual wheat cultivars by ‘fingerprinting’ the gliadins proteins in wheat using various analytical techniques including capillary electrophoresis (CE). CE offers advantages like high separation efficiency, and faster analysis. Further miniaturization of CE on microfluidic devices has enhanced the speed and efficiency of separation. Furthermore, it is possible to integrate multiple chemical analysis processes like sample preparation, separation and detection in a single microfluidics device. Microfluidic uses micron sized separation channels defined in a glass, quartz or polymer. This dissertation is focused on fabricating multilayer microfluidic devices from Poly(dimethylsiloxane) (PDMS) and using these devices to electrophoretically separate wheat gliadin proteins followed by detection using UV absorption in less than 5 min. PDMS is cheap, easy to fabricate and is optically transparent above ~230nm. Initial results of the UV absorbance detector developed for this device are presented.

Immune cell migration and chemotaxis plays a key role in immune response. Further research to study the mechanisms of immune cell migration and to develop clinical applications requires advanced experimental tools. Microfluidic devices can precisely apply chemical gradient signals to cells, which is advantageous in quantifying cell migratory response. However, most existing microfluidic systems are impractical to use without specialized facilities and research skills, which hinders their broad use in biological and medical research communities. In this thesis, we integrated several new developments in microfluidic gradient generating devices, compact imaging systems, on-chip cell isolation, cell patterning, and rapid data analysis, to provide an easy-to-use and practical solution for immune cell migration and chemotaxis experiments. Using these systems, we quantitatively studied neutrophil migration for both research and clinical applications. First, we developed a compact USB microscope-based Microfluidic Chemotaxis Analysis System (UMCAS), which integrates microfluidic devices, live cell imaging, environmental control, and data analysis to provide an inexpensive and compact solution for rapid microfluidic cell migration and chemotaxis experiments with real-time result reporting. To eliminate the lengthy cell preparation from large amounts of blood, we developed a simple all-on-chip method for magnetic isolation of untouched neutrophils directly from small volumes of blood, followed by chemotaxis testing on the same microfluidic device. Using these systems, we studied neutrophil migration in gradients of different chemoattractants, such as interleukin-8 (IL-8), N-formyl-methionyl-leucyl-phenylalanine (fMLP), and clinical sputum samples from Chronic Obstructive Pulmonary Disease (COPD) patients. Previous studies have shown that COPD is correlated with neutrophil infiltration into the airways through chemotactic migration. The thesis work is the first application of the microfluidic platform to quantitatively characterizing neutrophil chemotaxis to sputum samples from COPD patients. Our results show increased neutrophil chemotaxis to COPD sputum compared to control sputum from healthy individuals. The level of COPD sputum induced neutrophil chemotaxis was correlated with the patient’s spirometry data. Collectively, the research in this thesis provides novel microfluidic systems for neutrophil migration and chemotaxis analysis in both basic research and clinical applications. The developed microfluidic systems will find broad use in cell migration related applications. / May 2016

This diploma thesis deals with hyphenation of liquid chromatography with mass spectrometric detection based on microfluidic chip. Firstly, a miniaturized ion source for atmospheric-pressure chemical ionization (APCI), and atmospheric-pressure photoionization (APPI) was constructed. The main component of this source was a glass microfluidic chip. Geometry and the working conditions of the chip were optimized. Since both ion sources work under the same conditions, possible advantages resulting from APCI/APPI combination were investigated. Furthermore, the performance characteristics of the sources were evaluated, and compared to the conventional high flow-rate sources. The best performing source, APCI, was then hyphenated with HPLC using low flow-rate. A method for separation of fatty acids methyl esters using Supelco 37 standard was developed. The separation conditions were as follows: C18 reversed stationary phase, and acetonitrile containing 0.1 % formic acids was used as the mobile phase. A temperature gradient was used in order to enhance the speed of the separation. The limits of detection and quantitation of for selected analytes using the chip micro-APCI were calculated, and compared to the ones obtained using a commercially available micro-APCI source. The method was used for separation of...

Il existe un besoin industriel croissant de nouvelles technologies capables de manipuler des objets à l’échelle micrométrique (1-1000 μm). Dans cette thèse, une technique originale d’actionnement sans contact pour la manipulation d’objets à l’échelle micrométrique est proposée. Elle est basée sur les écoulements thermocapillaires convectifs induits par un laser pour manipuler des particules à l’interface fluide/gaz. Le laser chauffe localement la surface de l’eau, ce qui induit un gradient de tension de surface. Ce gradient génère un écoulement fluidique. Ces écoulements sont rapides et localisés, ce qui confère des performances intéressantes à cette technique d’actionnement. Les particules sont manipulées à l’interface fluide/gaz, où l’écoulement généré est le plus rapide.Pour assurer le positionnement précis d’une particule, des contrôleurs en boucle fermée sont implémentés dans le système. Ils sont basés sur les modèles développés dans cette thèse. Des essais expérimentaux montrent que le positionnement précis de particules peut être assuré. De plus, les forces d’interaction entre des particules placées à l’interface ont été étudiées, et une stratégie de contrôle a été proposée, en vue de la manipulation en parallèle de plusieurs particules. Tant les études analytiques et les simulations numériques que les tests expérimentaux soulignent l’intérêt des écoulements thermocapillaires pour la manipulation contrôle d’objets micrométriques. Cette technique est donc une alternative prometteuse aux approches classiques d’actionnement sans contact. / There is an industrial need for new technologies that can manipulate objects in the micrometric scale (1-1000 μm). In this thesis, an original non-contact actuation technique for the manipulation of microscale objects is proposed. The proposal is to use laser-induced thermocapillary convective flows to manipulate particles at the fluid/gas interface. These flows are generated when a surface tension stress is generated at the fluid/gas interface due to a thermal gradient. Laser heating is used because the generated thermal gradients produce fast, localized flows that improve the actuation performance. The particles are manipulated at the interface because the flow generated there is the fastest in the entire fluid.To ensure the precise positioning of a particle, closed-loop controllers are implemented in the system which are designed based on models proposed for the system. Experimental tests are performed that show that positioning precision can be ensured. In addition, the interaction forces between particles have been studied which is a preliminary step towards parallel manipulation. To counteract those forces during the manipulation, a different control strategy has been proposed, implemented and tested. Overall, the results obtained are comparable to the ones obtained with the other techniques. Therefore, the proposed technique can be considered as an attractive alternative that offers different advantages and disadvantages.

Magnetic nanoparticle (MNP) combined with biomolecules in a microfluidic system can be efficiently used in various applications such as mixing, pre-concentration, separation and detection. They can be either integrated for point-of care applications or used individually in the area of bio-defense, drug delivery, medical diagnostics, and pharmaceutical development. The interaction of magnetic fields with magnetic nanoparticles in microfluidic flows will allow simplifying the complexity of the present generation separation and detection systems. The ability to understand the dynamics of these interactions is a prerequisite for designing and developing more efficient systems. Therefore, in this work proof-of-concept experiments are combined with advanced numerical simulation to design, develop and optimize the magnetic microfluidic systems for mixing, separation and detection. Different strategies to combine magnetism with microfluidic technology are explored; a time-dependent magnetic actuation is used for efficiently mixing low volume of samples whereas tangential microfluidic channels were fabricated to demonstrate a simple low cost magnetic switching for continuous separation of biomolecules. A simple low cost generic microfluidic platform is developed using assembly of readily available permanent magnets and electromagnets. Microfluidic channels were fabricated at much lower cost and with a faster construction time using our in-house developed micromolding technique that does not require a clean room. Residence-time distribution (RTD) analysis obtained using dynamic light scattering data from samples was successfully used for the first time in microfluidic system to characterize the performance. Both advanced multiphysics finite element models and proof of concept experimentation demonstrates that MNPs when tagged with biomolecules can be easily manipulated within the microchannel. They can be precisely captured, separated or detected with high efficiency and ease of operation. Presence of MNPs together with time-dependent magnetic actuation also helps in mixing as well as tagging biomolecules on chip, which is useful for point-of-care applications. The advanced mathematical model that takes into account mass and momentum transport, convection & diffusion, magnetic body forces acting on magnetic nanoparticles further demonstrates that the performance of microfluidic surface-based bio-assay can be increased by incorporating the idea of magnetic actuation. The numerical simulations were helpful in testing and optimizing key design parameters and demonstrated that fluid flow rate, magnetic field strength, and magnetic nanoparticle size had dramatic impact on the performance of microfluidic systems studied. This work will also emphasize the importance of considering magnetic nanoparticles interactions for a complete design of magnetic nanoparticle-based Lab-on-a-chip system where all the laboratory unit operations can be easily integrated. The strategy demonstrated in this work will not only be easy to implement but also allows for versatile biochip design rules and provides a simple approach to integrate external elements for enhancing mixing, separation and detection of biomolecules. The vast applications of this novel concept studied in this work demonstrate its potential of to be applied to other kinds of on-chip immunoassays in future. We think that the possibility of integrating magnetism with microfluidic-based bioassay on a disposable chip is a very promising and versatile approach for point-of care diagnostics especially in resource-limited settings.