Master of Science / Department of Chemistry / Christopher T. Culbertson / A microfluidic device integrated with valves and a peristaltic pump was fabricated using multilayer soft lithography to analyze single cells. Fluid flow was generated and mammalian cells were transported through the channel manifold using the peristaltic pump. A laser beam was focused at the cross-section of the channels so fluorescence of individual labeled intact cells could be detected. Triggered by the fluorescence signals of intact cells, valves could be actuated so fluid flow was stopped and a single cell was trapped at the intersection. The cell was then rapidly lysed through the application of large electric fields and injected into a separation channel. Various conditions such as channel geometry, pumping frequency, control channel size, and pump location were optimized for cell transport. A Labview program was developed to control the actuation of the trapping valves and a control device was fabricated for operation of the peristaltic pump. Cells were labeled with a cytosolic dye, Calcein AM or Oregon Green, and cell transport and lysis were visualized using epi-fluorescent microscope. The cells were transported at rates of [simular to] 1mm/s. This rate was optimized to obtain both high throughput and single cell trapping. An electric field of 850-900 V/cm was applied so cells could be efficiently lysed and cell lysate could be electrophoretically separated. Calcein AM and Oregon Green released from single cells were separated and detected by laser-induced fluorescence. The fluorescence signals were collected by PMT and sampled with a multi-function I/O card. This analyzing method using microchip may be applied to explore other cellular contents from single cells in the future.

Doctor of Philosophy / Department of Chemistry / Christopher T. Culbertson / Microfluidic devices have a wide variety of biological applications. My Ph.D. dissertation focuses on three major projects. A) culturing a non-adherent immortal cell line within a microfluidic device under static and dynamic media flow conditions; B) designing and fabricating novel microfluidic devices for electrokinetic injecting analytes from a hydrodynamic fluid; and C) using this novel injection method to lyse single non-adherent cells by applying a high electric field across the cell at a microfluidic channel intersection. There are several potential advantages to the use of microfluidic devices for the analysis of single cells: First, cells can be handled with care and precision while being transported in the microfluidic channels. Second, cell culturing, handling, and analysis can be integrated together in a single, compact microfluidic device. Third, cell culturing and analysis in microfluidic devices uses only extremely small volumes of culturing media and analysis buffer. In this dissertation a non-adherent immortal cell line was studied under static media flow conditions inside a CO[subscript]2 incubator and under dynamic media flow conditions in a novel portable cell culture chamber. To culture cells they must first be trapped on a microfluidic device. To attempt to successfully trap cells, three different types of cellular traps were designed, fabricated and tested in polydimethylsiloxane (PDMS)-based microfluidic devices. In the first generation device, cubic-shaped traps were used. After 48 h of culturing in these devices the cell viability of 79 [plus or minus] 6 % (n = 3). In the second generation device, circular wells with narrow connecting channels were employed. However, after 12 h of culturing, no viable cells were found. While the second generation device was not capable of successfully culturing cells, it did demonstrate the importance of culturing under dynamic conditions which lead to next design. The third generation microfluidic device consisted of hydrodynamic shaped traps that were used to culture the cells in a less confined environment. The cell viability after 12 h in this design was 29 [plus or minus] 41% (n = 3). In addition to cell trapping, a novel electrokinetic injection method was developed for injecting analytes from a hydrodynamic flow into a separation channel that was followed by an electrokinetic separation. As the hydrodynamic flow could introduce some excess band broadening in the separation, the actual band broadening of an analyte was measured for different channel depths and hydrodynamic fluid flow rates. The results consistently showed that the separations performed on these devices were diffusion limited. Finally, using this novel injection method, single cell lysis was performed by applying a high voltage at the microfluidic channel intersection. The results of these studies may eventually be applied to help answer some fundamental questions in the areas of biochemistry and pharmaceutical science.

Doctor of Philosophy / Department of Chemistry / Christopher T. Culbertson / During the last five years, great advancements in microfluidics have been achieved with the development of “sample-in-answer-out” systems. Such systems have begun to realize the true potential of analytical miniaturization since the concept of the “micro-Total Analysis System” was first envisioned. These systems are characterized by the elegant integration of multiple fluid-handling channel architectures that enable serial execution of sample preparation, separation and detection techniques on a single device. While miniaturization and portability are often identified as key advantages for microfluidics, these highly integrated systems are heavily reliant upon large off-chip equipment, i.e. the microchip is often tethered to the laboratory via multiple syringe pumps, vacuum pumps, solenoid valves, gas cylinders and high voltage power supplies. In this dissertation, a procedure for the facile integration of dielectric elastomer (DE) actuators (called IDEAs) onto microfluidic devices is described. Poly(dimethylsiloxane) (PDMS) is commonly used as a microchip substrate because it is cheap and easy to fabricate, mechanically robust and optically transparent. The operation of an IDEA exploits the ability of PDMS to behave as a smart material and deform in the presence of an electric field. In Chapter 2, the fabrication of IDEA units on a standard microchip electrophoresis device is described. IDEA-derived injections were used to evaluate the physical performance of this novel actuator configuration. In Chapter 3, the analytical merits of IDEA-derived injections were evaluated. Sampling bias caused by electokinetic injection techniques has been problematic for conventional microchip electrophoresis systems due to the lack of fluid access. The hydrodynamic injections created by IDEA operation were found to be highly reproducible, efficient, and possess a negligible degree of sampling bias. In Chapter 4, the spatial characteristics of microchannel deformation due to IDEA actuation have been investigated using fluorescence microscopy. It was determined that the DE compresses more along the edge of the channel than in the middle of the channel. This information can be used to design a new generation of more efficient IDEAs.

Master of Science / Department of Diagnostic Medicine/Pathobiology / Shafiqul I. Chowdhury / Bovine herpesvirus type 1 (BHV-1) is an important pathogen of cattle that can cause severe respiratory tract infection known as infectious bovine rhinotracheitis (IBR), abortion in pregnant cows, and is an important component of the Bovine Respiratory Disease Complex (BRDC, “Shipping fever”). The ability of BHV-1 to transport anterogradely from neuron cell bodies in trigeminal ganglia to axon termini in the nasal and ocular epithelia of infected cattle complicates the control of the disease in both vaccinated and infected cattle populations. In calves and rabbits, Us9 deleted viruses have defective anterograde neuronal spread from cell bodies in the trigeminal ganglia to nerve termini in the nose and eye but retrograde spread remains unaffected. To characterize the neuronal spread of BHV-1, we developed primary neuronal cultures using the dorsal root ganglia (DRG) of rabbit embryos. We successfully used microfluidic chamber devices to isolate DRG in the somal compartment and allowed for efficient growth of axons into the axonal compartment. This enabled us to study axon mediated neuronal spread of infection as well as viral transport in axons. Thus, rabbit DRG neuronal culture was susceptible to BHV-1 mutant and wild-type infection, and the method allowed visualization of viral spread in chamber cultures using live cell imaging and fluorescent microscopy. Lastly, using the microfluidic chamber compartmentalized neuron culture system we showed that Us9 acidic domain-deleted and Us9 null mutant BHV-1 viruses had defective anterograde neuronal transport relative to BHV-1 wild type and/or Us9 rescued viruses.

Intracellular delivery of macromolecules is crucial for the success of many research and clinical applications. Several conventional intracellular delivery methods have been used for many years but are still inadequate for several applications because of the issues associated with toxicity, low-throughput, and/or difficulty to target certain cell types. In this study, we developed and evaluated new high-throughput intracellular delivery methods for the efficient delivery of macromolecules while maintaining high cell viability. First, we studied the feasibility of using an array of nanoneedles, with sharp tip diameters in the range of tens of nanometers, to physically make transient holes in cell membranes for intracellular delivery. Puncture loading and centrifuge loading methods were developed and assessed for the effect of various experimental parameters on cell viability and delivery efficiency of fluorescent molecules. In both methods, high-throughput intracellular delivery was feasible by creating transient holes in cell membranes with the sharp tips of the nanoneedles. The second physical intracellular delivery method we studied was a novel microfluidic device that created transient holes in the cell membrane by mechanical deformation and shear stress to the cell. We observed efficient delivery of fluorescent molecules and studied the effect of device design and flow pressure on the delivery efficiency compared to data in the literature. We accounted for cell loss and clogging in the microfluidic devices and determined the true loss of cell viability associated with this method. Lastly, we investigated the possibility of intracellular delivery using nanoparticles on a leukemia cell line. Among number of materials for nanoparticles tested, mesoporous silica/poly-L-lysine nanoparticles were selected for further intracellular delivery study based on cell viability and intracellular delivery capability. We demonstrated the co-delivery of protein and plasmid by encapsulating into and coating onto the surface of the nanoparticles, respectively, which would be advantageous for certain therapeutic strategies. In summary, this work introduced two new intracellular delivery methods involving nanoneedles and novel nanoparticles, and provided an early, independent assessment of microfluidic delivery, showing the strengths and weaknesses of each method. These methods can be further optimized for a number of laboratory and clinical applications with continued research.

Cell biophysical properties are a new class of biomarkers that can characterize cells into subgroups that indicate differences in phenotypes that may correlate with disease and cell state. Microfluidic biophysical cell sorters are platforms that utilize these newly developed biomarkers to expand biomedical capabilities for improvements in cell state detection and characterization. Cell biophysical properties are important indicators for cell state and function because they point to differences in cell structures, such as cytoskeletal arrangement and nuclear content. In particular, some diseases, such as cancer and malaria, can cause significant changes in cell biophysical properties. Therefore, cell biophysical properties have the potential to be used for disease diagnostics. Microfluidic systems which can interrogate these biophysical properties and exploit changes in biophysical properties to separate cells into subpopulations will provide important biomedical capabilities. In this combined theoretical and experimental investigation, we explore a new type of cell sorter which utilizes differences in biophysical properties of cells. These biophysical properties that can be utilized to sort cells include size, elasticity and viscosity. We invented a microfluidic system for continuous, label-free cell separation that utilizes variations in cell biophysical properties. A microfluidic channel is decorated by periodic diagonal ridges that are designed to compress flowing cells in rapid succession. The physical compression, in combination with hydrodynamic secondary flows induced by the ridged microfluidic channel, translates each cell perpendicular to the channel axis in proportion to its biophysical properties. Through careful experimental and computational studies, we found that the cell trajectories in the microfluidic cell sorter correlated to these biophysical properties. Furthermore, we examine the effect of channel design parameters under various experimental conditions to derive cell separation models that can be used to qualitatively predict cell sorting outcome. A variety of biophysical measurement tools, including atomic force microscopy and high-speed optical microscopy are used to directly characterize the heterogeneous population of cells before and after separation. Taken together, we describe the physical principles that our microfluidic approach can be effectively used to separate a variety of cell types. The major contribution is the creation and characterization of a novel microfluidic cell- sorting platform that utilizes cell biophysical properties to enrich cells into phenotypic subtypes. This innovative approach opens new ways for conducting rapid and low-cost cell analysis and disease diagnostics through biophysical markers.

Metamaterials are artificially designed composite materials which can exhibit unique and unusual properties such as the negative refractive index, negative phase velocity, etc. The concept of metamaterials becomes prevalent in the electromagnetic society since the first experimental implementation in the early 2000s. Many fascinated potential applications, e.g. super lens, invisibility cloaking, and novel antennas that are electrically small, have been proposed based on metamaterials. However, most of the applications still remain in theory and are not suitable for practical applications mainly due to the intrinsic loss and narrow bandwidth (large dispersion) determined by the fundamental physics of metamaterials .In this dissertation, we incorporate active gain devices into conventional passive metamaterials to overcome loss and even provide gain. Two types of active gain negative refractive index metamaterials are proposed, designed and experimentally demonstrated, including an active composite left-/right-handed transmission line and an active volumetric metamaterial. In addition, we investigate the non-Foster circuits for broadband matching of electrically small antennas. A rigorous way of analyzing the stability of non-Foster circuits by normalized determinant function is proposed. We study the practical factors that may affect the stability of non-Foster circuits, including the device parasitics, DC biasing, layouts and load impedance. A stable floating negative capacitor is designed, fabricated and tested. Moreover, it is important to resolve the sign of refractive index for active gain media which can be quite challenging. We investigate the analytical solution of a gain slab system, and apply the Nyquist criterion to analyze the stability of a causal gain medium. We then emphasize that the result of frequency domain simulation has to be treated with care. Lastly, this dissertation discusses another interesting topic about THz spectroscopy of live cells. THz spectroscopy becomes 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°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 medium with un-concentrated and concentrated cells.

Les biopiles enzymatiques sont considérés comme des systèmes potentiellement utilisables pour la production d'énergie renouvelable dans des marchés niches. Une biopile est constituée de deux électrodes associées à des enzymes, catalyseurs biologiques, qui permettent la production d'énergie électrique à partir de réactions chimiques d'oxydoréduction. Ce travail présente la réalisation d'une biopile alcool/oxygène, au sein de laquelle l'alcool est oxydé à l'anode par l'alcool déshydrogénase alors que l'oxygène moléculaire est réduit en eau à la cathode par l'enzyme laccase, en présence de médiateurs spécifiques. L'objectif de ce travail a été tout d'abord de développer des bioélectrodes avec des enzymes immobilisées de manière à minimiser la quantité de biocatalyseur et augmenter sa stabilité. Dans un second temps, l'assemblage de biocathodes et de bioanodes a permis de fabriquer des biopiles à alcool macroscopique et microfluidique. Différentes poudres de carbone combinées à des polymères ont été utilisées pour immobiliser les enzymes et les médiateurs par encapsulation selon diverses configurations. Des analyses électrochimiques ont permis de mettre en évidence l'influence importante de certains paramètres comme la nature du carbone et du polymère, le pH et la température sur les performances des bioélectrodes. Une fois assemblées dans les configurations classique ou microfluidique, ces bioélectrodes ont conduit à des systèmes électrochimiques de génération d'énergie délivrant une densité de puissance maximale de 300μW/cm2 à 0,61V pour la biopile macroscopique et de 45μW/cm2 à 0,5V pour le système microfluidique. / Enzymatic biofuel cells (BFC) are systems of great interest for the production of renewable energy in niche markets. A BFC consists of two electrodes associated with enzymes as catalysts allowing energy production from oxydoreduction reactions. This work is devoted to the development of an alcohol/oxygen BFC for which alcohol is oxidized at the anode by alcohol dehydrogenase while molecular oxygen is reduced to water at the cathode by laccase, in the presence of specific mediators. The objective of this work was first to develop bioelectrodes with immobilized enzymes in order to minimize the amount of biocatalyst and increase its stability. In a second step, biocathodes and bioanodes were assembled to make macroscopic and microfluidic alcohol BFCs. Various carbon powders combined to polymers were used to immobilize enzymes and mediators in various configurations by entrapment. Electrochemical analysis have highlighted the significant influence of certain parameters like the nature of polymer and carbon, the pH or the temperature on the bioelectrodes performances. Once assembled in classical or microfluidic configurations, these bioelectrode led to electrochemical energy generation systems delivering a maximum power density of 300μW/cm2 at 0,61V for the macroscopic BFC and 45μW/cm2 at 0,5V for the microfluidic system.

Zeolites are porous aluminosilicates that occur both naturally and synthetically, having numerous applications in catalysis, adsorption and separations. Despite over a half century of characterization and synthetic optimization of hundreds of frameworks, the exact mechanism of synthesis remains highly contested, with crystallization typically occurring under transport-limited regimes. In this work, a microcrystallization reactor working under segmented oscillatory flow has been designed to produce a semi-continuous flow of zeolite A. The fast injection of the reactants in a mixing section forms droplets of aqueous precursors in a stream of paraffin, dispersing microdroplets and avoiding any clog from occurring in the system. The crystallization occurred in the system at atmospheric pressure and isothermal conditions (65ºC). This allowed for a rather slow crystallization kinetics which was important to study and highlight the different crystallization mechanisms between flow and batch synthesis. The morphology, size distributions, crystallinity, and porosity were examined by ex-situ characterization of the samples by scanning electron microscopy, X-ray diffraction, Raman spectroscopy, and N2 Physisorption to support the conclusions drawn. The size distribution of the particles achieved in the flow reactor was conclusively narrower than the distribution achieved in the batch reactor. The average size of the crystals for both synthesis methods is reported as 400 nm and the crystallinity achieved was comparable between the two. However, the morphology was quite different between the two systems, the flow products having a much higher mesoporosity due to the presence of crystal aggregates at high crystallinity when compared to the batch crystals. Finally, extended crystallization times leads to a decline of the crystallinity of the product, which might be explained by the metastable state of zeolites in solution.

Zeolites are porous aluminosilicates that occur both naturally and synthetically, having numerous applications in catalysis, adsorption and separations. Despite over a half century of characterization and synthetic optimization of hundreds of frameworks, the exact mechanism of synthesis remains highly contested, with crystallization typically occurring under transport-limited regimes. In this work, a microcrystallization reactor working under segmented oscillatory flow has been designed to produce a semi-continuous flow of zeolite A. The fast injection of the reactants in a mixing section forms droplets of aqueous precursors in a stream of paraffin, dispersing microdroplets and avoiding any clog from occurring in the system. The crystallization occurred in the system at atmospheric pressure and isothermal conditions (65ºC). This allowed for a rather slow crystallization kinetics which was important to study and highlight the different crystallization mechanisms between flow and batch synthesis. The morphology, size distributions, crystallinity, and porosity were examined by ex-situ characterization of the samples by scanning electron microscopy, X-ray diffraction, Raman spectroscopy, and N2 Physisorption to support the conclusions drawn. The size distribution of the particles achieved in the flow reactor was conclusively narrower than the distribution achieved in the batch reactor. The average size of the crystals for both synthesis methods is reported as 400 nm and the crystallinity achieved was comparable between the two. However, the morphology was quite different between the two systems, the flow products having a much higher mesoporosity due to the presence of crystal aggregates at high crystallinity when compared to the batch crystals. Finally, extended crystallization times leads to a decline of the crystallinity of the product, which might be explained by the metastable state of zeolites in solution.