Thesis (M.S.)--Mississippi State University. Department of Chemistry. / Title from title screen. Includes bibliographical references.

Electrowetting on dielectric (EWOD) is a technique for reducing the apparent contact angle of a fluid droplet, which has many promising applications in the fields of optics, digital displays, and lab-on-a-chip research. In this thesis, a design is presented for a novel single circuit device for achieving continuous droplet motion, by using the current-rectifying properties of valve metals to create diode-like behavior. This contrasts with existing designs, which require an array of individual electrodes to achieve motion in discrete steps. We are able to demonstrate continuous droplet motion across a 28mm-long test strip with an applied voltage of 303 V and a velocity of 5.59 mm/s (at 370 V) using an ionic-fluid electrolyte (BMIM-PF6), and have achieved actuation at as low as 185 V, with a maximum observed velocity (at 300 V) of 13.8 mm/s using a 1M sodium sulfate solution.

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In this dissertation, an exosome capturing ciliary micropillar array microfluidics is introduced and applied to evaluate the response of resistant cancer cells under nanoparticle encapsulated chemotherapy. Cancer cells are able to develop different mechanisms to resist therapeutic treatment, frequently causing chemotherapy failure. Active drug expulsion is one of the usual resisting schemes to reduce intracellular drug accumulation to a non-effective level. Evidence has suggested a potential exosomal pathway is used by multi-drug resistant (MDR) cancer cells to expel drugs. Here I study the exosomes derived from MDR cancer cells treated by nanotherapeutics aiming to establish the correlation between nanotherapeutics and exosomal pathway for drug expulsion. The outcome would boost further understanding of cancer MDR, and in turn direct the development of pharmaceutical nanoparticles to overcome MDR cancer. To effectively isolate exosomes for drug expulsion evaluation, a ciliary micropillar structure is fabricated employing microelectromechanical systems (MEMS) and metal assisted chemical etching (MACE) techniques. The ciliary micropillar is fabricated in two major steps: deep silicon etch (DSE) for pillars followed by a MACE process to etch nanowires on the pillars. The concept of using MACE as an alternative to DSE is also explored to reduce fabrication cost. With optimized parameters, it shows a comparable result to DSE. COMSOL simulation revealed that ciliated micropillars exhibited a unique advantage as a unit structure for capturing small particles in fluid flow, according to particle filtration theory. A nanowire layer with high permittivity allows fluid streamlines to pass through, and increases interaction with particle carrying fluid to increase the probability of particle interception. Nanowires on the pillar can trap specific sized particles due to their characteristic dimension. Thanks to the weaker stability of porous silicon nanowires, trapped particles can then be released by dissolving these nanowires without damage to the particles themselves. A microfluidic chip is fabricated with an optimized circular micropillar arrangement for resistance reduction, and its particle filtration performance is demonstrated by processing model cell culture medium. The device is applied to study MDR cells’ response to micelle encapsulated paclitaxel treatment. Cell culture medium from sensitive and MDR variant of MDA-MB-231 cells treated with pure and capsulated drugs are processed through the device for exosome isolation. Drug volume in collected exosomes is determined after measurement. By measuring drug efflux through exosomes, it is determined that MDA-MB-231MDR cells do use an exosomal pathway to expel drugs, but other mechanisms are also at play. Nanoparticle encapsulation results in higher drug concentration in exosomes partly because the origin of exosomes and nanoparticle intake through endocytosis share some similar route. / text

ZnO nanostructures have been investigated for quite a long time. However, only recently they triggered much interest due to advances in materials synthesis and characterization, as well as emerging demand for new nanostructured materials in novel device implementations. A large part of the work was devoted to exploring new methodology for patterning growth sites and controlling nanowires morphology using the deposition methods that are compatible with integrated circuits (IC) processing. Microcontact printing was used to pattern the seeding layer, and, subsequently, ZnO nanowires through a resistless soft lithography process. When considering hydrothermal growth of ZnO nanowires in the framework of IC compatible techniques, it is favorable to keep the chemistry of the process constant, while tailoring morphological properties of ZnO nanowires through other means. Therefore, control over morphology of ZnO nanowires was realized by setting the physical properties of seeding layers. Atomic Layer Deposition (ALD) was used to deposit seeding layer required for hydrothermal growth and the effect of the physical properties of ALD thin films on resultant ZnO nanowires was studied. Opto-electrochemical properties of ZnO nanowires were studied in various electrolytes and performance of ZnO nanowires as an electrode material for multifunctional applications was investigated. Also, bulk nucleation and growth of novel aster-like nanostructures was investigated. These nanostructures may prove useful for creation of mechanically reinforced biocompatible polymers. Another key objective of the present work was to create strategies for controlled growth of ZnO nanowires on substrates previously unavailable for conventional hydrothermal growth of ZnO nanowires. The newly developed approach greatly facilitates growth of ZnO nanowires in confined microstructures, which greatly enhances the possibilities for the usage of ZnO nanowires in applications where they act as a porous electrode. These novel techniques open wide possibilities for improving performance of devices such as dye sensitized solar cells or supercapacitors.

<p>Electrowetting-on-dielectric (EWD) digital microfluidics is a droplet-based fluid handling technology capable of radically accelerating the pace of genome engineering research. EWD-based laboratory-on-chip (LoC) platforms demonstrate excellent performance in automating labor-intensive laboratory protocols at ever smaller scales. Until now, there has not been an effective means of gene transfer demonstrated in EWD microfluidic platforms. This thesis describes the theoretical and experimental approaches developed in the demonstration of an EWD-enabled electrotransfer device. Standard microfabrication methods were employed in the integration of electroporation (EP) and EWD device architectures. These devices enabled the droplet-based bulk transformation of E. coli with plasmid and oligo DNA. Peak on-chip transformation efficiencies for the EP/EWD device rivaled that of comparable benchtop protocols. Additionally, ultrasound induced in-droplet microstreaming was developed as a means of improving on-chip electroporation. The advent of electroporation in an EWD platform offers synthetic biologists a reconfigurable, programmable, and scalable fluid handling platform capable of automating next-generation genome engineering methods. This capability will drive the discovery and production of exotic biomaterials by providing the instrumentation necessary for rapidly generating ultra-rich genomic diversity at arbitrary volumetric scales.</p> / Dissertation

The ability to control the motion of particles and cells in microchannels has been a center of fascination since the advent of microfluidics. Entire fields have been created in order to accomplish separation, volume reduction and overall positioning of particles and cells within microfluidic devices in the fastest and most accurate manner possible. While most of these technologies rely on low Reynolds number operation, one technique entitled inertial focusing takes advantage of the inertia of the surrounding fluid and the interaction between a particle and the channel itself which cause the lateral migration of particles across streamlines to equilibrium positions within a flow. The major advantage of inertial microfluidics in biomedical and microfluidic applications is that it is inherently high throughput being dependent on inertia whereas most microfluidic concepts are dependent on low Reynolds number operation. / Engineering and Applied Sciences

The stochastic nature of intracellular chemistry guarantees that even genetically identical cells sharing an environment will differ in composition. The question of whether this chemical diversity translates into significant phenotypic individuality is tied to the relative timescales of the processes involved. In order for cells in a population to have distinct functional identities, they must maintain their states for an appreciable period of time. Quantification of these timescales requires accurate time-lapse measurements covering tens or even hundreds of generations, a technical hurdle that has left these questions largely underexplored. In this thesis I present three pieces of work that aim to provide a foundation for the study of fluctuation timescales in bacteria. In the first part, I describe modifications to a recently developed microfluidic platform for continuous culture of cells under constant conditions. These revised devices enable the high-throughput, long-term measurement of gene expression dynamics while eliminating several confounding experimental factors that interfere with timescale measurements. In the second part, I employ one of these devices to survey fluctuation timescales in ~50 reporters for Eshcerichia coli gene expression. Under rich conditions, all reporters exhibited nearly identical, rapid fluctuation dynamics that were captured by a simple model of gene expression. In contrast, under poor nutritional conditions gene expression states became correlated over several cell divisions. However, accounting for instantaneous growth rate fluctuations eliminated these slow timescales, revealing an exceedingly simple behavior. In the third part, I describe our work to dissect the stochastic transition between the solitary motile state and sessile multicellular state in exponentially growing Bacillus subtilis</italic.. By enforcing static environmental conditions, we uncover the cell's internal strategies for state switching. The transition to the multicellular state occurs without regard to the cell's state history, whereas commitment to the multicellular state is tightly timed. By manipulating the genetic circuit responsible for the switch, we also expose surprising functional modularity in the commitment. I believe that the striking range of gene expression timescales we observe--from the fast fluctuations in E. coli gene expression to the feedback-amplified noise in B. subtilis--will serve as a useful starting point for future studies.

As new therapeutic targets and drugs are discovered for B-cell lymphoma and other cancers, companion diagnostics are also needed to determine target engagement, therapeutic efficacy, and patient segmentation for clinical trials. We first employed synthetic chemistry to build a platform for modifying small molecule drugs into imaging probes, using the poly(ADP-ribose) polymerase 1 (PARP1) inhibitor AZD2281 (Olaparib) as a model for technology development. Our results showed that small-molecule companion imaging drugs can be used for fluorescence imaging in cells, as well as for pharmacokinetic studies and positron emission tomography (PET) imaging in vivo, without significantly perturbing their target binding properties or cellular uptake. To apply this approach to B-cell lymphoma drugs currently in clinical trials, we modified an irreversible inhibitor of Bruton's Tyrosine Kinase (BTK), PCI-32765 (Ibrutinib), with the fluorophore Bodipy FL (BFL), and used it for imaging in cells and in a mouse window-chamber xenograft model. The excellent co-localization of our probe (Ibrutinib-BFL) with BTK demonstrated its utility for studying additional BTK inhibitors and as a companion imaging probe. In parallel, we hypothesized that central nervous system (CNS) lymphoma diagnosis from paucicellular cerebrospinal fluid (CSF) samples could be improved with molecular profiling of putative lymphoma cells trapped in a customized microfluidic chip. Following fabrication and characterization of a polydimethylsiloxane (PDMS) diagnostic device containing an array of affinity-free single-cell capture sites, we were able to efficiently recover >90% of lymphocytes, perform immunostaining on chip, and apply an image-processing algorithm to group cells based on their molecular marker expression, such as kappa/lambda light chain restriction. Additionally, in combination with Ibrutinib-BFL or other imaging drugs, we demonstrated the potential for on-chip drug imaging for use in conjunction with drug development. Finally, we applied bioorthogonal conjugation chemistries on cellulose paper for potential applications in lowering the cost of drug screening. We anticipate that these approaches will enable direct, molecular information for personalized treatment decisions in B-cell lymphomas, as well as provide a roadmap for the development of companion diagnostic probes and devices for additional indications.

Interactions of cells with their chemical microenvironments are critical to many polarized processes, including differentiation, migration, and pathfinding. To investigate such cellular events, tools are required that can rapidly reshape the microscopic chemical landscapes presented to cultured cells. Existing chemical dosing technologies rely on use of pre-fabricated chemical gradients, thus offering static cell-reagent interactions. Such interactions are particularly limiting for studying migration and chemotaxis, during which cells undergo rapid changes in position, morphology, and intracellular signaling. This dissertation describes the use of laminar streams, containing cellular effector molecules, for precise delivery of effectors to selected subcellular regions. In this approach, cells are grown on an ultra-thin polymer membrane that serves as a barrier to an underlying reagent reservoir. By using a tightly-focused pulsed laser beam, micron-diameter pores can be ablated in the membrane upstream of desired subcellular dosing sites. Emerging through these pores are well-defined reagent streams, which dose the targeted regions. Multiple pores can be ablated to allow parallel delivery of effector molecules to an arbitrary number of targets. Importantly, both the directionality and the composition of the reagent streams can be changed on-the-fly under a second to present dynamically changing chemical signals to cells undergoing migration. These methods are applied to study the chemotactic responses of neutrophil precursor cells. The subcellular localization of the chemical signals emerging through pores is found to influence the morphological evolution of these motile cells as they polarize and migrate in response to rapidly altered effector gradients. / text