Browsing by Subject "Electrophoresis"
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Item Advancements In Microfluidics For Biotechnology Applications(2018-10) Agrawal, PranavMicrofluidic technology has made a huge impact in the field of biotechnology and life sciences. The advancements can be categorized into three aspects: understanding of physical phenomena at the microscale; development of tools for easy integration of different phenomena; and devising systems for various applications. This thesis highlights the ability of microfluidic technology in manipulating different biological entities by fabricating small feature sizes. In particular, we have focused on the development of new processes for three biotechnology applications – (i) long DNA sample preparation for genomic; (ii) delivery of genetic delivery vehicles for gene and cell therapy; and (iii) an in vitro model to study human gut. Each of these systems is developed in close collaboration with potential users and is aimed towards easy integration with the existing workflow. Long-read genomic applications such as genome mapping in nanochannels require long DNA that is free of small-DNA impurities. Chapter 2 reports a chip-based system based on entropic trapping that can simultaneously concentrate and purify a long DNA sample under the alternating application of an externally applied pressure (for sample injection) and an electric field (for filtration and concentration). In contrast, short DNA tends to pass through the filter owing to its comparatively weak entropic penalty for entering the nanoslit. The single-stage prototype developed here, which operates in a continuous pulsatile manner, achieves selectivity of up to 3.5 for λ-phage DNA (48.5 kilobase pairs) compared to a 2 kilobase pair standard based on experimental data for the fraction filtered using pure samples of each species. The device is fabricated in fused silica using standard clean-room methods, making it compatible for integration with long-read genomics technologies. Non-viral delivery vehicles are becoming a popular choice to deliver genetic materials for various therapeutic purposes, but they need engineering solution to improve and control the delivery process. In Chapter 3, we demonstrate a highly efficient method for gene delivery into clinically relevant human cell types, such as induced pluripotent stem cells (iPSCs) and fibroblasts, reducing the protocol time by one full day. To preserve cell physiology during gene transfer, we designed a microfluidic strategy, which facilitates significant gene delivery in short transfection time (<1 minute) for several human cell types. This fast, optimized and generally applicable cell transfection method can be used for rapid screening of different delivery systems and has significant potential for high-throughput cell therapy applications. Microfluidic in vitro models are being developed to mimic individual or combination of various human organ functions for systematic studies, and for better predictive models for clinical studies. In Chapter 4, we outline a microfluidic-based culture system to study host-pathogen interaction in the human gut. We demonstrate that the infection of Enterohemorrhagic Escherichia coli (EHEC) in epithelial cells are oxygen dependent and can be used to prolong co-culture of bacterial and epithelial cells. This work presents a large scope to study the factors influencing the infection, especially the commensal microbiome in the human gut. Overall, this thesis shows how the microfluidic system can be useful in solving real-life problems and envision further advancements in the field of biotechnology.Item Development, Characterization, and Applications of a 3D Printed micro Free-Flow Electrophoresis Device(2017-02) Anciaux, SarahMicro free-flow electrophoresis (μFFE) is a unique separation technique because of its continuous nature. Analytes are pressure driven through a planar separation channel, and an electric field applied laterally to the flow producing a spatial separation. Fabrication methods associated with μFFE devices hinder our ability to address the limitations of μFFE. This work focuses on a novel fabrication method to reduce the overall fabrication cost and time, followed by validating and characterizing the device. A novel μFFE device is fabricated in acrylonitrile butadiene styrene (ABS) by 3D printing two sides of the device and then acetone vapor bonding them while simultaneously inserting electrodes and clarifying the device. Fluorescent dyes are separated, and their limit of detection determined. After validation of the new fabrication method, a new device design is made with the sample inlet modified so that 2D nLC × μFFE separations can be performed. 2D nLC × μFFE separations of fluorescent dyes, proteins, and tryptic BSA digest are demonstrated. These samples allow comparison between the surface properties of glass and 3D printed devices. Peak asymmetries, widths, and the interface were investigated. Minimal surface adsorption is observed for fluorescent dyes, proteins, and peptides, unlike in glass devices. After investigating surface properties, an open edge device for coupling to mass spectrometry is designed and compared to its glass counterpart. A novel ionization method is demonstrated from a hydrophobic membrane and the open edge device is shown to have stable flow.Item Electrokinetic Phenomena and Singularity-Driven Flows in Nematic Liquid Crystals(2017-11) Conklin, ChristopherElectrokinetic phenomena, including electrophoresis and electroosmosis, provide a significant tool for engineering the transport of fluids and particles at microscopic scales. This thesis describes additional mechanisms for generating electrokinetic flow by using a nematic liquid crystal electrolyte. Under an applied electric field the anisotropic properties of the liquid crystal lead to separation of ionic impurities present in the fluid, which couple with the applied field to produce electrostatic forces that drive fluid and particle motion. This force is quadratic in the electric field, implying that systematic flow occurs even in the presence of an oscillating field. This thesis presents numerical and analytical investigations of this electrokinetic mechanism. We show that the charge density and fluid velocity of a system depends strongly on the topology of the liquid crystal orientation, and we present results for several distinct configurations, including periodic distortions, isolated disclinations, and particle suspensions. We also show that liquid crystal electrokinetic systems can be designed to mimic the behaviors of active nematics – collections of particles which can self-propel along a particular direction.Item Electrophoresis of large DNA with a sparse zinc oxide nanowire array.(2010-05) Araki, NoritoshiWe developed a simple inexpensive method to integrate ZnO nanowires into a microchannel using a combination of aqueous solution synthesis of ZnO nanowires and photolithography, which is used as a nanowire-embedded microfluidic device. The density of ZnO nanowires inside the microchannel is controllable by simply changing the concentration of the seed solution. We conducted a study of dynamic interactions between electric field driven !DNA and a single isolated ZnO nanowire using single molecule spectroscopy technique. The study shows that the hooking time is exponentially dependent on b/Rg, in agreement with a prediction by simulation work. We also find that the hooking probability for small values of b/Rg increases as the electric field strength increases.Item High Speed Separations of Complex Mixtures using nano-Liquid Chromatography Coupled with micro Free Flow Electrophoresis(2016-03) Geiger, MatthewMicro free flow electrophoresis (µFFE) is a separation technique which can be used for unique applications due to its continuous nature. Separations are performed in space, as opposed to time, as laminar flow drives analytes down the separation chamber and are separated laterally by an electric field. This continuous nature makes it an attractive option to be used as a second dimension in multidimensional separations. The major focus of this work will be the development of a 2D separation platform coupling a commercial nano-liquid chromatography (nLC) instrument with an all glass µFFE device followed by investigating factors which could affect the efficiency of the technique. A new µFFE device was designed and fabricated for coupling with nLC. High peak capacity separations of tryptic peptides of BSA demonstrated the power of the technique. Broadening in temporal and spatial dimensions were investigated since peak capacity is calculated using analyte peak width. The observation that the adsorption of analytes only affects broadening in the temporal dimension is critical for maximizing peak capacity. Finally, the effect of using fluorescent labels in 2D nLC × µFFE separations will be demonstrated. The impact of label choice can be seen in the peak capacity and orthogonality of separations of amino acids and peptides.Item Microfluidic assays for assessing oligonucleotide catalyst abundance and monitoring biomolecule concentration in real time(2023-10) Douma, CeciliaMicrofluidic platforms control and manipulate very small volumes of liquid, typically at the microliter or nanoliter scale. By replacing pipettes and flasks with microfluidic channels and chambers, routine laboratory processes can be scaled down and sped up. Microfluidic platforms can mix, react, incubate, separate, extract, and detect solutions with high throughput and reproducibility, measuring the natural world at physical scales and timescales that would be inaccessible using traditional laboratory techniques. This thesis describes the development of microfluidic assays to address two bioanalytical challenges. First, a droplet microfluidic platform was developed to quantify the abundance of catalytic molecules in pools of random-sequence DNA. Although catalytic oligonucleotides are attractive as sensors and therapeutic agents, the full scope of their catalytic activity is largely unknown. The microfluidic platform described here encapsulates a library of DNA sequences in droplets with a fluorogenic substrate. Droplets that contain a catalytic sequence will become fluorescent after a period of incubation, while droplets without a catalyst will remain dark. The frequency of catalysts in the original library can be calculated from the ratio of fluorescent and non-fluorescent droplets. This thesis describes the technical design of a droplet microfluidic platform, its performance in library screening experiments, and its application for the detection of a known DNA catalyst. A versatile microfluidic platform for oligonucleotide library screening could assess catalyst abundance across a wide variety of reactions and conditions, creating a new framework for understanding the catalytic potential of oligonucleotides. Second, an aptamer affinity assay was developed for continuous cytokine quantification using micro free-flow electrophoresis (µFFE). Affinity assays are a prominent tool for biomolecule quantification because of their excellent sensitivity and specificity. However, traditional affinity assays use discrete samples and are poorly suited for measuring dynamic changes in an analyte’s concentration. The ultimate aim of the aptamer assay is to continuously quantify cellular cytokine secretion in real time using µFFE, a continuous separation technique that can detect free aptamer and bound aptamer complexes in a flowing sample stream. This thesis describes the characterization of µFFE devices fabricated in cyclic olefin copolymer as well as initial development of a µFFE aptamer assay for continuous quantification of tumor necrosis factor α (TNFα).Item Understanding DNA Electrophoresis in Colloidal Crystals(2014-08) King, ScottThe electrophoretic separation of DNA (deoxyribose nucleic acid) has been a target of engineering and optimization since its inception. In the following pages, I describe an engineering investigation into the physics of DNA separation in colloidal crystals. Colloidal crystals are formed through self-assembly of micron-sized spheres, suspended as a colloidal suspension. In this work, we follow the pioneering separation work of Zeng and Harrison, seeking to better understand the properties that allow for the observed enhanced separations of small, <1 kilo base-pair (kbp) DNA and large (>10 kbp) DNA. I demonstrate some key insights required to fabricate these devices, then move on to evaluating their performance. In the first section I tackle the quality of the crystal and its potential effects on separation performance. In the second section, I attempt to explain the order of magnitude better separation behavior between agarose gels and colloidal crystals by evaluating the mobility regimes for large DNA. At the end of this work, I have included a discussion on the future place of colloidal crystals as a separation medium.