UMD Theses and Dissertations
Permanent URI for this collectionhttp://hdl.handle.net/1903/3
New submissions to the thesis/dissertation collections are added automatically as they are received from the Graduate School. Currently, the Graduate School deposits all theses and dissertations from a given semester after the official graduation date. This means that there may be up to a 4 month delay in the appearance of a given thesis/dissertation in DRUM.
More information is available at Theses and Dissertations at University of Maryland Libraries.
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Item ISOTHERMAL DNA DETECTION UTILIZING BICYCLIC AMPLIFICATION OF PADLOCK PROBES(2019) Zimmermann, Alessandra C.; Kahn, Jason D; White, Ian M; Biochemistry; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)As healthcare worldwide changes to more patient-centric models, medical diagnostics need to adapt to being used in settings outside of the central lab. Current strategies to bring diagnostics to the patient’s bedside involve miniaturizing complicated amplification techniques, such as polymerase chain reaction, or building convoluted microfluidic assays that are difficult to operate. Ideally, a patient-centric diagnostic would require little instrumentation or training to operate, for which isothermal amplification techniques are ideal. Recent developments in catalytic DNA have enabled novel ways of iterating on amplification strategies to detect medically-relevant target sequences in systems that require little manipulation to operate. In this thesis we improve upon the body of research on DNAzymes, catalytic DNAs that can self-cleave in the presence of a cofactor, used in concert with amplification techniques. We create a one-pot, bicyclic amplification assay capable of detecting single-stranded oligonucleotides, with straightforward extensions to double-stranded targets, multiplexing, and integration into advanced detection platforms. The target is detected through its hybridization to a circle template, using the sequence specificity of DNA to splint the ligation of this ‘Template I,’ with minimal detection of off-target sequences. The circular Template I is copied through rolling circle amplification (RCA), with the amplicon containing a DNAzyme that will self-cleave in the presence of copper ions. This generates a second primer in situ that can be used to prime a second, pre-ligated, Template II to elevate the RCA amplification scheme from a linear method to a polynomial one. This Circle II template can then be used in a variety of detection modalities. The second amplicon can be used to cleave a hybridized FRET probe through the same copper ion cleavage mechanism as the primer generation, resulting in real-time fluorescence tracking. Alternatively, the RCA of the second circle can produce G-quadruplexes, which can be visualized with ABTS as a colorimetric endpoint that can be seen by eye, reducing the need for peripheral electronics. Finally, this thesis demonstrates the performance of the bicyclic RCA system in a phase-change system providing sequential mixing of components separated by wax layers, allowing the assay to proceed without any user interaction other than heating.Item THREE-DIMENSIONAL BIOPATTERNING TECHNOLOGY AND APPLICATION FOR ENZYME-BASED BIOELECTRONICS(2018) Chu, Sangwook; Ghodssi, Reza; Electrical Engineering; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Integration of biomaterials with 3-D micro/nano devices and systems offers exciting opportunities for developing miniature bioelectronics with enhanced performances and advanced modes of operation. However, the limited wetting property of such small scale 3-D structures (Cassie-Baxter wetting) presents a potential challenge in these developments considering most biological materials require storage in buffered aqueous solutions due to their inherently narrow stability window. In this thesis research, an electrowetting-assisted 3-D biomanufacturing technology has been developed enabling highly selective and programmable biomolecular assembly on 3-D device components. The successful integration of microscale 3-D device structures created via conventional microfabrication techniques with a nanoscale molecular assembly of Tobacco mosaic virus (TMV), enabled hierarchical and modular material assembly approaches for creating highly functional and scalable enzyme-integrated microsystems components. The potential limitation in 3-D bio-device integration associated with the surface wettability has been investigated by adapting Si-based micropillar arrays (μPAs) as model 3-D device structures, and a cysteine-modified TMV (TMV1cys), as the biomolecular assembler which can functionalize onto electrode surfaces via a self-assembly. The comparative studies using μPAs of varying pillar densities have provided clear experimental evidence that the surface coverage of TMV1cys self-assembly on the μPA is strongly correlated with structural density, indicating the structural hydrophobicity as a key limiting factor for 3-D bio-device integration. The 3-D electro-bioprinting (3D-EBP) technology developed in this work leverages the hydrophobic surface wettability by adapting a capacitive wettability-control technique, known as electrowetting. The biological sample liquid was selectively introduced into the microcavities using a custom-integrated bioprinting system, allowing for patterning of the TMV1cys self-assembly on the μPA substrates without the limitations of the structural density. The functional integrity of the TMV1cys post 3D-EBP allowed conjugations of additional biological molecules within the 3-D substrates. Particularly in this work, immobilization of glucose oxidase (GOx) has been achieved via a hierarchical on-chip immobilization method incorporating 3D-EBP. Combined with the enhanced and scalable enzymatic reaction density on-chip and the electrochemical conversion strategies, the innovative 3D biomanufacturing technology opens up new possibilities for next-generation enzyme-based bioelectronics.Item A Microfluidic Programmable Array for Label-free Detection of Biomolecules(2011) Dykstra, Peter Hume; Ghodssi, Reza; Electrical Engineering; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)One of the most promising ways to improve clinical diagnostic tools is to use microfluidic Lab-on-a-chip devices. Such devices can provide a dense array of fluidic components and sensors at the micro-scale which drastically reduce the necessary sample volumes and testing time. This dissertation develops a unique electrochemical sensor array in a microfluidic device for high-throughput, label-free detection of both DNA hybridization and protein adsorption experiments. The device consists of a patterned 3 x 3 grid of electrodes which can be individually addressed and microfluidic channels molded using the elastomer PDMS. The channels are bonded over the patterned electrodes on a silicon or glass substrate. The electrodes are designed to provide a row-column addressing format to reduce the number of contact pads required and to drastically reduce the complexity involved in scaling the device to include larger arrays. The device includes straight channels of 100 micron height which can be manually rotated to provide either horizontal or vertical fluid flow over the patterned sensors. To enhance the design of the arrayed device, a series of microvalves were integrated with the platform. This integrated system requires rounded microfluidic channels of 32 micron height and a second layer of channels which act as pneumatic valves to pinch off selected areas of the microfluidic channel. With the valves, the fluid flow direction can be controlled autonomously without moving the bonded PDMS layer. Changes to the mechanism of detection and diffusion properties of the system were examined after the integration of the microvalve network. Protein adhesion studies of three different proteins to three functionalized surfaces were performed. The electrochemical characterization data could be used to help identify adhesion properties for surface coatings used in biomedical devices or for passivating sensor surfaces. DNA hybridization experiments were performed and confirmed both arrayed and sensitive detection. Hybridization experiments performed in the valved device demonstrated an altered diffusion regime which directly affected the detection mechanism. On average, successful hybridization yielded a signal increase 8x higher than two separate control experiments. The detection limit of the sensor was calculated to be 8 nM.