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.
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Item Optical and Thermal Systems for Automation of Point-of-Care Assays(2018) Goertz, John; White, Ian M; Bioengineering; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Modern medicine has detailed 70,000 different diagnoses; the 21st century challenge is bringing those diagnoses to over 7 billion people. This phenomenal feat requires precision biosensing strategies that minimize necessary training and manual effort while maximizing portability and affordability. Microfluidic strategies, both fabricated chips and paper-based devices, held the promise to facilitate point-of-care diagnostics but have been inadequate for many applications due to the trade-off between bulky pumps or limited control and complexity. This dissertation details novel strategies that control the progression of biochemical reactions with high functionality, portability, and ease-of-use. First, I will describe an amplified signaling reaction that leverages both positive and negative feedback loops to achieve optically-regulated control. This assay, termed “Peroxidyme-Amplified Radical Chain Reaction” enables naked-eye detection of catalytic reporter DNA structures at concentrations across five orders of magnitude down to 100 pM while eliminating the need for manual addition of hydrogen peroxide common to other such detection reactions. Next, I will describe the development of a platform for thermal regulation of generic reactions. To address the need for a broadly capable automation platform that provides equal utility in the lab and field alike, we recently developed “phase-change partitions”. In our system, purified waxes segregate reagents until incremental heating melts the partitions one by one, causing the now-liquid alkane to float and allowing the desired reagents to interact with the sample on demand. This tight control over reaction progression enabled us to construct hands-free detection systems for isothermal DNA amplification, heavy metal contamination, and antibiotic resistance profiling. My work has demonstrated a broadly capable suite of assay control systems with the potential to enable simple, inexpensive automation of a broad array of chemical and biological analysis across human medicine, environmental surveillance, and industrial chemical synthesis.Item Guanosine-borate hydrogels- Form and function(2015) Peters, Gretchen Marie; Davis, Jeffery T; Chemistry; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Due to their biocompatibility and stimuli-responsive nature, supramolecular hydrogels derived from natural products are attractive for a number of biomedical applications, including diagnostics, targeted drug delivery and tissue engineering. Nucleosides, the building blocks of nucleic acids, are desirable candidates for forming supramolecular gels as they readily engage in reversible, noncovalent interactions. Guanosine (G 1), in particular, is unique in that it has multiple faces for noncovalent interactions and can self-associate into stable higher-order assemblies, such as G4-quartets and G-quadruplexes. This self-assembly of G 1 and its derivatives into G4-quartets has long been known to induce hydrogelation. However, the requirement of excess salt and the propensity of G 1 to crystallize persist as limitations for G4-hydrogels. Thus, recent interest has focused on developing G4-hydrogels with improved lifetime stabilities and lower salt concentrations. The work described here focuses on a long-lived G4-hydrogel made from G 1 and 0.5 equiv. of KB(OH)4. Gelation occurs through the formation of guanosine-borate (GB) diesters and subsequent assembly into cation-templated G4•K+-quartets. The physical properties and stability of the GB hydrogel can be readily manipulated by varying the gelation components. For example, merely altering the identity of the cation drastically alters the gel’s physical properties. Namely, while GB hydrogels formed with K+ are self-supporting and robust, mixing G 1 with LiB(OH)4 results in a weak gel that readily dissociates upon physical agitation. Small molecules, such as cationic dyes and nucleosides, could be selectively incorporated into the GB hydrogel through reversible noncovalent and covalent interactions. One such dye and known G4-quartet binding ligand, thioflavin T (ThT) fluoresces in the presence of the GB hydrogel. The ThT fluorescence increases as a function of gelator concentration with a sharp increase correlating to the gel point. Thus, this ThT fluorescence assay is a new method for probing the formation of G4-hydrogels. Additionally, ThT acts as a molecular chaperone for Li+ GB hydrogelation. Substoichiometric amounts of ThT results in faster hydrogelation, increased gel strength and improved recovery of a hydrogel destroyed by external stress. Insights gained from this research have implications towards development of biomaterials, biomolecule sensing, and drug delivery.Item Lipophilic G-Quadruplexes: Structural Studies, Post-Assembly Modification, and Covalent Capture(2006-08-29) Kaucher, Mark Steven; Davis, Jeffery T; Chemistry; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)New nanostructures and functional materials are built through the self-assembly of guanosine. Both the size and regiochemistry of these noncovalent structures are controlled. Lipophilic G-quadruplexes are further stabilized through covalent capture techniques. These new nanostructures demonstrate the ability to bind cations and transport monovalent cation through phospholipid membranes. Diffusion NMR is demonstrated as a valuable technique in characterizing the size of lipophilic G-quadruplexes. Control over the size of self-assembled G-quadruplexes is demonstrated through modifying the guanosine nucleosides and the cation concentration. The solution structure of [G 8]16 4K+ 4pic- is determined to be a hexadecamer using diffusion NMR. Additionally, G 24 is also shown to form a hexadecamer G-quadruplex, which has an octameric intermediate structure. Two different octamers, a singly and doubly charged octamer, formed by G 29 are elucidated by diffusion NMR. The information gained from the diffusion NMR technique allowed for a better understanding of the self-assembly processes, especially regarding the roles of cation, anion and solvent. The use of a kinetically controlled exchange reaction to effect regioselective modification of a hydrogen-bonded assembly is discussed. The pseudo-regioselective exchange of isotopically labeled G 35-d into [G 8-h]16 4K+ 4pic- is demonstrated. Both the bound anion and cation can control the exchange of ligand into the different layers of a synthetic G-quadruplex. This regioselective exchange process allows for functionalized G-quadruplex structures to be built. Covalent capture of lipophilic G-quadruplex 60 with reactive groups on the periphery generates a unimolecular G-quadruplex 61. This unimolecular G-quadruplex 61 shows exceptional stability in nonpolar and polar solvents, even without the presence of cations. Furthermore, this unimolecular G-quadruplex transports monovalent cation across phospholipid membranes. The design of transmembrane transporters is of particular interest for their potential as new ion sensors, catalysts and anti-microbial agents.