Thesis (M.S.)--University of Wisconsin--Madison, 1969. / eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references.

Thesis (M.S.)--University of Wisconsin--Madison, 1968. / eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references.

No description available.

Diss. (sammanfattning) Uppsala : Uppsala universitet, 2006. / Härtill 4 uppsatser.

Thesis, (Ph. D.)--Oregon Graduate Institute of Science and Technology, 1997.

Many droplet microfluidic assays have been described in the literature over the last decade of research, however, there has been little reported industrial use of droplet microfluidics in drug discovery compound screening, and in particular that of P450 enzyme inhibition assays for profiling drug-drug interactions. This is partly for Intellectual Property reasons, since Pharmaceutical companies do not wish to give away trade secrets in a competitive market, but also because the technology is not yet 'proven' and remains in the proof-of-concept stage. In droplet microfluidics, where at least two liquid phases are encountered, it is important that leakage of material between phases is addressed. This effect has been extensively reported in the literature using fluorescent dyes, however there is very little evidence of research using large compound sets of diverse chemistry. This is probably because few researchers have access to the large pharmaceutical libraries necessary for this work. This project assessed the feasibility of translating a widely used microtitre plate-based P450 enzyme inhibition assay to droplet format; determined the extent of partitioning from droplets using a large pharmaceutical library set and attempted to model this behaviour, and thirdly, considered the pharmacological impact the droplet format may have on the assay. The P450 cytochrome 1A2 enzyme type (isoform) was chosen for translation to the micro-droplet format. Assays of this type are often conducted using fluorogenic substrates, making them favourable for relatively easy fluorescent detection in droplet format using simple optical detection assemblies. Oil selection was investigated to determine which oil systems would be better suited in respect of droplet formation. The use of surfactants in the oil phase and its impact on droplet formation was studied and the synthesis, preparation and characterisation of a custom perfluoropolyether (PFPE) surfactant ('AZF') conducted. Droplet chips were designed and fabricated to produce droplets of 200-300 µm diameter using novel channel designs and sealing techniques. The droplets were analysed by fluorescence spectroscopy using bespoke detector apparatus. Partitioning from aqueous to oil phase was studied for a small range of compounds and oils (with and without surfactant for fluorous oils). Partitioning was lowest using fluorous oils alone, and increased substantially when surfactant was included. Results from the large pharmaceutical test set suggested the percentage of compounds that may partition readily to the oil phase is low even when using surfactant. However, attempts to correlate this to known physicochemical properties and to develop a predictive model for fluorous solubility proved largely unsuccessful. Partitioning in the droplet chip using a droplet collection pooling method was difficult to quantify as a consequence of the profound impact turbulence had on partitioning. Miniaturisation of the P450 cytochrome inhibition assay to the droplet format initially gave poorly reproducible low signals. Possible causes included detector insensitivity, partitioning of reagent and/or fluorescent metabolite over longer incubation times, and binding of the 1A2 P450 cytochrome enzyme-protein at the droplet interface. Protein interaction at the droplet-oil boundary was studied by fluorescence labelling a protein contained in 200µm droplets and observing the extent of fluorescence localisation at the interface by epifluorescent and confocal fluorescence microscopy. The data from this work indicates a pronounced localisation of protein at the droplet interface, possibly leading to enzyme deactivation and the loss of signal seen for the assay in the droplet chip. A number of protein titrations were co-added to the droplets as 'blocking proteins' which were found to improve the reaction output, however were also noted to affect the pharmacology of the assay, noted by an order of magnitude shift in the reported IC50 for the test inhibitor used (fluvoxamine). The effects of compound leakage from droplets, and the possible detrimental impact on biological reagents by interaction at the droplet-oil interface, is a challenge that may limit widespread adoption of droplet MF systems in drug screening operations. Appropriate control measures and/or a means to reduce these effects are essential to enable accurate quantification with industrial drug discovery environments. The findings in this work highlight the challenges that have to be addressed for droplet microfluidic technology to be successfully incorporated into key areas of assay screening within drug discovery. In terms of further research, there is a significant requirement for the research community to delve further into these challenges and work closely with the industry sector to understand the beneficial role microfluidics can have and how to develop effective robust strategies the industry can easily adopt to progress this area of science.

Thesis (Ph.D.)--University of Nebraska-Lincoln, 2007. / Title from title screen (site viewed May 20, 2008). PDF text: 171 p. : ill. (some col.); 4 Mb. UMI publication number: AAT 3284263. Includes bibliographical references. Also available in microfilm and microfiche formats.

<p> The goal of this research project was the development of a protocol for preparation of optically clear organic/inorganic hybrid materials that was amenable to entrapment of lipophilic biomolecules. The protocol involved the acid-catalyzed hydrolysis of mixtures of tetraethylorthosilicate (TEOS) with organosilane precursors, including methyltriethoxysilane (MTES), dimethyldimethoxysilane (DMDMS) and propyltrimethoxysilane (PTMS) in the presence and absence of the polymer additives poly(ethylene glycol) or poly(vinyl alcohol).</p> <p> The effect of organosilane precursors and polymer additives on the optical clarity, hardness and hydration stability of the resulting materials was characterized. It was determined that there was a limit to the amount of organosilane that could be added before the materials exhibited unacceptable characteristics. These limits were 20.0% (v/v) for MTES, 10.0% (v/v) for PTMS, and 5.0% (v/v) for DMDMS. Addition of PEG to these materials at levels up to 10.0% (w/v) resulted in good material characteristics. However, addition of PVA produced opaque materials with poor material properties. The internal environment of the materials was also probed using the environmentally sensitive fluorescent probes 7-azaindole (7AI) and prodan. These studies showed that the method of hydrolysis of the silane precursors and the aging conditions had a dramatic effect on the resulting material.</p> <p> The hybrid materials were used to entrap human serum albumin (HSA) and lipase to determine the effect of organic content on the biological function of these biomolecules. Both biomolecules retained a portion of their native function when entrapped in sol-gel-derived materials, and it was found that both proteins showed enhanced function in the presence of MTES. In the case of lipase, it was also determined that addition of PEG 600 at 10.0% (w/v in the gelation buffer) provided a dramatic increase in activity compared to materials without this additive, likely owing to a direct effect of the PEG on the stability of the entrapped protein.</p> <p> Following studies using bulk glasses, a protocol was developed for the preparation of optically clear sol-gel-derived thin films that was amenable to entrapment of biomolecules. The optimal method involved dipcasting of co-hydrolyzed materials containing 1.0 to 3.0% PEG. By careful control of the viscosity of the casting solution and the rate of film deposition, it was possible to form very stable thin films with excellent physical characteristics. These films were used to entrap the pH-sensitive, ratiometric fluorescent probe dextran-SNARF-1, resulting in a prototype of a fluorimetric pH sensor. Co-entrapment of the probe and lipase into sol-gel-derived thin films resulted in a rapid, reagentless biosensor prototype that could monitor changes in pH due to the enzyme-catalyzed hydrolysis of triglycerides. These results demonstrate that species entrapped in sol-gel derived thin films are suitable for biosensor development.</p> / Thesis / Master of Science (MSc)