Browsing by Subject "Bioanalytical"
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Item Microfluidics for bioanalytical research : transitioning into point-of-care diagnostics(2014-12) Scida, Karen; Crooks, Richard M. (Richard McConnell)In this dissertation, three different microfluidic devices with bioanalytical applications are presented. From chapter to chapter, the bioanalytical focus will gradually become the development of a point-of-care sensor platform able to yield a reliable and quantitative response in the presence of the desired target. The first device consists of photolithographically-patterned gold on glass bipolar electrodes and PDMS Y-shaped microchannels for the controlled enrichment, separation from a mixture, and delivery of two charged dyes into separate receiving microchannels. The principle for the permanent separation of these dyes is based on the concept of bipolar electrochemistry and depended on the balancing/unbalancing of convective and electromigrating forces caused by the application of a potential bias, as well as the activation/deactivation of the bipolar electrodes. Two different bipolar electrode configurations are described and fluorescence is used to optimize their efficiency, speed, and cleanliness of delivery. The second device is a DNA sensor fabricated on paper by wax printing and folding to form 3D channels. DNA is detected by strand-displacement induced fluorescence of a single-stranded DNA. A multiplexed version of this sensor is also shown where the experiment results in “OR” and “AND” Boolean logic gate operations. In addition, the nonspecific adsorption of the reagents to cellulose is studied, demonstrating that significant reduction of nonspecific adsorption and increased sensitivity can be achieved by pre-treating the substrate with bovine serum albumin and by preparing all analyte solutions with spectator DNA. The third device, also made of paper, has a novel design and uses a versatile electrochemical detection method for the indirect detection of analytes via the direct detection of AgNP labels. A proof-of-concept experiment is shown where streptavidin-coated magnetic microbeads and biotin-coated AgNPs are used to form a composite model analyte. The paper device, called oSlip, and electrochemical method used are easily coupled so the resulting sensor has a simple user-device interface. LODs of 767 fM are achieved while retaining high reproducibility and efficiency. The fourth device is the updated version of the oSlip. In this case, the objective is to show the current progress and limitations in the detection of real analytes using the oSlip device. A sandwich-type immunoassay approach is used to detect human chorionic gonadotrophin (pregnancy hormone) present in human urine. Various optimization steps are performed to obtain the ideal reagent concentrations and incubation time necessary to form the immunocomposite in one step, that is, by mixing all reagents at the same time in the oSlip. Additionally, improvements to the electrochemical detection step are demonstrated.Item The Application of LC-ICP-MS to Study Metal Ion Homeostasis in Biological Systems(2014-12-10) McCormick, Sean P.Eukaryotic cells contain low-molecular-mass metal complexes (LMMMCs), defined as having masses between 200 ? 10,000 Da, but these so-called labile or chelatable metal pools are poorly defined in terms of structures and functions. LMMMCs are thought to participate in metal-ion regulation, trafficking, storage and/or signaling in cells. These cellular processes are often dysfunctional in metal-associated diseases. The objective of these studies was to detect and characterize LMMMCs in eukaryotic cells, organelles and tissues. A novel liquid chromatography system in a cold inert-atmosphere glove box was interfaced with an in-line inductively coupled plasma mass spectrometer, and this LC-ICP-MS system was used to detect LMMMCs in yeast cells, mitochondria, and vacuoles as well as in mouse brain and liver cells and mitochondria. In each biological system, this separations technique was applied to detect numerous LMMMCs. The molecular mass and concentration of such species were estimated. In yeast, the previously reported mismetallation of MnSOD2 was examined in the mutant strain ?mtm1. A combination of SEC and AEX chromatography revealed that the degree of mismetallation of the SOD2 protein, in which Fe replace Mn in the active site, was no greater in ?mtm1 cells than in WT cells. The mitochondria of such mutant cells did exhibit an intense chromatography peak of Mn corresponding to at mass of 2000 ? 3000 Da. Mitochondria from WT cells exhibited a similar species, but at much lower intensity. This was the only Mn species present, suggesting that it was the used to metallate apo-SOD2. Mitochondria isolated from WT yeast cells contained 6 Co, 3 Cu, 2 Mn, 5 Fe and 3 Zn LMMMCs and approximately 6 P- and S- LLM species. Some of the P- and S- LMMCs probably arose from compounds like ATP, ADP, etc. Molecular masses of the LMM Cu peaks were higher (> 5 kDa) than for the LMM complexes of other transition metals. Zinc, Mn, and Fe had multiple species of interest which demonstrate the presence and labiality of the metals in pools. The same separation system was utilized to examine mice brain LMM extracts were found to contain > 30 LMMMCs. Eleven Co, 2 Cu, 5 Mn, 4 Mo, 3 Fe and 2 Zn LLM complexes were detected. Most Cu and Zn complexes appeared to be protein-bound with masses ranging from 4?20 kDa. In these systems, Co was the only metal for which the aqueous complex was reproducibly observed. A second mouse study used the LC-ICP-MS system to examine the forms of iron present in mouse plasma. Chromatograms exhibited ~6 Fe-associated peaks that were assigned to ferritin, transferrin, and hemopexin, respectively; the other 3 peaks could not be assigned. The LC-ICP-MS experiment demonstrates that numerous Fe-containing species coexist with transferrin in healthy WT mouse plasma.