Microfluidic Interfacing for Primary Endocrine Tissue: Developing Bioanalytical Methodologies and Novel Fabrication Methods for Cell Culture and Analysis
Type of DegreePhD Dissertation
Chemistry and Biochemistry
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In order to bridge the gap between microfluidic designs and real world applications, microanalysis systems were developed using minimalistic design attributes which could be readily paired with compatible bioanalytical assays. Chapter 2 discusses monolithic fabrication methods which have been developed for incorporation of macroscale structures within microfluidic devices in order to accommodate on-chip cell culture and secretion sampling of primary, murine endocrine tissues. Rigid, polymer templates were hand constructed and served as molds in PDMS to generate millimeter scale fluidic reservoirs which could directly interface with microchannels. Initial hand fabricated templates were utilized for pancreatic islet and isolated adipocyte secretion sampling over 1 hour time periods where insulin and adiponectin concentrations were analyzed via ELISA. While these hand fabricated templates were proven adequate for cell culturing purposes, there were minor disadvantages associated with the templates. To improve upon the template fabrication process, designs were digitized for 3D printing purposes. 3D printing of templates provided not only more rapid and robust production of templates, but also allowed for easily designing templates with increased structural complexity. Microfluidic devices fabricated in this manner were used for temporally resolved sampling and quantization of insulin and glycerol secreted from pancreatic islets and adipose tissue explants. 3D templating methods were further utilized in Chapter 3 for real-time analysis of fatty acid uptake within adipose tissue explants. Real-time analysis was performed on-chip through a novel, fluorescence based assay designed in-house. This assay exploits the natural binding of fatty acids to the protein albumin and allows for homogenous monitoring of fatty acid uptake through fluorescently labeled fatty acid analogues and bovine serum albumin which was modified with covalently attached quencher molecules. Proximity immunoassays were developed and discussed in Chapter 4 for analysis of small volume samples such that microfluidic secretion samples could be analyzed without the need for expensive, high volume assays such as ELISA. Real-time quantitative PCR and square wave voltammetry were used as readout methods for proximity assays to quantitate insulin and leptin in cell secretion samples as well as serum. Chapter 5 represents a collaborative effort which branches out from the focus of endocrine tissue on-chip, while maintaining the theme of simplistic, low volume microanalysis systems. A droplet generating device was utilized for actinide metal-ligand binding analysis through UV-Vis specrtal shifts. Finally, Chapter 6 summarizes this work in its entirety and provides an outlook into future projects stemming from these topics.