The work in this dissertation presents microfluidic methods developed for the study of biological dynamics. The requirements for the methods development was to create approaches with the ability to perform dynamic cell stimulation, measurement, and sample preparation. The methods presented herein were initially developed for the study of pancreatic islet biology but are expected to be translatable to other applications. In another study, a method to interface transmission electron microscopy (TEM) with microfluidics methods was developed.
The primary biological topic of interest investigated was the mechanisms of inter-islet synchronization. To test this, a microfluidic device fabricated from poly(dimethylsiloxane) (PDMS) was used to culture and stimulate pancreatic islets. Intracellular calcium ([Ca2+]i) imaging was performed with a fluorescent indicator, Fura-2-acetoxymethyl ester (Fura-2 AM). Under constant glucose (11 mM), islets demonstrated asynchronous and heterogeneous [Ca2+]i oscillations that drifted in period. However, when exposed to a glucose wave (11+/– 1 mM, 5 min period) islets were entrained to a common and consistent [Ca2+]i oscillation mode. The effect of islet entrainment on cellular function was investigated by measuring gene expression levels with microarray profiling. Calcium-dependent genes were found to be differentially expressed. Furthermore, it was speculated that islet entrained produced a beneficial effect on cell function and upkeep.
While [Ca2+]i imaging is an acceptable proxy measurement for insulin, it is not a viable reporter for other islet peptides and direct measurement is desired. Electrophoretic affinity assays can be performed on a microfluidic device in a serial manner to measure peptide release from an on-chip cell culture in near real-time. Successful analysis of electrophoretic affinity assays depends strongly on the preservation of the affinity complex during separations. Elevated separation temperatures due to Joule heating promotes complex dissociation leading to a reduction in sensitivity. To address this limitation, a method to cool a glass microfluidic chip for performing an affinity assay for insulin was achieved by a Peltier cooler localized over the separation channel. The Peltier cooler allowed for rapid stabilization of temperatures, with 21 °C the lowest temperature that was possible to use without producing detrimental thermal gradients throughout the device. Kinetic capillary electrophoresis analysis was utilized as a diagnostic of the affinity assay and indicated that optimal conditions were at the highest attainable separation voltage, 6 kV, and the lowest separation temperature, 21 °C, leading to 3.4% dissociation of the complex peak during the separation. These optimum conditions were used to generate a calibration curve and produced 1 nM limits of detection (LOD), representing a 10-fold improvement over non-thermostated conditions.
To date, most approaches for measurement of rapid changes in insulin levels rely on separations, making the assays difficult to translate to non-specialist laboratories. To enable rapid measurements of secretion dynamics from a single islet in a manner that will be more suitable for transfer to non-specialized laboratories, a microfluidic online fluorescence anisotropy immunoassay was developed. A single islet was housed inside a microfluidic chamber and stimulated with varying glucose levels from a gravity-based perfusion system. The total effluent of the islet chamber containing the islet secretions was mixed with gravity-driven solutions of insulin antibody and cyanine-5 (Cy5) labeled insulin. After mixing was complete, a linearly polarized 635 nm laser was used to excite the immunoassay mixture and the emission was split into parallel and perpendicular components for determination of anisotropy. Key factors for reproducible anisotropy measurements, including temperature homogeneity and flow rate stability were optimized, which resulted in a 4 nM LOD for insulin with < 1% RSD of anisotropy values. The capability of this system for measuring insulin secretion from single islets was shown by stimulating an islet with varying glucose levels. As the entire analysis is performed optically, this system should be readily transferable to other laboratories.
To increase the number of analytes that can be simultaneously monitored by a fluorescence anisotropy immunoassay, frequency encoding was introduced. As a demonstration of the method, simultaneous competitive immunoassays for insulin and glucagon were performed by measuring the ratio of bound and free Cy5-insulin and fluorescein isothiocyanate (FITC)-glucagon in the presence of their respective antibodies. A vertically polarized 635 nm laser was pulsed at 73 Hz and used to excite Cy5-insulin, while a vertically polarized 488 nm laser pulsed at 137 Hz excited FITC-glucagon. The total emission was split into parallel and perpendicular polarizations and collected onto separate photomultiplier tubes. The signals from each channel were demodulated using a fast Fourier transform, resolving the contributions from each fluorophore. Anisotropy calculations were carried out using the magnitude of the peaks in the frequency domain. The method produced the expected shape of the calibration curves with LOD of 0.6 and 5 nM for insulin and glucagon, respectively. (Abstract shortened by ProQuest.)
|Advisor:||Roper, Michael G.|
|Commitee:||Bhide, Pradeep, Bleiholder, Christian, Marshall, Alan G.|
|School:||The Florida State University|
|Department:||Chemistry and Biochemistry|
|School Location:||United States -- Florida|
|Source:||DAI-B 79/07(E), Dissertation Abstracts International|
|Subjects:||Chemistry, Analytical chemistry|
|Keywords:||Biological dynamics, Electron microscopy, Immunoassay, Islet of Langerhans, Microfludics|
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