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. This methodology could readily be expanded to other biological systems and further multiplexed to monitor increased
numbers of analytes. In another study, a microfluidic platform was developed to prepare negatively stained grids for use in TEM. The
microfluidic device is composed of glass etched with readily fabricated features that facilitate the extraction of the grid post-staining and
maintains the integrity of the sample. Utilization of this device simultaneously reduced environmental contamination on the grids and
improved the homogeneity of the heavy metal stain needed to enhance visualization of biological specimens as compared to conventionally
prepared TEM grids. This easy-to-use TEM grid preparation device provides the basis for future developments of systems with more integrated
features, which will allow for high-throughput and dynamic structural biology studies. / A Dissertation submitted to the Department of Chemistry and Biochemistry in partial fulfillment of the
requirements for the degree of Doctor of Philosophy. / Fall Semester 2017. / October 9, 2017. / Biological Dynamics, Electron Microscopy, Immunoassay, Islet of Langerhans, Microfludics / Includes bibliographical references. / Michael G. Roper, Professor Directing Dissertation; Pradeep Bhide, University Representative; Alan G.
Marshall, Committee Member; Christian Bleiholder, Committee Member.
| Identifer | oai:union.ndltd.org:fsu.edu/oai:fsu.digital.flvc.org:fsu_604996 |
| Contributors | Mukhitov, Nikita (author), Bhide, Pradeep (university representative), Marshall, Alan G. (Alan George), 1944- (committee member), Bleiholder, Christian (committee member), Florida State University (degree granting institution), College of Arts and Sciences (degree granting college), Department of Chemistry and Biochemistry (degree granting departmentdgg) |
| Publisher | Florida State University |
| Source Sets | Florida State University |
| Language | English, English |
| Detected Language | English |
| Type | Text, text, doctoral thesis |
| Format | 1 online resource (199 pages), computer, application/pdf |
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