Electrophoresis Based Affinity Assays of Hormones and Its Application in Monitoring of Hormone Secretion from Islets of Langerhans
Yi, Lian (author)
Roper, Michael Gabriel (professor directing dissertation)
Chase, P. Bryant (university representative)
Dorsey, John G. (committee member)
Knappenberger, Kenneth L. (committee member)
Florida State University (degree granting institution)
College of Arts and Sciences (degree granting college)
Department of Chemistry and Biochemistry (degree granting department)
The work in this dissertation presents methods for study of glucose stimulated insulin secretion as well as other hormones involved in glucose homeostasis. A microfluidic system was developed to investigate the entrainment of insulin secretion from islets of Langerhans to oscillatory glucose levels. A gravity-driven perfusion system was integrated with a microfluidic system to deliver sinusoidal glucose waveforms to the islet chamber. Insulin levels in the perfusate were measured using an online competitive electrophoretic immunoassay with a sampling period of 10 s. The insulin immunoassay had a detection limit of 3 nM with RSDs of calibration points ranging from 2–8%. At 11 mM glucose, insulin secretion from single islets was oscillatory with a period ranging from 3–6 min. Application of a small amplitude sinusoidal wave of glucose with a period of 5 or 10 min, shifted the period of the insulin oscillations to this forcing period. Exposing groups of 6–10 islets to a sinusoidal glucose wave synchronized their behavior, producing a coherent pulsatile insulin response from the population. These results demonstrate the feasibility of the developed system for the study of oscillatory insulin secretion. A dual detection microscopy system was developed to simultaneously image intracellular messengers and monitor insulin secretion from islets of Langerhans. Glucose stimulated insulin secretion plays a critical role in glucose homeostasis, but the underlying mechanism is still unclear. To develop an automated system to simultaneously study the correlation between intracellular events and insulin secretion, fluorescence imaging and laser induced fluorescence detection for insulin immunoassay were integrated into a single microscopy system. Intracellular calcium ([Ca2+]i) was used as a representative secondary messenger and studied with insulin secretion from islets during exposure to constant and oscillatory glucose levels. Both of [Ca2+]i and insulin were oscillatory during constant glucose levels and entrained to a small amplitude sinusoidal wave of glucose (0.5 – 2 mM). [Ca2+]i and insulin oscillations were temporally but not necessarily quantitatively correlated. Oscillatory glucose waveforms amplified insulin oscillation amplitude without further increasing [Ca2+]i, which could be related to amplifying pathway. The developed system was also applied to study the effect of glucokinase activator 22 on [Ca2+]i and insulin secretion. These results indicated the robustness of the developed method, which can be potentially expanded to include other intracellular messengers to study the mechanism of glucose stimulated insulin secretion. A method was developed that allowed simultaneous monitoring of the acute secretory dynamics of insulin and islet amyloid polypeptide (IAPP) from islets of Langerhans using a microfluidic system with two-color detection. A flow-switching feature enabled changes in the perfusion media within 5 s, allowing rapid exchange of constant glucose concentrations delivered to groups of islets. The perfusate was continuously sampled by electroosmotic flow and mixed online with Cy5-labeled insulin, fluorescein isothiocyanate (FITC)-labeled IAPP, anti-insulin, and anti-IAPP antibodies in an 8.15 cm mixing channel maintained at 37 °C. The immunoassay mixture was injected for 0.3 s onto a 1.5 cm separation channel at 11.75 s intervals and immunoassay reagents detected using 488 and 635 nm lasers with two independent photomultiplier tubes for detection of the FITC and Cy5 signal. RSD of the bound-to-free immunoassay ratios ranged from 2 to 7% with LODs of 20 nM for insulin and 1 nM for IAPP. Simultaneous secretion profiles of the two peptides were monitored from groups of 4−10 islets during multiple step changes in glucose concentration. Insulin and IAPP were secreted in an approximately 10:1 ratio and displayed similar responses to step changes from 3 to 11 or 20 mM glucose. The ability to monitor the secretory dynamics of multiple peptides from islets of Langerhans in a highly automated fashion is expected to be a useful tool for investigating hormonal regulation of glucose homeostasis. The ability to detect picomolar concentrations of glucagon and amylin using fluorescently labeled mirror image aptamers, so-called Spiegelmers, is demonstrated. Using Spiegelmers as affinity probes, noncompetitive capillary electrophoresis affinity assays of glucagon and murine amylin were developed and optimized. The detection limit for glucagon was 6 pM and for amylin was 40 pM. Glucagon-like peptide-1 and -2 did not interfere with the glucagon assay, while the amylin assay showed cross-reactivity to calcitonin gene related peptide. The developed assays were combined with a competitive immunoassay for insulin to measure glucagon, amylin, and insulin secretion from batches of islets after incubation with different glucose concentrations. The development of these assays is an important step towards incorporation into an online measurement system for monitoring dynamic secretion from single islets.
affinity assay, capillary electrophoresis, hormone, islet of Langerhans, microfluidics
April 4, 2016.
A Dissertation submitted to the Department of Chemistry and Biochemistry in partial fulfillment of the Doctor of Philosophy.
Includes bibliographical references.
Michael G. Roper, Professor Directing Dissertation; P. Bryant Chase, University Representative; John G. Dorsey, Committee Member; Kenneth L. Knappenberger, Jr., Committee Member.
Florida State University
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