Monitoring Peptide Secretion Dynamics from Human Pancreatic Cells Using a Sandwich Assay and Microfluidics

Type II diabetes is a leading cause of death in the United States due to its high prevalence in adults with high blood pressure, obesity, other health problems. It affects ~10.5% of the U.S population, and this number continues to increase every year (projected to reach over 30% by 2025). Diabetes affects blood sugar regulation due to dysfunction in release patterns for insulin and glucagon. Currently, medication exists to help manage insulin concentrations in the body; however, no "cure" exists and much remains unknown regarding how processes go awry in a diabetic state. Specifically, a healthy human body releases insulin and glucagon in an oscillatory patten into the bloodstream in response to meals and periods of fasting to control blood glucose levels. However, in people with diabetes, the release of these hormones is disrupted, which can lead to a variety of symptoms and risk factors including seizers and death. The first steps in developing better therapeutics for people with diabetes is understanding the complex hormone release pattens in healthy human bodies as well as characterizing the disrupted release pattens in people with diabetes. Microfluidic devices have been introduced to characterize small concentrations of analytes (e.g., hormones) in liquid due to their ability to employ low-volume flow rates in highly controlled environments. Additionally, microfluidics can be coupled with highly sensitive immunoassay-based optical measurement systems to detect low concentrations of diabetes-related hormones such as insulin and glucagon. Microfluidics presents an unparalleled strength for understanding cellular function and communication because it allows determination of concentration changes for insulin and glucagon over very short time periods (e.g., 2-5 minutes), which gives insight into real-time hormone release within the body. The work described herein employs microfluidics coupled to various other analytical techniques to study glucagon and insulin release from healthy human pancreatic cells and provide increased understanding regarding their release pattens. Chapter 1 presents an introduction to the physiology and function of pancreatic cells in the human body and explains how immunoassay techniques are employed for characterization of pancreatic hormones. Chapter 2 introduces a novel microfluidic system to study glucagon secretion from human pancreatic cells using a Forster-resonance energy transfer (FRET)-based assay, which provides temporal resolution for glucagon release traces from ~30 islets. Finally, Chapter 3 builds upon the previous chapter and provides a method to simultaneously detect insulin and glucagon released from pancreatic cells in real time to characterize the release relationship of the two hormones.