Automated microfluidic device development for metabolism, nutrient uptake, and hormone secretion analyses of primary endocrine tissues
Type of DegreePhD Dissertation
DepartmentChemistry and Biochemistry
Restriction TypeAuburn University Users
MetadataShow full item record
Pancreatic islets secret the dominant endocrine hormone, insulin, which controls the metabolic function fo nearly all other organ systems. Additionally, adipose tissue (fat) is now understood to be a complex, multicellular endocrine organ with profound systemic effects, which also alters the function of many other organs. Although there has been renewed interest in insulin secretion and adipose tissue dynamics in response to meals high in refined carbohydrate and sugars, using standard approaches, we unfortunately have a limited view of the temporal relationships between glucose, insulin, and adipose function. Microfluidic technology offers novel features that can meet these needs. This dissertation work focuses on the development of automated microfluidic methods to aid in understanding metabolism, nutrient uptake, and hormone secretion of primary endocrine tissues. In Chapter 2, a digitally controllable, 16-channel microfluidic input/output multiplexer (µMUX) was developed for mimicking the circulation in the endocrine system. 3D-printed templates were designed to sculpt devices, creating millimeter scale reservoirs and confinement chambers to interface endocrine tissues to the channels. Dynamic insulin secretion profiles and fatty acid uptake/release were monitored in real-time on the µMUX, and quantitative measurement of proteins at attomole levels was achieved. This system has also revealed novel temporal information on insulin-dependent fatty acid transport machinery. The development of a real time fluorescence assay for monitoring of fatty acid uptake is discussed in Chapter 3. This assay was designed based on the natural binding of fatty acid to serum albumin. Quencher labeled bovine serum albumin was shown to mask the background fluorescence of Bodipy labeled fatty acid analogues and allow for homogeneous real-time measurement of fatty acid uptake by cells or tissues with minimal background fluorescence. The insulin-induced fatty acid uptake in adipose tissue explants was studied in the µMUX device using this assay. A synthesized CD-36/SR-B2 inhibitor has shown significant inhibition on the insulin effect on fatty acid uptake rate. An automated, droplet forming microfluidic system (PumpDrop) is introduce in Chapter 4 for continuous, high temporal resolution, on-chip secreted protein quantification. The PumpDrop system integrated precise peristaltic pumping, homogeneous immunoassay, lock-in droplet detection, and on-chip assay incubation with droplet storage channels. As a proof of concept, dynamic insulin secretion profiles from single islets of Langerhans was achieved at high temporal resolution using only an optical readout. This system should be directly translatable to other cells or tissues for secreteome dynamic studies. Chapter 5 highlights a study of protein coatings at aqueous-in-oil droplet surfaces. A biotinylated perfluorocarbon-surfactant was synthesized for microfluidic droplet formation, and the biotin moiety was shown to recruit streptavidin molecules to the aqueous-oil interface. This proof-of-concept paves the way for the development of interface binding based homogeneous bioassays within droplets, which could further improve the discrimination between signal and background in immunoassays. The final Chaper reviews the research contribution of this dissertation, and provides an outlook into future research stemming from these topics.