Development of thermally regulated homogeneous assays and droplet-based microfluidic tools for probing secretion dynamics from pancreatic and adipose tissues
Type of DegreePhD Dissertation
Chemistry and Biochemistry
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Cells can transmit signals and adapt to microenvironment variations rapidly with dynamic processes, such as biomolecule secretion for signal delivery. Dynamic, oscillatory secretion is expected to exist in many cell types, but only a few selected tissues have been well investigated. This gap in information is likely due to the shortage of simple bioassays and compatible fluidic tools to directly sample and detect these secretions at high temporal resolution. To address this need, we have developed an integrated microfluidic droplet system. Using on-chip valving, droplets with consistent volume could be generated without the need to connect to macroscopic plumbing such as syringe pumps for droplet formation or cell perfusion. Homogeneous immunoassays and enzyme-coupled assays were also developed and optimized for on-chip, indroplet readout and applied within the system to study dynamic secretions of proteins (insulin, asprosin) and small molecules (glycerol) from both primary tissues and cell spheroids at high temporal resolution (<4 s). Chapter 1 introduces the research background. The prevalence of diabetes and obesity, functional tissues (pancreatic and adipose tissue) and related bioactive molecules are discussed. Another major part of introduction reviews the detection of bioactive molecules, including microfluidic tools and bioassays. Microfluidics is a powerful tool to integrate multiple techniques such as cell/tissue culture, secretion sampling, and detection on a small scale device, with rapid developments in the area in recent years. Bioassays are geared toward biomolecule quantification, where both high sensitivity and specificity are ideal. Particularly for integrating onto microfluidics, however, assays with simple workflow are much more suitable. Ultimately, simple assays, such as the homogeneous immunoassays discussed herein, can recognize their targets in complicated matrices and transfer this recognition into an easily detectable signal such as direct fluorescence. In Chapter 2 and Chapter 3, homogeneous assays based on thermofluorimetric analysis (TFA) were developed for protein and small molecule quantification. TFA translates target levels quantitatively into multiplexable DNA melting transitions, allowing discrimination of the signal from the background, simultaneously. Chapter 2 discussed about TFA methods based on pairs of probes like antibody-oligo conjugated probes or aptamer pairs. Chapter 3 extends the TFA concept to study the binding of a single aptamer to protein targets, which can be used to quantify the targets or applied to monitor the progress of aptamer screening. Based on previous and continuing work in Prof. Easley’s lab, a microfluidic droplet system interfaced with enzymatic assays was developed and summarized in Chapter 4. The system was successfully adapted and utilized to detect the glycerol release from adipose tissue and encapsulated adipocytes. Interestingly, unique information was revealed about the dynamic function of adipose tissue, that glycerol release from the tissue was observed in a pulsatile pattern, distinctly different from adipocyte cell spheroids. In Chapter 5, we began to explore the dynamic secretion of newly-discovered adipokine, asprosin. TFA assays for human and mouse asprosin were developed, and the isothermal format of the mouse asprosin assay was integrated into the microfluidic droplet system for high temporal resolution sampling and detection of asprosin. The results show that asprosin secretion was responsive to glucose and insulin reductions within a few minutes, and dynamic secretion behavior was also detected with asprosin. As a newly-discovered hormone, the secretion mechanisms and dynamics of asprosin release from adipose tissue are currently unknown, and these results represent the first report of dynamic asprosin secretion to our knowledge. This dissertation concludes with Chapter 6, where we summarize the work and discuss the fact that further assay developments, combined with our novel microfluidic droplet-based sampling, should enable additional unique studies to be conducted on dynamic biological processes such as hormone or small-molecule secretion.