|dc.description.abstract||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
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.||en_US