|dc.description.abstract||Adipose tissue, playing an important role in the global epidemic obesity and diabetes, has attracted extensive attention in recent decades. However, quantitative measurement of dynamics of nutrient uptake and metabolic process in adipose tissue has not been accomplished at high temporal resolution (< 1 min) with traditional instruments and methods. Rapid developments in tissue engineering and microfluidic techniques have begun to meet the requirements of culturing and precisely bio-analyzing adipose tissue explants or adipocytes. Specially, our lab pioneered using automatic droplet-based microfluidics to reveal the metabolic dynamics of pancreatic islets and primary murine epididymal white adipose tissue (eWAT) at a temporal resolution of just a few seconds. In addition, droplet microchips should be powerful tools which significantly enhance the sensitivity of biomolecule (such as proteins, small molecules, nucleic acids, etc) detection with less labor and cost. This dissertation mainly focuses on the evolution of our automated droplet-based secretion sampling systems and their applications to quantification of analytes and the dynamic functions of eWAT.
In Chapter 1, we introduce obesity and adipose tissue. To deepen our understanding about adipose tissue, its inner structures and functions as well as its metabolic and uptake mechanisms are discussed in this chapter. Later the methodologies for quantifying biomolecules via heterogeneous and homogeneous immunoassays are reviewed herein. The last and most analytically important part of Chapter 1 is the detailed introduction of droplet-based microfluidics, which is viewed as an efficient tool to overcome several specific challenges in chemical and biological fields.
Chapter 2 describes the applications of our recently developed droplet microdevice which integrate salt-water electrodes, merging channels, and pneumatic valves. Firstly, the effects of frequency and magnitude of salt-water electrode voltage on droplet coalescence in this system were studied. Then, this automatic microfluidic system was proved to be a powerful platform for achieving precise serial dilution for pH regulation, and for exploring the assay responses of a homogeneous immunoassay via programmably generating and coalescing several nanoliter volume daughter droplets with a high electric field.
Chapter 3 highlights the integration of our µChopper approach with active valve-based pumps and salt-water electrodes for the first time, taking advantage of the benefits of programmable control of droplet generation and electrocoalescence. In this proof-of-concept work, we applied the device to real-time, continuous calibration of fluorescent labels, then we validated the system for continuous calibration of a homogeneous insulin immunoassay that exhibits a nonlinear response. With the significant savings in reagent use, assay cost, and user time that were incurred, this device provided a novel means to carry out economical measurements with precious reagents in a static or real-time manner.
Droplet-compatible nicking enzyme signal amplification (NESA) is discussed in Chapter 4. As we know, amplification methods enable improving detection sensitivity of biomolecules by several orders of magnitude compared to direct assays, and these benefits are combined with automation, multi-function, and high-throughput of our droplet system mentioned in Chapter 2. Herein, we developed a powerful microdevice by integrating droplet microdevice and isothermal NESA for analyte quantification. In this droplet-compatible NESA system, the limit of detection (LOD) of DNA and anti-digoxigenin were as low as the femtomolar level. This integrated system has the potential of measuring various types of targets at low concentrations.
In Chapter 5 and 6, two droplet-based system are introduced which are applied to resolve dynamics of uptake and metabolism of eWAT, a tissue of high functional importance in the global epidemics of obesity and diabetes. Here, we cultured and sampled from ex vivo eWAT explants with valve-automated droplet formation, then used on-chip mergers to combine tissue eluates with assay reagents downstream. For the first time, dynamics of both nutrient absorption (free fatty acid uptake) and lipolysis (glycerol secretion) from single eWAT explants were quantified at high temporal resolution (~9 s), revealing rapid reversal of uptake after treating with isoproterenol, without significant effects on lipolysis dynamics.
The final installment, Chapter 7, summaries this dissertation and provides an outlook into future research about the development of droplet microfluidics, analyte quantification and activities of tissue/cell monitoring on the novel platform of droplet microfluidics.||en_US