Development of Droplet Microfluidic and Micro Free-Flow Electrophoresis Assays for High-Throughput and Real-Time Biochemical Analyses

Abstract

The emergence of microfluidics and its evolution over the last four decades has transformed the field of chemical and biochemical analysis. Through miniaturization, assays are more efficient and more easily controlled, often leading to improved reproducibility and sensitivity. Expanding the analytical toolbox, microfluidics offer new opportunities for information-rich assays. This dissertation presents the development of two distinct microfluidic platforms capable of biochemical analyses at scales and timeframes not previously reported. First, a high-throughput droplet microfluidic platform was developed for the identification and characterization of high-efficiency catalytic oligonucleotides. While the existence of catalytic RNA and DNA has been known for decades, existing selection strategies introduce an inherent bias for single turnover catalyst identification. Here, a droplet microfluidic assay was created for the identification of high-turnover DNAzymes in the presence of trillions of non-catalytic sequences. Random sequence DNA libraries were confined in aqueous droplets alongside a fluorogenic substrate; if any random sequence were capable of efficient catalysis, fluorescent product will accumulate in-droplet. High-throughput laser-induced-fluorescence detection will reveal valuable information concerning catalytic frequency and kinetic properties. This dissertation details the development of the droplet microfluidic platform, its performance metrics, control assays with a protein enzyme and known DNAzyme, and early DNA library screening experiments. Second, an online affinity micro free-flow electrophoresis assay was developed for the continuous monitoring of biochemical messengers. Affinity assays are ubiquitous due to their exceptional affinity and selectivity for biochemical analytes. However, they are primarily single-time-point analyses, ill-equipped for the study of dynamic biological systems. Here, affinity assay technology was paired with micro free-flow electrophoresis (µFFE), a continuous separation technique. Affinity assay reagents were mixed online, and the bound and free species were continuously separated based on their electrophoretic mobilities. Real-time monitoring of Neuropeptide Y and insulin was shown, and the temporal response of the assay was optimized. The affinity µFFE method was applied to the characterization of insulin secretion from human islets, demonstrating real-time detection in the presence of a complex biological matrix.