Droplet-based Microfluidic Systems Based On Flow Injection Gradient Technique For Enzyme Inhibitor Screening And Enzyme Kinetic Study

Droplet microfluidics is the science and technology which generate and manipulate droplets in the nanoliter nanoliter to femtoliter range. Comparing with the continuous flow-based microfluidic systems, the droplet-based microfluidic systems offer advantages of rapid mixing, eliminated cross contamination, low sample/reagent consumption and high frequency of microreactor generation. In the past decade, various techniques of droplet generation, manipulation, fusion, splitting, sorting and detection have been developed, and applied in bioanalysis, protein crystallization screening, cell biology and high throughput screening. The aim of the present work is to develop droplet-based microfluidic systems capable of generating large-scale concentration gradient for enzyme inhibitor screening and enzyme kinetic study.In chapter1, the progress of microfluidic concentration gradient techniques under continuous flow mode and droplet-based mode, as well as their applications in cell biology, enzyme kinetics and high-throughput screening, are reviewed.In chapter2, the flow injection gradient technique (FIG) was coupled with droplet generation to generate droplets with large-scale concentration gradients. The droplet-based microfluidic system consisted of sample introduction system, micro chip, driven system and laser induced fluorescence detection system. Multiple modules including sampling probe, dispersion channel, reagent channel, droplet generation and detection were integrated into the microchip. The injected sample plug was dispersed in the flowing carrier to form a sample zone with continuous concentration gradients along the channel. The sample zone was then segmented into a series of droplets by a immiscible oil phase. The present system could be used to generate droplets with concentrations spanning3-4orders of magnitudes with a single nanoliter-scale injection. We applied it in β-gal inhibition assay to preliminarily demonstrate its application potentials in high throughput drug screening. In chapter3, the droplet-based flow injection gradient system built in chapter2was used to screen the caspase-1inhibitors. The assay was based on the inhibition of enzyme caspase-1by inhibitors of Ac-YVAD-CHO and Ac-(Nme)Tyr-Val-Ala-Asp-CHO, impeding the release of AFC from the substrate YVAD-AFC. A home-built laser induced fluorescence detection system with an excitation wavelength of405nm and emission wavelength of505nm was used to measure AFC produced in the droplets. With a single injection of3.3nL inhibitors, the IC50values of Ac-YVAD-CHO and Ac-(Nme)Tyr-Val-Ala-Asp-CHO were obtained.In chapter4, the droplet-based flow injection gradient system presented in chapter2was used in enzyme kinetic study for the measurement of Michaelis-Menten constant. With a single injection of13nL of substrate, the Michaelis-Menten constant of the β-gal catalyzed reaction was measured in less than1min.