基于流动注射梯度技术的液滴微流控系统及其在酶抑制剂筛选和酶反应动力学研究中的应用
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摘要
液滴微流控(Droplet-based microfluidics)是在微通道结构中生成和操控纳升至飞升级液滴的科学与技术。与连续流微流控系统相比,液滴微流控系统不仅具有混合速度快、抑制扩散、无交叉污染以及试剂和样品消耗量低等优点,而且微流控液滴的生成频率可高达数千赫兹,在高通量筛选上极具潜力。近十年来,随着微液滴的操纵、生成、分裂、分选、检测等技术的日趋成熟,液滴微流控系统在分析化学、蛋白质结晶筛选、细胞生物学、分子生物学、高通量筛选上的应用日益广泛。本文主要进行了微流控梯度液滴分析系统的研究,并将其应用于酶抑制剂筛选和酶反应动力学研究中。
第一章总结了微流控梯度技术的研究现状,包括各种用于连续流微流控系统与液滴微流控系统的梯度技术原理、特点及其在细胞生物学、酶反应动力学、高通量筛选等领域的应用。
第二章建立了一种基于流动注射梯度技术的液滴微流控系统。该系统由试样引入系统、微芯片、驱动系统与激光诱导荧光检测系统组成。微芯片上集成了取样探针、试样引入通道、试剂通道、液滴形成通道及液滴反应与检测通道。样品通过磨尖的取样探针注入微通道内,在流动的载流中由于对流和分子扩散形成具有连续轴向浓度梯度的样品区带。该样品区带在与试剂汇合后,被与水不互溶的油相间隔形成具有浓度梯度的液滴阵列。样品与试剂在液滴内混合、反应,其产物在流经检测点时被激光诱导荧光检测系统检测。该系统只需单次注射纳升级样品即可形成浓度梯度范围达3-4个数量级的液滴,具有很低的样品消耗。系统被成功地应用于基于β-半乳糖苷酶(β-galactosidase,(3-gal)的酶抑制分析,初步展示了本系统在高通量药物筛选中应用的潜力。
第三章用第二章建立的基于流动注射梯度技术的液滴微流控系统进行了Caspase-1酶抑制剂的筛选。Caspase-1催化荧光底物YVAD-AFC释放出游离的荧光染料分子AFC,抑制剂Ac-YVAD-CHO与Ac-(Nme)Tyr-Val-Ala-Asp-CHO对该酶反应有抑制作用。自行搭建了一套激发波长为405nm,发射波长为505nm的激光诱导荧光系统检测释放的游离染料分子AFC。该系统样品消耗仅为3.3nL。该系统被应用于Caspase-1抑制剂Ac-YVAD-CHO与Ac-(Nme)Tyr-Val-Ala-Asp-CHO半数抑制浓度(IC50)的测定。
第四章将第二章建立的基于流动注射梯度技术的液滴微流控系统,应用于酶动力学研究,测定了p-半乳糖苷酶催化荧光素双p-d-吡喃半乳糖苷反应的米氏常数(Km)。该方法具有简单、快速的特点,在1min内仅需一次注样操作就可测定出酶反应的米氏常数,耗样仅为13nL。
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.
第一章总结了微流控梯度技术的研究现状,包括各种用于连续流微流控系统与液滴微流控系统的梯度技术原理、特点及其在细胞生物学、酶反应动力学、高通量筛选等领域的应用。
第二章建立了一种基于流动注射梯度技术的液滴微流控系统。该系统由试样引入系统、微芯片、驱动系统与激光诱导荧光检测系统组成。微芯片上集成了取样探针、试样引入通道、试剂通道、液滴形成通道及液滴反应与检测通道。样品通过磨尖的取样探针注入微通道内,在流动的载流中由于对流和分子扩散形成具有连续轴向浓度梯度的样品区带。该样品区带在与试剂汇合后,被与水不互溶的油相间隔形成具有浓度梯度的液滴阵列。样品与试剂在液滴内混合、反应,其产物在流经检测点时被激光诱导荧光检测系统检测。该系统只需单次注射纳升级样品即可形成浓度梯度范围达3-4个数量级的液滴,具有很低的样品消耗。系统被成功地应用于基于β-半乳糖苷酶(β-galactosidase,(3-gal)的酶抑制分析,初步展示了本系统在高通量药物筛选中应用的潜力。
第三章用第二章建立的基于流动注射梯度技术的液滴微流控系统进行了Caspase-1酶抑制剂的筛选。Caspase-1催化荧光底物YVAD-AFC释放出游离的荧光染料分子AFC,抑制剂Ac-YVAD-CHO与Ac-(Nme)Tyr-Val-Ala-Asp-CHO对该酶反应有抑制作用。自行搭建了一套激发波长为405nm,发射波长为505nm的激光诱导荧光系统检测释放的游离染料分子AFC。该系统样品消耗仅为3.3nL。该系统被应用于Caspase-1抑制剂Ac-YVAD-CHO与Ac-(Nme)Tyr-Val-Ala-Asp-CHO半数抑制浓度(IC50)的测定。
第四章将第二章建立的基于流动注射梯度技术的液滴微流控系统,应用于酶动力学研究,测定了p-半乳糖苷酶催化荧光素双p-d-吡喃半乳糖苷反应的米氏常数(Km)。该方法具有简单、快速的特点,在1min内仅需一次注样操作就可测定出酶反应的米氏常数,耗样仅为13nL。
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.
引文
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