基于微流控液滴技术的基因定量分析和蛋白质配体筛选系统的研究

发布时间：2018-05-19 10:44

本文选题：微流控技术 + 液滴阵列　；参考：《浙江大学》2017年博士论文

【摘要】：微流控技术在微量、高通量、微型化、集成化和自动化等方面具有突出优势,近年来逐渐成为基因绝对定量分析和高通量筛选蛋白质-配体结合的重要技术平台,在生物学、医学、药学等众多领域获得了快速发展和广泛应用。第一章介绍了微流控数字PCR技术的发展和应用,根据不同的技术路线对现有的微流控数字PCR系统进行了分类和特点总结。此外,还介绍了目前蛋白质-配体亲和力分析技术的现状及应用。第二章中,我们结合液体操纵机器人与表面辅助液滴形成技术,发展了一种自动化、灵活的油下快速形成多体积二维液滴阵列的方法——表面辅助多因素流体间隔法(Surface assisted multifactor fluid segmentation,SAMFS)。SAMFS 法形成的液滴体积主要由泵流速和平移台的移动速度所决定,通过简单调节泵流速和平移台的移动速度即可实现液滴体积的灵活控制。基于SAMFS法,我们可实现多体积液滴阵列的形成,并将其用于构建多体积液滴数字PCR系统。该系统被应用于核酸的绝对定量分析以及不同乳腺癌细胞中HER2目标基因的表达水平分析,其检测动态范围横跨4个数量级。第三章中,我们发展了一种基于液滴阵列的蛋白质热稳定性分析系统,用于实现无标记、高通量、低消耗的蛋白质-配体结合筛选。该系统将液滴操纵机器人技术和二维平面液滴阵列技术相结合,自动化实现蛋白质液滴与大量不同种类的化合物液滴的混合操作,可在一块芯片上形成高密度的液滴反应器阵列。单个芯片一次实验即可实现100种化合物的筛选,单个筛选条件的蛋白质消耗量可降低至飞摩尔级。
[Abstract]:Microfluidic technology has outstanding advantages in micro, high throughput, miniaturization, integration and automation. In recent years, microfluidic technology has gradually become an important technology platform for gene absolute quantitative analysis and high throughput screening of protein-ligand binding. Medicine, pharmacy and many other fields have been rapidly developed and widely used. In the first chapter, the development and application of microfluidic digital PCR technology are introduced. The existing microfluidic digital PCR systems are classified and summarized according to different technical routes. In addition, the current status and application of protein-ligand affinity analysis techniques are also introduced. In the second chapter, we developed a kind of automation by combining liquid manipulation robot with surface assisted droplet forming technology. The rapid formation of a multi-volume two-dimensional droplet array under a flexible oil-surface assisted multi-factor fluid spacing method is mainly determined by the flow velocity of the pump and the moving velocity of the translational platform, and the volume of the droplet formed by the Surface assisted multifactor fluid Segmentation / SAMFS.SAMFS method is mainly determined by the flow rate of the pump and the moving velocity of the platform. The flexible control of droplet volume can be realized by simply adjusting the pump velocity and the moving speed of the translation table. Based on SAMFS method, we can realize the formation of multi-volume droplet array and use it to construct multi-volume droplet digital PCR system. The system has been applied to the absolute quantitative analysis of nucleic acid and the expression level of HER2 target gene in different breast cancer cells. The detection dynamic range is four orders of magnitude. In Chapter 3, we developed a protein thermal stability analysis system based on droplet array for protein ligand binding screening without labeling, high throughput and low consumption. The system combines droplet manipulating robot technology with two-dimensional planar droplet array technology to automate the hybrid operation of protein droplets and a large number of different kinds of compound droplets. A high density droplet reactor array can be formed on a chip. 100 compounds can be screened in a single chip, and the protein consumption of a single screening condition can be reduced to the flying mole level.
【学位授予单位】：浙江大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：Q78

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