面向核酸检测的生物传感器和液滴微流控系统的初步建立和研究

发布时间：2018-04-22 18:09

本文选题：核酸检测 + 等温扩增　；参考：《浙江大学》2017年硕士论文

【摘要】：核酸体外扩增和检测在病原体分析、分子诊断、肿瘤早期筛查、环境监测及法医鉴定等领域的重要性与日俱增,人们对于发展微型便捷、操作简单、灵敏高效、试剂能源低耗、价格经济的检测分析手段的需求也越来越高。在核酸检测的多种分析方法中,恒温扩增技术由于无需改变温度,只需简单的设备就可以实现特异性强、灵敏度高、成本低的核酸分析,所以在核酸检测中得到越来越多的发展和应用,电化学传感器与液滴微流控芯片相结合的核酸检测技术将同时具有电化学传感器操作简便、响应快以及液滴微流控耗样量少、高通量、无交叉污染等优点,有潜力发展出更加高效的核酸检测系统。在本论文中,我们研究了基于聚苯乙烯金电极的电化学DNA传感器在核酸检测中的性能,并且开发了一套基于液滴微流控技术的核酸检测系统,结合恒温扩增技术,实现了对DNA的有效检测。本论文共分为四章,主要内容如下:第一章综述了目前核酸检测的常用方法及其主要应用,介绍了电化学传感器和液滴微流控技术的原理及在核酸检测中的应用。第二章的工作中,我们采用了聚苯乙烯板取代传统的玻璃板作为基板,用化学镀金的方式制作了金电极,将其与基于镀膜-光刻法制备的玻璃基板金电极的电化学特性和固定巯基(-SH)DNA探针的能力进行对比和研究,解释了聚苯乙烯基板金电极的制作原理,以及其优于玻璃金电极的几个可能因素,并且用所制得的聚苯乙烯基板金电极进行DNA杂交反应和检测,检测限为7.2×10-11 M(单位符号M表示单位mol·L-1,下同)(S/N=3),为电化学DNA传感器的电极制备提供了一种比较好的电极制作工艺,有很大的推广价值。第三章的工作中我们制作了 PDMS-PDMS芯片以制备微液滴,设计了带凹槽夹层的三热块加热装置和蛇形毛细管通道以进行HRCA反应,搭建了基于荧光检测技术的检测平台,发挥三者各自的优势,建立了一套基于液滴微流控的核酸扩增及检测系统,并利用这个系统对DNA进行扩增和检测,检测限为7.5×10-10 M(S/N=3)。所设计的加热装置和加热通道材质配合优化后,有望用于非恒温技术PCR中,可以推广到更大的应用范围;液滴制备系统和检测系统经过改进,有潜力成为一种数字PCR或数字RCA的检测系统。第四章对本论文的工作进行了总结和展望,在接下来的工作中将结合电化学DNA传感器和液滴微流控技术并应用于核酸检测,希望能充分发挥两者的优势,实现对核酸更高效、更便捷的检测。
[Abstract]:Nucleic acid amplification and detection in vitro is becoming more and more important in the fields of pathogen analysis, molecular diagnosis, early tumor screening, environmental monitoring and forensic identification. The demand for price-economy means of detection and analysis is also getting higher and higher. Among the various analytical methods of nucleic acid detection, the constant temperature amplification technique can achieve nucleic acid analysis with strong specificity, high sensitivity and low cost because it does not need to change temperature. Therefore, more and more development and application have been made in nucleic acid detection. The nucleic acid detection technology combined with electrochemical sensor and droplet microfluidic chip will have the advantages of simple operation of electrochemical sensor, fast response and less sample consumption of droplet microfluidic control. High throughput, no cross-contamination and other advantages have the potential to develop a more efficient nucleic acid detection system. In this paper, we studied the performance of electrochemical DNA sensor based on polystyrene gold electrode in nucleic acid detection, and developed a nucleic acid detection system based on droplet microfluidic technology, combined with constant temperature amplification technology. The effective detection of DNA is realized. This paper is divided into four chapters. The main contents are as follows: in chapter 1, the methods and applications of nucleic acid detection are reviewed. The principle of electrochemical sensor and droplet microfluidic technology and its application in nucleic acid detection are introduced. In the second chapter, we used polystyrene instead of glass as substrate, and made gold electrode by electroless gold plating. The electrochemical characteristics of the gold electrode and the ability of immobilized thiol thiol -SHN DNA probe were compared with those of the glass substrate gold electrode based on film plating and photolithography, and the principle of the preparation of the gold electrode on the polystyrene substrate was explained. And some possible factors which are superior to the glass gold electrode, and the DNA hybridization reaction and detection with the prepared polystyrene substrate gold electrode were carried out. The detection limit is 7.2 脳 10 ~ (-11) M (the unit symbol M is the unit mol L ~ (-1), and the following is the same as S / N ~ (3)), which provides a good electrode preparation process for electrochemical DNA sensor, and has great value in popularization. In the third chapter, we made PDMS-PDMS chip to prepare microdroplets, designed a three-hot block heating device with grooves and a snake-shaped capillary channel for HRCA reaction, and set up a detection platform based on fluorescence detection technology. A system of nucleic acid amplification and detection based on droplet microfluidic control was established, and the detection limit of DNA was 7.5 脳 10 ~ (-10) m ~ (-1) m ~ (-1) S / N ~ (3 +). The designed heating device and heating channel material are optimized, which is expected to be used in the non-constant temperature technology PCR, and can be extended to a wider range of applications, the droplet preparation system and the detection system have been improved, Has the potential to become a digital PCR or digital RCA detection system. In the fourth chapter, the work of this paper is summarized and prospected. In the following work, the electrochemical DNA sensor and droplet microfluidic technology will be applied to nucleic acid detection, hoping to give full play to the advantages of the two and to achieve more efficient nucleic acid. More convenient detection.
【学位授予单位】：浙江大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：O629.74;O657.1

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