本文選題：核酸檢測 + 等溫擴增��； 參考：《浙江大學》2017年碩士論文

【摘要】：核酸體外擴增和檢測在病原體分析、分子診斷、腫瘤早期篩查、環(huán)境監(jiān)測及法醫(yī)鑒定等領域的重要性與日俱增,人們對于發(fā)展微型便捷、操作簡單、靈敏高效、試劑能源低耗、價格經(jīng)濟的檢測分析手段的需求也越來越高。在核酸檢測的多種分析方法中,恒溫擴增技術由于無需改變溫度,只需簡單的設備就可以實現(xiàn)特異性強、靈敏度高、成本低的核酸分析,所以在核酸檢測中得到越來越多的發(fā)展和應用,電化學傳感器與液滴微流控芯片相結(jié)合的核酸檢測技術將同時具有電化學傳感器操作簡便、響應快以及液滴微流控耗樣量少、高通量、無交叉污染等優(yōu)點,有潛力發(fā)展出更加高效的核酸檢測系統(tǒng)。在本論文中,我們研究了基于聚苯乙烯金電極的電化學DNA傳感器在核酸檢測中的性能,并且開發(fā)了一套基于液滴微流控技術的核酸檢測系統(tǒng),結(jié)合恒溫擴增技術,實現(xiàn)了對DNA的有效檢測。本論文共分為四章,主要內(nèi)容如下:第一章綜述了目前核酸檢測的常用方法及其主要應用,介紹了電化學傳感器和液滴微流控技術的原理及在核酸檢測中的應用。第二章的工作中,我們采用了聚苯乙烯板取代傳統(tǒng)的玻璃板作為基板,用化學鍍金的方式制作了金電極,將其與基于鍍膜-光刻法制備的玻璃基板金電極的電化學特性和固定巰基(-SH)DNA探針的能力進行對比和研究,解釋了聚苯乙烯基板金電極的制作原理,以及其優(yōu)于玻璃金電極的幾個可能因素,并且用所制得的聚苯乙烯基板金電極進行DNA雜交反應和檢測,檢測限為7.2×10-11 M(單位符號M表示單位mol·L-1,下同)(S/N=3),為電化學DNA傳感器的電極制備提供了一種比較好的電極制作工藝,有很大的推廣價值。第三章的工作中我們制作了 PDMS-PDMS芯片以制備微液滴,設計了帶凹槽夾層的三熱塊加熱裝置和蛇形毛細管通道以進行HRCA反應,搭建了基于熒光檢測技術的檢測平臺,發(fā)揮三者各自的優(yōu)勢,建立了一套基于液滴微流控的核酸擴增及檢測系統(tǒng),并利用這個系統(tǒng)對DNA進行擴增和檢測,檢測限為7.5×10-10 M(S/N=3)。所設計的加熱裝置和加熱通道材質(zhì)配合優(yōu)化后,有望用于非恒溫技術PCR中,可以推廣到更大的應用范圍;液滴制備系統(tǒng)和檢測系統(tǒng)經(jīng)過改進,有潛力成為一種數(shù)字PCR或數(shù)字RCA的檢測系統(tǒng)。第四章對本論文的工作進行了總結(jié)和展望,在接下來的工作中將結(jié)合電化學DNA傳感器和液滴微流控技術并應用于核酸檢測,希望能充分發(fā)揮兩者的優(yōu)勢,實現(xiàn)對核酸更高效、更便捷的檢測。
[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.
【學位授予單位】：浙江大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：O629.74;O657.1

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