高灵敏荧光核酸传感分析新方法及性能研究

发布时间：2018-05-08 22:43

  本文选题：DNA荧光生物传感器 + DNAzyme　； 参考：《青岛科技大学》2017年硕士论文

【摘要】：本论文主要基于核酸工具酶、DNAzymes辅助,靶标循环以及等温链置换扩增反应等信号放大策略,构筑了三种DNA荧光生物传感器,实现对核酸的高灵敏分析检测。主要内容包括:(1)基于靶标自循环和级联循环指数扩增策略(TR-CEA),构建了一个超灵敏、一步检测靶标DNA的生物传感器。整个传感体系由两个巧妙设计的发夹DNA链和两种酶构成,荧光探针(MB),包含一个切刻内切酶的识别片段,3’突出片段与茎环(HP)互补杂交,聚合酶聚合,释放出靶标DNA,引起靶标循环,置换下来的靶标继续参与到下一轮杂交过程。同时,双链DNA引发酶切割并沿切割处进行反向聚合,实现链置换扩增(SDA)过程。随后,MB的茎环结构打开,露出切刻内切酶的识别片段,从而发生切割和反向的SDA过程,被切割的MB产生荧光信号。对于靶标DNA的检测限估算达到0.61 fM,具有高灵敏度、重复性好的优点。(2)基于熵驱动靶标循环及DNAzyme辅助,构建了双信号放大检测核酸的生物传感器。在熵驱动靶标循环过程中,靶标与TP发生立足点介导的链置换反应(Toe-hold),置换下PP链,形成中间体I3,燃料链Fs与I3发生第二个Toe-hold链置换反应,先置换下R链,形成中间体I5,随后再置换下靶标,生成产物,并实现靶标循环。整个熵驱动反应是通过释放分子达到熵增加,产生了热力学推动。在循环Ⅱ过程中,由于循环I生成的产物包含有切割酶活性的Mg~(2+)-DNAzyme结构,能够识别切割MB,产生荧光信号。该传感器无酶辅助,成本低,背景信号低,具有良好的稳定性和特异性。(3)基于靶标引发的链置换聚合反应(CNDP)及Pb~(2+)-DNAzyme循环切割,构建了超灵敏检测p53基因的生物传感器。HP识别靶标DNA,促使HP的3’末端与PT杂交。从PT的3’端聚合延伸,替换下杂交的靶标DNA及PP链,并促进靶标与下一个HP杂交,实现靶标循环。在PT聚合产生的双链区域包含nick酶的识别位点,nick酶切割聚合产物,产生新的DNAzyme功能序列,同时此段切割生成的DNA链能够作为靶标类似物参与到靶标循环中。聚合酶和nick酶实现协同效应,聚合-切割-置换的过程反复进行,积累产生大量具有切割酶活性的Pb~(2+)-DNAzyme功能结构,切割MB,产生荧光信号。该传感器自主且有效地进行信号放大,操作简单,灵敏度较同类型传统的传感器高。
[Abstract]:In this paper, three kinds of DNA fluorescent biosensors were constructed based on the signal amplification strategies such as DNA zymes assisted by nucleic acid tools, target cycle and isothermal chain replacement amplification. The main contents include: (1) based on the target self-cycle and cascade cycle index amplification strategy, a super-sensitive, one-step biosensor for detecting target DNA was constructed. The whole sensing system consists of two cleverly designed hairpin DNA strands and two kinds of enzymes. The fluorescent probe is a fluorescent probe, which consists of a recognized fragment of the endonuclease, a 3 'protruding fragment, and a complementary hybridization with the HPs of the stem ring. The target DNA was released to cause the target cycle, and the replacement target continued to participate in the next round of hybridization. At the same time, double strand DNA initiated enzyme cleavage and reverse polymerization along the cleavage site to realize chain replacement amplification. The stem ring structure of MB was then opened to reveal the recognition fragment of the endonuclease, thus the cutting and reverse SDA process occurred, and the cut MB produced the fluorescence signal. The detection limit of target DNA is estimated to reach 0.61 fM, which has the advantages of high sensitivity and good repeatability. Based on entropy driven target cycle and DNAzyme aid, a biosensor for detecting nucleic acid with double signal amplification is constructed. In the course of entropy driven target cycle, the standing point mediated chain substitution reaction occurs between target and TP. PP chain is replaced to form intermediate I _ 3. The second Toe-hold chain substitution reaction occurs between fuel chain Fs and I _ 3, and then R chain is replaced. The intermediate I _ 5 was formed and then replaced with the target to produce the product and to realize the target cycle. The whole entropy-driven reaction is driven by the release of molecules to increase entropy, resulting in thermodynamics. In the process of cycle 鈪，

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