本文選題：DNA熒光生物傳感器 + DNAzyme��； 參考：《青島科技大學》2017年碩士論文

【摘要】：本論文主要基于核酸工具酶、DNAzymes輔助,靶標循環(huán)以及等溫鏈置換擴增反應等信號放大策略,構(gòu)筑了三種DNA熒光生物傳感器,實現(xiàn)對核酸的高靈敏分析檢測。主要內(nèi)容包括:(1)基于靶標自循環(huán)和級聯(lián)循環(huán)指數(shù)擴增策略(TR-CEA),構(gòu)建了一個超靈敏、一步檢測靶標DNA的生物傳感器。整個傳感體系由兩個巧妙設計的發(fā)夾DNA鏈和兩種酶構(gòu)成,熒光探針(MB),包含一個切刻內(nèi)切酶的識別片段,3’突出片段與莖環(huán)(HP)互補雜交,聚合酶聚合,釋放出靶標DNA,引起靶標循環(huán),置換下來的靶標繼續(xù)參與到下一輪雜交過程。同時,雙鏈DNA引發(fā)酶切割并沿切割處進行反向聚合,實現(xiàn)鏈置換擴增(SDA)過程。隨后,MB的莖環(huán)結(jié)構(gòu)打開,露出切刻內(nèi)切酶的識別片段,從而發(fā)生切割和反向的SDA過程,被切割的MB產(chǎn)生熒光信號。對于靶標DNA的檢測限估算達到0.61 fM,具有高靈敏度、重復性好的優(yōu)點。(2)基于熵驅(qū)動靶標循環(huán)及DNAzyme輔助,構(gòu)建了雙信號放大檢測核酸的生物傳感器。在熵驅(qū)動靶標循環(huán)過程中,靶標與TP發(fā)生立足點介導的鏈置換反應(Toe-hold),置換下PP鏈,形成中間體I3,燃料鏈Fs與I3發(fā)生第二個Toe-hold鏈置換反應,先置換下R鏈,形成中間體I5,隨后再置換下靶標,生成產(chǎn)物,并實現(xiàn)靶標循環(huán)。整個熵驅(qū)動反應是通過釋放分子達到熵增加,產(chǎn)生了熱力學推動。在循環(huán)Ⅱ過程中,由于循環(huán)I生成的產(chǎn)物包含有切割酶活性的Mg~(2+)-DNAzyme結(jié)構(gòu),能夠識別切割MB,產(chǎn)生熒光信號。該傳感器無酶輔助,成本低,背景信號低,具有良好的穩(wěn)定性和特異性。(3)基于靶標引發(fā)的鏈置換聚合反應(CNDP)及Pb~(2+)-DNAzyme循環(huán)切割,構(gòu)建了超靈敏檢測p53基因的生物傳感器。HP識別靶標DNA,促使HP的3’末端與PT雜交。從PT的3’端聚合延伸,替換下雜交的靶標DNA及PP鏈,并促進靶標與下一個HP雜交,實現(xiàn)靶標循環(huán)。在PT聚合產(chǎn)生的雙鏈區(qū)域包含nick酶的識別位點,nick酶切割聚合產(chǎn)物,產(chǎn)生新的DNAzyme功能序列,同時此段切割生成的DNA鏈能夠作為靶標類似物參與到靶標循環(huán)中。聚合酶和nick酶實現(xiàn)協(xié)同效應,聚合-切割-置換的過程反復進行,積累產(chǎn)生大量具有切割酶活性的Pb~(2+)-DNAzyme功能結(jié)構(gòu),切割MB,產(chǎn)生熒光信號。該傳感器自主且有效地進行信號放大,操作簡單,靈敏度較同類型傳統(tǒng)的傳感器高。
[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，

本文編號：1863438