本文選題：DNA熒光生物傳感器 + DNAzyme��； 參考：《青島科技大學(xué)》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(biāo)循環(huán)以及等溫鏈置換擴(kuò)增反應(yīng)等信號(hào)放大策略,構(gòu)筑了三種DNA熒光生物傳感器,實(shí)現(xiàn)對(duì)核酸的高靈敏分析檢測(cè)。主要內(nèi)容包括:(1)基于靶標(biāo)自循環(huán)和級(jí)聯(lián)循環(huán)指數(shù)擴(kuò)增策略(TR-CEA),構(gòu)建了一個(gè)超靈敏、一步檢測(cè)靶標(biāo)DNA的生物傳感器。整個(gè)傳感體系由兩個(gè)巧妙設(shè)計(jì)的發(fā)夾DNA鏈和兩種酶構(gòu)成,熒光探針(MB),包含一個(gè)切刻內(nèi)切酶的識(shí)別片段,3’突出片段與莖環(huán)(HP)互補(bǔ)雜交,聚合酶聚合,釋放出靶標(biāo)DNA,引起靶標(biāo)循環(huán),置換下來(lái)的靶標(biāo)繼續(xù)參與到下一輪雜交過(guò)程。同時(shí),雙鏈DNA引發(fā)酶切割并沿切割處進(jìn)行反向聚合,實(shí)現(xiàn)鏈置換擴(kuò)增(SDA)過(guò)程。隨后,MB的莖環(huán)結(jié)構(gòu)打開(kāi),露出切刻內(nèi)切酶的識(shí)別片段,從而發(fā)生切割和反向的SDA過(guò)程,被切割的MB產(chǎn)生熒光信號(hào)。對(duì)于靶標(biāo)DNA的檢測(cè)限估算達(dá)到0.61 fM,具有高靈敏度、重復(fù)性好的優(yōu)點(diǎn)。(2)基于熵驅(qū)動(dòng)靶標(biāo)循環(huán)及DNAzyme輔助,構(gòu)建了雙信號(hào)放大檢測(cè)核酸的生物傳感器。在熵驅(qū)動(dòng)靶標(biāo)循環(huán)過(guò)程中,靶標(biāo)與TP發(fā)生立足點(diǎn)介導(dǎo)的鏈置換反應(yīng)(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(gè)Toe-hold鏈置換反應(yīng),先置換下R鏈,形成中間體I5,隨后再置換下靶標(biāo),生成產(chǎn)物,并實(shí)現(xiàn)靶標(biāo)循環(huán)。整個(gè)熵驅(qū)動(dòng)反應(yīng)是通過(guò)釋放分子達(dá)到熵增加,產(chǎn)生了熱力學(xué)推動(dòng)。在循環(huán)Ⅱ過(guò)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(shí)別切割MB,產(chǎn)生熒光信號(hào)。該傳感器無(wú)酶輔助,成本低,背景信號(hào)低,具有良好的穩(wěn)定性和特異性。(3)基于靶標(biāo)引發(fā)的鏈置換聚合反應(yīng)(CNDP)及Pb~(2+)-DNAzyme循環(huán)切割,構(gòu)建了超靈敏檢測(cè)p53基因的生物傳感器。HP識(shí)別靶標(biāo)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(biāo)DNA及PP鏈,并促進(jìn)靶標(biāo)與下一個(gè)HP雜交,實(shí)現(xiàn)靶標(biāo)循環(huán)。在PT聚合產(chǎn)生的雙鏈區(qū)域包含nick酶的識(shí)別位點(diǎn),nick酶切割聚合產(chǎn)物,產(chǎn)生新的DNAzyme功能序列,同時(shí)此段切割生成的DNA鏈能夠作為靶標(biāo)類(lèi)似物參與到靶標(biāo)循環(huán)中。聚合酶和nick酶實(shí)現(xiàn)協(xié)同效應(yīng),聚合-切割-置換的過(guò)程反復(fù)進(jìn)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(hào)。該傳感器自主且有效地進(jìn)行信號(hào)放大,操作簡(jiǎn)單,靈敏度較同類(lèi)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(hào)：1863438