本文選題：電化學(xué)傳感　切入點(diǎn)：動(dòng)態(tài)DNA自組裝　出處：《青島科技大學(xué)》2017年碩士論文　論文類型：學(xué)位論文

【摘要】：本論文主要基于動(dòng)態(tài)DNA自組裝調(diào)控的依靠接近效應(yīng)的表面雜交策略構(gòu)筑了三種電化學(xué)DNA生物傳感器,實(shí)現(xiàn)了對(duì)與疾病相關(guān)的靶標(biāo)DNA高靈敏、高選擇性分析檢測(cè)。1.基于DNA燃料靶標(biāo)循環(huán)機(jī)制調(diào)控的依靠接近效應(yīng)的表面雜交電化學(xué)方法,用于一步、無(wú)酶、放大靈敏檢測(cè)靶標(biāo)DNA。其中DNA燃料靶標(biāo)循環(huán)機(jī)制由兩次Toe-hold介導(dǎo)的等溫鏈置換反應(yīng)(TSDR)所驅(qū)動(dòng)。在該策略中,DNA燃料靶標(biāo)循環(huán)機(jī)制的動(dòng)態(tài)DNA自組裝過(guò)程中,通過(guò)均相的靶標(biāo)特異識(shí)別和放大轉(zhuǎn)化,形成DNA腳手架-TS+FS形成的兩鏈復(fù)合物,促進(jìn)其自發(fā)依靠接近效應(yīng)與電極表面SH-DNA進(jìn)行穩(wěn)定雜交。此時(shí),由于信號(hào)分子Fc靠近電極表面,更加有利于與電極進(jìn)行電子傳遞與轉(zhuǎn)移,產(chǎn)生電化學(xué)響應(yīng)。該方法針對(duì)靶標(biāo)DNA可實(shí)現(xiàn)0.29 fM檢測(cè)限。2.基于雜交鏈?zhǔn)椒磻?yīng)HCR調(diào)控的依靠接近效應(yīng)的表面雜交進(jìn)行DNA動(dòng)態(tài)自組裝策略,建立一個(gè)動(dòng)態(tài)的、無(wú)酶的、靈敏的電化學(xué)核酸檢測(cè)新方法。首先,設(shè)計(jì)發(fā)夾探針HP1、HP2,其中HP2-5’標(biāo)記亞甲藍(lán)MB的突出堿基片段,可與電極表面固定的SH-DNA互補(bǔ)雜交。當(dāng)加入靶標(biāo)時(shí),通過(guò)連續(xù)的鏈置換反應(yīng),引發(fā)HP1、HP2發(fā)夾進(jìn)行級(jí)聯(lián)自組裝,最終形成線性的DNA串聯(lián)體。由于在線性串聯(lián)體中,發(fā)夾HP2-5’末端突出的堿基序列相互靠近,增加其在電極表面的局部濃度,從而更加有力于其依靠接近效應(yīng),與SH-DNA進(jìn)行表面雜交。此時(shí),MB信號(hào)分子靠近電極表面,產(chǎn)生電化學(xué)響應(yīng)。該方法針對(duì)靶標(biāo)DNA可實(shí)現(xiàn)50 aM檢測(cè)限。3.基于靶標(biāo)引發(fā)的等溫鏈置換擴(kuò)增反應(yīng)SDA以及接近效應(yīng)的表面雜交反應(yīng)策略,調(diào)控DNA動(dòng)態(tài)自組裝,實(shí)現(xiàn)對(duì)靶標(biāo)DNA的超靈敏檢測(cè)分析。引入酶輔助的等溫鏈置換反應(yīng)SDA,不僅可以實(shí)現(xiàn)靶標(biāo)循環(huán)信號(hào)放大;而且通過(guò)等溫鏈置換反應(yīng)SDA產(chǎn)生大量自由單鏈DNA可與信號(hào)探針SP互補(bǔ)雜交,使信號(hào)探針SP末端突出的堿基序列相互靠近,促進(jìn)其與SH-DNA依靠接近效應(yīng)發(fā)生穩(wěn)定的表面雜交,使MB信號(hào)分子靠近電極表面,產(chǎn)生信號(hào)響應(yīng),以此巧妙實(shí)現(xiàn)靶標(biāo)DNA的檢測(cè)。
[Abstract]:In this paper, three electrochemical DNA biosensors are constructed based on dynamic DNA self-assembly based surface hybridization strategy based on proximity effect, which is highly sensitive to disease-related target DNA. Highly selective analytical detection. 1. Surface hybridization electrochemical method based on proximity effect based on DNA fuel target cycle mechanism for one-step, non-enzymatic, The mechanism of DNA fuel target cycle is driven by two isothermal chain replacement reactions mediated by Toe-hold. In this strategy, the dynamic DNA self-assembly of the DNA fuel target cycle mechanism is carried out. Through the homogeneous target specific recognition and amplification transformation, a two-chain complex formed by DNA scaffolding TSFS was formed, which promoted its spontaneous stable hybridization with SH-DNA on the electrode surface by proximity effect. In this case, the signal molecule FC was close to the electrode surface, and the signal molecule FC was close to the electrode surface. This method can realize 0.29fM detection limit for target DNA. (2) dynamic self-assembly strategy of DNA based on surface hybridization based on proximity effect based on HCR regulation of hybrid chain reaction. A dynamic, non-enzymatic and sensitive electrochemical method for the detection of nucleic acid was established. Firstly, a hairpin probe HP1OHP2was designed, in which HP2-5 'labeled methylmethylene blue MB protruding base fragment could be hybridized with SH-DNA fixed on the electrode surface. When the target was added, By continuous chain replacement reaction, the HP1O HP2 hairpin is self-assembled in a cascade, and finally a linear DNA series is formed. Because the base sequence protruding at the HP2-5 'end of the hairpin is close to each other in the linear series, the local concentration of HP2-5' hairpin on the electrode surface is increased. Therefore, it is more powerful to hybridize with SH-DNA surface by proximity effect. At this time, the signal molecule of MB is close to the electrode surface, Electrochemical response was generated. The detection limit of 50 mm for target DNA could be achieved by this method. Based on the isothermal chain replacement amplification reaction SDA initiated by target and the surface hybridization strategy of proximity effect, the dynamic self-assembly of DNA was regulated. The enzyme assisted isothermal chain replacement reaction (SDAA) can not only amplify the cyclic signal of the target, but also produce a large number of free single-stranded DNA by isothermal chain replacement reaction, which can be hybridized with the signal probe SP. The protruding base sequence at the SP end of the signal probe is close to each other, which promotes the stable surface hybridization between the signal probe and the SH-DNA by means of proximity effect, and makes the MB signal molecule close to the electrode surface to produce signal response, so that the detection of target DNA can be accomplished cleverly.
【學(xué)位授予單位】：青島科技大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：O657.1

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本文編號(hào)：1635780