本文選題：電化學(xué)生物傳感器　切入點：Toehold　出處：《西南大學(xué)》2017年碩士論文　論文類型：學(xué)位論文

【摘要】：隨著基因診斷與治療、藥物篩選與作用機理探究、環(huán)境污染與食品安全監(jiān)測等諸多研究領(lǐng)域蓬勃迅速的發(fā)展,實現(xiàn)對病原蛋白和特定序列microRNA(miRNA)的方便、耗時少、高靈敏且高特異性分析檢測的意義是非常重要的。電化學(xué)DNA生物傳感器(DNA electrochemical biosensor)因為其自身構(gòu)造簡單、易實現(xiàn)便攜性、成本低廉、高靈敏且特異性強等優(yōu)點而備受關(guān)注。目前,對于研究者們來說,開發(fā)電化學(xué)DNA生物傳感器在生物和醫(yī)學(xué)科學(xué)領(lǐng)域已成為具有吸引力和極具挑戰(zhàn)的前沿性課題。粘性末端(Toehold)介導(dǎo)的鏈置換反應(yīng)(Toehold-mediated strand displacement reaction,TSDR)是構(gòu)建動態(tài)DNA納米機器的基礎(chǔ)反應(yīng)。本文結(jié)合TSDR輔助目標物循環(huán)放大技術(shù)構(gòu)建了三種電化學(xué)DNA生物傳感器,實現(xiàn)了對mi RNA和凝血酶的超靈敏檢測,具體研究工作如下:1.基于DNA分子機器實現(xiàn)無酶目標物循環(huán)放大和最小化背景噪聲的電化學(xué)檢測癌細胞中的MicroRNAMicroRNA(miRNA)表達水平的變化是可以作為診斷不同癌癥的有用生物標記物。在本項工作中,基于miRNA引發(fā)無酶目標物循環(huán)信號放大分子機器,我們開發(fā)了一個簡單新穎的電化學(xué)DNA生物傳感器實現(xiàn)高靈敏地檢測人乳腺癌細胞中的miRNA-21。相應(yīng)傳感器的制備是由3條單鏈DNA組成的雙鏈探針通過自組裝在金電極表面上,目標物miRNA-21結(jié)合到雙鏈探針的末端第一個toehold區(qū)域,將其中一條短DNA鏈置換下來,并暴露出第二個toehold區(qū)域用于隨后與亞甲藍(MB)修飾的燃料MB-DNA鏈進行雜交反應(yīng),MB-DNA鏈進一步置換出miRNA-21和另一短鏈DNA以激活分子機器的操作。結(jié)果,目標miRNA-21循環(huán)重復(fù)使用,導(dǎo)致許多MB-DNA燃料鏈連接到傳感器表面產(chǎn)生明顯放大的電流響應(yīng),實現(xiàn)對miRNA-21的高靈敏檢測,檢測限低至1.4 fmol/L。我們開發(fā)的傳感器對目標物表現(xiàn)出很強的序列特異性,同時可用于在癌細胞樣品中檢測miRNA-21。此外,不僅該傳感器的構(gòu)建有使用目標物循環(huán)放大技術(shù)避免了任何酶參與的優(yōu)點,而且還有高度最小化減少背景噪聲的優(yōu)點。使得該方法可以方便地監(jiān)測不同miRNA生物標志物,具有對各種癌癥的早期診斷中有巨大潛力。2.基于級聯(lián)鏈置換無酶介導(dǎo)目標物循環(huán)放大和免標記電化學(xué)檢測腫瘤細胞的MicroRNA痕量檢測miRNA在腫瘤細胞中的表達水平對于癌癥診斷具有非常重要意義。本項工作中,基于兩個級聯(lián)toehold介導(dǎo)的鏈置換反應(yīng)(TSDRs),我們設(shè)計了一個免標記和無酶輔助目標物循環(huán)放大的電化學(xué)方法檢測人乳腺癌細胞中的miRNA-21。含有被鎖定的G-四鏈體序列的“Y”型DNA探針通過自組裝在傳感器表面上。當(dāng)目標物miRNA-21存在的時候,引發(fā)第一個TSDR使“Y”型DNA探針解體的同時釋放活性G-四鏈體序列。隨后,DNA燃料鏈觸發(fā)第二個TSDR,使得miRNA-21循環(huán)再利用。通過級聯(lián)TSDR,傳感器表面上產(chǎn)生許多活性G-四鏈體序列,其與hemin結(jié)合以產(chǎn)生顯著放大的電流響應(yīng),實現(xiàn)靈敏檢測miRNA-21低至1.15 fmol/L。該傳感器表現(xiàn)出具有很強的選擇性,并且可以用于檢測來自人乳腺癌細胞樣品中的miRNA-21。3.基于結(jié)合目標物催化發(fā)夾自組裝(CHA)和TdT催化DNA聚合雙信號放大策略電化學(xué)檢測人血清中的凝血酶對蛋白質(zhì)生物標志物的特異和靈敏性檢測在生物醫(yī)學(xué)和生物分析應(yīng)用中具有非常重要的意義。該項工作中,實現(xiàn)了基于集成催化發(fā)夾自組裝(CHA)和末端脫氧核苷酸轉(zhuǎn)移酶(TdT)原位DNA聚合的雙放大信號放大技術(shù),高靈敏和免標記的電化學(xué)方法檢測人體血清中的凝血酶。當(dāng)目標物凝血酶的存在情況下,傳感器電極上捕獲的具有游離3'-OH末端的發(fā)夾DNA信號探針。隨后,在dGTP與dATP摩爾比為6:4的情況下,TdT可以催化信號探針的游離3'-OH末端延伸形成許多重復(fù)的G-四鏈體序列,G-四鏈體序列結(jié)合hemin并產(chǎn)生顯著放大的電流響應(yīng),實現(xiàn)超靈敏并且完全無標記的方式檢測凝血酶,靈敏度達到0.12 pmol/L。此外,我們開發(fā)的傳感器對凝血酶與其他干擾蛋白表現(xiàn)出具有良好的選擇性,并能在人血清樣品中檢測凝血酶。
[Abstract]:Along with the development of gene diagnosis and treatment research, drug screening and mechanism, the development of many research fields of environmental pollution and food safety monitoring is rapid, the realization of microRNA on pathogenic protein and specific sequence (miRNA) is convenient, less time-consuming, high sensitivity and high specificity of detection and analysis of the significance is very important. The electrochemical DNA biosensor (DNA electrochemical biosensor) because of its simple structure, easy portability, low cost, high sensitivity and high specificity is attracting more and more attention. At present, for researchers, the development of electrochemical DNA biosensors have become a frontier topic attractive and challenging in biological and medical sciences. Sticky ends (Toehold) chain replacement reaction mediated (Toehold-mediated strand displacement reaction, TSDR) is the basic reaction to construct the dynamic DNA nano machine. This paper combined with T SDR auxiliary target cycle amplification technology and constructs three kinds of electrochemical DNA biosensor, the ultra sensitive detection of MI RNA and thrombin, the main research work is as follows: 1. DNA molecular machine to achieve the target of non enzymatic electrochemical detection cycle amplification and minimization of background noise measurement in cancer cells based on MicroRNAMicroRNA (miRNA) expression level change can be used as a useful biomarker for the diagnosis of different cancers. In this work, based on the miRNA lead to non target enzyme signal amplification cycle molecular machine, we developed a simple and novel electrochemical DNA biosensor has high sensitive detection of miRNA-21. sensor in human breast cancer cells are prepared by double chain the probe consists of 3 single stranded DNA by self-assembly on gold electrode surface, the target miRNA-21 binding to the end of the first double chain probe toehold region, the In a short DNA chain replacement down, and expose the second toehold region for subsequent and methylene blue (MB) modified MB-DNA hybrid fuel chain reaction, MB-DNA chain further replacement of miRNA-21 and another short chain DNA to activate the molecular machine operation. As a result, the target miRNA-21 cycle of repeated use, resulting in many MB-DNA fuel chain connected to the sensor surface significantly enlarge the current response, high sensitive detection of miRNA-21 sensor, low detection limit to 1.4 fmol/L. we developed on the target sequence showed strong specificity, but also can be used in cancer cell samples for detection of miRNA-21. in addition, the sensor is used not only to build target circulating amplification technique avoids any enzymes involved in the advantages, but also highly minimize background noise. The method can conveniently monitor different miRNA biomarkers ,鍏鋒湁瀵瑰悇縐嶇檶鐥囩殑鏃╂湡璇婃柇涓湁宸ㄥぇ娼滃姏.2.鍩轟簬綰ц仈閾劇疆鎹㈡棤閰朵粙瀵肩洰鏍囩墿寰幆鏀懼ぇ鍜屽厤鏍囪鐢?shù)鍖栧妫�嫻嬭偪鐦ょ粏鑳?yōu)鐨凪icroRNA鐥曢噺媯，

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