本文關(guān)鍵詞： 電致化學(xué)發(fā)光 核酸信號(hào)放大技術(shù) 生物傳感器　出處：《西南大學(xué)》2017年碩士論文　論文類(lèi)型：學(xué)位論文

【摘要】：核酸信號(hào)放大技術(shù)是利用核酸擴(kuò)增、酶、脫氧核酶活性以及自組裝等技術(shù),將靶標(biāo)分子轉(zhuǎn)化為大量的核酸分子的輸出,從而實(shí)現(xiàn)對(duì)目標(biāo)分子檢測(cè)的信號(hào)放大的核酸反應(yīng)體系。核酸信號(hào)放大技術(shù)在生物傳感器領(lǐng)域扮演著重要的角色,不但在核酸分析中受到廣泛的應(yīng)用,而且已經(jīng)拓展到免疫傳感器和適體傳感器中。合理運(yùn)用核酸信號(hào)放大策略不僅可以提高傳感器檢測(cè)的靈敏度,還可以簡(jiǎn)化傳感器的操作、節(jié)省分析時(shí)間、甚至提高選擇性。電致化學(xué)發(fā)光(ECL)生物傳感器具有操作簡(jiǎn)單、分析快速、靈敏度高、準(zhǔn)確度高、選擇性好等優(yōu)點(diǎn),是一種極具潛力的理想分析工具。本文從ECL技術(shù)依賴(lài)電化學(xué)界面的特點(diǎn)出發(fā),設(shè)計(jì)適用于ECL技術(shù)的核酸信號(hào)放大策略,用于構(gòu)建操作便捷、成本低廉、響應(yīng)靈敏的ECL生物傳感器,實(shí)現(xiàn)對(duì)腫瘤標(biāo)志物的超靈敏檢測(cè),以滿(mǎn)足臨床診斷的需求。主要包括以下幾個(gè)方面的工作:1.基于phi29 DNA聚合酶調(diào)控的鏈置換擴(kuò)增構(gòu)建信號(hào)減小型ECL生物傳感器檢測(cè)microRNA的研究Phi29 DNA聚合酶是一種具有高保真連續(xù)鏈置換聚合活性的高性能DNA聚合酶,有可能用于調(diào)控鏈置換擴(kuò)增(SDA)并克服傳統(tǒng)的Klenow聚合酶不能用于長(zhǎng)鏈DNA擴(kuò)增且復(fù)制易出錯(cuò)的問(wèn)題。本文研究了由phi29 DNA聚合酶調(diào)控的目標(biāo)誘導(dǎo)循環(huán)SDA并將其應(yīng)用于構(gòu)建信號(hào)減小型ECL生物傳感器檢測(cè)microRNA。目標(biāo)microRNA觸發(fā)phi29 DNA聚合酶調(diào)控的SDA可以產(chǎn)生大量單鏈DNA的輔助探針,生成的輔助探針與傳感界面修飾的捕獲探針、標(biāo)記有二茂鐵的探針雜交形成三元“Y”型DNA結(jié)構(gòu),以此引入猝滅劑二茂鐵,使得ECL信號(hào)減小,從而實(shí)現(xiàn)對(duì)目標(biāo)microRNA的定量分析。由于引入目標(biāo)物引發(fā)循環(huán)SDA的高效信號(hào)放大技術(shù),該生物傳感器檢測(cè)miRNA-21的靈敏度得到顯著的提高,其線性范圍為10 amol·L-1到1.0pmol·L-1,檢測(cè)限低至3.3 amol·L-1。2.基于目標(biāo)物循環(huán)同步滾環(huán)擴(kuò)增信號(hào)放大策略構(gòu)建ECL生物傳感器檢測(cè)microRNA的研究滾環(huán)擴(kuò)增(RCA)是一種高效的等溫核酸擴(kuò)增技術(shù),也是生物傳感器構(gòu)建過(guò)程中常用的信號(hào)放大策略之一。通常,為提高傳感器的靈敏度,在引入RCA之前往往會(huì)級(jí)聯(lián)一個(gè)目標(biāo)物循環(huán)的信號(hào)放大策略,然而,由于這種級(jí)聯(lián)的信號(hào)放大策略是分步進(jìn)行目標(biāo)物循環(huán)與RCA,因此操作步驟繁瑣且RCA效率相對(duì)較低。本工作設(shè)計(jì)了新穎的目標(biāo)物循環(huán)同步RCA的信號(hào)放大策略并基于此構(gòu)建了超靈敏和簡(jiǎn)單的ECL生物傳感器用于microRNA的檢測(cè)。值得一提的是,我們巧妙地設(shè)計(jì)了由富含鳥(niǎo)嘌呤(G-rich)的區(qū)域和與引物雜交的區(qū)域組成的環(huán)形模板,在目標(biāo)物miR-21存在的情況下,環(huán)形模板的結(jié)合區(qū)域與引物、miR-21雜交,形成三元“P”結(jié)構(gòu),然后,從引物的3'端引發(fā)RCA。隨著RCA的進(jìn)行,目標(biāo)物miR-21被釋放并參與下一個(gè)RCA的引發(fā)。由于環(huán)形模板富含G堿基序列,因此目標(biāo)物循環(huán)同步RCA的產(chǎn)物具有串聯(lián)周期性富含胞嘧啶(C-rich)序列,以此作為配體進(jìn)一步原位電化學(xué)生成銀納米簇(Ag NCs)作為ECL信號(hào)探針。目標(biāo)物miR-21的濃度與Ag NCs的ECL強(qiáng)度呈正相關(guān)。該ECL分析法在miR-21濃度為100 amol·L-1至100 pmol·L-1的范圍展現(xiàn)出優(yōu)異的線性響應(yīng)和低至22 amol·L-1的檢測(cè)限。3.基于適體識(shí)別觸發(fā)發(fā)夾組裝無(wú)酶信號(hào)放大策略構(gòu)建ECL適體傳感器檢測(cè)黏蛋白的研究核酸適配體識(shí)別目標(biāo)物時(shí)往往伴隨著核酸二級(jí)結(jié)構(gòu)的轉(zhuǎn)換,這種特性使核酸信號(hào)放大策略在適體傳感器中同樣能夠發(fā)揮巨大的作用。酶輔助的目標(biāo)物循環(huán)信號(hào)放大策略利用酶的聚合或剪切活性使目標(biāo)物多次循環(huán)觸發(fā)適體識(shí)別過(guò)程,從而實(shí)現(xiàn)信號(hào)放大的目的。然而引入生物酶增加了傳感器的經(jīng)濟(jì)成本以及為滿(mǎn)足酶活性的苛刻檢測(cè)條件,這限制了酶在生物傳感器中的應(yīng)用范圍。本工作基于適體識(shí)別引起適體DNA二級(jí)結(jié)構(gòu)轉(zhuǎn)換及DNA自組裝技術(shù)觸發(fā)的目標(biāo)物循環(huán)的信號(hào)放大策略,結(jié)合多孔自增強(qiáng)Ru(II)聚合物空心納米微球作為ECL信號(hào)標(biāo)簽,構(gòu)建了ECL適體傳感器檢測(cè)黏蛋白MUC1。首先,設(shè)計(jì)了含MUC1適體序列的發(fā)夾型DNA識(shí)別探針,當(dāng)MUC1識(shí)別其適體序列時(shí),發(fā)夾型DNA識(shí)別探針打開(kāi)與MUC1形成適體耦合物并裸露出莖的部分,該裸露出莖的部分能夠引起另外兩個(gè)發(fā)夾DNA的循環(huán)組裝。該傳感器對(duì)MUC1在1.0 fg·mL-1至100 pg·mL-1濃度范圍內(nèi)具有靈敏的響應(yīng),其檢測(cè)限低至0.31 fg·mL-1。
[Abstract]:Nucleic acid amplification technology is the use of nucleic acid amplification, enzyme activity, DNAzyme and self-assembly technology, the target molecule to output a large number of nucleic acid molecules, nucleic acid reaction system so as to realize the signal of the target molecule detection. The nucleic acid amplification signal amplification technology plays an important role in the field of biosensors, not only widely the application in nucleic acid analysis, and has been extended to the immune sensor and the aptamer sensor. The rational use of nucleic acid amplification strategies can not only improve the sensitivity of the sensor, the sensor can also simplify operation, save the time of analysis, and improve the selectivity. Electrochemiluminescence (ECL) biosensor has the advantages of simple operation, rapid analysis, sensitivity high, high accuracy, good selectivity, is a potential ideal analysis tool. This article from the ECL technology on electrochemical industry The characteristics of the signal design for ECL nucleic acid amplification technology strategy for the construction of convenient operation, low cost, in response to the ECL sensitive bio sensors, the tumor markers of ultra sensitive detection to meet the needs of clinical diagnosis. The main work is as follows: 1. phi29 DNA polymerase chain replacement based on the control of the construction of small signal amplification by ECL biosensor for the detection of microRNA of Phi29 DNA polymerase is a high fidelity continuous strand displacement polymerization activity of the high performance DNA polymerase, may be used to control the strand displacement amplification (SDA) and overcome the disadvantages of traditional Klenow polymerase can not be used for long chain DNA amplification and replication error prone problems were systematically studied in this paper. Controlled by phi29 DNA polymerase induced cyclic SDA target and its application in the construction of small signal reduction ECL biosensor for the detection of microRNA. target microRNA touch The auxiliary probe phi29 DNA polymerase regulation SDA can produce a large number of single stranded DNA, generated by the auxiliary probe and modified sensor interface capture probe labeled with two probe hybridization of diferrocenyl formed three yuan of "Y" type DNA structure, as the introduction of quenching agent two ferrocene, the ECL signal is reduced, so as to realize the quantitative analysis of the target of microRNA. Due to the introduction of the target caused by cyclic SDA efficient signal amplification technology, the sensitivity of the biosensor for the detection of miRNA-21 was significantly improved, the linear range was 10 amol - L-1 to 1.0pmol - L-1, the detection limit is low to 3.3 amol - L-1.2. of amplified signal amplification strategy ECL biosensors for the detection of microRNA. Synchronous rolling circle amplification target based on cycle (RCA) is a kind of efficient isothermal nucleic acid amplification technology, and biosensors commonly used signal amplification strategy of process. Usually, for To improve the sensitivity of the sensor, prior to the introduction of the RCA are often a target loop cascade signal amplification strategy, however, because the signal of this cascade amplification strategy step by step target cycle with RCA, so the operation is complicated and the RCA efficiency is relatively low. The signal we design the target novel circular synchronous RCA amplification strategy and build on the ECL biosensor ultra sensitive and simple method for the detection of microRNA. It is worth mentioning that we cleverly designed by guanine rich (G-rich) region and the region of circular template and primer hybridization which, in the presence of target miR-21, and primer binding region miR-21, circular template hybridization, form the three element "P" structure, then raises the RCA. with RCA from the 3'end of the primer, causing the target miR-21 to be released and participate in the next RCA. Because of the ring Form G rich sequence, so the target product cycle synchronous RCA with series of periodic sequences, rich in cytosine (C-rich) as ligands in situ electrochemical further into silver nanoclusters (Ag NCs ECL) as a probe. The intensity of ECL was positively with the concentration of Ag NCs miR-21 of objects related to the ECL analysis. In the miR-21 concentration range of 100 amol to 100 pmol - L-1 - L-1 shows excellent linear response and low detection limit to 22 amol - L-1.3. aptamer recognition triggers hairpin assembly without enzyme signal amplification strategy research on the construction of ECL nucleic acid aptamer sensor detecting mucin aptamer target recognition is often accompanied by conversion the nucleic acid of two level structure based on the characteristics of the signal amplification strategy in nucleic acid aptamer sensor can also play a great role. The target signal amplification cycle enzyme assisted enzymatic polymerization or use strategy Shear activity make an object repeatedly triggered by aptameric recognition process, so as to realize signal amplification purposes. However the introduction of bio enzyme increased the economic cost of sensors and to meet the harsh conditions of enzyme activity detection, which limits the scope of application of enzyme in biosensor. The signal aptamer recognition by aptamer DNA two structural transfer and DNA based self-assembly technology trigger target cycle amplification strategy, combined with the porous self reinforced Ru (II) polymeric nano hollow spheres as ECL signal label, constructed the ECL aptamer sensor for the detection of MUC1. protein firstly designed hairpin DNA recognition probe containing MUC1 aptamer sequences, when MUC1 identification aptamer sequence, hairpin DNA recognition probe opens to form aptamer conjugate and exposed stem part and MUC1, the exposed portion of the stem can cause cycle another two hairpin DNA installed. The sensor has a sensitive response to MUC1 in the range of 1 FG. ML-1 to 100 pg. ML-1, and its detection limit is as low as 0.31 FG. ML-1.

【學(xué)位授予單位】：西南大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類(lèi)號(hào)】：TP212.3

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