基于核酸信号放大技术构建电致化学发光生物传感器的研究

发布时间：2018-01-31 02:55

本文关键词： 电致化学发光核酸信号放大技术生物传感器　出处：《西南大学》2017年硕士论文　论文类型：学位论文

【摘要】：核酸信号放大技术是利用核酸扩增、酶、脱氧核酶活性以及自组装等技术,将靶标分子转化为大量的核酸分子的输出,从而实现对目标分子检测的信号放大的核酸反应体系。核酸信号放大技术在生物传感器领域扮演着重要的角色,不但在核酸分析中受到广泛的应用,而且已经拓展到免疫传感器和适体传感器中。合理运用核酸信号放大策略不仅可以提高传感器检测的灵敏度,还可以简化传感器的操作、节省分析时间、甚至提高选择性。电致化学发光(ECL)生物传感器具有操作简单、分析快速、灵敏度高、准确度高、选择性好等优点,是一种极具潜力的理想分析工具。本文从ECL技术依赖电化学界面的特点出发,设计适用于ECL技术的核酸信号放大策略,用于构建操作便捷、成本低廉、响应灵敏的ECL生物传感器,实现对肿瘤标志物的超灵敏检测,以满足临床诊断的需求。主要包括以下几个方面的工作:1.基于phi29 DNA聚合酶调控的链置换扩增构建信号减小型ECL生物传感器检测microRNA的研究Phi29 DNA聚合酶是一种具有高保真连续链置换聚合活性的高性能DNA聚合酶,有可能用于调控链置换扩增(SDA)并克服传统的Klenow聚合酶不能用于长链DNA扩增且复制易出错的问题。本文研究了由phi29 DNA聚合酶调控的目标诱导循环SDA并将其应用于构建信号减小型ECL生物传感器检测microRNA。目标microRNA触发phi29 DNA聚合酶调控的SDA可以产生大量单链DNA的辅助探针,生成的辅助探针与传感界面修饰的捕获探针、标记有二茂铁的探针杂交形成三元“Y”型DNA结构,以此引入猝灭剂二茂铁,使得ECL信号减小,从而实现对目标microRNA的定量分析。由于引入目标物引发循环SDA的高效信号放大技术,该生物传感器检测miRNA-21的灵敏度得到显著的提高,其线性范围为10 amol·L-1到1.0pmol·L-1,检测限低至3.3 amol·L-1。2.基于目标物循环同步滚环扩增信号放大策略构建ECL生物传感器检测microRNA的研究滚环扩增(RCA)是一种高效的等温核酸扩增技术,也是生物传感器构建过程中常用的信号放大策略之一。通常,为提高传感器的灵敏度,在引入RCA之前往往会级联一个目标物循环的信号放大策略,然而,由于这种级联的信号放大策略是分步进行目标物循环与RCA,因此操作步骤繁琐且RCA效率相对较低。本工作设计了新颖的目标物循环同步RCA的信号放大策略并基于此构建了超灵敏和简单的ECL生物传感器用于microRNA的检测。值得一提的是,我们巧妙地设计了由富含鸟嘌呤(G-rich)的区域和与引物杂交的区域组成的环形模板,在目标物miR-21存在的情况下,环形模板的结合区域与引物、miR-21杂交,形成三元“P”结构,然后,从引物的3'端引发RCA。随着RCA的进行,目标物miR-21被释放并参与下一个RCA的引发。由于环形模板富含G碱基序列,因此目标物循环同步RCA的产物具有串联周期性富含胞嘧啶(C-rich)序列,以此作为配体进一步原位电化学生成银纳米簇(Ag NCs)作为ECL信号探针。目标物miR-21的浓度与Ag NCs的ECL强度呈正相关。该ECL分析法在miR-21浓度为100 amol·L-1至100 pmol·L-1的范围展现出优异的线性响应和低至22 amol·L-1的检测限。3.基于适体识别触发发夹组装无酶信号放大策略构建ECL适体传感器检测黏蛋白的研究核酸适配体识别目标物时往往伴随着核酸二级结构的转换,这种特性使核酸信号放大策略在适体传感器中同样能够发挥巨大的作用。酶辅助的目标物循环信号放大策略利用酶的聚合或剪切活性使目标物多次循环触发适体识别过程,从而实现信号放大的目的。然而引入生物酶增加了传感器的经济成本以及为满足酶活性的苛刻检测条件,这限制了酶在生物传感器中的应用范围。本工作基于适体识别引起适体DNA二级结构转换及DNA自组装技术触发的目标物循环的信号放大策略,结合多孔自增强Ru(II)聚合物空心纳米微球作为ECL信号标签,构建了ECL适体传感器检测黏蛋白MUC1。首先,设计了含MUC1适体序列的发夹型DNA识别探针,当MUC1识别其适体序列时,发夹型DNA识别探针打开与MUC1形成适体耦合物并裸露出茎的部分,该裸露出茎的部分能够引起另外两个发夹DNA的循环组装。该传感器对MUC1在1.0 fg·mL-1至100 pg·mL-1浓度范围内具有灵敏的响应,其检测限低至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.

【学位授予单位】：西南大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：TP212.3

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