基于级联信号放大策略的高灵敏电化学DNA生物传感检测研究

发布时间：2018-06-20 08:36

本文选题：电化学信号放大 + DNA生物传感器　；参考：《青岛科技大学》2016年硕士论文

【摘要】：本论文主要基于级联信号放大策略构筑了三种电化学DNA生物传感器,提出了基于靶标循环和后续扩增组合信号放大的有效构筑机制,对疾病相关的DNA、小分子等实现了高灵敏、高选择性分析检测。主要内容包括:1.基于聚合酶、切口酶等温扩增技术和酶/金纳米颗粒后续放大策略,构筑了一种高灵敏的DNA生物传感器。本论文设计了一个发夹DNA探针(HP),其含有与靶标DNA互补的3'端突出序列,切口酶的识别位点和用来终止聚合反应的烷烃间隔三部分。当靶标DNA存在时,在聚合酶和切口酶作用下,引发“聚合-切割-链置换”反应,打开HP;DNA标记的金纳米颗粒可与打开的HP杂交,金纳米上的DNA探针标记了生物素,其进一步与标记碱性磷酸酶(ALP)的亲和素亲和连接,进一步ALP催化1-萘基磷酸盐(1-NP)的生成1-萘酚,实现对靶标DNA双重信号放大的检测,检测限可低至0.065fM。2.基于Pb~(2+)-DNAzyme诱导切割,末端转移酶(TdTase)辅助的无模板聚合信号放大策略,发展了一种简单灵敏的电化学DNAzyme传感器,用于特异性检测Pb~(2+)。首先,将8-17 DNAzyme的发夹状底链(HP DNA)固定在电极上,它与靶标Pb~(2+)识别后催化切割底链,并露出3'-OH端。末端转移酶(TdTase)催化dUTP-biotin有序地添加到底链3'-OH端上,进而实现无模板扩增。利用生物素(biotin)与亲和素的亲和作用,将亲和素-碱性磷酸酶(SA-ALP)连接到电极上,进一步ALP催化1-萘基磷酸盐(1-NP)的生成1-萘酚,产生电化学信号,实现对靶标Pb~(2+)的高灵敏检测,检测限可达到0.043 nM。3.通过结合熵驱动靶标循环和DNA杂交链式反应组合放大策略,构筑了一种等温、无酶、免标记的高灵敏电化学DNA生物传感器,用于检测目标DNA。首先,在金电极表面自组装上DNA双链探针,该探针的末端突出区域作为toehold位点,其与靶标DNA(TD)特异性识别杂交,触发链置换反应,将辅助DNA探针(AP)置换下来并露出第二个toehold位点。这个toehold位点随后与燃料DNA链(FS)识别杂交,进而FS取代了TD和保护链(PP),靶标DNA循环利用。FS的突出序列进一步引发两个茎环DNA(HP-1、HP-2)交替杂交,形成周期性长链DNA的聚合体,作为信号载体;利用六氨基合钌配合物(RuHex)作为电化学指示剂,可以实现对靶标DNA的高灵敏检测。
[Abstract]:In this paper, three kinds of electrochemical DNA biosensors are constructed based on cascade signal amplification strategy, and an effective construction mechanism based on target cycle and subsequent amplification combined signal amplification is proposed, which is highly sensitive to disease-related DNA, small molecules and so on. High selectivity analysis and detection. The main contents include: 1. A highly sensitive DNA biosensor was constructed based on polymerase, notch enzyme isothermal amplification and subsequent amplification strategy of enzyme / gold nanoparticles. In this paper, a hairpin DNA probe (HPN) was designed, which contains 3 'protrusion sequence complementary to target DNA, recognition site of incision enzyme and alkane spacer used to terminate polymerization. In the presence of target DNA, "polymerization-cleaving-chain displacement" reaction was initiated under the action of polymerase and incision enzyme. The gold nanoparticles labeled with HPN DNA could be hybridized with open HP, and DNA probes on gold nanoparticles labeled biotin. It was further linked with the affinity of ALP, and then the 1-naphthol was synthesized by ALP. The detection limit of double signal amplification of target DNA could be as low as 0.065 fM.2. A simple and sensitive electrochemical DNAzyme sensor was developed based on the signal amplification strategy of template free polymeric signal assisted by PBN 2 + DNAzyme induced cleavage and terminal transferase (TdTase). Firstly, the 8-17 DNA zyme was fixed on the electrode, which was recognized with the target PbC-2), and then the base chain was dissected and the 3H-OH end was exposed. The HPS-DNA of 8-17 DNAzyme was immobilized on the electrode, and it was recognized with the target PbC-2). Terminal transferase (TdTase) catalyzes the ordered addition of dUTP-biotin onto the end of the 3- OH chain, which leads to the template free amplification of dUTP-biotin. By using the affinity of biotin) and avidin, SA-ALP) was connected to the electrode to further catalyze the formation of 1-naphthol from 1-naphthophosphate (1-NPP) by ALP, and to produce electrochemical signal. The detection limit can reach 0.043 nM.3. A highly sensitive electrochemical DNA biosensor was constructed by combining entropy driven target cycle and DNA hybridization chain reaction to detect target DNAs. First, a double-stranded probe was self-assembled on the surface of the gold electrode. The protruding region of the probe was used as the toehold site, which was specifically hybridized with the target DNA-TDs, triggered the strand replacement reaction, and replaced the auxiliary toehold probe with the second toehold site. The toehold site was then hybridized with the fuel DNA strand (FSs), and FS replaced the TD and the protective chain. The protruding sequence of the target DNA cycle using .FS further led to the alternant hybridization of the two stem rings, HP-1 and HP-2), forming a periodic long strand DNA polymer. As a signal carrier, RuHexis as an electrochemical indicator can be used to detect target DNA with high sensitivity.
【学位授予单位】：青岛科技大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：O657.1

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