摘要本文设计了一种结合了基于目标诱导构型变化的茎环结构探针和表面诱导延伸放大（SIEP）的新型电化学传感器策略。这种策略中使用了一种3'端标记巯基的茎环结构捕获探针，捕获探针通过硫金键固定到金电极的表面。巯基标记的寡聚乙二醇分子(SH-OEGs)可用于封闭电极上的空白位点，得到自清洁表面（ONS）。在传感器的初始状态，捕获探针的茎环结构阻止信号探针结合到捕获探针的5'端。与目标DNA进行杂交之后，目标DNA和信号探针杂交到捕获探针两侧，构成了一个“三明治”夹心结构。然后，末端脱氧核糖核酸转移酶（TdT）在信号探针的3'-OH端添加脱氧核糖核酸，这种DNA的延伸不需要模板参与。在TdT诱导的延伸反应中加入生物素标记的2'脱氧腺苷5'三磷酸，生物素标签就被加入到延伸出来的长DNA单链中。亲和素标记的辣根过氧化物酶通过亲和作用连接到生物素标签上，酶充当着信号转换和信号放大的角色。通过使用这种新型的策略，这种电化学传感器能够检测pM级的目标DNA，并且能够分辨出单个碱基的错配。64752

毕业论文关键词  构型变化；末端延伸放大；茎环结构；电化学；基因传感器

毕业设计说明书（论文）外文摘要

Title    Ultrasensitive Electrochemical DNA Sensor Based on the Target Induced Structural Switching and Surface-Initiated Enzymatic Polymerization.                       

Abstract

We herein report a novel strategy of electrochemical DNA (E-DNA) sensor based on the target induced structural switching of stem-loop probe and surface initiated enzymatic polymerization (SIEP). This DNA sensor employs a capture probe DNA with a stem-loop structure which is labeled with thiol at its 3' terminal to be immobilized at gold electrode via gold-thiol chemistry. Oligo (ethylene glycol) -terminated thiols (SH-OEGs) are then used to prepare an oligonucleotide-incorporated nonfouling surface (ONS). In the initial state of the sensor, the probe adopts the stem-loop structure, which shields 5' terminal of capture probe from being approached by the signal probe DNA. After hybridization, both the target and the signal probe flanked the capture probe forming a “sandwich” structure. After that, terminal deoxynucleotidyl transferase (TdT) is employed to catalyze the sequential addition of deoxynucleotides (dNTPs) at the 3'-OH group of signal probe DNA without template. During the TdT-mediated extension reaction, by using biotinlated 2'-Deoxyadenosine 5'-triphosphate (biotin-dATP), biotin labels are incorporated into the SIEP-generated long single-stranded DNA (ssDNA). Specific binding of avidin-horseradish peroxidase (Av-HRP) to the biotin label leads to enzyme turnover-based signal transduction. By using this new strategy, we demonstrate that the prototype E-DNA sensor has been able to detect as low as pM DNA targets with excellent differentiation ability for even single mismatches.

Keywords  target-induced conformational change; terminal extension amplification;   stem-loop; electrochemical; DNA sensor

目   次

1  绪论 1

1.1  电化学DNA传感器的工作原理 1

1.2  捕获探针设计 2

1.3  探针固定化 7

1.4  杂交检测 8

1.5  信号放大 10

1.6  电化学DNA传感器的应用 12

1.7  总结 13

2  基于目标响应结构和末端延伸放大的电化学DNA传感器的设计 基于目标响应结构与表面诱导延伸放大的电化学基因传感器:http://www.751com.cn/shengwu/lunwen_72063.html