基于表面接近杂交效应的高灵敏电化学核酸传感器构筑及性能研究

发布时间：2018-03-19 20:02

本文选题：电化学传感　切入点：动态DNA自组装　出处：《青岛科技大学》2017年硕士论文　论文类型：学位论文

【摘要】：本论文主要基于动态DNA自组装调控的依靠接近效应的表面杂交策略构筑了三种电化学DNA生物传感器,实现了对与疾病相关的靶标DNA高灵敏、高选择性分析检测。1.基于DNA燃料靶标循环机制调控的依靠接近效应的表面杂交电化学方法,用于一步、无酶、放大灵敏检测靶标DNA。其中DNA燃料靶标循环机制由两次Toe-hold介导的等温链置换反应(TSDR)所驱动。在该策略中,DNA燃料靶标循环机制的动态DNA自组装过程中,通过均相的靶标特异识别和放大转化,形成DNA脚手架-TS+FS形成的两链复合物,促进其自发依靠接近效应与电极表面SH-DNA进行稳定杂交。此时,由于信号分子Fc靠近电极表面,更加有利于与电极进行电子传递与转移,产生电化学响应。该方法针对靶标DNA可实现0.29 fM检测限。2.基于杂交链式反应HCR调控的依靠接近效应的表面杂交进行DNA动态自组装策略,建立一个动态的、无酶的、灵敏的电化学核酸检测新方法。首先,设计发夹探针HP1、HP2,其中HP2-5’标记亚甲蓝MB的突出碱基片段,可与电极表面固定的SH-DNA互补杂交。当加入靶标时,通过连续的链置换反应,引发HP1、HP2发夹进行级联自组装,最终形成线性的DNA串联体。由于在线性串联体中,发夹HP2-5’末端突出的碱基序列相互靠近,增加其在电极表面的局部浓度,从而更加有力于其依靠接近效应,与SH-DNA进行表面杂交。此时,MB信号分子靠近电极表面,产生电化学响应。该方法针对靶标DNA可实现50 aM检测限。3.基于靶标引发的等温链置换扩增反应SDA以及接近效应的表面杂交反应策略,调控DNA动态自组装,实现对靶标DNA的超灵敏检测分析。引入酶辅助的等温链置换反应SDA,不仅可以实现靶标循环信号放大;而且通过等温链置换反应SDA产生大量自由单链DNA可与信号探针SP互补杂交,使信号探针SP末端突出的碱基序列相互靠近,促进其与SH-DNA依靠接近效应发生稳定的表面杂交,使MB信号分子靠近电极表面,产生信号响应,以此巧妙实现靶标DNA的检测。
[Abstract]:In this paper, three electrochemical DNA biosensors are constructed based on dynamic DNA self-assembly based surface hybridization strategy based on proximity effect, which is highly sensitive to disease-related target DNA. Highly selective analytical detection. 1. Surface hybridization electrochemical method based on proximity effect based on DNA fuel target cycle mechanism for one-step, non-enzymatic, The mechanism of DNA fuel target cycle is driven by two isothermal chain replacement reactions mediated by Toe-hold. In this strategy, the dynamic DNA self-assembly of the DNA fuel target cycle mechanism is carried out. Through the homogeneous target specific recognition and amplification transformation, a two-chain complex formed by DNA scaffolding TSFS was formed, which promoted its spontaneous stable hybridization with SH-DNA on the electrode surface by proximity effect. In this case, the signal molecule FC was close to the electrode surface, and the signal molecule FC was close to the electrode surface. This method can realize 0.29fM detection limit for target DNA. (2) dynamic self-assembly strategy of DNA based on surface hybridization based on proximity effect based on HCR regulation of hybrid chain reaction. A dynamic, non-enzymatic and sensitive electrochemical method for the detection of nucleic acid was established. Firstly, a hairpin probe HP1OHP2was designed, in which HP2-5 'labeled methylmethylene blue MB protruding base fragment could be hybridized with SH-DNA fixed on the electrode surface. When the target was added, By continuous chain replacement reaction, the HP1O HP2 hairpin is self-assembled in a cascade, and finally a linear DNA series is formed. Because the base sequence protruding at the HP2-5 'end of the hairpin is close to each other in the linear series, the local concentration of HP2-5' hairpin on the electrode surface is increased. Therefore, it is more powerful to hybridize with SH-DNA surface by proximity effect. At this time, the signal molecule of MB is close to the electrode surface, Electrochemical response was generated. The detection limit of 50 mm for target DNA could be achieved by this method. Based on the isothermal chain replacement amplification reaction SDA initiated by target and the surface hybridization strategy of proximity effect, the dynamic self-assembly of DNA was regulated. The enzyme assisted isothermal chain replacement reaction (SDAA) can not only amplify the cyclic signal of the target, but also produce a large number of free single-stranded DNA by isothermal chain replacement reaction, which can be hybridized with the signal probe SP. The protruding base sequence at the SP end of the signal probe is close to each other, which promotes the stable surface hybridization between the signal probe and the SH-DNA by means of proximity effect, and makes the MB signal molecule close to the electrode surface to produce signal response, so that the detection of target DNA can be accomplished cleverly.
【学位授予单位】：青岛科技大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：O657.1

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