免固定电化学核酸传感分析新方法及性能研究

发布时间：2018-01-14 15:10

本文关键词：免固定电化学核酸传感分析新方法及性能研究　出处：《青岛科技大学》2017年硕士论文　论文类型：学位论文

【摘要】：本论文构建了三种新颖的免固定电化学DNA生物传感器,实现了对靶标分子核酸的高灵敏、高选择、新颖的检测,为电化学DNA生物传感器的研究开拓了新思路、新方法,有望应用于早期临床疾病的检测。1、提出了一个独特的基于外切酶Ⅲ催化释放亚甲基蓝标记的单核苷酸的免固定的电化学DNA生物传感策略,并且释放出的亚甲基蓝标记的单核苷酸可以与十二硫醇疏水的烷链相互结合,从而富集亚甲基蓝分子到十二硫醇单分子层上,产生电化学信号的响应。由于其不需固定并且是在均相中进行的生物识别和放大过程,因此实现了对于靶DNA简单、快速,高灵敏的检测,对于靶DNA的检测限低至1pM,并且此方法可以扩展到检测任何可促使亚甲基蓝信号分子释放的靶标分子,因此,为免固定电化学生物传感器的发展提供了广阔的空间。2、提出了一个基于T7核酸外切酶的辅助循环放大的免固定的电化学DNA生物传感策略,实现了对完全互补的靶标分子在均相溶液中的循环放大检测。当单碱基错配时无法实现均相中的反应,进而达到在电极表面富集情况的区别,实现了对错配碱基的检测,为基因单碱基突变的检测提供了新颖、高效、灵敏的检测策略。3、基于等温扩增技术和hemin/G-quadruplex DNAzyme构筑了一种高效、灵敏、方便、免固定检测靶标DNA的生物传感器。在靶标DNA存在时,基于等温扩增技术扩增出大量的产物链,产物链包括重要的两部分,一部分为链霉亲和素的适体链,另一部分为富G碱基链。首先通过电极表面的链霉亲和素和其适体结合,将产物链固定到电极表面,产物链上的富G碱基遇到K+和Hemin时就会形成G四链体,它具有辣根过氧化物酶的活性,其可以催化H2O2,通过测H2O2的电催化产生的电化学信号来实现对靶标DNA的检测。此策略通过聚合内切循环放大反应有助于G四链体的形成,从而可以对靶标DNA进行高灵敏检测。总之,本论文所构建的免固定的电化学DNA生物传感器可以扩充到其他生物分子的检测中,对今后的研究和诊断具有重大意义。
[Abstract]:In this paper, three novel immobilization free electrochemical DNA biosensors were constructed to detect target molecular nucleic acids with high sensitivity, high selectivity and novel detection. It opens up new ideas and methods for the study of electrochemical DNA biosensor and is expected to be used in the detection of early clinical diseases. 1. A novel immobilized electrochemical DNA biosensor strategy based on the catalytic release of methylene blue labeled single nucleotides was proposed. The released methylene blue labeled single nucleotides can bind to the hydrophobic alkane chain of 12 mercaptan, thus enriching the methylene blue molecule onto the 12 mercaptan monolayer. Because it does not need to be fixed and is carried out in the homogeneous biometric recognition and amplification process, it realizes simple, fast and highly sensitive detection for target DNA. The detection limit for target DNA is as low as 1 pm, and this method can be extended to detect any target molecule that can induce the release of methylene blue signal molecule. In order to provide a wide space for the development of electrochemical biosensor, a novel non-fixed electrochemical DNA biosensor strategy based on T7 nucleic acid exonuclease was proposed. The cyclic amplification detection of completely complementary target molecules in homogeneous solution is realized. When the single base mismatch is not realized, the reaction in homogeneous phase can not be realized, and then the difference of enrichment on the electrode surface is achieved. The detection of mismatch base is realized, which provides a novel, efficient and sensitive detection strategy for the detection of single base mutation of gene. Based on isothermal amplification technique and hemin/G-quadruplex DNAzyme, a kind of high efficiency, sensitivity and convenience was constructed. In the presence of target DNA, a large number of product chains were amplified based on isothermal amplification technique, which included two important parts. One is the aptamer chain of streptavidin and the other is the G-rich base chain. Firstly, the product chain is immobilized to the electrode surface through the binding of the streptavidin on the electrode surface to the aptamer. G-rich bases on the product chain will form G quadruplex when they encounter K and Hemin. It has the activity of horseradish peroxidase and can catalyze H _ 2O _ 2. The detection of target DNA is realized by measuring the electrochemical signal produced by electrocatalysis of H2O2. This strategy is helpful to the formation of G quadruplex by polymeric internal cycle amplification reaction. Therefore, the target DNA can be detected with high sensitivity. In a word, the immobilized electrochemical DNA biosensor can be extended to the detection of other biomolecules. It is of great significance for future research and diagnosis.
【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】：TP212.3;O657.1

【参考文献】