基于循环链置换聚合反应和超分支滚环扩增技术的超灵敏电化学生物传感器检测DNA
发布时间：2018-12-16 12:34
【摘要】：DNA检测在临床疾病的诊断与治疗、基因突变以及病原体检测等方面具有重要意义,因而建立一种简单、快速、灵敏的检测方法日益引人关注。近年,随着生物化学和分子生物学的结合,电化学生物传感器因其简单、易操作、响应快、成本低等优势在生物分子检测的研究中备受关注。为了提高检测方法的灵敏度,核酸等温扩增技术被研究发现并不断发展。本研究整合两种核酸等温扩增技术,利用电化学生物传感器的检测平台,对DNA进行超灵敏检测。本研究将循环链置换聚合反应和超分支滚环扩增两种核酸等温扩增技术相结合,构建了一个超灵敏的电化学生物传感器用于靶DNA的检测。当靶DNA存在时,分子信标与靶DNA特异性识别。两者结合后分子信标发生空间上的构象改变,由茎环结构变为链状结构。生物素标记的引物与分子信标暴露出来3’末端杂交,在d NTP和KF聚合酶的作用下延伸,引发循环链置换聚合反应。循环链置换聚合反应形成双链DNA。这些双链DNA有生物素标记,之后通过生物素-亲和素的结合作用将超分支滚环扩增的引物和模板锚定在传感器表面。在d NTP和phi29DNA聚合酶的作用下,超分支滚环扩增反应即可进行并在电极表面产生大量的长度不一的双链DNA。最后,检测探针与超分支滚环扩增的产物杂交结合,然后通过生物素与亲和素标记的碱性磷酸酶结合,催化底物α-NP产生电信号。构建的电化学生物传感器可以对浓度0.01 f M~10 p M的靶物质进行简单、快速、定量检测,最低检测限达到8.9 a M。特异性和灵敏性更高。该方法也可以用于其它DNA的检测。在疾病的早期诊断和预后以及生物医学研究方面也有很好的应用前景。
[Abstract]:DNA detection plays an important role in the diagnosis and treatment of clinical diseases, gene mutation and pathogen detection. Therefore, the establishment of a simple, rapid and sensitive detection method has attracted more and more attention. In recent years, with the combination of biochemistry and molecular biology, electrochemical biosensors have attracted much attention in the research of biomolecular detection for their advantages of simple, easy to operate, fast response and low cost. In order to improve the sensitivity of detection methods, isothermal amplification of nucleic acid has been developed. In this study, two techniques of nucleic acid isothermal amplification were used to detect DNA by electrochemical biosensor. In this study, a highly sensitive electrochemical biosensor was constructed for the detection of target DNA by combining cyclic chain replacement polymerization and hyperbranching ring-amplification with two isothermal amplification techniques of nucleic acid. When the target DNA exists, the molecular beacons are specifically identified with the target DNA. The conformation of the molecular beacon changed from the stem ring structure to the chain structure. Biotin labeled primers and molecular beacons were exposed to 3 'end hybridization and extended under the action of d NTP and KF polymerase to initiate cyclic chain replacement polymerization. Cyclic chain displacement Polymerization to form Double-stranded DNA. These double-stranded DNA were labeled with biotin, and the primers and templates of hyperbranching and rolling amplification were anchored on the surface of the sensor by the combination of biotin and avidin. Under the action of d NTP and phi29DNA polymerase, the hyperbranching rolling ring amplification reaction can be carried out and a large number of double-stranded DNA. of different lengths can be produced on the electrode surface. Finally, the probe was hybridized with the product of hyperbranching roller amplification, and then the electrical signal was generated by the binding of biotin and affinity labeled alkaline phosphatase to catalyze the generation of electrical signal by 伪-NP. The electrochemical biosensor can be used to detect the target material with a concentration of 0.01 F M ~ (10) p ~ (-1). The detection limit is 8.9 am. Specificity and sensitivity are higher. This method can also be used for other DNA detection. It also has a good prospect in early diagnosis and prognosis of disease and biomedical research.
【学位授予单位】：重庆医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R440;TP212.3
本文编号：2382350
[Abstract]:DNA detection plays an important role in the diagnosis and treatment of clinical diseases, gene mutation and pathogen detection. Therefore, the establishment of a simple, rapid and sensitive detection method has attracted more and more attention. In recent years, with the combination of biochemistry and molecular biology, electrochemical biosensors have attracted much attention in the research of biomolecular detection for their advantages of simple, easy to operate, fast response and low cost. In order to improve the sensitivity of detection methods, isothermal amplification of nucleic acid has been developed. In this study, two techniques of nucleic acid isothermal amplification were used to detect DNA by electrochemical biosensor. In this study, a highly sensitive electrochemical biosensor was constructed for the detection of target DNA by combining cyclic chain replacement polymerization and hyperbranching ring-amplification with two isothermal amplification techniques of nucleic acid. When the target DNA exists, the molecular beacons are specifically identified with the target DNA. The conformation of the molecular beacon changed from the stem ring structure to the chain structure. Biotin labeled primers and molecular beacons were exposed to 3 'end hybridization and extended under the action of d NTP and KF polymerase to initiate cyclic chain replacement polymerization. Cyclic chain displacement Polymerization to form Double-stranded DNA. These double-stranded DNA were labeled with biotin, and the primers and templates of hyperbranching and rolling amplification were anchored on the surface of the sensor by the combination of biotin and avidin. Under the action of d NTP and phi29DNA polymerase, the hyperbranching rolling ring amplification reaction can be carried out and a large number of double-stranded DNA. of different lengths can be produced on the electrode surface. Finally, the probe was hybridized with the product of hyperbranching roller amplification, and then the electrical signal was generated by the binding of biotin and affinity labeled alkaline phosphatase to catalyze the generation of electrical signal by 伪-NP. The electrochemical biosensor can be used to detect the target material with a concentration of 0.01 F M ~ (10) p ~ (-1). The detection limit is 8.9 am. Specificity and sensitivity are higher. This method can also be used for other DNA detection. It also has a good prospect in early diagnosis and prognosis of disease and biomedical research.
【学位授予单位】：重庆医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R440;TP212.3
【参考文献】
相关期刊论文 前1条
1 Jianjian Gao;Qingyun Zhang;Jianjun Xu;Lijuan Guo;Xuefeng Li;;Clinical significance of serum miR-21 in breast cancer compared with CA153 and CEA[J];Chinese Journal of Cancer Research;2013年06期
，本文编号：2382350
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