Hs-MB型电化学活性开关分子信标的研制及其应用研究
发布时间：2018-08-22 14:57
【摘要】：伴随着基因结构和基因功能研究的深入开展,尤其是“人类基因组项目”完成后,对于人类相关疾病的基因诊断和基因治疗的研究迈入了一个新的纪元,但其首要条件在于对致病基因碱基序列的识别和确定。而传统的DNA测序方法存在一些局限,比如,劳动强度大且消耗时间较长。但是随着Watson-Crick对碱基互补配对原则的深入探究,为分子生物学的相关研究和开发提供了一个全新的基因检测方法-DNA探针。其中,以杂交技术为基础的DNA探针技术,当结合一些标记方法,比如,荧光法,电化学法,化学发光法后,将会使DNA探针得到一定程度的优化使用,比如,免去了放射性物质对人体的危害；降低了标记物昂贵的价格；节省了操作时间等,具有特异性好,快速灵敏,操作简便和无污染等特点,且还具有分离纯化基因及分子识别的功能。目前这种类型的分子探针已经成为基因研究领域中最具有吸引力的课题之一。 最关键的问题在于怎样找出导致各种各样基因疾病的基因,并且具有高灵敏度和高选择性。目前检测DNA的错配差异性最有用的工具是分子信标,其和线形信标相比,具有高的目标选择性,因为信标分子特殊的茎环结构使得在检测前多了一步打开茎环结构进而才与目标物进行杂交反应的过程。因此在某种程度上提高了选择性。在本论文中,我们设计了一种新型的电化学分子信标,将之命名为“新型电化学活性开关分子信标”,其作用机理类似于传统的以荧光基团-猝灭基团为基础的分子信标。在此,我们使用了具有二聚能力的卟啉铁分子,并将其连接在分子信标两端,在没有目标分子存在时,连接在信标两端的完全相同的卟啉铁分子因为受到茎环的作用力而无限靠近,因此呈现二聚状态,此时表现出非电化学活性特征,电化学信号被抑制；当加入目标分子时,分子信标将于目标序列互补结合,此时分子灯塔被迫使打开,连接在其上的两个卟啉铁分子也同时被强制分开,破坏了卟啉铁的二聚体结构而使卟啉铁以单体状态存在,因此此时电信号恢复。这种类型的信标既具有传统荧光信标简单快捷、易于控制的特点,又具有电化学技术成本低廉、易于小型化、便于野外操作的优势。 论文分为三章： 第一章：绪论 以杂交技术为基础的DNA探针技术,结合一些标记方法后,比如,荧光方法,电化学方法,化学发光方法后,使DNA探针得到一定程度的优化,为分子生物学的相关研究和开发提供了一个全新的基因检测方法.分子信标技术以其高选择性及特异性成为目前基因研究领域中的热点之一。本章通过对分子信标技术,主要从荧光分子信标和电化学分子信标及卟啉铁的性质及应用的归纳及总结,详细说明了本论文所研究课题的理论基础和选题依据。最后阐述了本论文的目的意义及创新之处。 第二章：Hemin电化学开关行为的探讨和Hs-MB电化学活性开关分子信标的合成研究 众所周知,hemin是一种高度疏水性的分子,在中性和弱碱性溶液中具有较左的溶解性,大量的科研数据已经证明：hemin溶液的物理和化学性质随着它的聚集状态的改变而改变,因为hemin的结构中存在疏水性基团乙烯基,致使hemin的卟啉结构部分聚集趋势相当明显。本章通过实验证明hemin的单体和二聚体的电信号值存在很大的差异,可以将其用在新型电化学活性分子开关上。同时,通过采用各种方法对所合成的新型电化学活性开关分子信标进行表征。实验结果表明,合成出的分子信标良好,为新型信标在生物化学领域中的应用奠定了基础。 第三章：基于Hs-MB型电化学活性开关分子信标的应用研究 p53基因是一种重要的致癌基因。据文献报道,人类50%的肿瘤是因为存在p53基因的突变或缺失,因此p53丛因的检测就显得至关重要。在本章中,我们首先合成了一种Hs-MB型电化学活性开关分子信标,这种类型的信标分子在未加目标分子的情况下没有电化学信号,但当与完全互补杂交序列发生杂交反应后则产生电化学信号,并将其应用于p53基因的定性、定量检测及其SNP突变的识别。实验结果证明,电化学检测DPV的峰电流值与目标DNA的浓度之间有良好的线性关系,回归方程为y=8E-08x+2E-07(x为目标序列的浓度,y为响应的峰电流值,R=0.9861),得到其最低检测限为3nmol。且对一碱基错配序列与完全互补序列的响应信号有较明显的差异性,初步证明,该分子信标可以实现对目标分子的定量检测和SNP突变的识别上。
[Abstract]:With the development of the research on gene structure and function, especially after the completion of the Human Genome Project, the research on gene diagnosis and gene therapy of human related diseases has entered a new era, but the first condition is to identify and determine the base sequence of pathogenic genes. But as Watson-Crick explores the principle of base complementary pairing, it provides a new method for molecular biology research and development - DNA probes. For example, fluorescence method, electrochemical method, chemiluminescence method, will make the DNA probe to be optimized to a certain extent, for example, avoid the harm of radioactive substances to the human body; reduce the expensive price of markers; save operating time, etc., with good specificity, fast and sensitive, easy operation and pollution-free characteristics, and also with separation. Purification of genes and molecular recognition function. At present, this type of molecular probe has become one of the most attractive topics in the field of gene research.
The key problem is how to identify genes that cause a variety of genetic diseases with high sensitivity and selectivity. The most useful tool for detecting DNA mismatch differences is molecular beacons, which have high target selectivity compared with linear beacons because of the special stem-ring structure of beacon molecules that make them more prevalent. In this paper, a novel electrochemical molecular beacon named "novel electrochemical active switching molecular beacon" has been designed. Its mechanism of action is similar to that of traditional fluorescent group-quenching. Molecular beacons based on destroying groups. Here, we use a dimeric iron porphyrin molecule and link it to both ends of the molecule beacon. In the absence of a target molecule, the identical iron porphyrin molecule at both ends of the beacon is infinitely close to each other because of the action of the stem ring, thus showing a dimeric state. When the target molecule is added, the molecular beacon will be complementary to the target sequence. At this time, the molecular beacon is forced to open, and the two iron porphyrin molecules connected to it are also forced to separate, destroying the dimer structure of iron porphyrin, so that iron porphyrin exists in monomer state. This type of beacon not only has the characteristics of traditional fluorescent beacon which is simple, fast and easy to control, but also has the advantages of low cost, easy miniaturization and field operation.
The thesis is divided into three chapters.
Chapter 1: Introduction
DNA probe technology based on hybridization technology, combined with some labeling methods, such as fluorescence method, electrochemical method and chemiluminescence method, can optimize DNA probe to a certain extent, which provides a new method for the research and development of molecular biology. Molecular beacon technology has high selectivity and specificity. Sex has become one of the hotspots in gene research. In this chapter, we summarize the properties and applications of fluorescent molecular beacons, electrochemical molecular beacons and ferroporphyrin. The theoretical basis and the basis of topic selection are described in detail. Finally, the purpose and significance of this paper are discussed. Innovation.
Chapter 2: Investigation of Hemin's Electrochemical Switching Behavior and Synthesis of Hs-MB Electrochemical Switching Molecular Beacon
It is well known that hemin is a highly hydrophobic molecule with left solubility in neutral and weak alkaline solutions. A large number of scientific data have proved that the physical and chemical properties of hemin solution change with the change of its aggregation state, because there is hydrophobic group vinyl in the structure of hemin, resulting in the hemin's porphyrin junction. The experimental results show that there is a great difference in the electrical signal values of hemin monomers and dimers, which can be used in novel electrochemical active molecular switches. The molecular beacons were good, which laid the foundation for the application of new beacons in biochemistry.
The third chapter is about the application of Hs-MB based electrochemical active switch molecular beacon.
P53 gene is an important oncogene. It has been reported that 50% of human tumors are caused by mutation or deletion of p53 gene, so the detection of p53 cluster is very important. In this chapter, we first synthesized a Hs-MB type of electrochemical active switching molecular beacon. This type of beacon molecule is in the absence of target molecule. In this case, there is no electrochemical signal, but when the hybridization reaction occurs with the complete complementary hybridization sequence, the electrochemical signal is produced and applied to the qualitative and quantitative detection of p53 gene and the identification of SNP mutation. For y=8E-08x+2E-07 (x is the concentration of the target sequence, y is the peak current value of the response, R=0.9861), the minimum detection limit is 3 nmol. Moreover, the response signal of a base mismatch sequence to a complementary sequence is obviously different from that of a complete complementary sequence. It is preliminarily proved that the molecular beacon can realize the quantitative detection of the target molecule and the identification of SNP mutations.
【学位授予单位】：华东师范大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R346
[Abstract]:With the development of the research on gene structure and function, especially after the completion of the Human Genome Project, the research on gene diagnosis and gene therapy of human related diseases has entered a new era, but the first condition is to identify and determine the base sequence of pathogenic genes. But as Watson-Crick explores the principle of base complementary pairing, it provides a new method for molecular biology research and development - DNA probes. For example, fluorescence method, electrochemical method, chemiluminescence method, will make the DNA probe to be optimized to a certain extent, for example, avoid the harm of radioactive substances to the human body; reduce the expensive price of markers; save operating time, etc., with good specificity, fast and sensitive, easy operation and pollution-free characteristics, and also with separation. Purification of genes and molecular recognition function. At present, this type of molecular probe has become one of the most attractive topics in the field of gene research.
The key problem is how to identify genes that cause a variety of genetic diseases with high sensitivity and selectivity. The most useful tool for detecting DNA mismatch differences is molecular beacons, which have high target selectivity compared with linear beacons because of the special stem-ring structure of beacon molecules that make them more prevalent. In this paper, a novel electrochemical molecular beacon named "novel electrochemical active switching molecular beacon" has been designed. Its mechanism of action is similar to that of traditional fluorescent group-quenching. Molecular beacons based on destroying groups. Here, we use a dimeric iron porphyrin molecule and link it to both ends of the molecule beacon. In the absence of a target molecule, the identical iron porphyrin molecule at both ends of the beacon is infinitely close to each other because of the action of the stem ring, thus showing a dimeric state. When the target molecule is added, the molecular beacon will be complementary to the target sequence. At this time, the molecular beacon is forced to open, and the two iron porphyrin molecules connected to it are also forced to separate, destroying the dimer structure of iron porphyrin, so that iron porphyrin exists in monomer state. This type of beacon not only has the characteristics of traditional fluorescent beacon which is simple, fast and easy to control, but also has the advantages of low cost, easy miniaturization and field operation.
The thesis is divided into three chapters.
Chapter 1: Introduction
DNA probe technology based on hybridization technology, combined with some labeling methods, such as fluorescence method, electrochemical method and chemiluminescence method, can optimize DNA probe to a certain extent, which provides a new method for the research and development of molecular biology. Molecular beacon technology has high selectivity and specificity. Sex has become one of the hotspots in gene research. In this chapter, we summarize the properties and applications of fluorescent molecular beacons, electrochemical molecular beacons and ferroporphyrin. The theoretical basis and the basis of topic selection are described in detail. Finally, the purpose and significance of this paper are discussed. Innovation.
Chapter 2: Investigation of Hemin's Electrochemical Switching Behavior and Synthesis of Hs-MB Electrochemical Switching Molecular Beacon
It is well known that hemin is a highly hydrophobic molecule with left solubility in neutral and weak alkaline solutions. A large number of scientific data have proved that the physical and chemical properties of hemin solution change with the change of its aggregation state, because there is hydrophobic group vinyl in the structure of hemin, resulting in the hemin's porphyrin junction. The experimental results show that there is a great difference in the electrical signal values of hemin monomers and dimers, which can be used in novel electrochemical active molecular switches. The molecular beacons were good, which laid the foundation for the application of new beacons in biochemistry.
The third chapter is about the application of Hs-MB based electrochemical active switch molecular beacon.
P53 gene is an important oncogene. It has been reported that 50% of human tumors are caused by mutation or deletion of p53 gene, so the detection of p53 cluster is very important. In this chapter, we first synthesized a Hs-MB type of electrochemical active switching molecular beacon. This type of beacon molecule is in the absence of target molecule. In this case, there is no electrochemical signal, but when the hybridization reaction occurs with the complete complementary hybridization sequence, the electrochemical signal is produced and applied to the qualitative and quantitative detection of p53 gene and the identification of SNP mutation. For y=8E-08x+2E-07 (x is the concentration of the target sequence, y is the peak current value of the response, R=0.9861), the minimum detection limit is 3 nmol. Moreover, the response signal of a base mismatch sequence to a complementary sequence is obviously different from that of a complete complementary sequence. It is preliminarily proved that the molecular beacon can realize the quantitative detection of the target molecule and the identification of SNP mutations.
【学位授予单位】：华东师范大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R346
【参考文献】
相关期刊论文 前10条
1 曾百肇;李东辉;郑玉霞;刘杰;周性尧;;血红素的极谱行为研究[J];分析化学;1991年07期
2 郁宝铭,秦云峰,蒋家，
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