Hs-MB型電化學(xué)活性開(kāi)關(guān)分子信標(biāo)的研制及其應(yīng)用研究
發(fā)布時(shí)間：2018-08-22 14:57
【摘要】：伴隨著基因結(jié)構(gòu)和基因功能研究的深入開(kāi)展,尤其是“人類(lèi)基因組項(xiàng)目”完成后,對(duì)于人類(lèi)相關(guān)疾病的基因診斷和基因治療的研究邁入了一個(gè)新的紀(jì)元,但其首要條件在于對(duì)致病基因堿基序列的識(shí)別和確定。而傳統(tǒng)的DNA測(cè)序方法存在一些局限,比如,勞動(dòng)強(qiáng)度大且消耗時(shí)間較長(zhǎng)。但是隨著Watson-Crick對(duì)堿基互補(bǔ)配對(duì)原則的深入探究,為分子生物學(xué)的相關(guān)研究和開(kāi)發(fā)提供了一個(gè)全新的基因檢測(cè)方法-DNA探針。其中,以雜交技術(shù)為基礎(chǔ)的DNA探針技術(shù),當(dāng)結(jié)合一些標(biāo)記方法,比如,熒光法,電化學(xué)法,化學(xué)發(fā)光法后,將會(huì)使DNA探針得到一定程度的優(yōu)化使用,比如,免去了放射性物質(zhì)對(duì)人體的危害；降低了標(biāo)記物昂貴的價(jià)格；節(jié)省了操作時(shí)間等,具有特異性好,快速靈敏,操作簡(jiǎn)便和無(wú)污染等特點(diǎn),且還具有分離純化基因及分子識(shí)別的功能。目前這種類(lèi)型的分子探針已經(jīng)成為基因研究領(lǐng)域中最具有吸引力的課題之一。 最關(guān)鍵的問(wèn)題在于怎樣找出導(dǎo)致各種各樣基因疾病的基因,并且具有高靈敏度和高選擇性。目前檢測(cè)DNA的錯(cuò)配差異性最有用的工具是分子信標(biāo),其和線形信標(biāo)相比,具有高的目標(biāo)選擇性,因?yàn)樾艠?biāo)分子特殊的莖環(huán)結(jié)構(gòu)使得在檢測(cè)前多了一步打開(kāi)莖環(huán)結(jié)構(gòu)進(jìn)而才與目標(biāo)物進(jìn)行雜交反應(yīng)的過(guò)程。因此在某種程度上提高了選擇性。在本論文中,我們?cè)O(shè)計(jì)了一種新型的電化學(xué)分子信標(biāo),將之命名為“新型電化學(xué)活性開(kāi)關(guān)分子信標(biāo)”,其作用機(jī)理類(lèi)似于傳統(tǒng)的以熒光基團(tuán)-猝滅基團(tuán)為基礎(chǔ)的分子信標(biāo)。在此,我們使用了具有二聚能力的卟啉鐵分子,并將其連接在分子信標(biāo)兩端,在沒(méi)有目標(biāo)分子存在時(shí),連接在信標(biāo)兩端的完全相同的卟啉鐵分子因?yàn)槭艿角o環(huán)的作用力而無(wú)限靠近,因此呈現(xiàn)二聚狀態(tài),此時(shí)表現(xiàn)出非電化學(xué)活性特征,電化學(xué)信號(hào)被抑制；當(dāng)加入目標(biāo)分子時(shí),分子信標(biāo)將于目標(biāo)序列互補(bǔ)結(jié)合,此時(shí)分子燈塔被迫使打開(kāi),連接在其上的兩個(gè)卟啉鐵分子也同時(shí)被強(qiáng)制分開(kāi),破壞了卟啉鐵的二聚體結(jié)構(gòu)而使卟啉鐵以單體狀態(tài)存在,因此此時(shí)電信號(hào)恢復(fù)。這種類(lèi)型的信標(biāo)既具有傳統(tǒng)熒光信標(biāo)簡(jiǎn)單快捷、易于控制的特點(diǎn),又具有電化學(xué)技術(shù)成本低廉、易于小型化、便于野外操作的優(yōu)勢(shì)。 論文分為三章： 第一章：緒論 以雜交技術(shù)為基礎(chǔ)的DNA探針技術(shù),結(jié)合一些標(biāo)記方法后,比如,熒光方法,電化學(xué)方法,化學(xué)發(fā)光方法后,使DNA探針得到一定程度的優(yōu)化,為分子生物學(xué)的相關(guān)研究和開(kāi)發(fā)提供了一個(gè)全新的基因檢測(cè)方法.分子信標(biāo)技術(shù)以其高選擇性及特異性成為目前基因研究領(lǐng)域中的熱點(diǎn)之一。本章通過(guò)對(duì)分子信標(biāo)技術(shù),主要從熒光分子信標(biāo)和電化學(xué)分子信標(biāo)及卟啉鐵的性質(zhì)及應(yīng)用的歸納及總結(jié),詳細(xì)說(shuō)明了本論文所研究課題的理論基礎(chǔ)和選題依據(jù)。最后闡述了本論文的目的意義及創(chuàng)新之處。 第二章：Hemin電化學(xué)開(kāi)關(guān)行為的探討和Hs-MB電化學(xué)活性開(kāi)關(guān)分子信標(biāo)的合成研究 眾所周知,hemin是一種高度疏水性的分子,在中性和弱堿性溶液中具有較左的溶解性,大量的科研數(shù)據(jù)已經(jīng)證明：hemin溶液的物理和化學(xué)性質(zhì)隨著它的聚集狀態(tài)的改變而改變,因?yàn)閔emin的結(jié)構(gòu)中存在疏水性基團(tuán)乙烯基,致使hemin的卟啉結(jié)構(gòu)部分聚集趨勢(shì)相當(dāng)明顯。本章通過(guò)實(shí)驗(yàn)證明hemin的單體和二聚體的電信號(hào)值存在很大的差異,可以將其用在新型電化學(xué)活性分子開(kāi)關(guān)上。同時(shí),通過(guò)采用各種方法對(duì)所合成的新型電化學(xué)活性開(kāi)關(guān)分子信標(biāo)進(jìn)行表征。實(shí)驗(yàn)結(jié)果表明,合成出的分子信標(biāo)良好,為新型信標(biāo)在生物化學(xué)領(lǐng)域中的應(yīng)用奠定了基礎(chǔ)。 第三章：基于Hs-MB型電化學(xué)活性開(kāi)關(guān)分子信標(biāo)的應(yīng)用研究 p53基因是一種重要的致癌基因。據(jù)文獻(xiàn)報(bào)道,人類(lèi)50%的腫瘤是因?yàn)榇嬖趐53基因的突變或缺失,因此p53叢因的檢測(cè)就顯得至關(guān)重要。在本章中,我們首先合成了一種Hs-MB型電化學(xué)活性開(kāi)關(guān)分子信標(biāo),這種類(lèi)型的信標(biāo)分子在未加目標(biāo)分子的情況下沒(méi)有電化學(xué)信號(hào),但當(dāng)與完全互補(bǔ)雜交序列發(fā)生雜交反應(yīng)后則產(chǎn)生電化學(xué)信號(hào),并將其應(yīng)用于p53基因的定性、定量檢測(cè)及其SNP突變的識(shí)別。實(shí)驗(yàn)結(jié)果證明,電化學(xué)檢測(cè)DPV的峰電流值與目標(biāo)DNA的濃度之間有良好的線性關(guān)系,回歸方程為y=8E-08x+2E-07(x為目標(biāo)序列的濃度,y為響應(yīng)的峰電流值,R=0.9861),得到其最低檢測(cè)限為3nmol。且對(duì)一堿基錯(cuò)配序列與完全互補(bǔ)序列的響應(yīng)信號(hào)有較明顯的差異性,初步證明,該分子信標(biāo)可以實(shí)現(xiàn)對(duì)目標(biāo)分子的定量檢測(cè)和SNP突變的識(shí)別上。
[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.
【學(xué)位授予單位】：華東師范大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2011
【分類(lèi)號(hào)】：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.
【學(xué)位授予單位】：華東師范大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2011
【分類(lèi)號(hào)】：R346
【參考文獻(xiàn)】
相關(guān)期刊論文 前10條
1 曾百肇;李東輝;鄭玉霞;劉杰;周性堯;;血紅素的極譜行為研究[J];分析化學(xué);1991年07期
2 郁寶銘,秦云峰,蔣家，
本文編號(hào)：2197451
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