基于金纳米粒子修饰电极的DNA电化学传感器的研究
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摘要
癌症是多年来人类难以治愈的一种顽症,寻找能有效治疗癌症的新药是解决该问题的有效手段之一。进行癌症新药的临床筛选要花费大量的人力和物力,且耗时长久,因此需要寻找一种简单有效的体外筛选方法。科学家们已发现,所有的抗癌药物都能与DNA产生相互作用,因而在体外进行药物与DNA相互作用的研究是一项非常有意义的工作,可作为大量抗癌新药的初步筛选方法。
DNA生物传感器是一种能将目的DNA的存在转变为可检测的电、光、声等信号的传感装置。利用电化学原理检测基因的DNA电化学生物传感器是一种新的基因检测技术。它与传统的标记基因技术相比,具有快速、灵敏、操作简便、无污染等优点,并具有分子识别、基因分离纯化等功能。因此,在分子生物学和生物医学领域有着较大的实际意义。
纳米材料被认为是跨世纪材料研究领域的热点,有“21世纪最有前途的材料”之美誉。纳米颗粒的比表面积大、表面反应活性高、催化效率高、吸附能力强等优异性质,为生物医学研究提供了新的研究途径,同时也推动了化学和生物传感器的迅速发展。纳米粒子的独特性质与生物分子杂交反应的特异性和电化学检测方法的高灵敏性相结合,使其应用范围更加广阔。
本课题首先对一种基于Aunano-DNA修饰玻碳电极的新型DNA杂交体系进行了初步研究;其次合成了一种双核铜Schiff碱配合物,并对其结构进行了表征,同时研究了双核铜Schiff碱配合物与DNA的相互作用,以及其作为电化学杂交指示剂在DNA杂交体系中的应用研究。本论文的具体内容包括以下几方面:
第一章:综述
对DNA的研究是生命科学研究中一个极其重要的方面。本章综述了小分子化合物与DNA的相互作用、DNA电化学生物传感器原理(包括DNA探针及其分子识别原理和DNA在固体基质表面的固定化)及其应用等方面的内容。随后介绍了纳米粒子及纳米材料在分析化学中的一系列应用以及电化学生物传感器的发展方向。
第二章:基于Aunano-DNA修饰玻碳电极的新型DNA杂交方法
在Aunano-DNA修饰玻碳电极(GCE)上研究了DNA杂交。5’端巯基修饰探针被固定在GCE表面,探针修饰电极和杂交后的响应以亚甲基蓝(MB)作为杂交指示剂,通过DPV进行检测。在ctDNA存在下,金胶能有效的分散在GCE表面,从而使Aunano-DNA/GCE的活性位点大大增加,修饰和杂交后的响应信号增强,靶DNA的检测量也大大增加。靶DNA的检测线性范围在1.52×10-10到4.05×10-8 moll-1之间,检测限达到10?10 mol/L。
第三章:一种新型双核Cu(Ⅱ)配合物的合成及表征
以水杨醛、半胱氨酸和醋酸铜为原料,合成了希夫碱配体([Na3 (sal) (cys)]·2 H2O,Na3L)和双核铜配合物([Cu2(cys)(salic)]·2 H2O,Cu2 (Ⅱ) L),并通过元素分析、红外光谱、紫外光谱、热重分析、核磁共振方法对配合物的组成和结构进行了表征。实验结果显示,Cu(Ⅱ)与Schiff碱配体形成含三配位和四配位的双核铜配合物。
第四章:双核Cu(Ⅱ)配合物与DNA相互作用的研究
采用紫外可见光谱法和电化学方法初步研究了铜配合物与小牛胸腺DNA(ctDNA)的相互作用,并用差示脉冲伏安法对Cu2(Ⅱ)L作为杂交指示剂在DNA杂交体系中的应用做了初步研究。实验结果显示,双核Cu(Ⅱ)配合物与DNA的作用模式可能是插入与静电结合的混合作用模式;在DNA杂交体系中,Cu2(Ⅱ)L可用于识别不同序列的DNA。
Cancer is one of the most difficult problem to tackle with these years.One approach is to find new effective anticancer drugs. However, it takes a lot of expenditure on menpower and material resources to carry out the clinic choosing on the new drugs, and is also time-consuming. So there is a necessicity to find out a simple and effective way to make a choosing out of the body. It is discoveried that all anticancer drugs can interact with DNA,moreover, the study on the interaction between drugs and DNA out-of-body is quite a meaningful job. So the method based on the interaction can be used as the initial way to choose an effective drug from millions of new ones.
DNA biosensor is a transducer which can make the presence of target DNA change into the electronic, optic, sound signals and others can be detected. The biosensor which adopts the electrochemical theory to measure the gene is a novel genic measuring technique. Compared with traditional methods, electrochemistry biosensor provides rapid, sensitive, simple and pollution-free as well as inexpensive point-of-care detection for specific drugs. It also can recognize small molecule compounds, separate and purify gene and so forth. Therefore, it is of practical significance in molecule-biology and life science.
Recently, nano-material is a hot topic in the research areas. It is famous for the most promising material at the 21st century. It provides a new way for life science and contributes to the development of chemical sensor and biosensor owing to its features such as large relative surface area, high reaction activity, catalysis and strong absorption. The combination, among the special ability of nano-material, the speciality of biomolecule hybrid reaction and high sensitivity of electrochemical detection method, broadens the application fields.
In this article, a novel scheme of DNA hybridization based on Aunano-DNA modified glassy electrode has been studied first. Then, a new bi-nuclear copper Schiff base complex has been synthesized and characterized; its interaction with DNA and the usage as electrochemical indicator in DNA hybridization have been investigated. The details list as follows:
Chapter 1:General Introduction
Investigation of DNA is most important in the field of life sciences. This part mainly introduces the structure of DNA and its interaction with the small molecule, as well as the basic theory of DNA electrochemistry biosensor (including DNA probe and its recognition to small molecule as well as its modification onto solid subject surfaces) and its application, followed by the review about nano-material. At last, the paper describes the trends of development on DNA electrochemistry biosensor.
Chapter 2:A new scheme of hybridization based on the Aunano-DNA modified glassy carbon electrode
DNA hybridization is investigated on the Aunano-DNA modified Glassy carbon electrode (GCE) in this article. The thiol modified probe oligonucleotides (SH-ssDNA) at 5’-phosphate end, is assembled on the Aunano-DNA modified GCE surface. The Electrochemical responses of the probe immobilization and hybridization with target DNA are measured by differential pulse voltammetry (DPV) using methylene blue (MB) as an electroactive indicator. Gold nanoparticles can be dispersed effectively on the GCE surface in the presence of calf thymus DNA (ctDNA). Aunano-DNA modified GCE can greatly increase the active sites, enhances the response signal during immobilization and hybridization. The hybridization amount of target DNA can be greatly increased. The linear detection range of Aunano-DNA/GCE for the complementary 21-mer oligonucleotide (cDNA) is achieved from 1.52×10-10 to 4.05×10-8 moll-1. The detection limit can reach to the concentration of 10?10 mol/L.
Chapter 3:Synthesis and characterization of a novel bi-nuclear copper(II) complex
A novel bi-nuclear copper (Ⅱ) complex([Cu2(cys)(salic)]·2 H2O,Cu2 (Ⅱ) L), was synthesized with a Schiff base ligand derived from salicyladehyde and cysteine acid, and the characterization was determined by elemental analyses, infrared spectrum, ultraviolet-visible spectrum, thermal analyses and 1HNMR. The results show that three and four-coordinate numbers are contained in the bi-nuclear copper (Ⅱ) complex at the same time.
Chapter 4: Study on the interaction between bi-nuclear copper(II) complex and DNA
The interaction of bi-nuclear copper(II) complex (signified as Cu2(Ⅱ) L) with calf thymus DNA (ctDNA) is studied by UV and electrochemical techniques; the application of Cu2(Ⅱ) L as electrochemical indicator in DNA hybridization is carried out using different pulse voltammetry (DPV). The results have shown that copper complex has two nearly reversible redox peaks in the potential range of -0.3～0.3V in cyclic voltammetry (CV). The interaction model between DNA and Cu2(Ⅱ) L is partial intercalation coupled with electrostatic binding. The species of DNA can be distinguished when Cu2 (Ⅱ) L is used as the indicator in DNA hybridization system.
DNA生物传感器是一种能将目的DNA的存在转变为可检测的电、光、声等信号的传感装置。利用电化学原理检测基因的DNA电化学生物传感器是一种新的基因检测技术。它与传统的标记基因技术相比,具有快速、灵敏、操作简便、无污染等优点,并具有分子识别、基因分离纯化等功能。因此,在分子生物学和生物医学领域有着较大的实际意义。
纳米材料被认为是跨世纪材料研究领域的热点,有“21世纪最有前途的材料”之美誉。纳米颗粒的比表面积大、表面反应活性高、催化效率高、吸附能力强等优异性质,为生物医学研究提供了新的研究途径,同时也推动了化学和生物传感器的迅速发展。纳米粒子的独特性质与生物分子杂交反应的特异性和电化学检测方法的高灵敏性相结合,使其应用范围更加广阔。
本课题首先对一种基于Aunano-DNA修饰玻碳电极的新型DNA杂交体系进行了初步研究;其次合成了一种双核铜Schiff碱配合物,并对其结构进行了表征,同时研究了双核铜Schiff碱配合物与DNA的相互作用,以及其作为电化学杂交指示剂在DNA杂交体系中的应用研究。本论文的具体内容包括以下几方面:
第一章:综述
对DNA的研究是生命科学研究中一个极其重要的方面。本章综述了小分子化合物与DNA的相互作用、DNA电化学生物传感器原理(包括DNA探针及其分子识别原理和DNA在固体基质表面的固定化)及其应用等方面的内容。随后介绍了纳米粒子及纳米材料在分析化学中的一系列应用以及电化学生物传感器的发展方向。
第二章:基于Aunano-DNA修饰玻碳电极的新型DNA杂交方法
在Aunano-DNA修饰玻碳电极(GCE)上研究了DNA杂交。5’端巯基修饰探针被固定在GCE表面,探针修饰电极和杂交后的响应以亚甲基蓝(MB)作为杂交指示剂,通过DPV进行检测。在ctDNA存在下,金胶能有效的分散在GCE表面,从而使Aunano-DNA/GCE的活性位点大大增加,修饰和杂交后的响应信号增强,靶DNA的检测量也大大增加。靶DNA的检测线性范围在1.52×10-10到4.05×10-8 moll-1之间,检测限达到10?10 mol/L。
第三章:一种新型双核Cu(Ⅱ)配合物的合成及表征
以水杨醛、半胱氨酸和醋酸铜为原料,合成了希夫碱配体([Na3 (sal) (cys)]·2 H2O,Na3L)和双核铜配合物([Cu2(cys)(salic)]·2 H2O,Cu2 (Ⅱ) L),并通过元素分析、红外光谱、紫外光谱、热重分析、核磁共振方法对配合物的组成和结构进行了表征。实验结果显示,Cu(Ⅱ)与Schiff碱配体形成含三配位和四配位的双核铜配合物。
第四章:双核Cu(Ⅱ)配合物与DNA相互作用的研究
采用紫外可见光谱法和电化学方法初步研究了铜配合物与小牛胸腺DNA(ctDNA)的相互作用,并用差示脉冲伏安法对Cu2(Ⅱ)L作为杂交指示剂在DNA杂交体系中的应用做了初步研究。实验结果显示,双核Cu(Ⅱ)配合物与DNA的作用模式可能是插入与静电结合的混合作用模式;在DNA杂交体系中,Cu2(Ⅱ)L可用于识别不同序列的DNA。
Cancer is one of the most difficult problem to tackle with these years.One approach is to find new effective anticancer drugs. However, it takes a lot of expenditure on menpower and material resources to carry out the clinic choosing on the new drugs, and is also time-consuming. So there is a necessicity to find out a simple and effective way to make a choosing out of the body. It is discoveried that all anticancer drugs can interact with DNA,moreover, the study on the interaction between drugs and DNA out-of-body is quite a meaningful job. So the method based on the interaction can be used as the initial way to choose an effective drug from millions of new ones.
DNA biosensor is a transducer which can make the presence of target DNA change into the electronic, optic, sound signals and others can be detected. The biosensor which adopts the electrochemical theory to measure the gene is a novel genic measuring technique. Compared with traditional methods, electrochemistry biosensor provides rapid, sensitive, simple and pollution-free as well as inexpensive point-of-care detection for specific drugs. It also can recognize small molecule compounds, separate and purify gene and so forth. Therefore, it is of practical significance in molecule-biology and life science.
Recently, nano-material is a hot topic in the research areas. It is famous for the most promising material at the 21st century. It provides a new way for life science and contributes to the development of chemical sensor and biosensor owing to its features such as large relative surface area, high reaction activity, catalysis and strong absorption. The combination, among the special ability of nano-material, the speciality of biomolecule hybrid reaction and high sensitivity of electrochemical detection method, broadens the application fields.
In this article, a novel scheme of DNA hybridization based on Aunano-DNA modified glassy electrode has been studied first. Then, a new bi-nuclear copper Schiff base complex has been synthesized and characterized; its interaction with DNA and the usage as electrochemical indicator in DNA hybridization have been investigated. The details list as follows:
Chapter 1:General Introduction
Investigation of DNA is most important in the field of life sciences. This part mainly introduces the structure of DNA and its interaction with the small molecule, as well as the basic theory of DNA electrochemistry biosensor (including DNA probe and its recognition to small molecule as well as its modification onto solid subject surfaces) and its application, followed by the review about nano-material. At last, the paper describes the trends of development on DNA electrochemistry biosensor.
Chapter 2:A new scheme of hybridization based on the Aunano-DNA modified glassy carbon electrode
DNA hybridization is investigated on the Aunano-DNA modified Glassy carbon electrode (GCE) in this article. The thiol modified probe oligonucleotides (SH-ssDNA) at 5’-phosphate end, is assembled on the Aunano-DNA modified GCE surface. The Electrochemical responses of the probe immobilization and hybridization with target DNA are measured by differential pulse voltammetry (DPV) using methylene blue (MB) as an electroactive indicator. Gold nanoparticles can be dispersed effectively on the GCE surface in the presence of calf thymus DNA (ctDNA). Aunano-DNA modified GCE can greatly increase the active sites, enhances the response signal during immobilization and hybridization. The hybridization amount of target DNA can be greatly increased. The linear detection range of Aunano-DNA/GCE for the complementary 21-mer oligonucleotide (cDNA) is achieved from 1.52×10-10 to 4.05×10-8 moll-1. The detection limit can reach to the concentration of 10?10 mol/L.
Chapter 3:Synthesis and characterization of a novel bi-nuclear copper(II) complex
A novel bi-nuclear copper (Ⅱ) complex([Cu2(cys)(salic)]·2 H2O,Cu2 (Ⅱ) L), was synthesized with a Schiff base ligand derived from salicyladehyde and cysteine acid, and the characterization was determined by elemental analyses, infrared spectrum, ultraviolet-visible spectrum, thermal analyses and 1HNMR. The results show that three and four-coordinate numbers are contained in the bi-nuclear copper (Ⅱ) complex at the same time.
Chapter 4: Study on the interaction between bi-nuclear copper(II) complex and DNA
The interaction of bi-nuclear copper(II) complex (signified as Cu2(Ⅱ) L) with calf thymus DNA (ctDNA) is studied by UV and electrochemical techniques; the application of Cu2(Ⅱ) L as electrochemical indicator in DNA hybridization is carried out using different pulse voltammetry (DPV). The results have shown that copper complex has two nearly reversible redox peaks in the potential range of -0.3～0.3V in cyclic voltammetry (CV). The interaction model between DNA and Cu2(Ⅱ) L is partial intercalation coupled with electrostatic binding. The species of DNA can be distinguished when Cu2 (Ⅱ) L is used as the indicator in DNA hybridization system.
引文
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