发卡式DNA电化学发光生物传感器的研究
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摘要
基因结构与基因功能研究的不断深入已迅速推动了人类疾病的DNA诊断及基因治疗的研究。基因DNA分子序列的微小改变，基因突变及多态性如一个或几个核苷酸的取代、缺失或增多，就会导致遗传性状的改变或各种疾病的出现。分子信标是一种由寡聚核酸形成的发夹型分子，它包括一个环(loop)—干(stem)结构。这种DNA探针有很高的杂交特异性。分子信标因具有灵敏度高，特异结合性好等优点而被广泛应用于生物学和生物分析领域。在注重功能基因研究的后基因组时期，分子信标将会被更多地应用于基因突变引起的疾病的研究、活细胞中DNA的检测、蛋白质的检测及生物芯片研究等。大规模的基因检测要求建立更简单、快速、廉价、微型化的分析装置。许多新的生物技术的开发，为发展高灵敏度、高特异性的基因分析检测方法注入了活力，其中利用DNA双链的碱基互补配对原则发展起来的各种DNA生物传感技术，受到生物分析工作者的高度重视。
电化学发光是在电极上施加一定的电压使电极反应产物之间或电极反应产物与溶液中某组分之间进行化学反应而产生的一种光辐射。根据电化学发光的强度进行的分析方法称为电化学发光分析法。该方法具有化学发光法的灵敏度高、线性范围宽和仪器简单等优点，也具有电化学法容易控制等优点。电化学发光DNA生物传感器因其灵敏度高起而使其研究受到人们的关注。但是，现报道电化学发光DNA生物传感器存在背景信号大或目标物需要修饰等缺点。
本论文的目的是探索研究以发卡式DNA为分子识别物质，以钌联吡啶衍生物为电化学发光信号物质，构建高选择性简单的电化学发光DNA生物传感器的可行性，研究发卡式DNA探针的结构对传感器选择性的影响。
本论文由绪论、研究报告两部分组成。第一部分为绪论，介绍了DNA传感器原理、分类，总结了电化学发光分析体系和分析方法特点，分子信标的特性，综述了DNA检测方法的研究进展和应用，最后阐述了本论文的研究目的和内容。第二部分为研究报告，由两部分组成。研究报告的第一部分为发卡式DNA电化学发光生物传感器制作和基本性能的研究。提出用二(2，2'-联吡啶)(2，2'-吡啶-4，4'-二碳酸)琥珀酰胺钌(Ru(bpy)_2(dcbpy)NHS)标记发卡式DNA，制得探针Ru(bpy)_2(dcbpy)NHS-hairpin-DNA，将探针固定在金电极上制得电化学发光传感器，与待测的目标DNA杂交，然后在含有三丙胺(TPA)的杂交缓冲溶液中施加电压，记录电化学发光信号，通过电化学发光信号的变化对目标DNA进行检测。当不存在互补ss-DNA时，探针处于闭合状态，标记物钌联吡啶离电极很近，产生强的电化学发光信号；存在互补序列时，探针与目标ss-DNA发生杂交发应，环状结构被打开，形成刚性的直链ds-DNA，标记物远离电极，电化学发光强度降低。试验结果表明，发卡式DNA为探针与传统的直链DNA探针检测相比，可以极大的提高电化学发光传感器的选择性；电化学发光强度与目标DNA浓度的负对数在2.7×10~(-10)mol L~(-1)～5.4×10~(-6)mol L~(-1)范围内分段成线形关系，检出限为9.0×10~(-11)mol L~(-1)，对1.0×10~(-9)mol L~(-1)浓度的靶DNA进行7次测定，相对标准偏差为3.9％。
研究报告的第二部分研究了不同发卡式DNA电化学发光生物传感器的选择性问题。研究了电化学发光传感器对单碱基错配序列以及多碱基错配序列的识别，发现本文设计的传感器能够将单碱基错配序列从互补序列中区分开来。还研究了固定有不同环长探针传感器的选择性，将不同长度发卡式DNA的探针固定于金电极上，与待测目标进行杂交后，将杂交后的电极作为工作电极，在含有TPA的溶液中进行电化学发光测量。实验结果表明，探针中DNA的长度影响其在电极表面的固定量，且制作的传感器可以很好的检测多碱基错配系列。选择合适的发卡式DNA，提高传感器的选择性。
本研究工作主要以电化学发光为检测手段，结合发卡式DNA、DNA杂交技术、自组装技术制作了一种新的电化学发光DNA传感器。本论文的研究结果表明，以发卡式DNA为分子识别物质，以钌联吡啶衍生物为电化学发光信号物质，构建高选择性简单的电化学发光DNA生物传感器是可行的。发卡式DNA的使用可以提高电化学发光DNA传感器的选择性和灵敏度。这项工作将为电化学发光DNA传感器的研究提供一种新的思路，将促进电化学发光DNA传感器研究的发展。
Gene mutations and polymorphisms due to small genetic changes in DNA sequence, such as the replacement of one or several nucleic acid loss or increased, will lead to genetic change or the emergence of various diseases. Therefore, the analysis of human blood, body fluids and other specific DNA sequence, can be used to confirm the source of infection. DNA hairpins have been found to exhibit extraordinary stability, high selectivity and specificity compared to similar assays performed using single-stranded DNA. Wide-scale genetic testing requires the development of easy-to-use, fast, inexpensive, miniaturized analytical devices. Biosensors offer a promising alternative for nucleic acid assays duo to fast, cheap and simple.
Electrogenerated chemiluninescence (ECL) is the luminescence generated by relaxation of exited state molecules that are produced during an electrochemically initiated reaction. The ECL method allows a highly sensitive detection and control the ECL the reaction through modulation of applied potential without expensive instrumentation. The generation of light in the vicinity of the electrode gives better spatial control for the sensitive detection of analytes. High sensitivity can be achieved by optimizing the material, size and position of the electrodes. Molecular beacon is a kind of new fluorescence nucleic acid probe with a high sensitivity and specificity. The molecular beacon technology by its operation simple, high sensitivity and specificity may be carried on the real-time quantitative determination to the nucleic acid, and living specimen analysis. It can be used not only in the widespread application in biology research, but also in clinical applications.
This thesis includes review section and research section. In the review section, the principles and characteristics of different kinds of DNA biosensors are summarized. The basic principles, characteristics and systems of ECL, and characteristics of molecular beacon are introduced. The developments in applications of the molecular beacon in DNA sensors are reviewed, and the purpose and content of this thesis are presented. The aim of present work is to develop a high sensitivity and selectivity DNA biosensor. We combine the merit of hairpin DNA and the ECL technique with the nucleic acid hybridization technology to develop a new ECL sensor based on hairpin. It will be able to be used in the detection of DNA hybridization and mismatch rapidly and simply.
The research section contains two subunits. In the first subunit, a novel sensitive ECL sensor for the detection DNA hybridization based on hairpin probes was fabricated. The ECL sensor - "switch off" mode - was fabricated by self-assembling the ECL probe on a gold electrode through thiol group at its 5' terminal. In the absence of target single strand DNA (ss-DNA), the ECL probe on the electrode was in the folded configuration and its termini were held in close proximity to the electrode, thus resulting in a strong ECL signal. In the presence of target ss-DNA, the loop of the ECL probe on the electrode was converted into a rigid and linear double helix configuration due to a hybridization with a complementary target ss-DNA, allowed to remove the tag of Ru(bpy)_2(dcbpy)NHS away from the electrode surface and thus the ECL signal was dropped off. The decreased ECL signal was found to be related to the concentration of complementary target ss-DNA in a range from 2.7×10~(-10) mol L~(-1) to 5.4×10~(-6) mol L~(-1) with a detection limit of 9×10~(-11) mol L~(-1).
In the second subunit, the effect of the probe with different loops on the selectivity of ECL biosensors was investigated. It was found that mismatch target DNA can be discriminated from complementary one. This work demonstrated that the selectivity and specificity of ECL-based biosensor can be greatly improved using a hairpin DNA suitable of stem and loop length as a recognition element.
电化学发光是在电极上施加一定的电压使电极反应产物之间或电极反应产物与溶液中某组分之间进行化学反应而产生的一种光辐射。根据电化学发光的强度进行的分析方法称为电化学发光分析法。该方法具有化学发光法的灵敏度高、线性范围宽和仪器简单等优点，也具有电化学法容易控制等优点。电化学发光DNA生物传感器因其灵敏度高起而使其研究受到人们的关注。但是，现报道电化学发光DNA生物传感器存在背景信号大或目标物需要修饰等缺点。
本论文的目的是探索研究以发卡式DNA为分子识别物质，以钌联吡啶衍生物为电化学发光信号物质，构建高选择性简单的电化学发光DNA生物传感器的可行性，研究发卡式DNA探针的结构对传感器选择性的影响。
本论文由绪论、研究报告两部分组成。第一部分为绪论，介绍了DNA传感器原理、分类，总结了电化学发光分析体系和分析方法特点，分子信标的特性，综述了DNA检测方法的研究进展和应用，最后阐述了本论文的研究目的和内容。第二部分为研究报告，由两部分组成。研究报告的第一部分为发卡式DNA电化学发光生物传感器制作和基本性能的研究。提出用二(2，2'-联吡啶)(2，2'-吡啶-4，4'-二碳酸)琥珀酰胺钌(Ru(bpy)_2(dcbpy)NHS)标记发卡式DNA，制得探针Ru(bpy)_2(dcbpy)NHS-hairpin-DNA，将探针固定在金电极上制得电化学发光传感器，与待测的目标DNA杂交，然后在含有三丙胺(TPA)的杂交缓冲溶液中施加电压，记录电化学发光信号，通过电化学发光信号的变化对目标DNA进行检测。当不存在互补ss-DNA时，探针处于闭合状态，标记物钌联吡啶离电极很近，产生强的电化学发光信号；存在互补序列时，探针与目标ss-DNA发生杂交发应，环状结构被打开，形成刚性的直链ds-DNA，标记物远离电极，电化学发光强度降低。试验结果表明，发卡式DNA为探针与传统的直链DNA探针检测相比，可以极大的提高电化学发光传感器的选择性；电化学发光强度与目标DNA浓度的负对数在2.7×10~(-10)mol L~(-1)～5.4×10~(-6)mol L~(-1)范围内分段成线形关系，检出限为9.0×10~(-11)mol L~(-1)，对1.0×10~(-9)mol L~(-1)浓度的靶DNA进行7次测定，相对标准偏差为3.9％。
研究报告的第二部分研究了不同发卡式DNA电化学发光生物传感器的选择性问题。研究了电化学发光传感器对单碱基错配序列以及多碱基错配序列的识别，发现本文设计的传感器能够将单碱基错配序列从互补序列中区分开来。还研究了固定有不同环长探针传感器的选择性，将不同长度发卡式DNA的探针固定于金电极上，与待测目标进行杂交后，将杂交后的电极作为工作电极，在含有TPA的溶液中进行电化学发光测量。实验结果表明，探针中DNA的长度影响其在电极表面的固定量，且制作的传感器可以很好的检测多碱基错配系列。选择合适的发卡式DNA，提高传感器的选择性。
本研究工作主要以电化学发光为检测手段，结合发卡式DNA、DNA杂交技术、自组装技术制作了一种新的电化学发光DNA传感器。本论文的研究结果表明，以发卡式DNA为分子识别物质，以钌联吡啶衍生物为电化学发光信号物质，构建高选择性简单的电化学发光DNA生物传感器是可行的。发卡式DNA的使用可以提高电化学发光DNA传感器的选择性和灵敏度。这项工作将为电化学发光DNA传感器的研究提供一种新的思路，将促进电化学发光DNA传感器研究的发展。
Gene mutations and polymorphisms due to small genetic changes in DNA sequence, such as the replacement of one or several nucleic acid loss or increased, will lead to genetic change or the emergence of various diseases. Therefore, the analysis of human blood, body fluids and other specific DNA sequence, can be used to confirm the source of infection. DNA hairpins have been found to exhibit extraordinary stability, high selectivity and specificity compared to similar assays performed using single-stranded DNA. Wide-scale genetic testing requires the development of easy-to-use, fast, inexpensive, miniaturized analytical devices. Biosensors offer a promising alternative for nucleic acid assays duo to fast, cheap and simple.
Electrogenerated chemiluninescence (ECL) is the luminescence generated by relaxation of exited state molecules that are produced during an electrochemically initiated reaction. The ECL method allows a highly sensitive detection and control the ECL the reaction through modulation of applied potential without expensive instrumentation. The generation of light in the vicinity of the electrode gives better spatial control for the sensitive detection of analytes. High sensitivity can be achieved by optimizing the material, size and position of the electrodes. Molecular beacon is a kind of new fluorescence nucleic acid probe with a high sensitivity and specificity. The molecular beacon technology by its operation simple, high sensitivity and specificity may be carried on the real-time quantitative determination to the nucleic acid, and living specimen analysis. It can be used not only in the widespread application in biology research, but also in clinical applications.
This thesis includes review section and research section. In the review section, the principles and characteristics of different kinds of DNA biosensors are summarized. The basic principles, characteristics and systems of ECL, and characteristics of molecular beacon are introduced. The developments in applications of the molecular beacon in DNA sensors are reviewed, and the purpose and content of this thesis are presented. The aim of present work is to develop a high sensitivity and selectivity DNA biosensor. We combine the merit of hairpin DNA and the ECL technique with the nucleic acid hybridization technology to develop a new ECL sensor based on hairpin. It will be able to be used in the detection of DNA hybridization and mismatch rapidly and simply.
The research section contains two subunits. In the first subunit, a novel sensitive ECL sensor for the detection DNA hybridization based on hairpin probes was fabricated. The ECL sensor - "switch off" mode - was fabricated by self-assembling the ECL probe on a gold electrode through thiol group at its 5' terminal. In the absence of target single strand DNA (ss-DNA), the ECL probe on the electrode was in the folded configuration and its termini were held in close proximity to the electrode, thus resulting in a strong ECL signal. In the presence of target ss-DNA, the loop of the ECL probe on the electrode was converted into a rigid and linear double helix configuration due to a hybridization with a complementary target ss-DNA, allowed to remove the tag of Ru(bpy)_2(dcbpy)NHS away from the electrode surface and thus the ECL signal was dropped off. The decreased ECL signal was found to be related to the concentration of complementary target ss-DNA in a range from 2.7×10~(-10) mol L~(-1) to 5.4×10~(-6) mol L~(-1) with a detection limit of 9×10~(-11) mol L~(-1).
In the second subunit, the effect of the probe with different loops on the selectivity of ECL biosensors was investigated. It was found that mismatch target DNA can be discriminated from complementary one. This work demonstrated that the selectivity and specificity of ECL-based biosensor can be greatly improved using a hairpin DNA suitable of stem and loop length as a recognition element.
引文
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