基于Ru（bpy）_3～（2+）-SiO_2 NPs标记DNA探针构建电致化学发光生物传感器的研究
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摘要
脱氧核糖核酸(DNA)被称为生命体的遗传物质，研究发现人类的许多疾病都与DNA分子中碱基序列的变异有关。遗传物质通过翻译成蛋白质直接影响生命体。蛋白质是生命的基础，它参与生命体每一步反应和活动，承载着生命体中各种重要功能。蛋白质的表达异常是许多疾病的重要临床表现。因此，DNA特定序列的分析、DNA碱基突变的检测以及各类疾病表达蛋白(特别是低丰度蛋白)的分析对基因筛选、疾病的早期诊断和治疗都具有十分深远的意义。
虽然现有的DNA分析技术以及蛋白检测技术已经在很多方面得以应用，但是还是存在各种各样不尽如人意的地方。放射性同位素标记法存在着放射性污染等弊端，而非放射性标记法如荧光、化学发光和生物素标记法要么检测的灵敏度较低，要么标记过程繁琐复杂难以实现自动化或仪器价格昂贵。随着人类基因组学和蛋白质组学计划的发展，大量的分析迫切需要建立更加灵敏、准确、简便、快速并将之集于一身的检测方法。
电致化学发光(ECL)是由电化学反应而引发的一种化学发光(CL)现象，兼备电化学、化学发光的优点。如重现性好、选择性好、检测快速方便、仪器价格便宜等等，但其中最突出的优点为灵敏度高、检出限低。在生物传感器领域，想要利用好ECL技术，关键在于寻找优良的ECL标记物或标记方法。以核壳式纳米颗粒对生物分子进行标记作为一种新型的标记形式被广泛应用于生物分析中。由于包裹多个内容物分子而具有放大效应，可为分析带来了更高的灵敏度。
本论文根据DNA的碱基互补配对原则、核酸适配体对蛋白质的特异性识别能力，采用核壳式纳米颗粒作为标记物设计了电致化学发光生物传感器，将ECL方法、纳米颗粒带来的高灵敏度与生物识别的高选择性进行了有机的结合。
主要内容如下：
第一章绪论
简单的介绍了ECL的原理和特点，重点介绍了ECL两大发光体系的反应机理及其在分析中的应用。介绍了DNA生物传感器与核酸适配体生物传感器的构造、原理、特点以及分析应用，并总结了DNA分子在电极上的各种固定方法。介绍了SiO_2核壳式纳米颗粒作为标记物的优点以及在生物传感器中的应用。最后阐述了本论文的目的和意义，指出论文的创新之处以及主要研究内容。
第二章基于Ru(bpy)_3~(2+)-SiO_2 NPs标记DNA探针对DNA杂交进行电致化学发光检测
纳米颗粒的放大效应可为分析带来更高的灵敏度。本文以反相微乳法合成了SiO_2包裹的Ru(bpy)_3~(2+)纳米颗粒，将其标记在DNA上制备成探针，让探针与固定在PPy修饰的Pt电极表面的目标ssDNA进行杂交，在草酸体系中测定ECL杂交信号，实现对目标ssDNA的序列识别及含量的灵敏测定。实验表明，本方法能很好的区分与探针完全互补、三碱基错配、完全不互补的ssDNA序列，对完全互补的ssDNA其检测下限可达到1.0×10~(-13)mol／L(s／n=3)。
第三章基于Ru(bpy)_3~(2+)-SiO_2 NPs标记DNA探针构建核酸适配体传感器通过目标诱导替换实现凝血酶的电致化学发光特异性检测
本文利用目标诱导替换法检测凝血酶。在金电极上组装一段巯基修饰的凝血酶核酸适配体，将其与标记有Ru(bpy)_3~(2+)-SiO_2 NPs并与核酸适配体部分序列完全互补ssDNA探针进行杂交，测得I_(ECL1)。然后将此电极置于凝血酶溶液中进行培育。实验结果表明，凝血酶与核酸适配体之间的结合力强于核酸适配体与短链互补DNA之间的结合力，凝血酶可与适配体结合，将探针DNA替换下来，此时测得I_(ECL2)。利用替换前后的△I_(ECL)信号来定量反映目标蛋白——凝血酶的含量。此方法具有优异的特异性以及高灵敏度，对凝血酶检测限达1.0×10~(-15)mol／L(s／n=3)，并运用于实际样品的检测。
第四章基于Ru(bpy)_3~(2+)-SiO_2 NPs标记核酸适配体探针构建凝血酶电致化学发光生物传感器的研究
本文利用凝血酶的两段核酸适配体(aptamerⅠ以及aptamerⅡ)与凝血酶的高亲和识别作用结合电致化学发光技术设计了一种具有高灵敏度和高选择性的蛋白质传感器：将aptamerⅠ自组装到金电极表面，特异性“捕捉”凝血酶，进而结合另一段标记有Ru(bpy)_3~(2+)-SiO_2 NPs的aptamerⅡ探针构建三明治传感结构。在TPA体系中检测Ru(bpy)_3~(2+)的ECL信号对凝血酶进行定量检测。实验证明，此传感器同样具有优异的特异性，并拥有更高的灵敏度，对凝血酶检测限可达1.0×10~(-15)mol／L(s／n=3)。
Deoxyribonucleic Acid (DNA) is known as inherited substance in life body. With the increasing knowledge about human diseases it is found that the mutations of DNA base sequence are responsible for numerous inherited human diseases. Inherited substance directly impact on living things through being translated into protein. Proteins are involved in every life process and life activities, which play important roles in all kinds of life functions. Abnormal expression of the protein always suggest many clinical diseases. In order to select genes or make early diagnosis and cure for disease, much effort is needed to detect the specific DNA sequences, DNA mutation and the proteins (especially low-abundance protein) related to diseases.
Many detection techniques of DNA sequence and protein have been developed in recent years, but there is still a lot of dissatisfaction. The radioactive labels present many problems such as a potential hazard to analyst and environment. Non-radioactive labels such as fluorescent, chemiluminescent and biotin-avidin label probes also present many shortcomings such as low sensitivity or complex equipment or others. So it is necessary to develop another method for the more sensitive, easy-to-use, fast, inexpensive detection of specific DNA sequences and proteins to adapt to wide-scale genetic and protein testing requires.
Electrogenerated chemiluminescence (ECL) is a technique that combines chemiluminescence (CL) and electrochemistry (EC). ECL introduces both the merits of CL and EC, such as high sensitivity and selectivity, rapid and convenient operation and relatively simple instrumentation system. The most important issue for making good use of ECL technology in biosensor field is to find better ECL labels or label methods. The lumiophore-doped SiO_2 nanoparticles will be a good ECL labels for their high sensitivity in analysis.
This dissertation combines the principle of base-complementary, and the specific recognition of certain protein to design ECL biosensors based on SiO_2 NPs DNA probe with high sensitivity and unique selectivity. The dissertation is composed of four chapters as foliowings:
Chapter 1: Introduced the ECL method, pay attention to the two most primary type of ECL reaction and their application in analytical chemistry field. Review the DNA biosensor and the aptamer biosensor, including their principles, characteristics and
summarized the methods for the immobilization of ssDNA onto electrode surface. Introduced the advantages ;and the application of the SiO_2 nanoparticles which are used as tag. At last pointed out the purpose of the dissertation. Chapter 2: Ru(bpy)_3~(2+)-SiO_2 nanoparticles were prepared by water-in-oil(W/O) microemulsion method. A great deal of Ru(bpy)_3~(2+) was immobilized inside the nanoparticle, which could greatly enhance the ECL response and result in the increased sensitivity. Ru(bpy)_3~(2+)-SiO_2 NPs labled DNA probe was hybridized with target DNA immobilized on the surface of PPy modified Pt electrode. The hybridization events were evaluated by ECL measurements in oxalic acid solution and only the complementary sequence could give strong ECL signals. A three-base mismatch sequence and a non-complementary sequence had almost negligible responses. The assay allows detection at levels as low as 1.0×10~(-13)mol/L of the target DNA.
Chapter 3: Sensitive and .selective detection of thrombin via target protein-induced strand displacement of the ssDNA probe by △I_(ecl) is described. The hybridization between the ssDNA probe labeled by Ru(bpy)_3~(2+)-doped SNPs and the aptamer self-assembled on a microfebricated thin film gold electrode was evaluated by ECL measurements. Then, the eleetarode incubated with the thrombin analyte, and the binding event between the throflabin analyte and the aptamer was monitored by ECL measurements again. The △I_(ecl) of the two events can be used to quantify the thrombin. The assay has excellent selectivity because the aptamer could bind thrombin with high specificity. The assay allows detection at levels as low as 1.0 fM of the thrombin due to a. large number of Ru(bpy)_3~(2+) molecules inside SNPs labeled on DNA probe.
Chapter 4: Novel ECL protein biosensor in sandwich manner using the aptamers was developed. Two different aptamers (aptamer I and aptamer II), which recognize different positions of thrombin, were chosen to construct sandwich type sensing system for protein. Aptamer I was immobilized onto the gold electrode for capturing thrombin onto the electrode and aptamer II labled with Ru(bpy)_3~(2+)- SiO_2 NPs was used for detection. The increase of the ECL signal generated by Ru(bpy)_3~(2+)- SiO_2 NPs was observed in dependent manner of the concentration of thrombin added. The assay also has excellent selectivity and further more it has the better sensitivity. It could detect as low as 1.0 fM of the thrombin.
虽然现有的DNA分析技术以及蛋白检测技术已经在很多方面得以应用，但是还是存在各种各样不尽如人意的地方。放射性同位素标记法存在着放射性污染等弊端，而非放射性标记法如荧光、化学发光和生物素标记法要么检测的灵敏度较低，要么标记过程繁琐复杂难以实现自动化或仪器价格昂贵。随着人类基因组学和蛋白质组学计划的发展，大量的分析迫切需要建立更加灵敏、准确、简便、快速并将之集于一身的检测方法。
电致化学发光(ECL)是由电化学反应而引发的一种化学发光(CL)现象，兼备电化学、化学发光的优点。如重现性好、选择性好、检测快速方便、仪器价格便宜等等，但其中最突出的优点为灵敏度高、检出限低。在生物传感器领域，想要利用好ECL技术，关键在于寻找优良的ECL标记物或标记方法。以核壳式纳米颗粒对生物分子进行标记作为一种新型的标记形式被广泛应用于生物分析中。由于包裹多个内容物分子而具有放大效应，可为分析带来了更高的灵敏度。
本论文根据DNA的碱基互补配对原则、核酸适配体对蛋白质的特异性识别能力，采用核壳式纳米颗粒作为标记物设计了电致化学发光生物传感器，将ECL方法、纳米颗粒带来的高灵敏度与生物识别的高选择性进行了有机的结合。
主要内容如下：
第一章绪论
简单的介绍了ECL的原理和特点，重点介绍了ECL两大发光体系的反应机理及其在分析中的应用。介绍了DNA生物传感器与核酸适配体生物传感器的构造、原理、特点以及分析应用，并总结了DNA分子在电极上的各种固定方法。介绍了SiO_2核壳式纳米颗粒作为标记物的优点以及在生物传感器中的应用。最后阐述了本论文的目的和意义，指出论文的创新之处以及主要研究内容。
第二章基于Ru(bpy)_3~(2+)-SiO_2 NPs标记DNA探针对DNA杂交进行电致化学发光检测
纳米颗粒的放大效应可为分析带来更高的灵敏度。本文以反相微乳法合成了SiO_2包裹的Ru(bpy)_3~(2+)纳米颗粒，将其标记在DNA上制备成探针，让探针与固定在PPy修饰的Pt电极表面的目标ssDNA进行杂交，在草酸体系中测定ECL杂交信号，实现对目标ssDNA的序列识别及含量的灵敏测定。实验表明，本方法能很好的区分与探针完全互补、三碱基错配、完全不互补的ssDNA序列，对完全互补的ssDNA其检测下限可达到1.0×10~(-13)mol／L(s／n=3)。
第三章基于Ru(bpy)_3~(2+)-SiO_2 NPs标记DNA探针构建核酸适配体传感器通过目标诱导替换实现凝血酶的电致化学发光特异性检测
本文利用目标诱导替换法检测凝血酶。在金电极上组装一段巯基修饰的凝血酶核酸适配体，将其与标记有Ru(bpy)_3~(2+)-SiO_2 NPs并与核酸适配体部分序列完全互补ssDNA探针进行杂交，测得I_(ECL1)。然后将此电极置于凝血酶溶液中进行培育。实验结果表明，凝血酶与核酸适配体之间的结合力强于核酸适配体与短链互补DNA之间的结合力，凝血酶可与适配体结合，将探针DNA替换下来，此时测得I_(ECL2)。利用替换前后的△I_(ECL)信号来定量反映目标蛋白——凝血酶的含量。此方法具有优异的特异性以及高灵敏度，对凝血酶检测限达1.0×10~(-15)mol／L(s／n=3)，并运用于实际样品的检测。
第四章基于Ru(bpy)_3~(2+)-SiO_2 NPs标记核酸适配体探针构建凝血酶电致化学发光生物传感器的研究
本文利用凝血酶的两段核酸适配体(aptamerⅠ以及aptamerⅡ)与凝血酶的高亲和识别作用结合电致化学发光技术设计了一种具有高灵敏度和高选择性的蛋白质传感器：将aptamerⅠ自组装到金电极表面，特异性“捕捉”凝血酶，进而结合另一段标记有Ru(bpy)_3~(2+)-SiO_2 NPs的aptamerⅡ探针构建三明治传感结构。在TPA体系中检测Ru(bpy)_3~(2+)的ECL信号对凝血酶进行定量检测。实验证明，此传感器同样具有优异的特异性，并拥有更高的灵敏度，对凝血酶检测限可达1.0×10~(-15)mol／L(s／n=3)。
Deoxyribonucleic Acid (DNA) is known as inherited substance in life body. With the increasing knowledge about human diseases it is found that the mutations of DNA base sequence are responsible for numerous inherited human diseases. Inherited substance directly impact on living things through being translated into protein. Proteins are involved in every life process and life activities, which play important roles in all kinds of life functions. Abnormal expression of the protein always suggest many clinical diseases. In order to select genes or make early diagnosis and cure for disease, much effort is needed to detect the specific DNA sequences, DNA mutation and the proteins (especially low-abundance protein) related to diseases.
Many detection techniques of DNA sequence and protein have been developed in recent years, but there is still a lot of dissatisfaction. The radioactive labels present many problems such as a potential hazard to analyst and environment. Non-radioactive labels such as fluorescent, chemiluminescent and biotin-avidin label probes also present many shortcomings such as low sensitivity or complex equipment or others. So it is necessary to develop another method for the more sensitive, easy-to-use, fast, inexpensive detection of specific DNA sequences and proteins to adapt to wide-scale genetic and protein testing requires.
Electrogenerated chemiluminescence (ECL) is a technique that combines chemiluminescence (CL) and electrochemistry (EC). ECL introduces both the merits of CL and EC, such as high sensitivity and selectivity, rapid and convenient operation and relatively simple instrumentation system. The most important issue for making good use of ECL technology in biosensor field is to find better ECL labels or label methods. The lumiophore-doped SiO_2 nanoparticles will be a good ECL labels for their high sensitivity in analysis.
This dissertation combines the principle of base-complementary, and the specific recognition of certain protein to design ECL biosensors based on SiO_2 NPs DNA probe with high sensitivity and unique selectivity. The dissertation is composed of four chapters as foliowings:
Chapter 1: Introduced the ECL method, pay attention to the two most primary type of ECL reaction and their application in analytical chemistry field. Review the DNA biosensor and the aptamer biosensor, including their principles, characteristics and
summarized the methods for the immobilization of ssDNA onto electrode surface. Introduced the advantages ;and the application of the SiO_2 nanoparticles which are used as tag. At last pointed out the purpose of the dissertation. Chapter 2: Ru(bpy)_3~(2+)-SiO_2 nanoparticles were prepared by water-in-oil(W/O) microemulsion method. A great deal of Ru(bpy)_3~(2+) was immobilized inside the nanoparticle, which could greatly enhance the ECL response and result in the increased sensitivity. Ru(bpy)_3~(2+)-SiO_2 NPs labled DNA probe was hybridized with target DNA immobilized on the surface of PPy modified Pt electrode. The hybridization events were evaluated by ECL measurements in oxalic acid solution and only the complementary sequence could give strong ECL signals. A three-base mismatch sequence and a non-complementary sequence had almost negligible responses. The assay allows detection at levels as low as 1.0×10~(-13)mol/L of the target DNA.
Chapter 3: Sensitive and .selective detection of thrombin via target protein-induced strand displacement of the ssDNA probe by △I_(ecl) is described. The hybridization between the ssDNA probe labeled by Ru(bpy)_3~(2+)-doped SNPs and the aptamer self-assembled on a microfebricated thin film gold electrode was evaluated by ECL measurements. Then, the eleetarode incubated with the thrombin analyte, and the binding event between the throflabin analyte and the aptamer was monitored by ECL measurements again. The △I_(ecl) of the two events can be used to quantify the thrombin. The assay has excellent selectivity because the aptamer could bind thrombin with high specificity. The assay allows detection at levels as low as 1.0 fM of the thrombin due to a. large number of Ru(bpy)_3~(2+) molecules inside SNPs labeled on DNA probe.
Chapter 4: Novel ECL protein biosensor in sandwich manner using the aptamers was developed. Two different aptamers (aptamer I and aptamer II), which recognize different positions of thrombin, were chosen to construct sandwich type sensing system for protein. Aptamer I was immobilized onto the gold electrode for capturing thrombin onto the electrode and aptamer II labled with Ru(bpy)_3~(2+)- SiO_2 NPs was used for detection. The increase of the ECL signal generated by Ru(bpy)_3~(2+)- SiO_2 NPs was observed in dependent manner of the concentration of thrombin added. The assay also has excellent selectivity and further more it has the better sensitivity. It could detect as low as 1.0 fM of the thrombin.
引文
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