【作者】 郑静；

【导师】 何品刚； 方禹之；

【作者基本信息】 华东师范大学 ， 分析化学， 2007， 博士

【摘要】 基因诊断已经成为分子生物学和生物医学研究中的重要领域。人体、病毒和细菌核酸中特定碱基序列的检测在疾病诊断、食品污染、法医鉴定和环境监测等领域都将会发挥越来越重要的作用。大规模的基因检测要求建立更简便、迅速、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DNA检测方法以其灵敏度高、轻巧便宜、携带方便、耗能少、能与现代微电子技术联用，易于实现微型化等优点，受到了研究者们的广泛关注，俨然成为当今生物学、医学领域的前沿性课题。随着人类基因组计划（HGP）的顺利完成，生命科学从确定核酸（DNA、RNA）序列层次深入到功能基因组研究和蛋白质组学研究上来。如何构建高速度、高通量、集约化、有特异性的高灵敏蛋白质检测技术是目前蛋白质组学研究所面临的紧迫任务。传统蛋白质的检测主要利用抗体一抗原的特异相互作用。利用寡核苷酸间的严格的识别和亲和力而设计的人工合成寡核苷酸—适体（aptamer）的出现，使抗体抗原反应发生新的革命性变化，弥补了现有抗体的不足。也为传统免疫传感器发展开辟了一条新的道路。核酸适体对蛋白质的结合力和特异性可与抗原抗体间的作用力相媲美，且与抗体相比有许多优越性。因此利用核酸适体构建蛋白质的电化学检测方法已引起许多科学工作者的关注。蛋白质和核酸是组成生命的主要生物大分子，核酸具有传递遗传信息等功能，而蛋白质则贯穿所有的生命活动过程。有关核酸与蛋白质之间的相互作用的信息的获得，将有助于对一些生命过程的的研究，进而掌握生命活动和信息传递规律并实施调节及调控。纳米技术的出现为纳米材料在分析化学领域的发展和应用开辟了新的方向。纳米材料的优异性能例如比表面积大、反应活性高等为分析化学开辟了新的研究途径，纳米粒子的独特性能为生物检测奠定了基础，其和核酸适体的高度特异性，电化学方法的高灵敏性相结合，使得检测的灵敏度大大提高，使其应用范围更加广阔，将为生物分析化学开辟新的领域。本论文的目的就是研究基于纳米颗粒的新型电化学检测方法。利用纳米金的优异的电化学性能和纳米磁性颗粒的顺磁性，设计了几种新型的电化学DNA和凝血酶蛋白质检测方法，拓宽了后基因时代的研究领域，论文主要内容如下：第一章绪论首先介绍了DNA生物传感器和核酸适体生物传感器的原理、应用及其研究进展，其中着重介绍了电化学的DNA生物传感器的原理和研究进展，重点介绍了核酸适体生物传感器的原理和应用，对今后的发展方向和趋势进行了展望。接着介绍了纳米材料在分析化学中的应用。最后阐述了本论文的目的和意义。第二章基于核酸适体和纳米材料的凝血酶蛋白电化学特异性识别的研究本文介绍了一种结合核酸适体技术和纳米技术，以凝血酶蛋白为研究对象的高效、高灵敏、特异性识别蛋白质的电化学方法。利用金纳米颗粒标记的核酸适体以及被固定在磁性纳米颗粒上的核酸适体与凝血酶蛋白同时结合形成磁性颗粒／凝血酶／纳米金胶的三明治结构，利用磁性分离，将金胶纳米颗粒特异性地吸着到电极表面，通过检测电极上金胶的电化学信号，实现对凝血酶靶蛋白的检测。该方法对凝血酶蛋白具有很高的特异性识别能力，其检测不受其他蛋白质如牛血清白蛋白等存在的干扰，可应用于实际血浆中凝血酶的检测。由于利用磁性纳米颗粒使得分离、富集和测定在同一个自制的电化学反应池中进行，其操作不仅简单，而且检测的灵敏度得到提高。该蛋白质检测的线性范围为5.6×10^-12mol／L～1.12×10^-9mol／L，检测限可以达到1.42×10^-12mol／L。该方法对凝血酶蛋白有很好的特异性，其他蛋白如溶菌酶和牛血清白蛋白的存在对于检测没有影响。第三章利用互补核酸杂交富集金胶实现信号扩增的凝血酶蛋白质的特异性电化学检测研究在本文中，介绍了一种利用互补核酸杂交富集金胶实现信号扩增的特异性蛋白检测研究。以凝血酶蛋白为研究对象，利用凝血酶蛋白相对应的二段核酸适体，适体Ⅰ固定在磁性颗粒上用于特异性地捕获蛋白，适体Ⅱ标记金胶作为检测信标。由凝血酶蛋白和相对应的二段核酸适体构建三明治结构的特异性识别凝血酶的蛋白检测方法。另外，再通过信标金胶上过剩的核酸适体链与另一段标记有金胶的互补核酸进一步杂交，获得金胶的选择性聚集，实现信号扩增。通过信号扩增，此传感器的灵敏度大大提高，对凝血酶蛋白的检测下限可以达到4.52×10^-15mol／L。平行测定浓度为7.47×10^-14mol／L的凝血酶八次，其RSD为3.0％。第四章基于硫代三聚氢酸和金纳米颗粒形成网状结构实现信号扩增的超灵敏凝血酶蛋白质电化学检测的研究在本文中，介绍了一种利用金纳米颗粒和硫代三聚氢酸来实现信号扩增的超灵敏度和高度特异性的蛋白质的电化学检测方法。以凝血酶蛋白为研究对象，由凝血酶蛋白和相对应的二段核酸适体构建三明治结构的凝血酶蛋白特异性检测方法的基础上，再通过硫代三聚氢酸和金纳米颗粒的自组装作用，形成金纳米颗粒和硫代三聚氢酸的网状结构，获得金纳米颗粒的选择性聚集，实现信号扩增。通过信号扩增，该方法的灵敏度大大提高，对凝血酶蛋白的检测下限可以达到7.82 amol／L。该电化学检测方法对凝血酶蛋白有很好的特异性，其他蛋白如溶菌酶和牛血清白蛋白对于检测没有影响。可望用于其他蛋白的检测，疾病的诊断和其他的生物研究领域。第五章基于金纳米颗粒选择性聚集实现信号扩增特异性电化学检测p53基因的研究在本文中，介绍了一种利用金纳米颗粒的聚集实现信号放大作用的超灵敏和高度特异性的电化学方法用于人类p53肿瘤抑制剂基因研究。在实验中，根据p53基因的序列设计并合成了能特异性检测p53肿瘤抑制剂基因的二段探针，在一段探针上固定磁性颗粒以捕获并富集目标DNA，在另一段探针上标记金纳米颗粒作为检测信标。另外，再通过硫代三聚氢酸和金纳米颗粒的自组装作用，形成金纳米颗粒和硫代三聚氢酸的网状结构，获得金纳米颗粒的选择性聚集，实现信号扩增。用此法检测得到的目标野生型DNA，最低检测限为2.24×10^-17mol／L。在同样浓度的条件下，单碱基错配的突变型序列和完全互补的野生型序列的区分度为57.1％。第六章基于纳米金胶和磁性纳米颗粒和利用取代反应电化学特异性识别凝血酶蛋白质的研究本文介绍了一种基于取代反应的特异性识别凝血酶的电化学方法。利用金胶标记的互补核酸和固定在磁性纳米颗粒上的核酸适体的杂交，形成双链的核酸适体／互补核酸，在凝血酶蛋白的存在下，核酸适体与凝血酶蛋白特异性的结合，形成G-四分体的结构，使得原来的双链解离，标记有金胶的互补核酸游离出来，通过检测游离的互补核酸上标记的金的电化学信号，实现对凝血酶蛋白的特异性检测，其最低检测限可达1.22×10^-11mol／L。该方法十分简单有效，无需对蛋白进行标识，为蛋白质的检测提供了一种新的思路。第七章利用目标蛋白存在下的取代反应研究核酸适体-互补核酸和核酸适体-蛋白之间结合的热力学特性本文介绍了一种基于目标蛋白的取代反应来研究核酸适体-互补核酸和核酸适体-目标蛋白之间的相互作用，以凝血酶蛋白为研究对象，比较了凝血酶蛋白的两段核酸适体，核酸适体Ⅰ（aptamerⅠ）和核酸适体Ⅱ（aptamerⅡ）与互补核酸和目标蛋白作用的情况，并确定了核酸适体-互补核酸的解离反应和在目标蛋白存在下取代反应的平衡常数、标准焓变和标准熵变等热力学参数，结果表明该取代反应是一个熵驱动的自发过程，熵驱动从双链的核酸转变为核酸适体-目标蛋白的复合物。核酸适体与目标蛋白的特异性结合是一个放热过程。该特异性结合的主要作用是核酸适体与凝血酶之间的高度的分子构型的匹配、氢键力和沟槽作用。该热力学研究会对核酸适体-互补核酸和核酸适体-目标蛋白之间的结合过程的机理有一个更深的理解，将有助于我们进一步揭示核酸与蛋白这两种生命中最关键物质之间的相互作用和关系，对于我们更好地理解基本的生物过程和预测设计适体生物检测方法，发展用于疾病诊断的方法有着重要的意义。更多还原

【Abstract】 With the development of the Human Genome Project, Gene diagnosis has become an important area in molecular biology and biomedical research. The detection of specific DNA sequence in human body, viral and bacteria nucleic acid is playing a more and more important role in disease diagnose, food pollution, legal medical examination and environment detection. Wide-scale genetic testing requires the development of easy-to-use, fast, inexpensive, miniaturized devices. Many new biological technologies emerged and found their applications in this field. Among them, DNA detection technologies relying on the DNA base-pair recognition are rapidly developed and have received considerable attentions. Electrochemical DNA detection is a novel and developing technique that combining biochemical, electrochemical, medical and electronic techniques with the advantages of being simple, reliable, cheap, sensitive and selective for genetic detection, and has been a hot topic in the field of biochemistry and medicine.With the accomplishment of Human Genome Project and the rapid progress of proteomic strategies, protein detection has been a topic of significant interest. Intense research activities are carried out worldwide to develop rapid, simple, specific and sensitive detection devices for protein in medical diagnostics and biomedical research application, while the detection methodologies for protein based on antibodies cannot meet the demand of the human proteome. Now aptamers have been emerging as a new protein recognition element in wide range of bioassays. Aptamers have attracted a considerable attention due to their ability to bind target protein with high affinity and specificity and have many advantages over antibodies, including simpler synthesis, easier storage, reproducibility, and wider applicability. These properties make aptamers ideal candidates as protein recognition elements in a wide range of bioassays and for the development of diseases diagnostics.The emergence of nanotechnology is opening new horizons for the application of nanoparticles in analytical chemistry. Nanoparticles are of considerable interest owing to their unique physical and chemical properties, and offer excellent prospects for biosensor. The power and scope of such nanoparticles can be greatly enhanced by coupling them with the high specificity of the aptamer and the high sensitivity of the electrochemical recognitions.Nucleic acid and protein are the most important biomolecules in life. Nucleic acid is in charge of transferring genetic information and coding other biomolecules, while protein has run through the whole life process. The investigation on the interaction of the nucleic acids and protein has been one of the most important areas. The information of the detailed and fundamental nature of the interaction obtained will help to understand the organization of biological systems and contribute to control and adjust the life process.The goal of the present study is to design and optimize new electrochemical techniques with high sensitivity and selectivity. This paper combines the excellent characteristics of nanoparticles, base-complementary and the specific recognition of protein. The high sensitivity of electrochemical device coupled to their compatibility with modem micro-fabrication technologies, portability, low cost, minimal power requirements, and independence of sample turbidity, made them excellent candidates for DNA and protein detection and also it would broaden the research field of post-genetic time. The dissertation includes seven parts: Chapter One: introductionIn the beginning of this dissertation, the basic composition of a DNA biosensor and its classification were systematically reviewed, especially the design of a DNA electrochemical biosensor was introduced, together with the application and development of DNA biosensor. Secondly, we introduced the concept of the aptamer, emphatically reviewed the operational principle, classification, application and development trends of aptamer biosensor. Thirdly, nanoparticles were introduced, especially the application of gold nanoparticles and magnetic nanoparticles in the biosensor was presented. At last, the purpose and the significance of the dissertation were pointed out.Chapter Two: Study on an electrochemical detection for thrombin based on aptamers and nanoparticlesA high specific, sensitive electrochemical recognition of thrombin based on aptamers and nanoparticles was presented. Two different aptamers were chosen to construct a sandwich manner for detecting thrombin. Aptamer I was immobilized on nano magnetic particle for capturing thrombin, and aptamer II labled with nano gold was used for detection. The electrical current generated from gold after the formation of the complex of magnetic particle, thrombin and nano gold, and then an electrochemical cell designed by ourselves was used for separating, gathering, and electrochemical detecting. Through magnetic separation, high specific and sensitive detection of the target protein, thrombin, was achieved. Linear response was observed over the range 5.6 × 10¹² mol/L - 1.12 × 10^-9 mol/L, with a detection limit of 1.42×10^-12 mol/L. The presence of other protein as BSA did not affect the detection, which indicates that high selective recognition of thrombin can be achieved in complex biological samples such as human plasma.Chapter Three: An Amplified electrochemical biosensor for thrombin based on the enrichment of gold nanoparticles through the hybridization with the complementary oligonucleotideAn amplified electrochemical biosensor for thrombin based on the enrichment of the gold nanoparticles through the hybridization with the complememtary oligonucleotide was presented. Through the specific recognition for thrombin, a sandwich format of magnetic nanoparticle-aptamerI / thrombin / gold nanoparticle-aptamerII was fabricated. And the signal amplification was further implemented by the enrichment of gold nanoparticles through the hybridization with the gold labeled complementary oligonucleotide, the resulting hybridization is capable of realizing more gold nanoparticle markers attaching for one target protein, thrombin, and hence offer a remarkable amplification of detecting thrombin. A significant sensitivity enhancement was obtained for detection of thrombin with the signal amplification. This new strategy allows the detection of the target protein down to the 4.52 ×10^-15 mol//L. The relative standard deviation of eight replicate determinations of 7.47×10^-14 mol/L thrombin was 3.0%.Chapter Four: A new amplification strategy for ultrasensitive electrochemical detection with network-like thiocyanuric acid/gold nanoparticlesAn ultrasensitive and highly specific electrochemical detection for thrombin based on gold nanoparticles and thiocyanuric acid is presented. For this proposed aptasensor, aptamer I was immobilized on the magnetic nanoparticles, aptamer II was labeled with gold nanoparticles. Through the specific recognition for thrombin, a sandwich format of magnetic nanoparticle /thrombin / gold nanoparticle was fabricated, and the signal amplification was further implemented by forming network-like thiocyanuric acid /gold nanoparticles. A significant sensitivity enhancement had been obtained, and the detection limit was down to 7.82 aM. The presence of other proteins such as BSA and lysozyme did not affect the detection of thrombin, which indicates a high specificity of thrombin detection could be achieved. This electrochemical detection is expected to have wide applications in protein monitoring and disease diagnosis.Chapter Five: An ultrasensitive electrochemical approach for detection of p53 sequence based on the aggregation of gold nanoparticlesA new approach has been developed for the detection of human tumor suppression p53 gene. Two DNA sequences were designed, and the combined sequences are complementary to that of the target p53 sequence, one was immobilized on the magnetic nanoparticles for capturing the target p53 sequence, the other one is labeled with gold nanoparticles as probe, and the sandwich format was formed for detecting the p53 sequences. The ultrasensitive detection was achieved by the aggregation of gold nanoparticles with introducing thiocyanuric acid into the system, and the detection limit could go down to 2.24×10^-17 mol/L. The mutant type p53 DNA sequence could be obviously distinguished from the wide type p53 DNA sequence. Chapter Six: A displacement assay for protein based on gold nanoparticels and magnetic nanoparticlesA nanoparticle based displacement protocol for detection of thrombin is described. The assay relies on hybridizing the magnetic nanoparticle immobilized aptamer with gold nanoparticles labeled complementary DNA, followed by an incubation assay with the target protein. In the presence of thrombin, aptamer prefers to form G-quarter structure with the target protein, resulting in the release of the gold nanoparticle labeled DNA. A high sensitive detection of thrombin was achieved by the excellent electrochemical signal of gold, and its detection limit could go down to 1.22×10^-11 mol/L. The assay responds rapidly and specifically to the target protein andrequires no label of the protein and without other reagents.Chapter Seven: A Thermodynamic investigation into the binding affinitiesbetween aptamer-DNA and aptamer-protein based on the displacement reactionA thermodynamic investigantion into the binding affinities between aptamer-DNA and aptamer-protein based on the displacement reaction was presented. Some thermodynamic parameters such as the equilibrium constant, enthalpy and entropy of the dissociation reaction and the displacement reaction were evaluated. The results showed that the change of entropy played an important role in the conversion of the duplex DNA into aptamer-protein binding. And it was also deduced that the binding reaction of aptamer toward thrombin is exothermic, its high affinity and specificity are generally achieved by a combination of complementary molecular shapes, hydrogen bonding, and stacking interactions. This has been the first attempt to compare the stability of the duplex DNA and the DNA-protein complex, and it will provide an insight into how the conformation changes. The obtained thermal parameter will direct us to find out the basic principle for designing the aptamer based biosensor and to deeply understand the thermal properties of the aptamer toward protein, and it will be great importance of developing new methods for disease diagnosis.更多还原