【作者】 王振光；

【导师】 姜玮；

【作者基本信息】 山东大学 ， 分析化学， 2013， 硕士

【摘要】 单分子检测(Single-molecule detection, SMD)以单个分子为研究对象,是一种在单个分子水平上获取分子信息的检测手段。通过SMD可以获取在传统的种群平均方法中无法获得的分子个体信息,特别是在生物大分子的探测和研究中,SMD具有传统方法无法比拟的优势。单分子定量检测更是分析化学的终极目标,通过对一定体积中分子的数目进行清点、统计处理来定量检测溶液的浓度或分子的数量。与传统的依据强度进行定量的方法相比,单分子计数是根据目标分子的个数进行定量,更适宜于超低浓度下生物分子的定量检测。近年来,结构DNA纳米技术(Structural DNA nanotechnology)为分析化学提供了有力的工具和方法。结构DNA纳米技术即以DNA为原料,通过碱基配对、单链DNA的折叠构造出具有特定形状和功能的纳米结构。人们已经成功构造了各种二维和三维图形,研究工作除了构建材料外,还扩展到生物传感、纳米机械器件构建和DNA纳米计算等研究领域。鉴于单分子定量检测的重要性,我们利用结构DNA纳米材料构建了单分子定量检测新方法。本文共分为四个部分：第一章为绪论部分,概述了单分子检测及单分子定量检测的背景、原理、研究意义、检测方法以及现阶段存在的问题。并介绍了结构DNA纳米材料的发展过程以及在生物分析方面的应用。第二章首先设计了三个顶点氨基修饰一个顶点具有粘性末端的DNA四面体结构。三个氨基可以和盖玻片上的环氧基结合用于构建了四面体DNA修饰的基底,最后我们对基底抑制生物分子非特异性吸附能力进行了研究,结果表明四面体DNA修饰的基底可以有效的抑制非特异性吸附且优于文献报道的方法。第三章立足于单分子定量检测中非特异性分子吸附的问题,我们将构建的四面体DNA修饰基底用于DNA和蛋白质分子定量检测,该方法中固定在盖玻片上的四面体DNA具有粘性末端,粘性末端可以用于捕获目标物,我们首先将粘性末端设计为目标DNA的互补序列,将目标DNA捕获,加入生物素修饰的检测DNA及链酶亲和素包被的量子点形成三明治夹心结构。最后,通过落射荧光显微镜对与目标DNA一一对应的量子点进行成像、计数并定量检测。方法的线性范围是1.0×10－15mol/L—5.0×10-14mol/L,检出限为1.0×10-15mol/L。随后,将粘性末端设计为凝血酶的适体,用于捕获凝血酶。与DNA检测类似构建凝血酶-生物标记适体-量子点的复合结构,并成像计数,对凝血酶检测的线性范围是1.0×10-9mol/L—5.0×10-8mol/L,检出限为1.0×10-9mol/L。此外特异性分析及复杂基质影响研究结果表明该方法对目标物具有良好的选择性和复杂体系适用性,上述方法有望用于疾病的前期诊断。第四章设计了基于四面体DNA保护及基底富集定量检测单个DNA分子的方法。本章设计了让目标物分子在溶液中与其他分子结合,最后在基底上富集检测的分析策略。为了抑制非特异性吸附,我们在磁球上连接四面体DNA,加入目标物、生物素标记的检测DNA、链酶亲和素包被的量子点然后形成目标DNA与量子点一一对应的复合物；然后加入限制性内切酶,对检测DNA上的切割位点进行切割,使得量子点脱落并在生物素化的基底上富集,最后荧光成像检测。更多还原

【Abstract】 Single-molecule detection (SMD), which measures and gets informations form single molecule has attracted lots of attention in analytical chemistry and life science. We can get the small or rare differences between ostensibly identical molecules cannot be detected, especially for the investigation of dynamic and kinetics. As analytical chemists, the highest resolution measurement one can make is at the single molecule level. Through counting the number of molecules in certain volum, the concentration of molecules can be obtained; It is accomplished by counting target molecules one by one rather than measuring the signal intensity, which is suitable for ultralow concentrations of bio-molecules quantitation.Recently, Structural DNA nanotechnology has provided a powerful tool for analytical chemistry. We can get nanomaterials with different shape and functions, through base paring and folding of DNA strands. Up to now, a variety of2-D and3-D DNA nanomaterials have been constructed and they have been applied to biosensor, construction of nanodevice and DNA computing.As for the importance of SMD, we developed Quantitative SMD methods based on SDN. There are three parts in this article and the main contents are as follows:The first chapter is an introduction section to summarize the background, principle and the currents problems of SMD. The development and apllication of SDN have also been described.In the second chapter, a DNA tetrahedron structure with three amino groups and one pendant probes were positioned at the vertices was designed. Then, it was introduced onto the glass slide through the reaction between amino groups and epoxy groups. The pendant probes could be further used for carpturing target DNA. We also measured the nonspecific adsorption of quantum dots (QDs) on the substrate, which demonstrates that DNA tetrahedron modified substrates can prevent nonspecific adsorption better than repoted ones.In the third chapter, in order to solve the problem of nonspecific adsorption in SMD, we developed quantitative SMD methods using DNA tetrahedron modified substrates. Target DNA or proteins were carptured onto subtrate by DNA tetrahedron, which were then labeled with QDs. Fluorescent images were acquired by epi-fluorescence microscopy. Number of dots in those images was counted one by one for quantification. There was a linear relationship between number of dots and the logarithm concentration of target DNA in the range of1.0fM to50fM. Then, we designed a aptamer of thrombin in the DNA tetrahedron for capturing thrombin. QDs were also used to label thrombin for fluorescent imaging and counting. There was a linear relationship between number of dots and the logarithm concentration of target DNA in the range of1.0nM to50nM. The results showed above methods had a good selectivity to target molecules and it is possible to be used in the disease diagnosis.In chapter four, we designed a single DNA detection method based on the protection of DNA tetrahedron and the enriching on substrates. In this method, targets are designed to react with others in free solution and they are collected on substrates for counting. In order to control the nonspecific adsorption, DNA tetrahedron is introducted to the surface of magic beads. The complex of sandwich among DNA tetrahedron, target DNA and QDs can be easily formed, which can be cutted by DNA enzmy to release QDs. The released QDs are collected on substrates through the reaction between streptavidin and biotin on the substrates. The images of QDs on the substrates are obtained by epi-fluoreseeneemieroseopy.更多还原