新型DNA功能化材料的制备及其在核酸和蛋白检测中的应用

发布时间：2018-06-13 15:59

本文选题：自组装 + 金属有机骨架材料　；参考：《武汉大学》2015年博士论文

【摘要】：DNA作为一种智能高分子,因具备重要的生物学功能、生物相容性、分子识别能力以及可调控性,使其成为目前自然科学领域研究的热点。以DNA分子为模板或配体所制备的功能化纳米材料,兼备纳米材料特有的光电性质和在纳米维度上更好的调谐性。随着纳米生物技术在分析化学中的广泛应用,DNA功能化纳米材料已成为建立新型分析方法的有力工具。但是,现有的DNA功能化纳米材料存在制备步骤繁琐、稳定性差及生物毒性等缺点,因此建立新型的DNA功能化纳米材料制备方法并拓展其在生化分析领域的应用具有重要意义。本论文以建立DNA功能化纳米材料制备新方法为主线,分别从纳米材料的酶促合成、纳米材料的自组装、纳米材料的形貌调控和杂化纳米材料的构建等角度,对DNA功能化纳米材料的制备和性质进行了系统研究,并将所制备的DNA功能化纳米材料应用于核酸和蛋白质的分析检测,具体内容包括以下四个工作：1、提出了酶促合成铜银合金纳米簇的新方法。以焦磷酸盐与铜离子螯合形成的复合物为酶的底物,利用碱性磷酸酶对焦磷酸特异性的脱磷酸作用释放出铜离子。在DNA模板和银离子存在的条件下,以硼氢化钠为还原剂,制备得到了DNA功能化的铜银纳米合金簇。碱性磷酸酶作为一种通用型的抗体标记酶,在酶联免疫分析中有着广泛的应用。我们将所建立的合金簇酶促合成新方法与酶联免疫分析相结合,应用于肿瘤相关蛋白的检测。利用酶促合成与酶联免疫分析相结合,提高了DNA功能化金属团簇在蛋白质检测中的选择性,并且有效地克服了实际样品中高丰度蛋白对检测结果的干扰。2、建立了DNA功能化银纳米簇组装的新方法。以DNA滚环扩增反应的产物为模板,通过DNA的碱基互补配对,室温杂交得到了DNA功能化的银簇纳米线,实现了银纳米簇到银纳米线的形貌变化。在每个组装单元内,引入一条富含G碱基的DNA序列,利用富G碱基序列增强纳米银团簇发光的现象,可以在共聚焦显微镜下观察到银纳米簇组装前后,视野由暗到出现红色的银纳米线,表现出潜在的成像应用前景。当存在目标DNA分子时,形成银簇的组装体,基于滚环放大的扩增效应和银簇靠近富G碱基序列前后的荧光增强效应,实现了DNA分子的灵敏检测。3、提出了铜纳米颗粒的可控合成新方法。利用末端转移酶的无模板链延伸特性,在引物DNA的3’端延长得到聚胸腺嘧啶的DNA序列。当存在抗坏血酸和铜离子时,可在聚胸腺嘧啶序列部分还原得到红色荧光的铜纳米颗粒。研究发现,与改变酶反应时间相比,改变酶浓度对合成产物的影响更为明显。通过对合成所得到的铜纳米颗粒进行表征,发现改变末端转移酶的浓度可以对铜纳米颗粒的大小及其荧光寿命进行调谐。利用多聚核酸激酶与DNA的3’端磷酸基团的相互作用,建立多聚核酸激酶的灵敏检测方法。4、制备了DNA功能化的金属有机框架材料。金属化有机框架材料的配体和DNA分子中核苷之间存在π-π堆积作用,使得探针DNA吸附于金属有机骨架材料表面。金属框架材料中含有铜离子中心的单元结构能够有效猝灭所标记的荧光染料,当存在目标DNA分子时,形成的双链DNA结构使得染料远离金属框架材料表面,破坏了光致电荷转移效应,染料荧光恢复。我们采用同步荧光扫描的方法,实现了DNA功能化的金属有机框架材料对两种DNA分子的同时检测。
[Abstract]:As a kind of intelligent polymer, DNA has become a hot topic in the field of natural science because of its important biological functions, biocompatibility, molecular recognition ability and regulation. The functional nano materials prepared by DNA molecules as templates or ligands are also prepared by the specific photoelectric properties of nanomaterials and in nanometers. With the wide application of nano biotechnology in analytical chemistry, DNA functionalized nanomaterials have become a powerful tool for the establishment of new analytical methods. However, the existing DNA functionalized nanomaterials have the shortcomings of tedious preparation steps, poor stability and biological toxicity, so a new type of DNA functionalized nanomaterials has been established. It is of great significance to prepare and expand its application in the field of biochemical analysis. In this paper, a new method for the preparation of DNA functionalized nanomaterials is set up as the main line. The DNA functionalized nanomaterials are prepared from the aspects of enzymatic synthesis of nanomaterials, self-assembly of nanomaterials, morphology control of nanomaterials and the construction of hybrid nanomaterials. The preparation and properties were systematically studied, and the prepared DNA functionalized nanomaterials were applied to the analysis and detection of nucleic acids and proteins. The specific contents included four tasks: 1, a new method of enzymatic synthesis of copper and silver nanoclusters was proposed. The complexes formed by the chelating of pyrophosphate and copper ions were the substrates of the enzyme, and the alkaline phosphorus was used. Copper ions are released by the acid enzyme specific dephosphoric acid dephosphoric acid. Under the presence of DNA template and silver ion, a DNA functionalized copper silver nanoalloy cluster is prepared with sodium borohydride as a reductant. Alkaline phosphatase, as a generic antibody labeling enzyme, is widely used in ELISA. The new method of enzyme linked immunosorbent assay combined with enzyme linked immunoassay (ELISA) was applied to the detection of tumor related proteins. By combining enzyme synthesis with ELISA, the selectivity of DNA functional metal clusters in protein detection was improved, and the results were effectively overcome by the high abundance proteins in the actual samples. .2, a new method of DNA functionalized silver nanocluster assembly was established. The product of DNA rolling ring amplification reaction was used as a template. The silver cluster nanowires of DNA functionalized by hybridization at room temperature were obtained by the base complementary pairing of DNA. The morphology changes of silver nanowires to silver nanowires were realized. In each assembly unit, a DNA sequence rich in G base was introduced. By using the rich G base sequence to enhance the luminescence of nanoscale clusters, the silver nanowires can be observed under the confocal microscope before and after the assembly of silver nanoclusters. The potential imaging application prospects are shown from the dark to the appearance of red silver nanowires. When the target DNA molecule exists, the assembly of the silver cluster is formed, based on the amplification effect and silver of the roll ring amplification The fluorescence enhancement effect before and after the cluster near the rich G base sequence has realized the sensitive detection of.3 by the DNA molecule. A new method of controllable synthesis of copper nanoparticles was proposed. The DNA sequence of polythymine was extended at the 3 'end of the primer DNA by using the non template chain extension of the terminal transferase. When the ascorbic acid and copper ions were present, they could be in the polythorax. It was found that the influence of the change of the enzyme concentration on the synthesized product was more obvious than that of the change of the enzyme reaction time. By characterizing the copper nanoparticles obtained by the synthesis, it was found that the concentration of the terminal transferase could be changed to the size of the copper nanoparticles and the fluorescence longevity. The interaction between polynucleic acid kinase and the 3 'terminal phosphoric acid group of DNA was used to establish a sensitive detection method for polynucleic acid kinase (.4), and the DNA functionalized metal organic frame material was prepared. There was a pion pion accumulation between the ligand of the metallized organic frame material and the nucleosides in the DNA molecule, so that the probe DNA was adsorbed on the metal. The organic framework material surface. The element structure containing copper ion center in the metal frame material can effectively quench the labeled fluorescent dye. When the target DNA molecule exists, the double stranded DNA structure makes the dye far away from the surface of the metal frame material and destroys the photoinduced charge transfer effect and the dye fluorescence recovery. We use the synchronous fluorescence. Scanning method realized the simultaneous detection of two kinds of DNA molecules by DNA functionalized metalorganic frameworks.
【学位授予单位】：武汉大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：TB383.1

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