多元核—壳型荧光纳米生物标记物的制备与应用及噬菌体介导的新型免疫-PCR体系的建立

发布时间：2018-07-04 07:54

本文选题：荧光纳米 + 时间分辨　；参考：《厦门大学》2007年硕士论文

【摘要】： 本论文主要围绕超高灵敏免疫诊断新技术开展研究工作。时间分辨荧光免疫分析(TRFIA)以及免疫-PCR技术是现代免疫分析方法中两个极为引人注目的热点领域,特别适合于临床上极微量抗原、抗体的检测及病原微生物痕量检测等。本论文选题于这两个研究方向,并开展相关的研究工作。全文主要包括三个部分:综述了各种类型的高灵敏免疫检测技术;新型稀土鳌合剂2,9-双[N,N-双(羧甲基)氨甲基]-1,10-(菲咯啉){BBCAP}的合成及多元核-壳型荧光纳米生物标记物的制备;天然噬菌体介导的免疫-PCR模型的建立。第一章为绪论,介绍了各种类型的高灵敏免疫检测技术,并简要介绍高灵敏检测技术发展的最新成果。第二章主要为稀土螯合剂BBCAP的合成及多元核壳型荧光纳米标记物的制备、表征及应用。首先合成了一种新型稀土螯合剂BBCAP,该螯合剂具有良好的水溶性,即可与铕(Eu)、铽(Tb)等稀土金属离子形成强荧光络合物,不需要加入增强液,且与稀土离子所形成的络合物荧光强度十分稳定。将纳米荧光材料制备技术与时间分辨荧光免疫测定法相结合,建立了以荧光纳米颗粒为生物标记物的高灵敏度时间分辨荧光免疫分析方法。稀土螯合剂BBCAP与3-氨丙基三甲氧基硅烷(APTMS)室温搅拌下反应2 h,合成功能化APTMS-BBCAP复合体,可以与Eu(III)、Tb(III)进行螯合,形成了APTMS-BBCAP-Eu(III)/Tb(III)前躯体。将前躯体加入到油包水型(W/O)微乳液体系,通过优化乳液中各组分的配比和氨水的量来控制纳米颗粒的大小和四乙氧基硅烷的水合化、聚合速度,得到了直径在35-55 nm之间的硅胶包裹稀土络合物荧光纳米颗粒,该荧光纳米颗粒具有较好的分散性,光谱性质与前驱体基本相同,但其抗光漂白能力要高于前驱体,更远高于有机荧光染料。控制Tb(III)及Eu(III)二者之间的比例,制备出一系列多元核-壳型荧光纳米颗粒,配合紫外灯及合适的滤光片可以分辨出从绿光到红光间一系列的颜色差异。多元型核-壳荧光纳米颗粒能够方便地引入氨基、巯基、环氧基活性基团等,经过表面修饰后的荧光纳米颗粒,可以与抗体、抗原、寡核糖核苷酸等生物大分子交联。这种多元核-壳型荧光纳米作为生物标记物在免疫分析,细胞成像及DNA杂化等领域将具有广阔的应用空间。我们选取了其中一种Tb(III)纳米颗粒,采用“三明治”夹心法对人血清中乙肝表面抗原(HBsAg)进行了检测,以空白的3倍相对偏差除以工作曲线的斜率得出本体系检测限为35 pg/ml(3σ/s)。免疫分析应用结果表明核-壳型荧光纳米作为一种生物标记物用于时间分辨荧光生物分析具有进一步深入研究的价值。第三章描述了新型免疫PCR体系的建立及评价。选取具有天然纳米结构的双链DNA噬菌体T7,通过异型双功能交联剂Sulfo-SMCC活化,使其形成具有巯基反应活性的T7噬菌体。抗HBsAg抗体可经Traut’s Reagent引进巯基,形成巯基化抗体。二者交联,经超滤纯化,得到了T7噬菌体表面带有抗体分子的T7-Antibody复合物。以此作为检测探针,初步建立了一种超灵敏的免疫-PCR体系。通过采用1%戊二醛处理聚丙烯PCR管,使得免疫反应和PCR反应能在同一管进行。实验结果表明,HBsAg在1000 ng/ml-0.01 ng/ml范围内呈现良好的线性关系(R=0.98),检测限达到0.1 pg/ml。实验检测了29份阳性血清和45份阴性血清,其结果与ELISA结果吻合。结果证实了该噬菌体介导的免疫PCR检测具有可行性和应用价值。
[Abstract]:This paper mainly focuses on the research of ultra high sensitive immunodiagnostic technology. Time resolved fluoro immunoassay (TRFIA) and immune -PCR technology are two most attractive areas of modern immunoassay. It is especially suitable for the clinical extreme microantigen, anti body detection and trace detection of pathogenic microorganism. Wen Xuan, which consists of these two research directions, and carried out the relevant research work. The full text includes three parts: a summary of various types of high sensitive immunoassay techniques; the synthesis of a new rare earth chelating mixture 2,9- double [N, N- double (Suo Jiaji) ammonia methyl]-1,10- (phenanthrene) {BBCAP} and the preparation of multiple nuclear shell fluorescent nanoscale biomarkers; The establishment of an immunized -PCR model mediated by phage.
The first chapter is an introduction, introducing various types of highly sensitive immunoassay techniques, and briefly introducing the latest development of highly sensitive detection technology.
The second chapter mainly consists of the synthesis of rare earth chelating agent BBCAP and the preparation, characterization and application of multiple core shell fluorescent nanoparticles. First, a new type of rare earth chelating agent, BBCAP, has been synthesized. The chelating agent has good water solubility and can form strong fluorescent complexes with rare earth metals such as europium (Eu) and terbium (Tb). The fluorescence intensity of the complex formed by rare earth ions is very stable. A highly sensitive time resolved fluoro immunoassay for the fluorescent nanoparticles as biomarkers is established by combining the preparation of nanofluorescence materials with the time resolved fluorometric immunoassay method. The rare earth chelating agent BBCAP and 3- ammonia propyl trimethoxane (APTMS) chamber A functional APTMS-BBCAP complex can be synthesized by the reaction of 2 h under warm stirring, which can be chelated with Eu (III), Tb (III) and form a APTMS-BBCAP-Eu (III) /Tb (III) precursor. The body is added to the oil package (W/O) microemulsion system. The size of the nanoparticles and four ethoxy silicon are controlled by optimizing the composition of the components in the emulsion and the amount of ammonia water. In the hydration and polymerization rate of alkane, the silica gel coated rare earth complex fluorescent nanoparticles with a diameter of 35-55 nm are obtained. The fluorescent nanoparticles have better dispersibility and the spectral properties are basically the same as those of the precursors, but their photobleaching ability is higher than that of the precursors and is much higher than the organic fluorescent dye. The control of Tb (III) and Eu (III) two A series of multi core shell type fluorescent nanoparticles are prepared. With the UV lamp and the suitable filter, a series of color differences can be distinguished from green light to red light. The multi type nuclear shell fluorescent nanoparticles can easily be introduced into the amino, sulfhydryl, epoxy group and so on. The surface modified fluorescent nanoparticles can be easily introduced. It can be cross linked with biological macromolecules, such as antibodies, antigens, oligosaccharide nucleotides, and so on. This multicore shell fluorescent nanometer, as a biomarker, will have wide application space in the fields of immunoassay, cell imaging and DNA hybridization. We have selected one of the Tb (III) nanoparticles and sandwich sandwich method to the human serum B The detection of liver surface antigen (HBsAg) was carried out. The detection limit of the system was 35 pg/ml (3 Sigma /s) by dividing the slope of the blank 3 times relative deviation by the slope of the work curve. The results of immunoassay showed that the nuclear shell fluorescent nanoparticles were used as a biomarker for the time resolved fluorescence biological analysis.
The third chapter describes the establishment and evaluation of a new immune PCR system. A double stranded DNA phage T7 with natural nanoscale structure is selected to form a T7 phage with sulfhydryl reactive activity through the activation of a heterogeneous bifunctional crosslinking agent Sulfo-SMCC. The anti HBsAg antibody can be induced by Traut 's Reagent to form sulfhydryl group and to form mercapto antibody. The cross-linking of the anti HBsAg antibody can be cross linked. T7-Antibody complex with antibody molecules on the surface of T7 phage was obtained by ultrafiltration. As a detection probe, a super sensitive immune -PCR system was established. Using 1% glutaraldehyde to treat polypropylene PCR tubes, the immune response and PCR reaction could be carried out on the same tube. The experimental results showed that HBsAg was in 1000 ng/ml-0.01 ng/. A good linear relationship (R=0.98) was presented in the range of ML. The detection limit reached 0.1 pg/ml. and 29 positive sera and 45 negative sera were detected. The results were in agreement with the ELISA results. The results confirmed the feasibility and application value of the phage mediated immune PCR detection.
【学位授予单位】：厦门大学
【学位级别】：硕士
【学位授予年份】：2007
【分类号】：R392

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