本文選題：熒光納米 + 時(shí)間分辨��； 參考：《廈門大學(xué)》2007年碩士論文

【摘要】： 本論文主要圍繞超高靈敏免疫診斷新技術(shù)開展研究工作。時(shí)間分辨熒光免疫分析(TRFIA)以及免疫-PCR技術(shù)是現(xiàn)代免疫分析方法中兩個(gè)極為引人注目的熱點(diǎn)領(lǐng)域,特別適合于臨床上極微量抗原、抗體的檢測(cè)及病原微生物痕量檢測(cè)等。本論文選題于這兩個(gè)研究方向,并開展相關(guān)的研究工作。全文主要包括三個(gè)部分:綜述了各種類型的高靈敏免疫檢測(cè)技術(shù);新型稀土鰲合劑2,9-雙[N,N-雙(羧甲基)氨甲基]-1,10-(菲咯啉){BBCAP}的合成及多元核-殼型熒光納米生物標(biāo)記物的制備;天然噬菌體介導(dǎo)的免疫-PCR模型的建立。 第一章為緒論,介紹了各種類型的高靈敏免疫檢測(cè)技術(shù),并簡(jiǎn)要介紹高靈敏檢測(cè)技術(shù)發(fā)展的最新成果。 第二章主要為稀土螯合劑BBCAP的合成及多元核殼型熒光納米標(biāo)記物的制備、表征及應(yīng)用。首先合成了一種新型稀土螯合劑BBCAP,該螯合劑具有良好的水溶性,即可與銪(Eu)、鋱(Tb)等稀土金屬離子形成強(qiáng)熒光絡(luò)合物,不需要加入增強(qiáng)液,且與稀土離子所形成的絡(luò)合物熒光強(qiáng)度十分穩(wěn)定。將納米熒光材料制備技術(shù)與時(shí)間分辨熒光免疫測(cè)定法相結(jié)合,建立了以熒光納米顆粒為生物標(biāo)記物的高靈敏度時(shí)間分辨熒光免疫分析方法。稀土螯合劑BBCAP與3-氨丙基三甲氧基硅烷(APTMS)室溫?cái)嚢柘路磻?yīng)2 h,合成功能化APTMS-BBCAP復(fù)合體,可以與Eu(III)、Tb(III)進(jìn)行螯合,形成了APTMS-BBCAP-Eu(III)/Tb(III)前軀體。將前軀體加入到油包水型(W/O)微乳液體系,通過優(yōu)化乳液中各組分的配比和氨水的量來控制納米顆粒的大小和四乙氧基硅烷的水合化、聚合速度,得到了直徑在35-55 nm之間的硅膠包裹稀土絡(luò)合物熒光納米顆粒,該熒光納米顆粒具有較好的分散性,光譜性質(zhì)與前驅(qū)體基本相同,但其抗光漂白能力要高于前驅(qū)體,更遠(yuǎn)高于有機(jī)熒光染料�？刂芓b(III)及Eu(III)二者之間的比例,制備出一系列多元核-殼型熒光納米顆粒,配合紫外燈及合適的濾光片可以分辨出從綠光到紅光間一系列的顏色差異。多元型核-殼熒光納米顆粒能夠方便地引入氨基、巰基、環(huán)氧基活性基團(tuán)等,經(jīng)過表面修飾后的熒光納米顆粒,可以與抗體、抗原、寡核糖核苷酸等生物大分子交聯(lián)。這種多元核-殼型熒光納米作為生物標(biāo)記物在免疫分析,細(xì)胞成像及DNA雜化等領(lǐng)域?qū)⒕哂袕V闊的應(yīng)用空間。我們選取了其中一種Tb(III)納米顆粒,采用“三明治”夾心法對(duì)人血清中乙肝表面抗原(HBsAg)進(jìn)行了檢測(cè),以空白的3倍相對(duì)偏差除以工作曲線的斜率得出本體系檢測(cè)限為35 pg/ml(3σ/s)。免疫分析應(yīng)用結(jié)果表明核-殼型熒光納米作為一種生物標(biāo)記物用于時(shí)間分辨熒光生物分析具有進(jìn)一步深入研究的價(jià)值。 第三章描述了新型免疫PCR體系的建立及評(píng)價(jià)。選取具有天然納米結(jié)構(gòu)的雙鏈DNA噬菌體T7,通過異型雙功能交聯(lián)劑Sulfo-SMCC活化,使其形成具有巰基反應(yīng)活性的T7噬菌體�？笻BsAg抗體可經(jīng)Traut’s Reagent引進(jìn)巰基,形成巰基化抗體。二者交聯(lián),經(jīng)超濾純化,得到了T7噬菌體表面帶有抗體分子的T7-Antibody復(fù)合物。以此作為檢測(cè)探針,初步建立了一種超靈敏的免疫-PCR體系。通過采用1%戊二醛處理聚丙烯PCR管,使得免疫反應(yīng)和PCR反應(yīng)能在同一管進(jìn)行。實(shí)驗(yàn)結(jié)果表明,HBsAg在1000 ng/ml-0.01 ng/ml范圍內(nèi)呈現(xiàn)良好的線性關(guān)系(R=0.98),檢測(cè)限達(dá)到0.1 pg/ml。實(shí)驗(yàn)檢測(cè)了29份陽(yáng)性血清和45份陰性血清,其結(jié)果與ELISA結(jié)果吻合。結(jié)果證實(shí)了該噬菌體介導(dǎo)的免疫PCR檢測(cè)具有可行性和應(yīng)用價(jià)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.
【學(xué)位授予單位】：廈門大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2007
【分類號(hào)】：R392

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