本文選題：熒光納米 + 時間分辨　； 參考：《廈門大學》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開展研究工作。時間分辨熒光免疫分析(TRFIA)以及免疫-PCR技術是現(xiàn)代免疫分析方法中兩個極為引人注目的熱點領域,特別適合于臨床上極微量抗原、抗體的檢測及病原微生物痕量檢測等。本論文選題于這兩個研究方向,并開展相關的研究工作。全文主要包括三個部分:綜述了各種類型的高靈敏免疫檢測技術;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記物的制備;天然噬菌體介導的免疫-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技術,并簡要介紹高靈敏檢測技術發(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記物的制備、表征及應用。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熒光絡合物,不需要加入增強液,且與稀土離子所形成的絡合物熒光強度十分穩(wěn)定。將納米熒光材料制備技術與時間分辨熒光免疫測定法相結合,建立了以熒光納米顆粒為生物標記物的高靈敏度時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)室溫攪拌下反應2 h,合成功能化APTMS-BBCAP復合體,可以與Eu(III)、Tb(III)進行螯合,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,更遠高于有機熒光染料�？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記物在免疫分析,細胞成像及DNA雜化等領域?qū)⒕哂袕V闊的應用空間。我們選取了其中一種Tb(III)納米顆粒,采用“三明治”夾心法對人血清中乙肝表面抗原(HBsAg)進行了檢測,以空白的3倍相對偏差除以工作曲線的斜率得出本體系檢測限為35 pg/ml(3σ/s)。免疫分析應用結果表明核-殼型熒光納米作為一種生物標記物用于時間分辨熒光生物分析具有進一步深入研究的價值。 第三章描述了新型免疫PCR體系的建立及評價。選取具有天然納米結構的雙鏈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活性的T7噬菌體�？笻BsAg抗體可經(jīng)Traut’s Reagent引進巰基,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復合物。以此作為檢測探針,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,使得免疫反應和PCR反應能在同一管進行。實驗結果表明,HBsAg在1000 ng/ml-0.01 ng/ml范圍內(nèi)呈現(xiàn)良好的線性關系(R=0.98),檢測限達到0.1 pg/ml。實驗檢測了29份陽性血清和45份陰性血清,其結果與ELISA結果吻合。結果證實了該噬菌體介導的免疫PCR檢測具有可行性和應用價值。
[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.
【學位授予單位】：廈門大學
【學位級別】：碩士
【學位授予年份】：2007
【分類號】：R392

【參考文獻】

相關期刊論文 前8條

1 彭洪尚;吳長峰;黃世華;McNeill Jason;;小尺寸銪(Ⅲ)配合物熒光納米顆粒的制備[J];發(fā)光學報;2006年05期

2 何曉曉;陳基耘;王柯敏;譚蔚泓;秦迪嵐;;吲哚類菁染料Cy3嵌入的核殼熒光納米顆粒的制備及其性質(zhì)研究[J];高等學校化學學報;2006年10期

3 何曉曉,王柯敏,譚蔚泓,肖丹,李軍,羊小海;基于生物熒光納米顆粒的新型熒光標記方法及其在細胞識別中的應用[J];科學通報;2001年16期

4 李朝輝,王柯敏,譚蔚泓,李軍,付志英,王益林,劉劍波,羊小海;硅殼包被的核殼型量子點熒光納米顆粒的制備及其在細胞識別中的應用[J];科學通報;2005年13期

5 黃松,牛春,姜洪杰;免疫PCR實驗技術條件的探討[J];首都醫(yī)科大學學報;2000年02期

6 徐平西,肖華勝,鞠躬;一種簡易的免疫PCR方法的建立[J];生物化學與生物物理進展;1999年04期

7 王先良,王小利,蘇艷華,張遲,張莉君,李芳,李柏生,李曉波,徐淑雷,李媛媛,徐順清;一種新的基于適體和酶切保護分析的超靈敏蛋白質(zhì)檢測方法[J];中國生物化學與分子生物學報;2005年03期

8 朱詩國,唐珂,向娟娟,呂紅斌,李小玲,聶新民,周后德,沈守榮,李桂源;生物熒光氧化硅納米顆粒的研制與應用[J];物理化學學報;2003年04期

，

本文編號：2095364