【作者】 叶志强；

【导师】 袁景利；

【作者基本信息】 中国科学院研究生院（大连化学物理研究所） ， 分析化学， 2004， 博士

【摘要】 时间分辨荧光免疫分析是一种超高灵敏度的分析方法,作为取代放射性免疫分析法的一种手段,该方法已经在临床检测和医学与生命科学的研究中得到了较广泛的应用。在本学位论文的研究中,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,通过制备出几种纳米尺度的稀土荧光微粒,建立了以纳米稀土荧光微粒为标记物的高灵敏度时间分辨荧光免疫分析方法。利用油包水型（W/O）微乳液法制备了硅胶包裹BHHT-Eu³⁺和BPTA-Tb³⁺两种稀土配合物的纳米荧光颗粒。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。得到了几种直径在25-50 nm之间的硅胶包裹稀土配合物纳米荧光微粒。它们的光谱性质和其前驱体基本相同,但其抗光漂白能力要高于前驱体,更远高于有机荧光染料。一种新型无毒的、更稳定的引进活性基团的方法用于纳米微粒的表面修饰。氨丙基三乙氧基硅被稳固地键合在纳米微粒表面后,首先将牛血清白蛋白（BSA）键合在纳米微粒表面,然后再将BSA包裹的纳米微粒与链亲和素（SA）键合。这种方法不但使得纳米微粒和SA 稳定地连接在一起,而且保证了SA 在固相免疫分析中有很高的生物素亲和性。在上述工作的基础上,利用标记了SA 的硅胶包裹BHHT-Eu³⁺和BPTA-Tb³⁺纳米荧光微粒分别建立了人乙肝表面抗原（HBsAg）和前列腺特异抗原（PSA）的夹心型时间分辨荧光免疫测定方法。它们的最低检出浓度分别为74 pg/ml（HBsAg）和7.0 pg/ml （PSA）,结果显示两种检测方法均有很高的灵敏度。PSA 测定结果与临床检测结果相比较,相关系数为99%,说明方法具有很好的实用性。制备了表面带有活性基团的硅胶包覆BPTA-Tb³⁺纳米荧光微粒。通过简单的操作即可标记蛋白质。这种纳米微粒标记抗人甲胎蛋白（AFP）抗体后,被用于测定人血清中的AFP 含量。方法的线性范围为0.1-100 ng/ml, 检测下限为0.1 ng/ml。所有样品测定的相对标准偏差均小于9.0%, 样品添加回收率的测定更多还原

【Abstract】 Time-resolved fluoroimmunoassay （TR-FIA） is an ultrasenstive analyticalmethod, instead of radioimmunoassay, it has been widely used in diagnostics andclinical analyses. In this doctoral dissertation, several kinds of fluorescent lanthanidechelate-doped silica （or ZrO₂） nanoparticles were prepared, characterized, anddeveloped as new fluorescence probes for improving the sensitivity of TR-FIA, andseveral highly sensitive TR-FIA methods were established. The experimental resultsare described in the following.The nanoparticles were prepared by controlling hydrolysis of tetraethylorthosilicate （TEOS） with ammonium hydroxide in a water-in-oil （W/O）microemulsion containing aqueous solution of fluorescent lanthanide chelate{N,N,N1,N1-[2,6-bis（3’-aminomethyl-1’-pyrazolyl）-phenylpyridine]tetrakis（acetate）-Tb3+ or 4,4’-bis（1”,1”,1”,2”,2”,3”,3”-heptafluoro-4”,6”-hexanedion-6”-yl）-o-terphenyl-Eu³⁺}, surfactant, co-surfactant and oil phase. The diameters of thenanoparticles are in range of 25-50 nm with narrow size distribution. The results offluorometric measurement show that the fluorescence of the nanoparticles is highlyphotostable and long-lived.A stable and nontoxic method was established for the surface modification andprotein immobilization of the nanoparticles. After the nanoparticle’s surface wassilanized by 3-aminopropyl-triethoxyl silane, the modified nanoparticles wereconjugated to bovine serum albumin （BSA）, and then the BSA-coated nanoparticleswere covalently conjugated to streptavidin （SA）. It was found that thenanoparticle-labeled SA prepared by this method remains high biological activityandstability for a long time. Based on these works, the sandwich-type TR-FIA methods of human hepatitis Bsurface antigen （HBsAg） and prostate specific antigen （PSA） were established byusing silica-coated europium（III） chelate nanoparticles and silica-coated terbium（III）chelate nanoparticles as the fluorescence probes, respectively. The results show thattwo kinds of assays are highly sensitive with the detection limits of 74.0 pg/ml forHBsAg and 7.0 pg/ml for PSA, respectively. The correlation coefficient of themeasurement results for PSA in 24 human serum samples is 0.998 between thepresent method and general clinical immunoassaymethod. Novel fluorescent Tb3+ chelate-doped silica nanoparticles having surface aminogroups were prepared and characterized. After the nanoparticles were conjugated toanti-human α-fetoprotein antibody, the TR-FIA of α-fetoprotein （AFP） in humanserum was carried out by using the nanoparticle-labeled AFP antibody. The assayresponse is linear from 0.10 to ¹00 ng/ml with a detection limit of 0.10 ng/ml. Thecoefficient variations （CVs） of the method are less than 9.0%, and the recoveries arein the range of 84-98% for serum sample measurements. The preparation and application of ZrO2-based fluorescent terbium nanoparticleswere explored. The fluorescent nanoparticles having uniform size, 33 ±4 nm indiameter, were prepared by controlling condensation of Zr（OCH2CH3）4 and ZrOCl2.After nanoparticle’s surface was coated with BSA by physical absorption, thenanoparticles were covalently bound to SA. The result of TR-FIA shows that theZrO2-based fluorescent terbium nanoparticles are also useful as a new type offluorescence probe.更多还原