Preparation Of Fluorescent Lanthanide Nanoparticles And Their Application To Time-Resolved Fluoroimmunoassay

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.