Preparation, Characterization, and Time-Resolved Fluorometric Application of Silica-Coated Terbium(III) Fluorescent Nanoparticles

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Abstract
Novel silica-coated terbium(III) chelate fluorescent nanoparticles have been prepared and characterized as a new type of fluorescence probe for highly sensitive time-resolved fluorescence bioassay. The preparation was carried out in a water-in-oil microemulsion containing a strongly fluorescent Tb3+ chelate, N,N,N1,N1-[2,6-bis(3‘-aminomethyl-1‘-pyrazolyl)-phenylpyridine]tetrakis(acetate)−Tb3+, Triton X-100, hexanol, and cyclohexane by controlling hydrolysis of tetraethyl orthosilicate. The nanoparticles are spherical and uniform in size, 42 ± 3 nm in diameter, strongly fluorescent, and highly photostable and have enough of a long fluorescence lifetime (1.52 ms) for time-resolved fluorescence measurement. A stable and nontoxic method was developed for the surface modification and protein immobilization of the nanoparticles. As a model of application, the nanoparticle-labeled streptavidin was prepared and used in a sandwich-type time-resolved fluoroimmunoassay of human prostate-specific antigen (PSA) by using a 96-well microtiter plate as the solid-phase carrier. The method gives a detection limit of 7.0 pg/mL for the PSA assay.