Development of functionalized fluorescent europium nanoparticles for biolabeling and time-resolved fluorometric applications

Abstract

A covalent binding-copolymerization method was developed to prepare silica-based fluorescent europium nanoparticles that can be used for biolabeling and highly sensitive time-resolved fluorescence bioassays. The nanoparticles were prepared in a water-in-oil (W/O) microemulsion consisting of a conjugate of (3-aminopropyl)triethoxysilane bound to a fluorescent Eu³⁺ chelate, 4,4′-bis(1″,1″,1″,2″,2″,3″,3″-heptafluoro-4″,6″-hexanedion-6″-yl)chlorosulfo-o-terphenyl-Eu³⁺ (APS-BHHCT-Eu³⁺), free (3-aminopropyl)triethoxysilane (APS), tetraethyl orthosilicate (TEOS), Triton X-100, n-octanol, water, and cyclohexane by copolymerization of APS-BHHCT-Eu³⁺, APS, and TEOS with aqueous ammonia. Characterization by transmission electron microscopy and fluorometric methods indicate that the nanoparticles are spherical and uniform in size, 36 ± 4 nm in diameter, highly photostable, and strongly fluorescent, having a fluorescence quantum yield of 50.6% and a long fluorescence lifetime of 384 µs. The amino groups directly introduced to the surface of the nanoparticles by using free (3-aminopropyl)triethoxysilane in the nanoparticle preparation made the surface modification and bioconjugation of the nanoparticles easier. The nanoparticles were used for streptavidin labeling, and the nanoparticle-labeled streptavidin was used in sandwich-type time-resolved fluoroimmunoassays (TR-FIA) of carcinoembryonic antigens (CEA) and hepatitis B surface antigens (HBsAg) in human sera. The methods give detection limits of 1.9 pg ml⁻¹ for CEA, and 23 pg ml⁻¹ for HBsAg. The concentrations of HBsAg in 30 human serum samples were determined, and the results were compared with those independently determined by an established TR-FIA method using the BHHCT-Eu³⁺-labeled streptavidin. A good correlation was obtained with a correlation coefficient of 0.993.