Biotinylation of TiO2 Nanoparticles and Their Conjugation with Streptavidin

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Abstract
Photoactive TiO2 can be used to mediate a variety of disinfection processes. It was postulated that TiO2 particles could be directed to specific targets of interest using biotin/streptavidin linkages. Biotinylated TiO2 nanoparticles (anatase) were obtained by treating TiO2 nanoparticles with 3-aminopropyltriethoxysilane (APTS) in anhydrous DMSO, followed by reaction with N-hydroxysuccinimidobiotin. 29Si CP-MAS NMR, 13C CP-MAS NMR, and FTIR spectra showed that biotin was covalently bound to the TiO2 surface. Transmission electron microscopy (TEM) demonstrated that prolonging the silanization reaction times led to increasingly thick silsesquioxane coating layers of up to approximately 10 nm. The specific surface area (SSA) of the TiO2 particles decreased from 16 m2 g-1 before treatment to 9.1 m2 g-1 after aminosilanization and to 8.4 m2 g-1 after biotinylation, as measured by nitrogen adsorption. Amino surfaces modified for 4, 16, and 26 h had total amino group densities ranging from 2.9 to 26 to 66 nm-2, respectively, whereas accessible surface amino group densities ranged from 2.7 to 10 to 17 nm-2 as shown from nitrogen adsorption, polyelectrolyte titration, conductiometric titration, and biotin assays. Not all the amino groups were accessible for biotinylation: the densities of active biotin were found to be 2.1, 7.0, and 11.5 nm-2. The ability of the attached biotin to bind to streptavidin was demonstrated by confocal microscopy with the use of fluorescently labeled streptavidin−FITC. Although streptavidin was readily able to bind to biotinylated TiO2 particles, it did not act as a strong flocculating agent for the biotinylated TiO2 particles. The implications of these observations, with respect to particle accessibility to tethered streptavidin, are discussed.