Covalent conjugation of avidin with dye-doped silica nanopaticles and preparation of high density avidin nanoparticles as photostable bioprobes

Progress in biomedical imaging depends on the development of bioprobes with a high sensitivity and stability. Fluorescent silica nanoparticles (NPs) covalent conjugation of avidin has been proposed for cancer cells imaging by fluorescence microscopy. Uniform silica NPs were prepared using water-in-oil (W/O) microemulsion methods and primary amine groups were introduced onto the surface of the NPs by condensation of tetraethyl orthosilicate (TEOS). Optically stable organic dyes, tris(2,2'-bipyridyl) dichlororuthenium(II) hexahydrate (Rubpy), were doped inside the silica NPs. The amine functions were transferred to carboxyl groups coupled with a linker elongation. Avidin was immobilized at the surface of the NPs by covalent binding to the carboxyl linkers. The binding capacity of the avidin-covered NPs for ligand biotin was quantified by titration with biotin(5-fluorescein) conjugate to 1.25 biotin binding sites/100 nm(2). We used biotinylated antibody and cell recognition by fluorescence microscopy imaging technique. The lung carcinoma cells were identified easily with high efficiency using these antibody-coated NPs. By comparison with fluorescein isothiocyanate (FITC), dye-doped silica NPs display dramatically increased stability of fluorescence as well as photostability, as compared to the common organic dye, when under continuous irradiation. (C) 2012 Elsevier B.V. All rights reserved.