Controlled Surface Functionalization of Silica Nanospheres by Covalent Conjugation Reactions and Preparation of High Density Streptavidin Nanoparticles

Silica nanoparticles with a diameter of 100 nm were covalently modified at their surface by adjustable amounts of amine and carboxyl functional groups. Bioconjugation studies of two proteins, streptavidin and streptactin, with the functional nanoparticles resulted in optimum binding of the proteins to a long-chain carboxyl-terminated linker. The surface functionalization of the nanoparticles was monitored by a variety of independent methods, including zeta-potential measurements, dynamic light scattering (DLS), scanning electron microscopy (SEM), particle charge detection titrations (PCD) and elemental analysis. At the surface of the nanoparticles, a functional surface group density of 1.8 amino groups per nm2 was realized. The amine functions were quantitatively transferred to carboxyl groups coupled with a linker elongation. Streptavidin was immobilized by covalent binding to the carboxyl linkers and resulted in a protein density at the surface of the nanoparticles that was three times higher than the highest binding densities at nanoparticles published to date. The binding capacity of the streptavidin-covered nanoparticles for ligand biotin was quantified by titration with biotin-4-fluorescein to 2.5 biotin binding sites per 100 nm2.