Preparation of near-IR fluorescent nanoparticles for fluorescence-anisotropy-based immunoagglutination assay in whole blood

A class of novel core/shell near-IR fluorescent nanoparticles have been prepared through co-hydrolysis of a hydrophobic silicon alkoxide, hexadecyltrimethoxysilane, and tetraethyl orthosilicate as the dye-doped core, followed by the formation of a hydrophilic shell via hydrolysis of tetraethyl orthosilicate in a water-in-oil microemulsion. The co-hydrolysis of hexadecyltrimethoxysilane and tetraethyl orthosilicate produces a highly hydrophobic core for the entrapment of a low-cost near-IR fluorescence dye, methylene blue. Experimental investigation of this particular core/shell nanoparticle in comparison with conventional dye-doped silica nanoparticles demonstrates that the hydrophobic core enables the doped dye to exhibit enhanced fluorescence and show improved stability to dye leaching and exogenous quenchers. In contrast to rhodamine B doped silica nanoparticles, the near-IR fluorescent nanoparticles also show negligible background fluorescence and low inner-filtration interference in complex biological systems such as whole blood. This advantage is utilized for the development of an immunoagglutination assay method based on fluorescence-anisotropy measurement for the detection of alpha fetoprotein (AFP) in whole-blood samples. The results reveal that increase in fluorescence anisotropy is linearly correlated with AFP concentration in the range 1.9-51.9 ng mL(-1).