Preparation of Near‐IR Fluorescent Nanoparticles for Fluorescence‐Anisotropy‐Based Immunoagglutination Assay in Whole Blood

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Abstract
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.