Bioconjugation of functionalized fluorescent YVO4:Eu nanocrystals with BSA for immunoassay

Novel fluorescent YVO₄:Eu nanocrystals were ∼15 nm in diameter and had been functionalized with phosphorous polyacrylic acids at the surface during the synthesis, which illuminated their potential in bioassays. In this paper, bovine serum albumin (BSA) was covalently coupled to activated nanocrystals from primary amine groups by EDC and sulfo-NHS chemistry successfully. Reaction condition, including concentration, pH value and separation methods, were carefully chosen and well discussed. Binding site number was determined simply by measuring BSA concentration using optimized microplate bicinchonic acid (BCA) assay and nanocrystals concentration according to their fluorescence emission in the monodisperse bioconjugate suspension. Transmission electron microscopy (TEM) imaging showed the monodispersity of the bioconjugates. Fluorescent characteristics were also retained. Immunoblots verified that BSA was covalently coupled to nanocrystals and remained immunoreactive. Finally, the two-step heterogeneous time-resolved fluoroimmunoassay (TR-FIA) of rabbit anti-BSA polyclonal antibody (PcAb) was operated to further validate the bioconjugates.