Resonance Energy Transfer in Steady-State and Time-Decay Fluoro-Immunoassays for Lanthanide Nanoparticles Based on Biotin and Avidin Affinity

Compared with conventional organic fluorophores, lanthanide-doped nanoparticles as donor and gold nanoparticles as quencher-like acceptor in energy transfer studies offer many technological advantages; in particular, the long-lifetime donor endows the fluorescence resonance energy transfer (FRET) assay with great signal-to-noise ratio, and the quencher acceptor allows monitoring of the donor channel alone. The LaPO₄:Ce,Tb nanoparticles with high quantum yield in aqueous solution are readily paired with differently sized gold nanoparticles through the well-established binding affinity of biotin and avidin. The spectra overlap between the emission of lanthanide nanoparticles and absorption of gold nanoparticles meets the prerequisite for energy transfer, and the resonance energy transfer process is characterized by both steady-state and time-decay luminescent measurements. With the emission contributions from Tb³⁺ ions located at the surface and inside the lanthanide nanoparticles taken into account, the comparison of the donor emission in the presence and absence of acceptors in combination with lifetime changes are used to determine luminescence resonance energy transfer (LRET) efficiency more accurately. The results indicate that the Tb³⁺ ions located at/near the surface of the nanoparticles play a more important role than Tb³⁺ ions inside the nanoparticles in the LRET process. The present study introduces the possibility of multiplexing donor/acceptor pairs if sufficient spectral separation is achieved.