Abstract
A novel europium ligand 2, 2’, 2’’, 2’’’-(4, 7-diphenyl-1, 10-phenanthroline-2, 9-diyl) bis (methylene) bis (azanetriyl) tetra acetic acid (BC-EDTA) was synthesized and characterized. It shows an emission spectrum peak at 610 nm when it is excited at 360 nm, with a large Stock shift (250 nm). It is covalently coated on the surface of a bare silica nanosphere containi free amino groups, using 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride and N-Hydroxysuccinimide. We also observed an interesting phenomenon that when BC-EDTA is labeled with a silica nanosphere, the chelate shows different excitation spectrum peaks of about 295 nm. We speculate that the carboxyl has a significant influence on its excitation spectrum. The BC-EDTA/Eu3+coated nanosphere could be used as a fluorescent probe for time-resolved fluorescence immunoassay. We labeled the antibody with the fluorescent nanosphere to develop a nanosphere based hepatitis B surface antigen as a time-resolved fluorescence immunoassay reagent, which is very easy to operate and eliminates potential contamination of Eu3+ contained in the environment. The analytical and functional sensitivities are 0.0037 μg/L and 0.08 μg/L (S/N≥2.0) respectively. The detection range is 0.08-166.67 μg/L, which is much wider than that of ELISA (0.2-5μg/L). It is comparable to the commercial dissociation-enhanced lanthanide fluoro-immunoassay system (DELFIA) reagents (0.2-145μg/L). We propose that it can fulfill clinical applications.
Highlights
Multiple studies reported that lanthanide and its chelate can be applied in time-resolved fluorescence immunoassay (TrFIA)[1, 2]
The BC-EDTA/Eu3+ coated nanosphere (Fig 8) didn’t show significant difference in size and shape compared with the bare silica nanosphere (Fig 6)
We found an interesting phenomenon where the maxima excitation wavelength of a BC-EDTA nanosphere is shortened compared with a bare one, and peaks at about 295 nm (Fig 9)
Summary
Multiple studies reported that lanthanide and its chelate can be applied in time-resolved fluorescence immunoassay (TrFIA)[1, 2]. It is widely used in clinical immunoassay, such as in DELFIA reagents (Perkinelmer Inc.). Europium and its chelate have many features that make them suitable for TrFIA, the maximum excitation wavelength of Eu3+ fluorescent complexes is in the UV region and the emission maximum is about 610 nm, which is ion-specific. This large Stokes shift can avoid the interference of excitation light. The emission band of Eu3+ fluorescent complexes is very narrow, with the full width at half maximum (FWHM) being about 10
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