| Time-resolved fluorescence (TRF) bioassay technique based on long-lived fluorescence of lanthanide probes is an ultrasenstive bioanalytical method, and has been widely used in clinical diagnostics and biotechnology. For most luminescent lanthainde biolabels, one of the major drawbacks is that the optical excitation window is limited to the UV region, which has stronger phototoxicity on the living biological samples. Although the excitation wavelengths for several Eu3+ complexes can be extended into the visible region, water insolubility, poor stability in polar solvents, low luminescence quantum yield and/or the lack of active groups of these complexes make them unsuitable be used for biolabeling.In this work, to prepare a visible-light-excited ternary Eu3+ ternary complex, 4,4'-bis(1",1",1 ",2",2",3",3"-heptafluoro-4",6"-hexanedion-6"-yl)-chlorosulfo-o-terphenyl (BHHCT)-Eu3+-2-(N,N-diethylanilin-4-yl)-4,6-bis(3,5-dimethylpyrazol-1-yl)-l,3,5-triazine (DPBT), two ligands (BHHCT and DPBT) were conjugated to Eu3+ synchronously. Conjugating bovine serum albumin (BSA) with the visible-light-sensitized Eu3+ complex, a Eu3+ complex-BSA conjugate having good water solubility, visible-light excitability (>380 nm), longer luminescence lifetime (0.46 ms) and larger quantum yield (27%) was prepared. Then, the Eu3+ complex-BSA conjugate was bound with streptavidin(SA), and successfully used for immuno-staining and time-resolved luminescence imaging detection of three environmental pathogens including giardia lamblia, cryptosporidium muris, cryptosporidium parvum in the water sample.
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