Development of a visible-light-sensitized europium complex for time-resolved fluorometric application

The time-resolved luminescence bioassay technique using luminescent lanthanide complexes as labels is a highly sensitive and widely used bioassay method for clinical diagnostics and biotechnology. A major drawback of the current technique is that the luminescent lanthanide labels require UV excitation (typically less than 360 nm), which can damage living biological systems and is holding back further development of time-resolved luminescence instruments. Herein we describe two approaches for preparing a visible-light-sensitized Eu³⁺ complex in aqueous media for time-resolved fluorometric applications: a dissociation enhancement aqueous solution that can be excited by visible light for ethylenediaminetetraacetate (EDTA)-Eu³⁺ detection and a visible-lightsensitized water-soluble Eu³⁺ complex conjugated bovine serum albumin (BSA) for biolabeling and timeresolved luminescence bioimaging. In the first approach, a weakly acidic aqueous solution consisting of 4,4′bis(1″,1″,1″, 2″,2″,3″,3″-heptafluoro-4″,6″- hexanedion6″-yl)-o-terphenyl (BHHT), 2-(N,N-diethylanilin-4-yl)4,6-bis(3, 5-dimethylpyrazol-l-yl)-l,3,5-triazine (DPBT), and Triton X-100 was prepared. This solution shows a strong luminescence enhancement effect for EDTA-Eu ³⁺ with a wide excitation wavelength range from UV to visible light (a maximum at 387 nm) and a long luminescence lifetime (520 μs), to provide a novel dissociation enhancement solution for time-resolved luminescence detection of EDTA-Eu³⁺. In the second approach, a ternary Eu ³⁺ complex, 4, 4′-bis(1″,1″,1″,2″, 2″,3″,3″-heptafluoro-4″,6″-hexanedion-6″-yl) -chlorosulfo-o-terphenyl (BHHCT)-EU³⁺-DPBT, was covalentIy bound to BSA to form a water-soluble BSA-BHHCTEu³⁺-DPBT conjugate. This biocompatible conjugate is of the visible-light excitable feature in aqueous media with a wide excitation wavelength range from UV to visible light (a maximum at 387 nm), a long luminescence lifetime (460 μs), and a higher quantum yield (27%). The conjugate was successfully used for streptavidin (SA) labeling and time-resolved luminescence imaging detection of three environmental pathogens, Giardialamblia, Cryptosporidiummuris, and Cryptosporidium parvum, in water samples. Our strategy gives a general idea for designing a visible-light-sensitized Eu³⁺ complex for time-resolved luminescence bioassay applications.