Preparation of visible-light-excited luminescence enhancement solutions for time-resolved luminescence detection of europium biolabel

Abstract

Dissociation enhanced lanthanide fluoroimmunoassay (DELFIA) technique based on EDTA-Eu³⁺ derivative biolabels is the most widely used time-resolved luminescence bioassay technique for clinical diagnosis, but its major drawback is that the conventional luminescence enhancement solution of EDTA-Eu³⁺ requires UV excitation (<360 nm). In this work, three new visible-light-excited luminescence enhancement solutions are developed and their luminescence response behaviors to EDTA-Eu³⁺ are systematically investigated. The new solutions were prepared by co-dissolving a newly synthesized tetradentate β-diketone, 1,2-bis[8′-(1′′,1′′,1′′,2′′,2′′,3′′,3′′-heptafluoro-4′′,6′′-hexanedion-6′′-yl)-naphth-2′-yl]-benzene (BHHNB), and one of three derivatives of triazine, 2-(N,N-diethylanilin-4-yl)-4,6-bis(3,5-dimethylpyrazol-1-yl)-1,3,5-triazine (DPBT), 2-(N,N-diethylanilin-4-yl)-4,6-bis(3-methylpyrazol-1-yl)-1,3,5-triazine (MPBT) or 2-(N,N-diethylanilin-4-yl)-4,6-bis(pyrazol-1-yl)-1,3,5-triazine (BPT), in a weakly acidic aqueous buffer at pH 3.2 containing 0.1% Triton X-100. These solutions showed sensitive and rapid luminescence responses to non-luminescent EDTA-Eu³⁺ by the formation of the ternary Eu³⁺ complexes, BHHNB-Eu³⁺–DPBT, BHHNB-Eu³⁺–MPBT and BHHNB-Eu³⁺–BPT. These complexes have long luminescence lifetimes (>500 μs) and a wide excitation wavelength range from UV to visible light with the excitation peaks at 390, 400 and 420 nm, respectively, which enabled the solutions to be used as visible-light-excited luminescence enhancement solutions for the highly sensitive time-resolved luminescence detection of EDTA-Eu³⁺.