Preparation And Application Of Novel Visible-Light-Excited Luminescence Enhancement Solutions

The dissociation enhanced lanthanide fluoroimmunoassay (DELFIA) system using EDTA-Eu3+complex derivatives as biolabels is the most widely used time-resolved (or time-gated) luminescence bioassay system for clinical diagnostics, but it requires a UV lamp (<360nm) as the excitation source because of the limitation of the luminescence enhancement solution of EDTA-Eu3+used in the system. This limitation has affected the effective use of time-resolved luminescence bioassay technique for living biological systems.In this work, three aqueous luminescence enhancement solutions that can be used for visible-light-excited time-resolved luminescence detection of EDTA-Eu3+were prepared, and their luminescence response properties to EDTA-Eu3+were systematically investigated. The new solutions were prepared by co-dissolving a newly synthesized tetradentate β-diketone,1,2-bis[8’-(1",1",1"1",2",2",3",3"-heptafluoro-4",6"-hexanedion-6"-yl)-naphth-2’-yl]-benzene (BHHNB), and one of three4,6-bis(pyrazol-1-yl)-1,3,5-triazine derivatives,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) or2-(N,N-diethylanilin-4-yl)-4,6-bis(pyrazol-1-yl)-1,3,5-triazine (BPT), in a weakly acidic aqueous buffer at pH3.2containing0.1%Triton X-100. These solutions displayed strong luminescence response behaviors to non-luminescent EDTA-Eu3+by the formations of the ternary Eu3+complexes, BHHNB-Eu3+-DPBT, BHHNB-Eu3+-MPBT and BHHNB-Eu3+-BPT, with long luminescence lifetimes (>500μs) and a wide excitation wavelength range from UV to visible light with the excitation peaks at390,400and420nm, respectively. These unique luminescence responses enabled the solutions to be used as the visible-light-excited luminescence enhancement solutions for the highly sensitive time-resolved luminescence detection of EDTA-Eu3+, and the dose-dependent luminescence enhancements in the three systems showed good linearities in the concentration range of EDTA-Eu3+from0.1nmol/L to1.0μmol/L with the detection limits of～0.1nmol/L. Based on these properties,a novel strategy that visible light can be used as the excitation source for time-resolved luminescence detection of EDTA-Eu3+was proposed.