Stable and highly fluorescent europium(III) chelates for time-resolved immunoassays

Derivatives of 4-[2-(4-isothiocyanatophenyl)ethynyl]-2,6,-bis{[N,N-bis(carboxymethyl)-amino]methyl}pyridine europium(III) (1) bearing one (6) or two (7) additional iminodiacetate coordinating arms have been synthesized. 6 and 7 were significantly more stable than 1 as evidenced by competition experiments with ethylenediaminetetraacetic acid (EDTA) and 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA). While the luminescence quantum yield of 1 remained modest, the other two complexes displayed substantial luminescence efficiency. The introduction of a supplementary iminodiacetate arm in 6 brought important improvements to both the stability and the luminescence properties of the Eu complex. In contrast, although 7 is more luminescent than 1, the introduction of a second iminodiacetate coordinating arm brings no further benefit on the photophysical properties. The most promising results were obtained with the nine-dentate chelate 6 and its Eu complex, which was conjugated to biotin and applied within the frame of a bioaffinity immunoassay of human C-reactive protein.