Europium and terbium chelators as candidate substrates for enzyme-labelled time-resolved fluorimetric immunoassays

Abstract

Thirty-three candidate flurogenic chelators for Eu³⁺ and Tb³⁺ were examined. Ten were found to form highly fluorescent complexes with Eu³⁺ and five with Tb³⁺. In all cases, the fluorescence observed was lanthanide-specific, long-lived and was monitored by microsecond time-resolved fluorimetry. The fluorogenic chelators could be quantified, in the presence of an excess of lanthanide, at levels of < 1 × 10^–8 mol l^–1 in all but one case. Two new fluorogenic chelators could form ternary complexes with Eu³⁺ and Tb³⁺, in the presence of ethylenediaminetetraacetic acid. The structures of the chelators identified are such that enzyme substrates could be developed and used for enzyme-labelled time-resolved fluorimetric immunoassays. It was found that one of the new chelators identified, 4-methylumbelliferyl phosphate, which forms fluorescent, long-lived complexes with Eu³⁺, could be split by alkaline phosphatase to phosphate and 4-methylumbelliferone, which does not form fluorescent complexes with Eu³⁺. Based on this, highly sensitive immunoassays for thyroid stimulating hormone and thyroxine in human serum were developed. It is anticipated that some of the identified chelators could be transformed into enzyme substrates suitable for highly sensitive, enzyme-labelled time-resolved fluorimetric immunoassays.