Abstract

To meet the continual demands of more-sensitive immunoassays, the synthesis of novel luminescent Eu(III) chelate labels having similar substituted 4-(phenylethynyl)pyridine chromophores in three different chelate structure classes are reported. Significantly enhanced luminescence intensities were obtained, evidently caused by the intra-ligand charge transfer (ILCT) mediated sensitization, but the alternative ligands triplet state process cannot be ruled out. Based on the present study, even quite small changes on the chelate structure, and, especially, on the substituents’ donor/acceptor strength on both ends of 4-(phenylethynyl)pyridine subunits have an unpredictable effect on the luminescence. The highest observed brightness was 16,400 M−1 cm−1 in solution and 69,500 M−1 cm−1 on dry surface, being 3.4 and 8.7 fold higher compared to the reference chelate. The new label chelates provide solutions for improved assay sensitivity up-to tenfold from the present concepts.

Highlights

  • Time-resolved fluorometry (TRF) employing long-lifetime emitting luminescent lanthanide chelates has been applied in many specific binding assays, such as immunoassays, DNA hybridization assays, receptor-binding assays, enzymatic assays, bio-imaging such as immunocytochemical, immunohisto-chemical assays or cell based assays to measure the wanted analyte at very low concentration [1,2,3]

  • Microwave heating at 100 ◦ C for 30–45 min in diethyl amine (DEA) and dimethylformamide (DMF) gave appropriate yields of products

  • Seven new Eu(III) labeling chelates having two to three 4-(phenylethynyl)pyridine chromophors in three different basic structural designs were synthesized and their key photophysical properties were studied in this work

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Summary

Introduction

Time-resolved fluorometry (TRF) employing long-lifetime emitting luminescent lanthanide chelates has been applied in many specific binding assays, such as immunoassays, DNA hybridization assays, receptor-binding assays, enzymatic assays, bio-imaging such as immunocytochemical, immunohisto-chemical assays or cell based assays to measure the wanted analyte at very low concentration [1,2,3]. Lanthanide chelates have special properties offering proper alternative markers in bio-affinity assays: (i) the difference between the excitation and emission wavelengths is large; (ii) normally, they have long emission life-time compared to the background, and, the time-resolved fluorescence (TR-FIA) measurement techniques together with narrow emission lines eliminate the fluorescence background almost completely; (iii) the concentration quenching is small and enables using wide dynamic assay range without additional dilutions, and (iv) several different sample matrixes e.g., whole blood, serum and plasma can be used for the assays. It has to have efficient cation emission i.e., intense brightness (excitation coefficient x quantum yield, εΦ)

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