Synthesis of a highly fluorescent beta-diketone-europium chelate and its utility in time-resolved fluoroimmunoassay of serum total thyroxine.
A new highly fluorescent beta-diketone-europium chelate was synthesized and employed as a tracer to develop a time-resolved fluoroimmunoassay (TRFIA) for detection of serum total thyroxine (T4). The tetradentate beta-diketone chelator, 1,10-bis(thiophene-2'-yl)-4,4,5,5,6,6,7,7-octafluorodecane-1,3,8,10-tetraone (BTOT), was structurally composed of two units of thenoyltrifluoroacetone (TTA) derivatives but expressed fluorescence that was greatly enhanced, as compared to the original TTA molecules, in the presence of excess amount of Eu3+. The luminescence properties of the europium chelate of BTOT werestudied in aqueous solution. Chlorosulfonylation of BTOT afforded 1, 10-bis(5'-chlorosulfo-thiophene-2'-yl)-4,4,5,5,6,6,7,7-octafluorodecane-1,3,8,10-tetraone (BCTOT), which could be coupled to proteins (i.e., streptavidin or the BSA-T4 conjugate) and used as a tracer for TRFIA. Although the BCTOT-Eu complex could be detected at a very low level (approximately 1.07 x 10(-12) mol/L) in buffered aqueous solution (50 mmoVLTris-HCl; pH, 8.0), the application of the chelate label in direct serum T4 TRFIA experienced a problem of matrix interference, which was probably caused by some unknown chelating components in the samples as a result of the fact that the fluorescence of the BCTOT-Eu chelate was prone to quenching or enhancement by some chelating reagents. To remove this problem, an indirect serum T4 TRFIA was proposed with the use of BCTOT-Eu-labeled streptavidin (SA) as signal generation reagent. The concentrations of T4 in 27 human serums were determined by indirect T4 TRFIA, and the assay results correlated well with those obtained by commercial Coming-CLIA (r = 0.955) and Wallac-DELFIA (r 0.965).
Wu
Feng-Bo
FB
Department of Isotope, China Institute of Atomic Energy, 102413, P.O. Box 275-39, Beijing, People's Republic of China. wufb@etang.com
Han
Shi-Quan
SQ
Zhang
Chao
C
He
You-Feng
YF
D002614:Chelating Agents / Q000138:chemical synthesis / Q000737:chemistry*; D005063:Europium / Q000737:chemistry*; D015200:Fluoroimmunoassay; D006801:Humans; D007202:Indicators and Reagents; D007659:Ketones / Q000737:chemistry*; D008024:Ligands; D013974:Thyroxine / Q000097:blood*
D002614:Chelating Agents; D007202:Indicators and Reagents; D007659:Ketones; D008024:Ligands; D005063:Europium; D013974:Thyroxine