title: Activated phosphonated trifunctional chelates for highly sensitive lanthanide-based FRET immunoassays applied to total prostate specific antigen detection.
abstract: The first example of an activated phosphonated trifunctional chelate (TFC) is presented, which combines a non-macrocyclic coordination site for lanthanide coordination based on two aminobis-methylphosphonate coordinating arms, a central bispyrazolylpyridyl antenna and an N-hydroxysuccinimide ester in para position of the central pyridine as an activated function for the labeling of biomaterial. The synthesis of the TFC is presented together with photo-physical studies of the related Tb and Eu complexes. Excited state lifetime measurements in H2O and D2O confirmed an excellent shielding of the cation from water molecules with a hydration number of zero. The Tb complex provides a high photoluminescence (PL) quantum yield of 24% in aqueous solutions (0.01 M Tris-HCl, pH 7.4) and a very long luminescence lifetime of 2.6 ms. The activated ligand was conjugated to different biological compounds such as streptavidin, and a monoclonal antibody against total prostate specific antigen (TPSA). In combination with AlexaFluor647 (AF647) and crosslinked allophycocyanin (XL665) antibody (ABs) conjugates, homogeneous time-resolved Fluorescence Resonance Energy Transfer (FRET) immunoassays of TPSA were performed in serum samples. The Tb donor-dye acceptor FRET pairs provided large Förster distances of 5.3 nm (AF647) and 7.1 nm (XL665). A detailed time-resolved FRET analysis of Tb donor and dye acceptor PL decays revealed average donor-acceptor distances of 4.2 nm (AF647) and 6.3 nm (XL665) within the sandwich immunocomplex and FRET efficiencies of 0.79 and 0.68, respectively. Very low detection limits of 1.4 ng mL(-1) (43 pM) and 2.4 ng mL(-1) (74 pM) TPSA were determined using a KRYPTOR fluorescence immunoanalyzer. These results demonstrate the applicability of our novel Tb-bioconjugates for highly sensitive clinical diagnostics.
authors.affiliation: Laboratoire d'Ingénierie Moléculaire Appliquée à l'Analyse, IPHC, UMR 7178 CNRS/UdS, ECPM, 25 rue Becquerel, 67087 Strasbourg Cedex, France.
authors.forename: Katia
authors.initials: K
authors.lastname: Nchimi-Nono
authors.forename: K David
authors.initials: KD
authors.lastname: Wegner
authors.forename: Stina
authors.initials: S
authors.lastname: Lindén
authors.forename: Alexandre
authors.initials: A
authors.lastname: Lecointre
authors.forename: Laurence
authors.initials: L
authors.lastname: Ehret-Sabatier
authors.forename: Shakir
authors.initials: S
authors.lastname: Shakir
authors.forename: Niko
authors.initials: N
authors.lastname: Hildebrandt
authors.forename: Loïc J
authors.initials: LJ
authors.lastname: Charbonnière
mesh_terms: D002614:Chelating Agents / Q000138:chemical synthesis / Q000737:chemistry*; D031541:Fluorescence Resonance Energy Transfer; D007118:Immunoassay; D028581:Lanthanoid Series Elements / Q000737:chemistry*; D015394:Molecular Structure; D009942:Organometallic Compounds / Q000138:chemical synthesis / Q000737:chemistry*; D009943:Organophosphorus Compounds / Q000737:chemistry*; D017430:Prostate-Specific Antigen / Q000032:analysis*
chemical_list: D002614:Chelating Agents; D028581:Lanthanoid Series Elements; D009942:Organometallic Compounds; D009943:Organophosphorus Compounds; D017430:Prostate-Specific Antigen