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.

Importance of Outer‐Sphere and Aggregation Phenomena in the Relaxation Properties of Phosphonated Gadolinium Complexes with Potential Applications as MRI Contrast Agents

“…The 1 HNMR spectrum of the La III complex recorded at 298 Kshows only five signals (Figure3), which suggests an effective C 2v symmetry of the complex in solution. Thisi sc onfirmed by the 13 Cs pectrum, which consists of five signals for the 11 carbon nuclei of the ligand backbone.…”

“…The solution 1 H, 13 Ca nd 31 PNMR spectra of the diamagnetic LaL 4 and LuL 4 complexesw ere recorded in D 2 Os olution at pD 10.9. At this pD, the predominant species in solution is expected to be the fully deprotonated form, as demonstrated by potentiometric measurements (see above).…”

“…The 13 CNMR resonanced ue to the methylenephosphonate groups is split into ad oublet of doublets by spin coupling to [ c] Precipitation of the complexes precludes accurate determination. The hydrolysisc onstants of the free Ln 3 + cations have been taken into account in the statistical processing (logK LaOH = À8.83;l ogK NdOH = À8.20;l ogK EuOH = À7.78;l og K GdOH = À7.85;l ogK TbOH = À7.66;l ogK ErOH = À7.54;l og K LuOH = À7.29).…”

“…[11] Finally,p hosphonate functions induce large second-sphere interactions with water molecules or cations in solution. [12] Within the frame of our search for highly efficient complexes for molecular imaging, we have been interested in the design of polyphosphonated chelates for luminescence, [7,13] or radiochemicala pplications. [14] In this study,w er eport on the coordination behavior of as eries of bis-,t ris-and tetraphosphonated ligands( Scheme 1) with lanthanide cations and we particularly inspect the influence of the number of phosphonic coordinating units on the relaxation properties of the corresponding Gd III chelates.…”

A time-resolved luminescent competitive assay to detect L-selectin using aptamers as recognition elements

“…When combining those properties with time-gated time-resolved luminescence signal acquisition, the background signal resulting from undesired effects, such as biosample autofluorescence, short-living fluorescence of fluorophores as well as scattered light are reduced significantly. In recent years, FRET systems based on the lanthanide complexes as donors have been used for multiplexed diagnostics [40,41], as molecular ruler [42,43], for investigations of the plasma influence on FRET signal [44], and in biotin-streptavidin assays [17,16] as well as for the determination of macromolecular biomarkers, such as prostate specific antigen (PSA) [47,48], carcinoembryonic antigen (CEA) [49], CARM1 activators [50], and total protein concentration [51].…”