Photophysical evaluation of a new functional terbium complex in FRET-based time-resolved homogenous fluoroassays

Abstract

A new functional luminescent lanthanide complex (LLC) has been synthesized with terbium as a central lanthanide ion and biotin as a functional moiety. Unlike in typical lanthanide complexes assembled via carboxylic moieties, in the presented complex, four phosphate groups are chelating the central lanthanide ion. This special chemical assembly enhances the complex stability in phosphate buffers conventionally used in biochemistry. The complex synthesis strategy and photophysical properties are described as well as the performance in time-resolved Förster Resonance Energy Transfer (FRET) assays. In those assays, this biotin-LLC transferred energy either to acceptor organic dyes (Cy5 or AF680) labelled on streptavidin or to quantum dots (QD655 or QD705) surfacefunctionalised with streptavidins. The permanent spatial donor-acceptor proximity is assured through strong and stable biotin-streptavidin binding. The energy transfer is evidenced from the quenching observed in donor emission and from a decrease in donor luminescence decay, both associated with simultaneous increase in acceptor intensity and in the decay time. The dye-based assays are realised in TRIS and in PBS, whereas QD-based systems are studied in borate buffer. The delayed emission analysis allows for quantifying the recognition process and for auto-fluorescence-free detection, which is particularly relevant for application in bioanalysis. In accordance with Förster theory, Förster-radii (R 0 ) were found to be around 60 Å for organic dyes and around 105 Å for QDs. The FRET efficiency (Z) reached 80% and 25% for dye and QD acceptors, respectively. Physical donor-acceptor distances (r) have been determined in the range 45-60 Å for organic dye acceptors, while for acceptor QDs between 120 Å and 145 Å. This newly synthesised biotin-LLC extends the class of highly sensitive analytical tools to be applied in the bioanalytical methods such as time-resolved fluoroimmunoassays (TR-FIA), luminescent imaging and biosensing.

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

“…3, the changes in the decay time are comparable to other Tb complex-based FRET systems which were investigated using time-resolved spectroscopy [17,51]. The major problem with data interpretation is associated with the fluorescence signal in the acceptor channel.…”

“…The influence of the so called "donor leak" into the acceptor channels can be minimized significantly using the Differential Correction Method [17,51]. Therefore, the decay signals have been processed using the Equation (6) …”

“…Mainly organic dyes [15] or quantum dots [16,17] were used to realize FRET aptamer-based systems; however, other lumiophores such as up-converting [18], gold [19] or fluorophore-stained nanoparticles [20] were also used. Aptamer-based FRET sensing systems have been employed for recognizing poisonous cations, e.g.…”

“…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].…”

Rare earth based nanostructured materials: synthesis, functionalization, properties and bioimaging and biosensing applications

“…Other sensing assays based on different resonance ETs, and consisting of nanostructured systems containing REs have been reported in the literature, especially those involving lanthanide complexes [356]. For example, cadmium selenide QDs conjugated with europium complexes and biotin have been used for the detection and quantification of streptavidin [357]; a similar strategy was employed with the conjugation of biotin-terbium complexes with streptavidin-functionalized QDs [358]; semiconductor nanorods (quantum rods) conjugated with terbium complexes with long photoluminescence lifetimes have been suggested to be a powerful tool for homogeneous optical biosensing [359]; QDs conjugated with terbium-labeled monoclonal primary antibodies against prostate specific antigen have been employed for FRET immunoassays for prostate specific antigen [360]; polystyrene NPs labeled with Eu 3+ chelates and coated with Alexa Fluor 680 labeled γ-globulin can be used for the analysis of nucleic acid samples [361]; and transfers between terbium complexes, organic dyes and QDs (multistep FRET) have also been reported [362]. Eu chelates can also be conjugated with peptides for monitoring kinase catalyzed tyrosine phosphorylation through a quenching resonance energy transfer (QRET) between the Eu chelate and a soluble quencher molecule.…”