QTR-FRET: Efficient background reduction technology in time-resolved förster resonance energy transfer assays

A novel homogeneous assay system QTR-FRET (Quencher modulated Time-Resolved Förster Resonance Energy Transfer) combining quenching resonance energy transfer (QRET) and time-resolved Förster resonance energy transfer (TR-FRET) was developed to reduce background signal in the conventional energy transfer applications. The TR-FRET functionality is often limited by the lanthanide donor background signal leading to the use of low donor concentration. QTR-FRET reduces this background by introducing soluble quencher molecule, and in this work the concept functionality was proven and compared to previously introduced QRET and TR-FRET technologies. Comparison was performed with three different Eu³⁺-chelates exhibiting different luminescent lifetime and stability. The side-by-side comparison of the three signaling systems and Eu³⁺-chelates was demonstrated in a model assay with Eu³⁺-chelate conjugated biotin and streptavidin (SA) or Cy5-SA conjugate. Comparison of the methodologies showed increased signal-to-background ratios when comparing QTR-FRET to TR-FRET, especially at high Eu³⁺-biotin concentrations. Quenching the non-bound Eu³⁺-biotin improved the assay performance, which suggests that an improved assay performance can be attained with the QTR-FRET method. QTR-FRET is expected to be especially useful for Eu³⁺-labeled ligands with low affinity or assays requiring high Eu³⁺-ligand concentration. The QTR-FRET indicated potential for multi-analyte approaches separately utilizing the direct QRET-type Eu³⁺-chelate signal and energy transfer signal readout in a single-well. This potential was hypothesized with Avi-KRAS nucleotide exchange assay as a second biologically relevant model system.