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

Emmiliisa Vuorinen; Harri Härmä; Kari Kopra; Markku Syrjänpää; Qi Wang; Sakari Kulmala

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

Emmiliisa Vuorinen
Harri Härmä
Kari Kopra
Markku Syrjänpää
Qi Wang
Sakari Kulmala
Katso/Avaa
Final draft (1.191Mb)
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ELSEVIER
doi:10.1016/j.aca.2019.09.045
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Julkaisun pysyvä osoite on:
https://urn.fi/URN:NBN:fi-fe2021042821201
Tiivistelmä
A novel homogeneous assay system QTR-FRET (Quencher modulated Time-Resolved Forster Resonance Energy Transfer) combining quenching resonance energy transfer (QRET) and time-resolved Forster 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.
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