A1 Vertaisarvioitu alkuperäisartikkeli tieteellisessä lehdessä
QTR-FRET: Efficient background reduction technology in time-resolved förster resonance energy transfer assays
Tekijät: Markku Syrjänpää, Emmiliisa Vuorinen, Sakari Kulmala, Qi Wang, Harri Härmä, Kari Kopra
Kustantaja: ELSEVIER
Julkaisuvuosi: 2019
Journal: Analytica Chimica Acta
Tietokannassa oleva lehden nimi: ANALYTICA CHIMICA ACTA
Lehden akronyymi: ANAL CHIM ACTA
Vuosikerta: 1092
Aloitussivu: 93
Lopetussivu: 101
Sivujen määrä: 9
ISSN: 0003-2670
eISSN: 1873-4324
DOI: https://doi.org/10.1016/j.aca.2019.09.045
Rinnakkaistallenteen osoite: https://research.utu.fi/converis/portal/detail/Publication/42894260
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.
Ladattava julkaisu This is an electronic reprint of the original article. |  |
