Homogeneous assay based on low quantum yield Sm(III)-donor and anti-Stokes' shift time-resolved fluorescence resonance energy-transfer measurement

Non-overlapping fluorescence resonance energy-transfer (nFRET) is a highly sensitive, time-resolved, assay technology. It utilizes lanthanide chelates as donors together with non-overlapping acceptor fluorophores, which have their absorption maximum energetically at a higher level than the emittive transitions of the donor. In this report the nFRET was studied using a low quantum yield Sm(III)-chelate as the donor in a homogeneous dF508-DNA hybridization assay. Despite the low quantum yield, Sm(III) could function as an efficient donor in nFRET, which resulted in strong energy-transfer induced acceptor emission in the assay. Further, the induced acceptor emission of the assay showed non-Förster-type decay characteristics and allowed the sensitive anti-Stokes' shift FRET measurement, in which the induced acceptor emission was measured at a wavelength region shorter than the main emittive transitions of the donor. The detection limit for the dF508 DNA-target was 12.0 pM (S/B = 2). The assay sensitivity was surprisingly similar with the corresponding Eu-based nFRET-assay, despite the approximately 90-fold lower quantum yield of the Sm-donor. The results confirm the exceptional properties of the nFRET induced acceptor signal and support the theory of energy-transfer taking place from the excited energy levels above the emittive energy level of the lanthanide donor. We conclude that nFRET-technique is well suited for highly sensitive DNA assays and that the assay sensitivity is not limited by the emittive quantum yield of the donor.