Homogeneous Assay Based on Anti-Stokes' Shift Time-Resolved Fluorescence Resonance Energy-Transfer Measurement

Abstract

We report here a novel, time-resolved, lanthanide-based energy-transfer assay utilizing nonoverlapping acceptor fluorophores, which have their absorption energetically at a higher level than the emittive transitions of the donor. The technique was studied by comparing a series of nonoverlapping acceptors in a homogeneous DNA model assay utilizing Eu3+ chelate as a donor. The assay provides strong energy-transfer enhanced acceptor emission and enables the anti-Stokes' shift FRET measurement, in which the induced acceptor emission is at shorter wavelength than the donor emission. This results in high sensitivity, and 0.8 pM detection limit was measured for the DNA target. The acceptor signal of the assay is characterized by exceptional lifetime properties and is not strictly following the Forster's theory. The mechanism of nonoverlapping energy transfer is considered, and we propose that when nonoverlapping acceptors are utilized, the energy transfer arises from the upper 5D2 and 5D1 excited states of europium. The assumption was studied using a simplified energy level scheme of the Eu3+ donor and the acceptors, and a correlation between the acceptor emission behavior and the energy level scheme was found.

Fluorescence in Nanobiotechnology: Sophisticated Fluorophores for Novel Applications

“…Binding of GTP‐Tb to the catalytic site of Rab21‐GFP could be monitored by FRET‐quenching of the Tb‐donors and FRET‐sensitization of the GFP‐acceptors due to spatial proximity (the Tb millisecond lifetime was quenched to approximately 60 μs, which could also be found for the sensitized GFP emission). The GTP‐Eu‐Rab21‐YFP pair showed a phenomenon, which was named non‐overlapping FRET (nFRET) 153. As the emitting energy transitions (from the 5 D 0 state) for Eu do not overlap with the absorption energies of YFP (or Alexa Fluor dyes in case of the first nFRET publication153), it was assumed that the ET occurs from the higher (non‐emitting) energy levels ( 5 D 2 and 5 D 1 ) of Eu.…”

“…The GTP-Eu-Rab21-YFP pair showed a phenomenon, which was named non-overlapping FRET (nFRET). [ 153 ] As the emitting energy transitions (from the 5 D 0 state) for Eu do not overlap with the absorption energies of YFP (or Alexa Fluor dyes in case of the fi rst nFRET publication [ 153 ] ), it was assumed that the ET occurs from the higher (non-emitting) energy levels ( 5 D 2 and 5 D 1 ) of Eu. As the quenched (for Eu) and sensitized (for YFP) fl uorescence lifetimes (two-exponential decays with ca.…”

Luminescent lanthanide reporters: new concepts for use in bioanalytical applications

“…According to the traditional definition of FRET, yet another requirement is a substantial spectral overlap between the donor emission and acceptor excitation spectra. However, according to recent studies, this is not an absolute requirement for non-radiative energy transfer, since a phenomenon termed non-overlapping FRET (nFRET; also called anti-Stokes shift FRET) has been described (Laitala and Hemmilä, 2005a;Laitala and Hemmilä, 2005b). The nFRET phenomenon is based on non-radiative energy transfer between a lanthanide donor and a spectrally non-overlapping acceptor.…”

Lanthanide Nanoparticules as Photoluminescent Reporters

“…In principle, LRET from europium(III) with an emission peak at 615 nm and measurement of the sensitized emission at 730 nm would require acceptor dyes with an exceptionally large Stokes' shift, over 100 nm, although this is not necessarily required with luminescent lanthanides as efficient energy transfer has been demonstrated with conventional far-red-emitting fluorophores. It has been proposed and shown that with long-lifetime fluorescent donors, FRET is efficient even when the donor emission and the acceptor absorption spectrum do not overlap significantly (nonoverlapping FRET) [128][129][130]. In cases where maximum overlap of the absorption spectra of the donors with the major emission band of europium(III) at 615 nm is desirable, several acceptor dyes, e.g., AlexaFluor 680 and 700 (Invitrogen) and Cy5 (GE Healthcare), are commercially available.…”