Why is TR-FRET so sensitive?

TR-FRET is a combination of Time Resolved Fluorescence (TRF) and Forster's (Fluorescent) Resonance Energy Transfer (FRET) technologies.

In TR-FRET the donor molecule is a Lanthanide metal such as Europium. Upon excitation, the energy from the donor (resonance energy) is transferred (donated) to an acceptor (Quencher or fluorescent dye) provided that both molecules are in close proximity.
The acceptor in turn emits light at its corresponding emission wavelength.

Low background with improved assay sensitivity

Long-lived Fluorescence

Biological samples (biological fluids, serum, cells, tissues) typically generate background fluorescence that is detected during a conventional FRET assay, with low signal-to-noise ratio.

With TR (time-resolved) and TR-FRET assays, the fluorescent signal of the lanthanide metals is long-lived and can be measured after the background fluorescence of the biological sample has decayed, resulting in low background with improved assay sensitivity.

Donor fluorophores used in conventional FRET assays have a short stokes shift, with overlapping emission and excitation peaks. This results in background interference from the excitation fluorescence during emission detection. In TR-FRET, the lanthanide donor has a large stokes shift resulting in minimal crosstalk between excitation and emission wavelengths, and a maximal assay sensitivity.

Fig 1. Stokes shift absorption and emission peaks for Europium

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