Time-resolved fluorescence energy transfer

The FRET aspect of the technology is driven by several factors, including spectral overlap and the proximity of the fluorophores involved, wherein energy transfer occurs only when the distance between the donor and the acceptor is small enough.

Certain life science applications take advantage of the unique fluorescence properties of lanthanide ion complexes (Ln(III) chelates or cryptates).

These are well-suited for this application due to their large Stokes shifts and extremely long emission lifetimes (from microseconds to milliseconds) compared to more traditional fluorophores (e.g. fluorescein, allophycoyanin, phycoerythrin, and rhodamine).

This method (commonly referred to as time-resolved fluorometry or TRF) involves two fluorophores: a donor and an acceptor.

(donor excitation ⇒ acceptor emission) As noted in the table above, fluorescent energy transfer from Europium to allophycocyanin can be used in a time resolved manner, particularly in biomolecular screening assays.

Through the design of the high-throughput screening assay, the materials are mixed, and if the enzyme does act on the peptide, all components will bind their respective targets and FRET will occur.

('cross-talk' in this instance refers to overlapping spectral profiles, which could result in false-positives, false-negatives, or reduced sensitivity depending on the assay design.