Detection of multivalent interactions through two-tiered energy transfer

A method based on two-tiered fluorescence resonant energy transfer (FRET) h as been developed for selective and sensitive detection of species involved in a multivalent interaction. Pentavalent binding between cholera toxin an d ganglioside GM1 is used as a model system to demonstrate the advantage of the two-tiered FRET over one-stage FRET in both conventional fluorimeter a nd how cytometer, In the system, three fluorescent probes (namely, fluoresc ence donor, acceptor, and intermediate) are covalently tagged to receptors, and the intermediate is used to bridge the energy transfer between the don or and acceptor even though the donor's fluorescence spectrum does not over lap with absorption spectrum of the acceptor. One of the most significant i mprovements of the scheme over one-stage FRET is a dramatic decrease in the background fluorescence of the acceptor fluorescence, which, theoretically and practically, increases the detection sensitivity. (C) 2001 Academic Pr ess.