Photochromism-Fret (phFRET): Modulation of Fluorescence Resonance Energy Transfer by A Photochromic Acceptor

We have designed a new method for the quantitative determination of fluorescence resonance energy transfer (FRET) based on the modulation of the fluorescence emission of a donor by a photochromic acceptor. Light induces a change in the structure and thus the absorption properties of the acceptor. Only one of the absorption spectra overlaps with the emission of the donor, thereby providing the means for switching the process of FRET on and off reversibly. A direct evaluation of the change in the donor quantum yield due to FRET is possible. A test molecule was devised consisting of a Lucifer Yellow donor bound covalently to a 6-nitroBIPS acceptor via a cysteine linker molecule. Photoisomerisation of the acceptor by near-UV irradiation from the spiropyran to the merocyanine form potentiated the FRET process, as evidenced by quenching of the donor. This effect disappeared upon thermal reversion to the initial spiropyran form. Photokinetic simulations were also carried out.