Ratiometric fluorescent probes with a self-immolative spacer for real-time detection of β-galactosidase and imaging in living cells

Abstract Ratiometric fluorescent probes with a self-immolative spacer for β -galactosidase ( β -gal) were developed. They function by β -gal-cleaving the β -galactoside bond of fluorescent substrates, followed by self-immolation to liberate the amino group of fluorophore. Thus, a remarkable variation in the photophysical properties was observed and the corresponding ratiometric detection of β -gal was realized. Our studies demonstrated that the GNPN exhibited high sensitivity for recognition of β -gal, with a detection limit as low as 0.17 U L −1 . GNPN can rapidly quantify β -gal enzyme activity; the emission ratio F 545 / F 475 for the GNPN reached maxima after approximately 4 min, which was one of the shortest response time ever reported. Furthermore, we demonstrated that these probes possess excellent biocompatibility and can be used to visualize the endogenous β -gal in ovarian cancer cells.