Development of highly sensitive fluorescent probes for the detection of β-galactosidase activity - application to the real-time monitoring of senescence in live cells

We report the development of four novel fluorescent probes to monitor the activity of the β-galactosidase enzyme (β-gal), in vitro and in living cells. The fluorophores are based on a 6-amino-styryl-benzothiazole push-pull core and display a strong ICT emission. The probes encompass the fluorescent motif that is connected to a β- d -galactopyranoside moiety through a self-immolative benzyl carbamate linker ( βGal-1-4 ). The screening of four different fluorophores enabled us to access new light-up and two-band ratiometric reporters. The four probes, βGal-1-4 , exhibited an extremely fast response and over 200-fold fluorescence enhancement ( βGal-1 ) following the enzymatic cleavage of the β- d -galactopyranoside unit. This rapid and extremely sensitive response allowed the detection of senescence-associated β-galactosidase (SA-β-gal) activity; a widely used biomarker of senescence. More importantly, βGal-1 also enabled us to monitor, in real-time, the emergence of senescence in live cells , i.e. the phenotypic transformation from normal to senescent cell. These findings underpin the fact that βGal-1 may find useful applications in biomedical research. Importantly, βGal-1 is suitable for epifluorescence and confocal microscopies, and flow cytometry techniques, which are among the most common analytical tools in biology. Highly sensitive fluorescent reporters for the detection and monitoring of β-galactosidase activity in live cells.