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

Abstract

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.

A general strategy to the intracellular sensing of glycosidases using AIE-based glycoclusters

“…Compared to traditional colorimetric and enzyme-linked immunosorbent assays, fluorescent probes have been viewed as a more privileged technique for the detection of enzymatic activities due to their high sensitivity, simplicity in design and preparation, and the ability for the in-situ imaging of enzymatic activities in cells and in vivo. [9][10][11][12][13][14] The rationale by which glycosidase probes are designed typically relied on the conjugation of one molecule of monosaccharide to the phenol group of a donor-acceptor (D-A) type fluorogen (Table S1). This glycosylation reaction quenches the intrinsic fluorescence of the phenol dye through modulation of the intramolecular charge transfer (ICT) or photo-induced electronic transfer (PeT) process.…”

A General Strategy to the Intracellular Sensing of Glycosidases using AIE-Based Glycoclusters

“…Compared to the traditional colorimetric biochemical tests and the enzyme-linked immunosorbent assay, fluorescent probes have been viewed as a more privileged technique for the detection of enzymatic activities due to their high sensitivity, simplicity in design and preparation, and the ability for the in-situ imaging of enzymatic activities in cells and in vivo. [9][10][11][12][13][14] The rationale by which glycosidase probes are designed typically relied on the conjugation of one molecule of monosaccharide to the phenol group of a donor-acceptor (D-A) type fluorogen (Table S1). This glycosylation quenches the intrinsic fluorescence of the phenol dye through modulation of the intramolecular charge transfer (ICT) or photo-induced electronic transfer (PeT) process.…”

Visualization of intracellular lipid droplets using lipophilic benzothiazole-based push-pull fluorophores at ultralow concentration

“…[24][25][26][27] In our laboratory, we have recently developed a new set of environment responsive D-π-A fluorophores and demonstrated their high biocompatibility, fine-tuned spectroscopic properties, and their smooth chemical transformation into molecular probes for biological applications. [28,29] The molecular structures of our fluorophores encompass an electron donating 6-aminofunctionalized benzothiazole scaffold (D) which is connected via a single vinyl linker to different electron acceptor aryl groups (A) (Fig. 1).…”

“…[28] Thereafter, these fluorophores were successfully employed to develop highly sensitive probes to monitor β-galactosidase activity during early stages of cellular senescence. [29] Figure 1. General structure of the benzothiazole-based styryl fluorophores previously reported by our group.…”

“…General structure of the benzothiazole-based styryl fluorophores previously reported by our group. [28,29] Herein, we disclose the photophysical characterization of these novel structures by UV-Vis absorption and steady-state fluorescence emission spectroscopies and exemplify their use for intracellular lipid droplet visualization by confocal microscopy. (Table 1).…”