Development of fluorescent probes for on-site sensing and long-term tracking of specific biomarkers is particularly desirable for the early detection of diseases. However, available small-molecule probes tend to facilely diffuse across the cell membrane or remain at the activation site but always suffer from the aggregation-caused quenching (ACQ) effect. Here we report an enzyme-activatable aggregation-induced emission (AIE) probe QM–βgal, which is composed of a hydrophilic β-galactosidase (β-gal)-triggered galactose moiety and a hydrophobic AIE-active fluorophore QM–OH. The probe is virtually non-emissive in aqueous media, but when activated by β-gal, specific enzymatic turnover would liberate hydrophobic AIE luminogen (AIEgen) QM–OH, and then highly fluorescent nanoaggregates are in situ generated as a result of the AIE process, allowing for on-site sensing of endogenous β-gal activity in living cells. Notably, taking advantage of the improved intracellular retention of nanoaggregates, we further exemplify QM–βgal for long-term (∼12 h) visualization of β-gal-overexpressing ovarian cancer cells with high fidelity, which is essential for biomedicine and diagnostics. Thus, this enzyme-activatable AIE probe not only is a potent tool for elucidating the roles of β-gal in biological systems, but also offers an enzyme-regulated liberation strategy to exploit multifunctional probes for preclinical applications.

Intramolecular charge transfer (ICT) is a fundamental mechanism that enables the development of numerous fluorophores and probes for bioimaging and sensing. However, the electron-withdrawing targets (EWTs)-induced fluorescence quenching is a long-standing and unsolved issue in ICT fluorophores, and significantly limits the widespread applicability. Here we report a simple and generalizable structural-modification for completely overturning the intramolecular rotation driving energy, and thus fully reversing the ICT fluorophores’ quenching mode into light-up mode. Specifically, the insertion of an indazole unit into ICT scaffold can fully amplify the intramolecular rotation in donor-indazole-π-acceptor fluorophores (fluorescence OFF), whereas efficiently suppressing the rotation in their EWT-substituted system (fluorescence ON). This molecular strategy is generalizable, yielding a palette of chromophores with fluorescence umpolung that spans visible and near-infrared range. This strategy expands the bio-analytical toolboxes and allows exploiting ICT fluorophores for light-up sensing of EWTs including N-acetyltransferases and nerve agents.

β-Galactosidase (β-gal), a typical hydrolytic enzyme, is a vital biomarker for cell senescence and primary ovarian cancers. Developing precise and rapid methods to monitor β-gal activity is crucial for early cancer diagnoses and biological research. Over the past decade, activatable optical probes have become a powerful tool for real-time tracking and in vivo visualization with high sensitivity and specificity. In this review, we summarize the latest advances in the design of β-gal-activatable probes via spectral characteristics and responsiveness regulation for biological applications, and particularly focus on the molecular design strategy from turn-on mode to ratiometric mode, from aggregation-caused quenching (ACQ) probes to aggregation-induced emission (AIE)-active probes, from near-infrared-I (NIR-I) imaging to NIR-II imaging, and from one-mode to dual-mode of chemo-fluoro-luminescence sensing β-gal activity.