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.

Water-soluble, long wavelength fluorescent aggregation-induced emission (AIE)-active materials are in great demand for high contrast biosensing and bioimaging. The substitution position effects of the sulfonate group on the basis of two quinoline-malononitrile (QM) derivatives (EDS and EDPS) provide insight into efficient modulation in the hydrophilicity, emitting color, and specific AIE characteristics. EDS shows a unique AIE behaviour in aqueous solution, but EDPS does not. The abnormal non-fluorescence aggregation for EDS in pure water is capsule-like with loose packing characteristics, but still has enough cavities or free volume to consume the radiative energy, resulting in nearly no fluorescence. When binding with the protein BSA, the sulfonate unit as a conformation function group (CFG) plays a vital role in altering its initial loose ensemble into tightly compact aggregation with light-up AIE characteristics. By cell tracking, dynamic light scattering (DLS) and transmission electron microscopy (TEM), the key role of sulfonate groups in the conformation alteration has been well demonstrated for the first time. Moreover, EDS is successfully exploited in a label-free real time AIE fluorescent assay for trypsin detection and inhibitor screening. The hydrophilic sulfonate group from the different substitution position in the AIE-active QM building blocks provides an effective way to tailor the intermolecular aggregation associated with molecular stacking, especially for in situ cell tracking and real-time trypsin detection.

The advance of cancer imaging requires innovations to establish novel fluorescent scaffolds that are excitable and emit in the near-infrared region with favorable Stokes shifts. Nevertheless, the lack of probes with these optimized optical properties presents a major bottleneck in targeted cancer imaging. By coupling of boron dipyrromethene platforms to enzymic substrates via a self-immolative benzyl thioether linker, we here report a strategy toward enzyme-activated fluorescent probes to satisfy these requirements. This strategy is applicable to generate various BODIPY-based probes across the NIR spectrum via introducing diverse electron-withdrawing substituents at the 3-position of the BODIPY core through a vinylene unit. As expected, such designed probes show advantages of two-channel ratiometric fluorescence and light-up NIR (I and II) emission with large Stokes shifts upon enzyme activation, enabling targeted cancer cell imaging and accurate tumor location by real-time monitoring of enzyme activities. This strategy is promising in engineering activatable molecular probes suitable for precision medicine.