A neutral multivalent AIE-TICT scaffold: An off-on, wash-free fluorogenic strategy for biosensing glycan-protein...

Fluorescent probes for wash-free imaging of weak or transient interactions, such as glycan-glycan-binding protein (GBP) recognition, remain limited by poor responsiveness and high background emission. A modular, photophysically robust, and selective platform must be established.

Herein, we report a tetraphenylethene (TPE)-julolidine framework, referred to as the viscosity-sensitive TPE-julolidine (VTJ) scaffold, that operates through two mechanisms: aggregation-induced emission and twisted intramolecular charge transfer. Glycan-conjugated VTJ probes enable selective imaging of cancer cells and mycobacteria. Upon specific glycan-GBP engagement, VTJ glycoprobes accumulate in subcellular compartments and undergo target-induced molecular stacking, restricting intramolecular motion and exhibiting tenfold "off-to-on" fluorescence enhancement without washing. Di-glycan probes amplify signals through multivalency, improving labeling efficiency and sensitivity in cell-based imaging. VTJ-Di-Galactose and VTJ-Di-N-acetylneuraminic acid could selectively label hepatocellular carcinoma cell line 2 (HepG2) and human pancreatic epithelioid carcinoma cell line (PANC-1) cells, respectively, whereas VTJ-Mono-Trehalose was internalized into Mycobacterium smegmatis mc²155 through Mycobacterium smegmatis porin A and was discovered to fluoresce upon its metabolic incorporation into the cell wall. Notably, VTJ glycoprobes exhibited concentration-dependent color shifts in live cells, reflecting dynamic stacking and enabling visualization of molecular crowding.

VTJ is the first fluorogenic scaffold for selective labeling of living systems through diverse glycan ligation. Its neutral, photostable framework and color-tunable behavior support modular bioconjugation for imaging applications. Notably, it has strong potential for integration with dynamic matrices (e.g., three-dimensional hydrogels) and can provide new insights into cancer cell migration and metastatic processes.