A neutral multivalent AIE-TICT scaffold: An off-on, wash-free fluorogenic strategy for biosensing glycan-protein interactions in live cells and mycobacteria

Abstract

Background: 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.

Results: 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.

Significance: 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.

Keywords: Aggregation-induced emission (AIE); Bacterial metabolism; Cancer cell detection; Fluorogenic probe; Glycan–protein interaction; Twisted intramolecular charge transfer (TICT).