Beyond Thioflavin-T: Excited State Intramolecular Proton Transfer-Active 2-Phenylbenzimidazoles for Sensitive and Selective Detection of Insulin Amyloid Aggregates via a Unique Binding Mechanism

We introduce a novel series of excited state intramolecular proton transfer (ESIPT)-active 2-phenylbenzimidazole fluorophores (<b>1</b>-<b>3</b>) as highly selective turn-on probes for insulin amyloid aggregates (IAAs). Synthesized via an eco-friendly, metal-free route, these probes show high specificity for IAAs over native insulin and Thioflavin-T (ThT). Density functional theory (DFT) calculations confirm the intrinsic ESIPT mechanism, while photophysical studies reveal that a specific binding mode restricts molecular motion, enhancing quantum yield and fluorescence lifetime. Crucially, the lead compound (<b>3</b>) binds to a unique site distinct from ThT, as proven by competitive binding and Förster Resonance Energy Transfer (FRET) assays. It also exhibits superior affinity, a low detection limit, the ability to modulate aggregation kinetics, and excellent biocompatibility. This work moves beyond ThT, positioning the ESIPT-active 2-phenylbenzimidazole scaffold as a promising theranostic tool for amyloid-related pathologies such as Type-2 diabetes.