Xiang_2026_Anal.Chem__

Acetylcholinesterase (AChE) is a cholinergic enzyme that hydrolyzes the neurotransmitter acetylcholine (ACh). Dysregulated AChE activity is closely linked to neurodegenerative diseases and cancer. Current chemical probes for AChE detection mostly rely on single-modality fluorescence readouts, limiting their utility. Herein, we developed a set of activity-based multimodal probes, termed AFP1, AFP2, AFP3, and AFPP, which combine the complementary strengths of near-infrared fluorescence (NIRF) imaging, photoacoustic (PA) detection, and proteome profiling. These probes offer a comprehensive toolset for deciphering the AChE code in complex biological systems. AFP1/2/3 enable continuous monitoring of AChE activity via NIRF/PA detection, while AFPP represents the first trimodal probe for simultaneous NIRF/PA imaging and proteome profiling in live systems. Notably, the usage of AFP2 and AFPP (1) highlighted the superior capability of PA imaging for deep-tissue studies in live animals, (2) revealed significantly elevated AChE activity in microglia compared to astrocytes in the depressive brain, and (3) identified a functional link between AChE and adhesion G protein-coupled receptor B2 and B3 (ADGRB2 and ADGRB3) in both cellular and murine models of depression. Our study not only provides powerful molecular tools for studying cholinergic systems but also reveals novel therapeutic targets for depression intervention.