Chemiluminescent Probe Enables Enhanced Visualization of Acute Ischemic Stroke via Acetylcholinesterase Activation

Acute ischemic stroke (AIS) leads to severe hypoxia-induced brain injury, yet timely management is hampered by the lack of imaging modalities that simultaneously offer speed, sensitivity, and precision, owing to the scarcity of stable and reliable biomarkers. Acetylcholinesterase (AChE), markedly upregulated under anaerobic stress, has emerged as a promising hypoxia biomarker; however, its potential for AIS visualization remains unexplored. Herein, we report a chemiluminescent AChE-responsive probe, AhCL, constructed by functionalizing Schaap’s dioxetane with a quaternary ammonium–N,N-formate moiety. The probe demonstrates high selectivity toward AChE with minimal interference from common physiological analytes. Under hypoxic conditions, it enables ultrasensitive detection of endogenous AChE, achieving a detection threshold as low as 863 cells. In an AIS mouse model, in situ imaging with AhCL produced a 3–23-fold enhancement in high-contrast chemiluminescent signals across differentially hypoxia-exposed brain tissues within 1 h, clearly distinguishing tissue hypoxia levels. Notably, AChE activation allowed the probe to effectively differentiate hypoxia-induced brain injury, potentially providing critical guidance for clinical intervention. This study establishes AChE as a robust biomarker for AIS and offers a potential tool for timely diagnosis and assessment.