Enzyme‐Mediated In Situ Self‐Assembly AIE Probes: Mapping the Distribution of Acetylcholinesterase in Aging Mice Brain

Abstract Acetylcholinesterase (AChE) exhibits profound association with the underlying pathological mechanisms of aging‐related disorders. However, revealing the spatial distribution of AChE among whole‐brain remains an uncharted territory, posing a substantial challenge. Herein, a dual regulation strategy to obtain an aggregation‐induced emission (AIE)‐activable probe for high‐fidility mapping of AChE in the brain is proposed. The well‐tailored probe (AChE‐QM‐2) consists of four components: AChE‐specific cleavage unit (dimethylcarbamate), self‐immolative linker ( p ‐hydroxybenzyl alcohol), AIE framework (Py‐QM), and electron‐donor group (EDG). The hydrophilic pyridine salt of AChE‐QM‐2 improves its dispersity, collaborating with EDG to lower its Δ E S1‐T1 and HOMO‐LUMO energy gap, resulting reduced fluorescence quantum yield ( Φ f ) and initial off‐fluorescence state. Moreover, optimizing EDGs further enhance the probe's responsive performance and extend its wavelength. Upon AChE‐activization, AChE‐QM‐2 undergoes deionization, resulting in molecule aggregation and increased Φ f , further triggering amplified AIE signal. AChE‐QM‐2 has successfully realized monitoring of endogenous AChE in PC12 cells, tissues and living mice. Furthermore, utilizing light‐sheet microscopy, spatial mapping of the intracranial AChE, providing a highly specialized visualization of its localization within the brain, is achieved for the first time. This study satisfactorily demonstrates a valuable strategy for designing ultra‐sensitive AIE enzymatic probes, highlighting their potential for precise analysis in fundamental life science research.