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However, in situ quantitative chemical analysis of single\u2010vesicle storage and release dynamics at single\u2010cell level remains a major technical challenge, hindering mechanistic understanding of cholinergic synaptic plasticity across physiological and pathological states. To address this, we developed an Ti\n                    <jats:sub>3<\/jats:sub>\n                    C\n                    <jats:sub>2<\/jats:sub>\n                    T\n                    <jats:sub>x<\/jats:sub>\n                    MXene\/enzyme\u2010functionalized (M@E@CF) nanosensor, enabling real\u2010time monitoring of vesicular ACh storage and exocytotic release in primary cholinergic neurons and human spinal cord organoids. Our findings reveal a sub\u2010quantal release mode in both mouse and human\u2010derived neurons. To further elucidate the regulatory principles of exocytosis kinetics, we classified single\u2010vesicle exocytosis patterns based on signal peak shapes using a 1D convolutional neural network (1D\u2010CNN) deep learning model, uncovering significant differences in the number of released molecules and kinetic parameters across modes. Critically, in Down syndrome models, we observed significantly reduced single vesicle ACh storage and release alongside an elevated release fraction, concurrent with shortened fusion pore durations during exocytosis. Thus, the M@E@CF nanosensor platform establishes a versatile tool for spatiotemporal investigation of neurotransmitter storage and release dynamics, providing a critical technical foundation for exploring physiological functions and pathological mechanisms of the cholinergic system.\n                  <\/jats:p>","DOI":"10.1002\/anie.202520854","type":"journal-article","created":{"date-parts":[[2026,1,10]],"date-time":"2026-01-10T14:09:41Z","timestamp":1768054181000},"update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":3,"title":["A Novel Acetylcholine Nanosensor for Single Vesicle Storage and Sub\u2010Quantal Exocytosis in Living Neurons and Organoids"],"prefix":"10.1002","volume":"65","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-6292-4108","authenticated-orcid":false,"given":"Wanying","family":"Zhu","sequence":"first","affiliation":[{"name":"School of Pharmacy Nanjing Medical University  Nanjing 211166 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