A Novel Acetylcholine Nanosensor for Single Vesicle Storage and Sub‐Quantal Exocytosis in Living Neurons and Organoids

Abstract Acetylcholine (ACh) is a critical neurotransmitter that regulates diverse physiological functions, such as cognition and muscle contraction, through synaptic transmission. However, in situ quantitative chemical analysis of single‐vesicle storage and release dynamics at single‐cell 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 3 C 2 T x MXene/enzyme‐functionalized (M@E@CF) nanosensor, enabling real‐time monitoring of vesicular ACh storage and exocytotic release in primary cholinergic neurons and human spinal cord organoids. Our findings reveal a sub‐quantal release mode in both mouse and human‐derived neurons. To further elucidate the regulatory principles of exocytosis kinetics, we classified single‐vesicle exocytosis patterns based on signal peak shapes using a 1D convolutional neural network (1D‐CNN) 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.