Nanotip Acetylcholine Biosensor Reveals Cholinergic Differentiated SH-SY5Y Cells Release Partial Vesicle Content During Exocytosis

Acetylcholine (ACh) is a key neurotransmitter involved in cognitive function, motor control, and synaptic modulation, yet its electrochemical inactivity and the rapid kinetics of exocytosis have hindered real-time quantal detection. Previous micrometer-scale enzymatic ACh biosensors enabled sub-millisecond extracellular recordings but were too large for placement at synapses and unsuitable for intracellular recordings. Here we present a short, ultrafast, low-noise amperometric ACh biosensor based on a thin, needle-shaped carbon fiber nanotip electrode functionalized with gold nanoparticles and enzymes. This miniaturized design allows precise positioning at neurite release sites and minimally invasive insertion into the cell cytoplasm, enabling both high-temporal resolution monitoring of exocytosis and quantification of intracellular ACh vesicle content. We applied this platform to differentiated human cholinergic SH-SY5Y neuroblastoma cells, an established yet underutilized model for studying cholinergic signaling and neurodegenerative diseases. The nanotip sensor successfully captured amperometric signals corresponding to both detection of intracellular vesicle ACh content and exocytotic release events. Comparative analysis revealed that average intracellular vesicle bursting events released 1.8-fold higher ACh than exocytosis events, indicating the predominance of partial exocytosis in these cells. However, the most frequent intracellular vesicle bursts released only about half the ACh content compared to the presumed full exocytosis events, suggesting that partial release mechanism might also be dominant during intracellular vesicle bursting. Together, these results demonstrate the unique capability of the nanotip ACh biosensor to probe fusion pore dynamics at subcellular resolution and provide new insights into the quantal nature of cholinergic signaling in human neuronal models.