Nanotip acetylcholine biosensor reveals cholinergic differentiated SH-SY5Y cells release partial vesicle content during exocytosis

Acetylcholine (ACh) is a central neurotransmitter in cognitive function, motor control, and synaptic modulation, yet its electrochemical inactivity and the rapid kinetics of exocytosis have hindered real-time quantal measurements. Micrometer-scale enzymatic ACh biosensors previously enabled sub-millisecond extracellular recordings but were too large for synaptic positioning and intracellular recordings. Here we present a short, ultrafast and low-noise amperometric ACh biosensor based on a needle-shaped carbon fiber nanotip electrode functionalized with gold nanoparticles and enzymes. The miniaturized geometry allows precise placement at neurite release sites and minimally invasive insertion into the cell cytoplasm, enabling high-temporal resolution monitoring of presynaptic exocytosis together with quantification of intracellular ACh vesicle content. We applied this platform to differentiated human cholinergic SH-SY5Y neuroblastoma cells, an established yet underutilized cell model for cholinergic signaling. The nanotip sensor successfully captured amperometric spikes from both intracellular vesicle burst events and presynaptic ACh release. Intracellular events released a larger amount of ACh than presynaptic exocytosis events, indicating a predominance of partial exocytosis mode in these cells. These results demonstrate the nanotip ACh biosensor as a unique tool for probing fusion pore dynamics at subcellular resolution and for providing quantitative insight into the quantal nature of cholinergic signaling in human neuronal models.