Neuronal communication occurs through the rapid, calciumtriggered release of neurotransmitters from synaptic vesicles fusing with the cell plasma membrane. This process, known as exocytosis, happens in less than a millisecond, 2 allowing neurotransmitters released to bind to receptors at neighboring neurons and transmit chemical signals. The amount and frequency of neurotransmitter released are important factors in determining the communication strength between neurons and in shaping synaptic plasticity. This process underlies fundamental brain functions such as learning and memory, and is also involved in the pathophysiology of brain disorders such as drug abuse and addiction, which are not yet fully understood. Acetylcholine (ACh) is a key excitatory neurotransmitter in the central and peripheral nervous systems (CNS and PNS), essential for regulating heart rate, controlling muscle contraction, and contributing to cognitive function. It also serves as a neuromodulator of glutamatergic and GABAergic synapses in the CNS. The impairment of the cholinergic system has been associated with several neurological and psychiatric disorders, such as Alzheimer's disease and schizophrenia. Investigations of cholinergic neurotransmission and its role in complex cognitive behavior, have led to the development of various analytical techniques for studying ACh signaling, such as in vivo microdialysis, capillary electrophoresis, mass spectrometry, neuroimaging, and photoelectrochemical biosensing. However, for an in depth understanding of the regulatory aspects of ACh neurotransmission at synapses, methods that spatially and temporally can capture the dynamics of rapid synaptic vesicle ACh release during neuronal activity are needed. Synaptic neurotransmission can be studied using electrochemical methods where carbon fiber amperometry is commonly used due to its straightforward application and superior temporal resolution (µseconds). By placing carbon fiber micro/nanoelectrodes at release sites of single cells, redox currents in response to detection of neurotransmitters released from single exocytosis events can be recorded. This method enables the recording of vesicle fusion pore-controlled release dynamics of single synaptic exocytosis events that occur on sub-millisecond timescale. 12 Further, amperometry is quantitative and the number of molecules released during exocytosis can be determined, resulting in a measure of synaptic signaling strength. The integrated charge (Q) from individual current spike detected, can be converted into the number of molecules released using Faraday's law. 12 However, its applicability is limited to electroactive neurotransmitters, such as catecholamines and serotonin, which can be readily oxidized at the electrode surface. For non-electroactive neurotransmitters like ACh and glutamate, redox reactions at the carbon surface are not possible. To address this, a chemically selective biosensor can be created by modifying the electrode surface with enzymes capable of converting non-electroactive neurotransmitters into detectable reporting molecules, usually hydrogen peroxide (H2O2). While these biosensors are sensitive and selective, their detection capabilities have remained limited to sub-second time resolutions, which is too slow for monitoring individual exocytosis events. Insufficient technology for direct quantitative measurements of ACh then has led to varying estimates of ACh vesicle quantal size, ranging from 400 to 22,000 ACh molecules, depending on the biological model system and analysis techniques used. To overcome this challenge, our lab introduced a novel biosensor design concept that improves sensor speed by limiting enzyme coatings to a molecular monolayer. This approach minimizes diffusion distances for enzymatic electroactive reporter molecules to reach the electrode surface for detection. Compared to traditional sensors with thick enzyme layers, our minimal enzyme-coating concept has reduced response times by up to 3 orders of magnitude. With a 33-µm carbon fiber biosensor, we have captured ACh release from artificial cells in tens of milliseconds, and glutamate from exocytotic release in brain tissue and isolated synaptic vesicles in sub-milliseconds. In this work, we tailored our ultrafast sensor concept for single cell ACh presynaptic measurements by immobilizing the sequential enzymes acetylcholinesterase (AChE) and choline oxidase (ChO) onto a 5 μm carbon fiber disc electrode surface (Figure ), providing dimensions for precise positioning against single cell structures. To functionalize the carbon microelectrode surface, maximizing its sensing surface area, enhancing detection efficiency of H2O2 and providing a scaffold for the ultrathin enzyme coating, a dense layer of gold nanoparticle (AuNP) hemispheres, approximately 80 nm in diameter, was electrodeposited, creating a surface topology resembling to cauliflower (Figure , Figure ). The enzymes AChE and ChO were then immobilized onto the AuNP-modified sensor surface at a molar ratio of 1:10, based on a previous study optimizing ACh sequential enzymatic catalysis (Figure ). The ACh biosensor created was then placed in contact to axon terminals of differentiated cholinergic human SH-SY5Y neuroblastoma cells (Figure ,F and Figure ). In Figure , and detailed in Table , when a constant potential of -0.5V versus a Ag/AgCl reference electrode was applied to the ACh sensor surface, the sensor successfully captured spontaneous bursts of isolated reduction current transients corresponding to individual ACh exocytotic events at the submillisecond timescale (n = 18 cells). The average frequency of ACh release was measured at 28 ± 5 Hz (n = 16 cells), with the error representing the standard error of the mean (SEM) and the average current spike from these recordings shows the typical shape characteristics of fusion pore regulated exocytosis with a rapid Trise and slower Tfall (Figure ). To demonstrate how the ACh biosensor can be used to investigate the drug effect on ACh signaling, single cholinergic cells were exposed to 0.1 μM bafilomycin A1 (Figure ,H), a V-ATPase inhibitor known to also activate release of intracellular calcium stores, potentially affecting fusion pore dynamics and exocytosis activity. By recording ACh release from a single release site before and after a 10-min local administration of the drug using a glass microinjection pipette, an increase in both the exocytosis activity (170%) and quantal release (120%) was observed (Figure , Figure and Table ). This finding is consistent with a previous study on catecholamine signaling in chromaffin cells and demonstrates the applicability of this technology for presynaptic studies. The high temporal resolution of our recordings (50 kHz) enabled detection of a diversity of shapes and time courses for individual current spikes. This spike heterogeneity indicates that the fusion pore is highly regulated by factors controlling its opening and closing and leads to presynaptic tuning of ACh release in these cells. Figure presents the spike shapes identified and shows that the highest abundance of spikes corresponds to "sharp spikes" (42%). Many of these spike types have been previously identified with comparable prevalence in brain tissue recordings of octopamine in Drosophila melanogaster and glutamate in rodents. In these recordings, a novel category of sharp spike was detected, adding to the six previously observed spike types associated with partial, full and multi-release in glutamatergic exocytosis. This spike, predominant at 9%, exhibits a sharp simple shape (depicted in Figure ). Compared to the other current spike types, it has approximately twice the current amplitude (Imax) and result in a similar total charge detected as spikes representing full exocytosis mode (Figure ). Hence, we classify these spikes as 'full sharp' spikes. To accurately quantify the number of ACh molecules by exocytosis release, an appropriate calibration of the sensor signal is needed. Here we used a liposome-based calibration strategy, pioneered by our lab for quantifying glutamate. This method involves immersing the ACh sensor into a solution of large unilamellar liposomes (LUVs) preloaded with ACh and performing amperometric recording of single LUV content. By applying a potential to the biosensor, individual LUVs in contact with the sensor surface stochastically rupture and the released ACh is detected as current spikes. Figure and B display a typical redox current time trace, detecting ACh release from single LUVs pre-filled with a 400 mM ACh solution, demonstrating detection of single current spikes on sub-millisecond timescale (Figure , Table ). Extruded LUVs (~180 nm in diameter) pre-filled with various ACh concentrations (200 mM to 600 mM) underwent electroanalysis using the ACh sensor.