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Acetylcholine (Ach) is one of the earliest studied neurotransmitters and is an excitatory neurotransmitter for many peripheral nerves such as motor nerves, preganglionic fibers of the autonomic nervous system, and postganglionic fibers of the parasympathetic nervous system. Acetylcholine reacts with the substrate solution to generate a brown compound through a color reaction. The absorbance OD value is measured, and the color depth is proportional to the concentration of acetylcholine.