Ex Vivo Electrochemical Monitoring of Cholinergic Signaling in the Mouse Colon Using an Enzyme-Based Biosensor

Cholinergic signaling, i.e., neurotransmission mediated by acetylcholine, is involved in a host of physiological processes, including learning and memory. Cholinergic dysfunction is commonly associated with neurodegenerative diseases, including Alzheimer’s disease. In the gut, acetylcholine acts as an excitatory neuromuscular signaler to mediate smooth muscle contraction, which facilitates peristaltic propulsion. Gastrointestinal dysfunction has also been associated with Alzheimer’s disease. This research focuses on the preparation of an electrochemical enzyme-based biosensor to monitor cholinergic signaling in the gut and its application for measuring electrically stimulated acetylcholine release in the mouse colon ex vivo. The biosensors were prepared by platinizing Pt microelectrodes through potential cycling in a potassium hexachloroplatinate (IV) solution to roughen the electrode surface and improve adhesion of the multienzyme film. These electrodes were then modified with a permselective poly(m-phenylenediamine) polymer film, which blocks electroactive interferents from reaching the underlying substrate while remaining permeable to small molecules like H₂O₂. A multienzyme film containing choline oxidase and acetylcholinesterase was then drop-cast on these modified electrodes. The sensor responds to acetylcholine and choline through the enzymatic production of H₂O₂, which is electrochemically oxidized to produce an increase in current with increasing acetylcholine or choline concentration. Important figures of merit include a sensitivity of 190 ± 10 mA mol^–1 L cm^–2, a limit of detection of 0.8 μmol L^–1, and a batch reproducibility of 6.1% relative standard deviation at room temperature. These sensors were used to detect electrically stimulated acetylcholine release from mouse myenteric ganglia in the presence and absence of tetrodotoxin and neostigmine, an acetylcholinesterase inhibitor.