Selective Detection of Acetylcholine against Choline and In Vivo Measurement in the Mouse Brain Using the Micropipet-Supported Liquid/Liquid Interface Electrode

Acetylcholine (ACh) is a neurotransmitter that plays critical roles in human health and diseases. To better understand ACh signaling in the brain, developing analytical capabilities for its selective and quantitative measurement in real time is essential. While electrochemical amperometry offers exceptional temporal resolution, most in vivo electrochemical ACh sensors have limited selectivity, such as against choline (the product of ACh hydrolysis) and ascorbic acid. Here, we report a micropipet-supported ITIES (interface between two immiscible electrolyte solutions), which demonstrated the selective detection of ACh against choline, ascorbic acid, and other neurotransmitters using chronoamperometry. The detection of ACh is based on its ion transfer across a water/oil interface, which was formed between an aqueous solution and a 2-nitrophenyl octyl ether (NPOE) solution containing an ACh ionophore, termed water/NPOE-ionophore ITIES. The ionophore was heptakis-(2,6-di-O-methyl)-β-cyclodextrin (DM-β-CD). The addition of DM-β-CD in the NPOE phase resulted in an easier ACh transfer at the water/NPOE-ionophore ITIES compared with that at the ITIES without the ionophore, suggesting the presence of ionophore-facilitated ion transfer in addition to the direct ion transfer at the water/NPOE-ionophore ITIES. We observed a linear detection of ACh on the water/NPOE-ionophore ITIES. When implanted in the cortex of the brain of a live mouse, the water/NPOE-ionophore ITIES tracked the dynamic concentration changes of the injected ACh in the brain. The measuring techniques are broadly applicable to quantifying real-time ACh release in the brain with negligible interference, enabling a better understanding of neurological disorders and diseases.