Liquid/liquid junction microelectrodes for monitoring cholinergic transmitter in live mice brain in vivo

Acetylcholine (ACh) is an important neurotransmitter and biomarker for neurological disorders. The quantitative detection of ACh in vivo is critical but remains a challenge. In this work, we developed a novel micrometer-sized electrode based on interface between two immiscible electrolyte solutions (ITIES) to achieve in vivo measurement of ACh at high spatiotemporal resolution. The fabricated microITIES electrode was tested in vitro for ACh sensing using electrochemical methods including cyclical voltammetry and i-t amperometry in artificial cerebrospinal fluid (ACSF) solution. An increase in current was observed in both CV and i-t at -0.25 V (vs E_{1/2, TEA}). Both CV and i-t showed a high sensitivity and a linear response with the linear range starting from as low as 0.5 μM. Then the electrode was applied for in vivo measurement of ACh in the living mouse brain. The electrode was implanted in the cortex of the mouse brain via stereotaxic surgery. The electrode was tested for exogenously applied ACh in vivo by local injection of ACh (500 nL, 0.5 M) twice. Repeated cyclic voltammograms were recorded before, during, and after both injections; the cyclic voltammograms showed a significant increase in current at ACh detection potential. The electrode was also tested for endogenously released ACh in vivo by the local injection of a high concentration KCl solution (500 nL and 1000 nL, 100 mM) to stimulate ACh release. Similarly, repeated cyclic voltammograms were recorded before, during, and after both injections; a significant increase in current at the ACh detection potential in the cyclic voltammograms was observed following each injection of KCl. These results validated the capability of the introduced microITIES electrode to measure exogenous and endogenous ACh in vivo.