ONLINE ELECTROCHEMICAL SENSOR FOR SELECTIVE CONTINUOUS MEASUREMENT OFACETYLCHOLINE IN CULTURED BRAIN-TISSUE

An on-line acetylcholine (ACh) sensor was developed in order to determ ine extracellular ACh concentrations without interference from choline (Ch). The sensor is composed of a small-volume enzymatic prereactor ( 22-mu L inner volume) in which choline oxidase and catalase are immobi lized in series. Carbon electrodes were modified with an acetylcholine esterase (AChE), choline oxidase (ChOx), and osmium poly(vinylpyridin e)-based redox polymer containing horseradish peroxidase (Os-gel-HRP). The sensor sensitivity was 43.7 nA/mu M (+/-0.15, n = 3) for ACh unde r optimized conditions. Almost no response was seen when 100 mu M Ch w as continuously injected. The detection limit for ACh with the sensor was comparable to that obtained using liquid chromatography with elect rochemical detection combined with an enzymatic reactor. The electrica l stimulation of cultured rat hippocampal tissue resulted in an extrac ellular ACh increase of 20 nM (+/-11 nM, n = 3). This increase was obs erved continuously with our online sensor combined with a microcapilla ry sampling probe located very close to the tissue. The continuous mea surement of ACh and Ch using a split disk carbon film dual electrode i n which one electrode surface was modified with ChOx/Os-gel-HRP and th e other with AChE-ChOx/Os-gel-HRP bilayer film was also demonstrated t o improve the response time by eliminating the prereactor.