Analytica Chimica Acta, zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHG 179 (1986) 439-444 Elsevier Science Publishers B.V., Amsterdam - Printed in The Netherlands Short Communication AMPEROMETRIC ACETYLCHOLINE AND CHOLINE SENSORS WITH IMMOBILIZED ENZYMES MARCO MASCINI* and DANILA MOSCONE Dipartimento di Scienze e Tecnologie Chimiche, II Universitci di Roma, Tor Vergata, 00172 Roma (Italy) (Received 6th September 1985) Summary. Acetylcholine and choline sensors are prepared by immobilizing enzymes on nylon net attached to a hydrogen peroxide sensor. Choline oxidase is used for the choline sensor, acetylcholinesterase and choline oxidase are used for acetylcholine. The platinum/ silver electrode pair is polarized at +0.6 V. The assembly is protected with an acetate cel- lulose membrane to enhance selectivity. The ranges measured are l-10 pmol 1-l in 0.1-l ml of sample. The response times are l-2 min. Acetylcholine is the first documented neurotransmitter and choline is its metabolite. There is considerable interest in the in vivo measurement of such neurotransmitters [ 1, 21. Voltammetric measurements of oxidizable com- pounds in extracellular fluid provide an active area of research [3, 41, but acetylcholine and choline are not oxidizable. Ion-selective microelectrodes have been described [5] but the sensitivity (around 0.1 mmol zyxwvutsrqponmlkji 1-l) and selec- tivity are inadequate for clinical purposes. The assembly of choline electrodes based on immobilized choline oxidase and an oxygen electrode [6, 71 and a chemically modified platinum electrode [ 81 have been reported. The coupling between the oxygen sensor and choline oxidase was exploited for the deter- mination of phospholipids in several biological matrices [9] . Acetylcholine and choline in nerve tissue have been measured reliably by liquid chromatog- raphy with electrochemical detection, but a lengthy clean-up is needed before introduction of the sample [lo, 111. A simultaneous determination of both acetylcholine and choline in a single sample with two sensors, one selective for choline and the other for acetyl- choline, is described below. Membranes with immobilized enzymes are coupled with amperometric hydrogen peroxide sensors. Both batch and flow procedures are possible. Micromolar concentrations and volumes as small as 0.1 ml can be handled. For the choline-selective sensor, choline oxidase is immobilized on nylon net; for the acetylcholine-selective sensor, choline oxi- dase and acetylcholine esterase are immobilized on opposite sides of the net. These sensors are very selective and can be used for determinations of these substances in nerve tissue extract. 0003-2670/86/$03.50 0 1986 Elsevier Science Publishers B.V.