Development of Choline and Acetylcholine Pt Microelectrodes

Choline (Ch) and acetylcholine (Ach) microenzyme sensors were developed based on the immobilization of choline oxidase (ChO) and acetylcholinesterase (AchE) at the tip of a 25-μm Pt wire sealed in glass. Several immobilization procedures were tested, including code-position of the enzyme/s with an electropolymer and cross-linking with glutaraldehyde. The various electropolymers used were 1,2-diaminobenzene, resorcinol, 4-hydroxybenzenesulfonic acid, and a combination of two polymers, 1,2-diaminobenzene and resorcinol. An inner membrane constructed from cellulose acetate (CA) was deposited prior to immobilization with glutaraldehyde. The analytical characteristics of the microelectrodes, including optimization of immobilization procedures, calibration curves, pH response curves, stability, and selectivity toward possible electroactive compounds found in the brain extracellular fluid, were determined. The best microelectrodes were prepared by cross-linking the enzymes with glutaraldehyde on top of the inner CA membrane. The responses are linear in the concentration range 5.0 × 10−7-1.0 × 10−4 M Ch and 5.0 × 10−7-9.3 × 10−5 M Ach. The time to reach 95% steady-state current was 15-20 s. The CA-coated Ch microelectrodes were useful for measurement of changes in Ch concentration in artificial brain extra-cellular fluid.