Optimization of multienzyme flow reactors for determination of acetylcholine

Immobilized enzyme reactors have been used with high-performance liquid chromatography (HPLC) and electrochemical detection to detect acetylcholine and choline in brain tissue samples. Acetylcholine and choline eluting from the LC column are introduced into a reactor containing immobilized acetylcholinesterase, which hydrolyzes acetylcholine to choline. The product is converted by a second enzyme, choline oxidase, to hydrogen peroxide, which is determined amperometrically. Several novel immobilization techniques including immobilization through enzyme-specific antibodies were used to immobilize these enzymes to retain maximum activity. Improved detection limits were observed when the enzymes were immobilized through the avidin-biotin linkage. Better sensitivity and detection limit were obtained when both enzymes were immobilized together on the same support through the avidin-biotin linkage than when they were separately immobilized and used in two columns. The postcolumn system was applied to brain tissue extracts.