Acetylcholine and choline amperometric enzyme sensors characterized in vitro and in vivo.

Acetylcholine (ACh) and choline (Ch) are important neuroactive molecules, yet detection of these substances in vivo presents significant analytical challenges. New multienzyme amperometric biosensors are presented here with measurement of physiologically relevant levels of ACh and Ch in vivo. Poly(m-(1,3)-phenylenediamine) (pmPD) electropolymerized on a platinum iridium wire (Pt) served as a template for immobilization of enzymes. A multienzyme layer containing choline oxidase (ChOx) and ascorbic acid oxidase (AAO) for a Ch sensor or ChOx, acetylcholinesterase (AChE), and AAO for a ACh/Ch sensor was immobilized with bovine serum albumin by cross-linking with glutaraldeyhyde. The pmPD enzyme sensors displayed enhanced sensitivity, stability, and selectivity compared to the same multienzyme systems immobilized to solvent cast Nafion and cellulose acetate-modified Pt. Sensor response was linear up to 100 microM ACh or Ch. Detection limits were 0.66 +/- 0.46 microM ACh and 0.33 +/- 0.09 microM Ch, and response times were <1 s. Selectivity for Ch and ACh relative to potential interferences and pharmacological agents commonly used to examine cholinergic physiology was demonstrated. Temperature and pH dependence and the effect of storage conditions on sensor sensitivity and selectivity were determined. Exogenous and endogenous Ch and ACh were measured in the rat brain in vivo.