Enzymatically amplified voltammetric sensor for microliter sample volumes of salicylate

A new voltammetric sensing strategy for salicylate employing two enzymes and applicable to microliter sample volumes is demonstrated. The method involves the use of the enzyme salicylate hydroxylase to convert salicylate to catechol, which is oxidized at a carbon electrode. The product of this oxidation reaction, o-quinone, is then reduced by a second enzyme, glucose oxidase, to regenerate catechol. Reoxidation of catechol results in a signal that is amplified due to repeated cycling of catechol molecules between the oxidized and reduced states. This chemistry is implemented in two configurations. (i) A paper disk into which both enzymes have been absorbed is mounted on a coplanar three-electrode assembly for aqueous experiments. Determination of salicylate in a nonprescription dermatological product is demonstrated. (ii) A small solution volume confined directly on the coplanar electrodes is used for determination of salicylate in whole blood. The advantages of the use of two enzymes and of monitoring steady-state catalytic currents are discussed.