Uricase-catalyzed oxidation of uric acid using an artificial electron acceptor and fabrication of amperometric uric acid sensors with use of a redox ladder polymer

Electrochemical oxidation of uric acid catalyzed by uricase (uric acid oxidase, UOx; EC 1.7.3.3) was studied using several redox compounds including 5-methylphenazinium (MP) and 1-methoxy-5-methylphenazinium (MMP) as electron accepters for UOx, which does not contain any redox cofactor. It was found that MP and MMP were useful to mediate electrons from UOx to an electrode in the enzymatic oxidation of uric acid. A novel redox polymer, poly(N-methyl-o-phenylenediamine) (poly-MPD), containing the MP units was also found to possess the mediation ability for UOx, and poly-MPD was immobilized together with UOx onto an electrode substrate covered with a self-assembled monolayer of 2-aminoethanethiolate with use of glutaraldehyde as a binding agent. The resulting electrode (poly-MPD/UOx/Au) exhibited amperometric responses to uric acid with very fast response of similar to 30 s, allowing reagentless amperometric determination in a concentration range covering that in the blood of a healthy human being. Kinetic parameters of the apparent Michaelis constant and the maximum current response obtained at the poly-MPD/UOx/Au suggested that electrochemical oxidation of uric acid was controlled by diffusion of uric acid into the enzyme film and that the redox polymer worked well in mediating between active sites of UOx molecules and the electrode substrate.