Electropolymerization Conditions of Methylene Green Used as an Electron Transfer Mediator for Coenzyme-dependent Oxidoreductases

Biosensors and biofuel cells are human-friendly and eco-friendly technologies that utilize enzymatic redox reactions. To operate in a solution flow environment, enzymes and electron mediators must be immobilized on the electrodes. The state of the electron mediator at an enzyme-immobilized electrode affects not only the electron transfer between the enzyme and the electrode but also the retention and orientation of the enzyme. Phenazine dyes, often used as electron mediators, can be easily immobilized on an electrode by electropolymerization. However, little is known about what kind of polymer films result from the conditions of electropolymerization. Here, we show that the electropolymerized film of methylene green (MG), a phenazine dye, changes from a uniformly distributed mesoscale structure to a nonuniformly dispersed state of spherical particles as polymerization progresses. Furthermore, we found that polyMG films with a uniform surface are suitable for enzyme-immobilized electrodes. More MG was deposited on the electrode under conditions of wide potential sweeps, slower scan rates, and more cycles of electrolysis. Uniform surfaces were observed in films with dissipation (ΔD), a measure of film softness as measured by EQCM-D, of less than approximately 7 × 10−6. These results indicate that the electrochemical polymerization conditions of MG can control the surface morphology of the polymerized film, as well as the properties of the enzyme-immobilized electrode. This finding could be applied to NAD-dependent and FAD-dependent glucose dehydrogenases, in which MG is known to function as a mediator, and could contribute to improving the current of biosensors and biofuel cells in which they are used.