The third technique for enzyme immobilization is employing micellar polymer Nafion® for enzyme entrapment within the pore structure of the membrane, as shown in Figure 12.5. However, commercial Nafion® has not been successful at immobilizing enzymes at the surface of biofuel cell electrodes because Nafion® forms an acidic membrane that decreases the lifetime and activity of the enzyme. Researchers have been successful in maintaining the activity of glucose oxidase enzymes immobilized in Nafion® by diluting the Nafion® suspension [20];

Enzyme casting layer

I

FIGURE 12.4 Enzyme immobilization by sandwich technique.

however, this approach did not form stable and uniform films. The most recent method employed by Karyakina and coworkers was to neutralize the Nafion® casting solution and dilute the solution to a lesser degree in ethanol; however, both of these approaches have problems with maintaining activity of enzymes for extended times. As the pH environment in the solution around the Nafion® membrane decreases, protons will exchange back into the membrane and re­acidify the membrane [20].