It is of great interest to develop new non-damaging immobilization techniques of enzyme for the development of stable biofuel cells. In fact, it is very difficult to propose a technique that does not affect the stability of biomolecules. Enzymes are proteins which possess tridimensional structures in which active centers are insulated. To keep the stability of the molecule and to preserve its catalytic efficiency it is necessary not to modify this tridimensional structure and particularly not to affect the environment of the active center. Different combining techniques can be used to immobilize enzymes onto the surface of solid electrodes:

— immobilization into a polymer network

— adsorption on an electrode material

— covalent grafting to an electrode

— immobilization within a membrane.

The first technique consists in immobilizing enzyme in an electropolymerized thin film. It is a very simple technique since it only needs to dip the electrode into a solution containing both monomers and biomolecules. Then the growth of polymer film can be realized by different ways: chronoamperometry (Brunel et al., 2007), chronopotentiometry or cyclic voltammetry (Fei et al., 2007). Different monomers such as pyrrole (Habrioux et al., 2008), aniline (Timur et al., 2004) or phenol (Bartlett et al., 1992) can be electropolymerized. This kind of films can be either conductive or not. The main advantage in using tridimensional conductive films lies in their ability to transfer electrons. Moreover, to increase the number of enzymatic molecules immobilized close to the electrode surface, a first adsorption step of enzymatic molecules can be performed (Merle et al., 2009). Other non-electropolymerized films can be used for enzyme holding. Currently, both chitosan and Nafion® are commonly used (Habrioux et al., 2010; Klotzbach et al., 2008). These two polymers possess surfactant properties interesting to immobilize enzymes in micellar structures (Moore et al., 2004). Moreover, the hydrophobic/hydrophilic property of the polymers can be tuned by modifying the chemical structure of these molecules (Klotzbach et al., 2008; Thomas et al., 2003). It is also possible to simply use retention properties of the Nafion® film for buffering its sulfonic groups (Habrioux et al., 2010). The main problem associated with the use of these polymers lies in the non-control of the film thickness. One of the most promising immobilization techniques has been proposed by Heller’s group. This approach consists in immobilizing enzymes in an osmium-based redox polymer (Mao et al., 2003) which is able to swell in contact with water. It acts both as an immobilizing network and an electrochemical mediator. The whole structure of the film leads to very fast electron transfer between the active centers of enzymes and the electrode surface. Another smart technique consists in covalent grafting of enzyme to the electrode surface. Thus Merle et al. (Merle et al., 2008) realized the grafting of amino groups on a carbon electrode before coupling these functions with amino-groups of enzymes using glutaraldehyde. This seems to confer a remarkable stability to the resulting electrode. Another well-known approach has been proposed by Willner et al. (Willner et al., 1996) that consisted in the reconstitution of the enzyme after the grafting of its active center on a gold electrode.