BOD is naturally capable of catalyzing the oxidation of bilirubin into biliverdin and to simultaneously reduce dioxygen (Shimizu et al., 1999). BOD is very similar to laccase. Performances of BOD electrodes are greatly related to the amino-acids sequence around T1 site of the enzyme (Li et al., 2004). It is clearly reported that the most efficient BOD enzyme comes from Myrothecium verrucaria. Redox potential of its T1 site is included between 650 and 750 mV vs. NHE, and the enzyme is thermally stable up to 60 °C (Mano et al., 2002b). It is thus possible to use it at physiological temperature without denaturing the protein. To build efficient BOD electrodes intended in working at physiological pH value, it is judicious to use positively charged mediator molecules since the isoelectric point of BOD is close to pH = 4. Actually, during oxygen reduction reaction, the use of an osmium based redox polymer has lead to performances such as 880 pA cm-2 at 0.3 V vs. Ag/ AgCl (physiological conditions) at a scan rate of 1 mV s-1 (Mano et al., 2002a). Additionally, the redox osmium based hydrogel conferred a very favorable environment to stabilize BOD since 95% of the initial activity of a BOD electrode can be preserved after three weeks storage (Mano et al., 2002a). This remarkable stability probably results in auspicious electrostatic interactions between the swelling matrix and the enzyme. Performances of BOD electrodes are furthermore unaffected in the presence of chloride ions. In fact BOD remains active for chloride concentrations lower than 1 M (Mano et al., 2002a). This property is of major interest for the development of implantable microscale glucose/O2 biofuel cells using BOD as cathode catalysts. The major encountered problem with BOD electrodes is the relative lack of stability of the enzyme in physiological serum. Cupric centers of BOD are indeed capable of binding with one urea oxidation product, oxidation reaction catalyzed by the enzyme (Kang et al., 2004). This phenomenon can nevertheless be limited by spreading a Nafion® film on the catalyst (Kang et al., 2004). It is moreover reported that chemically modified Nafion® is capable of constituting a favorable environment to stabilize BOD (Topcagic & Minteer, 2006). Consequently, it seems of interest to immobilize BOD in Nafion® films. A promising technique for the development of efficient BOD electrodes has already been reported in literature (Habrioux et al., 2010). It consists in firstly adsorbing BOD/ABTS2- (2,2-azinobis-3-ethylbenzothiazoline-5-sulfonic acid) complex on a carbon powder, Vulcan XC 72 R in order to increase both enzyme loading, the stability of the protein and the quality of the percolating network in the whole thickness of the polymer film. Actually, to realize the electrochemical reaction, a triple contact point (between the catalytic system, the electrolyte and the electronic conductor) is required. Once the catalytic

system is adsorbed, a buffered Nafion® solution is added. The whole system is then immobilized onto a solid carbon electrode (Fig. 3).

Fig. 3. Method used for the preparation of BOD cathodes according to the process described in Ref. (Habrioux et al., 2010)

Previous studies have shown the interest lying in the use of ABTS2- as redox mediator in combination with multicopper oxidases. One of them was carried out by Karnicka et al. who have shown that wiring laccase to glassy carbon through a ABTS2-/carbon nanotube system was a very efficient pathway to reduce molecular oxygen into water (Karnicka et al., 2008). The combination of ABTS2- with BOD is also known to exhibit a high electrochemical activity towards oxygen reduction reaction (Tsujimura et al., 2001). These observations are confirmed by electrochemical studies performed on electrodes previously described (Fig. 3).

Curves of Fig.4 clearly show the interest of such electrodes that exhibit a catalytic current from potentials as high as -50 mV vs. O2/H2O (0.536 V vs. SCE). Furthermore the half-wave potential is only 100 mV lower than the reversible redox potential of O2/H2O. This value is in good agreement with that reported by Tsujimura et al. (0.49 V vs. Ag/AgCl/KCl(sat.) at pH = 7.0) (Tsujimura et al., 2001). Let’s notice that the half-wave potential value is very close to the redox potential of T1 site of BOD (0.46 V vs. SCE). This has already been explained by the fact that the reaction between ABTS2- and BOD is an uphill one (Tsujimura et al., 2001).

Fig. 5. Electrochemical activity of BOD/ABTS2-/Nafion® electrode: dependence of the current value at 0.2 V vs. SCE with oxygen concentration

The current linearly increases with the oxygen concentration from low values to around 35%. This linearity suggests that the reaction is of a first order with oxygen concentration thereby, the Koutecky-Levich plots can be considered. Assuming that the rate determining step is an enzymatic intramolecular electron transfer step, it is possible to express the current density of a BOD/ ABTS2-/Nafion® electrode working in an air saturated solution as follows (Schmidt et al., 1999):

(2)

In Eq.2, represents the diffusion limiting current density expressed by Levich equation:

j“ = 0.2nFD23v _16C^/q (3)

In Eq.3, n is the number of electrons exchanged, D the diffusion coefficient, C0 is the oxygen concentration, Q is the rotation rate, F the Faraday constant and nis the kinematic viscosity. Then, jifilm corresponds to the limitation due to oxygen diffusion in the catalytic film and jLads is the limiting current density due to oxygen adsorption on the catalytic site. Since these two last contributions to the total current density do not depend on Q, it is impossible to separate them. They will be described according to Eq.4.

1 — JL _/L

ads film

jL jL jL

In Eq.2, n is the overpotential (n = E-Eeq), j0 the exchange current density, a the transfer coefficient, R = 8.31 J mol-1 K-1, F=96500 C mol-1 and T the temperature. 0 and 0c are the

covering rates of the active sites of the enzyme at E and Eeq, respectively. We will assume that Q » Qc for all potential values. From Eq.2, when Q^<x>, the limit of 1/j can be expressed as follows:

In Eq.5, when n^®, 1/jk^1/jL. It is thus possible to determine jL value by extrapolating and reporting the 1/jk values as a function of the potential value E. Transforming Eq.5 (Grolleau et al., 2008), it becomes as follows:

Under these experimental conditions, calculated values for both Tafel slope and exchange current density are respectively of 69 mV / decade and 25 pA cm-2. The high value obtained for j0 confirms the ability of BOD/ ABTS2-/Nafion® system to activate molecular oxygen in a physiological type medium. Moreover, it also certifies that the oxygen reduction reaction starts at very high potentials. The reference catalyst classically used to reduce molecular oxygen is platinum. It can be noticed that under similar conditions, the exchange current density is only of 5 pA cm-2 when we used platinum nanoparticles as catalyst. This clearly shows the great interest lying in these electrodes to reduce oxygen in glucose/O2 biofuel cells. Nowadays, the ma—or problem encountered with these electrodes is the lack of stability of the redox mediator (ABTS2-) (Tsujimura et al., 2001).