Except the lack of stability of enzyme molecules due to their proteic nature, one of the major problems encountered with enzymatic electrodes concerns electron transfer between the enzyme and the electrode surface. In the next part we will describe the different electron transfer mechanisms occurring between an enzyme and the electrode as well as the immobilization techniques of the protein.

1.1 Electron transfer between enzyme and electrode

Enzymes are proteins which have high molecular weights. The active sites of these molecules are located in the organic matrix at a depth of several angstroms from the surface. It is thus easy to understand that kinetically fast electron transfer between enzymes and electrodes surface is difficult to obtain because of great insulation of the active centers (Armstrong et al., 1985). Different strategies have been used by the past to make efficient electrical connections between the enzyme and the electrode surface. Corresponding electron transfer mechanisms can be arranged in two different classes: mediated electron transfer (MET) and direct electron transfer (DET).

The major interest in directly transferring electrons between enzymes and electrodes is to reduce the electrode overpotential which is of particular importance for biofuel cells applications. DET is possible as soon as the distance between the active center of the enzyme and the electrode surface is in the order of a tunneling one (Degani & Heller, 1987). Different evidences for DET between enzymes classically used in glucose/O2 biofuel cells and electrodes have already been given. Actually, laccase (Gupta et al., 2004), bilirubin oxidase (Shleev et al., 2005) and glucose oxidase (Wang et al., 2009) are capable of exhibiting non­negligible catalytic current densities without the presence of a redox mediator.

In the case of MET, a redox molecule acts as a substrate and is able to transfer electrons between the electrode surface and the active center of the enzymatic molecule. Let’s notice that current densities obtained with MET are generally higher that what can be delivered in the case of DET. However, to get efficient MET, the redox mediator must possess some properties which can be deduced from Marcus theory as it was already mentioned by Rusling et al. (Rusling et al., 2008). This theory is used to describe outer sphere electron transfer between an electron donor (D) and an electron acceptor (A) as depicted in Fig. 1.

Fig. 1. Curves presenting potential energy of reactants (R) and products (P) (A5—D5+) as a function of reaction coordinates (RC).

The rate (k) of electron transfer can be described as follows (Eq.1.) by an Arrhenius type law.

f (ag+xA

I 4RTX I

k = AKe1 J (1)

where A is the collision frequency, K is the electronic transmission factor, AG° is the Gibbs free energy, R is the gas constant and T the temperature. X is the reorganization energy (energetic cost associated to the reorganization of both solvent and molecules and necessary to proceed in electronic transfer between the donor and the acceptor). From this relation it can be deduced that to have an efficient electron transfer between enzyme and mediator, it is essential that the redox mediator used presents a highly reversible redox system to minimize X value. It is also fundamental to minimize the AG° value. Thus it is very important that formal potentials of mediator and enzyme are close. Moreover, since active centers of enzyme are greatly insulated in high molecular weight molecules it is necessary to use small mediator molecules to reduce the distance of electron transfer and to guarantee a high k value.