*2.2. Direct Electron Transfer*

Direct electron transfer has a significant advantage in comparison to mediated electron transfer [64] as the use of mediators can cause voltage losses stemming from the difference between the redox potentials of the enzyme and the mediator. In addition, the development and miniaturization of biofuel cells can be easier as no membrane or compartment is required [51,64,65]. Some oxidoreductases, such as multi copper oxidases, can undergo direct electron transfer with the electrode. In direct electron transfer, the distance (d) between the electrode and cofactor plays a key role in the rate of electron transfer. In an electron transfer reaction, the heterogeneous rate constant k◦ depends on the distance between the mediator and the electrode, (Equation (1)) [66,67]:

$$\mathbf{k}^{\circ} = \mathbf{k}^{\circ}\_{\text{maximum}} \exp(-\beta \mathbf{d}) \tag{1}$$

β where β is the decay coefficient, d is the distance between the redox centre of the enzyme and the electrode and k◦ maximum is the rate constant at the closest approach. The orientation of enzymes on the electrode surface can affect d so it is crucial that enzymes have the optimal orientation on the surface for direct electron transfer [67]. In general, the distance should be less than 2 nm. At longer distances, the rate of electron transfer between the electrode and cofactor is too low [35]. DET studies of multi-copper oxidases such as laccase, cellobiose dehydrogenase and pyrroloquinoline quinone-dependent glucose dehydrogenases have been widely reported [34]. Interprotein electron

transfer occurs readily in multi-copper oxidases, minimizing the distance for electron transfer and increasing the rate of electron transfer [68]. For example; the copper T<sup>1</sup> site is the primary electron acceptor site and then transfers electrons via an intermolecular mechanism to other copper sites, where O<sup>2</sup> is reduced [69]. The greatest challenge in direct electron transfer is to enable electrical communication between the electrode and enzyme. Some methods of enzyme immobilisation can improve DET [35]. For example, Lee et al. used a gold-binding peptide (GBP) to bind glucose dehydrogenase which facilitates enzyme orientation on the surface of the gold electrode, decreasing the distance between flavin adenine dinucleotide (FAD) and the electrode surface [70]. Cross-linked hydrogels can be used for enzyme immobilisation on the surface of an electrode. Liu et al. used an agarose hydrogel to immobilise haemoglobin, myoglobin and horseradish peroxidase on the electrode surface whereby the proteins could undergo direct electron transfer [71]. Kuk et al. reported the electroenzymatic reduction of CO<sup>2</sup> using NADH-free formate dehydrogenase immobilised on a conductive polyaniline hydrogel. The hydrogel amplified the electron transfer between the electrode and enzyme and moreover, it increased enzyme loading [72]. Hickey et al. reported successful direct electron transfer between enzymes (laccase and nitrogenase) and an electrode using carbon electrodes modified with multi-walled carbon nanotubes and poly(ethylenimine) attached to pyrene moieties. The system was used for the catalytic reduction of O<sup>2</sup> and the production of NH<sup>3</sup> from N<sup>2</sup> [73]. Much of the work on the direct electron transfer of redox enzymes has focused on biosensors and biofuel cells as DET offers a number of advantages. DET enables the use of reagentless biosensors, an important advantage for such devices [74] while with biofuel cells, the absence of a mediator reduces voltage losses arising from differences in the redox potentials of the mediator and the redox enzyme.

### **3. Immobilisation Strategies**

There are five general methods of immobilising enzymes; physical adsorption, covalent binding, immobilisation via ionic interactions, cross linking and entrapment in a polymeric gel or capsule [75]. Physical adsorption is the easiest method of immobilisation [33,34]. Sakai et al. used a glassy carbon electrode modified with 4-mercaptopyridine and gold nanoparticles to adsorb formate dehydrogenase for the oxidation of HCOO<sup>−</sup> oxidation and the reduction of CO<sup>2</sup> [76]. However, due to weak interactions, physisorbed enzymes can leach from the electrode surface. Covalent binding onto electrode surfaces provides very stable enzyme immobilisation [75,77]. The most commonly used electrodes in this method are gold and carbon electrodes [34]. The immobilisation of bilirubin oxidases on nanoporous gold electrodes was performed by attaching -COOH via diazonium surface coupling to the electrode surface which was then used for the covalent coupling of bilirubin oxidases [78]. Immobilisation via ionic interactions is commonly used and is dependent on parameters such as pH and the concentration of salt [75]. The crosslinking of enzymes occurs via a bifunctional agent such as glutaraldehyde which results in enzyme aggregation, with the enzymes acting as their own carrier [75]. Encapsulation techniques entail the entrapment of enzymes in the pores of polymers, hydrogels and sol–gels. Polymers are crossed linked in the presence of enzymes, encapsulating the enzymes in the polymers [34,79]. Redox polymers (Section 2.1) were widely used for the immobilisation of enzymes, with immobilisation occurring via electrostatic interactions, cross linking or/and encapsulation. These polymers are used for the construction of cathodes and anodes in biofuel cells as they enable the successful electrical connection of enzymes, providing a stable means of attachment and can be miniaturized [80,81]. On account of these advantages, redox polymers are successfully and widely used for the construction of biofuel cells [82], biosensors [83], biosupercapacitors [84] and bioelectrosynthesis [85]. As an example of a biocatalytic system, Alsaoub et al. constructed a biosupercapacitor using an Os complex modified polymer to immobilize glucose oxidase and flavin adenine dinucleotide (FAD)-dependent glucose dehydrogenase at the anode and bilirubin oxidase at the cathode [86].
