Planar electrodes

77 In the first studies, MCOs have been immobilized on various kinds of carbon electrodes: glassy carbon (GC),235 various types of graphite (low density graphite (LDG),

spectroscopic graphite (SPG), pyrolytic graphite (PG), basal plane or edge plane highly oriented pyrolytic graphite (HOPG)…),203,210,215,221,235 _{and screen printed carbon.}279 _Except

glassy carbon, carbon surfaces are however rarely strictly planar. While it is clear that DET catalysis has been observed with different MCOs simply adsorbed on most of these materials, it is difficult to compare their efficiency as enzyme support. Indeed, it is well-known that carbon electrodes differ from one another as far as microstructure, roughness, and chemical functions are concerned.

To give an idea of the magnitude of currents or current densities recorded with MCOs adsorbed on carbon electrodes, we list below some values obtained at 0 V vs. Ag/AgCl. Tv- laccase adsorbed on edge plane graphite gave currents of  40 µA. In that case, the maximum rate at which O2 could be reduced, deduced from the intercept of a Koutecky Levich plot,

indicated a turnover rate of ca. 20 s-1.211 Rv-laccase was adsorbed on two carbon materials

(RWII and RW0), and the higher electrocatalytic current, 60 µA.cm-2 at neutral pH, was

observed with RWII. It was attributed to a higher porosity and the presence of more oxygen functions. With laccases of different origins, T. ochracea, C. maxima, C. unicolor, C. fulvocinerea, adsorbed on SPG or HOPG, currents were in the range 2-20 µA.210 It is

interesting to note that Tv-laccase adsorbed on HOPG was observed by STM, revealing that the enzyme was not homogeneously distributed at the electrode surface and probably forming aggregates.271

In the very first study of DET by a BOD, Mv-BOD was also adsorbed on different electrode materials: GC, HOPG and PFC (plastic formed carbon). PFC and edge-plane HOPG gave the best results (200 µA.cm-2 with HOPG at pH 7), even exceeding the highest current

78 and/or nanostructure governed the surface ET kinetics and adsorption property of BOD, but no more details were provided.235 In the following reports, the current densities diverged. The

differences cannot be assigned merely to different operating conditions; they may be explained by the fact that, as already mentioned, the electrode surface was not well characterized. For example  25 µA.cm-2 at pH 7 were recorded with Mv-BOD adsorbed on

HOPG,245 and  30 µA.cm-2 for the same enzyme adsorbed on SPG.215 Tt-BOD adsorbed on

SPG led to 20 µA.cm-2 _{at pH 7}203 _{and  0.5 mA.cm}-2 _{under O}

2 at pH 4,204 i.e. the results

differed by more than one order of magnitude. Although it is not possible to identify the exact origin of this difference, we can note that it is much higher than those observed in studies of pH influence performed in homogeneous solution.

DET on carbon electrodes was also routinely used to characterize less widespread or newly discovered MCOs.166,181,280,281 Interestingly, the proof of concept of some EBFCs was

established with enzymatic planar carbon cathodes. Tv-laccase adsorbed on graphite provided the cathode of the first totally enzymatic H2/O2 BFC.282,283 Similarly, the first mediator- and

cofactor-free EBFC based on cellulose dehydrogenase (CDH) operating in neutral chloride- containing buffers as well as in serum relied on Mv-BOD adsorbed on SPG electrode as a cathode.241 A cathode based on Mv-BOD adsorbed on a LDG electrode was also combined to

a bi-enzymatic anode for the 6 e--oxidation of glucose in a membrane-less EBFC operating in

a buffer mimicking human physiological fluid.284 This is detailed further in section 6.

4.1.1.2 Other planar electrodes

DET was also achieved for different MCOs on bare or modified gold surfaces. Since this is explored in details in section 3.4.4. and 4.1.4., we only remind here some current densities obtained when MCOS were randomly immobilized.221 For most laccases, no DET

79 current was recorded when they were adsorbed on bare gold. Rv-laccase on MPA-modified gold gave 84 µA.cm-2 at pH 7.214 With Mv-BOD, very different values ranging between 5 and

more than 100 µA.cm-2 were recorded on bare gold electrodes.17,225,228,231

Some less conventional materials have been proposed, like boron doped diamond (BDD) or semiconductors. BDD has a wide working potential window, low background current, high stability, tunable hydrophilicity and optical transparency. Direct O2 reduction by

Cerrena unicolor laccase was demonstrated on BDD285 with the enzyme either in solution or

immobilized in a liquid crystalline cubic phase at the electrode. However, DET currents were very low (< 1 µA.cm-2_{) while much higher currents were achieved upon addition of a redox}

mediator (25 µA.cm-2). This proved that the enzyme orientation was not optimal.285 Tv-

laccase was immobilized on p-type nanostructured silicon (111) as a model semiconductor.286

Under illumination with visible light, the material was theoretically able to supply electrons for the enzyme activation. O2 reduction started about 200 mV more positive when laccase was

present, but still at a very low potential (-0.24 V vs. SCE). The plateau current was 30 µA.cm-

2_.286

4.1.1.3 Strategies for increasing the enzyme loading

To overcome the limitation in current densities, and reach values usable in EBFC, the surface concentration of enzymes on electrode surfaces had to be improved. A study by Ikeda et al. showed that multiple layers of enzymes could be connected in DET configuration on a planar electrode.252 Multilayers of Mv-BOD were entrapped electrostatically in poly-L-lysine

(PLL) on a PFC electrode. In O2 saturated buffer, the process was diffusion controlled. A

plateau at 845 µA.cm-2 was reached upon stirring at 1400 rpm, while only 300 µA.cm-2 were

recorded when BOD was simply adsorbed on this PFC electrode. Calculations according to the model presented in section 3 indicated that the reaction layer was 0.2 µm-thick, i.e. much smaller than the real thickness of the BOD-PLL film (50 µm). HET standard rate constant was

80 19 s-1, i.e. about 10 times smaller than for adsorbed BOD (178 s-1). These two calculations

highlight that this design was not optimized, although it allowed to connect more enzymes and to reach higher currents.252

Another option for immobilizing more enzymes is to increase the roughness of the electrode surface. Following this strategy, graphitized carbon cloth treated with H2SO4 was

developed for the covalent binding of laccase,287 while Mv-BOD was adsorbed on thorn-like

nanostructures with an average roughness of 3.5-4 Å homogeneously distributed on a nano- carbon film (Fig 21).288

Figure 21. (A) AFM topographical images and height line profiles at points indicated by the arrows. Top: before UV/ozone irradiation. Down: after irradiation for three hours. The thorn height distribution is centered at 2,25 nm. (B) Voltammograms of BOD physically adsorbed on original and nanostructured carbon films with different surface wettabilities. 50 mM PB pH 7.0, scan rate 20 mV.s-1 and electrode area 0,0314 cm2. Top: real scale and down: enlargement of the dotted square in Top. Adapted with permission from [288]. Copyright 2017 American Chemical Society.

81 In both cases, currents increased compared to the planar electrode: 0.27 mA.cm-2 were

reached at 0 V in the first case, supposedly due to higher number of electrochemically active laccases287, and 102 µA.cm-2 in the second thanks to the nanostructure.288 The importance of

the roughness has also been highlighted with Mv-BOD immobilized on a planar electrode together with platinum group metal-free catalysts. Current densities did not only increase because of the inherent properties of the catalysts, but the contribution of the increased roughness was also important, providing better results when pores within the ratio 3-5 nm where formed. No significant increase occurred with larger pores (12-20 and 40-80 nm).289

Alternatively, a large variety of materials can be used to enlarge the electroactive specific surface area (ESSA) of the electrode. Matrices based on nanomaterials with a high surface/volume ratio have been developed. Typical nanomaterials are carbon nanotubes, nanoparticles or nanofibers (resp. CNTs, CNPs or CNFs), and gold nanoparticles (AuNPs). Various metallic nanoparticles (Fe, Pt...) have also been described. Ideally, it would be beneficial to find materials with hierarchical porosity allowing Efficiently immobilizing enzymes and preventing mass transport limitations. Therefore, highly porous 3D materials like mesoporous gold or carbon with the adequate and tailored porosity have also been engineered. In the following of this section we will focus on studies where the nano- structuration of the electrode has been evaluated, first with carbon and then with gold-based materials.