*3.6. Multi-Layer Assembly, Multilayered Lipid Membrane Stacks, 3D Structure Electrode*

An important aspect to improve the performance of bioelectrocatalysis is catalyst loading on the electrode surface. For membrane proteins, this can be achieved via two alternative strategies: multi-layer (membrane) assembly (Figure 2d,i,j) and/or 3D electrode structure (Figure 2k). For a more detailed discussion on layer-by-layer assembly approach for redox proteins, we also refer to [65,81]. Multilayer immobilisation within a redox polymer matrix was already discussed in Section 3.4. As an example of a layer-by-layer approach (Figure 2i), PSI has been co-assembled with cytochrome *c* as mediator using DNA as an anionic polymer [82]. The purple bacteria RC was also co-assembled into a multilayer architecture by alternating layers of RC with a cationic polymer poly(dimethyldiallylammonium chloride) (PDDA) [83,84]. Our group used poly-l-lysine (PLL) as an electrostatic polymer to construct multilayered lipid membrane stacks (Figure 2j) [85,86]. Two membrane proteins, *E. coli* cytochrome *bo*<sup>3</sup> and *R. eutropha* MBH, were incorporated into these lipid membranes stacks. Lipophilic quinones, the natural substrates of cytochrome *bo*<sup>3</sup> and MBH, diffuse freely between the multilayered membranes and mediated electron transfer between the proteins and the electrode. Catalytic activity was shown to increase linearly with the number of membrane layers for at least up to 5 layers [86].

The high surface area of so-called 3D electrodes can be used to immobilise proteins at high loading [87]. Mesoporous metal electrodes have been shown to increase protein loading compared to planar electrodes, e.g., rough silver electrode for bacteria RC [88] and nanoporous gold electrode for PSI [89]. A mesoporous WO3-TiO<sup>2</sup> film electrode has been reported for the entrapment for bacterial RCs [90] and mesoporous indium tin oxide (ITO) electrodes have been used to immobilise PSII [91]. The ITO electrode can be modified with SAM to covalently bind and orientate PSII with the electron acceptor side of towards the electrode surface, enhancing electron transfer kinetics and electrode stability [92]. A hierarchically structured, inverse opal, mesoporous (IO-meso) ITO electrode was later developed to provide even larger surface areas [93]. These electrodes were combined with redox polymers to electrically wire PSII with the 3D structure of IO-meso ITO, yielding photocurrent densities of up to ~410 µA cm−<sup>2</sup> [94]. Mesoporous ITO electrodes were also applied to co-immobilise cytochrome *c* and PSI and photocurrent densities >150 µA·cm−<sup>2</sup> were achieved [95]. The effect of pore size was studied by comparing photocurrents between mesoporous (20–100 nm) and macroporous (5 µm) electrodes using 2,6-dichlorophenolindophenol (DCPIP) and ascorbate as redox mediator for PSI. The macroporous electrode showed three times higher photocurrent than the mesoporous electrode [96]. The authors observed that the macroporous electrode increased the active surface area twice compared to the mesoporous electrode with the same PSI mass loading. They concluded that the increase in photocurrent was due to multilayers of PSI deposited along pore walls and the macropores enhanced the MET within a single pore.
