*3.2. SAM Modified Electrode*

An electrode surface can be modified to promote protein immobilisation and control the orientation of the protein on the surface. A common method to functionalise metallic electrode surfaces, in particular gold electrodes, is to form a self-assembly monolayer (SAM) of thiols (e.g., alkanethiols) (Figure 2b). By changing the length of the thiol compound (e.g., the alkyl chain), the distance between the protein and electrode can be controlled. More importantly, by changing the terminal group of the SAM (e.g., *n*-hydroxy-alkanethiol or *n*-amino-alkanethiol), the surface chemistry of the electrode can be controlled. As protein binding to surfaces is typically governed by van-der-Waals and electrostatic interactions, the surface chemistry strongly influences the orientation of proteins on the surface [42,43].

Besides tuning the chemistry of the electrode surface, proteins can be genetically engineered to control their orientation on the electrode through affinity interactions. RCs from *Rhodobacter sphaeroides* in LDAO detergent have been immobilised on SAM-modified electrodes terminated by Ni–NTA groups. By genetically engineering a poly-histidine tag (His7) at the C-terminus of the M-subunit of the RC, the primary donor of RC was positioned to face the electrode [44]. The histidine tag can also be engineered on H subunit of RCs to achieve the opposite orientation [45]. The Ni-NTA modified gold electrodes have also been used to immobilise His-tagged PSII solubilised in DDM buffer [46]. A different strategy that has been explored is to modify the gold electrode surface with an amine terminated SAM for further reaction with terephthaldialdehyde (TPDA). The TPDA modified SAM reacts with lysine residues from PSI to form covalent imine bonds [47]. Such an approach does not create the same orientational control compared to the His-tag/NTA coupling. Irrespective of selective orientation on the surface, mediators are often required for efficient electron exchange with membrane proteins. Small proteins like cytochrome *c* are widely used as mediators for PSI and purple bacteria RC [48]. Water-soluble redox mediators such as 2,6-dichloro-1,4-benzoquinone (PSII), 2,6-dichlorophenolindophenol (PSI), sodium ascorbate (PSI) and ferricyanide (PSI) are also commonly used to facilitate efficient electron transfer between the protein and electrode [49].

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**Figure 2.** Overview of the strategies for membrane protein modified electrodes (not to scale). Proteins absorbed on (**a**) an unmodified electrode, (**b**) a SAM modified electrode and (**c**) a nanoparticle modified electrode. (**d**) Immobilisation of membrane proteins within a redox polymer. Examples of lipid membrane-modified electrodes: (**e**) a hybrid bilayer lipid membrane (hBLM), (**f**) a solid supported bilayer lipid membrane (sBLM), (**g**) a tethered bilayer lipid membrane (tBLM) and (**h**) a protein tethered bilayer lipid membrane. (ptBLM). (**i**) Layer-by-layer deposition of alternating charged films. (**j**) Multilayered lipid membrane stacks. (**k**) 3D structure electrode for protein immobilisation.
