*3.3. Nanoparticle Modified Electrode*

Nanoparticles (NPs) have been shown to enhance interfacial electron transfer between a variety of electrodes and proteins [50] and gold NP-modified electrodes have been successfully used to study membrane proteins (Figure 2c), such as terminal oxidases: *E. coli* cytochrome *bd* (a quinol oxidase) [51], *Paracoccus denitrificans* cytochrome *aa*<sup>3</sup> [52] and *Thermus thermophilus* cytochrome *ba*<sup>3</sup> [53] (two cytochrome *c* oxidases). The use of NPs facilitated DET between the electrode and protein, enabling more in-depth studies on enzyme activity. The size of the NPs significantly affects the electron transfer rates and smaller particles reduced the requirement of overpotential for O<sup>2</sup> reduction activity by *E. coli* cytochrome *bo*<sup>3</sup> [54]. The group of Hellwig has recently studied the role of the surface charge of thiol-modified gold NPs, the length of the thiols and the effect of phospholipid composition on the interaction and DET between NP and the membrane enzyme, cytochrome *bd* [55]. Both cytochromes *bo*<sup>3</sup> and *bd* used in the aforementioned studies were isolated and stabilised in DDM buffer. Besides gold NP, various other particles have been used for protein immobilisation [56]. The Armstrong group used graphite microparticles to pair electron donor and acceptor membrane enzymes [57]. [NiFe] membrane-bound hydrogenase was reconstituted with either *E. coli* nitrate reductase (NarGHI) or *E. coli* fumarate reductase (FrdAB; not a membrane protein) on single microparticles; both systems catalysed the reductions of nitrate or fumarate, respectively, by hydrogen. In 2015, Duca et al. [58] demonstrated a cascade electrochemical reduction of nitrate to ammonia by immobilising *E. coli* respiratory nitrate reductase (NarGHI) on an electrode (in polyoxyethylene 9-dodecyl ether detergent) with Pt or Rh nanoparticles.
