4.4.2. Atomic Force Microscopy

Atomic force microscopy (AFM) is a scanning probe microscopic technique based on a nanoprobe, a tip placed at the end of a long and narrow cantilever, that interacts with the sample surface to measure the topography of a surface [137]. Although AFM has been widely used to study lipid membranes [138–140], membrane proteins [141–144] and electrochemical systems with soluble proteins [145–147], AFM is less often used to study membrane proteins on electrode surfaces. In 2006, we used tapping-mode AFM to analyse the distribution of cytochrome *bo*<sup>3</sup> on a tethered bilayer lipid membrane (tBLM) on stripped gold electrode [75]. AFM combined with Polarisation Modulation Infrared Reflection-Absorption Spectroscopy (PM-IRRAS) was used to study the different orientation of membrane-bound *Aquifex aeolicus* (Aa) [NiFe] hydrogenase immobilised on hydrophilic and hydrophobic SAM on gold electrodes. This work highlighted that on charged or hydrophilic interfaces, H<sup>2</sup> oxidation proceeds through both direct and mediated electron transfer processes, while on hydrophobic surfaces, a mediator is required [148]. In 2014, Gutiérrez-Sanz et al. [149] characterised the functional reconstitution of respiratory complex I on SAM on gold electrode. Their AFM study showed the formation of a phospholipid bilayer on SAM modified gold electrode and protrusions of 6–8 nm height were observed which were ascribed to the hydrophilic arm of complex I as this arm extends outside the membrane.
