Creation of a gold nanoparticle based electrochemical assay for the detection of inhibitors of bacterial cytochrome bd oxidases

Membrane Protein Modified Electrodes in Bioelectrocatalysis

“…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 3 [52] and Thermus thermophilus cytochrome ba 3 [53] (two cytochrome c oxidases). The use of NPs facilitated DET between the electrode and protein, enabling more in-depth studies on enzyme activity.…”

Supramolecular electrode assemblies for bioelectrochemistry

“…40 Terminal oxidases (the membrane proteins cytochrome c oxidase and cytochrome bd oxidase) were also immobilised on the same modified gold nanoparticles and both non-turnover and catalytic signals were observed by cyclic voltammetry. 41–43 Furthermore, immobilisation of cytochrome bo 3 (another terminal oxidase) was compared on gold nanoparticles with different diameters, where smaller nanoparticles required lower overpotentials to reduce cytochrome bo 3 . 43 The latter suggests that smaller particles have enhanced electronic coupling to the redox active sites of complex enzymes, presumably because smaller particles are able to come nearer the redox sites that are buried underneath a structured protein surface.…”

Stabilization of the Highly Hydrophobic Membrane Protein, Cytochrome bd Oxidase, on Metallic Surfaces for Direct Electrochemical Studies

“…The study of the oxygen reaction via direct electrochemistry on gold nanoparticles (NPs) modified with thiols was recently presented by our group, with focus on the influence of pH, protein concentration and quinone content [ 22 ]. The catalytic activity of the immobilized proteins was found optimal at pH 7 and for concentrations in the range of 0.6–1.0 mg/mL.…”

“…The catalytic activity of the immobilized proteins was found optimal at pH 7 and for concentrations in the range of 0.6–1.0 mg/mL. The surface coverage of active protein on the electrode was then 0.1 pmol·cm −1 , and their turnover rate (k cat ) 100 s −1 ± 25%, as determined from a Koutecky–Levich plot [ 22 ]. The long-term protein film stability after immobilization was also investigated previously in our group.…”

“…The response of the modified electrode with time showed that after two hours only 5% of the activity was lost, while after 120 h the enzyme still kept 50% of the catalytic signal. The observed loss in the catalytic current was likely due to partial enzyme desorption or deactivation of the membrane protein with time [ 22 ].…”