Assisted lipid deposition by reductive electrochemical aryldiazonium grafting and insertion of the antiport NhaA protein in this stable biomimetic membrane

Quartz crystal microbalance studies have been carried out to monitor the fusion of lipid vesicles (pure 1,2-dimyristoyl-sn-glycero-3-phosphocholine, DMPC) and mixed vesicles (DMPC and 4-decylaniline). In order to increase the stability of the lipid deposits onto the electrodes, we have developed an original approach involving electrografting of adsorbed mixed vesicles. Aryldiazonium salts generated in situ from 4-decylaniline (4DA) present in adsorbed and fused mixed vesicles at the electrode surface allow their cathodic reduction and subsequent grafting. The stability of the supported lipid deposit has been shown to significantly increase from less than one day for pure DMPC to about two weeks with the lipid deposition assisted by electrochemical grafting. In this stable lipid deposit, the insertion of the sodium/proton antiporter membrane protein (NhaA) or its inactive mutant has been carried out by fusion of proteoliposomes. This has been followed by characterization of the inserted protein activity by cyclic voltammetry onto an electrode previously modified by an adsorbed pH sensor (2-anthraquinone sulfonate). Activation of the protein function by sodium ions leads to a shift of the interfacial pH and confirms the integrity of the immobilized NhaA.