Self-assembled monolayers (SAMs) comprised of a mixture of a cholesterol functionalized thiol derivative and a short chain ethyleneoxythiol derivative have been used to attach phospholipid bilayers to gold surfaces. The cholesterol derivatives serve as “anchoring units” and are inserted into the lower leaflet of the attached bilayer. The short chain ethyleneoxy derivatives are present to promote a disordered hydrophilic region beneath the bilayer. The bilayers were formed by incubation of the SAMs with small unilamellar vesicles. On single component hydrophobic surfaces a single lipid layer was adsorbed, while on mixed SAMs containing the cholesterol anchoring units, and single component hydrophilic surfaces, a lipid bilayer was adsorbed. The kinetics of bilayer formation was followed using surface plasmon resonance spectroscopy, and showed dramatic differences depending on the SAM composition.

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An electrode surface is presented that enables the characterization of redox-active membrane enzymes in a native-like environment. An ubiquinol oxidase from Escherichia coli, cytochrome bo(3) (cbo(3)), has been co-immobilized into tethered bilayer lipid membranes (tBLMs). The tBLM is formed on gold surfaces functionalized with cholesterol tethers which insert into the lower leaflet of the membrane. The planar membrane architecture is formed by self-assembly of proteoliposomes, and its structure is characterized by surface plasmon resonance (SPR), electrochemical impedance spectroscopy (EIS), and tapping-mode atomic force microscopy (TM-AFM). The functionality of cbo(3) is investigated by cyclic voltammetry (CV) and is confirmed by the catalytic reduction of oxygen. Interfacial electron transfer to cbo(3) is mediated by the membrane-localized ubiquinol-8, the physiological electron donor of cbo(3). Enzyme coverages observed with TM-AFM and CV coincide (2-8.5 fmol.cm(-)(2)), indicating that most-if not all-cbo(3) on the surface is catalytically active and thus retains its integrity during immobilization.