Fast formation of low-defect-density tethered bilayers by fusion of multilamellar vesicles

Abstract

A facile and reproducible preparation of surface-supported lipid bilayers is essential for fundamental membrane research and biotechnological applications. We demonstrate that multilamellar vesicles fuse to molecular-anchor-grafted surfaces yielding low-defect-density, tethered bilayer membranes. Continuous bilayers are formed within 10min, while the electrically insulating bilayers with <0.1μm defect density can be accomplished within 60min. Surface plasmon resonance spectroscopy indicates that an amount of lipid material transferred from vesicles to a surface is inversely proportional to the density of an anchor, while the total amount of lipid that includes tethered and transferred lipid remains constant within 5% standard error. This attests for the formation of intact bilayers independent of the tethering agent density. Neutron reflectometry (NR) revealed the atomic level structural details of the tethered bilayer showing, among other things, that the total thickness of the hydrophobic slab of the construct was 3.2nm and that the molar fraction of cholesterol in lipid content is essentially the same as the molar fraction of cholesterol in the multilamellar liposomes. NR also indicated the formation of an overlayer with an effective thickness of 1.9nm. These overlayers may be easily removed by a single rinse of the tethered construct with 30% ethanol solution. Fast assembly and low residual defect density achievable within an hour of fusion makes our tethered bilayer methodology an attractive platform for biosensing of membrane damaging agents, such as pore forming toxins.

Inerolysin and vaginolysin, the cytolysins implicated in vaginal dysbiosis, differently impair molecular integrity of phospholipid membranes

“…The formation of tBLMs is a multistep process that includes the deposition of an anchoring self-assembled monolayer (SAM), preparation of MLVs and the fusion of these MLVs to the anchoring SAM. Vesicle suspensions were prepared from 1, 2–dioleoyl–sn–glycero–3–phosphocholine (DOPC) and cholesterol from Avanti Polar Lipids Inc. (USA) at various molar rates as described previously 53 . Briefly, the lipids were dissolved in 99% chloroform (Sigma-Aldrich) to a concentration of 10 mM.…”

“…The tBLMs were prepared as describe previously 53 . Briefly, freshly gold-coated substrates were immersed in 0.05 mM ethanolic solution of the tether WC14 (20–tetradecyloxy-3,6,9,12,15,18,22–heptaoxahexatricontane–1–thiol; synthesized in–house), and β-mercaptoethanol (ßME) (Sigma-Aldrich, St. Louis, MO) mixed at molar ratio 30:70 to form mixed self-assembled monolayers (SAMs) 20 .…”

Electrochemical Biosensors Based on Membrane-Bound Enzymes in Biomimetic Configurations

“…The hydrophilic part provides an ion and water reservoir underneath the bilayer. In the second step, the outer layer is anchored by VF [9,40] or rapid solvent exchange [41]. Alternatively, the composition of the lipid tethered bilayer can be modified by vesicle exchange [42].…”

Association of Model Neurotransmitters with Lipid Bilayer Membranes

“…1) were prepared as follows. Self-assembled monolayers (SAMs) composed of βME and HC18 (7:3 in solution) were formed on gold-covered solid substrates, followed by bilayer completion through rapid solvent exchange (23) or vesicle fusion (24). For SPR, we used microscopy slides made of glass (Thermo Fisher Scientific, Waltham, MA) or sapphire (Rubicon, Bensonville, IL).…”