Microcavity-Supported Lipid Membranes: Versatile Platforms for Building Asymmetric Lipid Bilayers and for Protein Recognition

Abstract

Microcavity supported lipid bilayers (MSLB) are contact-free membranes suspended across aqueousfilled pores that maintain the lipid bilayer in a highly fluidic state and free from frictional interactions with substrate. Such platforms offer the prospect of liposome-like fluidity with the compositional versatility and addressability of supported lipid bilayers and thus offer significant opportunity for modelling membrane asymmetry, protein-membrane interactions and aggregation at the membrane interface. Herein, we evaluate their performance by studying the effect of transmembrane lipid asymmetry on lipid diffusivity, membrane viscosity and cholera toxin-ganglioside recognition across six symmetric and asymmetric membranes including binary compositions containing both fluid and gel phase, and ternary phase separated membrane compositions. Fluorescence lifetime correlation spectroscopy (FLCS) was used to determine the lateral mobility of lipid and protein, and electrochemical impedance spectroscopy (EIS) enabled detection of protein-membrane assembly over the nanomolar range. Transmembrane leaflet asymmetry was observed to have profound impact on membrane electrochemical resistance where the resistance of a ternary symmetric phase separated bilayer was found to be at least 2.6 times higher than the asymmetric bilayer with analogous composition at the distal leaflet but where the lower leaflet comprised only 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC). Similarly, the diffusion coefficient for MSLBs was observed to be 2.5 fold faster for asymmetric MSLBs where the lower leaflet is DOPC alone. Our results demonstrate that interplay of lipid packing across both membrane leaflets and concentration of GM1 both affect the extent of cholera toxin aggregation and consequent diffusion of the cholera-GM1 aggregates. Given that true biomembranes are both fluidic and asymmetric, MSLBs offer the opportunity for building greater biomimicry into biophysical models and the approach described demonstrates the value of MSLBs in studying aggregation and membrane associated multivalent interactions prevalent in many carbohydrates mediated processes.

The Impact of Membrane Composition and Co‐Drug Synergistic Effects on Vancomycin Association with Model Membranes from Electrochemical Impedance Spectroscopy

“…Fabrication of gold microcavity array electrodes with hexagonally packed pores of 1 μm diameter and depth 0.5 μm, and the assembly of pore spanning lipid bilayers span across the arrays were carried out as reported earlier by us. [44,45] The detailed methods are provided in SI ( Figures S1-S3, SI) and a schematic representation of such a biomimetic platform is shown in Scheme 1a. The impedance of the MSLBs of each composition was assessed in triplicate, as well as their temporal stability.…”

“…Gold microcavity array electrodes (~1 × 1 cm 2 ) prepared by polystyrene microsphere lithography technique as reported previously by us [44,45,64,65] and were selectively modified with a self-assembled monolayer (SAM) of 1 mM 6-Mercapto-1-hexanol (MH). The detailed fabrication and characterization methods are also presented in SI.…”

“…The MSLB functions as the working electrode enabling electrochemical measurements of drug-membrane interaction and protein-lipid detection. [44,45] The lipid bilayer is assembled over an aqueous reservoir by a combination of both Langmuir-Blodgett and vesicle disruption to permit asymmetric/symmetric lipid bilayer assembly in a robust, relatively quick and facile manner. [41,43] Herein, we exploit MSLB platforms, to study by, electrochemical impedance spectroscopy, the interactions of vancomycin with 5 symmetric biomembranes of increasing complexity; DOPC, DOPC : Chol and DOPC : SM : Chol, eukaryotic membranes, a mammalian plasma membrane mimetic fusogenic composition coined "Nature's own" by Lentz et al [46,47] (referred to forthwith as MPM) expected to promote membrane curvature and E. coli extract comprised of DOPE, DOPG and cardiolipin (CA) as bacterial membranes.…”

Interaction of Miltefosine with Microcavity Supported Lipid Membrane: Biophysical Insights from Electrochemical Impedance Spectroscopy

“…The fabrication of the microcavity pore array across ã 1 × 1 cm 2 gold electrode was achieved via controlled gold electrodeposition through polystyrene sphere template as previously described by us [36,[51][52][53][54]. Following the fabrication of the micropore array, the SAM modified electrodes were applied to layer by layer transfer of two lipid monolayers using the LB method to form the final MSLB.…”

“…Most importantly, in the case of buffer filled pore supported bilayers, they offer the advantage of a receiver aqueous reservoir, that SLBs lack, in contact with proximal leaflet, that mimics the cytoplasm [47][48][49][50]. We recently reported such microcavity array supported lipid bilayers (MSLB) formed on the gold substrate and applied as working electrodes for electrochemical measurements to study drug-membrane and protein-membrane interaction [36,[51][52][53][54].…”