On-Demand Formation of Supported Lipid Membrane Arrays by Trehalose-Assisted Vesicle Delivery for SPR Imaging

Abstract

The fabrication of large-scale, solid-supported lipid bilayer (SLB) arrays has traditionally been an arduous and complex task, primarily due to the need to maintain SLBs within an aqueous environment. In this work, we demonstrate the use of trehalose vitrified phospholipid vesicles that facilitate on-demand generation of microarrays, allowing each element a unique composition, for the label-free and high-throughput analysis of biomolecular interactions by SPR imaging (SPRi). Small, unilamellar vesicles (SUVs) are suspended in trehalose, deposited in a spatially defined manner, with the trehalose vitrifying on either hydrophilic or hydrophobic SPR substrates. SLBs are subsequently spontaneously formed on-demand simply by in situ hydration of the array in the SPR instrument flow cell. The resulting SLBs exhibit high lateral mobility, characteristic of fluidic cellular lipid membranes, and preserve the biological function of embedded cell membrane receptors, as indicated by SPR affinity measurements. Independent fluorescence and SPR imaging studies show that the individual SLBs stay localized at the area of deposition, without any encapsulating matrix, confining coral, or boundaries. The introduced methodology allows individually addressable SLB arrays to be analyzed with excellent label-free sensitivity in a real-time, high-throughput manner. Various protein-ganglioside interactions have been selected as a model system to illustrate discrimination of strong and weak binding responses in SPRi sensorgrams. This methodology has been applied toward generating hybrid bilayer membranes on hydrophobic SPR substrates, demonstrating its versatility toward a range of surfaces and membrane geometries. The stability of the fabricated arrays, over medium to long storage periods, was evaluated and found to be good. The highly efficient and easily scalable nature of the method has the potential to be applied to a variety of label-free sensing platforms requiring lipid membranes for high-throughput analysis of their properties and constituents.

Keywords: HBM; SLB; SPR; SPR imaging; devitrification; microarray.

Figure 1

Schematic diagram showing the process of vesicle deposition, desiccation, and devitrification upon hydration of the trehalose matrix on the modified SPR sensor chips. Each SPR chip is modified with ca. 10 nm of silica, applied by plasma-enhanced chemical vapor deposition to increase hydrophilicity and provide a fusogenic surface for the SUVs. The devitrification process releasing the SUVs takes place in the SPR flow cell environment.

Figure 2

FRAP analysis of supported lipid bilayers formed using direct, traditional vesicle fusion and trehalose assisted deposition methods on microscope coverslips and SiO₂-modified SPR surfaces. Calculated values are the result of N = 3 experiments. (a) Fluorescence microscopy images showing bleaching and recovery of fluorescence due to redistribution of lipids over time. Scale bars represent 20 μm. (b) FRAP recovery curve of the devitrified membrane on modified SPR surface. (c) Diffusion coefficients. (d) Mobile fractions.

Figure 3

SPR studies of vesicle fusion upon devitrification of trehalose and preservation of embedded cargo activity. (a) Flow rate effects on devitrification of trehalose, release of SUVs, and formation of supported lipid bilayers. (b) Formation of supported lipid bilayer from trehalose released SUVs containing GM1 and subsequent CT binding response, followed by a comparative study on the identical chip of the same system generated by traditional vesicle fusion, showing excellent agreement. (c) Responses of membranes formed by vesicle injection methods to cholera toxin injections. (d) Responses of membranes formed by vesicle preservation and devitrification methods to cholera toxin injections.

Figure 4

SPR imaging study of membrane arrays formed using trehalose deposited and preserved vesicles. (a) Spatial confinement of lipids before, during, and after rehydration. The middle image exhibits the buffer front. (b) SPR difference image comparing bare silica surface and membrane covered surface. (c) SPR imaging sensorgrams comparing cholera toxin binding to SLBs of various compositions on the same SPR imaging substrate.

Figure 5

Hybrid bilayer membrane formation on SPR substrates using trehalose assisted vesicle delivery. (a) Scheme of deposition, desiccation, and hydration on hydrophobic rendered SPR surface. (b) SPR difference image comparing bare C18 surface and membrane-covered surface. (c) SPR imaging sensorgrams comparing cholera toxin binding to HBMs of various compositions on the same SPR imaging substrate.