Multi-Stacked Supported Lipid Bilayer Micropatterning through Polymer Stencil Lift-Off

Abstract

Complex multi-lamellar structures play a critical role in biological systems, where they are present as lamellar bodies, and as part of biological assemblies that control energy transduction processes. Multi-lamellar lipid layers not only provide interesting systems for fundamental research on membrane structure and bilayer-associated polypeptides, but can also serve as components in bioinspired materials or devices. Although the ability to pattern stacked lipid bilayers at the micron scale is of importance for these purposes, limited work has been done in developing such patterning techniques. Here, we present a simple and direct approach to pattern stacked supported lipid bilayers (SLBs) using polymer stencil lift-off and the electrostatic interactions between cationic and anionic lipids. Both homogeneous and phase-segregated stacked SLB patterns were produced, demonstrating that the stacked lipid bilayers retain lateral diffusivity. We demonstrate patterned SLB stacks of up to four bilayers, where fluorescence resonance energy transfer (FRET) and quenching was used to probe the interactions between lipid bilayers. Furthermore, the study of lipid phase behaviour showed that gel phase domains align between adjacent layers. The proposed stacked SLB pattern platform provides a robust model for studying lipid behaviour with a controlled number of bilayers, and an attractive means towards building functional bioinspired materials or devices.

Keywords: fluorescence microscopy; micropatterning; phase separation; polymer stencil lift-off; stacked supported lipid bilayer.

Figure 1

Illustration of the PSLO patterning process for stacked SLBs. Parylene film is deposited onto a clean substrate, and photoresist is patterned using photolithography. After reactive ion etching of Parylene and removal of photoresist, small unilamellar lipid vesicles are deposited on the stencils and incubated to form SLBs. The final step is to lift off the stencil, revealing stacked SLB patterns. Inset: side view illustration of a double lipid bilayer with cationic and anionic lipids, and fluorescence images of a two-bilayer stack.

Figure 2

Epifluorescence images of four-bilayer homogeneous stacked SLB micropatterns. The first and third bilayers were composed of DOPC:DOTAP:DHPE-OG and can be observed in green channel. Second and fourth bilayers were composed of DOPC:DMPS:DHPE-LR, and can be observed in red channel (middle). The bottom row presents an overlay of both red and green channels. All images were acquired at the same magnification.

Figure 3

Quantitative fluorescence intensity analysis of stacked SLB formation. (a) Raw fluorescence intensity of green (DHPE-OG) and red (DHPE-LR) channels of the four-bilayer SLB patterns after each stacking step; (b) normalized intensity of each stacking step (green channel normalized to bilayer 1 and red channel normalized to bilayer 2 values); and (c) ratio of the raw intensities of red to green channels at each stacking step.

Figure 3

Quantitative fluorescence intensity analysis of stacked SLB formation. (a) Raw fluorescence intensity of green (DHPE-OG) and red (DHPE-LR) channels of the four-bilayer SLB patterns after each stacking step; (b) normalized intensity of each stacking step (green channel normalized to bilayer 1 and red channel normalized to bilayer 2 values); and (c) ratio of the raw intensities of red to green channels at each stacking step.

Figure 4

Epifluorescence images showing patterns of two-bilayer SLB stacks consisting of a homogeneous bottom bilayer and a phase-segregated top bilayer. The first lipid bilayer (composed of DOPC: DOTAP: DHPE-OG) is observed in the green channel, while the second lipid bilayer (composed of DOPC: DSPC: DOPS: DHPE-LR) can be observed in the red channel. The formation of the second bilayer leads to significant fluorescence intensity changes in the first bilayer. The overlaid image of the two bilayers shows the fluorescence patterns are complimentary, due to FRET.

Figure 5

Epifluorescence images of stacked phase-segregated SLB patterns. The first lipid bilayer was composed of DOPC:DSPC:DOTAP:DHPE-OG (green channel), while the second lipid bilayer was composed of DOPC:DSPC:DOPS:DHPE-LR (red channel). Insets show zoomed-in areas of the patterned bilayers. (a) When the first bilayer showed good phase segregation (green), the second lipid bilayer would phase segregate in the same pattern (red). The fluorescence intensity of first bilayer would decrease due to FRET, and the overlaid image of the two channels showed alignment of gel phase domains in the two bilayers; (b) When the first bilayer did not phase-segregate, the second SLB would still show gel-phase domains. The first bilayer appeared dimmer in areas that overlapped with the liquid disordered phase of the second bilayer. Image contrast has been adjusted to facilitate the visualization of the overlapping patterns in the stacked lipid bilayers.