Free-Standing Lipid Membranes

HOPG revealed well organized films with a two-dimensional network of molecular or-dered domains. The domains display the threefold symmetry of the HOPG lattice.

The film thickness for unpolymerized monolayers was much smaller than the chain length of the lipid. Thus, a lying configuration of the lipids is assumed. The film thickness is consistent with the lipid chains lying parallel to the HOPG basal plane.

The distance between neighboring, repetitive lamellae confirmed the accepted model of a head-to-head configuration. Polymerization resulted in a rigid film and prevented (DiynePC) or reduced (PTPE) the ability of self assembling.

tem-perature.²⁵³ Mechanical behavior of pore-spanning membranes of DPhPC has been studied by AFM in aqueous environments.²⁵⁴ There, highly ordered silicon substrate exhibiting pore radii ranging from 225–600 nmwere utilized as a supporting material for free-standing lipid membranes.

Investigation with HIM

In contrast to membrane experiments in aqueous environments, a free-standing lipid membrane in air is much more fragile. Membranes of DPhPC are primarily stabilized by weak van der Waals interactions.²⁵⁵ In this context, intact membranes of DPhPC were not to be expected in HIM.

Figure 4.16: Free-standing monolayer of DPhPC on a Quantifoil grid. LB transfer at 40 mN m⁻¹ and 16^◦C. (A) Very few intact pore suspending membranes were found (red circle). (B) In a close-up, the membrane burst during the scan and created an (artificial) image of a half intact/broken membrane.

(C) A further scan revealed the complete ruptured membrane.

Inspected in the HIM (4.16), free-standing membranes of DPhPC were found to be very unstable and disrupted instantly. AFM measurements were not performed.

Investigation of UV polymerized lipid membranes of DiynePC and PTPE in the HIM revealed successful formation of robust pore-spanning monolayers up to 8×2µm². In contrast to the DPhPC monolayers, the membranes were found to remain intact for several hours both under laboratory and high vacuum conditions.

Fig. 4.17 shows polymerized membranes of DiynePC and PTPE taken by HIM, where apertures of various sizes are suspended. In rare cases, the polymerized monolayers were locally damaged during the scan with higher energy per unit area, allowing to investigate the behavior of forming defects. Although punctures were occurring, most parts of the membrane remained intact. Images of DiynePC membranes (Fig. 4.17A)

revealed a high beam resistance and stability. The geometry of defects (marked by a red circle) was irregular, indicating a high-grade cross-linked and stiff membrane.

Figure 4.17: Images of polymerized DiynePC (A) and PTPE (B) lipid monolayers on a Quantifoil TEM grid. UV polymerization time: 45 min. Most membranes of DiynePC were intact, but some exhibited elongated ruptures (A) (e.g.

red circle), ion dose: 7.25×10¹³cm⁻². Membranes of PTPE (B) were intact, one pore (blue circle) is not covered, defects (red circle) are circular, ion dose: 5.44×10¹²cm⁻².

In comparison, membranes of polymerized PTPE (Fig. 4.17B) showed less defects, which were exclusively circular and smooth in shape (red circle). One pore (blue circle) was not covered. While membranes of DiynePC got only locally damaged during a close-up scan, small ruptures of PTPE membranes ended up in a much larger defect of invariably circular shape. The HIM images highly support the assumed model of a stiffer DiynePC membrane, attributed to the higher amount of diacetylene groups per molecule in comparison to polymerized PTPE membranes.

Investigation with AFM

Membrane-covered and uncovered voids were also imaged with AFM. Successful forma-tion of pore-spanning polymerized PTPE membranes can be seen in Fig. 4.18, where several apertures of a Quantifoil TEM grid are covered by polymerized PTPE. Aper-tures up to 8×2µm² could be scanned. Although there was a moderate interaction of the AFM-tip with the sample under ambient conditions, the membranes were kept intact during scanning. The line scan profile for PTPE taken from Fig. 4.18B reveals

a distinct downward bending of the membrane during AFM scanning as a consequence of the applied forces by the AFM cantilever and gravitational forces.

Figure 4.18: AFM scan of polymeric pore-spanning PTPE. (A) Several membranes up to 2µm in diameter are intact. (B) Close-up of a2µmmembrane, which bent down to 70 nmalong the line profile (inset). This is the same sample as in Fig. 4.17B.

Figure 4.19: AFM scan of polymeric pore-spanning DiynePC. (A) Several membranes up to 4×2µm² in diameter were found to be intact. Oscillations of the membrane due to the tapping image mode and resulting resonance are visible. (B) An intact membrane of 8×2µm² is shown. In contrast to PTPE, the DiynePC membrane exhibited a constant height level with no sagging. This is the same sample as in Fig. 4.17A.

Pore-spanning membranes of polymerized DyinePC are shown in Fig. 4.19, where sev-eral intact membranes were covering the apertures of a Quantifoil TEM grid. Again, apertures up to 8×2µm² could be scanned successfully. In contrast to PTPE mem-branes, the line scan profile for the polymerized DiynePC membrane in Fig. 4.19A exhibits a constant height level with oscillations of the membrane due to the tapping image mode and resulting resonance. These observations can be explained with a stiffer and more rigid DiynePC membrane, which complies with the higher density of polymerizable diacetylene groups available.

Conclusion

Free-standing polymerized lipid membranes of DiynePC and PTPE were prepared and characterized by HIM and AFM. Due to covalent bonds in the polymeric mem-brane, advanced mechanical properties allowed pore-spanning membranes in the order of micrometers, not achievable with ordinary lipid membranes. In contrast, unpoly-merized DPhPC could not form stable membranes. The AFM investigation clearly revealed robust membranes for DiynePC and PTPE with different characteristics, and the mechanical properties could be attributed to the amount of diacetylene groups per molecule. This is also supported by AFM images of adlayers of DiynePC and PTPE on HOPG, where polymerized DiynePC exhibited a rigid structure after polymeriza-tion showing no alignment to the underlying crystalline structure of the HOPG. In contrast, polymerized PTPE organized into lamellae, indicating a flexible structure.