Dry Two-Step Self-Assembly of Stable Supported Lipid Bilayers on Silicon Substrates

Abstract

Artificial membranes are models for biological systems and are important for applications. We introduce a dry two-step self-assembly method consisting of the high-vacuum evaporation of phospholipid molecules over silicon, followed by a subsequent annealing step in air. We evaporate dipalmitoylphosphatidylcholine (DPPC) molecules over bare silicon without the use of polymer cushions or solvents. High-resolution ellipsometry and AFM temperature-dependent measurements are performed in air to detect the characteristic phase transitions of DPPC bilayers. Complementary AFM force-spectroscopy breakthrough events are induced to detect single- and multi-bilayer formation. These combined experimental methods confirm the formation of stable non-hydrated supported lipid bilayers with phase transitions gel to ripple at 311.5 ± 0.9 K, ripple to liquid crystalline at 323.8 ± 2.5 K and liquid crystalline to fluid disordered at 330.4 ± 0.9 K, consistent with such structures reported in wet environments. We find that the AFM tip induces a restructuring or intercalation of the bilayer that is strongly related to the applied tip-force. These dry supported lipid bilayers show long-term stability. These findings are relevant for the development of functional biointerfaces, specifically for fabrication of biosensors and membrane protein platforms. The observed stability is relevant in the context of lifetimes of systems protected by bilayers in dry environments.

Keywords: artificial membranes; atomic force microscopy; bio-silica interfaces; force spectroscopy; high resolution ellipsometry; phase transitions; physical vapor deposition; self-assembly; supported lipid bilayers.

Figure 1

(a) AFM topography image of DPPC/SiO₂/Si taken directly after evaporation. (b) Height distribution of the topography map showing the three distinct levels at 4 nm, 8 nm and 12 nm. (c) Adhesion map of the same region. (d) Adhesion distribution centred at 9 nN.

Figure 2

(a) Force curve on the first bilayer (level 1). (b) Force curve on the second bilayer (level 2). (c) Force curve on the third bilayer (level 3).

Figure 3

Ellipsometric changes in polarizer angle ΔP of DPPC bilayer, measured during (a) the first, (b) the second and (c) the third heating–cooling cycle. Vertical dashed lines indicate the phase transition temperatures as described in the text. The ΔP signal of the following heating–cooling cycles show the same characteristics of figures (b) and (c), leading to the conclusion that the structure formed after the first heating–cooling cycle remains stable.

Figure 4

Adhesion maps obtained using QI^TM mode for (a) 296 K (b) 308 K and (c) 323 K. (d) Normalized adhesion taken for three different samples as a function of temperature.

Figure 5

Topographic AFM images from DPPC/SiO₂/Si samples at RT: (a) directly after evaporation and before the first temperature cycle, (b) after the first and (c) sixth temperature cycle performed within the ellipsometer. (d) Topography image directly after evaporation and before temperature cycling within the AFM, (e) after the first temperature cycle while scanning with AFM, and (f) after the first temperature cycle in a contiguous region to the scanned area.

Figure 6

AFM topography images of the DPPC bilayer taken at RT in air during consecutive measurements in the same scan region (2 µm × 2 µm) to induce the reduction of the island-like DPPC bilayer surface area, applying two different tip forces, (a–c): 1 nN; (d–f): 3 nN.

Figure 7

Surface area reduction during consecutive scans at RT using two different tip forces applied in the same scan regions. Scans 1–5 are taken at 1 nN, while scans 6–10 are taken at 3 nN.