NCAFM2023 Programme Booklet

VISUALIZATION OF HYDRATION LAYERS BENEATH LIPID BILAYERS SUPPORTED ON MICA BY USING FM-AFM

Hiroaki Kominami, Hirofumi Yamada, and Kei Kobayashi

Department of Electronic Science and Engineering, Kyoto University, Kyoto 615-8510, Japan. Email: keicoba@iic.kyoto-u.ac.jp

Lipid bilayers supported on substrates, namely supported lipid bilayers (SLBs), have been widely used as a platform for various biological studies. They form flat membranes on substrate, and the thickness typically ranges from 1 nm to 2 nm. It has been suggested that the SLBs on mica substrate has a fluidity because of the existence of the hydration layer at the lipid-mica interfaces. However, it has been difficult to directly visualize the hydration layers beneath the SLBs on mica. Frequency-modulation atomic force microscopy (FM-AFM) is capable of investigation of hydration structure at a solid–liquid interface[2]. Here, the hydration layers at the lipid–mica interfaces were investigated by using FM-AFM. In this study, we used 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) lipid bilayers. DPPC was dissolved in 10 mM HEPES solution containing 150 mM NaCl, 10 mM CaCl2 with concentration of 0.2 mg/mL. The DPPC solution was deposited onto a fleshly cleaved mica substrate. After 10 minutes, the substrate was gently rinsed 10 mM HEPES solution containing 150 mM NaCl, 10 mM CaCl2. Figure 1(a) shows a two-dimensional frequency shift map obtained at the edge of the lipid bilayer on mica. Frequency shift curves on lipid bilayer and mica substrate, which were extracted from the map as indicated by the red and blue arrows in Fig. 1(a), are shown in Fig. 1(b), respectively. As shown in Fig. 1(b), the frequency shift increased drastically during penetration of the lipid bilayer. We observed oscillations in the frequency shift with a spacing of about 0.25 nm, before the tip reached the mica surface. On the other hand, at the mica–liquid interfaces, the oscillatory features were also observed. This result suggests the existence of the water layers between the lipid and mica, which explains the fluidity of the SLBs.

Fig. 1 (a) Two-dimensional frequency shift map at the edge of the lipid bilayer on mica. (b) Frequency shift curves penetrating lipid bilayer (red) and frequency shift curve on mica substrate (blue). References [1] C. M. Ajo-Franklin, C. Yoshina-Ishii, S. G. Boxer, Langmuir, 2005, 21 , 4976. [2] K. Kobayashi, N. Oyabu, K. Kimura, S. Ido, K. Suzuki, T. Imai, K. Tagami, M. Tsukada, H. Yamada, J. Chem. Phys., 2013, 138 , 184704.

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