Preparation and atomic force microscopy observation of lipid membranes on micro-patterned self-assembled monolayers Yasuhiro Nishimura ⁎, Takaaki Manaka, Mitsumasa Iwamoto Department of Physical Electronics, Tokyo Institute of Technology, 2-12-1 O-okayama, Meguro-ku, Tokyo 152-8552, Japan abstract article info Article history: Received 17 December 2007 Received in revised form 20 September 2008 Accepted 30 October 2008 Available online 10 November 2008 Keywords: Hybrid bilayer membrane Self-assembled monolayer Vesicle fusion Self-healing Using micro-contact printing (μCP) method, lipid membranes were deposited on the surface of micro- patterned self-assembled monolayers. Height and phase mode atomic force microscopy (AFM) images showed that the resulting deposited films were flat and that a micro-patterned composite bilayer system was constructed. It was also shown that the use of crystalline phase lipid membrane is effective for the preparation of the micro-patterned composite bilayer system of membranes with flat surface. © 2008 Elsevier B.V. All rights reserved. 1. Introduction Membranes have attracted considerable attention in science and engineering. Some types of membranes are very sensitive to their chemical environment, e.g. NO 2 , CO 2 , which can results in changes in the membranes physical and chemical properties. Singer and Nicolson proposed the famous fluid mosaic model of bio-mem- branes in 1972 [1]. Since then many experimental and theoretical studies have been conducted to clarify the relationship between function and structure of membranes. Also, the study of membrane has been motivated by possible applications in electronics, e.g. as chemical- and bio-sensors, and as an element of self-healing effect system [2,3]. Hence development of techniques available for preparing artificial bilayer system is needed. Among them are the Langmuir–Blodgett method and vesicle fusion method. However, these methods are not sufficient for the fabrication of in-plane- designed bilayer system. In the present study, using micro-contact printing (μCP) method [4,5], lipid membranes are deposited on self- assembled monolayers (SAM) that are micro-patterned with lines and spaces [6]. We observed the surface of the prepared micro- patterned composite membrane in solution by using atomic force microscopy (AFM). Cyclic-voltammetry (CV) was used to examine the insulating property of the prepared composite system of bilayer. 2. Experiments The lipid bilayer was prepared as follows: A 100-nm thick Au- layer was evaporated onto glass substrate (1.3 cm×3.9 cm (width×length)) coated with a 20-nm thick adhesion layer of Cr. After UV-ozone treatment of the evaporated Au-layer surface, micro-patterned SAM of 1-hexadecanthiol (HXDT from Wako Pure Chemicals, Fig. 1(a)) was deposited with a μCP method, using a poly (dimethylsiloxane) (PDMS, Sylgard 184 from Dow Corning) stamp [4,5]. A 2 mM ethanol solution containing HXDT was used as ink in the deposition. The PDMS stamp with a droplet of the HXDT solution was dried in air for 30 min, and then contacted with Au- coated substrate for 10 s in pure water. The Au-coated substrate was peeled off from the stamp that was taken out from pure water. Finally the resulting substrate was dried in N 2 gas. After preparation of the micro-patterned SAM (p-SAM), lipid layer was deposited using vesicle fusion method [7–10], where a chloroform solution containing 15 mg-lipid/ml was used for extrusion [11]. Briefly, lipids (1,2-Distearoyl-sn-glecero-3-phospho- choline (DSPC supplied from Sigma Aldrich Japan, Fig. 1(b)) and 1,2- Dioleoyl-sn-glecero-3-phosphocholine (DOPC supplied from Sigma Aldrich Japan, Fig. 1(c)) were dissolved in chloroform solution, then a lipid film was prepared using vesicle fusion method under a dry N 2 gas flow. The resulting lipid film was desiccated in a vacuum vessel overnight. The desiccated lipid film was hydrated by adding a buffer solution containing 150 mM-NaCl, 2 mM-CaCl 2 and 10 mM-HEPES (pH 7.0). After that, multilammelar vesicles were prepared. The temperature of buffer solution was 60 °C, above the gel–liquid crystal transition temperature (Tc) for DSPC (Tc: 55 °C). On the other hand, Thin Solid Films 517 (2009) 3227–3229 ⁎ Corresponding author. E-mail address: iwamoto.m.ac@m.titech.ac.jp (Y. Nishimura). 0040-6090/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.tsf.2008.10.113 Contents lists available at ScienceDirect Thin Solid Films journal homepage: www.elsevier.com/locate/tsf