micromachines Article Efficient Lipid Bilayer Formation by Dipping Lipid-Loaded Microperforated Sheet in Aqueous Solution Nobuo Misawa 1,† , Satoshi Fujii 1, Koki Kamiya 1,‡ , Toshihisa Osaki 1,2 and Shoji Takeuchi 1,2,3,* 1 Artificial Cell Membrane Systems Group, Kanagawa Institute of Industrial Science and Technology, 3-2-1 Sakado, Takatsu-ku, Kawasaki, Kanagawa 213-0012, Japan; [email protected] (N.M.); [email protected] (S.F.); [email protected] (K.K.); [email protected] (T.O.) 2 Institute of Industrial Science, The University of Tokyo, 4-6-1 Komaba, Meguro-ku, Tokyo 153-8505, Japan 3 Department of Mechano-Informatics, Graduate School of Information Science and Technology, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8656, Japan * Correspondence: [email protected] † Present address: School of Veterinary Medicine, Azabu University, 1-17-71 Fuchinobe, Chuo-ku, Sagamihara, Kanagawa 252-5201, Japan. ‡ Present address: Division of Molecular Science, Graduate School of Science and Technology, Gunma University, 1-5-1 Tenjin-cho, Kiryu, Gunma 376-8515, Japan. Abstract: This paper describes a method for a bilayer lipid membrane (BLM) formation using a perforated sheet along with an open chamber. Microscopic observation of the formed membrane showed a typical droplet interface bilayer. We proved that the formed membrane was a BLM based on electrical measurements of the membrane protein α-hemolysin, which produces nanopores in BLMs. Unlike the conventional approach for BLM formation based on the droplet contact method, this method provides aqueous surfaces with no organic solvent coating layer. Hence, this method is suitable for producing BLMs that facilitate the direct addition of chemicals into the aqueous phase. Keywords: bilayer lipid membrane; droplet interface bilayer; membrane protein; α-hemolysin; ion channel current Citation: Misawa, N.; Fujii, S.; Kamiya, K.; Osaki, T.; Takeuchi, S. Efficient Lipid Bilayer Formation by Dipping Lipid-Loaded 1. Introduction Microperforated Sheet in Aqueous Bilayer lipid membranes (BLMs) are widely used as artificial cell membranes for Solution. Micromachines 2021, 12, 53. research on plasma membranes, membrane proteins, and various membrane-associated https://doi.org/10.3390/mi12010053 biomolecules [1–4]. Artificial cell membranes are applicable to platforms for the functional Received: 14 December 2020 analysis of membrane proteins and biosensing applications [5–7]. Furthermore, artificial Accepted: 4 January 2021 membranes integrating a nanopore membrane protein have been recently used for DNA Published: 5 January 2021 sequencing [8] and have been studied for protein sequencing [9]. Over the years, the “painting method” [10] and “monolayer-folding method” [11] have been frequently used Publisher’s Note: MDPI stays neu- and regarded as simple methods for obtaining BLMs. In recent years, the “droplet contact tral with regard to jurisdictional clai- method” [12–14] has received significant attention for BLM production as an easier tech- ms in published maps and institutio- nique that generates a droplet interface bilayer (DIB) in organic solvents containing lipid nal affiliations. molecules. The droplet contact method is generally carried out using aqueous droplets submerged in organic solvents. Hence, DIBs are formed in organic solvents. This configuration makes it difficult to inject some chemicals, such as drug candidates; in addition, the aqueous Copyright: © 2021 by the authors. Li- phase from the outside impedes the fundamental studies of membrane proteins. Similarly, censee MDPI, Basel, Switzerland. for the development of odorant sensors, the oil layer on DIBs inhibit the penetration of This article is an open access article volatile compounds, which are mostly lipophilic, into an aqueous phase. The addition distributed under the terms and con- ditions of the Creative Commons At- of chemicals and the exchange of the isolated aqueous phase can be conducted using a tribution (CC BY) license (https:// pumping system or superabsorbent polymer with fluidic channels connected to a BLM creativecommons.org/licenses/by/ formation chamber [15,16]. The system requires an accurate regulation of the solution 4.0/). injection and/or ejection to prevent damage to the BLMs owing to a pressure imbalance. Micromachines 2021, 12, 53. https://doi.org/10.3390/mi12010053 https://www.mdpi.com/journal/micromachines Micromachines 2021, 12, x FOR PEER REVIEW 2 of 9 Micromachines 2021, 12, 53 2 of 9 BLM formation chamber [15,16]. The system requires an accurate regulation of the solu‐ tion injection and/or ejection to prevent damage to the BLMs owing to a pressure imbal‐ Forance. gas For absorption gas absorption into the into aqueous the aqueous phase, a hydrogelphase, a hydrogel is applicable is applicable as a mediating as a mediating material betweenmaterial the between atmosphere the atmosphere and BLM [ 6and,17 ].BLM However, [6,17]. theHowever, reproducible the reproducible formation of formation a BLM atof the a BLM interface at the between interface the between solution the and solution gel is a and laborious gel is a process. laborious process. Herein, we propose a method of BLM formation involving a promising system for a Herein, we propose a method of BLM formation involving a promising system for a chemical addition and gas absorption into an aqueous phase in contact with the BLMs. To chemical addition and gas absorption into an aqueous phase in contact with the BLMs. To establish such a BLM formation system equipped with an air–water interface, only a small establish such a BLM formation system equipped with an air–water interface, only a small amount of organic solvent along with an open chamber device is used. In this study, as amount of organic solvent along with an open chamber device is used. In this study, as a a first proof of concept for BLM formation, as indicated in Figure1, we carry out a BLM first proof of concept for BLM formation, as indicated in Figure 1, we carry out a BLM formation by dipping a perforated sheet loaded with a lipid solution into a buffer solution. formation by dipping a perforated sheet loaded with a lipid solution into a buffer solution. We fluorinate the surface of the device for a reproducible membrane formation, and then We fluorinate the surface of the device for a reproducible membrane formation, and then we microscopically observe the membrane and measure the electric current in conjunction we microscopically observe the membrane and measure the electric current in conjunction with the membrane proteins to ascertain whether the membranes are decent BLMs. with the membrane proteins to ascertain whether the membranes are decent BLMs. Figure 1. Schematic sectional images of bilayer lipid membrane (BLM) formation by dipping a Figure 1. Schematic sectional images of bilayer lipid membrane (BLM) formation by dipping a perforated sheet coated with a lipid solution into an aqueous solution. A through‐slit in the cham‐ perforated sheet coated with a lipid solution into an aqueous solution. A through-slit in the chamber ber bottom allows penetration of the sheet. BLM is spontaneously formed at the interface of the bottomtwo isolated allows compartments. penetration of the sheet. BLM is spontaneously formed at the interface of the two isolated compartments. Micromachines 2021, 12, 53 3 of 9 2. Materials and Methods 2.1. Materials As the device material, we used a polymethylmethacrylate (PMMA) film and plate (i.e., Acryplen and Acrylight, purchased from Mitsubishi Chemical Co., Tokyo, Japan) for fabrication of the perforated sheets and an open chamber device, respectively. An amphiphobic coating reagent (SFCOAT, SFE-DP02H) was obtained from AGC SEIMI Chemical Co., Ltd. (Kanagawa, Japan) for surface treatment of the PMMA film. Lipid molecules dissolved in chloroform, 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), and 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) (Avanti Polar Lipids, Inc., Alabaster, AL, USA) were purchased as commercialized products, and n-decane (Merck KGaA, Darmstadt, Germany) was used as the solvent for the lipid solution pre- pared in this study. A nanopore-forming protein α-hemolysin (αHL) was used (Merck KGaA). A concentrated stock solution of αHL (20 µM) was prepared by dissolution of αHL lyophilized powder using ultrapure water from a Milli-Q system (Millipore, MA, USA). Ag/AgCl electrodes (0.4-mm diameter AG-401355, The Nilaco Co., Tokyo, Japan) were pre- pared in advance by dipping in a commercial bleach agent overnight. Other chemicals were obtained from Wako Pure Chemical Industries, Ltd. (Osaka, Japan) and Sigma-Aldrich Co. (St. Louis, MO, USA). All reagents were used without further purification. Micromachines 2021, 12, x FOR PEER2.2. REVIEW Device Fabrication 4 of 9 We fabricated perforated T-shaped PMMA (Acryplen) sheets with a thickness of 75 µm and pore diameters of 500, 600, 700, and 800 µm. An open chamber device made of PMMA (Acrylight)sheet loaded was with processed the lipid into solution a gourd-shaped was manually well (3-mm dipped depth into andthe buffer 24.7 mm solution2 area) in with the aopen narrow chamber through-slit, using tweezers. as observed Owing in to the the top transparency view of Figure of the2b. PMMA, The clearance we could gap observe of thethe narrow through slit‐hole was of 1 the mm. sheet For using easy a observation microscope of (YDZ the‐ formed3F, Yashima BLMs, Optical the inside Co., Ltd., wall To of ‐ thekyo, chamber Japan) from was partiallyoutside the planarized. chamber even The device when the parts sheet (well was chip, submerged perforated in the sheet, aqueous and Ag/AgClsolution inside electrodes) the chamber. were manually In addition, assembled we investigated using tweezers. the effect All of PMMA fluorination parts wereof the fabricatedperforated using sheet a on milling the liquid machine retention (MM-100, in the Modiachamber Systems and the Co., efficiency Ltd., Saitama, of the BLM Japan) for‐ withmation. 0.5- and 1-mm diameter end mills.