JP2011149868A - Lipid duplex film, self-support film used for forming the same, and micro-flow path device with the same - Google Patents
Lipid duplex film, self-support film used for forming the same, and micro-flow path device with the same Download PDFInfo
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Abstract
Description
本発明は、安定な脂質二重膜、これを形成するために用いることができる、透孔を有する自己支持性フィルム及びその製造方法、並びに該脂質二重膜を具備するマイクロ流路デバイスに関する。 The present invention relates to a stable lipid bilayer membrane, a self-supporting film having pores that can be used to form the membrane, a method for producing the same, and a microchannel device including the lipid bilayer membrane.
生物を構成する細胞や、細胞内に存在するミトコンドリア、ゴルジ体、小胞体等の各種オルガネラ、細胞核等は、外側が生体膜で覆われており、この生体膜は、基本的に脂質二重膜から構成されている。生理活性を有する様々なタンパク質、すなわち、レセプターや酵素等がこの脂質二重膜を貫通する形で脂質二重膜上に保持されている。これらの膜貫通タンパク質は、生体内で重要な役割を果たしている。特に、細胞膜上に存在する各種レセプターは、生体内に存在するリガンドと結合することにより、様々な生理学的反応を引き起こす引き金になることがわかっている。このため、レセプターの機能を亢進する各種リガンドや、レセプターの機能を阻害する阻害剤等が医薬品として用いられており、また、新たな医薬品として利用可能な天然又は人工のリガンドや阻害剤が研究されている。 Cells that make up living organisms, various organelles such as mitochondria, Golgi bodies, and endoplasmic reticulum, cell nuclei, etc. are covered with a biological membrane on the outside. This biological membrane is basically a lipid bilayer membrane. It is composed of Various proteins having physiological activity, that is, receptors, enzymes, and the like, are retained on the lipid bilayer membrane in a form that penetrates the lipid bilayer membrane. These transmembrane proteins play an important role in vivo. In particular, it has been found that various receptors present on cell membranes trigger various physiological reactions by binding to ligands present in the living body. For this reason, various ligands that enhance the function of the receptor, inhibitors that inhibit the function of the receptor, and the like are used as pharmaceuticals, and natural or artificial ligands and inhibitors that can be used as new pharmaceuticals have been studied. ing.
これらの膜貫通タンパク質や、そのリガンド、阻害剤等を開発するためには、生体内と同じ状態、すなわち、膜貫通タンパク質が生体膜に保持された状態で各種測定を行うことが望まれる。従来、膜貫通タンパク質が生体膜に保持された状態を模するべく、透孔を塞ぐ形で脂質二重膜を形成し、この脂質二重膜に膜貫通タンパク質を保持させ、この状態で各種測定が行われている（非特許文献１）。 In order to develop these transmembrane proteins, their ligands, inhibitors and the like, it is desired to perform various measurements in the same state as in the living body, that is, in a state where the transmembrane protein is held on the biological membrane. Conventionally, in order to mimic the state where transmembrane proteins are retained on biological membranes, a lipid bilayer is formed by closing the pores, and the transmembrane protein is retained on this lipid bilayer, and various measurements are performed in this state. (Non-patent Document 1).
しかしながら、従来から各種測定に用いられている脂質二重膜は、外部の圧力変化や機械的振動、電気的な刺激等により破壊されやすく、形成後、各種測定を行うための準備段階や測定中に脂質二重膜が破壊されて測定ができなくなることがしばしば起きる（非特許文献２）。従って、形成後に安定に保持できる脂質二重膜が求められている。 However, conventional lipid bilayer membranes used for various measurements are easily destroyed by external pressure changes, mechanical vibrations, electrical stimulation, etc., and after formation, during the preparation stage and during measurement In many cases, the lipid bilayer is destroyed and measurement becomes impossible (Non-patent Document 2). Accordingly, there is a need for a lipid bilayer that can be stably retained after formation.
従って、本発明の目的は、安定性が高くて破壊されにくい脂質二重膜を提供することである。また、本発明の目的は、該脂質二重膜の形成に有用な自己支持性フィルム、及び該脂質二重膜を含むマイクロ流路デバイスを提供することである。 Accordingly, an object of the present invention is to provide a lipid bilayer that is highly stable and difficult to break. In addition, an object of the present invention is to provide a self-supporting film useful for forming the lipid bilayer membrane and a microchannel device including the lipid bilayer membrane.
従来から脂質二重膜の形成に用いられている透孔は、その孔径が数十μｍ〜数百μｍである（非特許文献３及び非特許文献４）。本願発明者らは、従来から用いられている、透孔の内壁にその周縁部が接し、該透孔を塞ぐ脂質二重膜において、透孔の孔径を十分小さくすることにより脂質二重膜の安定性が高まるのではないかと予測した。しかしながら、脂質二重膜を形成することが可能な透孔であって、孔径が１μｍ未満のものは全く知られておらず、このような小さな孔径を有する透孔を形成する方法も知られていない。 The pores conventionally used for forming the lipid bilayer membrane have a pore diameter of several tens of μm to several hundreds of μm (Non-patent Documents 3 and 4). In the lipid bilayer membrane in which the peripheral edge is in contact with the inner wall of the through-hole, which has been conventionally used, and closes the through-hole, the present inventors have made the pore size of the through-hole small enough to reduce the lipid bilayer membrane. We predicted that stability would increase. However, there are no known pores capable of forming a lipid bilayer membrane having a pore diameter of less than 1 μm, and methods for forming such pores having such a small pore diameter are also known. Absent.
本願発明者らは、鋭意研究の結果、自己支持性フィルムに透孔を形成し、次いでこの自己支持性フィルムに、物質を蒸着すると、透孔の内壁にも該物質が被着して透孔の孔径が縮小されることを見出した。そして、この方法により形成した、孔径が１μｍ未満の透孔に脂質二重膜を形成すると、公知の脂質二重膜よりも安定に保持されることを実験的に確認し、本発明を完成した。 As a result of diligent research, the inventors of the present application formed a through hole in the self-supporting film, and then deposited a substance on the self-supporting film, and the substance was deposited on the inner wall of the through-hole. It has been found that the pore diameter of the material is reduced. Then, it was experimentally confirmed that when a lipid bilayer is formed in a pore having a pore diameter of less than 1 μm formed by this method, the lipid bilayer is more stably maintained than the known lipid bilayer, and the present invention has been completed. .
すなわち、本発明は、孔径が１μｍ未満の透孔を有する自己支持性フィルムを提供する。また、本発明は、孔径が１μｍ以上の透孔を有する自己支持性フィルムを準備する工程と、該自己支持性フィルム上に被着可能な化合物を、蒸着により少なくとも前記透孔の内壁上に被着させ、それによって前記透孔の孔径を１μｍ未満に縮小する、上記本発明の自己支持性フィルムの製造方法を提供する。さらに、本発明は、孔径が１μｍ未満の透孔の内壁にその周縁部が接し、該透孔を塞ぐ脂質二重膜を提供する。さらに本発明は、第１のマイクロ流路を具備する第１のマイクロ流路チップと、前記第１のマイクロ流路と少なくとも一部が接する第２のマイクロ流路を具備する第２のマイクロ流路チップとを具備し、前記第１のマイクロ流路と前記第２のマイクロ流路の境界に、脂質二重膜を形成するための、孔径が１μｍ未満の透孔が形成されているマイクロ流路デバイスを提供する。さらに本発明は、上記本発明のマイクロ流路デバイス中の前記透孔の内壁にその周縁部が接し、該透孔を塞ぐ脂質二重膜が形成されている、脂質二重膜マイクロ流路デバイスを提供する。 That is, this invention provides the self-supporting film which has a through-hole whose hole diameter is less than 1 micrometer. The present invention also provides a step of preparing a self-supporting film having pores having a pore diameter of 1 μm or more, and a compound that can be deposited on the self-supporting film is deposited on at least the inner walls of the pores by vapor deposition. The method for producing a self-supporting film of the present invention is provided, in which the hole diameter of the through-hole is reduced to less than 1 μm. Furthermore, the present invention provides a lipid bilayer membrane in which a peripheral edge thereof is in contact with an inner wall of a pore having a pore diameter of less than 1 μm to block the pore. Furthermore, the present invention provides a second micro flow path including a first micro flow path chip including a first micro flow path, and a second micro flow path at least partially in contact with the first micro flow path. A microfluid having a channel chip and having a pore diameter of less than 1 μm for forming a lipid bilayer membrane at the boundary between the first microchannel and the second microchannel Provide a road device. Furthermore, the present invention is a lipid bilayer microchannel device in which a peripheral portion is in contact with an inner wall of the through hole in the microchannel device of the present invention, and a lipid bilayer membrane is formed to block the through hole. I will provide a.
本発明により、形成後に破壊されにくく安定な脂質二重膜が提供された。本発明の脂質二重膜は安定であり、また、容易に再現性良く形成可能である。従って、本発明の脂質二重膜を利用して、膜貫通タンパク質やそのリガンド等の性質を調べる実験を容易に行うことができるようになった。従って、本発明は、創薬のためのスクリーニング等に好適に用いることができる。 The present invention provides a stable lipid bilayer that is difficult to break after formation. The lipid bilayer membrane of the present invention is stable and can be easily formed with good reproducibility. Therefore, it has become possible to easily conduct experiments for examining the properties of transmembrane proteins and their ligands using the lipid bilayer membrane of the present invention. Therefore, the present invention can be suitably used for screening for drug discovery.
１０ マイクロ流路デバイス
１２ 上部マイクロ流路チップ
１４ 下部マイクロ流路チップ
１６ 上部マイクロ流路
１８ 下部マイクロ流路
２０ セパレーター
２２ 自己支持性フィルム
２４ 脂質二重膜
２６ タンパク質分子
DESCRIPTION OF SYMBOLS 10 Microchannel device 12 Upper microchannel chip 14 Lower microchannel chip 16 Upper microchannel 18 Lower microchannel 20 Separator 22 Self-supporting film 24 Lipid bilayer membrane 26 Protein molecule
上記のとおり、本発明の脂質二重膜を形成するために好適に用いることができる自己支持性フィルムは、孔径が１μｍ未満の透孔を有するものである。ここで、「自己支持性」とは、透孔を形成した状態のフィルムを単独で、フィルムの一部分を把持してフィルムを持ち上げることができることを意味する。このように小さな透孔を有する自己支持性フィルムはこれまでに知られていない。また、「透孔」とは、フィルムの片側の表面の開口部から、他方側の表面の開口部に直線的に開いている貫通孔を意味し、ろ紙やメンブレンフィルターのような多孔性材料の孔は包含されない。透孔の表面形状（すなわち、開口部の平面形状）は、円形が最も好ましいが、円に近い（好ましくは長径と短径の比率が0.8〜1.2程度の範囲に入る）楕円でも問題はない。孔径は、直径（楕円形の場合は長径）を意味する。孔径は、１μｍ未満であり、好ましくは1nm〜900nm 、さらに好ましくは、100nm〜800nmである。孔径は電子顕微鏡観察により容易に測定可能である。自己支持性フィルムの膜厚（後述のように、化合物をフィルム全体に蒸着する場合には、蒸着後の膜厚）は、特に限定されないが、通常0.5μｍ〜20μｍ程度、好ましくは2μｍ〜10μｍ程度である。自己支持性フィルムは、上記透孔を複数個有していてもよい。また、本発明の自己支持性フィルムの表面上には、金等の金属を蒸着して電極として利用したり、半導体を塗布することで透明電極として使用可能である。 As described above, the self-supporting film that can be suitably used for forming the lipid bilayer membrane of the present invention has a pore having a pore diameter of less than 1 μm. Here, “self-supporting” means that the film in a state where the through holes are formed can be lifted by holding a part of the film alone. No self-supporting film having such small pores has been known so far. The term “through hole” means a through hole that opens linearly from the opening on one surface of the film to the opening on the other surface, and is made of a porous material such as a filter paper or a membrane filter. Holes are not included. The surface shape of the through hole (that is, the planar shape of the opening) is most preferably circular, but there is no problem with an ellipse that is close to a circle (preferably the ratio of the major axis to the minor axis is in the range of about 0.8 to 1.2). The pore diameter means a diameter (long diameter in the case of an ellipse). The pore diameter is less than 1 μm, preferably 1 nm to 900 nm, more preferably 100 nm to 800 nm. The pore diameter can be easily measured by electron microscope observation. The film thickness of the self-supporting film (when the compound is deposited on the entire film as will be described later) is not particularly limited, but is usually about 0.5 μm to 20 μm, preferably about 2 μm to 10 μm. It is. The self-supporting film may have a plurality of the through holes. Further, on the surface of the self-supporting film of the present invention, a metal such as gold is vapor-deposited and used as an electrode, or by applying a semiconductor, it can be used as a transparent electrode.
自己支持性フィルムの材質としては、孔径数μｍ程度の透孔を開けることができ、蒸着により化合物（後述）を被着することができるものであれば、何ら限定されるものではない。たとえばガラスや金属の小孔に蒸着を行うことにより、ナノメートルサイズの微小孔の作製が可能である。蒸着する化合物と同じ材料でフィルムを構成すると、フィルムを一体的に形成することができ、フィルムの耐久性や取扱性が高まるので好ましい。もっとも、蒸着する化合物以外の材料でフィルムを形成することも可能であり、この場合の材料の例としては、例えば、スピンコート可能な高分子材料（ポリオレフィン系、ポリエチレン系）等を挙げることができる。 The material of the self-supporting film is not particularly limited as long as it can open holes having a pore diameter of about several μm and can deposit a compound (described later) by vapor deposition. For example, nanometer-sized micropores can be produced by vapor deposition in small holes of glass or metal. It is preferable that the film is made of the same material as the compound to be deposited because the film can be formed integrally and the durability and handling of the film are increased. However, it is also possible to form a film with a material other than the compound to be deposited, and examples of the material in this case include a spin coatable polymer material (polyolefin type, polyethylene type) and the like. .
上記自己支持性フィルムは、以下の方法により製造することができる。 The self-supporting film can be produced by the following method.
まず、孔径が数μｍ、好ましくは２μｍ〜８μｍ程度の透孔を有する自己支持性フィルムを準備する。この程度の孔径の透孔であれば、フォトリソグラフィー等の公知の方法で形成可能であり、この方法については下記実施例に具体的に記載する。なお、次の工程で、フィルム全体に蒸着を施す場合には、蒸着前のフィルムの膜厚は、通常、１μｍ〜２０μｍ程度、好ましくは２μｍ〜１０μｍ程度である。 First, a self-supporting film having through holes having a pore diameter of several μm, preferably about 2 μm to 8 μm is prepared. If it is a through-hole of such a hole diameter, it can be formed by a known method such as photolithography, and this method will be specifically described in the following examples. In the next step, when vapor deposition is performed on the entire film, the film thickness of the film before vapor deposition is usually about 1 μm to 20 μm, preferably about 2 μm to 10 μm.
次に、自己支持性フィルム上に被着可能な化合物を、蒸着により少なくとも前記透孔の内壁上に被着させ、それによって前記透孔の孔径を１μｍ未満に縮小する。蒸着する化合物は、蒸着により、先に準備した透孔を有する自己支持性フィルム上に被着することができる化合物であればいずれの化合物であってもよく、好ましい例として、パラキシリレン系ポリマーを挙げることができる。これらのうち、パラキシレン系ポリマーは、等方蒸着が可能であり、透孔の内壁にもフィルムの両面にも均一にポリマーが被着（堆積）していく性質を有しており、また、耐熱性及び耐薬品性に優れているので好ましい。さらに、上記のとおり、蒸着に供される自己支持性フィルムも同種のパラキシレン系ポリマーで形成しておくと、蒸着後の自己支持性フィルムは一体的であり、耐久性及び取扱性が優れている。 Next, a compound that can be deposited on the self-supporting film is deposited on at least the inner wall of the through hole by vapor deposition, thereby reducing the hole diameter of the through hole to less than 1 μm. The compound to be vapor-deposited may be any compound as long as it is a compound that can be deposited on the self-supporting film having the previously prepared pores by vapor deposition, and a preferred example is a paraxylylene polymer. be able to. Among these, the paraxylene-based polymer can be isotropically vapor-deposited, and has the property that the polymer is uniformly deposited (deposited) on both the inner wall of the through-hole and on both sides of the film. Since it is excellent in heat resistance and chemical resistance, it is preferable. Furthermore, as described above, if the self-supporting film to be subjected to vapor deposition is also formed of the same kind of para-xylene polymer, the self-supporting film after vapor deposition is integrated, and has excellent durability and handleability. Yes.
パラキシレン系ポリマーは、下記一般式で表わされる繰返し単位から成るポリマーであり、パリレン(Parylene)の商品名で市販されているので、市販品を好ましく用いることができる。 The paraxylene-based polymer is a polymer composed of repeating units represented by the following general formula, and is commercially available under the trade name of Parylene, so that a commercially available product can be preferably used.
（式中、Ｘは水素原子又はフッ素原子、R1及びR2は互いに独立して水素原子又は塩素原子を表す）。 (In the formula, X represents a hydrogen atom or a fluorine atom, and R 1 and R 2 each independently represent a hydrogen atom or a chlorine atom).
パラキシレン系ポリマーは、そのモノマーを真空チャンバー内で支持体に蒸着すると、支持体上で重合が起きてポリマーとなる。ポリマーの分子量は、蒸着量や蒸着時間に依存して変化し、最大５０万程度である。モノマーは、蒸着により、種々の材質から成る支持体上に被着（堆積）することができ、支持体がパラキシリレン系ポリマーから形成されている場合のみならず、上記した種々の材質の支持体上に被着可能である。 When the paraxylene-based polymer is vapor-deposited on a support in a vacuum chamber, polymerization occurs on the support to become a polymer. The molecular weight of the polymer varies depending on the deposition amount and the deposition time, and is about 500,000 at the maximum. The monomer can be deposited (deposited) on a support made of various materials by vapor deposition, and not only when the support is made of a paraxylylene polymer, but also on the support made of various materials described above. It can be attached to.
蒸着は、蒸着される化合物を少なくとも前記透孔の内壁上に被着させればよいが、蒸着前の自己支持性フィルム全体に蒸着を施すことが簡便で好ましい。自己支持性フィルム全体に蒸着を施すと、透孔の内壁上に化合物が被着されるので、透孔の孔径が縮小され、これと同時に自己支持性フィルムの両側の表面にも被着されるので、フィルムの膜厚も同時に大きくなる。 The vapor deposition may be performed by depositing at least the compound to be vapor deposited on the inner wall of the through hole, but it is simple and preferable to deposit the entire self-supporting film before vapor deposition. When vapor deposition is performed on the entire self-supporting film, the compound is deposited on the inner wall of the through-hole, so that the hole diameter of the through-hole is reduced, and at the same time, it is deposited on both surfaces of the self-supporting film. Therefore, the film thickness increases simultaneously.
パラキシレンモノマーを真空チャンバー内で自己支持性フィルム上に蒸着する場合、パリレン（商品名）の使用説明書に記載された方法に従って蒸着を行うことができ、蒸着温度は、通常室温、蒸着時間は通常30分間〜180分間、好ましくは30分間〜60分間である。 When paraxylene monomer is deposited on a self-supporting film in a vacuum chamber, deposition can be performed according to the method described in the instruction manual of Parylene (trade name). The deposition temperature is usually room temperature and the deposition time is Usually, it is 30 minutes to 180 minutes, preferably 30 minutes to 60 minutes.
本発明の脂質二重膜は、孔径が１μｍ未満の透孔の内壁にその周縁部が接し、該透孔を塞ぐ脂質二重膜である。該透孔は、必ずしも自己支持性フィルムに形成された透孔でなくてもよく、他の支持体上に支持されている層であってもよい。もっとも、上記した本発明の自己支持性フィルムに設けられた透孔であれば、該フィルムを任意の場所に単独で移動させることができ、後述するマイクロ流路デバイスへの組込み等を容易に行うことができるので好ましい。 The lipid bilayer membrane of the present invention is a lipid bilayer membrane in which the peripheral edge is in contact with the inner wall of a pore having a pore diameter of less than 1 μm and closes the pore. The through hole is not necessarily a through hole formed in the self-supporting film, and may be a layer supported on another support. But if it is a through-hole provided in the above-mentioned self-supporting film of this invention, this film can be moved independently to arbitrary places, and the incorporation to the microchannel device mentioned later etc. is performed easily. This is preferable.
透孔の内壁にその周縁部が接し、該透孔を塞ぐ脂質二重膜は、脂質二重膜を構成する脂質溶液を、単に透孔に施すだけで形成することができる。脂質二重膜を構成する脂質としては、脂質二重膜、すなわち、親水性領域と疎水性領域を１分子中に有する脂質分子が、疎水性領域を内側、親水性領域を外側に向けて２層に並んだ膜を形成できる脂質であれば特に限定されないが、生体膜における反応を模するためには、生体膜と同じか類似したものが好ましく、この分野において従来から広く用いられているリン脂質、例えば、ジフィタノイルフォスファチジルコリン(diphytanoyl phosphatidylcholine, DPhPC)、ジパルミトイルフォスファチジルコリン(dipalmytoyl phosphatidylcholine)、パルミトイルオレオイルフォスファチジルコリン(1-Palmitoyl 2-Oleoyl phosphatidylcholine, POPC)、ジオレオイルフォスファチジルコリン(Dioleoyl phosphatidylcholine, DOPC)等を好ましい例として挙げることができる。これらの多くは市販されているので、市販品を好ましく用いることができる。 A lipid bilayer membrane whose peripheral edge is in contact with the inner wall of the through-hole and plugs the through-hole can be formed by simply applying a lipid solution constituting the lipid bilayer membrane to the through-hole. As the lipid constituting the lipid bilayer membrane, a lipid bilayer membrane, that is, a lipid molecule having a hydrophilic region and a hydrophobic region in one molecule, has a hydrophobic region on the inside and a hydrophilic region on the outside. The lipid is not particularly limited as long as it is a lipid capable of forming a membrane arranged in layers, but in order to simulate a reaction in a biological membrane, the same or similar one as that of a biological membrane is preferable. Lipids such as diphytanoyl phosphatidylcholine (DPhPC), dipalmytoyl phosphatidylcholine, palmitoyl oleoylphosphatidylcholine (POPC), POPC A preferred example is oil phosphatidylcholine (DOPC). Since many of these are commercially available, commercially available products can be preferably used.
脂質二重膜の形成に用いられる溶液中のリン脂質の濃度は、脂質二重膜が形成可能な濃度であれば特に限定されないが、通常、5g/L〜20g/L程度、好ましくは7g/L〜15g/L程度である。また、リン脂質溶液の溶媒は、特に限定されないが、有機溶媒が好ましく、n-デカンのような脂肪族炭化水素溶媒が好ましい。また、リン脂質は、この溶液中でリポソームを形成してもよく、この場合には、用いられる液はリポソーム懸濁液になる。 The concentration of the phospholipid in the solution used for forming the lipid bilayer membrane is not particularly limited as long as the lipid bilayer membrane can be formed, but is usually about 5 g / L to 20 g / L, preferably 7 g / L. It is about L-15g / L. The solvent of the phospholipid solution is not particularly limited, but an organic solvent is preferable, and an aliphatic hydrocarbon solvent such as n-decane is preferable. Phospholipids may also form liposomes in this solution, in which case the liquid used is a liposome suspension.
本発明の脂質二重膜は、該脂質二重膜に保持された状態におけるタンパク質の性質や機能を調べたり、該タンパク質に結合して、その生理活性を変化させるリガンドをスクリーニングしたりその性質を調べたりする各種測定に好適に用いられるものであるので、脂質二重膜は、タンパク質を含んでいることが好ましく、特に生体内で生体膜に保持された状態で機能している膜貫通タンパク質が好ましい。脂質二重膜に保持するタンパク質としては、各種レセプターや酵素を挙げることができ、例としては、アルファーヘモリシン、グラミシジン、アラメチシンなどのペプチドタンパク質類、ABCトランスポータタンパク質等を挙げることができるがこれらに限定されるものではない。 The lipid bilayer membrane of the present invention can be used to examine the properties and functions of proteins in the state retained on the lipid bilayer membrane, to screen for ligands that bind to the protein and change its physiological activity, The lipid bilayer membrane preferably contains a protein because it is suitably used for various measurements to be investigated, and in particular, a transmembrane protein functioning in a state retained in the biological membrane in vivo. preferable. Examples of proteins retained in the lipid bilayer include various receptors and enzymes. Examples include peptide proteins such as alpha-hemolysin, gramicidin, and alamethicin, and ABC transporter proteins. It is not limited to.
タンパク質を保持する脂質二重膜は、上記したリン脂質溶液にタンパク質を溶解しておくことにより形成することもできるし、先に上記のとおり脂質二重膜を形成し、その後、タンパク質の溶液を該脂質二重膜に施すことによっても形成することができる。リン脂質溶液がリポソームを含むリポソーム懸濁液である場合には、タンパク質を保持するリポソームの懸濁液を用いることにより形成することができる。これらの溶液中のタンパク質の濃度は、特に限定されるものではなく、適宜選択することができるが、通常、1nM〜1mM程度、好ましくは0.1μM〜100μM程度である。 The lipid bilayer membrane that retains the protein can be formed by dissolving the protein in the phospholipid solution described above. Alternatively, the lipid bilayer membrane is formed as described above, and then the protein solution is added. It can also be formed by applying to the lipid bilayer membrane. When the phospholipid solution is a liposome suspension containing liposomes, it can be formed by using a suspension of liposomes retaining proteins. The concentration of the protein in these solutions is not particularly limited and can be appropriately selected, but is usually about 1 nM to 1 mM, preferably about 0.1 μM to 100 μM.
本発明の脂質二重膜は、上記各種測定に供するために、一方の表面が第１のマイクロ流路に接し、他方の表面が第２のマイクロ流路に接していることが好ましい。本発明は、上記した本発明の脂質二重膜を組み込むことができるマイクロ流路デバイス及び該脂質二重膜を組み込んだマイクロ流路デバイスをも提供する。 In order to use the lipid bilayer membrane of the present invention for the above various measurements, it is preferable that one surface is in contact with the first microchannel and the other surface is in contact with the second microchannel. The present invention also provides a microchannel device that can incorporate the above-described lipid bilayer membrane of the present invention and a microchannel device incorporating the lipid bilayer membrane.
本発明の脂質二重膜を形成するためのマイクロ流路デバイスは、第１のマイクロ流路を具備する第１のマイクロ流路チップと、前記第１のマイクロ流路と少なくとも一部が接する第２のマイクロ流路を具備する第２のマイクロ流路チップとを具備し、前記第１のマイクロ流路と前記第２のマイクロ流路の境界に、脂質二重膜を形成するための孔径が１μｍ未満の透孔が形成されている。好ましい態様では、前記透孔が、自己支持性フィルムに設けられた透孔であり、該自己支持性フィルムにより前記第１のマイクロ流路と前記第２のマイクロ流路の接続部分が隔てられている。そして、このマイクロ流路デバイスの前記透孔に、上記した方法により脂質二重膜を形成する。そうすると、上記したように、脂質二重膜の一方の表面が第１のマイクロ流路に接し、他方の表面が第２のマイクロ流路に接するようにすることができる。脂質二重膜の両表面が、互いに分離された異なるマイクロ流路に接するようにした場合には、各マイクロ流路に任意の液を流すことにより、脂質二重膜の両側をそれぞれ任意の液と接触させることができる。生体内の細胞では、細胞の内側と外側で環境が異なっている場合が多く、脂質二重膜の両側をそれぞれ任意の液と接触させることを可能にすることにより、より的確に生体内の環境を模することが可能となり、タンパク質を生体内により近い環境においてその挙動を調べることが可能となり好ましい。 A microchannel device for forming a lipid bilayer membrane according to the present invention includes a first microchannel chip having a first microchannel, and a first microchannel chip that is at least partially in contact with the first microchannel. And a second microchannel chip having two microchannels, and a pore diameter for forming a lipid bilayer membrane at a boundary between the first microchannel and the second microchannel. A through-hole of less than 1 μm is formed. In a preferred embodiment, the through hole is a through hole provided in a self-supporting film, and the connection portion between the first microchannel and the second microchannel is separated by the self-supporting film. Yes. Then, a lipid bilayer membrane is formed in the through hole of the microchannel device by the method described above. Then, as described above, one surface of the lipid bilayer membrane can be in contact with the first microchannel, and the other surface can be in contact with the second microchannel. When both surfaces of the lipid bilayer membrane are in contact with different microchannels separated from each other, an arbitrary liquid is allowed to flow on each side of the lipid bilayer membrane. Can be contacted with. For cells in a living body, the environment is often different between the inside and outside of the cell, and it is possible to bring both sides of the lipid bilayer membrane into contact with an arbitrary liquid, so that the environment in the living body can be more accurately determined. It is possible to simulate the behavior of the protein in an environment closer to the living body.
上記本発明の脂質二重膜を組み込んだマイクロ流路デバイスの好ましい一具体例を、図１に基づき説明する。図１Ａは、本発明の好ましい一具体例になるマイクロ流路デバイスの模式斜視図、図１Ｂは、該マイクロ流路デバイス中の１枚の本発明の自己支持性フィルムの透孔部分付近を拡大した模式拡大斜視図である。また、図２は、該マイクロ流路デバイス中の１枚の本発明の自己支持性フィルムの透孔部分に、タンパク質を保持した脂質二重膜が形成されている様子を示す模式拡大断面図である。 A preferred specific example of the microchannel device incorporating the lipid bilayer membrane of the present invention will be described with reference to FIG. FIG. 1A is a schematic perspective view of a microchannel device according to a preferred embodiment of the present invention, and FIG. 1B is an enlarged view of the vicinity of a through-hole portion of one self-supporting film of the present invention in the microchannel device. FIG. FIG. 2 is a schematic enlarged cross-sectional view showing a state in which a lipid bilayer holding a protein is formed in the through-hole portion of one self-supporting film of the present invention in the microchannel device. is there.
図１Ａに示すように、本具体例になるマイクロ流路デバイス１０は、第１のマイクロ流路チップである上部マイクロ流路チップ１２と、第２のマイクロ流路チップである下部マイクロ流路チップ１４を具備する。上部マイクロ流路チップ１２には、第１のマイクロ流路である３本の上部マイクロ流路１６が形成されている。下部マイクロ流路チップ１４には、第２のマイクロ流路である下部マイクロ流路１８が形成されている。各マイクロ流路は、例えば、幅及び深さが共に400μｍ程度の細い溝である。下部マイクロ流路チップ上には、１枚のプラスチックシートから成るセパレーター２０（図２参照）が載置され、これにより、上部マイクロ流路１６と下部マイクロ流路１８が隔てられる。上部マイクロ流路チップ１２と下部マイクロ流路チップ１４を重ね合わせると、上部マイクロ流路１６と下部マイクロ流路１８とがセパレーター２０を介して接するようになっている。すなわち、これらを重ね合わせると、セパレーター２０が上部マイクロ流路１６の底面となり、同時に下部マイクロ流路１８の上面となる。セパレーター２０には３つの開口部が設けられ、各開口部にはそれぞれ上記した本発明の自己支持性フィルム２２が載置される。ここで、自己支持性フィルム２２の透孔は、セパレーター２０の開口部に位置し、従って、自己支持性フィルム２２の透孔は、上部マイクロ流路１６に開口し、同時にセパレーター２０の開口部を介して下部マイクロ流路１８にも開口している。すなわち、セパレーター２０の開口部は、上部マイクロ流路１６と下部マイクロ流路１８とが接続される接続部分を構成しており、ここでは両流路は自己支持性フィルム２２により隔てられており、上記のとおり、自己支持性フィルム２２の透孔２２は両流路に開口している。所望により、上部マイクロ流路１６と下部マイクロ流路１８にはそれぞれAg/AgCl等から成る電極を配置し、これらの間に直流電圧を負荷して各流路に電位を付与することもできる（図２参照）し、膜を介して流れる膜電流を測定することもできる。 As shown in FIG. 1A, the microchannel device 10 according to this example includes an upper microchannel chip 12 that is a first microchannel chip and a lower microchannel chip that is a second microchannel chip. 14. In the upper microchannel chip 12, three upper microchannels 16 that are first microchannels are formed. The lower microchannel chip 14 is formed with a lower microchannel 18 which is a second microchannel. Each microchannel is, for example, a thin groove having a width and a depth of about 400 μm. A separator 20 (see FIG. 2) made of a single plastic sheet is placed on the lower microchannel chip, whereby the upper microchannel 16 and the lower microchannel 18 are separated. When the upper micro-channel chip 12 and the lower micro-channel chip 14 are overlapped, the upper micro-channel 16 and the lower micro-channel 18 are in contact with each other through the separator 20. That is, when these are overlapped, the separator 20 becomes the bottom surface of the upper microchannel 16 and at the same time, the upper surface of the lower microchannel 18. The separator 20 is provided with three openings, and the above-described self-supporting film 22 of the present invention is placed in each opening. Here, the through hole of the self-supporting film 22 is located at the opening of the separator 20, and thus the through hole of the self-supporting film 22 opens into the upper microchannel 16, and at the same time the opening of the separator 20 is opened. The lower microchannel 18 is also opened. That is, the opening of the separator 20 constitutes a connecting portion to which the upper micro-channel 16 and the lower micro-channel 18 are connected. Here, both channels are separated by the self-supporting film 22, As described above, the through holes 22 of the self-supporting film 22 are open to both flow paths. If desired, electrodes made of Ag / AgCl or the like may be disposed in the upper microchannel 16 and the lower microchannel 18, respectively, and a DC voltage may be applied between them to apply a potential to each channel ( The membrane current flowing through the membrane can also be measured.
上部マイクロ流路チップ１２と下部マイクロ流路チップ１４を重ね合わせた状態で、上部マイクロ流路１６に上記したリン脂質溶液を流すと、上記のとおり、自己支持性フィルム２２の透孔に脂質二重膜２４が形成される（図２参照）。次に、上部マイクロ流路１６に上記したタンパク質溶液を流すと、タンパク質分子２６が脂質二重膜２４中に入りこみ、脂質二重膜２４に保持される（図２参照）。 When the above-described phospholipid solution is allowed to flow through the upper micro-channel 16 in a state where the upper micro-channel chip 12 and the lower micro-channel chip 14 are superposed, as described above, lipid 2 is introduced into the through-holes of the self-supporting film 22. A heavy film 24 is formed (see FIG. 2). Next, when the protein solution described above is allowed to flow through the upper microchannel 16, the protein molecules 26 enter the lipid bilayer membrane 24 and are retained on the lipid bilayer membrane 24 (see FIG. 2).
以上により、タンパク質分子２６を保持した脂質二重膜２４の両面が、それぞれ上部マイクロ流路１６及び下部マイクロ流路１８と接する状態となる。この状態で、上部マイクロ流路１６及び下部マイクロ流路１８にそれぞれ、実験の目的に応じた所望の液を流すことにより、脂質二重膜２４の両側をそれぞれ実験に適した所望の環境に置くことができる。 As described above, both surfaces of the lipid bilayer membrane 24 holding the protein molecules 26 are in contact with the upper microchannel 16 and the lower microchannel 18, respectively. In this state, by flowing a desired liquid according to the purpose of the experiment through the upper microchannel 16 and the lower microchannel 18, respectively, both sides of the lipid bilayer membrane 24 are respectively placed in a desired environment suitable for the experiment. be able to.
このようなマイクロ流路デバイスを用いて、例えば創薬のためのスクリーニングを行う場合には、例えば次のように行うことができる。すなわち、各チャンネルにターゲットとなる膜タンパク質を再構成し、上下の流路にそれぞれ所望の薬剤を任意の量導入することにより、創薬スクリーニングを行うことができる。 When screening for drug discovery, for example, using such a microchannel device, for example, it can be performed as follows. That is, drug discovery screening can be performed by reconstituting a target membrane protein in each channel and introducing an arbitrary amount of a desired drug into the upper and lower channels.
以下、本発明を実施例に基づきより具体的に説明する。もっとも、本発明は下記実施例に限定されるものではない。 Hereinafter, the present invention will be described more specifically based on examples. However, the present invention is not limited to the following examples.
実施例１
１． 透孔を有する自己支持性フィルムの作製
図３に模式的に示すフォトリソグラフィー法により、孔径800nmの透孔を有する、パラキシリレン系ポリマーから成る自己支持性フィルムを作製した。
Example 1
1. Production of self-supporting film having through-holes A self-supporting film made of paraxylylene-based polymer having through-holes having a pore diameter of 800 nm was produced by a photolithography method schematically shown in FIG.
まず、シリコンウェハを真空チャンバー内に置き、パラキシリレン系モノマー（商品名パリレンC、上記一般式において、XがH2、R1がCl、R2がH）を蒸着してシリコンウェハ上に厚さ５μｍのパラキシリレン系ポリマーから成るフィルムを形成した。蒸着温度は24℃、蒸着時間は50分であった。次に、形成したパラキシリレン系ポリマーフィルム上にアルミニウムを真空蒸着し（蒸着温度：室温、蒸着時間：1分）、厚さ５μｍのアルミニウム層を形成した（図３の１）。 First, a silicon wafer is placed in a vacuum chamber, and a paraxylylene monomer (trade name Parylene C, in the above general formula, X is H 2 , R 1 is Cl, and R 2 is H) is deposited on the silicon wafer. A film composed of 5 μm paraxylylene-based polymer was formed. The deposition temperature was 24 ° C. and the deposition time was 50 minutes. Next, aluminum was vacuum-deposited on the formed paraxylylene polymer film (deposition temperature: room temperature, deposition time: 1 minute) to form an aluminum layer having a thickness of 5 μm (1 in FIG. 3).
アルミニウム層上にフォトレジストをスピンコートし、CADにより設計したマスクを用いUV光を露光することにフォトレジストを感光、その後現像し、直径５μｍの孔をあけた（図３の２）。UV露光の条件は、UVの照射エネルギー量が24ｍW/cm2 (@405nm)、露光時間は、6.5秒であった。その後混酸処理することによりアルミニウム層にも直径５μｍの孔をあけた。 A photoresist was spin-coated on the aluminum layer, and the photoresist was exposed to UV light using a CAD-designed mask and then developed, and a hole having a diameter of 5 μm was formed (2 in FIG. 3). The UV exposure conditions were a UV irradiation energy amount of 24 mW / cm 2 (@ 405 nm) and an exposure time of 6.5 seconds. Thereafter, a hole having a diameter of 5 μm was formed in the aluminum layer by performing a mixed acid treatment.
次に、パリレン層を酸素プラズマによりエッチングした（図３の３）。具体的な条件は次の通りであった。酸素量10mL/min、25W。 Next, the parylene layer was etched by oxygen plasma (3 in FIG. 3). Specific conditions were as follows. Oxygen amount 10mL / min, 25W.
次に、アルミニウム層を混酸処理（60℃、1分）により溶解除去した（図３の４）。 Next, the aluminum layer was dissolved and removed by mixed acid treatment (60 ° C., 1 minute) (4 in FIG. 3).
自己支持性フィルムの一端をピンセットで把持してシリコンウェハから剥離し、孔径５μｍの透孔を有する自己支持性フィルムを得た（図３の５）。このように、フィルムは一端を把持して剥離して移動させることができたので、自己支持性であった。得られた自己支持性フィルムの寸法は、3mm x 3mmのものを用いた。 One end of the self-supporting film was grasped with tweezers and peeled from the silicon wafer to obtain a self-supporting film having through-holes with a pore diameter of 5 μm (5 in FIG. 3). Thus, the film was self-supporting because it was able to grip and peel off one end and move it. The obtained self-supporting film had a size of 3 mm × 3 mm.
剥離した自己支持性フィルムを真空チャンバーに入れ、上記と同じパラキシリレン系モノマーを蒸着した（室温、40分）。これにより、自己支持性フィルムの透孔内壁を含む全面にパラキシリレン系ポリマーがさらに被着され、透孔の孔径が縮小された（図３の６）。 The peeled self-supporting film was placed in a vacuum chamber, and the same paraxylylene monomer as above was deposited (room temperature, 40 minutes). As a result, the paraxylylene polymer was further adhered to the entire surface including the inner wall of the through hole of the self-supporting film, and the hole diameter of the through hole was reduced (6 in FIG. 3).
縮小後の透孔及びその近傍の走査電子顕微鏡写真を図４に示す。図４に示すように、縮小後の孔径は約800nmであった。なお、蒸着時間を長くすれば、孔径をさらに小さくすることができる。 FIG. 4 shows a scanning electron micrograph of the through-hole after reduction and the vicinity thereof. As shown in FIG. 4, the pore diameter after reduction was about 800 nm. In addition, if the vapor deposition time is lengthened, the hole diameter can be further reduced.
３． マイクロ流路デバイスの作製
図１及び図２に示す、上記したマイクロ流路デバイスを作成した。上部マイクロ流路チップ１２、下部マイクロ流路チップ１４及びセパレーター２０はいずれもポリメチルメタクリレート(PMMA)で形成した。上部マイクロ流路チップ１２及び下部マイクロ流路チップ１４の寸法は、40mm x 25 mm x 3 mmであった。また、全てのマイクロ流路の幅及び深さは400μｍであった。セパレーター２０の３つの開口部の寸法は、直径1mmであり、この開口部に各自己支持性フィルム２２の透孔が重なるように自己支持性フィルム２２を載置した（図２参照）。さらに、上部マイクロ流路１６及び下部マイクロ流路１８中にAg/AgCl電極をそれぞれ配置し、これらの間に電圧をかけるパッチクランプアンプと接続できるようにした。上部マイクロ流路チップ１２、下部マイクロ流路チップ１４、セパレーター２０を重ね合わせた状態で125℃で３０分間熱処理し、これらを接着した。
3. Production of microchannel device The above-described microchannel device shown in FIGS. 1 and 2 was produced. The upper microchannel chip 12, the lower microchannel chip 14 and the separator 20 were all formed of polymethyl methacrylate (PMMA). The dimensions of the upper microchannel chip 12 and the lower microchannel chip 14 were 40 mm × 25 mm × 3 mm. Moreover, the width and depth of all the microchannels were 400 μm. The dimensions of the three openings of the separator 20 were 1 mm in diameter, and the self-supporting films 22 were placed so that the through holes of the respective self-supporting films 22 overlapped with the openings (see FIG. 2). Furthermore, an Ag / AgCl electrode is disposed in each of the upper microchannel 16 and the lower microchannel 18 so that it can be connected to a patch clamp amplifier that applies a voltage therebetween. The upper micro-channel chip 12, the lower micro-channel chip 14 and the separator 20 were superposed and heat-treated at 125 ° C. for 30 minutes, and these were bonded.
作製したマイクロ流路デバイスの上部マイクロ流路及び下部マイクロ流路に1.0M KCl溶液を流しながら、両流路に電圧をかけ、両流路間の電気抵抗（すなわち、透孔の電気抵抗）を測定した。その結果、図５に示すような直線関係が得られ、電気抵抗は0.32MΩであった。この値は、
Rp = (4ρL)/(πd2)
（式中、Rpは透孔の電気抵抗、ρは電解質の比抵抗、Lは透孔の長さ、dは透孔の孔径）
で表わされる理論値（非特許文献５）と概ね一致していた。
While flowing 1.0M KCl solution through the upper microchannel and lower microchannel of the fabricated microchannel device, voltage was applied to both channels, and the electrical resistance between both channels (ie, the electrical resistance of the through holes) It was measured. As a result, a linear relationship as shown in FIG. 5 was obtained, and the electric resistance was 0.32 MΩ. This value is
R p = (4ρL) / (πd 2 )
(Where R p is the electrical resistance of the through-hole, ρ is the specific resistance of the electrolyte, L is the length of the through-hole, and d is the diameter of the through-hole)
It almost coincided with the theoretical value represented by (Non-Patent Document 5).
４． 脂質二重膜の作製
作製したマイクロ流路デバイスの上部マイクロ流路チップ１２と下部マイクロ流路チップ１４を重ね合わせた状態で、上部マイクロ流路１６に、ポンプ（マイクロシリンジとチューブから成る手動式のもの、図示せず）により脂質溶液を流した。脂質溶液は、10mgのDPhPC(米国Avanti Polar Lipids社製）を1mLのn-デカンに溶解したものである。これにより、自己支持性フィルムの透孔に脂質二重膜が形成された。
4). Manufacture of lipid bilayer membrane In the state where the upper microchannel chip 12 and the lower microchannel chip 14 of the prepared microchannel device are overlapped, a pump (manual type consisting of a microsyringe and a tube is placed in the upper microchannel 16. The lipid solution was allowed to flow through (not shown). The lipid solution is 10 mg of DPhPC (manufactured by Avanti Polar Lipids, USA) dissolved in 1 mL of n-decane. Thereby, the lipid bilayer membrane was formed in the through-hole of the self-supporting film.
形成された脂質二重膜は、両マイクロ流路間に10mVの電圧をかけた状態で、２４℃で２４時間以上安定に維持された。従来から用いられている、孔径約50μｍの透孔に形成された脂質二重膜は、同条件で５時間程度しか維持されない。よって、本発明により、脂質二重膜の安定性が大幅に向上したことが確認された。 The formed lipid bilayer membrane was stably maintained at 24 ° C. for 24 hours or more with a voltage of 10 mV applied between both microchannels. Conventionally used lipid bilayer membranes having a pore diameter of about 50 μm are maintained only for about 5 hours under the same conditions. Therefore, it was confirmed that the stability of the lipid bilayer membrane was greatly improved by the present invention.
次に上部マイクロ流路１６に、α−ヘモリシン(α-HL)溶液を流した。α-HL（モノマー）は、Staphylococcus aureus由来のもの（米国Sigma社製）を用い、1.0M KCl、10mM PBS、pH7.4中に0.3μMの濃度で溶解してα-HL溶液とした。α-HLは、ヘプタマーを形成し、脂質二重膜中に保持されてイオンチャネルを形成することが知られており、そのチャネルコンダクタンスは1.0M KCl中で約1nSである（非特許文献６）。この間、上部マイクロ流路と下部マイクロ流路にそれぞれ配置した電極間の電流、すなわち、脂質二重膜を介して流れる電流（膜電流）を測定した。膜電流は、上記両電極に接続されたパッチクランプ増幅器（patch-clamp amplifier、CEZ-2400、日本光電社製）を用いて測定した。上部マイクロ流路１６内に配置したAg/AgCl電極は、記録電極であり、下部マイクロ流路１８内に配置したAg/AgCl電極は、接地電極であった。電流は、デジタルデータ獲得システム(Digidata 1322A及びpCLAMP ver.9, 米国Molecular Devices社製）を用いて記録した。 Next, an α-hemolysin (α-HL) solution was passed through the upper microchannel 16. α-HL (monomer) was derived from Staphylococcus aureus (manufactured by Sigma, USA) and dissolved in 1.0 M KCl, 10 mM PBS, pH 7.4 at a concentration of 0.3 μM to obtain an α-HL solution. α-HL is known to form a heptamer and be retained in a lipid bilayer to form an ion channel, and its channel conductance is about 1 nS in 1.0 M KCl (Non-patent Document 6). . During this time, the current between the electrodes arranged in the upper microchannel and the lower microchannel, that is, the current flowing through the lipid bilayer (membrane current) was measured. The membrane current was measured using a patch-clamp amplifier (CEZ-2400, manufactured by Nihon Kohden Co., Ltd.) connected to both electrodes. The Ag / AgCl electrode disposed in the upper microchannel 16 was a recording electrode, and the Ag / AgCl electrode disposed in the lower microchannel 18 was a ground electrode. The current was recorded using a digital data acquisition system (Digidata 1322A and pCLAMP ver.9, manufactured by Molecular Devices, USA).
結果を図６に示す。図６に示されるように、イオンチャネルコンダクタンスは、約1nSであり、非特許文献６記載の値と一致しており、α-HLが再構成された状態で脂質二重膜に適切に組み込まれてイオンチャネルを形成したこと、及び脂質二重膜に破れがなく、透孔が脂質二重膜で隙間なく覆われていたことが確認された。α-HLの組込み実験は複数回行ったが、いずれの場合も同様にイオンチャネルの形成が確認された。このことから、本発明の脂質二重膜にタンパク質を再現性良く保持できることが確認された。 The results are shown in FIG. As shown in FIG. 6, the ion channel conductance is about 1 nS, which is consistent with the value described in Non-Patent Document 6, and is appropriately incorporated into the lipid bilayer membrane in a state where α-HL is reconstituted. Thus, it was confirmed that the ion channel was formed and that the lipid bilayer membrane was not torn, and the pores were covered with the lipid bilayer membrane without any gaps. The α-HL integration experiment was performed several times, and in all cases, formation of ion channels was confirmed. From this, it was confirmed that the protein can be retained with good reproducibility in the lipid bilayer membrane of the present invention.
Claims (18)
Priority Applications (1)
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| JP2014030382A (en) * | 2012-08-03 | 2014-02-20 | Univ Of Tokyo | Microfluidic device and method for forming lipid bilayer membrane |
| JP2015077559A (en) * | 2013-10-17 | 2015-04-23 | 公益財団法人神奈川科学技術アカデミー | Lipid double membrane forming instrument |
| WO2019142784A1 (en) | 2018-01-19 | 2019-07-25 | 地方独立行政法人神奈川県立産業技術総合研究所 | Partition wall for formation of lipid bilayer membrane, and method for producing same |
| US10576456B2 (en) * | 2014-06-30 | 2020-03-03 | Arizona Board Of Regents On Behalf Of The University Of Arizona | Systems and methods of preparing stabilized lipid assemblies |
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| WO2019142784A1 (en) | 2018-01-19 | 2019-07-25 | 地方独立行政法人神奈川県立産業技術総合研究所 | Partition wall for formation of lipid bilayer membrane, and method for producing same |
| US11607871B2 (en) | 2018-01-19 | 2023-03-21 | Kanagawa Institute Of Industrial Science And Technology | Partition wall for formation of lipid bilayer membrane, and method for producing same |
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