JP2014100672A - Formation method of lipid double membrane and instrument for the same - Google Patents
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Abstract
Description
本発明は、脂質二重膜の形成方法及びそのための器具に関する。 The present invention relates to a method for forming a lipid bilayer membrane and a device therefor.
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。脂質二重膜の形成方法として、浅い円筒状の凹部（ウェル）を２個隣接して配置し、かつ、２個のウェルが互いに接する境界部分を空隙（一辺数mm程度）としたダブルウェルチャンバーを用いる方法が知られている（非特許文献１〜３）。この方法は、このダブルウェルチャンバーの各ウェルに脂質溶液を充填し、次いで、各ウェルに水系緩衝液を添加して脂質溶液中に緩衝液の液滴を形成させると、ウェルの境界部分で脂質溶液と緩衝液との界面が形成され、この部分に脂質二重膜が形成されることを利用するものである。 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. As a method of forming a lipid bilayer membrane, a double well chamber in which two shallow cylindrical recesses (wells) are arranged adjacent to each other, and a boundary portion where the two wells are in contact with each other is a void (about several mm on a side) A method of using is known (Non-Patent Documents 1 to 3). In this method, each well of the double well chamber is filled with a lipid solution, and then an aqueous buffer solution is added to each well to form droplets of the buffer solution in the lipid solution. This utilizes the fact that an interface between a solution and a buffer solution is formed, and a lipid bilayer is formed in this portion.
本願発明者らは、さらに、従来の脂質二重膜の形成方法に比べて、脂質二重膜をより高い再現性で形成することができ、形成された脂質二重膜をより安定的に維持する方法として、孔径が１μｍ未満の透孔を有する自己支持性フィルムの透孔に脂質二重膜を形成する方法（特許文献１）や、孔径が５００ nm〜５００μmの１個又は複数の貫通孔を有する隔壁を介して隔てられた２つのウェルのそれぞれに脂質二重膜形成性脂質溶液を添加し、各ウェルに水系緩衝液を添加して脂質溶液中に緩衝液の液滴を形成させ、この状態で放置して、貫通孔の部分に脂質二重膜を形成する方法（特許文献２）を開発している。 Furthermore, the inventors of the present application can form a lipid bilayer membrane with higher reproducibility than the conventional method of forming a lipid bilayer membrane, and maintain the formed lipid bilayer membrane more stably. As a method of forming a lipid bilayer in the perforation of a self-supporting film having a perforation having a pore diameter of less than 1 μm (Patent Document 1), or one or a plurality of through-holes having a pore diameter of 500 nm to 500 μm A lipid bilayer-forming lipid solution is added to each of two wells separated by a septum having, and an aqueous buffer is added to each well to form buffer droplets in the lipid solution, A method of forming a lipid bilayer membrane in the through-hole portion by leaving it in this state (Patent Document 2) has been developed.
公知の方法では、脂質二重膜が壊れた場合には、そのシステムで再生することは困難であるという問題がある。また、脂質二重膜に接する液滴を交換することが困難である。 The known method has a problem that when the lipid bilayer is broken, it is difficult to regenerate the system. In addition, it is difficult to exchange droplets in contact with the lipid bilayer membrane.
従って、本発明の目的は、脂質二重膜が壊れた場合に、容易に脂質二重膜を再生することができる、脂質二重膜の形成方法及びそのための器具を提供することである。また、本発明の目的は、脂質二重膜に接する液滴を容易に交換することができる、脂質二重膜の形成方法及びそのための器具を提供することである。 Accordingly, an object of the present invention is to provide a method for forming a lipid bilayer membrane and a device therefor that can easily regenerate the lipid bilayer membrane when the lipid bilayer membrane is broken. Moreover, the objective of this invention is providing the formation method of a lipid bilayer membrane, and the instrument for it which can replace | exchange the droplet which touches a lipid bilayer membrane easily.
本願発明者らは、鋭意研究の結果、脂質一重膜で囲包された液滴を外力により分割し、生じた液滴に一重膜を再生させ、脂質一重膜で囲包された複数の分割液滴を生成させ、得られた分割液滴の少なくとも１個を、同様に生成させた他の分割液滴と接触させ、その接触面に脂質二重膜を形成することにより、脂質二重膜に接する液滴を容易に交換することができ、また、脂質二重膜が壊れた場合には、容易に脂質二重膜を再生することができることを見出し、本発明を完成した。 As a result of diligent research, the inventors of the present application divide a droplet surrounded by a lipid monolayer by external force, regenerate the single membrane into the generated droplet, and a plurality of divided liquids surrounded by a lipid monolayer By forming at least one of the obtained divided droplets into contact with other similarly generated divided droplets and forming a lipid bilayer on the contact surface, a lipid bilayer is formed. It was found that the droplets in contact with each other can be easily exchanged, and when the lipid bilayer membrane is broken, the lipid bilayer membrane can be easily regenerated, and the present invention has been completed.
すなわち、本発明は、脂質一重膜で囲包された液滴を、脂質一重膜で囲包された他の液滴と機械的な外力で接触させ、その接触面に脂質二重膜を形成することを含む、脂質二重膜の形成方法を提供する。特に好ましい実施形態では、本発明は、脂質一重膜で囲包された液滴を外力により複数の液滴に分割し、生じた液滴に一重膜を再生させ、脂質一重膜で囲包された複数の分割液滴を生成させる工程と、得られた分割液滴の少なくとも１個を、同様に生成させた他の分割液滴と接触させ、その接触面に脂質二重膜を形成する工程とを含む、上記本発明の脂質二重膜の形成方法を提供する。 That is, according to the present invention, a droplet surrounded by a lipid monolayer is brought into contact with other droplets surrounded by a lipid monolayer by a mechanical external force to form a lipid bilayer on the contact surface. A method for forming a lipid bilayer membrane. In a particularly preferred embodiment, the present invention divides a droplet surrounded by a lipid monolayer into a plurality of droplets by an external force, and regenerates the monolayer by the generated droplet, and is surrounded by a lipid monolayer. A step of generating a plurality of divided droplets, a step of contacting at least one of the obtained divided droplets with another similarly generated divided droplet, and forming a lipid bilayer on the contact surface; A method for forming the lipid bilayer membrane of the present invention.
また、本発明は、周縁部に複数の凹部を有する回転可能な円板であって、各凹部は、水平方向の外側に向かって開口している円板と、前記円板に外接する固定板であって、その内周縁部に、前記複数の凹部にそれぞれ接する位置に複数の凹部を有し、各凹部は、水平方向の内側に向かって開口している固定板とを具備し、前記円板内の各凹部の開口部と、前記固定板内の各凹部の開口部を位置合わせすることによりそれぞれ１個の凹部が形成される、脂質二重膜形成器具を提供する。 Further, the present invention is a rotatable disc having a plurality of recesses in the peripheral portion, each recess having a disc opening toward the outside in the horizontal direction, and a fixed plate circumscribing the disc The inner circumferential edge has a plurality of recesses at positions in contact with the plurality of recesses, and each recess has a fixing plate that opens inward in the horizontal direction, and the circle Provided is a lipid bilayer membrane forming device in which one recess is formed by aligning the opening of each recess in the plate and the opening of each recess in the fixed plate.
さらに本発明は、凹部を有する基板と、該凹部の一部分を被覆する液滴スプリッターとを具備し、該液滴スプリッターは、鉛直方向の壁により囲包されて成り、互いに分断可能な複数のセパレーターを含み、各セパレーターは、鉛直方向に延びる、対向する一対の壁を具備し、該セパレーターのうちの少なくとも一部は、前記凹部の、液滴スプリッターにより被覆されていない領域上まで水平方向にスライド可能である、脂質二重膜形成器具を提供する。 The present invention further includes a substrate having a recess and a droplet splitter that covers a part of the recess, and the droplet splitter is surrounded by a vertical wall and is separated from each other. Each separator has a pair of opposing walls extending in the vertical direction, at least a portion of which is horizontally slid onto an area of the recess not covered by the droplet splitter Provided is a lipid bilayer forming device that is possible.
本発明により、脂質二重膜が壊れた場合には、容易に脂質二重膜を再生することができる、脂質二重膜の形成方法及びそのための器具が初めて提供された。また、組成の異なる別の液滴を分割した分割液滴同士を接触させる場合には、本発明の方法により脂質二重膜に接する液滴を容易に交換することができる。 According to the present invention, for the first time, a method for forming a lipid bilayer membrane and a device therefor are provided, which can easily regenerate a lipid bilayer membrane when the lipid bilayer membrane is broken. Moreover, when contacting the divided droplets obtained by dividing other droplets having different compositions, the droplets in contact with the lipid bilayer membrane can be easily exchanged by the method of the present invention.
上記の通り、本発明の好ましい実施形態の方法では、先ず、脂質一重膜で囲包された液滴を形成する。脂質一重膜で囲包された液滴は、周知の方法に従い、容易に形成することができる。例えば、脂質二重膜形成性脂質溶液に水又は水溶液を添加することにより容易に脂質一重膜を形成することができる。ここで、脂質二重膜形成性脂質としては、リポソームの作製に用いられている周知のリン脂質でよく、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。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〜30g/L程度、好ましくは10g/L〜20g/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。なお、脂質一重膜により囲包される液滴は、公知の方法又は本発明の方法により形成された脂質二重膜が壊れることにより生じたものであってもよい。この場合には、壊れた脂質二重膜を、本発明の方法により容易に再生することができる。 As described above, in the method according to a preferred embodiment of the present invention, first, a droplet surrounded by a lipid monolayer is formed. A droplet surrounded by a lipid monolayer can be easily formed according to a known method. For example, a lipid monolayer can be easily formed by adding water or an aqueous solution to a lipid bilayer-forming lipid solution. Here, the lipid bilayer-forming lipid may be a well-known phospholipid used for the preparation of liposomes, and in order to simulate the reaction in the biological membrane, the same or similar to the biological membrane is preferable, Conventionally widely used phospholipids in this field, such as diphytanoyl phosphatidylcholine (diphytanoyl phosphatidylcholine, DPhPC), dipalmytoyl phosphatidylcholine, palmitoyl oleoylphosphatidylcholine (1- Preferred examples include Palmitoyl 2-Oleoyl phosphatidylcholine (POPC), dioleoylphosphatidylcholine (DOPC), and the like. Since many of these are commercially available, commercially available products can be preferably used. 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 30 g / L, preferably 10 g / L. It is about L-20g / 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. In addition, the droplet surrounded by the lipid single membrane may be produced by a known method or a lipid bilayer formed by the method of the present invention being broken. In this case, the broken lipid bilayer membrane can be easily regenerated by the method of the present invention.
次に、脂質一重膜で囲包された液滴を外力により複数の液滴に分割する。好ましい分割の方法の具体例は後述する。３個以上の液滴に分割することもできるが、２個の液滴に分割することが簡便であり、後述するスピン型の本発明の器具を用いて容易に行うことができるので好ましい。もっとも、後述するスライド型の本発明の器具を用いれば、液滴を３個以上に容易に分割することも可能である。分割した液滴は、分割により引き裂かれた部分が脂質一重膜を欠いているが、短時間放置することにより脂質一重膜が再生され、液滴の全体が脂質一重膜により囲包される。 Next, the droplet surrounded by the lipid single membrane is divided into a plurality of droplets by an external force. Specific examples of preferable dividing methods will be described later. Although it can be divided into three or more droplets, it is preferable to divide into two droplets because it is simple and can be easily performed using the spin-type instrument of the present invention described later. However, if a slide-type instrument of the present invention described later is used, it is possible to easily divide the droplet into three or more. The part of the divided droplet that has been torn by the division lacks the lipid monolayer, but when left for a short time, the lipid monolayer is regenerated and the entire droplet is surrounded by the lipid monolayer.
次に、分割により生じた分割液滴を、同様に生成した他の分割液滴と接触させる。この場合、互いに接触させる２個の分割液滴は、それぞれ異なる液滴を分割して生じたものであってもよいし、同一の液滴を分割して生じたものであってもよい。前者の場合、接触させる分割液滴が、それぞれ異なる組成の液を内包するものであってもよく、この場合には、脂質二重膜に接する液滴を交換することができる。また、後者の場合で、もともと脂質二重膜が形成されていたが、その脂質二重膜が壊れることにより脂質一重膜で囲包された液滴となった液滴に適用すれば、壊れた脂質二重膜を容易に再生することができる。 Next, the divided droplets generated by the division are brought into contact with other divided droplets similarly generated. In this case, the two divided droplets brought into contact with each other may be generated by dividing different droplets, or may be generated by dividing the same droplet. In the former case, the divided droplets to be contacted may contain liquids having different compositions, and in this case, the droplets in contact with the lipid bilayer membrane can be exchanged. Also, in the latter case, a lipid bilayer was originally formed, but if the lipid bilayer was broken, it was broken when applied to a droplet surrounded by a lipid monolayer. The lipid bilayer membrane can be easily regenerated.
上記本発明の方法により脂質二重膜を形成した後、形成された脂質二重膜を介して接触する液滴を、脂質二重膜の部分で外力により分割し、生じた液滴に一重膜を再生させ、脂質一重膜で囲包された複数の分割液滴を生成させ、得られた分割液滴の少なくとも１個を、同様に生成させた他の分割液滴と接触させ、その接触面に脂質二重膜を形成することもできる。すなわち、本発明の方法により脂質二重膜を形成した後、これを意図的に分断し、生じた液滴を上記本発明の方法に付すこともできる。さらにこれを繰り返すことにより、脂質二重膜の形成と破壊を繰り返すことができ、新たに脂質二重膜を形成する際に、異なる組成の液を内包するもの同士を接触させることにより、脂質二重膜に接する液滴を次々に交換していくことができる。 After forming the lipid bilayer membrane by the method of the present invention, a droplet contacting through the formed lipid bilayer membrane is divided by an external force at the lipid bilayer portion, and a single membrane is formed on the resulting droplet. To generate a plurality of divided droplets surrounded by a lipid monolayer, and at least one of the obtained divided droplets is brought into contact with another similarly generated divided droplet, and the contact surface It is also possible to form a lipid bilayer. That is, after forming a lipid bilayer membrane by the method of the present invention, this can be intentionally divided, and the resulting droplets can be subjected to the method of the present invention. Furthermore, by repeating this, formation and destruction of the lipid bilayer membrane can be repeated, and when a lipid bilayer membrane is newly formed, the lipid bilayer membrane is brought into contact with each other by bringing in contact with those containing liquids of different compositions. The droplets in contact with the heavy film can be exchanged one after another.
本発明の方法により形成された脂質二重膜は、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、α−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、各種イオンチャンネル、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。タンパク質が水溶性の場合、上記水溶液としてタンパク質の水溶液を用いることが好ましく、水系緩衝液中にタンパク質を含む水溶液を用いることがさらに好ましい。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〜10μM程度である。なお、タンパク質は、接触させる２つの液滴のいずれか一方に含まれていればよいが、両方の液滴中に含まれていてもよい。また、脂質二重膜に保持するタンパク質が脂溶性の場合には、膜タンパク質を発現させたプロテオリポソームを、本発明の方法により形成された脂質二重膜に融合させることで使用可能である。 The lipid bilayer formed by the method of the present invention is used to examine the properties and functions of proteins in the state retained in the lipid bilayer, and to screen for ligands that bind to the proteins and change their physiological activities. Therefore, the lipid bilayer membrane preferably contains a protein, and particularly functions in a state of being held in the biological membrane in the living body. Preferred are transmembrane proteins. Examples of the protein retained in the lipid bilayer include various receptors and enzymes. Examples include peptide proteins such as α-hemolysin, gramicidin, and alamethicin, various ion channels, ABC transporter protein, and the like. However, it is not limited to these. When the protein is water-soluble, it is preferable to use an aqueous protein solution as the aqueous solution, and it is more preferable to use an aqueous solution containing the protein in an aqueous buffer solution. 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 10 μM. The protein only needs to be contained in one of the two droplets to be contacted, but may be contained in both droplets. When the protein retained in the lipid bilayer is lipid-soluble, it can be used by fusing a proteoliposome expressing the membrane protein to the lipid bilayer formed by the method of the present invention.
タンパク質がα−ヘモリシンやアラメチシン等のイオンチャネルを形成するタンパク質である場合、脂質二重膜にタンパク質が正しく保持されると、保持されたタンパク質の部分がイオンチャネルを形成して、２つのウェルが電気的に導通される。従って、２つのウェル間に所定の電圧をかけ、脂質二重膜を介して流れる電流を測定することにより、タンパク質が脂質二重膜中に正しく保持された脂質二重膜が形成されたことを確認することができる。また、タンパク質に種々の物質を作用させることにより、タンパク質の構造を変化させてイオンチャネルを閉じたり開いたりする物質をスクリーニングすることもでき、このような物質は何らかの生理活性を発揮する可能性があり、医薬品の候補となり得る。また、この本発明の方法により形成された脂質二重膜を用いれば、簡単に再現性良く膜タンパク質の解析が可能であることから、今後、膜タンパク質の機能解析に関する研究効率を飛躍的に向上させることが期待できる。 When the protein is a protein that forms an ion channel such as α-hemolysin or alamethicin, when the protein is correctly retained in the lipid bilayer membrane, the retained protein part forms an ion channel, and the two wells Electrically conducted. Therefore, by applying a predetermined voltage between the two wells and measuring the current flowing through the lipid bilayer, it was confirmed that a lipid bilayer in which the protein was properly retained in the lipid bilayer was formed. Can be confirmed. In addition, by causing various substances to act on proteins, it is also possible to screen for substances that change the protein structure to close or open ion channels, and such substances may exhibit some physiological activity. Yes, it can be a drug candidate. In addition, if the lipid bilayer membrane formed by the method of the present invention is used, it is possible to easily analyze membrane proteins with good reproducibility, and in the future, the efficiency of research on functional analysis of membrane proteins will be dramatically improved. You can expect
次に、本発明の好ましい方法及びそれに用いられる好ましい器具を説明する。なお、本明細書及び特許請求の範囲において、「上」、「鉛直」、「水平」等の語は、器具に液を入れて使用する際の使用状態を基準にした方向を示す。 Next, a preferred method of the present invention and a preferred instrument used therefor will be described. In the present specification and claims, terms such as “upper”, “vertical”, and “horizontal” indicate directions based on the state of use when a liquid is put into an instrument.
第１の好ましい方法は、周縁部に複数の凹部を有する回転可能な円板であって、各凹部は、水平方向の外側に向かって開口している円板と、前記円板に外接する固定板であって、その内周縁部に、前記複数の凹部にそれぞれ接する位置に複数の凹部を有し、各凹部は、水平方向の内側に向かって開口している固定板とを具備し、前記円板内の各凹部の開口部と、前記固定板内の各凹部の開口部を位置合わせすることによりそれぞれ１個の凹部が形成される、脂質二重膜形成器具（スピン型）を用いる方法である。以下、この方法の好ましい具体例を図面に基づき説明する。 A first preferred method is a rotatable disc having a plurality of recesses in the peripheral edge, each recess being open toward the outside in the horizontal direction and a fixing that circumscribes the disc A plate having a plurality of recesses at positions in contact with the plurality of recesses on the inner peripheral edge thereof, each recess comprising a fixed plate that opens toward the inside in the horizontal direction, A method using a lipid bilayer membrane forming device (spin type), wherein one recess is formed by aligning the opening of each recess in the disk and the opening of each recess in the fixed plate. It is. Hereinafter, preferred specific examples of this method will be described with reference to the drawings.
図１は、この具体例に用いられる脂質二重膜形成器具の模式的斜視図である。器具は、基板１０を含む。この具体例では、基板１０は、上記した固定板を構成するものである。基板１０の中央付近には、円板１２が回転可能に枢支されている。円板１２は、基板１０の中央付近に設けられた円板収容凹部内に収容されており、円板１２の上面は、基板１０の上面より上部にある。円板１２は、一方向に回転可能であればよいが、両方向に回転可能、すなわち、回動自在に枢支されている方が便利である。円板１２の周縁部には複数の凹部１４が設けられている。図示の例では、４個の凹部１４が設けられているが、この個数は４個に限定されるものではなく、通常、２個〜１６個程度である。各凹部１４は、水平方向の外側に向かって開口している（開口部１４ａ）。図示の例では、各凹部１４の底部は、基板１０中の円板収容凹部の上面により構成されている。 FIG. 1 is a schematic perspective view of a device for forming a lipid bilayer membrane used in this specific example. The instrument includes a substrate 10. In this specific example, the substrate 10 constitutes the fixed plate described above. Near the center of the substrate 10, a disk 12 is pivotally supported. The disc 12 is accommodated in a disc accommodating recess provided near the center of the substrate 10, and the upper surface of the disc 12 is above the upper surface of the substrate 10. The disk 12 only needs to be rotatable in one direction, but it is more convenient that the disk 12 can be rotated in both directions, that is, pivotally supported. A plurality of recesses 14 are provided at the peripheral edge of the disc 12. In the example shown in the figure, four recesses 14 are provided, but this number is not limited to four, and is usually about two to sixteen. Each recess 14 is open toward the outside in the horizontal direction (opening 14a). In the example shown in the figure, the bottom of each recess 14 is constituted by the upper surface of the disc housing recess in the substrate 10.
円板１２に外接する、基板１０の内周縁部には、凹部１４のそれぞれ接する位置に複数の凹部１６が設けられている。各凹部１６は、水平方向の内側に向かって開口している（開口部１６ａ）。すなわち、円板１２内の各凹部１４と、基板１０内の各凹部１６が接している位置に来るように円板１２を位置合わせすると、各凹部１４の開口部１４ａと、各凹部１６の開口部１６ａとは接することになり、各凹部１４と各凹部１６とによりそれぞれ１個の凹部が形成される。図１の右上部分には、円板１２を回転させて、各凹部１４と各凹部１６とが接していない状態が示されている。基板１０には、この器具を用いて形成した脂質二重膜を用いて各種実験を行う場合に便利な、各凹部に接続する電極２０が接続される。なお、特に限定されないが、凹部１４及び凹部１６の直径は、例えば、2 mm〜 10 mm程度、開口部１４ａと開口部１６ａの長さは、例えば1 mm〜5 mm程度、円板１２の直径は、例えば5 mm〜20 mm程度、円板１２の厚さは、例えば1 mm〜 5 mm程度でよい。 A plurality of concave portions 16 are provided at positions where the concave portions 14 are in contact with each other at the inner peripheral edge portion of the substrate 10 that circumscribes the disc 12. Each recess 16 opens toward the inner side in the horizontal direction (opening 16a). That is, when the disk 12 is aligned so that each recess 14 in the disk 12 and each recess 16 in the substrate 10 are in contact with each other, the opening 14a of each recess 14 and the opening of each recess 16 are located. Each of the recesses 14 and each of the recesses 16 forms one recess. In the upper right part of FIG. 1, a state is shown in which the circular plate 12 is rotated and the concave portions 14 and the concave portions 16 are not in contact with each other. Connected to the substrate 10 is an electrode 20 connected to each recess, which is convenient when various experiments are performed using a lipid bilayer membrane formed using this instrument. Although not particularly limited, the diameter of the recess 14 and the recess 16 is, for example, about 2 mm to 10 mm, and the length of the opening 14a and the opening 16a is, for example, about 1 mm to 5 mm. May be about 5 mm to 20 mm, for example, and the thickness of the disc 12 may be about 1 mm to 5 mm, for example.
次に、この器具を用いて脂質二重膜を形成する方法を図２を参照して説明する。図２は、器具を上面から見た模式図である。先ず、図２の１（左上の図）に示す通り、脂質二重膜形成性溶液を基板１０の凹部に注入して、円板１２を各凹部１４の開口部と、各凹部１６の開口部とがそれぞれ接し、各凹部１４と各凹部１６とによりそれぞれ１個のひょうたん形の凹部が形成されるように配置する。 Next, a method of forming a lipid bilayer using this instrument will be described with reference to FIG. FIG. 2 is a schematic view of the instrument as seen from above. First, as shown in 1 of FIG. 2 (upper left figure), a lipid bilayer-forming solution is injected into the recesses of the substrate 10, and the disc 12 is opened in the recesses 14 and in the recesses 16. Are arranged such that one gourd-shaped recess is formed by each recess 14 and each recess 16.
次に、図２の２（右上の図）に示すように、各ひょうたん形の凹部に水溶液（例えば、水系緩衝液内に、実験に必要なタンパク質等を溶解したもの等）を注入する。そうすると、図２の２の右下の枠内に模式的に示すように、各ひょうたん形の凹部内において、脂質一重膜で囲包されたひょうたん形の液滴が生成する。このとき、各ひょうたん形の凹部に、組成が異なる水溶液を注入しておくこともできる。 Next, as shown in 2 of FIG. 2 (upper right figure), an aqueous solution (for example, a solution in which a protein or the like necessary for an experiment is dissolved in an aqueous buffer solution) is injected into each gourd-shaped recess. Then, as schematically shown in the lower right frame of 2 in FIG. 2, a gourd-shaped droplet surrounded by a lipid monolayer is generated in each gourd-shaped recess. At this time, aqueous solutions having different compositions can be poured into each gourd-shaped recess.
次に、図２の３（右下の図）に示すように、円板１２を時計回りに回転させる。そうすると、各各ひょうたん形の凹部は分断され、脂質一重膜で囲包されたひょうたん形の液滴はそれぞれ２個の液滴に分割される。分割直後は、凹部１４の開口部１４ａ及び凹部１６の開口部１６ａの部分に脂質一重膜は存在しないが、直ちにこの部分に脂質一重膜が自動的に形成され、図２の３の右下の枠内に模式的に示すように、各凹部１４内及び各凹部１６内でそれぞれ、脂質一重膜で囲包された分割液滴が形成される。 Next, as shown in 3 of FIG. 2 (lower right figure), the disk 12 is rotated clockwise. Then, each gourd-shaped recess is divided, and the gourd-shaped droplets surrounded by the lipid monolayer are each divided into two droplets. Immediately after the division, a lipid monolayer does not exist in the opening 14a of the recess 14 and the opening 16a of the recess 16, but immediately a lipid monolayer is automatically formed in this portion. As schematically shown in the frame, divided droplets surrounded by a lipid monolayer are formed in each recess 14 and each recess 16.
次に、図２の４（左下の図）に示すように、各凹部１４が、最初に接していた各凹部１６のそれぞれ隣の各凹部１６と接する位置に来るまで、円板１２をさらに時計回りに回転させる。すなわち、図２の１の位置から時計回りに９０度の位置に来るまで回転させる。そうすると、各凹部１４及び各凹部１６内でそれぞれ形成されていた、脂質一重膜で囲包された分割液滴同士が各開口部１４ａ及び各開口部１６ａの位置で互いに接触し、図２の４の右下の枠内に模式的に示すように、この位置に脂質二重膜が自動的に形成される。ここで、上記図２の２の段階で、各ひょうたん形の凹部に注入する水溶液として、組成が異なる水溶液を注入しておくと、図２の４の段階では、それぞれ異なる組成の液を内包する分割液滴同士が接触し、これらの間で脂質二重膜が形成されることになる。 Next, as shown in 4 of FIG. 2 (lower left figure), the disk 12 is further rotated until each recess 14 comes to a position in contact with each recess 16 adjacent to each recess 16 that was initially in contact. Rotate around. That is, it is rotated from the position 1 in FIG. 2 until it reaches a position of 90 degrees clockwise. As a result, the divided droplets surrounded by the lipid monolayer formed in each of the recesses 14 and 16 come into contact with each other at the positions of the openings 14a and the openings 16a, respectively. As shown schematically in the lower right frame, a lipid bilayer membrane is automatically formed at this position. Here, if an aqueous solution having a different composition is injected as an aqueous solution to be injected into each gourd-shaped recess in step 2 in FIG. 2, the solutions having different compositions are included in step 4 in FIG. The divided droplets come into contact with each other, and a lipid bilayer membrane is formed between them.
この状態で形成された脂質二重膜を用いて所望の測定を行い、それで実験を終了してもよいが、この状態で所望の測定を行った後、さらに、円板１２を回転させ、各ひょうたん形の凹部を分断してもよい。そうすると、形成されていた脂質二重膜は分断され、各開口部１４ａ及び各開口部１６ａの位置には、脂質一重膜で囲包された液滴が収容される状態、すなわち、図２の３の状態になる。この状態からさらに時計回りに円板１２を回転させる。各凹部１４が、最初に接していた各凹部１６の隣の隣の各凹部１６と接触する位置に来るまで時計回りに円板１２を回転させる（すなわち、最初の位置から１８０度回転させた位置まで回転させる）と、図２の４に示すように、脂質一重膜で囲包された分割液滴同士が各開口部１４ａ及び各開口部１６ａの位置で互いに接触し、図２の４の右下の枠内に模式的に示すように、この位置に脂質二重膜が自動的に形成される。ここで、上記図２の２の段階で、各ひょうたん形の凹部に注入する水溶液として、組成が異なる水溶液を注入しておくと、図２の４の段階では、先に脂質二重膜を形成した際とはまた異なる組成の液を内包する分割液滴同士が接触し、これらの間で脂質二重膜が形成されることになる。 The desired measurement may be performed using the lipid bilayer membrane formed in this state, and the experiment may be terminated. However, after performing the desired measurement in this state, the disk 12 is further rotated, The gourd-shaped recess may be divided. Then, the formed lipid bilayer membrane is divided, and the positions surrounded by the lipid monolayer are accommodated at the positions of the openings 14a and 16a, that is, 3 in FIG. It becomes the state of. From this state, the disk 12 is further rotated clockwise. The disk 12 is rotated clockwise until each concave portion 14 comes into contact with each adjacent concave portion 16 adjacent to the first concave portion 16 (that is, a position rotated 180 degrees from the initial position). 2), the divided liquid droplets surrounded by the lipid monolayer come into contact with each other at the positions of the openings 14a and 16a, and the right side of 4 in FIG. As shown schematically in the lower frame, a lipid bilayer membrane is automatically formed at this position. Here, when an aqueous solution having a different composition is injected as an aqueous solution to be injected into each gourd-shaped recess in step 2 in FIG. 2, a lipid bilayer is first formed in step 4 in FIG. Then, the divided droplets containing the liquid having a different composition come into contact with each other, and a lipid bilayer membrane is formed between them.
この状態で必要な測定を行い、所望により、さらに円板１２を回転して、上記と同様の操作を行うことも可能であり、それによって、これまでとはさらに別の組成を有する分割液滴同士を接触させてそれらの間で脂質二重膜を形成することができる。 It is also possible to perform necessary measurements in this state, and further rotate the disc 12 to perform the same operation as described above, if desired, whereby divided droplets having a composition different from the conventional ones. They can be brought into contact with each other to form a lipid bilayer membrane between them.
なお、上記具体例では、円板１２をずっと時計回りに回しているが、円板１２を回転させて凹部１４と凹部１６とを分断し、脂質一重膜を再生後、円板１２を逆回転させて元の凹部同士を接触させてもよい。この方法によれば、例えば、一旦形成された脂質二重膜が何らかの理由で壊れた際に、単に円板１２を少し回転させ、元に戻すという極めて簡単な操作により、先に壊れた脂質二重膜と同じ脂質二重膜を再生することができる。また、円板１２は、モーターにより自動的に回転させるようにしてもよい。 In the above specific example, the disk 12 is rotated clockwise all the time, but the disk 12 is rotated to divide the recess 14 and the recess 16, and after regenerating the lipid monolayer, the disk 12 is rotated backward. The original recesses may be brought into contact with each other. According to this method, for example, when the formed lipid bilayer membrane is broken for some reason, the previously broken lipid bilayer is simply rotated by rotating the disk 12 a little and returning it to the original state. The same lipid bilayer as the bilayer can be regenerated. Further, the disk 12 may be automatically rotated by a motor.
本発明の方法を実施する第２の好ましい方法は、凹部を有する基板と、該凹部の一部分を被覆する液滴スプリッターとを具備し、該液滴スプリッターは、鉛直方向の壁により囲包されて成り、互いに分断可能な複数のセパレーターを含み、各セパレーターは、鉛直方向に延びる、対向する一対の壁を具備し、該セパレーターのうちの少なくとも一部は、前記凹部の、液滴スプリッターにより被覆されていない領域上まで水平方向にスライド可能である、脂質二重膜形成器具（スライド型）を用いる方法である。以下、この方法の好ましい具体例を図面に基づき説明する。 A second preferred method of carrying out the method of the present invention comprises a substrate having a recess and a droplet splitter covering a portion of the recess, the droplet splitter being surrounded by a vertical wall. A plurality of separators that can be separated from each other, each separator having a pair of opposing walls extending in a vertical direction, at least a part of the separators being covered by a droplet splitter of the recess. This is a method of using a lipid bilayer membrane formation device (slide type) that can slide in a horizontal direction up to a region that is not. Hereinafter, preferred specific examples of this method will be described with reference to the drawings.
図３は、この具体例に用いられる脂質二重膜形成器具の模式的斜視図である。器具は、基板２２を含み、基板２２には凹部２４が形成されている。凹部２４上には液滴スプリッター２６が配置され、該液滴スプリッター２６が凹部２４の一部分を被覆している。該液滴スプリッター２６は、鉛直方向の壁２６ａにより囲包されて成り、互いに分断可能な複数のセパレーター２７を含む（図示の例では４つのセパレーター）。各セパレーター２７は、鉛直方向に延びる、対向する一対の壁２７ａを具備する（図３の右上の枠内参照）。図示の例では、４つのセパレーター２７のうち、一番端の１つを除き、頂部が頂板２７により覆われており、この頂板２７を介して対向する一対の壁２７ａ同士が連結され、従って、各セパレーター２７は、液滴スプリッター２６の長手方向に直行する方向に、水平方向から見て断面コの字形の形状を有している。各頂板２７ｂには、空気孔２８が設けられている。一端のセパレーターには頂板がなく、解放されており、この開口が水注入口３０を構成している。両端のセパレーターには、前記対向する壁２７ａを連結する側板２７ｃが、液滴スプリッター２６の長手方向に直行する方向の最端部に設けられており、両端のセパレーターに液を充填しても各セパレーターから液がこぼれ出ないようになっている。さらに、各セパレーター同士が接する部分の手前側には、短冊状の側板２７ｄが壁２７ａと直行する方向に設けられている（図３の右上の枠内参照）。側板２７ｄは、脂質一重膜を切断しやすくするためのもので、設けなくても問題はない。基板２２には、この器具を用いて形成した脂質二重膜を用いて各種実験を行う場合に便利な、各セパレーター内部に接続する電極３２が接続される。なお、図３の右上の枠内は、４個のセパレーター２７のうち、隣接していない２個を水平方向にスライドさせた後の状態を示す。セパレーター２７のサイズは、特に限定されないが、例えば、一辺が１mm〜10mm程度の立方体でよく、凹部２４の広さは、所望の個数のセパレータを収容し、スライドさせることができる大きさでよく、凹部２４の深さは、特に限定されないが、例えば、1mm〜5mm程度である。 FIG. 3 is a schematic perspective view of a lipid bilayer membrane forming instrument used in this specific example. The instrument includes a substrate 22 in which a recess 24 is formed. Droplet splitter 26 is disposed on the recess 24, said droplets splitter 26 covers the portion of the recess 24. The droplet splitter 26 is surrounded by a vertical wall 26a and includes a plurality of separators 27 (four separators in the illustrated example) that can be separated from each other. Each separator 27 includes a pair of opposing walls 27a extending in the vertical direction (see the upper right frame in FIG. 3). In the illustrated example, the top part is covered with the top plate 27 except for one of the four separators 27, and a pair of opposing walls 27a are connected via the top plate 27. Each separator 27 has a U-shaped cross section when viewed from the horizontal direction in a direction perpendicular to the longitudinal direction of the droplet splitter 26 . Each top plate 27 b is provided with an air hole 28. The separator at one end has no top plate and is open, and this opening constitutes the water inlet 30. The separators at both ends are provided with side plates 27c that connect the opposing walls 27a at the extreme ends in the direction perpendicular to the longitudinal direction of the droplet splitter 26. The liquid does not spill out from the separator. Further, a strip-shaped side plate 27d is provided in a direction perpendicular to the wall 27a on the front side of the portion where the separators are in contact with each other (see the upper right frame in FIG. 3). The side plate 27d is for facilitating cutting of the lipid monolayer, and there is no problem even if it is not provided. Connected to the substrate 22 is an electrode 32 connected to the inside of each separator, which is convenient when various experiments are performed using a lipid bilayer membrane formed using this instrument. Note that the upper right frame in FIG. 3 shows a state after two non-adjacent of the four separators 27 are slid in the horizontal direction. The size of the separator 27 is not particularly limited. For example, the separator 27 may be a cube having a side of about 1 mm to 10 mm, and the width of the recess 24 may be a size that can accommodate and slide a desired number of separators. Although the depth of the recessed part 24 is not specifically limited, For example, it is about 1 mm-5 mm.
次に、この器具を用いて脂質二重膜を形成する方法を図４を参照して説明する。図４は、器具を上方から見た平面模式図であり、明瞭性のために頂板２７ｂは省かれている。図４の１に示すように、先ず、凹部２４に脂質二重膜形成性溶液を入れ、上から液滴スプリッター２６を被せる。 Next, a method of forming a lipid bilayer using this instrument will be described with reference to FIG. FIG. 4 is a schematic plan view of the instrument as viewed from above, and the top plate 27b is omitted for clarity. As shown in 1 of FIG. 4, first, the lipid bilayer-forming solution is put in the recess 24 and the droplet splitter 26 is covered from above.
次に、水注入口３０（図３参照）から、水又は水溶液（以下、単に「水」）を注入し、液滴スプリッター２６内に水を充填する。そうすると、水は、脂質二重膜形成性溶液よりも重いので、下に沈み、脂質二重膜形成性溶液と側面でも接触し、図４の右側の拡大枠２に示される通り、液滴スプリッター２６内に脂質一重膜で囲包された液滴が形成される。なお、図４の右側の拡大枠２に示される脂質一重膜の２個の出っ張りは、短冊型の側板２７ｄにより形成される突起である。 Next, water or an aqueous solution (hereinafter simply referred to as “water”) is injected from the water injection port 30 (see FIG. 3), and the droplet splitter 26 is filled with water. Then, since the water is heavier than the lipid bilayer-forming solution, it sinks down and comes into contact with the lipid bilayer-forming solution also on the side surface, and as shown in the enlarged frame 2 on the right side of FIG. A droplet surrounded by a monolayer of lipid is formed in 26 . Note that the two protrusions of the lipid monolayer shown in the enlarged frame 2 on the right side of FIG. 4 are protrusions formed by the strip-shaped side plate 27d.
次に、図４の３に示すように、液滴スプリッター２６を構成する４個のセパレーターのうち、水注入口３０を形成したセパレーターと、その隣の隣のセパレーターをそのままの位置に置いたまま、残る２個のセパレーターを、凹部２４の液滴スプリッター２６により覆われていない部分に向けてスライドさせる。そうすると、図４の３に示すように、脂質一重膜で囲包された液滴が４個の液滴に分割される。各液滴の分断された面には、分割直後は脂質一重膜が存在しないが、直ちにこの部分に脂質一重膜が自動的に形成され、図４の３の右側の枠内に模式的に示すように、各セパレーター２７内でそれぞれ、脂質一重膜で囲包された分割液滴が形成される。 Next, as shown in 3 of FIG. 4, among the four separators constituting the droplet splitter 26 , the separator in which the water injection port 30 is formed and the adjacent separator next to the separator are left in the same positions. Then, the remaining two separators are slid toward the portion of the recess 24 not covered by the droplet splitter 26 . If it does so, as shown to 3 of FIG. 4, the droplet enclosed by the lipid single membrane will be divided | segmented into four droplets. On the divided surface of each droplet, there is no lipid monolayer immediately after the division, but immediately a lipid monolayer is automatically formed in this portion, and is schematically shown in the right frame of 3 in FIG. Thus, in each separator 27, a divided droplet surrounded by a lipid monolayer is formed.
次に、図４の４に示すように、スライドさせた２個のセパレーターを元の位置に戻す。そうすると、各セパレーター間で、脂質一重膜同士が接触し、この部分に脂質二重膜が形成される。図示の例では、セパレーターが４個あるので、それらの各境界において、合計３つの脂質二重膜が形成される。 Next, as shown by 4 in FIG. 4, the two slid separators are returned to their original positions. If it does so, lipid single membranes will contact between each separator, and a lipid bilayer membrane will be formed in this part. In the example shown in the figure, since there are four separators, a total of three lipid bilayers are formed at their respective boundaries.
なお、スライド型の器具では、セパレーターの形状を変化させることで、上下左右どの方向にも脂質二重膜を形成可能であり、有機溶媒中でなら、どこにでも脂質二重膜を形成可能である。また、複数の液滴を結合させることで、脂質二重膜を介した液滴ネットワークを形成できる。さらに、様々な液滴を用意しておけば、自由な組合せで液滴ネットワークを形成可能である。 In addition, in a slide-type instrument, by changing the shape of the separator, a lipid bilayer can be formed in any direction, up, down, left, or right, and a lipid bilayer can be formed anywhere in an organic solvent. . Moreover, the droplet network through a lipid bilayer membrane can be formed by combining a plurality of droplets. Furthermore, if various droplets are prepared, a droplet network can be formed in any combination.
なお、上記実施形態では、１個の脂質一重膜に囲包された液滴を先ず分割し、生じた分割液滴同士を接触させているが、本発明の方法は、これに限定されるものではなく、それぞれ予め生成した脂質一重膜に囲包された液滴２個同士を機械的外力により接触させてもよい。例えば、上記スピン型の器具を用い、凹部１４と凹部１６とが接していない状態で、各凹部内で脂質一重膜に囲包された液滴を形成し、円板１２を回転させて凹部１４と凹部１６とを接触させてもよい。 In the above embodiment, the droplet surrounded by one lipid single membrane is first divided and the resulting divided droplets are brought into contact with each other. However, the method of the present invention is limited to this. Instead, two droplets surrounded by a previously formed lipid monolayer may be brought into contact with each other by mechanical external force. For example, using the spin-type instrument, in a state where the recess 14 and the recess 16 are not in contact with each other, a droplet surrounded by a lipid monolayer is formed in each recess, and the disc 12 is rotated to rotate the recess 14. And the recess 16 may be brought into contact with each other.
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
図１及び図２に示したスピン型の器具を作製した。各凹部１４及び各凹部１６の直径は、４mm、円板１４の厚さは３mmであった。円板１２を取り外した円板収容凹部に、卵黄フォスファチジルコリンのn-デカン溶液（20mg/mL)50μLを入れ、円板１２を回動自在に装着し、各凹部１４と各凹部１６が接してそれぞれひょうたん形の凹部が形成される位置に円板を回転した。この状態で、合計４個の各ひょうたん形の凹部に、10nMのα−ヘモリシン（αHL)及び1M KClを含む緩衝液をそれぞれ40μLずつ滴下した。円板１２を９０度回転させ、各凹部１４を、最初に接していた凹部１６に隣接する凹部１６と接触させ、再度ひょうたん形の凹部を形成した。各凹部に接続した電極を用いて各凹部間に100 mVの電圧をかけ、各凹部間に流れる電流を経時的に測定した。
Example 1
The spin-type instrument shown in FIGS. 1 and 2 was produced. The diameter of each recess 14 and each recess 16 was 4 mm, and the thickness of the disk 14 was 3 mm. 50 μL of egg yolk phosphatidylcholine n-decane solution (20 mg / mL) is placed in the disc housing recess from which the disc 12 has been removed, and the disc 12 is rotatably mounted. The disc was rotated to a position where a gourd-shaped recess was formed in contact with each other. In this state, 40 μL each of a buffer solution containing 10 nM α-hemolysin (αHL) and 1M KCl was dropped into a total of four gourd-shaped recesses. The circular plate 12 was rotated 90 degrees, and each concave portion 14 was brought into contact with the concave portion 16 adjacent to the concave portion 16 that was initially in contact with each other to form a gourd-shaped concave portion again. A voltage of 100 mV was applied between the recesses using electrodes connected to the recesses, and the current flowing between the recesses was measured over time.
結果を図５に示す。図５に示すように、４つのひょうたん形の凹部に形成された脂質二重膜に、α−ヘモリシンによりチャネルが形成され、電流が流れることが、全ての凹部について確認された。電流は、形成されるチャネルの個数の増大に伴い、階段的に大きくなった。 The results are shown in FIG. As shown in FIG. 5, it was confirmed for all the recesses that a channel was formed by α-hemolysin in the lipid bilayer membrane formed in the four gourd-shaped recesses, and current flowed. The current increased stepwise as the number of channels formed increased.
実施例２
実施例１と同じ器具を用い、基本的に実施例１と同じ操作を行った。ただし、４つのひょうたん形の凹部のうち、隣接する２個に実施例１と同じ、α−ヘモリシン水溶液を滴下し、他の１個に100μMのs7βCD（β−シクロデキストリン）水溶液を滴下し、残りの１個に10nM α−ヘモリシンと50merの一本鎖ポリチミジル酸(ssDNA)（25μM）の水溶液を滴下した。この状態で、図６のb)に示す、白い点線で囲まれたひょうたん形の凹部（α−ヘモリシン水溶液を滴下）において、凹部１４と凹部１６の間に流れる電流を実施例１と同様に測定した。
Example 2
Using the same instrument as in Example 1, the same operation as in Example 1 was basically performed. However, the same α-hemolysin aqueous solution as in Example 1 was dropped on two adjacent gourd-shaped recesses, and 100 μM s7βCD (β-cyclodextrin) aqueous solution was dropped on the other one, and the rest An aqueous solution of 10 nM α-hemolysin and 50-mer single-stranded polythymidylic acid (ssDNA) (25 μM) was added dropwise to one of these. In this state, the current flowing between the concave portion 14 and the concave portion 16 was measured in the same manner as in Example 1 in the gourd-shaped concave portion (α-hemolysin aqueous solution dropped) surrounded by a white dotted line shown in FIG. did.
円板１２を反時計回りに９０度回転させ、共にα−ヘモリシン水溶液を含む分割液滴間に脂質二重膜を形成した。図６に示すように、α−ヘモリシンによりチャネルが形成され、電流が流れることが確認された。 The disk 12 was rotated 90 degrees counterclockwise, and a lipid bilayer membrane was formed between the divided droplets containing both α-hemolysin aqueous solutions. As shown in FIG. 6, it was confirmed that a channel was formed by α-hemolysin and current flowed.
次に、円板１２をさらに９０度反時計回りに回転させると、α−ヘモリシン水溶液を含む分割液滴が、s7βCD水溶液を含む分割液滴と接し、この間に脂質二重膜が形成された。α−ヘモリシンによりチャネルが形成され、電流が流れることが確認されたが、s7βCDがチャネル内を通過する間は、チャネルがs7βCDにより閉塞されて電流がときどき流れなくなることも確認された（図６のc)。 Next, when the disk 12 is further rotated 90 degrees counterclockwise, the divided droplets containing the α-hemolysin aqueous solution come into contact with the divided droplets containing the s 7 βCD aqueous solution, and a lipid bilayer membrane is formed therebetween. It was. It was confirmed that a channel was formed by α-hemolysin and current flowed, but while s 7 βCD passed through the channel, it was confirmed that the channel was blocked by s 7 βCD and the current occasionally stopped flowing. (FIG. 6c).
次に、円板１２をさらに９０度反時計回りに回転させると、α−ヘモリシン水溶液を含む分割液滴が、ssDNA水溶液を含む分割液滴と接し、この間に脂質二重膜が形成された。α−ヘモリシンによりチャネルが形成され、電流が流れることが確認されたが、ssDNAがチャネル内を通過する間は、チャネルがssDNAにより閉塞されて電流がときどき流れなくなることも確認された（図６のd)。 Next, when the disk 12 was further rotated 90 degrees counterclockwise, the divided droplets containing the α-hemolysin aqueous solution contacted the divided droplets containing the ssDNA aqueous solution, and a lipid bilayer membrane was formed therebetween. It was confirmed that a channel was formed by α-hemolysin and current flowed, but while ssDNA passed through the channel, it was also confirmed that the channel was blocked by ssDNA and sometimes the current did not flow (Fig. 6). d).
次に、円板１２をさらに９０度反時計回りに回転させると、α−ヘモリシン水溶液を含む分割液滴が、同じくα−ヘモリシン水溶液を含む分割液滴と接し（最初の状態に戻る）、α−ヘモリシンによるチャネルが形成されて電流が定常的に流れることが確認された（図６のe)。 Next, when the disk 12 is further rotated counterclockwise by 90 degrees, the divided droplets containing the α-hemolysin aqueous solution come into contact with the divided droplets also containing the α-hemolysin aqueous solution (return to the initial state), and α -It was confirmed that a channel due to hemolysin was formed and current steadily flowed (e in FIG. 6).
実施例３
図３及び図４に示すスライド型の脂質二重膜形成器具を作製した。セパレータは、１辺が5mmの立方体であり、凹部２４の深さは1mmであった。凹部２４に卵黄フォスファチジルコリンのn-デカン溶液（20mg/mL)100μLを入れ、一方、水注入口３０から、水100μLを注入し、注入した水が４つのセパレーター内で均等に配分されるまで待った。4つのセパレータをそれぞれ個別に分断し（この時水も一緒に分断される）、再び元の位置に戻すと、輸送された液滴の接触界面に脂質二重膜を形成し、さらに脂質二重膜を介した液滴のネットワークを形成することができた。
Example 3
The slide-type lipid bilayer membrane forming device shown in FIGS. 3 and 4 was produced. The separator was a cube having a side of 5 mm, and the depth of the recess 24 was 1 mm. 100 μL of egg yolk phosphatidylcholine n-decane solution (20 mg / mL) is put into the recess 24, while 100 μL of water is injected from the water inlet 30, and the injected water is evenly distributed in the four separators. I waited until. When the four separators are individually separated (water is also separated at this time) and returned to the original position, a lipid bilayer membrane is formed at the contact interface of the transported droplets. A network of droplets through the membrane could be formed.
実施例４
セパレータの数が２個、凹部２４に入れた卵黄フォスファチジルコリンのn-デカン溶液の量が40μL、水注入口３０から注入した水の量が50μLであることを除き、実施例３と同様な操作を行った。元の位置に戻したセパレータ間に形成された脂質二重膜を介して流れる電流を経時的に測定した。
Example 4
Example 3 except that the number of separators is 2, the amount of n-decane solution of egg yolk phosphatidylcholine placed in the recess 24 is 40 μL, and the amount of water injected from the water inlet 30 is 50 μL. The operation was performed. The current flowing through the lipid bilayer formed between the separators returned to the original position was measured over time.
結果を図７に示す。形成された脂質二重膜に、α−ヘモリシンによりチャネルが形成され、電流が流れることが、確認された。電流は、形成されるチャネルの個数の増大に伴い、階段的に大きくなった。 The results are shown in FIG. It was confirmed that in the formed lipid bilayer membrane, a channel was formed by α-hemolysin and current flowed. The current increased stepwise as the number of channels formed increased.
１０ 基板
１２ 円板
１４ 凹部
１４ａ 凹部開口部
１６ 凹部
１６ａ 凹部開口部
２０ 電極
２２ 基板
２４ 凹部
２６ 液滴スプリッター
２６ａ 壁
２７ セパレーター
２７ａ 壁
２７ｂ 頂板
２７ｃ 側板
２７ｄ 側板
２８ 空気孔
３０ 水注入口
３２ 電極
DESCRIPTION OF SYMBOLS 10 Substrate 12 Disc 14 Recess 14a Recess opening 16 Recess 16a Recess opening 20 Electrode 22 Substrate 24 Recess
26 droplet splitter 26a wall 27 separator 27a wall 27b top plate 27c side plate 27d side plate 28 air hole 30 water inlet 32 electrode
Claims (12)
前記円板に外接する固定板であって、その内周縁部に、前記複数の凹部にそれぞれ接する位置に複数の凹部を有し、各凹部は、水平方向の内側に向かって開口している固定板とを具備し、
前記円板内の各凹部の開口部と、前記固定板内の各凹部の開口部を位置合わせすることによりそれぞれ１個の凹部が形成される、脂質二重膜形成器具を用い、
前記円板内の各凹部の開口部と、前記固定板内の各凹部の開口部とを位置合わせしてそれぞれ１個の凹部を形成した状態で、各凹部内において脂質一重膜で囲包された液滴を形成する工程と、
前記円板を回転させて、各凹部内で形成された液滴をそれぞれ２個の液滴に分割し、生じた各液滴に脂質一重膜を再生させ、脂質一重膜で囲包された複数の分割液滴を生成させる工程と、
前記円板をさらに回転又は逆回転させて、円板内の各凹部を、先に接触していた固定板内凹部と同一又は異なる固定板内凹部と位置合わせしてそれぞれ１個の凹部を形成し、それによって、各凹部内の分割液滴同士を接触させ、この接触面において脂質二重膜を形成する、請求項１〜６のいずれか１項に記載の方法。 A rotatable disc having a plurality of recesses at the peripheral edge, each recess being a disc that is open toward the outside in the horizontal direction;
A fixing plate that circumscribes the disk, and has a plurality of recesses at positions that contact the plurality of recesses on the inner peripheral edge thereof, and each recess is fixed to open inward in the horizontal direction. A board,
Using a lipid bilayer membrane forming device in which one recess is formed by aligning the opening of each recess in the disc and the opening of each recess in the fixed plate,
In the state where one recess is formed by aligning the opening of each recess in the disk and the opening of each recess in the fixed plate, each recess is surrounded by a lipid monolayer. Forming a liquid droplet;
The disk is rotated to divide the droplets formed in each recess into two droplets, and a single lipid membrane is regenerated in each of the generated droplets. Generating divided droplets of:
The disk is further rotated or reversely rotated so that each recessed part in the disk is aligned with the recessed part in the fixed plate that is the same as or different from the recessed part in the fixed plate that has been in contact with the disk. The method according to claim 1, wherein the divided liquid droplets in each recess are brought into contact with each other, and a lipid bilayer membrane is formed on the contact surface.
該凹部の一部分を被覆する液滴スプリッターとを具備し、
該液滴スプリッターは、鉛直方向の壁により囲包されて成り、互いに分断可能な複数のセパレーターを含み、各セパレーターは、鉛直方向に延びる、対向する一対の壁を具備し、該セパレーターのうちの少なくとも一部は、前記凹部の、液滴スプリッターにより被覆されていない領域上まで水平方向にスライド可能である、脂質二重膜形成器具を用い、
前記凹部内に、脂質溶液を入れる工程と、
前記液滴スプリッターに水又は水溶液を注入して該液滴スプリッター内において、脂質一重膜で囲包された液滴を形成する工程と、
該液滴スプリッターの前記複数のセパレーターのうちの少なくとも一部のセパレーターを、該液滴スプリッターにより被覆されていない領域上まで水平方向にスライドさせ、一方、水平方向にスライドさせるセパレーターに隣接するセパレーターはスライドさせず、それによって、前記液滴を複数の液滴に分割し、生じた各液滴に脂質一重膜を再生させ、脂質一重膜で囲包された複数の分割液滴を生成させる工程と、
水平方向にスライドさせたセパレーターを元の位置に戻し、それによって、各セパレーター内の分割液滴同士を接触させ、この接触面において脂質二重膜を形成する、請求項１〜３及び６のいずれか１項に記載の方法。 A substrate having a recess;
A droplet splitter covering a portion of the recess,
The droplet splitter includes a plurality of separators surrounded by vertical walls and separable from each other, and each separator has a pair of opposing walls extending in the vertical direction. Using a lipid bilayer forming device that is slidable horizontally, at least in part, onto a region of the recess that is not covered by a droplet splitter,
Placing a lipid solution in the recess;
Injecting water or an aqueous solution into the droplet splitter to form a droplet surrounded by a lipid monolayer in the droplet splitter;
At least a part of the separators of the droplet splitter is horizontally slid onto an area not covered by the droplet splitter, while the separator adjacent to the separator to be slid horizontally is Non-sliding, thereby dividing the droplet into a plurality of droplets, regenerating a lipid monolayer on each resulting droplet, and generating a plurality of segmented droplets surrounded by the lipid monolayer; and ,
The separator slid in the horizontal direction is returned to its original position, whereby the divided liquid droplets in each separator are brought into contact with each other, and a lipid bilayer membrane is formed on this contact surface. The method according to claim 1.
前記円板に外接する固定板であって、その内周縁部に、前記複数の凹部にそれぞれ接する位置に複数の凹部を有し、各凹部は、水平方向の内側に向かって開口している固定板とを具備し、
前記円板内の各凹部の開口部と、前記固定板内の各凹部の開口部を位置合わせすることによりそれぞれ１個の凹部が形成される、脂質二重膜形成器具。 A rotatable disc having a plurality of recesses at the peripheral edge, each recess being a disc that is open toward the outside in the horizontal direction;
A fixing plate that circumscribes the disk, and has a plurality of recesses at positions that contact the plurality of recesses on the inner peripheral edge thereof, and each recess is fixed to open inward in the horizontal direction. A board,
A lipid bilayer membrane forming device in which one recess is formed by aligning the opening of each recess in the disk and the opening of each recess in the fixed plate.
該凹部の一部分を被覆する液滴スプリッターとを具備し、
該液滴スプリッターは、鉛直方向の壁により囲包されて成り、互いに分断可能な複数のセパレーターを含み、各セパレーターは、鉛直方向に延びる、対向する一対の壁を具備し、該セパレーターのうちの少なくとも一部は、前記凹部の、液滴スプリッターにより被覆されていない領域上まで水平方向にスライド可能である、脂質二重膜形成器具。 A substrate having a recess;
A droplet splitter covering a portion of the recess,
The droplet splitter includes a plurality of separators surrounded by vertical walls and separable from each other, and each separator has a pair of opposing walls extending in the vertical direction. A device for forming a lipid bilayer, at least partially slidable horizontally over a region of the recess that is not covered by a droplet splitter.
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