JP2012081405A - Method for forming lipid bilayer membrane, and apparatus for the same - Google Patents
Method for forming lipid bilayer membrane, and apparatus for the same Download PDFInfo
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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.
上記した方法は、簡便に脂質二重膜を形成できる方法であるが、創薬スクリーニング等においては、自動化装置を用いて多数の脂質二重膜を同時に形成し、形成された多数の脂質二重膜を用いて同時並行的にスクリーニングを行うことができれば、スクリーニングの効率を高めることができ、有利である。このような自動化を達成するためには、脂質二重膜が自動化装置（ロボット）を用いた場合でも再現性良く形成され、かつ、一旦形成された脂質二重膜は、自動化装置内での輸送や測定処理中に破壊されることなく安定に存在することが望まれる。 The above-described method is a method that can easily form a lipid bilayer. In drug discovery screening, etc., a large number of lipid bilayers are formed by simultaneously forming a large number of lipid bilayers using an automated device. If screening can be performed in parallel using a membrane, the efficiency of screening can be increased, which is advantageous. In order to achieve such automation, the lipid bilayer membrane is formed with good reproducibility even when an automated device (robot) is used, and once formed, the lipid bilayer membrane is transported within the automated device. It is desirable to exist stably without being destroyed during the measurement process.
従って、本発明の目的は、脂質二重膜を再現性よく形成することができ、形成された脂質二重膜が安定に維持される、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, which can form a lipid bilayer membrane with good reproducibility and maintain the formed lipid bilayer membrane stably. That is.
本願発明者らは、鋭意研究の結果、特定のサイズ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、この状態で放置すると、貫通孔の部分に脂質二重膜が再現性よく形成され、かつ、一旦形成された脂質二重膜が安定に維持されることを見出し、本発明を完成した。 As a result of earnest research, the inventors of the present application added a lipid bilayer-forming lipid solution to each of two wells separated by a partition wall having a through-hole of a specific size, and an aqueous buffer solution was added to each well. To form a buffer droplet in the lipid solution, and when left in this state, a lipid bilayer membrane is formed in the through-hole part with good reproducibility, and the once formed lipid bilayer membrane The present invention was completed by finding that it was stably maintained.
すなわち、本発明は、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する工程と、各ウェルに水又は水溶液を添加して前記脂質溶液中に水又は水溶液の液滴を形成させる工程と、この状態で放置して前記貫通孔の部分に脂質二重膜を形成させる工程とを含む、脂質二重膜の形成方法を提供する。また、本発明は、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つのウェルを具備する脂質二重膜形成器具を提供する。 That is, the present invention includes a step of adding a lipid bilayer-forming lipid solution to each of two wells separated by a partition wall having one or a plurality of through holes having a pore diameter of 500 nm to 500 μm, Adding water or an aqueous solution to the well to form droplets of water or an aqueous solution in the lipid solution; and allowing the well to stand in this state to form a lipid bilayer membrane in the through-hole portion. A method of forming a lipid bilayer membrane is provided. The present invention also provides a lipid bilayer membrane forming device comprising two wells separated by a partition wall having one or a plurality of through holes having a pore diameter of 500 nm to 500 μm.
本発明により、脂質二重膜を簡便に再現性よく形成することができ、形成された脂質二重膜が安定に維持される、新規な脂質二重膜の形成方法及びそのための器具が提供された。本発明の方法によれば、脂質二重膜を簡便に再現性よく形成することができるので、ロボットを用いて多数の脂質二重膜を同時並行的に形成することが可能になる。また、形成された脂質二重膜は安定に維持されるので、脂質二重膜を用いた各種試験を自動化装置により再現性よく行うことが可能である。 The present invention provides a novel method for forming a lipid bilayer membrane and a device therefor, in which the lipid bilayer membrane can be easily formed with good reproducibility and the formed lipid bilayer membrane is stably maintained. It was. According to the method of the present invention, a lipid bilayer membrane can be easily formed with good reproducibility, and therefore a large number of lipid bilayer membranes can be simultaneously formed using a robot. In addition, since the formed lipid bilayer membrane is stably maintained, various tests using the lipid bilayer membrane can be performed with an automated device with good reproducibility.
１０ 基板
１２ 隔壁（パリレン樹脂フィルム）
１４ ウェル
１６ ウェル
１８ 貫通孔
２０ 脂質溶液
２２ マイクロピペット
２４ 水又は水溶液の液滴
２６ シリコンウェハ
２８ パリレン樹脂フィルム
３０ アルミニウム膜
３２ フォトレジスト膜
３４ ポリメチルメタクリレート樹脂フィルム
３６ AgCl電極
３８ 銀電極
10 Substrate 12 Bulkhead (Parylene resin film)
14 well 16 well 18 through-hole 20 lipid solution 22 micropipette 24 droplet of water or aqueous solution 26 silicon wafer 28 parylene resin film 30 aluminum film 32 photoresist film 34 polymethyl methacrylate resin film 36 AgCl electrode 38 silver electrode
本発明の方法では、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つのウェルを用いる。貫通孔の孔径は、好ましくは１０μm〜２００μmである。孔径が上記範囲よりも小さい場合も大きな場合も、脂質二重膜形成の再現性が低くなり、また、上記範囲よりも大きな場合には形成された脂質二重膜の安定性が低下する。また、貫通孔の形状は、脂質二重膜を再現性良く形成し、かつ、安定に維持する観点及び貫通孔の形成の容易さの観点から円形が最も好ましいが、脂質二重膜が形成可能であれば、他の形状、例えば楕円形や正多角形等の多角形を採用することも可能である。楕円形の場合、孔径は長径を意味し、多角形の場合にはそれに外接する円の直径を孔径とする。 In the method of the present invention, two wells separated by a partition having one or a plurality of through holes having a hole diameter of 500 nm to 500 μm are used. The diameter of the through hole is preferably 10 μm to 200 μm. When the pore size is smaller or larger than the above range, the reproducibility of the formation of the lipid bilayer membrane is low, and when it is larger than the above range, the stability of the formed lipid bilayer membrane is lowered. The shape of the through-hole is most preferably circular from the viewpoint of forming the lipid bilayer membrane with good reproducibility and maintaining stability and the ease of formation of the through-hole, but the lipid bilayer membrane can be formed. If so, it is possible to adopt other shapes, for example, a polygon such as an ellipse or a regular polygon. In the case of an ellipse, the hole diameter means a long diameter, and in the case of a polygon, the diameter of a circle circumscribing it is taken as the hole diameter.
上記隔壁で隔てられた２つのウェルから構成されるチャンバーを「ダブルウェルチャンバー」(DWC)と呼ぶことがある。本発明で用いることができるDWCの好ましい１例を図１に模式的に示す。図１の(a)は平面図であり、(b)は(a)中のb-b'線切断部端面図である。なお、図１は、発明の理解のためにDWCを模式的に示すものであり、各構成要素の寸法比率は実物とは大きく異なる。 A chamber composed of two wells separated by the partition wall may be referred to as a “double well chamber” (DWC). A preferred example of DWC that can be used in the present invention is schematically shown in FIG. 1A is a plan view, and FIG. 1B is an end view taken along the line bb ′ in FIG. FIG. 1 schematically shows a DWC for understanding the invention, and the dimensional ratio of each component is greatly different from the actual one.
図１に模式的に示す具体例では、基板１０中に、２つのウェル１４及び１６が形成され、それらの境界が隔壁１２により隔てられている。隔壁１２には、貫通孔１８（図１の(b)参照）が設けられている。図１に示す例では、ウェルの平面形状が基本的に円形であり、２つの円が接する境界部分のみが直線状になっているが、ウェルの形状は限定されるものではなく、上記範囲の孔径の貫通孔を有する隔壁によって隔てられていれば、他の形状でも問題はない。ウェルのサイズは、特に限定されないが、後述のように水又は水溶液の液滴を脂質溶液中に形成した際に脂質溶液が液滴によって圧迫されやすくなるように孔径が2mm〜8mm程度、さらに好ましくは3mm〜5mm程度、深さは孔径の50%〜200%、さらに好ましくは50%〜100%程度が好ましいが、この範囲よりも大きくても小さくても本発明の方法を実施することが可能である。隔壁に設けられた貫通孔の数は１個でも複数個でもよく、通常、１個〜１０個程度、好ましくは１個〜６個程度である。貫通孔が複数存在する場合には、必ずしも全ての貫通孔において脂質二重膜が形成されるわけではなく、脂質溶液が液滴によって強く圧迫される貫通孔においてのみ脂質二重膜が形成される。 In the specific example schematically shown in FIG. 1, two wells 14 and 16 are formed in the substrate 10, and their boundaries are separated by a partition wall 12. The partition wall 12 is provided with a through hole 18 (see FIG. 1B). In the example shown in FIG. 1, the planar shape of the well is basically circular, and only the boundary portion where the two circles contact is linear, but the shape of the well is not limited and is in the above range. Other shapes are not a problem as long as they are separated by a partition wall having a through hole having a hole diameter. The size of the well is not particularly limited, but when the water or aqueous solution droplets are formed in the lipid solution as described later, the pore size is preferably about 2 mm to 8 mm so that the lipid solution is easily pressed by the droplets, more preferably Is preferably about 3 mm to 5 mm, and the depth is preferably 50% to 200% of the pore diameter, more preferably about 50% to 100%, but the method of the present invention can be carried out even if it is larger or smaller than this range It is. The number of through holes provided in the partition wall may be one or more, and is usually about 1 to 10 and preferably about 1 to 6. When there are a plurality of through-holes, lipid bilayers are not necessarily formed in all through-holes, but lipid bilayers are formed only in through-holes in which the lipid solution is strongly pressed by droplets. .
なお、上記DWCは、ウェル１４を透孔として設けた基板部材と、ウェル１６を透孔として設けた基板部材と、貫通孔を形成した自己支持性のポリマーフィルム等から構成される隔壁１２と、各ウェルの底部を構成する底板とを接着剤で接着することにより容易に作製可能である。これについては下記実施例において具体的に説明する。 The DWC includes a substrate member provided with the well 14 as a through hole, a substrate member provided with the well 16 as a through hole, a partition wall 12 formed of a self-supporting polymer film having a through hole, and the like. It can be easily produced by bonding the bottom plate constituting the bottom of each well with an adhesive. This will be specifically described in the following examples.
本発明の方法では、上記した２つのウェル１４及び１６のそれぞれに、脂質二重膜形成性脂質溶液を添加して各ウェルに該脂質溶液を充填する。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。また２つのウェルに異なる種類の脂質を導入することにより、簡便に非対称膜を形成可能なことも本発明の特徴である。 In the method of the present invention, a lipid bilayer-forming lipid solution is added to each of the two wells 14 and 16, and each well is filled with the 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. Another feature of the present invention is that an asymmetric membrane can be easily formed by introducing different types of lipids into two wells.
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。 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.
次に、各ウェルに水又は水溶液を添加して前記脂質溶液中に水又は水溶液の液滴を形成させる。この様子を図２に模式的に示す。図２は、各ウェルに充填された脂質溶液２０内にマイクロピペット２２を挿入して水又は水溶液の液滴２４を形成する様子を示す。図２は、一方のウェル中に液滴２４が形成され、もう一方のウェルに液滴２４を形成しつつある状態を模式的に示すものである。この工程で液滴を形成する液は、純水であってもよいし、水系緩衝液（主たる溶媒が水である緩衝液）のような水溶液であってもよい。各ウェルに添加する水又は水溶液の量は、特に限定されないが、脂質二重膜を効率良く形成する観点から、各ウェルに充填されている脂質溶液の体積の１倍〜５倍程度が好ましく、さらに好ましくは1.5倍〜３倍程度である。 Next, water or an aqueous solution is added to each well to form droplets of water or an aqueous solution in the lipid solution. This is schematically shown in FIG. FIG. 2 shows a state in which a micropipette 22 is inserted into the lipid solution 20 filled in each well to form a droplet 24 of water or an aqueous solution. FIG. 2 schematically shows a state in which a droplet 24 is formed in one well and the droplet 24 is being formed in the other well. The liquid that forms the droplets in this step may be pure water or an aqueous solution such as an aqueous buffer solution (buffer solution whose main solvent is water). The amount of water or aqueous solution added to each well is not particularly limited, but from the viewpoint of efficiently forming a lipid bilayer membrane, preferably about 1 to 5 times the volume of the lipid solution filled in each well, More preferably, it is about 1.5 to 3 times.
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、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、２つのウェルのいずれか一方に添加される水溶液中に含まれていればよいが、両方のウェルに添加される水溶液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デバイス中の脂質膜に融合させることで使用可能である。 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 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. In addition, protein should just be contained in the aqueous solution added to either one of two wells, but may be contained in the aqueous solution added to both wells. In addition, when the protein retained in the lipid bilayer membrane is fat-soluble, it can be used by fusing a proteoliposome expressing the membrane protein to the lipid membrane in this device.
脂質溶液内で上記液滴を形成すると、脂質溶液が圧迫されて隔壁に押しつけられ、この部分で脂質溶液の層と水層とがある程度の圧力で接することとなる。この際に隔壁の貫通孔内に脂質二重膜が形成される。脂質二重膜は、水又は水溶液の液滴を形成した状態で、通常３分間〜１時間程度放置することにより、隔壁の貫通孔を塞ぐ形で形成される。 When the droplets are formed in the lipid solution, the lipid solution is compressed and pressed against the partition wall, and the lipid solution layer and the aqueous layer come into contact with each other at a certain pressure. At this time, a lipid bilayer membrane is formed in the through hole of the partition wall. The lipid bilayer membrane is formed so as to block the through-holes of the partition walls by leaving it for about 3 minutes to 1 hour, usually in the form of droplets of water or an aqueous solution.
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広げ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。従って、２つのウェル間の電流を測定することが望まれる場合が少なくない。下記実施例で具体的に説明するように、本発明の方法に用いられるDWCは、各ウェルの底部を構成する底板の上面に電極を配置し、この電極をウェル外に接続することにより容易に各ウェルを配線することができる。公知の方法では、各ウェルの上部から電極を浸漬して電気的測定を行っていたが、本発明の好ましい態様では、電極はウェル底部に配置されるので、自動装置を用いた測定が容易になり、さらに機械的刺激が軽減されるという利点がもたらされる。 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 applying various substances to proteins, it is also possible to screen for substances that change the structure of the protein to close or widen the ion channel, and such substances may exhibit some physiological activity. Yes, it can be a drug candidate. Thus, it is often desirable to measure the current between two wells. As will be described in detail in the following examples, the DWC used in the method of the present invention can be easily arranged by arranging an electrode on the upper surface of the bottom plate constituting the bottom of each well and connecting this electrode outside the well. Each well can be wired. In the known method, an electrode is immersed from the top of each well and electrical measurement is performed. However, in a preferred embodiment of the present invention, the electrode is arranged at the bottom of the well, so that measurement using an automatic device is easy. And has the advantage that mechanical irritation is reduced.
DWCは、１枚の基板に１個だけ形成してもよいが、複数形成することもできる。DWCが１枚の基板中に複数個形成された基板を用い、複数のDWC内で同時並行して脂質二重膜を形成してもよい。この場合には、創薬スクリーニング等の測定も各DWCで同時並行して行うことが可能となり、創薬スクリーニング等の効率を大幅に高めることができる。 Although only one DWC may be formed on one substrate, a plurality of DWCs may be formed. A lipid bilayer may be formed simultaneously in a plurality of DWCs using a substrate in which a plurality of DWCs are formed in one substrate. In this case, measurements such as drug discovery screening can be performed simultaneously in each DWC, and the efficiency of drug discovery screening can be greatly increased.
本発明はまた、上記DWCを具備する脂質二重膜形成器具をも提供するものである。この脂質二重膜形成器具の好ましい態様は上記した通りである。 The present invention also provides a device for forming a lipid bilayer membrane comprising the above DWC. The preferred embodiment of the lipid bilayer membrane forming device is as described above.
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.
１． 隔壁の作製
上記の通り、隔壁としては、自己支持性のポリマーフィルムに上記貫通孔を設けたものを好ましく用いることができる。隔壁が自己支持性のポリマーフィルムであれば、DWCを具備する基板を容易に組み立てることができるので有利である。
1. As described above, as the partition wall, a self-supporting polymer film provided with the through-holes can be preferably used. If the partition wall is a self-supporting polymer film, it is advantageous because a substrate having a DWC can be easily assembled.
この実施例では、パリレン（ポリ-p-キシリレン）樹脂にフォトリソグラフィーにより貫通孔を形成したものを作製し、隔壁とした。以下、図３に基づき説明する。なお、パリレン樹脂フィルムをフォトリソグラフィーによりパターニングする手法は周知であり、各工程は常法により容易に行うことができる。 In this example, a parylene (poly-p-xylylene) resin having through holes formed by photolithography was prepared as a partition wall. Hereinafter, a description will be given with reference to FIG. In addition, the method of patterning a parylene resin film by photolithography is well-known, and each process can be easily performed by a conventional method.
図３の(1)に示すように、シリコンウェハ２６上にパリレン樹脂膜２８（厚さ10μm）を化学蒸着によりコーティングした。次に、アルミニウム膜３０及びフォトレジスト膜３２を順次コーティングした。次に貫通孔を形成する部分に選択的に紫外線を露光し、現像してフォトレジスト膜３２に透孔を形成し、このフォトレジスト膜３２をマスクとして下層のアルミニウム膜３０を混酸アルミ液を用いたウェットエッチング法により食刻してアルミニウム膜３０に透孔を形成した。次に酸素プラズマエッチングによりパリレン樹脂膜２８に貫通孔（図１中の１８に対応）を形成した (図１(2))。アルミニウム膜３０を混酸アルミ液で除去した（図１(3))。貫通孔が形成されたパリレン樹脂フィルム２８をシリコンウェハ２６から剥離して隔壁を得た。なお、貫通孔の孔径が１０μm、３０μm、５０μm、１００μm、１５０μm、２５０μm、５００μmである７種類のパリレン樹脂フィルムを作製した。また、各フィルム中、貫通孔は５個形成した。５個の透孔は、正方形の４個の頂点のそれぞれと、正方形の中心にそれぞれ位置するように配置した。 As shown in (1) of FIG. 3, a parylene resin film 28 (thickness 10 μm) was coated on the silicon wafer 26 by chemical vapor deposition. Next, an aluminum film 30 and a photoresist film 32 were sequentially coated. Next, ultraviolet rays are selectively exposed to a portion where a through hole is to be formed, and developed to form a through hole in the photoresist film 32. Using the photoresist film 32 as a mask, the lower aluminum film 30 is used as a mixed acid aluminum liquid. A through-hole was formed in the aluminum film 30 by etching using a wet etching method. Next, through holes (corresponding to 18 in FIG. 1) were formed in the parylene resin film 28 by oxygen plasma etching (FIG. 1 (2)). The aluminum film 30 was removed with a mixed acid aluminum solution (FIG. 1 (3)). The parylene resin film 28 in which the through holes were formed was peeled from the silicon wafer 26 to obtain partition walls. Seven types of parylene resin films having through-hole diameters of 10 μm, 30 μm, 50 μm, 100 μm, 150 μm, 250 μm, and 500 μm were prepared. Moreover, five through-holes were formed in each film. The five through holes were arranged so as to be located at each of the four vertices of the square and at the center of the square.
なお、この実施例の方法では、パリレン樹脂を用いたが、フォトリソグラフィーによるパターニングが可能で自己支持性フィルムを与えることができる他のポリマー（例えばポリイミド等）も用いることができる。また、本発明で規定される貫通孔の孔径であれば、フォトリソグラフィー以外の手法、例えば、マイクロドリルを用いた機械的な手法により形成することも可能である。この場合には、フォトリソグラフィーができない自己支持性の種々のポリマーフィルムも利用可能である。 In addition, although the parylene resin was used in the method of this Example, other polymers (for example, polyimide etc.) which can be patterned by photolithography and can provide a self-supporting film can also be used. Moreover, if it is the hole diameter of the through-hole prescribed | regulated by this invention, it is also possible to form by methods other than photolithography, for example, the mechanical method using a micro drill. In this case, various self-supporting polymer films which cannot be photolithography can be used.
２．DWCを具備する基板の作製
基本的に図１に模式的に示す形状及び構造のDWCを作製した。具体的な構成を図４に模式的に示す。図４の左側の上の図は、平面図であり、その下が側面図である。側面図は、分解組み立て図になっている。右側の図は、隔壁部分の拡大図である。
2. Production of Substrate Comprising DWC Basically, a DWC having the shape and structure schematically shown in FIG. 1 was produced. A specific configuration is schematically shown in FIG. The upper diagram on the left side of FIG. 4 is a plan view, and the lower diagram is a side view. The side view is an exploded view. The figure on the right is an enlarged view of the partition wall.
以下、作製方法を説明する。厚さ3mmのアクリル板10aにドリルで透孔14'を開けた。透孔14'は、最終的にウェル１４（図１参照）を構成するものであるので、その平面形状は、図１に示すウェル１４と同様である。同様に、厚さ3mmのアクリル板10bにドリルで透孔16'を開けた。透孔16'は、最終的にウェル１６（図１参照）を構成するものであるので、その平面形状は、図１に示すウェル１６と同様である。透孔14'及び16'の円形部の直径は4mmであった。アクリル板10aと10bの間に上記１で作製したパリレン樹脂フィルム（隔壁）１２を挟み込んだ。この際、パリレン樹脂フィルム１２を補強するために、パリレン樹脂フィルム１２の両側にそれぞれポリメチルメタクリレート樹脂フィルム３４を添えて挟み込んだ。ポリメチルメタクリレート樹脂フィルム３４は、孔径1.5mmの透孔を有しており、この透孔の中にパリレン樹脂フィルム１２中の５個の貫通孔が位置するように配置した。この状態で、アクリル板10a、ポリメチルメタクリレート樹脂フィルム３４、パリレン樹脂フィルム１２、もう一方のポリメチルメタクリレート樹脂フィルム３４、アクリル板10bを接着剤で接着した。得られた板の下方から、透孔14'及び16'を塞ぐ底板10cを貼り合わせ、透孔14'及び16'の底部を形成してウェル１４及び１６を具備する基板１０（図１）を形成した。この際、底板10cの端部に銀電極３８を形成し、さらにその端部にAg/AgClペーストを塗布してAg/AgCl電極３６を形成し、Ag/AgCl電極を構成した。なお、Ag/AgCl電極３６及び銀電極３８の一部は、各ウェルの底部に露出する位置に形成した。各銀電極３８はさらにDWCの外部に接続した。このようにして、各ウェルの底部に電極を配設したDWCを有する基板を作製した。 Hereinafter, a manufacturing method will be described. A through hole 14 'was drilled in a 3mm thick acrylic plate 10a. Since the through-hole 14 ′ finally constitutes the well 14 (see FIG. 1), the planar shape thereof is the same as that of the well 14 shown in FIG. Similarly, a through-hole 16 ′ was drilled in a 3 mm thick acrylic plate 10b. Since the through-hole 16 ′ finally constitutes the well 16 (see FIG. 1), the planar shape thereof is the same as the well 16 shown in FIG. The diameters of the circular portions of the through holes 14 ′ and 16 ′ were 4 mm. The parylene resin film (partition wall) 12 produced in 1 above was sandwiched between the acrylic plates 10a and 10b. At this time, in order to reinforce the parylene resin film 12, the polymethyl methacrylate resin film 34 was attached to both sides of the parylene resin film 12 and sandwiched. The polymethyl methacrylate resin film 34 has a through hole having a hole diameter of 1.5 mm, and the five through holes in the parylene resin film 12 are arranged in the through hole. In this state, the acrylic plate 10a, the polymethyl methacrylate resin film 34, the parylene resin film 12, the other polymethyl methacrylate resin film 34, and the acrylic plate 10b were bonded with an adhesive. From the lower side of the obtained plate, a bottom plate 10c for closing the through holes 14 'and 16' is bonded to form a bottom portion of the through holes 14 'and 16' to form the substrate 10 having the wells 14 and 16 (FIG. 1). Formed. At this time, a silver electrode 38 was formed at the end of the bottom plate 10c, and an Ag / AgCl paste was applied to the end to form an Ag / AgCl electrode 36, thereby constituting an Ag / AgCl electrode. Part of the Ag / AgCl electrode 36 and the silver electrode 38 was formed at a position exposed at the bottom of each well. Each silver electrode 38 was further connected to the outside of the DWC. In this way, a substrate having a DWC in which an electrode was disposed at the bottom of each well was produced.
なお、上記では、理解を容易にするために１個のDWCに着目して説明したが、実際に作製した基板は、縦長のアクリル板10a中に、同じ形状の透孔14'を縦に８個並べたものと、同様に縦長のアクリル板10b中に、同じ形状の透孔16'を縦に８個並べたものとの間に上記の通りパリレン樹脂フィルム１２とポリメチルメタクリレート樹脂フィルム３４を挟んで貼り合わせたものを２枚作製し、それらをさらに貼り合わせて合計１６個のDWCを１枚の基板中に有するものであった。従って、１６個のDWCについて同時に電気的測定が可能である。そして、各ウェルへの脂質溶液や水溶液の添加は、８つのマイクロピペットを持つロボットにより自動的に行った。また、上記のようにパリレン樹脂フィルムは、貫通孔の孔径が異なる７種類のフィルムを作製したので、基板もこれらの７種類のパリレン樹脂フィルムを配置した７種類の基板を作製した。 In the above description, for ease of understanding, the description has been given focusing on one DWC. However, in the actually manufactured substrate, the vertically-shaped acrylic plate 10a has eight through holes 14 'of the same shape in the vertical direction. The parylene resin film 12 and the polymethylmethacrylate resin film 34 are placed as described above between the one arranged in parallel and the one in which eight pieces of the same shape of the through holes 16 'are arranged vertically in the vertically long acrylic plate 10b. Two sheets were bonded together, and these were further bonded together to have a total of 16 DWCs in one substrate. Therefore, electrical measurement can be performed on 16 DWCs simultaneously. The lipid solution or aqueous solution was automatically added to each well by a robot having eight micropipettes. Moreover, since the parylene resin film produced seven types of films having different through-hole diameters as described above, seven types of substrates were prepared in which these seven types of parylene resin films were arranged.
３．脂質二重膜の形成
卵黄フォスファチジルコリン（EggPC)のデカン溶液(20 mg/mL)８μLずつを各ウェルに加えた。各DWCの一方のウェルに１８μLのリン酸緩衝液(0.1M KCl、溶媒は水)をマイクロピペットで加え液滴を形成した。他方のウェルには、上記緩衝液に0.3μMのα−ヘモリシンを含む水溶液１８μLをマイクロピペットで加え液滴を形成した。この状態で放置することにより、下記電気化学的測定データから明らかなようにパリレン樹脂フィルムの貫通孔部分に、α−ヘモリシンを保持する脂質二重膜が形成された。
3. Formation of lipid bilayer 8 μL of yolk phosphatidylcholine (EggPC) in decane (20 mg / mL) was added to each well. To one well of each DWC, 18 μL of phosphate buffer (0.1 M KCl, solvent is water) was added with a micropipette to form droplets. In the other well, 18 μL of an aqueous solution containing 0.3 μM α-hemolysin was added to the above buffer with a micropipette to form droplets. By leaving in this state, a lipid bilayer membrane holding α-hemolysin was formed in the through-hole portion of the parylene resin film, as is apparent from the following electrochemical measurement data.
４．電気化学的測定
α−ヘモリシンは、生体内では生体膜中に保持されてイオンチャネルを形成するタンパク質であるので、脂質二重膜中に適切に保持されればイオンチャネルを形成して、脂質二重膜の両側が電気的に導通される。従って、DWCの２つのウェル間に直流電圧をかけると、電流が測定される。図５に示すように各DWCを配線し、各ウェル間を流れる電流を経時的に測定した。上記したように作製した基板は１６個のDWCを有するので、１６個のDWCの全てについて同じ測定を行った。なお、図５には示していないが、回路中には、１００ｍＶの印加電圧がかけられている。
4). Electrochemical measurement Since α-hemolysin is a protein that is retained in the biological membrane and forms an ion channel in the living body, it forms an ion channel when properly retained in the lipid bilayer membrane, and the lipid bilayer. Both sides of the heavy membrane are electrically connected. Therefore, when a DC voltage is applied between the two wells of the DWC, the current is measured. As shown in FIG. 5, each DWC was wired and the current flowing between each well was measured over time. Since the substrate fabricated as described above has 16 DWCs, the same measurement was performed for all 16 DWCs. Although not shown in FIG. 5, an applied voltage of 100 mV is applied in the circuit.
結果を図６に示す。α−ヘモリシンを適切に保持する脂質二重膜が貫通孔に形成されると、イオンチャネルが形成されるので、電流が流れる。さらに、上記の通り、貫通孔は５個存在するので、α−ヘモリシンを適切に保持する脂質二重膜が形成された貫通孔の数が増えるごとに電流は増大するので、電流の大きさはステップ状に増大する。なお、図６に示す結果は、貫通孔の孔径が１５０μmであるパリレン樹脂フィルムを有する基板を用いた場合の結果である。 The results are shown in FIG. When a lipid bilayer membrane that appropriately retains α-hemolysin is formed in the through-hole, an ion channel is formed, and thus a current flows. Furthermore, as described above, since there are five through-holes, the current increases as the number of through-holes formed with lipid bilayers that appropriately retain α-hemolysin increases. Increase in steps. In addition, the result shown in FIG. 6 is a result at the time of using the board | substrate which has a parylene resin film whose hole diameter of a through-hole is 150 micrometers.
図６に示すように、１６個のDWCのうち、１４個（4chと16ch以外）でα−ヘモリシンを適切に保持する脂質二重膜が形成された。従って、本発明により、再現性よく脂質二重膜が形成されることが明らかになった。 As shown in FIG. 6, a lipid bilayer membrane that appropriately retains α-hemolysin was formed in 14 (other than 4ch and 16ch) out of 16 DWCs. Therefore, it became clear that the lipid bilayer membrane was formed with good reproducibility by the present invention.
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