JP6376636B2 - Lipid bilayer forming device - Google Patents
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- JP6376636B2 JP6376636B2 JP2013216009A JP2013216009A JP6376636B2 JP 6376636 B2 JP6376636 B2 JP 6376636B2 JP 2013216009 A JP2013216009 A JP 2013216009A JP 2013216009 A JP2013216009 A JP 2013216009A JP 6376636 B2 JP6376636 B2 JP 6376636B2
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Description
本発明は、脂質二重膜を形成するための器具に関する。 The present invention relates to a device for forming a 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。また、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。 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. In drug discovery screening, etc., if an automated apparatus is used to simultaneously form a large number of lipid bilayer membranes and screening can be performed simultaneously using the large number of lipid bilayer membranes formed, It can increase the efficiency and 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.
これを達成するために、本願出願人は先に、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及びこの方法に用いられる器具を発明し、特許出願した（特許文献１）。特許文献１に記載されている方法によれば、前記貫通孔の部分に脂質二重膜が再現性良く形成され、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。しかしながら、特許文献１記載の方法に用いられる脂質二重膜形成器具は、各ウェルをそれぞれ異なる基板内に形成し、隔壁を一対の補強フィルムの間に挟み、合計２個の基板と、合計２枚の補強フィルムと、隔壁とを接着剤で貼り合わせ、さらに各ウェルの底部を塞ぐ基板を接着剤で貼り合わせて作製している。 In order to achieve this, the applicant of the present invention has previously described a lipid bilayer-forming lipid in 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. A step of adding a solution, a step of adding water or an aqueous solution to each well to form droplets of water or an aqueous solution in the lipid solution, and leaving in this state to form a lipid bilayer membrane in the portion of the through-hole Invented a method of forming a lipid bilayer membrane and a device used in this method, including a step of forming a patent (Patent Document 1). According to the method described in Patent Document 1, a lipid bilayer membrane is formed with good reproducibility in the portion of the through-hole, and once formed, the lipid bilayer membrane is being transported and measured in an automated apparatus. It exists stably without being destroyed. However, in the lipid bilayer membrane forming device used in the method described in Patent Document 1, each well is formed in a different substrate, a partition wall is sandwiched between a pair of reinforcing films, and a total of two substrates and 2 in total. A sheet of reinforcing film and a partition wall are bonded together with an adhesive, and a substrate that closes the bottom of each well is bonded with an adhesive.
特許文献１記載の脂質二重膜形成器具は、作製に手間がかかり、また、隔壁を新たな隔壁と交換することはできない。このため、使用により隔壁が劣化した場合や、新品の隔壁を用いて実験を行いたい場合には、器具全体を新たに作製する必要があり、不便でコストもかかるものであった。 The device for forming a lipid bilayer membrane described in Patent Document 1 takes time and effort, and the partition cannot be replaced with a new partition. For this reason, when the partition wall deteriorates due to use or when it is desired to conduct an experiment using a new partition wall, it is necessary to newly prepare the entire device, which is inconvenient and costly.
従って、本発明の目的は、簡単に作製することができ、かつ、隔壁を交換することが可能な脂質二重膜形成器具を提供することである。 Accordingly, an object of the present invention is to provide a device for forming a lipid bilayer membrane that can be easily produced and can exchange a partition wall.
本願発明者らは、鋭意研究の結果、隔壁を一対の支持フィルムで挟持して一体化した隔壁カセット内に隔壁を保持すると共に、ダブルウェルチャンバーを構成する一対のウェルが当接する部分に溝を形成し、この溝に前記隔壁カセットを挿入して、前記一対のウェルを隔壁で仕切るようにすれば、一対のウェルを単一の基板内に形成することができ、隔壁で仕切るために接着操作が不要となり、かつ、溝に挿入した隔壁カセットを新たな隔壁カセットと交換することにより器具形成後でも簡便に隔壁が交換可能となることに想到し、本発明を完成した。 As a result of diligent research, the inventors of the present application have held the partition wall in an integrated partition cassette sandwiched by a pair of support films and formed grooves in the portions where the pair of wells constituting the double well chamber abut. If the partition cassette is inserted into the groove and the pair of wells are partitioned by the partition, the pair of wells can be formed in a single substrate, and the bonding operation is performed to partition the partition by the partition. The present invention has been completed by conceiving that the partition wall can be easily replaced even after the device is formed by replacing the partition wall cassette inserted into the groove with a new partition wall cassette.
すなわち、本発明は、基板と、該基板内に形成された、互いに当接する一対のウェルと、該一対のウェル間に配置された隔壁カセットとを具備し、該隔壁カセットは、孔径が５００ nm〜５００μmの１個又は複数の貫通孔を有する隔壁と、該隔壁を挟持する一対の支持フィルムとを具備し、該隔壁と該一対の支持フィルムとは一体化されており、各支持フィルムには、前記隔壁の前記１個又は複数の貫通孔を内包する透孔が形成されて各支持フィルムの各透孔と前記隔壁の前記１個又は複数の貫通孔とが全体として貫通しており、前記基板の前記一対のウェルの当接部分には溝が設けられており、前記隔壁カセットは、該溝に着脱自在に挿入され、前記隔壁カセットが該溝に挿入されている状態で、前記一対のウェルが前記隔壁を介して互いに接触し、前記隔壁の前記１個又は複数の貫通孔が前記一対のウェルの双方に開口している脂質二重膜形成器具を提供する。 That is, the present invention comprises a substrate, a pair of wells formed in the substrate, and a partition cassette disposed between the pair of wells, and the partition cassette has a hole diameter of 500 nm. A partition wall having one or a plurality of through holes of ˜500 μm and a pair of support films sandwiching the partition wall, and the partition walls and the pair of support films are integrated, A through hole is formed to enclose the one or more through holes of the partition wall, and the through holes of each support film and the one or more through holes of the partition wall pass through as a whole, A groove is provided in a contact portion between the pair of wells of the substrate, and the partition cassette is detachably inserted into the groove, and the pair of partition cassettes is inserted into the groove. Wells are connected to each other through the partition wall. Touch to provide the one or more through holes both in the opening to have a lipid bilayer membrane formation device of the pair of wells of the partition wall.
本発明によれば、特許文献１記載の脂質二重膜形成器具と比べて、遙かに簡便に脂質二重膜形成器具を作製することができ、器具の量産にも適しており、また、器具を形成し、脂質二重膜形成に使用した後、容易に隔壁を交換することができる。 According to the present invention, compared with the lipid bilayer membrane forming device described in Patent Document 1, it is possible to produce a lipid bilayer membrane forming device much more easily, which is suitable for mass production of the device, After the device is formed and used for lipid bilayer formation, the septum can be easily replaced.
本発明の脂質二重膜形成器具の好ましい具体例の模式平面図を図１に示す。図１に模式的に示す具体例では、基板１０中に、互いに当接する一対のウェル１４及び１６が形成され、これらの境界には溝１２が形成されている。溝１２には、後述する隔壁カセットが挿入される。基板の材質は特に限定されないが、アクリル板等のプラスチック板を好適に用いることができる。なお、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、後述する所定の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脂質二重膜を形成することが可能である。 A schematic plan view of a preferred specific example of the device for forming a lipid bilayer membrane of the present invention is shown in FIG. In the specific example schematically shown in FIG. 1, a pair of wells 14 and 16 that are in contact with each other are formed in the substrate 10, and a groove 12 is formed at the boundary between them. A partition cassette described later is inserted into the groove 12. The material of the substrate is not particularly limited, but a plastic plate such as an acrylic plate can be preferably used. 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 will be described later. Other shapes are not a problem as long as they are separated by a predetermined partition wall. 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 about 3 mm to 5 mm, and the depth is preferably about 50% to 200% of the pore diameter, more preferably about 50% to 100%. However, it is possible to form a lipid bilayer membrane that is larger or smaller than this range. It is.
図２に、本発明の脂質二重膜形成器具に用いられる隔壁カセット１８の好ましい１具体例の模式分解斜視図を示す。隔壁カセット１８は、隔壁２０と、該隔壁２０を挟持する一対の支持フィルム２２、２４とを具備する。隔壁２０には、１個又は複数の貫通孔２０ａが形成されている。図２に図示される例では５つの貫通孔２０ａが形成されている。なお、隔壁２０に設けられた貫通孔２０ａ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２０ａ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。貫通孔の孔径は、通常、好ましくは、５００ｎｍ〜５００μm、さらに好ましくは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。隔壁は、例えば、パリレン（ポリ-p-キシリレン）樹脂フィルムのような、好ましくは自己支持性のフィルムにフォトリソグラフィーにより貫通孔を形成することにより作製することができる。もっとも、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、貫通孔の上記範囲の孔径であれば、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。さらに、孔径が１μm未満の貫通孔は、フォトリソグラフィー等により孔径が１μm以上の貫通孔を形成し、この貫通孔の内壁に樹脂を蒸着して孔径を小さくすることにより形成することができる（特開２０１１−１４９８６８号公報）。なお、隔壁の厚さは、特に限定されないが、通常、５μｍ〜２０μｍ程度であり、市販のパリレン樹脂フィルムを好適に利用することができる。 FIG. 2 is a schematic exploded perspective view of a preferred specific example of the partition wall cassette 18 used in the lipid bilayer membrane forming device of the present invention. The partition cassette 18 includes a partition 20 and a pair of support films 22 and 24 that sandwich the partition 20. The partition wall 20 is formed with one or a plurality of through holes 20a. In the example shown in FIG. 2, five through holes 20a are formed. In addition, the number of the through-holes 20a provided in the partition wall 20 may be one or plural, and is usually about 1 to 10 and preferably about 1 to 6. When there are a plurality of through-holes 20a, lipid bilayer membranes are not necessarily formed in all through-holes, but lipid bilayer membranes are formed only in through-holes in which the lipid solution is strongly pressed by droplets. The The diameter of the through hole is usually preferably 500 nm to 500 μm, more 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. The partition walls can be produced by forming through holes on a preferably self-supporting film such as a parylene (poly-p-xylylene) resin film, for example, by photolithography. However, other polymers (such as polyimide) that 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 said range of a through-hole, 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. Furthermore, a through hole having a hole diameter of less than 1 μm can be formed by forming a through hole having a hole diameter of 1 μm or more by photolithography or the like, and depositing a resin on the inner wall of the through hole to reduce the hole diameter (special feature). No. 2011-149868). In addition, although the thickness of a partition is not specifically limited, Usually, it is about 5 micrometers-20 micrometers, and a commercially available parylene resin film can be utilized suitably.
隔壁２０と一対の支持フィルム２２、２４とは一体化されている。ここで、「一体化されている」とは、隔壁２０と一対の支持フィルム２２、２４とが互いの位置関係を維持した状態で固定されていることを意味し、これら３つの部材は常に行動を共にする。固定は、例えば、隔壁２０を支持フィルム２２、２４の間に挟み込んだ状態で支持フィルム２２、２４を接着剤で接着することにより行うことができる。隔壁２０も接着剤で支持フィルム２２、２４に接着してもよいが、接着することなく、支持フィルム２２、２４の間に挟持するだけでも隔壁２０を摩擦力により固定することが可能である。支持フィルム２２と２４は、接着剤で容易に接着できるが、熱融着等、他の方法で貼り合わせてもよい。支持フィルム２２と２４には、それぞれ透孔２２ａ、２４ａが形成されている。透孔２２ａ、２４ａは、隔壁２０の前記１個又は複数の貫通孔２０ａを内包するものであり、従って、各支持フィルム２２、２４の各透孔２２ａ、２４ａと隔壁２０の１個又は複数の貫通孔２０ａとが全体として貫通している。その結果、隔壁カセット１８を上記溝１２に挿入した状態では、１個又は複数の貫通孔２０ａはそれぞれ、ウェル１４及び１６の双方に開口している（図３参照）。なお、支持フィルム２２、２４は、任意の材料で構成することができ、例えば、ポリメチルメタクリレート樹脂フィルムのようなプラスチックフィルムを好適に利用することができる。また、支持フィルム２２、２４の厚さは、限定されないが、通常、0.1 mm〜0.3 mm程度である。隔壁カセット１８を上記溝１２に挿入した状態の断面図を図３に模式的に示す。図３は、図１の3-3'線切断部端面図である。ただし、図１には図示されていない隔壁カセット１８も図示されている。なお、明瞭性のため、隔壁２０の貫通孔２０ａは１つだけ図示しており、また、各部材のサイズの比率は実物とは大きく異なる。 The partition wall 20 and the pair of support films 22 and 24 are integrated. Here, “integrated” means that the partition wall 20 and the pair of support films 22 and 24 are fixed in a state in which the mutual positional relationship is maintained, and these three members always act. Together. The fixing can be performed, for example, by adhering the support films 22 and 24 with an adhesive in a state where the partition wall 20 is sandwiched between the support films 22 and 24. The partition wall 20 may also be adhered to the support films 22 and 24 with an adhesive, but the partition wall 20 can be fixed by frictional force only by being sandwiched between the support films 22 and 24 without being bonded. The support films 22 and 24 can be easily bonded with an adhesive, but may be bonded together by other methods such as heat fusion. Through holes 22a and 24a are formed in the support films 22 and 24, respectively. The through holes 22a and 24a contain the one or more through holes 20a of the partition wall 20. Therefore, the through holes 22a and 24a of the support films 22 and 24 and one or more of the partition walls 20 are included. The through hole 20a penetrates as a whole. As a result, in the state where the partition cassette 18 is inserted into the groove 12, one or a plurality of through holes 20a are opened in both the wells 14 and 16 (see FIG. 3). In addition, the support films 22 and 24 can be comprised by arbitrary materials, For example, a plastic film like a polymethylmethacrylate resin film can be utilized suitably. Moreover, although the thickness of the support films 22 and 24 is not limited, Usually, it is about 0.1 mm-0.3 mm. A sectional view of the partition cassette 18 inserted into the groove 12 is schematically shown in FIG. 3 is an end view taken along the line 3-3 ′ of FIG. However, a partition cassette 18 not shown in FIG. 1 is also shown. For the sake of clarity, only one through hole 20a of the partition wall 20 is shown, and the size ratio of each member is greatly different from the actual one.
各ウェルの底面には、電極（図示せず）を設けることができる。電極は、後述する方法により隔壁の貫通孔に脂質二重膜を形成した後、該脂質二重膜を介して各ウェル間に流れる電流を測定することなどに役立つ。この場合、電極は、ウェル外部の電流計等に接続されるが、この電極とウェル外とを接続する配線を、基板１０の裏面上に形成することが好ましい。特許文献１記載の器具では、基板の上面に配線を設けているが、裏面に配線を設けることにより、デザインの自由度が大きくなり、量産化しやすくなる。 An electrode (not shown) can be provided on the bottom surface of each well. The electrode is useful for, for example, measuring a current flowing between each well through the lipid bilayer after forming a lipid bilayer in the through-hole of the partition wall by a method described later. In this case, the electrode is connected to an ammeter or the like outside the well, but it is preferable to form a wiring connecting the electrode and the outside of the well on the back surface of the substrate 10. In the instrument described in Patent Document 1, wiring is provided on the upper surface of the substrate. However, providing wiring on the back surface increases the degree of freedom in design and facilitates mass production.
上記した脂質二重膜形成器具は、ドリルにより上側基板にウェル１４、１６となる透孔と溝１２を形成し、下側から底板を貼り付け、溝１２に上記隔壁カセット１８を挿入することにより形成することができる。この場合、上側基板と底板により基板１０が形成されることになる。さらに、各ウェルの底部に電極を配設し、これをウェル外と接続することにより、例えば、各ウェル間を流れる電流を測定することが可能になる。この電極の配設及び接続方法の１具体例を図４に基づき説明する。図４のａ及びｂに示すとおり、上記した２つのウェル１４及び１６の中央部にそれぞれスルーホール１４ａ及び１６ａを形成する。なお、図４のｂは、図４のａにおけるｂ−ｂ’切断部端面図である。次に、図４のｃに示すように、基板１０の裏面にAg/AgClペースト１７を全面塗布し、スルーホール１４ａ及び１６ａ内にもAg/AgClペースト１７を充填する。次に、基板１０の裏面上のAg/AgClペースト１７を拭き取る。これにより、スルーホール１４ａ及び１６ａ内のみにAg/AgClペーストが残留し、これによって、ウェル１４及び１６の底部にそれぞれ電極１７’が形成される（図４のｄ）。電極１７’は、スルーホール１４ａ及び１６ａをそれぞれ介して基板１０の裏側にも露出している。従って、例えば、図４のｅに示すように、基板１０の裏面上に例えば、銀蒸着等により配線１９を形成して、ウェル外と接続することができる。各配線１９の端部には、所望によりスルーホールを形成して、例えば、該スルーホールの内部並びに該スルーホールの表面及び裏面周縁部にAg/AgClペーストを塗布してターミナル２１とすることができる（下記実施例参照）。この場合、ターミナル２１は、基板１０の表面側にも形成されるので、微小電流測定アンプ等の測定機器を基板の表面側にも接続することができる。このように、基板の裏面側に配線を形成することにより、配線作製の自由度を大きくすることができ、量産に有利である。特に、後述のように、１枚の基板に複数のウェル対を形成する場合には、基板表面に配線を形成するよりも遙かに配線がしやすくなる。 The above-described lipid bilayer membrane-forming device is formed by forming through holes and grooves 12 to become wells 14 and 16 on the upper substrate by a drill, attaching a bottom plate from the lower side, and inserting the partition cassette 18 into the groove 12. Can be formed. In this case, the substrate 10 is formed by the upper substrate and the bottom plate. Furthermore, by arranging an electrode at the bottom of each well and connecting it to the outside of the well, for example, it is possible to measure the current flowing between the wells. One specific example of this electrode arrangement and connection method will be described with reference to FIG. As shown in FIGS. 4a and 4b, through-holes 14a and 16a are formed in the central portions of the two wells 14 and 16, respectively. 4B is an end view taken along the line bb ′ in FIG. 4A. Next, as shown in FIG. 4c, an Ag / AgCl paste 17 is applied over the entire back surface of the substrate 10, and the through holes 14a and 16a are filled with the Ag / AgCl paste 17 as well. Next, the Ag / AgCl paste 17 on the back surface of the substrate 10 is wiped off. As a result, the Ag / AgCl paste remains only in the through holes 14a and 16a, and thereby electrodes 17 'are formed on the bottoms of the wells 14 and 16, respectively (d in FIG. 4). The electrode 17 ′ is also exposed on the back side of the substrate 10 through the through holes 14a and 16a. Therefore, for example, as shown in e of FIG. 4, the wiring 19 can be formed on the back surface of the substrate 10 by, for example, silver vapor deposition and connected to the outside of the well. A through hole is formed at the end of each wiring 19 as desired. For example, an Ag / AgCl paste is applied to the inside of the through hole and the front and back peripheral portions of the through hole to form the terminal 21. Yes (see examples below). In this case, since the terminal 21 is also formed on the surface side of the substrate 10, a measuring device such as a minute current measuring amplifier can be connected to the surface side of the substrate. Thus, by forming the wiring on the back surface side of the substrate, the degree of freedom of wiring fabrication can be increased, which is advantageous for mass production. In particular, as will be described later, when a plurality of well pairs are formed on a single substrate, wiring is much easier than when wiring is formed on the substrate surface.
上記した本発明の脂質二重膜形成器具を使用して脂質二重膜を形成する方法自体は、特許文献１に記載された公知の方法と同様である。すなわち、上記した２つのウェル１４及び１６（以下、この一対のウェルをダブルウェルチャンバー又はDWCと呼ぶことがある）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、卵黄フォスファチジルコリン、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することもできる。 The method itself of forming a lipid bilayer membrane using the above-described lipid bilayer membrane forming device of the present invention is the same as the known method described in Patent Document 1. That is, a lipid bilayer-forming lipid solution is added to each of the two wells 14 and 16 (hereinafter, the pair of wells may be referred to as a double well chamber or DWC), and the lipid is added to each well. Fill the 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 used phospholipids in this field, such as egg yolk phosphatidylcholine, diphytanoyl phosphatidylcholine (DPhPC), dipalmitoyl phosphatidylcholine, palmitoyl oleoylphosphat Preferable examples include fatidylcholine (1-Palmitoyl 2-Oleoylphosphatidylcholine, POPC), dioleoylphosphatidylcholine (DOPC), and the like. Since many of these are commercially available, commercially available products can be preferably used. Moreover, 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。添加はマイクロピペットを用いて容易に行うことができる。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. The addition can be easily performed using a micropipette. 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.
本発明の器具により形成される脂質二重膜は、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 formed by the device of the present invention is used to examine the properties and functions of proteins in the state retained in the lipid bilayer membrane, or to screen for ligands that bind to the protein and change its physiological activity. 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, an aqueous protein solution is preferably used as the aqueous solution, and an aqueous solution containing the protein in an aqueous buffer is more preferably used. 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。このための電極及び配線については上記したとおりである。 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. The electrode and wiring for this purpose are as described above.
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。また、１つのウェルの両側にそれぞれウェルを配置したり、１つのウェルを囲むように３つのウェルを配置したりすることにより、単一のウェルが、複数のウェルと同時にそれぞれDWCを形成するようにウェルを配置することも可能である。 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. In addition, by arranging wells on both sides of one well or by arranging three wells so as to surround one well, a single well forms a DWC simultaneously with a plurality of wells. It is also possible to arrange the wells.
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 脂質二重膜形成器具の作製
１． 隔壁の作製
市販のパリレン樹脂フィルム（厚さ１０μm）に、特許文献１に記載された方法により、フォトリソグラフィーを用いて直径100μｍ又は150μmの貫通孔を図２に示すように５個形成した。
Example 1 Production of a device for forming a lipid bilayer membrane Production of Partition Walls Five through-holes having a diameter of 100 μm or 150 μm were formed in a commercially available parylene resin film (thickness 10 μm) by photolithography using the method described in Patent Document 1, as shown in FIG.
２． DWCの作製
厚さ３mmの第１のアクリル板（上側基板）に、ドリルで図１に示すDWCと溝を形成した。DWCの直径は4mm、溝の長さは6 mm、幅は0.45 mmであった。次いで、アクリル板の下部に第２のアクリル板（底板）を貼り付けてDWCの底部を形成した。次に図４に示すように、各ウェルの底部にスルーホールを形成し、基板裏面にAg/AgClペーストを塗布して各スルーホール内にAg/AgClペーストを充填して各ウェルの底部に電極を形成した。基板裏面に、上記電極と接続する配線を銀蒸着により形成した。各配線の端部には、底板にスルーホールを形成し、スルーホール内部に及びその表面及び裏面の周縁部にAg/AgClおよびAgペーストを塗布してターミナルとした。
2. Production of DWC A DWC and a groove shown in FIG. 1 were formed on a first acrylic plate (upper substrate) having a thickness of 3 mm by a drill. The diameter of the DWC was 4 mm, the groove length was 6 mm, and the width was 0.45 mm. Next, a second acrylic plate (bottom plate) was attached to the lower part of the acrylic plate to form the bottom of the DWC. Next, as shown in FIG. 4, a through hole is formed at the bottom of each well, an Ag / AgCl paste is applied to the back of the substrate, an Ag / AgCl paste is filled into each through hole, and an electrode is formed at the bottom of each well. Formed. A wiring connected to the electrode was formed on the back surface of the substrate by silver vapor deposition. At the end of each wiring, through holes were formed in the bottom plate, and Ag / AgCl and Ag paste were applied to the inside of the through holes and to the peripheral portions of the front and back surfaces to form terminals.
実施例２ 脂質二重膜の形成
EXTRASYNTHESEより購入した卵黄フォスファチジルコリン(EggPC)のデカン溶液(SIGMA Aldrich, 20 mg/ml) 3μLずつを各ウェルに滴下した。続いて、一方のウェルに16 μLのバッファ(リン酸緩衝液 1M KCl)を，もう一方にチャネルを形成する膜タンパク質であるアルファヘモリシン（αHL、SIGMA Aldrich, 30 nM)が混合されたバッファを滴下した。器具にTecella社の微小電流測定アンプであるPICO 2を接続し、ウェル間に120mVの電圧を印加し、サンプリングレート500 kHz、フィルター約50 kHzの条件下で電流測定を行いながらしばらく静置すると、脂質二分子膜が形成されαHLが再構成されチャネル電流が確認された。結果を図５に示す。図５中、横軸は時間、縦軸はウェル間の電流を示す。
Example 2 Formation of a lipid bilayer membrane
3 μL of a yolk phosphatidylcholine (EggPC) decane solution (SIGMA Aldrich, 20 mg / ml) purchased from EXTRASYNTHESE was added dropwise to each well. Next, 16 μL of buffer (phosphate buffer 1 M KCl) is added to one well, and the other is mixed with alpha hemolysin (αHL, SIGMA Aldrich, 30 nM), which is a membrane protein that forms a channel. It was dripped. Connect PICO 2 which is a micro current measurement amplifier from Tecella to the instrument, apply a voltage of 120mV between the wells, leave it for a while while measuring current under the condition of sampling rate 500 kHz, filter about 50 kHz, A lipid bilayer was formed, αHL was reconstituted, and channel current was confirmed. The results are shown in FIG. In FIG. 5, the horizontal axis represents time, and the vertical axis represents the current between wells.
１０ 基板
１２ 溝
１４ ウェル
１６ ウェル
１７ Ag/AgClペースト
１８ 隔壁カセット
１９ 配線
２０ 隔壁
２１ ターミナル
２２ 支持フィルム
２４ 支持フィルム
10 substrate 12 groove 14 well 16 well 17 Ag / AgCl paste
18 Bulkhead cassette 19 Wiring 20 Bulkhead 21 Terminal 22 Support film 24 Support film
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