JP2016165233A - Cell-capturing chip and cell-capturing method - Google Patents
Cell-capturing chip and cell-capturing method Download PDFInfo
- Publication number
- JP2016165233A JP2016165233A JP2015045829A JP2015045829A JP2016165233A JP 2016165233 A JP2016165233 A JP 2016165233A JP 2015045829 A JP2015045829 A JP 2015045829A JP 2015045829 A JP2015045829 A JP 2015045829A JP 2016165233 A JP2016165233 A JP 2016165233A
- Authority
- JP
- Japan
- Prior art keywords
- cell
- hole
- hydrophilicity
- substrate
- plasma
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Abstract
Description
本発明は、単位細胞毎に細胞の性状や機能などの解析を行うための細胞捕捉チップ及び細胞捕捉方法に関する。 The present invention relates to a cell trapping chip and a cell trapping method for analyzing cell properties and functions for each unit cell.
従来、下記特許文献１〜４に開示されているように、基板に多数の孔を２次元配列して、各孔に細胞を捕捉することで、孔の座標により細胞を特定してその性状や機能の分析を行うことが知られている。
特許文献１において、Ｓｉ基板に細胞を捕捉する碗状凹部とその底部に連続する吸引孔から成る多数の貫通孔を２次元配列した細胞捕捉チップが知られている。この細胞捕捉チップでは、吸引孔からの吸引により細胞が碗状凹部に捕捉される。また、特許文献２によると、特許文献１に開示された構造と同様に、碗状凹部とその底部に連続する径のより小さな貫通孔とを有した構造において、碗状凹部の内壁面の一部は疎水処理が、貫通孔の内壁面には親水処理が施されている。貫通孔に液体が入り込む毛細管現象を利用して細胞を碗状凹部に捕捉するものである。
Conventionally, as disclosed in the following Patent Documents 1 to 4, a plurality of holes are two-dimensionally arranged on a substrate, and cells are captured in each hole. It is known to perform functional analysis.
In Patent Document 1, there is known a cell trapping chip in which a plurality of through-holes including a bowl-shaped recess for trapping cells on a Si substrate and suction holes continuous at the bottom thereof are two-dimensionally arranged. In this cell trapping chip, cells are trapped in the bowl-shaped recess by suction from the suction hole. Further, according to Patent Document 2, similar to the structure disclosed in Patent Document 1, in a structure having a bowl-shaped recess and a through-hole having a smaller diameter continuous with the bottom, one of the inner wall surfaces of the bowl-shaped recess is provided. The part is subjected to hydrophobic treatment, and the inner wall surface of the through hole is subjected to hydrophilic treatment. The cells are trapped in the bowl-shaped recess using a capillary phenomenon in which liquid enters the through-hole.
また、特許文献３においては、基板をＰＥＴ樹脂として、２次元配列させる孔を、細胞を取り入れる側の開口の径が液体を流出させる側の開口の径よりも小さい反すり鉢状とし、細胞懸濁液を孔に導入する流路内面を非イオン性活性剤で処理することが行われている。これにより孔の吐出口から吸引することによる細胞の破壊の防止と、導入流路に細胞が付着することが防止されている。 Further, in Patent Document 3, the substrate is made of PET resin, and the holes to be two-dimensionally arranged are formed in a mortar shape in which the diameter of the opening on the cell receiving side is smaller than the diameter of the opening on the liquid discharging side, Treatment of the inner surface of the flow channel for introducing the liquid into the holes with a nonionic active agent is performed. This prevents the destruction of the cells by suction from the discharge port of the hole and prevents the cells from adhering to the introduction channel.
さらに、特許文献４においては、細胞を捕捉する孔を２次元配列させたマイクロアレイチップにおいて、基板をポリスチレンなどの疎水性材料で構成した場合には、基板の表面を酸素プラズマ処理などの親水化処理を施すことが望ましく、基板をガラスなどの親水材料で構成した場合には、基板表面を親水化処理することが必要ではないとしている。 Further, in Patent Document 4, in a microarray chip in which holes for capturing cells are two-dimensionally arranged, when the substrate is made of a hydrophobic material such as polystyrene, the surface of the substrate is subjected to a hydrophilic treatment such as oxygen plasma treatment. In the case where the substrate is made of a hydrophilic material such as glass, it is not necessary to hydrophilize the substrate surface.
しかしながら、上記特許文献１、３に開示のマイクロアレイチップは、孔の吐出口から吸引することで、細胞を孔に捕捉するチップであるので、細胞に損傷を与えるという問題があった。また、特許文献２によると細胞を捕捉する径の大きな碗状凹部とその底部に連続する径のより小さな貫通孔とを設けることが必要であり、さらには、碗状凹部には疎水処理、貫通孔には親水処理を必要とするために、加工が複雑になるという問題がある。また、特許文献４に開示のマイクロアレイチップは、樹脂製基板の表面にプラズマを照射して、接触角が所定範囲に制御された親水性を持たせることで、基板表面に細胞懸濁液を滴下して孔に細胞を捕捉するようにしている。しかしながら、基板表面における細胞懸濁液の流動性は向上しても、孔内部での親水性については示唆がなく、細胞の捕捉効率が未だ高くないという問題がある。 However, since the microarray chip disclosed in Patent Documents 1 and 3 is a chip that captures a cell in a hole by suction from the discharge port of the hole, there is a problem in that the cell is damaged. Further, according to Patent Document 2, it is necessary to provide a large bowl-shaped recess for capturing cells and a through-hole having a smaller diameter continuous with the bottom thereof. Since the hole requires hydrophilic treatment, there is a problem that processing becomes complicated. In addition, the microarray chip disclosed in Patent Document 4 irradiates plasma on the surface of a resin substrate to give hydrophilicity with a contact angle controlled within a predetermined range, thereby dropping a cell suspension on the surface of the substrate. And trap the cells in the pores. However, even though the fluidity of the cell suspension on the substrate surface is improved, there is no suggestion about the hydrophilicity inside the pores, and there is a problem that the cell capture efficiency is not yet high.
そこで、本発明は、上記の課題を解決するためになされたものであり、細胞に損傷を与えることなく細胞を捕捉し、細胞捕捉チップの孔における細胞捕捉率を向上させることを目的とする。 Therefore, the present invention has been made to solve the above-described problems, and an object of the present invention is to capture cells without damaging the cells and improve the cell capture rate in the pores of the cell capture chip.
本発明は、単一細胞を基板上に捕捉する細胞捕捉チップにおいて、基板と、基板の両面を構成する第１面から第２面に向けて貫通し、基板面に対して２次元配列された貫通孔とを有し、貫通孔は第１面から第２面に向けて径が連続して小さくなる円錐台形状であって、少なくともその貫通孔の内側面はプラズマ照射により、照射前の基板の親水性よりも高い親水性を有することを特徴とする細胞捕捉チップである。 The present invention relates to a cell trapping chip for capturing a single cell on a substrate, which penetrates from the first surface to the second surface constituting both surfaces of the substrate and the substrate, and is two-dimensionally arranged with respect to the substrate surface. The through hole has a truncated cone shape with a diameter continuously decreasing from the first surface toward the second surface, and at least the inner surface of the through hole is irradiated with plasma so that the substrate before irradiation. It is a cell capture chip characterized by having hydrophilicity higher than the hydrophilicity of.
本発明において、プラズマ照射による親水処理は、ラジカル（酸素ラジカルなど）による表面洗浄による表面からの疎水基の離脱、付着した不純物の除去、表面への親水基の付着、表面改質などの概念を含む処理である。また、基板には、ガラス基板の他、ステンレス、シリコン、ＰＥＴなどの樹脂を用いることができる。
本発明において、第２面はプラズマ照射による親水処理が施されていることが望ましい。このときに、貫通孔の内側面の親水性と第２面の親水性との共同作用により、細胞懸濁液の貫通孔への流入と、溶媒の貫通孔からの排出を促進でき、細胞の捕捉率を向上させることができる。また、貫通孔の内側面における親水性は、第２面に近い程、高いことが望ましい。また、貫通孔の内側面は、第２面に近い程、親水基の密度が大きいことが望ましい。これらの親水度又は親水基の分布のときに捕捉率はさらに向上するものと予測される。これらの親水度や親水基の分布は、第２面の側からのプラズマ照射により形成されると考えられる。また、貫通孔の内側面の親水性は、第１面及び第２面の両側からのプラズマ照射により形成されたものでも良い。さらに、第１面は、貫通孔が配列され、細胞懸濁液を滴下する領域は親水性を有し、他の領域はその領域に比べて疎水性を有することが望ましい。両面からプラズマを照射して、且つ、第１面に親水性に差を持たせる場合に、捕捉率は最大となる。
In the present invention, the hydrophilic treatment by plasma irradiation is based on the concepts such as removal of hydrophobic groups from the surface by surface cleaning with radicals (oxygen radicals, etc.), removal of attached impurities, attachment of hydrophilic groups to the surface, surface modification, etc. It is processing to include. In addition to a glass substrate, a resin such as stainless steel, silicon, or PET can be used as the substrate.
In the present invention, the second surface is preferably subjected to a hydrophilic treatment by plasma irradiation. At this time, the joint action of the hydrophilicity of the inner surface of the through-hole and the hydrophilicity of the second surface can promote the inflow of the cell suspension into the through-hole and the discharge of the solvent from the through-hole. The capture rate can be improved. Further, it is desirable that the hydrophilicity on the inner surface of the through hole is higher as it is closer to the second surface. In addition, it is desirable that the inner surface of the through hole has a higher density of hydrophilic groups as it is closer to the second surface. It is expected that the capture rate is further improved when the degree of hydrophilicity or the distribution of hydrophilic groups is present. These hydrophilicity and hydrophilic group distributions are considered to be formed by plasma irradiation from the second surface side. The hydrophilicity of the inner surface of the through hole may be formed by plasma irradiation from both sides of the first surface and the second surface. Furthermore, it is desirable that the first surface has through holes arranged therein, the region where the cell suspension is dropped is hydrophilic, and the other region is more hydrophobic than that region. The capture rate is maximized when plasma is irradiated from both sides and the first surface has a difference in hydrophilicity.
他の発明は、方法発明であり、単一細胞を基板上に捕捉する細胞捕捉方法において、基板の両面を構成する第１面から第２面に向けて貫通し、直径が第１面から第２面に向けて小さくなる円錐台形状である、基板面に対して２次元配列された貫通孔を有する基板に対して、少なくとも第２面からプラズマを照射して、貫通孔の内面に親水処理を施し、基板の第１面に細胞懸濁液を滴下して、貫通孔に単一細胞を捕捉することを特徴とする細胞捕捉方法である。 Another invention is a method invention, in the cell capturing method for capturing a single cell on a substrate, penetrating from a first surface to a second surface constituting both surfaces of the substrate, and having a diameter from the first surface to the second surface. A substrate having through-holes that are two-dimensionally arranged with respect to the substrate surface, which has a truncated cone shape that decreases toward two surfaces, is irradiated with plasma from at least the second surface, and the inner surface of the through-holes is subjected to a hydrophilic treatment. And a cell suspension is dropped onto the first surface of the substrate to capture a single cell in the through-hole.
この方法発明においては、細胞懸濁液は、貫通孔の内側面における親水性を利用して貫通孔の第２面側に導くことができる。また、第２面にも親水性処理が施されていることから、貫通孔から溶媒の排出を促進させることができる。また、貫通孔は、第２面に近い程、親水性を高くすることで、捕捉率を向上させ得ると考えられる。また、プラズマ照射は、第１面及び第２面の両側から照射されても良い。両面から照射する場合に細胞の捕捉率を最も大きくすることができる。さらに、第１面におけるプラズマ照射は、細胞懸濁液を滴下する領域のみ実行して、その領域の親水性を他の領域よりも高くすることが望ましい。 In this method invention, the cell suspension can be guided to the second surface side of the through hole by utilizing the hydrophilicity on the inner surface of the through hole. In addition, since the hydrophilic treatment is applied to the second surface, the discharge of the solvent from the through hole can be promoted. Moreover, it is thought that a through-hole can improve a capture rate by making hydrophilic property high, so that it is near the 2nd surface. Further, the plasma irradiation may be performed from both sides of the first surface and the second surface. When irradiating from both sides, the cell capture rate can be maximized. Furthermore, it is desirable that the plasma irradiation on the first surface is performed only in the region where the cell suspension is dropped, and the hydrophilicity of the region is made higher than that in other regions.
本発明によれば、貫通孔は第１面から第２面に向けて径が連続して小さくなる円錐台形状であって、少なくともその貫通孔の内側面はプラズマ照射により、照射前の基板の親水性よりも高い親水性を有している。このため、細胞懸濁液を貫通孔の内部に容易に導き、溶媒を排出して細胞を貫通孔に効果的に捕捉することができ、細胞の捕捉率を向上させることができる。吸引などの外力を用いていないので、細胞に損傷を与えることがない。 According to the present invention, the through-hole has a truncated cone shape whose diameter continuously decreases from the first surface toward the second surface, and at least the inner surface of the through-hole is formed by plasma irradiation, so that the substrate before irradiation is irradiated. It has higher hydrophilicity than hydrophilicity. For this reason, a cell suspension can be easily guide | induced to the inside of a through-hole, a solvent can be discharged | emitted, a cell can be effectively capture | acquired by a through-hole, and the capture rate of a cell can be improved. Since external force such as suction is not used, cells are not damaged.
また、第２面がプラズマ照射による親水処理が施されていると、貫通孔に流入した細胞懸濁液の溶媒の第２面への排出を促進させることができる。
また、貫通孔の内側面における親水性を第２面に近い程、高くすることが、第２面側の開口から溶媒を排出することを促進し、細胞の捕捉率をより向上させているものと推定される。
In addition, when the second surface is subjected to a hydrophilic treatment by plasma irradiation, the discharge of the solvent of the cell suspension that has flowed into the through hole to the second surface can be promoted.
In addition, increasing the hydrophilicity on the inner surface of the through-hole closer to the second surface promotes the discharge of the solvent from the opening on the second surface side and further improves the cell capture rate. It is estimated to be.
方法発明においては、基板の第１面から第２面に向けて径が連続して小さくなる円錐台形状の貫通孔の内側面を、少なくとも第２面からプラズマを照射して、貫通孔の内面に親水処理を施し、基板の第１面に細胞懸濁液を滴下して、貫通孔に単一細胞を捕捉するようにしている。このため、細胞懸濁液が貫通孔の内部を流れ易くなり、細胞の捕捉率が向上するする。また、貫通孔の吐出口から吸引するなどの外力を用いていないので、捕捉する細胞を損傷させることがない。 In the method invention, the inner surface of the frustoconical through hole whose diameter continuously decreases from the first surface to the second surface of the substrate is irradiated with plasma from at least the second surface, and the inner surface of the through hole The substrate is subjected to a hydrophilic treatment, and a cell suspension is dropped onto the first surface of the substrate so that a single cell is captured in the through hole. For this reason, it becomes easy for the cell suspension to flow inside the through hole, and the capture rate of the cells is improved. In addition, since an external force such as suction from the discharge port of the through hole is not used, the captured cells are not damaged.
また、細胞懸濁液は、貫通孔の内側面における親水性が第２面に近い程高いことを利用して貫通孔の第２面側に導くことにより、貫通孔の第２面側の開口からの溶媒の排出が促進されるので、細胞の捕捉率をより向上させることができる。 The cell suspension is guided to the second surface side of the through hole by utilizing the fact that the hydrophilicity on the inner surface of the through hole is closer to the second surface, thereby opening the second surface side of the through hole. Since the discharge of the solvent from the cell is promoted, the cell capture rate can be further improved.
以下、本発明を具体的な一実施例に基づいて説明する。本発明は下記の実施例に限定されるものではない。 Hereinafter, the present invention will be described based on a specific example. The present invention is not limited to the following examples.
［細胞捕捉チップの構成］
本実施例は基板にガラス基板を用いた例である。図１は、細胞捕捉チップ１の平面図であり、図２はその断面図ある。細胞捕捉チップ１は、ガラス基板１０に後述する貫通孔を形成し、プラズマ処理を施すことにより形成される。ガラス基板１０は、細胞懸濁液を滴下して細胞を捕捉する貫通孔２０が２次元配列された捕捉領域Ｓ１と、貫通孔２０の存在しない非捕捉領域Ｓ２とを有している。厚さ１４０μｍのガラス基板１０の捕捉領域Ｓ１において、ｘ軸方向に１０列、ｙ方向に１０行の２次元配列された１００個の貫通孔２０が形成されている。ガラス基板１０は表面である第１面１１と裏面である第２面１２とを有し、貫通孔２０は第１面１１上の第１開口２１と第２面１２上の第２開口２２とを有している。第１面１１は細胞懸濁液を滴下する面、第２面１２は細胞懸濁液を滴下しない面で溶媒を排出する側の面である。貫通孔２０は円錐台形状であり、第１開口２１の直径は２５μｍ、第２開口２２の直径は６μｍ、高さは１４０μｍである。この貫通孔２０の内側面２３と、第１面１１の捕捉領域Ｓ１、又は、第２面１２には後述するプラズマ照射により親水性処理が施されている。
[Configuration of cell capture chip]
In this embodiment, a glass substrate is used as the substrate. FIG. 1 is a plan view of the cell trapping chip 1, and FIG. 2 is a cross-sectional view thereof. The cell trapping chip 1 is formed by forming a through-hole described later in the glass substrate 10 and performing a plasma treatment. The glass substrate 10 has a capture region S1 in which through holes 20 for capturing cells by dropping a cell suspension are two-dimensionally arranged, and a non-capture region S2 in which the through holes 20 are not present. In the capture region S1 of the glass substrate 10 having a thickness of 140 μm, 100 through-holes 20 that are two-dimensionally arranged in 10 columns in the x-axis direction and 10 rows in the y-direction are formed. The glass substrate 10 has a first surface 11 that is a front surface and a second surface 12 that is a back surface, and the through hole 20 includes a first opening 21 on the first surface 11 and a second opening 22 on the second surface 12. have. The first surface 11 is a surface on which the cell suspension is dripped, and the second surface 12 is a surface on which the cell suspension is not dripped, and the surface on which the solvent is discharged. The through hole 20 has a truncated cone shape. The diameter of the first opening 21 is 25 μm, the diameter of the second opening 22 is 6 μm, and the height is 140 μm. The inner surface 23 of the through-hole 20 and the capturing region S1 of the first surface 11 or the second surface 12 are subjected to hydrophilic treatment by plasma irradiation described later.
［貫通孔の製法］
貫通孔２０の形成は次のようにレーザを用いて行った。レーザには、第３高調波Ｎｄ：ＹＶＯ４レーザ装置から出射される高繰り返しパルスレーザ（波長３５５ｎｍ、繰り返し周波数１１０ｋＨｚ、２８Ｗ）を用いた。レーザ装置から出射されたレーザパルス（パルス幅２０ｎｓ、パワー７Ｗ、ビーム径３．５ｍｍ）を、対物レンズでガラス基板１０の第１面１１に集光させた。１つの貫通孔２０当たりの照射時間は約３．５ｍｓであり、ガラス基板１０をＸＹステージに固定して、貫通孔２０を加工する毎にＸＹステージを任意に動かすことによってピッチ２００μｍで１０×１０に２次元配列された貫通孔群を作成した。
[Manufacturing method of through hole]
The through-hole 20 was formed using a laser as follows. As the laser, a high repetition pulse laser (wavelength 355 nm, repetition frequency 110 kHz, 28 W) emitted from the third harmonic Nd: YVO4 laser device was used. A laser pulse (pulse width 20 ns, power 7 W, beam diameter 3.5 mm) emitted from the laser device was condensed on the first surface 11 of the glass substrate 10 by an objective lens. The irradiation time per one through-hole 20 is about 3.5 ms, and the glass substrate 10 is fixed to the XY stage, and the XY stage is arbitrarily moved every time the through-hole 20 is processed. A through-hole group arranged in a two-dimensional manner was created.
第１面１１上のレーザビームのスポット径を調整することで、貫通孔２０の第１開口２１の直径を制御することができる。第１開口２１の直径は、２０〜２５μｍφ、第２開口２２の直径は５〜１０μｍφとすることができた。このようにガラス基板１０の第１面１１側からレーザを照射するだけで、第１面１１上に第１開口２１の直径が、第２面１２側の第２開口２２の直径よりも大きくなる円錐台形状の貫通孔２０を形成できた。 By adjusting the spot diameter of the laser beam on the first surface 11, the diameter of the first opening 21 of the through hole 20 can be controlled. The diameter of the first opening 21 could be 20-25 μmφ, and the diameter of the second opening 22 could be 5-10 μmφ. In this way, the diameter of the first opening 21 on the first surface 11 becomes larger than the diameter of the second opening 22 on the second surface 12 simply by irradiating the laser from the first surface 11 side of the glass substrate 10. A through-hole 20 having a truncated cone shape could be formed.
なお、貫通孔２０の第１面１１での第１開口２１の半径と第２面１２での第２開口２２の半径との差の基板の厚さに対する比は、実験で用いたガラス基板では、０．０６８であるが、０．０１７以上、０．１２３以下の範囲の任意の値とすることが望ましい。この比の範囲は、貫通孔２０の内側面２３と第１面１１との成す角で表現すると、実験で用いたガラス基板では、３．８８°であり、１°以上、７°以下の範囲である。これらの範囲にある時に細胞の捕捉率を高くすることができる。内側面２３の傾斜が大きくなり過ぎると細胞を内側面２３に捕捉し難く、内側面２３の傾斜が緩やかになり過ぎると、単一細胞の捕捉が困難になると思われる。さらに、ガラス基板１０の厚さの貫通孔２０の第１面１１における第１開口２１の直径に対する比は、実験で用いたガラス基板では、５．６であり、２以上、１０以下とすることが望ましい。この比はアスペクト比であるが、第１開口２１の直径や基板の厚さ（貫通孔の長さ）が捕捉率に関与するものと想定される。この範囲にある時に細胞の捕捉率を高くすることができる。また、貫通孔２０の第１面１１での第１開口２１の直径は捕捉する細胞の最大長の１倍以上、２倍以下とすることが望ましく、第２開口２２の直径は捕捉する細胞の最小長の０．５倍以上、１倍以下とすることが望ましい。これは、血球系細胞・がん細胞の直径が概ね１０μｍ〜２０μｍの範囲に分布することに由来し、細胞懸濁液を第１面１１側から第２面１２側に効率よく導き、細胞を貫通孔２０により効果的に捕捉するためである。したがって、第１開口２１の直径は、１０μｍ以上、４０μｍ以下、より望ましくは１５μｍ以上、３０μｍ以下が望ましく、第２開口２２の直径は、５μｍ以上、２０μｍ以下、より望ましくは５μｍ以上、１７μｍ以下が望ましい。 The ratio of the difference between the radius of the first opening 21 at the first surface 11 of the through hole 20 and the radius of the second opening 22 at the second surface 12 to the thickness of the substrate is the same as that of the glass substrate used in the experiment. 0.068, but an arbitrary value in the range of 0.017 or more and 0.123 or less is desirable. The range of this ratio is 3.88 ° in the glass substrate used in the experiment, expressed as an angle formed by the inner surface 23 of the through-hole 20 and the first surface 11, and ranges from 1 ° to 7 °. It is. When in these ranges, the cell capture rate can be increased. If the inclination of the inner side surface 23 becomes too large, it is difficult to trap the cells on the inner side surface 23, and if the inclination of the inner side surface 23 becomes too gentle, it will be difficult to capture a single cell. Furthermore, the ratio of the thickness of the glass substrate 10 to the diameter of the first opening 21 in the first surface 11 of the through hole 20 is 5.6 in the glass substrate used in the experiment, and should be 2 or more and 10 or less. Is desirable. Although this ratio is an aspect ratio, it is assumed that the diameter of the first opening 21 and the thickness of the substrate (the length of the through hole) are involved in the capture rate. When in this range, the cell capture rate can be increased. In addition, the diameter of the first opening 21 in the first surface 11 of the through hole 20 is desirably 1 to 2 times the maximum length of the cell to be captured, and the diameter of the second opening 22 is the size of the cell to be captured. It is desirable that the minimum length be 0.5 times or more and 1 time or less. This is because the diameter of blood cells and cancer cells is distributed in the range of about 10 μm to 20 μm, and the cell suspension is efficiently guided from the first surface 11 side to the second surface 12 side, This is because it is effectively captured by the through hole 20. Therefore, the diameter of the first opening 21 is preferably 10 μm or more and 40 μm or less, more preferably 15 μm or more and 30 μm or less, and the diameter of the second opening 22 is 5 μm or more and 20 μm or less, more preferably 5 μm or more and 17 μm or less. desirable.
［親水性処理とその評価］
次に、ガラス基板１０の貫通孔２０の開口の径が小さい側の第２面１２にプラズマを照射した後、貫通孔２０の開口の径が大きい側の第１面における貫通孔２０の配列上に水を１μＬ滴下して、水滴の接触角を測定した。プラズマの照射条件は、大気圧、Ａｒ流量２．１３ＳＬＭ、Ｎ2 流量５．０ＳＬＭ、電圧９０Ｖであり、処理時間を０〜５ｍｉｍまで変化させた。比較のために貫通孔の形成されていないガラス基板（No. １）、貫通孔を有するがプラズマを照射しないガラス基板（No. ２）、貫通孔を有するガラス基板であって、プラズマ照射時間が、それぞれ、１、２、３、４、５ｍｉｎのガラス基板（No. ３、No. ４、No. ５、No. ６、No. ７）を準備した。なお、水滴の下には、１０×１０個の貫通孔が存在する。なお、微量に酸素を含ませると、酸素ラジカルの作用により親水性が向上するので望ましい。
[Hydrophilic treatment and its evaluation]
Next, after irradiating the second surface 12 with the smaller diameter of the opening of the through hole 20 of the glass substrate 10 with plasma, on the arrangement of the through holes 20 on the first surface with the larger diameter of the through hole 20 1 μL of water was dropped on the sample, and the contact angle of the water droplet was measured. The plasma irradiation conditions were atmospheric pressure, Ar flow rate 2.13 SLM, N 2 flow rate 5.0 SLM, voltage 90 V, and the treatment time was changed from 0 to 5 mim. For comparison, a glass substrate having no through hole (No. 1), a glass substrate having a through hole but not irradiated with plasma (No. 2), a glass substrate having a through hole, and a plasma irradiation time 1, 2, 3, 4, 5 min glass substrates (No. 3, No. 4, No. 5, No. 6, No. 7) were prepared. Note that there are 10 × 10 through holes under the water droplets. Note that it is desirable to include a small amount of oxygen because hydrophilicity is improved by the action of oxygen radicals.
水滴滴下直後の水滴の初期接触角θ1 と滴下後２ｍｉｍ経過後の水滴の接触角θ2 を測定し、接触角の変化率Δθ（％）を求めた。ただし、変化率Δθは、（１）式で表される。
その結果を図３に示す。No. １とNo. ２のガラス基板を比較すると、接触角の変化率に差異はない。すなわち、プラズマを照射していない場合には、貫通孔２０の存在は接触角の変化に影響を与えないということが分かる。プラズマを照射した場合には、接触角の変化率は、No. １とNo. ２のガラス基板に比べて大きくなっている。すなわち、水滴滴下２ｍｉｎ後の接触角θ2 が初期接触角θ1 に比べて約７０％程低下していることが分かる。そして、接触角の変化率は、プラズマの照射時間が１ｍｉｎ以上の場合に、それ以上長くなっても、ほとんど変化していないことが理解される。したがって、プラズマ照射時間は少なくとも１ｍｉｎあれば良い。 The result is shown in FIG. When the glass substrates of No. 1 and No. 2 are compared, there is no difference in the change rate of the contact angle. That is, it can be seen that the presence of the through hole 20 does not affect the change in the contact angle when the plasma is not irradiated. When the plasma is irradiated, the change rate of the contact angle is larger than that of the glass substrates of No. 1 and No. 2. That is, it can be seen that the contact angle θ 2 after 2 minutes of dripping the water drops by about 70% compared to the initial contact angle θ 1 . It is understood that the change rate of the contact angle hardly changes even when the plasma irradiation time is longer than 1 min, even if the plasma irradiation time is longer than that. Therefore, the plasma irradiation time may be at least 1 min.
この実験から、プラズマを照射していない場合には、貫通孔が存在しても、接触角の変化率が小さいことから、水滴はその貫通孔に浸水し難いことが分かる。また、プラズマを照射した場合には、接触角の変化率が大きく、接触角が水滴滴下後の経過時間に対して減衰することから、時間の経過に伴い水が貫通孔に浸入し易いことが分かる。プラズマは第２面から照射しているので、第１面の親水性は、プラズマを照射していないガラス基板（No. ２）と、第２面からプラズマを照射したガラス基板（No. ３〜No. ７）とで差異はない。それであるにもかかわらず、接触角の変化率は、ガラス基板（No. ３〜No. ７）の方が、ガラス基板（No. ２）に比べて圧倒的に大きい。このことから、第２面からのプラズマ照射により、貫通孔の内側面の親水性が向上するために、貫通孔への水の浸入が促進されたと理解することができる。また、第２面からのプラズマ照射により、第１面上の水滴の貫通孔へ浸入容易性が向上していることは、図４に示す水滴の画像からも理解される。図４からは、減衰して安定した接触角は１３．６°である。なお、この接触角は、貫通孔への浸水を促進するには、１０°以下となることがなお望ましい。 From this experiment, it can be seen that when the plasma is not irradiated, even if the through hole exists, the rate of change of the contact angle is small, so that the water droplet is not easily immersed in the through hole. In addition, when the plasma is irradiated, the rate of change of the contact angle is large, and the contact angle attenuates with respect to the elapsed time after dropping the water droplets, so that water can easily enter the through-hole as time passes. I understand. Since the plasma is irradiated from the second surface, the hydrophilicity of the first surface is determined by the glass substrate not irradiated with plasma (No. 2) and the glass substrate irradiated with plasma from the second surface (No. 3 to No. 3). No difference from No. 7). Nevertheless, the change rate of the contact angle is overwhelmingly larger in the glass substrate (No. 3 to No. 7) than in the glass substrate (No. 2). From this, it can be understood that the plasma irradiation from the second surface improves the hydrophilicity of the inner side surface of the through hole, so that the penetration of water into the through hole is promoted. It can also be understood from the image of the water droplet shown in FIG. 4 that the ease of intrusion into the through hole of the water droplet on the first surface is improved by the plasma irradiation from the second surface. From FIG. 4, the attenuated and stable contact angle is 13.6 °. This contact angle is more preferably 10 ° or less in order to promote water immersion into the through hole.
［捕捉実験］
次に、細胞の貫通孔への捕捉率を推定するために、直径１０μｍの蛍光ビーズを水に溶解して一定濃度のビーズ懸濁液を製造した。ビーズの個数密度は１０００／５．５μＬである。準備したガラス基板は、表１に示すように、No. １１〜No. １８の８個であり、何れも図１に示す貫通孔２０の２次元配列を有している。
Next, in order to estimate the capture rate of the cells into the through-hole, fluorescent beads having a diameter of 10 μm were dissolved in water to produce a bead suspension having a constant concentration. The number density of beads is 1000 / 5.5 μL. As shown in Table 1, the prepared glass substrates are eight, No. 11 to No. 18, and each has a two-dimensional array of through holes 20 shown in FIG.
１. プラズマ照射の差異によるガラス基板の種類
No. １１〜No. １３のガラス基板は、ビーズ懸濁液を滴下する第１面において、貫通孔の存在しない非捕捉領域Ｓ２（図１）にはテープを貼付してプラズマが第１面に照射されないようにした基板である。ただし、プラズマ照射後にビーズ懸濁液を第１面に滴下する時には、そのテープを剥がしている。したがって、第１面からプラズマを照射したNo. １１とNo. １３のガラス基板においては、第１面の捕捉領域Ｓ１の親水度はプラズマ照射前のガラス基板の親水度より高く、非捕捉領域Ｓ２の親水度はテープの糊が付着するためプラズマ照射前のガラス基板の親水度よりも低い。また、No. １２のガラス基板においては、第１面にはプラズマを照射していないので、第１面の捕捉領域Ｓ１の親水度はプラズマ照射前の親水度に等しく、非捕捉領域Ｓ２の親水度はテープの糊の付着によりプラズマ照射前のガラス基板の親水度よりも低い。いずれにしても、No. １１〜No. １３のガラス基板の第１面においては、捕捉領域Ｓ１の親水度は非捕捉領域Ｓ２の親水度よりも高い。また、No. １２のガラス基板は第２面にプラズマを照射しているが、第２面には照射領域を制限するためのテープの貼付は行っていない。No. １１のガラス基板は第１面側からのみプラズマを照射し、No. １２のガラス基板は第２面側からのみプラズマを照射し、No. １３のガラス基板は、第１面側からプラズマを照射し、その後第２面側からもプラズマを照射した基板である。以下、これらのNo. １１〜No. １３のガラス基板を、以下の説明を簡略にするために、便宜上、有親水度差ガラス基板という。
1. Types of glass substrates due to differences in plasma irradiation
The glass substrates of No. 11 to No. 13 have a first surface on which the bead suspension is dropped, a tape is applied to the non-capturing region S2 (FIG. 1) where no through-holes are present, and plasma is applied to the first surface. This is a substrate that is not irradiated. However, when the bead suspension is dropped onto the first surface after plasma irradiation, the tape is peeled off. Therefore, in the glass substrates of No. 11 and No. 13 irradiated with plasma from the first surface, the hydrophilicity of the capturing region S1 on the first surface is higher than the hydrophilicity of the glass substrate before plasma irradiation, and the non-capturing region S2 The hydrophilicity of is lower than the hydrophilicity of the glass substrate before plasma irradiation because the glue of the tape adheres. Further, in the glass substrate of No. 12, since the first surface is not irradiated with plasma, the hydrophilicity of the capture region S1 on the first surface is equal to the hydrophilicity before the plasma irradiation, and the hydrophilicity of the non-capture region S2 The degree of adhesion is lower than the hydrophilicity of the glass substrate before the plasma irradiation due to adhesion of the adhesive on the tape. In any case, on the first surface of the glass substrates No. 11 to No. 13, the hydrophilicity of the capturing region S1 is higher than the hydrophilicity of the non-capturing region S2. Moreover, although the glass substrate of No. 12 has irradiated the 2nd surface with the plasma, the sticking of the tape for restrict | limiting an irradiation area | region is not performed to the 2nd surface. The glass substrate of No. 11 is irradiated with plasma only from the first surface side, the glass substrate of No. 12 is irradiated with plasma only from the second surface side, and the glass substrate of No. 13 is plasma from the first surface side. The substrate is then irradiated with plasma from the second surface side. Hereinafter, these glass substrates of No. 11 to No. 13 are referred to as hydrophilicity difference glass substrates for convenience in order to simplify the following description.
No. １４〜No. １６のガラス基板は、プラズマ照射時に、第１面及び第２面にテープの貼付を行っていない基板である。したがって、No. １４〜No. １６のガラス基板の第１面において捕捉領域Ｓ１と非捕捉領域Ｓ２とで親水度の差は存在しない。No. １４のガラス基板は第１面側からのみプラズマを照射し、No. １５のガラス基板は第２面側からのみプラズマを照射し、No. １６のガラス基板は、第１面側からプラズマを照射し、その後第２面側からもプラズマを照射した基板である。以下、これらのNo. １４〜No. １６のガラス基板を、便宜上、均一親水度ガラス基板という。No. １７、No. １８のガラス基板は、プラズマを照射しない基板である。 The glass substrates of No. 14 to No. 16 are substrates on which no tape is applied to the first surface and the second surface during plasma irradiation. Accordingly, there is no difference in hydrophilicity between the capture region S1 and the non-capture region S2 on the first surface of the glass substrates No. 14 to No. 16. The glass substrate of No. 14 is irradiated with plasma only from the first surface side, the glass substrate of No. 15 is irradiated with plasma only from the second surface side, and the glass substrate of No. 16 is plasma from the first surface side. The substrate is then irradiated with plasma from the second surface side. Hereinafter, these glass substrates of No. 14 to No. 16 are referred to as a uniform hydrophilic glass substrate for convenience. The glass substrates of No. 17 and No. 18 are substrates that are not irradiated with plasma.
２．ビーズ懸濁液の滴下による捕捉実験
貫通孔を有したこれらのガラス基板上の第１面の捕捉領域Ｓ１にビーズ懸濁液を滴下し、１時間経過後に、貫通孔に捕捉されたビーズの個数を蛍光顕微鏡により計数し、ビーズの捕捉率を求めた。ただし、捕捉率βは、（２）式で定義される。ビーズは、細胞とほぼ同じ大きさとするため、直径１０μｍの蛍光ビーズとした。ビーズ懸濁液における個数密度は、１０００／５．５μＬである。
３．第１面における親水度が捕捉率に与える影響
No. １４、No. １６の均一親水度ガラス基板のビーズ捕捉率は、それぞれ、No. １１、No. １３の有親水度差ガラス基板のビーズ捕捉率に比べて小さい。特に、第１面にのみプラズマを照射したガラス基板（No. １１、No. １４）では、No. １１の有親水度差ガラス基板の捕捉率は、No. １２の均一親水度ガラス基板の捕捉率に対して、２．５倍大きい。また、両面にプラズマを照射したガラス基板（No. １３、No. １６）においては、No. １３の有親水度差ガラス基板の捕捉率は、No. １６の均一親水度ガラス基板の捕捉率に対して、１．７倍大きい。
3. Effect of hydrophilicity on the first surface on the capture rate
The bead capture rates of the uniform hydrophilic glass substrates of No. 14 and No. 16 are smaller than the bead capture rates of the hydrophilicity difference glass substrates of No. 11 and No. 13, respectively. In particular, in the glass substrates (No. 11 and No. 14) irradiated with plasma only on the first surface, the capture rate of the No. 11 hydrophilicity difference glass substrate is that of the No. 12 uniform hydrophilicity glass substrate. 2.5 times greater than the rate. Moreover, in the glass substrate (No. 13, No. 16) which irradiated plasma on both surfaces, the capture rate of the hydrophilicity difference glass substrate of No. 13 is equal to the capture rate of the uniform hydrophilicity glass substrate of No. 16. On the other hand, it is 1.7 times larger.
このことから、ビーズ懸濁液を滴下する第１面にのみプラズマを照射した場合には、捕捉率に与える効果は、貫通孔の内側面での親水性の向上によるよりも、第１面上の捕捉領域Ｓ１のみ親水性を向上させたことによる方が大きいことを意味している。すなわち、有親水度差ガラス基板は、第１面の平坦な面における捕捉領域Ｓ１の親水性が非捕捉領域Ｓ２の親水性に比べて高いために、液滴が広がらずに貫通孔に効果的に浸入するためであると考えられる。また、均一親水度ガラス基板は、捕捉領域Ｓ１と非捕捉領域Ｓ２との親水度に差がないために、液滴が広がり貫通孔に流れ込む効率が低下するものと考えられる。 From this, when the plasma is irradiated only on the first surface on which the bead suspension is dropped, the effect on the capture rate is higher on the first surface than on the improvement in hydrophilicity on the inner surface of the through hole. This means that only the trapping region S1 has a larger hydrophilicity. That is, since the hydrophilicity of the capture region S1 on the flat surface of the first surface is higher than the hydrophilicity of the non-capture region S2, the hydrophilicity-difference glass substrate is effective for the through-holes without spreading the droplets. This is thought to be due to intrusion. Further, since the uniform hydrophilic glass substrate has no difference in hydrophilicity between the capturing region S1 and the non-capturing region S2, it is considered that the efficiency of the liquid droplet spreading and flowing into the through hole is lowered.
これに対して、第２面側からのみプラズマを照射した場合には、有親水度差ガラス基板と均一親水度ガラス基板とで、捕捉率の差異が小さく、均一親水度ガラス基板の方がやや大きい。上記の第１面における液滴の広がりによる捕捉率低下の考察からすれば、有親水度差ガラス基板No. １２の方が、均一親水度ガラス基板No. １５よりも捕捉率は高いはずである。第１面側からプラズマを照射する場合に比べて第２面側からプラズマを照射した方が、捕捉率の向上に大きく寄与しており、その効果が、均一親水度ガラス基板No. １５の第１面における液滴の広がりによる捕捉率の低下を補償しているものと思われる。 On the other hand, when the plasma is irradiated only from the second surface side, the difference in capture rate between the hydrophilicity-difference glass substrate and the uniform hydrophilicity glass substrate is small, and the uniform hydrophilicity glass substrate is slightly more large. From the above consideration of the decrease in the capture rate due to the spread of droplets on the first surface, the hydrophilicity-difference glass substrate No. 12 should have a higher capture rate than the uniform hydrophilicity glass substrate No. 15. . Compared with the case where the plasma is irradiated from the first surface side, the direction where the plasma is irradiated from the second surface side greatly contributes to the improvement of the trapping rate. It seems to compensate for the decrease in capture rate due to the spread of droplets on one surface.
４．プラズマ照射面と捕捉率との関係
有親水度差ガラス基板No. １２の親水度は、有親水度差ガラス基板No. １１の親水度の１．１倍である。均一親水度ガラス基板No. １５の親水度は均一親水度ガラス基板No. １４の親水度の３．０倍である。プラズマの照射による効果は、何れか一方の面に照射するのであれば、第１面に照射するよりは第２面に照射した方が高いことを意味している。この原因には、次のことが考えられる。第１面側からプラズマを照射した場合には、第１面及び貫通孔の内側面に親水性処理が施される。第２面側からプラズマを照射した場合には、第２面及び貫通孔の内側面に親水性処理が施される。
4). Relationship Between Plasma Irradiation Surface and Capture Rate The hydrophilicity of hydrophilicity difference glass substrate No. 12 is 1.1 times the hydrophilicity of hydrophilicity difference glass substrate No. 11. The hydrophilicity of the uniform hydrophilic glass substrate No. 15 is 3.0 times the hydrophilicity of the uniform hydrophilic glass substrate No. 14. The effect of plasma irradiation means that if one surface is irradiated, the second surface is irradiated more than the first surface. The following can be considered as the cause. When the plasma is irradiated from the first surface side, hydrophilic treatment is performed on the first surface and the inner surface of the through hole. When the plasma is irradiated from the second surface side, hydrophilic treatment is performed on the second surface and the inner surface of the through hole.
したがって、第１には、第２面の親水度が向上したために、貫通孔の第２開口から溶媒が第２面に回り込み、溶媒の流出を促進しているためであると考えられる。第２には、貫通孔の内側面における親水度の分布が、プラズマを第１面側から照射した場合と第２面側から照射した場合とで異なっており、第２面側からプラズマを照射した時の親水度の分布の方が貫通孔への懸濁液の引き込みと排出が促進されるためであると考えられる。貫通孔の内側面での親水度の分布が異なるとすれば、プラズマの照射側に近い方が親水度は高いと考えられる。よって、第２面側からプラズマを照射した時には、貫通孔の内側面では第１開口から第２開口に向かうに連れて親水度が高くなっている、又は、親水基密度が高くなっているものと考えられる。これにより、貫通孔に流入した懸濁液のうち溶媒を第２開口から外部に排出する作用が強くなり、捕集率が向上したものと思われる。 Therefore, the first reason is considered to be that the hydrophilicity of the second surface is improved, so that the solvent wraps around the second surface from the second opening of the through hole and promotes the outflow of the solvent. Second, the distribution of hydrophilicity on the inner surface of the through hole is different between when the plasma is irradiated from the first surface side and when the plasma is irradiated from the second surface side, and the plasma is irradiated from the second surface side. The distribution of the hydrophilicity at this time is considered to be because the suspension is drawn into and discharged from the through hole. If the hydrophilicity distribution on the inner surface of the through hole is different, it is considered that the hydrophilicity is higher near the plasma irradiation side. Therefore, when the plasma is irradiated from the second surface side, the hydrophilicity increases from the first opening to the second opening on the inner surface of the through hole, or the hydrophilic group density increases. it is conceivable that. Thereby, it is considered that the action of discharging the solvent from the second opening to the outside out of the suspension flowing into the through-hole is enhanced, and the collection rate is improved.
５．両面からプラズマを照射した場合の捕捉率への影響
有親水度差ガラス基板No. １３の親水度は、均一親水度ガラス基板No. １６の親水度の１．７倍である。これは、第１面において捕捉領域Ｓ１の親水度が非捕捉領域Ｓ２の親水度よりも可なり大きいために、液滴の広がりが抑制されるので第１開口から懸濁液の流入が促進されるためであると考えられる。また、有親水度差ガラス基板No. １３の親水度は、有親水度差ガラス基板No. １２の親水度の１．３倍である。これは第１面の捕捉領域Ｓ１の親水度が向上したために、第１開口から懸濁液の流入が促進されるためであると考えられる。
5. Effect on capture rate when plasma is irradiated from both sides The hydrophilicity of hydrophilicity-difference glass substrate No. 13 is 1.7 times that of uniform hydrophilicity glass substrate No. 16. This is because, on the first surface, the hydrophilicity of the trapping region S1 is much larger than the hydrophilicity of the non-capturing region S2, so that the spread of the liquid droplets is suppressed and the inflow of the suspension is promoted from the first opening. This is considered to be because of this. Further, the hydrophilicity of the hydrophilicity-difference glass substrate No. 13 is 1.3 times the hydrophilicity of the hydrophilicity-difference glass substrate No. 12. This is considered to be because the inflow of the suspension is promoted from the first opening because the hydrophilicity of the capturing region S1 on the first surface is improved.
一方、均一親水度ガラス基板No. １６の親水度は均一親水度ガラス基板No. １５の親水度より低い。これは、第１面にプラズマを照射したために、逆に、捕捉率が低下したことを意味する。第１面の親水度の分布に差がないために、液滴が大きくひろがり、貫通孔の第１開口からの懸濁液の流入が低下するためと思われる。 On the other hand, the hydrophilicity of the uniform hydrophilic glass substrate No. 16 is lower than the hydrophilicity of the uniform hydrophilic glass substrate No. 15. This means that, since the first surface was irradiated with plasma, the capture rate was decreased. This is probably because there is no difference in the distribution of hydrophilicity on the first surface, so that the droplets expand widely and the inflow of the suspension from the first opening of the through hole decreases.
６．細胞の捕捉率
以上のことから、直径１０μｍのビーズの捕捉率の実験から、同様な大きさの細胞において同様な捕捉率の特性が得られると考えられる。
次に、細胞の捕捉率を測定した。用いた細胞は、H1299(ヒト非小細胞肺癌細胞である。染色方法はCalcein-AM(495/515nm) を用いた。観察方法は共焦点顕微鏡による観察で貫通孔に捕捉された蛍光細胞の数を計測した。共焦点顕微鏡による平面画像と断面画像により単一細胞が貫通孔に捕捉されていることを確認した。貫通孔における捕捉位置は第２開口（吐出口）から貫通孔の全長の約１／３位置であった。
6). From the above, it is considered that the same capture rate characteristics can be obtained for cells of the same size from experiments on the capture rate of beads having a diameter of 10 μm.
Next, the cell capture rate was measured. The cells used were H1299 (human non-small cell lung cancer cells. The staining method was Calcein-AM (495/515 nm). It was confirmed that a single cell was captured in the through-hole by a plane image and a cross-sectional image by a confocal microscope.The capture position in the through-hole was about the entire length of the through-hole from the second opening (discharge port). The position was 1/3.
細胞懸濁液の個数濃度を変化させて、捕捉率を測定した。個数濃度は、２５０／３μＬ、５００／３μＬ、１０００／３μＬである。用いたガラス基板は、両面にプラズマを照射した有親水度差ガラス基板No. １３である。なお、１液滴は３μＬであるので、１液滴中の細胞の個数と上記の個数密度は等しい。捕捉率を図６に示す。細胞懸濁液の細胞の個数密度が１０００／３μＬの場合の捕捉率は８５％と、高い捕捉率を示している。 The capture rate was measured by changing the number concentration of the cell suspension. The number concentration is 250/3 μL, 500/3 μL, and 1000/3 μL. The glass substrate used is a hydrophilicity difference glass substrate No. 13 in which plasma is irradiated on both sides. Since one droplet is 3 μL, the number of cells in one droplet is equal to the above number density. The capture rate is shown in FIG. When the cell number density of the cell suspension is 1000/3 μL, the capture rate is 85%, indicating a high capture rate.
以上、説明したように、本発明によると、基板の第１面の捕捉領域に細胞懸濁液を滴下するだけで、貫通孔の第２開口（吐出口）から吸引することなく、親水性だけで単位細胞毎に貫通孔に捕捉することができる。この結果、細胞に損傷を与えることがない。特に、細胞懸濁液を滴下する面とは反対側の第２面の親水度を向上させることで、細胞の捕捉率が向上されたものと想定される。 As described above, according to the present invention, only the cell suspension is dropped into the capturing region of the first surface of the substrate, and only the hydrophilicity is not sucked from the second opening (discharge port) of the through hole. Thus, each unit cell can be captured in the through hole. As a result, the cells are not damaged. In particular, it is assumed that the cell capture rate is improved by improving the hydrophilicity of the second surface opposite to the surface on which the cell suspension is dropped.
細胞毎の性状、機能の分析に用いることができる。 It can be used to analyze the properties and functions of each cell.
１０…ガラス基板
１１…第１面
１２…第２面
２０…貫通孔
２１…第１開口
２２…第２開口
２３…内側面
DESCRIPTION OF SYMBOLS 10 ... Glass substrate 11 ... 1st surface 12 ... 2nd surface 20 ... Through-hole 21 ... 1st opening 22 ... 2nd opening 23 ... Inner surface
Claims (10)
基板と、
前記基板の両面を構成する第１面から第２面に向けて貫通し、基板面に対して２次元配列された貫通孔とを有し、
前記貫通孔は前記第１面から第２面に向けて径が連続して小さくなる円錐台形状であって、少なくともその貫通孔の内側面はプラズマ照射により、照射前の前記基板の親水性よりも高い親水性を有することを特徴とする細胞捕捉チップ。 In a cell capture chip that captures a single cell on a substrate,
A substrate,
Penetrating from the first surface constituting the both surfaces of the substrate toward the second surface, and having two-dimensionally arranged through holes with respect to the substrate surface,
The through hole has a truncated cone shape whose diameter continuously decreases from the first surface toward the second surface, and at least the inner surface of the through hole is exposed to plasma, and the hydrophilicity of the substrate before irradiation is reduced. A cell trapping chip characterized by having high hydrophilicity.
基板の両面を構成する第１面から第２面に向けて貫通し、直径が前記第１面から第２面に向けて小さくなる円錐台形状である、基板面に対して２次元配列された貫通孔を有する基板に対して、少なくとも前記第２面からプラズマを照射して、前記貫通孔の内面に親水処理を施し、
前記基板の前記第１面に細胞懸濁液を滴下して、前記貫通孔に単一細胞を捕捉することを特徴とする細胞捕捉方法。 In a cell capture method for capturing a single cell on a substrate,
Two-dimensionally arrayed with respect to the substrate surface, which has a truncated cone shape penetrating from the first surface to the second surface constituting both surfaces of the substrate and having a diameter that decreases from the first surface to the second surface. A substrate having a through hole is irradiated with plasma from at least the second surface, and the inner surface of the through hole is subjected to a hydrophilic treatment,
A cell capturing method, wherein a cell suspension is dropped onto the first surface of the substrate to capture a single cell in the through hole.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP2015045829A JP2016165233A (en) | 2015-03-09 | 2015-03-09 | Cell-capturing chip and cell-capturing method |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP2015045829A JP2016165233A (en) | 2015-03-09 | 2015-03-09 | Cell-capturing chip and cell-capturing method |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| JP2016165233A true JP2016165233A (en) | 2016-09-15 |
Family
ID=56897051
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP2015045829A Pending JP2016165233A (en) | 2015-03-09 | 2015-03-09 | Cell-capturing chip and cell-capturing method |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JP2016165233A (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107603849A (en) * | 2017-09-14 | 2018-01-19 | 中国科学院半导体研究所 | Unicellular RT pcr chips and preparation method thereof |
-
2015
- 2015-03-09 JP JP2015045829A patent/JP2016165233A/en active Pending
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107603849A (en) * | 2017-09-14 | 2018-01-19 | 中国科学院半导体研究所 | Unicellular RT pcr chips and preparation method thereof |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| US10661277B2 (en) | Microfluidic device and method of making the same | |
| US20200354781A1 (en) | Cell analysis device, apparatus, and cell analysis method using same | |
| US20150004687A1 (en) | Cell trapping device | |
| CN109701298B (en) | Super-affinity super-hydrophilic-super-hydrophobic gas copper foil with gradually-changed wettability, preparation method thereof and positioning and collecting device | |
| JP6919181B2 (en) | Droplet dispensing device, droplet dispensing method, and manufacturing method of adherend | |
| Li et al. | Comparison of structures and hydrophobicity of femtosecond and nanosecond laser-etched surfaces on silicon | |
| US20170321183A1 (en) | Cell Isolation Method and Cell Trapping Filter | |
| RU2017133063A (en) | APPLICATION OF A POROUS CAPILLARY MEMBRANE FOR DETERMINING THE NUMBER OF AMPLIFICATION PRODUCTS BY TYPE OF Rolling RING | |
| JP2016165233A (en) | Cell-capturing chip and cell-capturing method | |
| US20180334700A1 (en) | Live-cell seeding method for microarrays | |
| CN109415674B (en) | Cell filtering filter | |
| JP2008116211A (en) | Cell separator and method for separating cells using the same | |
| US20210060459A1 (en) | Filtration device and filtration method | |
| US20210339246A1 (en) | Ultrafast particle sorting | |
| JP2018033434A (en) | Tip for cell capturing, method of producing same and cell capturing method | |
| Bruhn et al. | Dual-pore glass chips for cell-attached single-channel recordings | |
| CN110564588B (en) | Structure and chip for capturing peripheral blood circulation tumor cells and application method of structure and chip | |
| JP6128620B2 (en) | Base material for controlling movement direction of animal cell, cell identification method and cell separation method using the base material | |
| US20230166261A1 (en) | Particle sorting systems and methods | |
| CN109797089A (en) | Screening system and method, chip and its manufacturing method and system | |
| JP2014138970A (en) | Substrate | |
| JP2005525812A (en) | Cell culture equipment | |
| JP2023165540A (en) | Method for producing open microchannel device | |
| JP2013081438A (en) | Device and method of cell development, and cell observation system |