JPWO2004008132A1 - Biomolecule separation cell, method for producing the same, and DNA sorting apparatus - Google Patents

Abstract

The electrophoresis cell has a substrate 2, and a groove 3 is formed on the upper surface of the substrate 2. The substrate 2 is formed of a transparent material so that the inside of the substrate 2 can be seen through. The groove 3 includes a separation matrix 5 consisting of a number of columns 4. The upper surface of the substrate 2 and the groove 3 are covered with a transparent cover 6. Further, the electrophoresis cell 1 is provided with two sample injection ports 7 and two electrodes 8. Each electrode 8 is inserted into the groove 3 through the corresponding sample inlet 7. Both electrodes 8 are connected to a DC power supply 10 via lead wires 9. According to the electrophoresis cell 1, it is possible to overcome the drawbacks of an agarose gel, polyacrylamide gel, or a conventional artificial separation matrix used for electrophoresis analysis.

Description

  The present invention relates to a biomolecule separation cell such as an electrophoresis cell, a method for producing the same, and a DNA fractionation apparatus using the electrophoresis cell.

Biomolecules such as DNA extracted from biological samples, proteins, and fragments thereof are essential elements for medical examination, medical diagnosis, and biological examination. Diagnosis of diseases, detection of microorganisms, determination of genes, and the like are performed by biochemically analyzing these biomolecules and examining their molecular structures. These biomolecules are characterized by high molecular weight polymers. Electrophoresis is used as the most common analysis method. In electrophoresis, biomolecules are separated in a separation matrix to which a voltage is applied, due to differences in molecular size (molecular weight) and charge. As an electrophoresis method, a method using agarose gel (agar gel) or polyacrylamide gel as a separation matrix has been established. In this method, DNA and protein are separated while passing through a network of molecular chains of agarose gel or polyacrylamide gel. The mesh holes in these gels are estimated to be between 5 and 200 nm. However, these gels are heterogeneous in microstructure and unstable in morphology. For this reason, the handling is difficult and there are the following problems.
(Problem of gel)
(1) Since the form is unstable, storage stability is poor and handling is poor.
(2) It is always necessary to include moisture.
(3) The separation speed is low.
(4) A relatively large amount of sample at the μg level is required.
(5) It cannot be reused.
(6) Highly sensitive optical detection cannot be performed.
(7) Miniaturization is difficult.
In order to solve these problems, a hard gel for electrophoresis using a semiconductor lithography technique has been recently proposed. Specifically, for example, an artificial microstructure as an obstacle is formed on a silicon substrate or a glass substrate, and this is used as an alternative to a gel network, or an optically transparent Artificial separation matrixes formed by microfabrication molding of plastic have been proposed.
Examples of the former include a report by Volkmuth et al. Published in Nature, Vol. 358, pages 602 to 602 published in 1992, and analytical chemistry published in 2001. Examples include hard gels disclosed in a report by Bakajin et al., Published in Journal of Analytical Chemistry, Vol. 73, pages 6053-6056. In these reports, the authors describe a separation matrix with fine artificial obstacles made of silicon dioxide or glass targeted for the analysis of DNA having a length of 50-200 kilobase pairs (kilobase pairs). Has proposed. This separation matrix consists of regularly arranged struts with a diameter of several μm. The sample DNA is separated according to its size in the separation matrix by applying an electric field with alternating orientation and angle, and is observed or detected by a fluorescence microscope placed at the sample outlet. The
On the other hand, an example of the latter is an artificial separation matrix using plastic disclosed in Japanese Patent Application Laid-Open No. 11-508042 related to Yager et al. This artificial separation matrix is different from the separation matrix based on a silicon substrate or a glass substrate in the following points.
(Difference)
(1) An organic polymer is used as a separation matrix material.
(2) The inter-array distance of the artificial microstructure is 100 nm or less, preferably 10 to 30 nm.
(3) A polymer is polymerized in a microfabricated mold to produce a separation matrix.
However, these conventional techniques have the following problems or problems. That is, when silicon or glass is used for the separation matrix for electrophoresis, there are the following problems.
(problem)
(1) Silicon and glass have poor processability, are expensive and are not suitable for mass production.
{Circle around (2)} The diameter and gap of the support are several μm or more, but these values are about 100 times larger than the agarose gel or polyacrylamide gel, and are not suitable for analysis of small DNA.
On the other hand, the artificial separation matrix using plastic has the following problems.
(problem)
{Circle around (1)} Plastic molding technology at the nanometer (nm) to micrometer (μm) level when producing the separation matrix has not been established.
{Circle around (2)} The spacing between the support pillars is as narrow as 100 nm or less, hydrated DNA molecules are easily clogged, and smooth separation is difficult.
(3) In the manufacturing technology using a micro-processed mold, the plastic peeling process and the fine pattern transfer process have not been put into practical use at present and are not put into practical use.

The present invention has been made to solve the above-mentioned conventional problems, and overcomes the above-mentioned drawbacks in, for example, agarose gels and polyacrylamide gels used in electrophoretic analysis, or conventional artificial separation matrices. It is an object of the present invention to provide a separation matrix or a biomolecule separation cell such as an electrophoresis cell and a production method thereof. Furthermore, it aims at providing the application technique of this biomolecule separation cell.
The biomolecule separation cell according to the present invention separates or detects biomolecules in a liquid sample, for example, by electrophoresis. The biomolecule separation cell includes a substrate having a groove (flow channel) formed on the surface, a separation matrix, a cover, a buffer solution filling the groove, and at least two electrodes. The separation matrix is arranged in a partial region of the groove, and is composed of a plurality of pillars in which the narrowest part between the pillars is 0.1 to 5 μm. The cover covers the surface of the substrate and the groove and is in close contact with the upper surface of the support column. The electrode is in direct contact with the buffer solution and a voltage is applied to the buffer solution. The substrate and the separation matrix are made of a polymer material.
In this biomolecule separation cell, an artificial micro-obstacle separation matrix is used as an alternative to gel for electrophoresis, for example. For this reason, the separation matrix can be reduced in size and weight, and can be stored in a dry state. That is, even with a small amount of sample, biomolecules such as DNA can be separated or detected quickly and with high sensitivity.
In this biomolecule separation cell, an optimum structure can be selected according to the size and properties of the biomolecule to be separated. That is, the degree of freedom in design is great. For this reason, the cross section of a support | pillar can be made into various shapes, such as a circle, an ellipse, a polygon, or the polygon which rounded or cut off the corner | angular part, for example.
Part or all of the substrate and the separation matrix are preferably formed of, for example, an elastomer, plastic, or the like, which is a polymer material that is inexpensive and easy to mass-produce. As the elastomer, for example, it is preferable to use a silicone rubber which is a general material and is inexpensive and has good moldability. As the plastic, for example, it is preferable to use an acrylic resin, a polystyrene resin, a polycarbonate resin, or the like, which is a general material and is inexpensive and has good moldability.
The groove width is preferably set to 20 to 1000 microns, the groove depth is preferably set to 1 to 50 μm, and the groove length is preferably set to 0.1 to 50 mm. In this way, biomolecules such as DNA are electrophoresed uniformly and smoothly. For this reason, an electrophoresis cell capable of highly sensitive detection can be realized.
The arrangement density of the columns is preferably 1000 pieces / mm ² or more. In the present invention, for example, the basic principle is that the DNA migration speed is affected when DNA passes through the submicron-order gap of the support column, so the number of gaps greatly affects the resolution. And if the arrangement density of support | pillars is 1000 pieces / mm < ² > or more, isolation | separation of DNA below 1 million base pairs is possible.
If the cover is provided with a sample inlet for injecting a biomolecule sample into the groove, the separation matrix is not in the vicinity of the sample inlet, or there are no regions or columns where the arrangement density of the columns is lower than other regions It is preferable to have a region. For example, DNA does not easily enter the groove through the gap at the top of the support column, but in this way, DNA can be easily injected.
In one manufacturing method of the biomolecule separation cell according to the present invention, first, a silicon substrate, a glass substrate, or a polymer substrate is subjected to fine processing to form a mold having a negative pattern corresponding to the grooves and the support columns. Next, a liquid elastomer or plastic, or a monomer or prepolymer as a raw material for the elastomer or plastic is poured into the mold and solidified. Thereafter, the solidified product is separated from the template to obtain a biomolecule separation cell including a substrate and a separation matrix. According to this manufacturing method, the microstructure of the template can be faithfully transferred, and mass production of electrophoresis cells becomes possible.
In another manufacturing method of the biomolecule separation cell according to the present invention, first, a silicon substrate, a glass substrate, or a polymer substrate is subjected to fine processing to form a press die having a negative pattern corresponding to the groove and the column. To do. Next, after the plastic heated above the glass transition temperature is pressed against a press mold, the temperature is lowered and cured. Thereafter, the cured product is separated from the press mold to obtain a biomolecule separation cell including a substrate and a separation matrix. According to this manufacturing method, since the fine structure of the template can be transferred faithfully and at high speed, mass production of electrophoresis cells becomes possible.
A DNA sorting apparatus according to the present invention uses a biomolecule cell according to the present invention to separate biomolecules in a liquid sample by electrophoresis, and from the liquid sample containing DNA supplied into the groove, The DNA is fractionated (separated) or fractionated based on properties and structure. As described above, by using the electrophoresis cell according to the present invention for DNA analysis and medical examination, genetic examination, which has been poor in operability and requires a long time for analysis, is performed quickly and with high sensitivity. be able to.

The invention will be more fully understood from the following detailed description and the accompanying drawings. In the accompanying drawings, common constituent elements are denoted by the same reference numerals.
FIG. 1 is an overall perspective view (perspective view) of an electrophoresis cell according to the present invention having a separation matrix formed of a polymer material.
FIG. 2 is an enlarged perspective view showing a part of the electrophoresis cell or separation matrix shown in FIG.
FIG. 3 is an elevational sectional view of the electrophoresis cell shown in FIG.
FIG. 4A to FIG. 4E are three-dimensional schematic diagrams showing various pillar shapes and arrangement forms.
5A to 5E are top views of the separation matrix, showing various post arrangements.
6A to 6C are a perspective view and two elevation cross-sectional views showing the structure of the sample inlet and the separation matrix, respectively.
7A to 7G are views showing a method for producing a template for producing a separation matrix and a method for producing an electrophoresis cell using this template.
FIG. 8 is a schematic diagram showing a configuration or layout of a fluorescence microscope for electrophoresis cell and its attached devices.
FIG. 9 is a graph showing the relationship between the number of DNA bases in the electrophoresis cell and the electrophoresis speed.
FIG. 10 is a diagram showing the movement of DNA in the gap between the columns.
FIG. 11 is a schematic diagram showing a schematic configuration of one DNA sorting apparatus using the electrophoresis cell according to the present invention.
12A and 12B are schematic views showing a schematic configuration of another DNA sorting apparatus using the electrophoresis cell according to the present invention.

すなわち、アガロースゲルなどの網目の物理的な大きさは１０〜１００ｎｍである。しかし、実際には、糖鎖分子やポリアクリルアミド分子鎖の影響、あるいはＤＮＡに水和した水分子の影響が加わっている。アガロースやポリアクリルアミドの場合、これらの水分子は、ＤＮＡや、分離マトリックスである網目と相互作用を及ぼし合い、これによりＤＮＡの分離が円滑に行われる。これらの分子鎖は弾力性があるため、ＤＮＡなどの水和水の効果を利用し、あるいは水分子の影響をうまく吸収しているものと推察される。これらの水和水の分子レベルでの大きさは、現時点では殆どわかっていないが、１００ｎｍレベルの隙間では、水和水の影響でＤＮＡが浸入しにくいものと考えられる。逆浸透圧法や高圧液体クロマトグラフィなどにより、人工的に隙間に物理的な力（圧力など）を加えれば、水の透過は可能である。しかし、この場合、分離すべきＤＮＡ等の生体分子が破壊されるおそれがある。
他方、本発明にかかる分離マトリックス５では、ギャップ距離の上限値は５μｍとされる。以下、この理由を説明する。表２は、Ｔ４ＤＮＡとλＤＮＡとについて、ギャップ距離とＤＮＡの移動速度との関連性を調べた結果を示している。表２によれば、ギャップ距離が５μｍを超えると、移動速度差が確実には得られないことがわかる。そこで、ギャップ距離の上限値は５μｍとされる。

（支柱の配置密度）
以下、支柱４の配置密度を説明する。分離マトリックス５における支柱４の配置密度は、基本的には任意に設定することができる。ただし、本発明にかかる分離マトリックス５は、ＤＮＡの通過速度が支柱ギャップによって影響されるといった原理に基づいているので、ＤＮＡの分離能は、ＤＮＡが通過する支柱ギャップの数に比例して向上する。本願発明者による実験によれば、５０〜１００Ｖ／ｃｍの条件での５０〜２００ｋｂｐ（キロ塩基対）のＤＮＡの電気泳動による移動速度は、平均５０μｍ／秒であった。そして、１秒間に通過する支柱４（直径１５μｍの円柱）は約３個であり、支柱ギャップの数は６個であった。このとき、１０００塩基対あたり約０．１μｍの移動距離の差（塩基対が短いほど速く移動する）が観察された。溝３中の分離マトリックス５の長さを５ｍｍとすれば、このＤＮＡは約１００秒で分離マトリックス５を通過し、５ｍｍ先の検出ポイントで、１０００塩基対あたり約１０μｍの移動距離の差をもたらすことになる。検出系の光学分解能を５μｍとすれば、この移動距離の差は、十分検出可能である。
この場合、ＤＮＡは、その移動距離１ｍｍあたり６０個の支柱４と約１２０個の支柱ギャップとを通過する。電界の向きと垂直な支柱ギャップにはＤＮＡが泳動しないものと仮定すれば、支柱ギャップの数は支柱４の数のほぼ２倍となる。したがって、支柱４の配置密度は約４０００個／ｍｍ^２となり、支柱ギャップの配置密度は約１４０００個／ｍｍ^２となる。このＤＮＡよりも短いＤＮＡは、より早く泳動するので、支柱４の配置密度又は支柱ギャップの配置密度は、それぞれ、４０００個／ｍｍ^２又は１４０００個／ｍｍ^２よりも高くなければならない。表３に、上記実験を、Ｔ４ＤＮＡとλＤＮＡを用いて、さらに種々の支柱４の配置密度で繰り返し行い、支柱４の配置密度とＤＮＡの移動速度差との関連性を求めた結果を示す。表３によれば、分離マトリックス５の支柱４の配置密度は、１０００個／ｍｍ^２以上であることが必要であることがわかる。

（基板及び分離マトリックスの材質）
以下、基板２及び分離マトリックス５の材料について説明する。基板２及び分離マトリックス５の材料は、安価でありかつ量産しやすい高分子材料であれば、とくには制約はない。ただし、これらの材料は、光学的な検出が容易であることから、透明であるのが望ましい。したがって、やわらかい材料としては例えばエラストマを用いることができ、硬い材料としては例えばプラスチックを用いることができる。エラストマである最も一般的な透明素材としては、シリコンゴム、ブタジエンゴム、イソプレンゴム、フッ素系ゴム、スチレン系透明ゴムなどの透明合成ゴムと、これらのブレンドゴムとがあげられる。また、性能としては、次の条件を満たせばよい。
密度：〜１ｇ／ｍｌ
給水率：０．１％以下
全光線透過率：８５％以上
成形収縮率：１％以下
曲げ弾性率：３００Ｍｐａ以上
硬化手法はどのようなものでもよいが、成形性の観点から、化学開始剤や架橋剤により硬化を開始させる手法、あるいは熱硬化や光などのエネルギー線で硬化させる手法が好ましい。
他方、プラスチック材料は、励起光や蛍光を透過させるものであるのが好ましい。このような材料としては、例えば、ポリスチレン系樹脂、ポリスルホン系樹脂、アクリル系樹脂、ポリカーボネート系樹脂、ポリオレフィン系樹脂、塩素含有ポリマ、ポリエーテル系樹脂、ポリエステル系樹脂などをあげることができる。これらの樹脂は、単独、共重合、ブレンド、プレポリマのいずれの形態でもよい。成形の点では熱可塑性樹脂が好ましいが、熱硬化性又はエネルギ線硬化性の架橋重合体でもよい。また、基板２と溝３と分離マトリックス５とは同時に同一材料で成形するのが望ましい。ただし、接着が十分であれば、別々の材料を張り合わせたり、はめ込んでもよい。この場合、基板２の材料としては、例えば、セラミックス、ガラス、シリコン、ポリマーアロイなどを用いることができる。
（カバーの材料）
以下、カバー６の材料について説明する。カバー６の材料は、励起光及び蛍光を十分に透過させることができ、カバー６の下面が基板２、溝３及び分離マトリックス５（支柱４）の上面と十分に密着して液漏れを生じさせず、かつＤＮＡ等の生体分子を吸着しないものであれば、とくには制約されない。例えば、ガラス、プラスチック、エラストマなどを用いることができる。カバー６の表面は親水性であるのが好ましいが、疎水性であってもよい。カバー６の厚さは、任意に設定することができる。ただし、高倍率の検出系の光学焦点を考慮すれば、カバー６の厚さは２ｍｍ以下であるのが好ましい。
（材料の表面処理）
以下、基板２、分離マトリックス５及びカバー６の材料の表面処理について説明する。基板２の表面は、疎水性であり、水との接触角度が４５度以上であるのが望ましい。他方、溝３の表面、分離マトリックス５（支柱４）の表面、及びカバー６の溝３を覆う部分の表面は、緩衝液が接触するのが容易となり、かつ毛細管現象で緩衝液が支柱４の隙間を自発的に満たすのが容易となるよう、親水性であるのが好ましい。したがって、これらの表面に親水化処理を施すのが好ましい。親水化はどのような方法で行ってもよい。具体的には、例えば、プラズマ処理、プラズマ重合、コロナ放電、コーティングポリマ等の親水性化学物質のコーティング、物理的な微細構造による親水化処理などを用いることができる。
コーティングポリマとしては、例えばポリヒドロキシメチルメタクリレート、ポリアクリルアミド、セルロース系ポリマなどがあげられる。ここで、注意すべきことは、これらのコーティングポリマが電気泳動中に溶け出さないように架橋処理をする必要があるということである。また、電気浸透流や電気泳動をイオン性物質の種類や濃度で制御することが可能であるので、緩衝液にイオン性物質などを加えることにより、同様の効果を奏することができる。
溝３の表面、分離マトリックス５（支柱４）の表面、及びカバー６の溝３を覆う部分の表面は、基板２の表面と同様に疎水性であっても、電気泳動セル１を使用することは可能である。一般に、代表的なエラストマやプラスチックは、表面処理をしなければ疎水性である。これらをそのまま使えば、毛細管現象で緩衝液を、溝３内あるいは分離マトリックス５内（支柱４の隙間）に満たすことはできない。したがって、この場合、緩衝液の中に電気泳動セル１を浸漬して脱気処理を行う必要がある。しかし、一旦、このようにして溝３内あるいは分離マトリックス５内に緩衝液を満たせば、この後本発明の効果を十分に発揮することができる。なお、電気泳動セル１が全体として疎水性であれば、例えばエラストマとプラスチックとを用いる場合、これらの密着性が高まる。さらに、生体分子の非特異的吸着が減少し、電気泳動セル１が汚れにくくなるといった効果も得られる。
（電気泳動セルの製造方法）
以下、電気泳動セル１の具体的な製造方法を説明する。電気泳動セル１は、およそ次のような手順で製造される。電気泳動セル１の材料はいずれも高分子材料であるが、これらの高分子材料は成形により加工される。高分子材料の成形方法としては、圧縮成形法、射出成形法、押出し成形法、吹込み成形法、鋳造成形法、うす物成形法などが知られているが、ナノメータ〜サブミクロンのレベルの微細構造を成形することができれば、どのような方法を用いてもよい。この実施の形態では、次の２つの方法が用いられる。すなわち、１つは、微細加工したシリコン（Ｓｉ）を鋳型として、エラストマをキャスティングして微細構造を転写するといった方法である。もう１つは、微細加工したシリコンの成形型に、ガラス転移温度以上に加熱した熱可塑性ポリマを押し付けて微細構造を転写するといった方法である。
鋳型の材料としては、例えば、ガラス、シリコン、セラミック、金属、プラスチックなどが考えられるが、ナノメータ〜サブミクロンのレベルのネガパターンを微細加工することができれば、どのようなものでもよい。鋳型を製作する方法としては、マスクを作製してドライエッチングで加工するといった方法や、レジストをそのままリソグラフィにより微細加工するといった方法を用いることができる。ただ、この実施の形態では、剥離性を向上させるため、シリコンのエッチング技術を用いている。この方法は、ネガパターンの微細構造表面を滑らかにすることができ、これにより剥離性や微細構造の転写能をより高めることができる。
（鋳型の製作）
以下、図７Ａ〜図７Ｇを参照しつつ、鋳型を製作する方法と、該鋳型を用いて、分離マトリックス５を含む溝３を備えた基板２（以下、「デバイス」という。）を製作する方法とを、より具体的に説明する。
まず、図７Ａに示すように、シリコンウエハ１１を準備する。続いて、図７Ｂに示すように、シリコンウエハ１１の上面に感光性有機膜１２を塗布する。次に、図７Ｃに示すように、感光性有機膜１２を伴ったシリコンウエハ１１の上に、光を通す領域１４ａと光を通さない領域１４ｂとが所定のパターンで形成されたマスク１４を配置する。そして、マスク１４を介して感光性有機膜１２の所定の領域に光１３を照射し、現像液を用いて現像する。例えば感光性有機膜１２として、感光した部分が溶解するいわゆるポジ型感光性有機膜を用いた場合は、光を通す領域１４ａに対応する部位では感光性有機膜１２が除去され、穴部１５が形成される。
次に、図７Ｄに示すように、シリコンウエハ１１にエッチングを施す。エッチング手法としては、既存技術である誘導電磁結合プラズマ反応性プラズマエッチングを用いる。反応ガスとしては、フロロカーボンを主体としたガスを用いる。このエッチングにより、感光性有機膜１２に形成された穴部１５に対応する部位、すなわち感光性有機膜１２で覆われていない部位ではシリコンウエハ１１がエッチングされ、凹部１６が形成される。この後、感光性有機膜１２をアセトンなどの有機溶媒を用いて溶解させる。これにより、図７Ｅに示すように、シリコンからなる鋳型１７が得られる。
そして、この鋳型１７を用いてデバイスを製作する。具体的には、まず、図７Ｆに示すように、シリコンからなる鋳型１７に、シリコーンラバー溶融液１８を塗布する。なお、鋳型１７の表面には、予めシランカップリング剤を塗布するなどして、疎水性を保つ処理を施しておく。この疎水性を保つ処理により、後記の剥離工程における、シリコーンラバーと鋳型１７との離型性を向上させることができる。
シリコーンラバー溶解液１８を室温で固化させた後、固化物を鋳型１７から剥離する。これにより、シリコーンラバーからなるデバイス（シリコーンラバー構造体）、すなわち分離マトリックス５を含む溝３を備えた基板２が得られる。さらに、図７Ｇに示すように、デバイスに、所定の位置に試料注入口（貫通穴）を備えたカバー６を配置する。なお、カバー６としては、ガラス基板やプラスチック基板を用いることができる。そして、カバー６をデバイスに圧着することにより、カバー６とシリコーンラバーからなるデバイスとが密着した電気泳動セル１が完成する。シリコーンラバーは粘着性を有するので、カバー６をデバイスに圧着するだけで、ガラス基板やプラスチック基板からなるカバー６とデバイスとをシールすることができ、液漏れの問題は生じない。
このように、非常に簡便な手法で、鋳型１７ないし電気泳動セル１を製造することができる。また、シリコンからなる鋳型１７は、繰り返して使用することができるので、電気泳動セル１の生産性や生産コストを改善することができる。
上記デバイス、すなわち分離マトリックス５を含む溝３を備えた基板２は、上記の製造方法と異なる手法で製造してもよい。例えば、シリコンからなるプレス型に熱をかけながら、プラスチック板をプレス型に押し付けて加圧成形すれば、プラスチックからなるデバイスを得ることができる。この後、デバイスにカバー６を取り付ければ、電気泳動セル１が完成する。このようにプラスチックを用いたデバイスないし電気泳動セル１では、シリコーンラバーを用いた場合に比べてその強度が改善される。
また、シリコンからなる鋳型１７に代えて、厚膜の感光性有機膜からなる鋳型を用いてもよい。この場合、例えば厚膜の感光性有機膜を光照射によりパターニングし、パターニングされた感光性有機膜をそのまま鋳型として用いればよい。この手法によれば、図７Ｄに示すシリコンウエハ１１のエッチング工程を省くことができるので、生産性をさらに向上させることができる。
（ＰＤＭＳチップの製造方法）
以下、代表的なシリコーンラバーであるＰＤＭＳ（ポリジメチルシロキサン）をデバイスの材料として用いて、実際に電気泳動セル１を製造した過程ないし結果を説明する。この製造方法では、シリコンウエハからなるネガパターンの鋳型は、微細加工技術により形成された。この鋳型は、分離マトリックスを含む溝に対応する型面を有している。この鋳型を、直径９０ｃｍのプラスチックシャーレ内に配置し、その上部からＰＤＭＳを注入した。ＰＤＭＳは、東レ・ダウコーニング社から購入したもの（商標名「ＳＹＬＧＡＲＤ」）を用いた。
シリコンウエハ（シリコン基板）には、あらかじめシランカップリング剤で表面処理を行った。シリコンウエハの表面に、アセトニトリルに４％のジメチルジクロロシランを溶解させた溶液を塗布した後、該シリコンウエハに、１００℃で３０分間、乾燥処理を施した。１００ｍｌのビーカに約４０ｍｌのＰＤＭＳを入れ、さらに４ｍｌのキャタリストを加えてよく混合した後、このビーカをデシケータに入れて、３０分間、真空ポンプで脱気した。
この後、シリコンウエハが入ったプラスチックシャーレに、２０ｍｌの脱気したＰＤＭＳを入れ、このプラスチックシャーレをデシケータに入れて、３０分間、真空ポンプで脱気した。分離マトリックスを含む溝から気泡を十分に追い出した後、デシケータに空気を入れて、プラスチックシャーレを取り出した。そして、脱気済みのＰＤＭＳを、シリコンウエハの上に、約５〜７ｍｍの厚さで積層（重層）した。ＰＤＭＳが積層されたプラスチックシャーレを、４０度の恒温ボックスに入れてＰＤＭＳを重合させた。４８時間以上経過した後、プラスチックシャーレを取り出した。そして、必要とする分離マトリックスの部分の周りに、カッターで切り込みを入れ、重合したＰＤＭＳを、シリコンウエハからなる鋳型からゆっくり剥離して、ＰＤＭＳチップを得た。
ＰＤＭＳチップ（幅５ｍｍ、長さ２０ｍｍ、厚さ５ｍｍ）に、カバーとして同一寸法（幅５ｍｍ、長さ２０ｍｍ、厚さ１ｍｍ）のポリスチレン板をかぶせた。密着性が悪い場合は、ＰＤＭＳチップの溝側表面に、酸素プラズマ処理（酸素圧力１．０ｋＰａ、励起電力１００ｗ、処理時間５分）や紫外線処理（２０ｗ紫外線ランプ、処理時間２０分、距離１０ｃｍ）を施して、密着性を高めればよい。カバーには、直径１ｍｍの試料注入口（穴）を、溝に沿って２つ形成した。そして、白金線（直径０．２ｍｍ）からなる電極を、先端近傍部が試料注入口を通って溝内の緩衝液に直接触れるように配線した（図１参照）。なお、白金蒸着により電極を形成した場合も、白金線を用いた場合と同様の効果を奏するのはもちろんである。
ＤＮＡの電気泳動用の緩衝液としては、ＴＢＥ緩衝液（８９ｍＭ Ｔｒｉｓ−ｂｏｒａｔｅ、２ｍＭ ＥＤＴＡ）を用いた。ＰＤＭＳチップに酸素プラズマ処理を施した場合、ＰＤＭＳチップ表面は親水性となるので、緩衝液は分離マトリックスの支柱の間隙に、毛管現象により侵入する。このような親水化処理を行わない場合は、一方の試料注入口から溝内に緩衝液を供給しつつ、他方の試料注入口を注射器で吸引し、緩衝液を分離マトリックスに充填すればよい。あるいは、緩衝液の中にＰＤＭＳチップを浸漬し、真空デシケータの中で減圧して、緩衝液を分離マトリックスに充填すればよい。後者の場合、カバーは脱気後にＰＤＭＳチップに慎重にかぶせる必要がある。
（電気泳動の実施）
以下、本発明にかかる電気泳動セルを用いて実際に電気泳動を行った過程及び結果を説明する。生体分子試料としてＤＮＡ試料を用いた。ＤＮＡ試料としては、λＤＮＡと、Ｔ４ファージＤＮＡと、λＤＮＡを制限酵素Ｘｂｏ−Ｉで約半分に切断した１／２λＤＮＡとを用いた。これらのＤＮＡの塩基数は、それぞれ、４８５００、１６６０００、２４０００であった。これらのＤＮＡをＴＢＥ緩衝液１μｌあたり０．０５μｇ含み、かつ蛍光染色試料としてＹＯＰＲＯ−Ｉ（モレキュラープローブ社製）、サイバーグリーン（宝酒造社製）又はサイバーゴールド（宝酒造社製）を１μＭ含むＤＮＡ混合液を試料として用いた。この試料を、１回の電気泳動あたり１μＬ使用した。そして、カバーに形成された２つの試料注入口のいずれか一方に試料を注入して電気泳動を行った。電気泳動用の電源は、０〜１０００Ｖ、１Ａ定格のＡＴＴＯクロスパワー１０００を用いた。この電源で、白金線からなる電極の両端に５０〜１００Ｖ／ｃｍの電圧を印加した。電流は１０ｍＡ以下であった。ＤＮＡは、ＴＢＥ緩衝液中で負に帯電しているので、正極である電極に向かって泳動する。溝の長さが１ｃｍの場合、電気泳動時間を２〜５分間とした。
（電気泳動の観察）
以下、電気泳動の観察手法の一例を説明する。図８は、ＤＮＡの電気泳動を観察し、又は記録するための電気泳動評価装置の概略構成を示している。
図８に示すように、この電気泳動評価装置は、蛍光顕微鏡２０を備えている。この蛍光顕微鏡２０には、サンプル２１（電気泳動セル）を載せるサンプル台２２と、励起側フィルタ（図示せず）を備えた光源２３と、ダイクロイックミラー２４と、ＤＮＡを観察するための観察窓２５と、光路を切り替える切り替えミラー２６と、光を電気信号に変換する光電子増倍管２７と、ビデオカメラ２８（高感度ＣＣＤカメラ）とが設けられている。また、この電気泳動評価装置は、画像を記録するＶＴＲ２９（ビデオ・テープ・レコーダ）と、パーソナルコンピュータ３０と、ディスプレイ３１とを備えている。
蛍光顕微鏡２０においては、光源２３から放射され、励起側フィルタにより波長が４２０〜４９０ｎｍに調整された光Ｌ１は、ダイクロイックミラー２４によって反射された後、サンプル２１に照射される。その結果、サンプル内のＤＮＡは蛍光を放射する。この蛍光は、吸収フィルタ（図示せず）によって波長が５２０ｎｍの光Ｌ２に調整される。この光Ｌ２は、ダイクロイックミラー２４によって、観察窓２５に向かう光Ｌ３と、光電子増倍管２７又はビデオカメラ２８に向かう光Ｌ４とに分割される。
かくして、観察窓２５を覗くことにより、蛍光を発しているＤＮＡ分子が、電気泳動により分離マトリックス内（支柱の隙間）を通過する様子を観察することができる。光Ｌ４は、切り替えミラー２６で光路を切り替えることにより、光電子増倍管２７又はビデオカメラ２８に入力される。光電子増倍管２７は、この光Ｌ４を電気信号に変換して、外部機器に出力する。高感度ＣＣＤカメラであるビデオカメラ２８は、ＤＮＡ分子が電気泳動により分離マトリックス内を通過する様子を撮影し、得られた画像をＶＴＲ２９に送る。ＶＴＲ２９はこの画像を記録する。ＶＴＲ２９に記録されている画像は、パーソナルコンピュータ３０を用いて解析することができ、またディスプレイ３１に表示することができる。
（塩基数と移動速度との関係）
以下、ＤＮＡの塩基数と、電気泳動によるＤＮＡの移動速度（電気泳動速度）との関係を説明する。
図９は、次の条件における、ＤＮＡの塩基数とＤＮＡの移動速度との関係を示している。
溝の幅：２００μｍ
溝の深さ：１０μｍ
分離マトリックスの長さ：１０ｍｍ
支柱の形状：円柱、
支柱の直径：１５μｍ
支柱の隙間：約１μｍ
支柱の配置密度：４０００個／ｍｍ^２
電圧：５０Ｖ／ｃｍ
緩衝液：ＴＢＥ緩衝液
ＤＮＡ濃度：５０ｎｇ／μｌ
染色剤：サイバーグリーン１ｍＭ
図９から明らかなとおり、電気泳動によるＤＮＡの移動速度は、ＤＮＡの塩基数に依存して顕著に変化している。したがって、人工的な支柱と隙間とからなる本発明にかかる分離マトリックスは、アガロースゲルやポリアクリルアミドゲルの網目と同様の効果を奏し、これらのゲルに代替することができることが分かる。
（ＤＮＡの移動モデル）
図１０は、円柱形の支柱４と、支柱４の隙間を通り抜けるＤＮＡの運動モデルとを示している。図１０に示すように、障害物である支柱４の隙間が、この隙間を通過するＤＮＡの運動に対して、エントロピ障壁の役割を果たしていると考えられる。すなわち、球状のＤＮＡ分子は、支柱４の間隙を通過する際、一時的に、熱力学的に低エントロピのコンフォメーションをとり、変形して間隙を通り抜ける。これが、電気泳動によるＤＮＡの移動速度に影響していると考えられる。
（本発明にかかる電気泳動セルの応用例）
以上、本発明にかかる基本的な電気泳動セルについて、その形状、材質、製造方法、ＤＮＡの分離の原理、ＤＮＡの運動モデル等を詳細に説明したが、以下では、このような基本的な電気泳動セルを応用したＤＮＡ分取装置（生体試料分取装置）を説明する。
（ＤＮＡ分取装置１：応用例１）
図１１は、本発明にかかる電気泳動セルを応用した１つのＤＮＡ分取装置を示している。図１１に示すように、このＤＮＡ分取装置では、分離マトリックス５が、支柱の配置密度ないし支柱の間隙（ピラー間隙）が異なる５つのピラーエリアＡ〜Ｅに区分されている。ここで、ピラーエリアＡ、Ｂ、Ｃ、Ｄ、Ｅは、この順に、支柱の配置密度が大きくなっている（支柱の間隙が小さくなる）。そして、このＤＮＡ分取装置は、陰極を伴った１つの試料注入口３３と、それぞれピラーエリアＡ〜Ｅに設けられ陽極を伴った５つの試料回収口３４ａ〜３４ｅとを備えている。
かくして、このＤＮＡ分取装置では、例えば、分子量の異なるＤＮＡを含むサンプルを試料注入口３３に供給すれば、分子量が大きいＤＮＡは、支柱の配置密度が大きいピラーエリア（ピラーエリアＥ側）を通過しにくいので、支柱の配置密度が小さいピラーエリア（ピラーエリアＡ側）に多く集まり、このピラーエリアの試料回収口から回収される。したがって、ピラーエリアＡ、Ｂ、Ｃ、Ｄ、Ｅに存在するＤＮＡ、ないし試料回収口３４ａ〜３４ｅで回収されるＤＮＡは、後者ほど分子量が小さくなる。よって、サンプル中のＤＮＡを、分子量に応じて５種類に分取（分画）することができる。
（ＤＮＡ分取装置２：応用例２）
図１２Ａ及び図１２Ｂは、本発明にかかる電気泳動セルを応用したもう１つのＤＮＡ分取装置を示している。図１２Ａ及び図１２Ｂに示すように、このＤＮＡ分取装置では、分離マトリックス５は、平面視で、縦方向（Ｘ１−Ｘ２方向）及び横方向（Ｙ１−Ｙ２方向）について２次元的に互いに離間して配置された多数の角柱状の支柱４で構成されている。また、このＤＮＡ分取装置は、陰極を伴った１つの試料注入口３５と、それぞれ陽極を伴った６つの試料回収口３６ａ〜３６ｆとを備えている。
この分離マトリックス５では、ある支柱４と、Ｘ２側でこの支柱４とＸ１−Ｘ２方向に隣り合うの２つの支柱４との間に形成されＹ１−Ｙ２方向に伸びる間隙において、Ｙ１側に位置する間隙３８は広く、Ｙ２側に位置する間隙３９は狭くなっている。他方、Ｙ１−Ｙ２方向に隣り合う支柱４間でＸ１−Ｘ２方向に伸びる間隙４０は、すべて広くなっている。なお、Ｘ１−Ｘ２方向に伸びる間隙４０のＸ２側の端部と、Ｙ１−Ｙ２方向に伸びる両間隙３８、３９との接続部には、略矩形の空間部４１が形成されている。
かくして、このＤＮＡ分取装置では、例えば、分子量の異なるＤＮＡを含むサンプルを試料注入口３５に供給すれば、分子量が大きいＤＮＡは、狭い間隙３９を通過することができないので、例えばＲ１で示すように、Ｙ１−Ｙ２方向（横方向）に関しては、Ｙ１方向には移動せず、Ｙ２方向にのみ移動してゆく。これに対して、分子量が小さいＤＮＡは、Ｙ１方向及びＹ２方向のどちらへでも移動することができる。しかし、前記のとおり、分子量が大きいＤＮＡがほとんどＹ２方向に移動するので、その影響により、分子量は小さいＤＮＡは、例えばＲ２で示すように、Ｙ１方向に移動する傾向が強くなる。したがって、分離マトリックス５内において、試料回収口３６ａ〜３６ｆで回収されるＤＮＡは、後者ほど分子量が小さくなる。よって、サンプル中のＤＮＡを、分子量に応じて６種類に分取（分画）することができる。
（医療診断への応用）
本発明にかかる電気泳動セルないしＤＮＡ分取装置（生体試料分取装置）は、医療診断あるいは遺伝子多型検出に応用することができる。例えば、ヒトの遺伝子には、個体ごとに塩基配列にわずかな違い（遺伝子多型）が存在する。そして、その違いは、遺伝子制御領域やイントロン領域、あるいは蛋白質などをコードしている領域にあるといわれている。遺伝子制御領域やイントロン領域の違いは、遺伝子の発現量が個体によって異なることを示している。また、蛋白質などをコードしている領域は、酵素の活性が個体によって異なることを示している。個体差は遺伝子多型の組合せで決まるともいわれている。例えば、薬の代謝には個体差があり、これが薬の薬効や副作用に深く関連している。すなわち、これらの薬効や副作用には、個体が持つ薬代謝遺伝子群の遺伝子多型が大きく影響している。
遺伝子多型には種々のものがあるが、現在、最も数が多く、大量解析が容易であるといわれているのは、１塩基多型（ＳＮＰｓ：ｓｉｎｇｌｅ ｎｕｃｌｅｏｔｉｄｅ ｐｏｌｙｍｏｒｐｈｉｓｍｓ）である。ＳＮＰｓは個体間で１遺伝子暗号（１塩基）が他の塩基に置き換わっている部分で、ヒトのゲノムの中には３００〜１０００万個あると予想されている。そのため、薬代謝遺伝子群の遺伝子多型情報は、医薬品の開発、薬物の有効利用、副作用防止等に役立つであろうと期待されている。
ＳＮＰｓ解析手法としては、現在，制限酵素法、ＳＳＣＰ（ｓｉｎｇｌｅ−ｓｔｒａｎｄ ｃｏｎｆｏｒｍａｔｉｏｎ ｐｏｌｙｍｏｒｐｈｉｓｍｓ）解析法、直接シーケンシング法、電気化学検出法などが知られている。この中で電気泳動により分析を行うものは、制限酵素法、ＳＳＣＰ解析法、直接シーケンシング法である。これらの迅速な分離や検出に、本発明にかかる分離マトリックスを利用した電気泳動セルを応用することができる。本発明にかかる電気泳動セルを用いることにより、少量のサンプルで迅速にＳＮＰｓを検出することができる。この中でも、ＳＳＣＰ法は、ＰＣＲ技術を利用して増幅し、立体構造の変化を電気泳動で検出するので、本発明に好適である。
本明細書では、ＤＮＡや生体分子をターゲットにしているが、ターゲットは生体分子に限られるわけではない。環境化学物質や高分子化合物の分離や検出にも応用することができる。さらに、最近話題にのぼっている化学マイクロデバイスやＬａｂ ｏｎ ａｃｈｉｐなどのマイクロ流路における分離や検出にも、この分離マトリックス及びその製造方法が役立つものと予想される。
以上、本発明は、その特定の実施の形態に関連して説明されてきたが、このほか多数の変形例及び修正例が可能であるということは当業者にとっては自明なことであろう。それゆえ、本発明は、このような実施の形態によって限定されるものではなく、添付の請求の範囲によって限定されるべきものである。  Hereinafter, embodiments of the present invention will be specifically described with reference to the accompanying drawings. However, in each figure, common members, that is, members having substantially the same configuration and function are denoted by common reference numerals, and redundant description is omitted in principle.
  (Outline of electrophoresis cell)
  First, an outline of the electrophoresis cell according to the present invention will be described. FIG. 1 shows the overall structure of the basic form of an electrophoresis cell according to the present invention. FIG. 2 shows an enlarged part of the electrophoresis cell, and FIG. 3 shows an elevational section of the electrophoresis cell.
  As shown in FIGS. 1 to 3, the electrophoresis cell 1 has a substrate 2, and a groove 3 is formed on the upper surface of the substrate 2. The substrate 2 is not necessarily transparent, but is preferably transparent because the inside of the substrate 2 can be seen through. The groove 3 includes a separation matrix 5 composed of a large number of pillars 4 (pillars). The upper surface of the substrate 2 and the groove 3 are covered with a transparent cover 6. Further, the electrophoresis cell 1 is provided with two sample injection ports 7 and two electrodes 8 (positive electrode and negative electrode). Each electrode 8 is inserted into the groove 3 through the corresponding sample inlet 7. That is, the sample injection port 7 also serves as an electrode insertion hole. In addition, both electrodes 8 are connected to a DC power supply 10 via lead wires 9.
  In the electrophoresis cell 1, each support | pillar 4 which comprises the separation matrix 5 has a geometric cross section. The upper surface of each column is in close contact with the lower surface of the cover 6. The groove 3 containing the separation matrix 5 is filled with a buffer solution for electrophoresis, and therefore there is a buffer solution in the gap between the columns 4. Each electrode 8 inserted into the groove 3 is in direct contact with the buffer solution in the groove 3. When a biomolecule is present in the buffer solution, when a voltage is applied between both electrodes 8, the biomolecule is charged, so that it moves toward one of the electrodes 8 depending on the state of the charge. To do. For example, if the biomolecule is DNA (deoxyribonucleic acid), since it is negatively charged in the buffer solution, the positive electrode 8 (in the direction in which the electrodes 8 extend) (the direction connecting the electrodes 8). In FIG. 1, it moves toward the left electrode).
  The shape and thickness of the substrate 2 are not particularly limited, and can be a desired shape according to the application and purpose. However, since the molding is easy and the strength is high, the substrate 2 is preferably plate-shaped. When the material of the substrate 2 is an elastomer, the thickness of the substrate 2 is preferably 3 mm or more in order to ensure adhesion with the cover 6. A plurality or a plurality of grooves 3 including the separation matrix 5 may be provided on the surface of the substrate 2. The grooves 3 serving as a flow path for biomolecules such as DNA may cross each other in order to facilitate sample injection and mixing with other biomolecules such as DNA or reagents. In addition, a circular or polygonal portion is formed in the groove 3 (flow path) to control the resistance of the groove 3 to the liquid and the moving speed of the liquid, and perform fluid adjustment at the micro level. May be.
  (Groove structure)
  Hereinafter, a specific configuration or function of the groove 3 will be described. In the example shown in FIGS. 1 to 3, the groove 3 has a linear shape extending in the longitudinal direction of the substrate 2. However, the shape of the groove 3 is not limited to this, and may be, for example, U-shaped, S-shaped, or circular. However, in this case, since the electric field distribution is not parallel to the groove 3 at the curved portion of the groove 3, it is necessary to devise the shape of the groove 3 to perform electric field correction and detection correction.
  The electrophoresis cell 1 according to the present invention can be easily miniaturized, but the width of the groove 3 is preferably 1 μm or more, and more preferably 20 μm or more. This is because, when the width of the groove 3 is narrower than 1 μm, the detection sensitivity is lowered, a high-magnification optical instrument is required for detection of the biomolecule, and the detection cost increases. The width of the groove 3 is preferably 1000 μm or less, and more preferably 300 μm or less. When the width of the groove 3 exceeds 1000 μm, the uniformity of electrophoresis is impaired and the effect of the present invention is reduced. However, when the present invention is applied to a separation matrix 5 having a two-dimensional extension including a plurality of sets of electrodes 8, for example, a separation matrix 5 for pulse field electrophoresis, the width of the groove 3 may exceed 1000 μm. Good.
  The depth of the groove 3 is preferably 1 μm or more. This is because if the depth of the groove 3 is shallower than this, the effect of the present invention is reduced and the detection sensitivity is lowered. Further, the depth of the groove 3 is preferably 500 μm or less, and more preferably 50 μm or less. If the depth of the groove 3 exceeds 500 μm, vertical diffusion of biomolecules such as DNA occurs, and the effect of the present invention is reduced, and it becomes difficult to focus optically when detecting biomolecules, and the detection sensitivity decreases. It is because there is a possibility of doing.
  The length of the groove 3 and the length of the separation matrix 5 can be arbitrarily set. Specifically, for example, it can be determined in the range of 0.1 to 50 mm according to the target of the biomolecule to be separated and the separation ability required for each target. However, the upper limit of these lengths may be limited by the size of a silicon wafer that will be a mold described later.
  (Configuration of electrode)
  Hereinafter, a specific configuration or function of the electrode 8 will be described. In this electrophoresis cell 1, the electrode 8 is a platinum wire having a diameter of 0.2 mm, and is connected to the lead wire 9 of the power supply 10 by soldering. However, the electrode 8 may be linear, plate-like, or film-like (deposited film) as long as the terminal can be in contact with the buffer in the groove 3, and the size and thickness are arbitrary. Can be set to The electrode 8 may be made of any material as long as a voltage of 500 V can be applied, a current of several tens of mA can be applied, and electrolysis or elution of the material does not occur. Examples of such a material include platinum, gold, carbon, and a conductive polymer.
  In this electrophoresis cell 1, most of the electrode 8 made of platinum wire is disposed on the upper surface of the cover 6, and the vicinity of the tip is inserted into the groove 3 through the sample inlet 7 provided in the cover 6. ing. However, the electrode 8 may be directly bonded or deposited on the cover 6. Further, wiring may be performed from an external support through the sample injection port 7 or through the substrate 2. Note that the vicinity of the tip of the electrode 8 may be inserted into the groove 3 through an electrode insertion hole separately formed in the cover 6 instead of passing through the sample injection port 7.
  In the electrophoresis cell 1, a DC voltage in the range of 0.1 to 500 V / cm is applied to the electrode 8. However, a pulse voltage may be applied. Further, when a plurality of sets of electrodes 8 are provided, the direction of current may be periodically changed.
  In the electrophoresis cell 1, as a buffer solution for electrophoresis that fills the flow path in the groove 3 and the gap between the columns 4 constituting the separation matrix 5, when the biomolecule to be separated is DNA, TBE buffer (89 mM Tris-borate, 2 mM EDTA) or TAE buffer (40 mM Tris-acetate, 1 mM EDTA) is used. The pH of the buffer is around 8. When the biomolecule to be separated is a protein, an optimum buffer for electrophoresis is used according to the type of the protein.
  (Structure of the support)
  Hereinafter, the specific structure thru | or function of the support | pillar 4 are demonstrated. In the electrophoresis cell 1, as is apparent from FIG. 2, the shape (hereinafter simply referred to as “top” or cross section (cross section cut along a plane perpendicular to the central axis of the column) of each column 4 constituting the separation matrix 5. “Cross sectional shape”) is circular. However, the cross-sectional shape of the support column 4 does not need to be circular, and can be an arbitrary shape.
  For example, as shown in FIGS. 4A to 4E, the cross-sectional shape of the support column 4 is a rectangle with rounded corners (FIG. 4A), an ellipse (FIG. 4B), a square or rhombus (FIG. 4C), and a hexagon (FIG. 4D). ) Or a rectangle (FIG. 4E). Alternatively, other polygons or polygons with corners cut off may be used. Furthermore, as long as the flow of biomolecules such as DNA is smooth, the cross-sectional shape may be a star shape, a geometric shape having protrusions, or a shape having no fixed shape. The cross-sectional shapes of the columns 4 need not be the same, and the shapes described above may be mixed. For example, the cross-sectional shape of the support column 4 may be an ellipse in the vicinity of the sample injection port 7 and a hexagonal shape in other portions.
  The cross-sectional area of each column 4 can be arbitrarily set as long as the dimension of the column 4 in the groove width direction is smaller than the width of the groove 3. However, when it is necessary to make the vertical flow of biomolecules such as DNA uniform, the cross-sectional area of the column 4 is preferably uniform in the vertical direction (column axis direction). Further, when separating biomolecules in the depth direction of the groove 3, the support column 4 may have a tapered shape that becomes narrower in the vertical direction. If molding is possible, the support column 4 may have a three-dimensional geometric shape, for example, a shape in which a plurality of spheres continuously form a support column.
  (Place arrangement)
  Hereinafter, the specific arrangement | positioning form of the support | pillar 4 is demonstrated. In this electrophoresis cell 1, as is apparent from FIGS. 1 and 2, the support columns 4 are arranged in an arrangement form in which a plurality of support columns 4 spaced apart at equal intervals in the groove width direction are arranged at equal intervals in the groove longitudinal direction. Has been. However, the arrangement form of the support columns 4 does not need to be such and can be arbitrary. In order to exert the function as the separation matrix 5, the support columns 4 are arranged in a predetermined region of the groove 3, but the arrangement form or arrangement form is regular as long as biomolecules such as DNA flow smoothly. However, it is not necessary. However, if the arrangement of the columns 4 is not regular, the columns 4 need to be arranged randomly in order to generate a uniform sample flow.
  5A to 5E show five specific examples of the arrangement form of the pillars 4 in the groove 3 (top view of the substrate 2). The arrangement shown in FIG. 5A is a two-dimensional array pattern that can move biomolecules two-dimensionally in plan view. In this arrangement, four sample inlets 7 and two sets (four) of electrodes 8 are arranged around the rectangular groove 3. The arrangement form shown in FIG. 5B is an arrangement pattern in which the arrangement density of the columns 4 or the gaps between the columns 4 change stepwise in the longitudinal direction of the grooves 3. The arrangement form shown in FIG. 5C is an arrangement pattern in which the columns 4 are arranged in a row in the groove 3. The arrangement form shown in FIG. 5D is an arrangement pattern in which the columns 4 are arranged in a row in the groove 3 and the wall surface of the groove 3 functions as a column. In other words, the separation matrix 5 is composed of independent columns 4 and columns 4 ′ that are part of the wall surface of the groove 3. The arrangement shown in FIG. 4E is an arrangement pattern in which a plurality of columns 4 are arranged in the groove 3, and two sample injection ports 7 are connected to one end in the longitudinal direction of the groove 3.
  As described above, when the present invention is applied to a separation matrix for pulse field electrophoresis, separation of a two-dimensional array pattern including a plurality of sample injection ports 7 and a plurality of sets of electrodes 8 as shown in FIG. 5A, for example. A matrix 5 is required. In order to facilitate the injection of the sample into the groove 3, the arrangement density of the columns 4 may be lowered near the sample injection port 7 (see FIG. 5B). Further, a concentration region for biomolecules such as DNA may be provided, or a gate for selectively separating biomolecules may be provided. Furthermore, a one-dimensional or two-dimensional gradient may be given to the gap between the support columns 4.
  The struts 4 may be arranged so as to contact other plural struts 4 or may be arranged so as to contact continuously in the arrangement direction. The support column 4 may be in contact with the side surface of the groove 3. Further, the wall surface of the groove 3 may be repeatedly protruded into the groove with a shape corresponding to the support column 4, and the protruding portion may be used as the support column. In the simplest case, the separation matrix 5 is composed of only the protrusions on one wall surface of the groove 3 or only the protrusions on both wall surfaces. In this case, the electrophoresis cell 1 is a simple flow path device that does not have an independent support structure.
  (Sample inlet)
  Hereinafter, a specific configuration and function of the sample injection port 7 will be described. Only one sample injection port 7 for injecting a sample containing a biomolecule into the groove 3 may be provided, or a plurality or a plurality of sample injection ports 7 may be provided. When a plurality of sample injection ports 7 are provided, the electrophoresis cell 1 exhibits a function of mixing a plurality of biomolecules in addition to a function of separating biomolecules (see FIG. 5E).
  In addition, as shown in FIG. 6A, in order to easily inject a sample containing a biomolecule such as DNA into the groove 3, a region where the support column 4 is not disposed in the groove 3 is provided at a portion where the sample injection port 7 is disposed. It is preferable to provide a region where the arrangement density of the columns 4 is low.
  As shown in FIG. 6B, when the columns 4 are provided at the same arrangement density as the other parts at the part where the sample inlet 7 is arranged, the sample is introduced onto the separation matrix 5 and then the arrow P1. As shown by the curve, it flows in a curve and enters the separation matrix 5. In this case, since the resistance to the flow of the sample is large, it is difficult to smoothly inject the sample into the groove 3.
  On the other hand, as shown in FIG. 6C, when the column 4 is not provided at the site where the sample inlet 7 is disposed, the sample is introduced into the space where the column 4 does not exist, and then as shown by the arrow P2. It flows linearly and enters the separation matrix 5 from the side of the column 4. In this case, since the resistance to the sample flow is small, the sample can be smoothly injected into the groove 3.
  (Biomolecular separation principle)
  Hereinafter, the principle of separating biomolecules in the electrophoresis cell 1 will be described. The interval between the columns 4 is the most important factor for the separation of biomolecules. In the following, for the sake of convenience, the narrowest part of the gap between the columns is referred to as a “column gap”, and the column gap is not enlarged or the distance is referred to as a “gap distance”. The gap distance must be large enough for the biomolecule to be separated to pass through. And the gap distance functions as an obstacle to the passage of biomolecules and can influence the passage speed of biomolecules depending on the parameters defined by the size of the molecule, eg the radius of inertia of the molecule Must be a thing. The separation principle based on the size (molecular weight) of a biomolecule such as DNA in the separation matrix 5 according to the present invention is the same as that in an agarose gel or polyacrylamide gel. Therefore, it can be said that the electrophoresis cell 1 according to the present invention replaces the role of the gel network in electrophoresis with the separation matrix 5 composed of a plurality of support columns 4.
  In general, DNA in a buffer is a linear polymer in a random coil state, and these displacement lengths can be expressed as an average square end-to-end distance. For example, Paul J., famous for polymer solution theory. According to Flor J. Flory, the displacement length R of DNA is defined by the following equation 1.
        R = 0.3 × (number of bases)^1/2[Nm] ……………………………… Formula 1
  According to Equation 1, the size (displacement length or radius of inertia) of DNA increases in proportion to the 1/2 power of the number of bases. The role of the agarose gel or polyacrylamide gel network in electrophoresis is to be an obstacle to the passing DNA. Therefore, if the size of the DNA and the gap distance of the separation matrix 5 are on the same order, the movement of the DNA molecular chain passing through the support gap is affected by the gap distance, and the support gap serves as an entropy barrier. It is considered a thing. That is, when DNA passes through a support gap that is narrower than its size, the DNA must be in a thermodynamically low entropy conformation (deformed shape). This is the principle by which a difference occurs in the electrophoresis speed of DNA depending on the molecular weight of DNA.
  (Post gap)
  Hereinafter, the support gap or the gap distance will be described in more detail. As inferred from the above principle, if the gap distance is about the same as the size of DNA, the strut gap becomes an obstacle to the movement of DNA, and separation depending on the molecular weight of DNA can be achieved. Usually, the size of the agarose or polyacrylamide gel network used for the separation of oligonucleotides by electrophoresis and the sequencing of 10 to 1000 bases of DNA is 10 to 100 nm (for example, published in 2000, “ (See the report by Yoshinobu Baba described in “Protein / Nucleic Acid / Enzyme”, Vol. 45, No. 1, pp. 76-84). Therefore, when separating DNA fragments of 1000 bases or less, it can be said that it is essential that the gap distance is 100 nm or less (see, for example, JP-T-11-508042).
  On the other hand, in the separation matrix 5 according to the present invention, the optimum gap distance is 0.1 to 5 μm. Hereinafter, the reason will be described. Table 1 shows the results of examining the relationship between gap distance and DNA mobility for three DNAs having different numbers of bases. According to Table 1, it can be seen that T4 DNA (166 kbp) and λDNA (48.5 kbp) do not easily pass through the support gap when the gap distance is 0.1 μm or less. This is presumably because the radius of inertia of DNA is larger than the actual size of DNA due to the influence of water molecules hydrated to DNA.

  That is, the physical size of a mesh such as agarose gel is 10 to 100 nm. However, in reality, the influence of sugar chain molecules and polyacrylamide molecular chains or the influence of water molecules hydrated to DNA is added. In the case of agarose and polyacrylamide, these water molecules interact with DNA and the mesh that is the separation matrix, whereby the DNA is smoothly separated. Since these molecular chains are elastic, it is presumed that they utilize the effects of hydration water such as DNA or absorb the influence of water molecules well. The size of these hydrated waters at the molecular level is hardly known at the present time, but it is considered that DNA is difficult to enter due to the influence of hydrated water in the gap of 100 nm level. Permeation of water is possible if a physical force (pressure, etc.) is artificially applied to the gaps by reverse osmosis or high pressure liquid chromatography. However, in this case, biomolecules such as DNA to be separated may be destroyed.
  On the other hand, in the separation matrix 5 according to the present invention, the upper limit of the gap distance is 5 μm. Hereinafter, the reason will be described. Table 2 shows the results of examining the relationship between the gap distance and the DNA movement speed for T4 DNA and λDNA. According to Table 2, it can be seen that when the gap distance exceeds 5 μm, a difference in moving speed cannot be obtained with certainty. Therefore, the upper limit value of the gap distance is set to 5 μm.

  (Pole arrangement density)
  Hereinafter, the arrangement density of the columns 4 will be described. The arrangement density of the columns 4 in the separation matrix 5 can basically be set arbitrarily. However, since the separation matrix 5 according to the present invention is based on the principle that the passage speed of DNA is affected by the support gap, the DNA separation performance is improved in proportion to the number of support gaps through which the DNA passes. . According to the experiment by the inventors of the present application, the migration speed by electrophoresis of DNA of 50 to 200 kbp (kilo base pairs) under the condition of 50 to 100 V / cm was an average of 50 μm / second. The number of support columns 4 (cylinder with a diameter of 15 μm) passing through in 1 second was about 3, and the number of support column gaps was 6. At this time, a difference in moving distance of about 0.1 μm per 1000 base pairs (moving faster as base pairs are shorter) was observed. If the length of the separation matrix 5 in the groove 3 is 5 mm, this DNA passes through the separation matrix 5 in about 100 seconds, and causes a difference in movement distance of about 10 μm per 1000 base pairs at a detection point 5 mm ahead. It will be. If the optical resolution of the detection system is 5 μm, this difference in moving distance can be sufficiently detected.
  In this case, DNA passes through 60 struts 4 and about 120 strut gaps per 1 mm of the movement distance. If it is assumed that DNA does not migrate in the column gap perpendicular to the direction of the electric field, the number of column gaps is almost twice the number of columns 4. Therefore, the arrangement density of the columns 4 is about 4000 pieces / mm.²The arrangement density of the support gap is about 14,000 pieces / mm.²It becomes. Since DNA shorter than this DNA migrates faster, the arrangement density of the columns 4 or the arrangement density of the column gaps is 4000 pieces / mm, respectively.²Or 14,000 pieces / mm²Must be higher than. Table 3 shows the results obtained by repeatedly performing the above experiment using T4 DNA and λDNA at various arrangement densities of the support columns 4 and determining the relationship between the arrangement density of the support columns 4 and the difference in DNA movement speed. According to Table 3, the arrangement density of the columns 4 of the separation matrix 5 is 1000 pieces / mm.²It turns out that it is necessary above.

  (Material of substrate and separation matrix)
  Hereinafter, materials of the substrate 2 and the separation matrix 5 will be described. The material of the substrate 2 and the separation matrix 5 is not particularly limited as long as it is a polymer material that is inexpensive and easily mass-produced. However, these materials are preferably transparent because they are easy to detect optically. Therefore, for example, an elastomer can be used as the soft material, and a plastic can be used as the hard material. The most common transparent materials that are elastomers include transparent synthetic rubbers such as silicon rubber, butadiene rubber, isoprene rubber, fluorine rubber, styrene transparent rubber, and blended rubbers thereof. Moreover, what is necessary is just to satisfy the following conditions as performance.
      Density: ~ 1g / ml
      Water supply rate: 0.1% or less
      Total light transmittance: 85% or more
      Mold shrinkage: 1% or less
      Flexural modulus: 300Mpa or more
  Any curing method may be used, but from the viewpoint of moldability, a method of starting curing with a chemical initiator or a crosslinking agent, or a method of curing with an energy beam such as heat curing or light is preferable.
  On the other hand, the plastic material is preferably one that transmits excitation light and fluorescence. Examples of such materials include polystyrene resins, polysulfone resins, acrylic resins, polycarbonate resins, polyolefin resins, chlorine-containing polymers, polyether resins, polyester resins, and the like. These resins may be in the form of homopolymers, copolymers, blends or prepolymers. A thermoplastic resin is preferable in terms of molding, but a thermosetting or energy ray curable crosslinked polymer may be used. Further, it is desirable that the substrate 2, the grooves 3 and the separation matrix 5 are simultaneously formed from the same material. However, if the adhesion is sufficient, different materials may be bonded or fitted. In this case, as the material of the substrate 2, for example, ceramics, glass, silicon, polymer alloy, or the like can be used.
  (Cover material)
  Hereinafter, the material of the cover 6 will be described. The material of the cover 6 can sufficiently transmit excitation light and fluorescence, and the lower surface of the cover 6 is sufficiently in close contact with the upper surface of the substrate 2, the groove 3, and the separation matrix 5 (support 4) to cause liquid leakage. As long as it does not adsorb biomolecules such as DNA, it is not particularly limited. For example, glass, plastic, elastomer and the like can be used. The surface of the cover 6 is preferably hydrophilic, but may be hydrophobic. The thickness of the cover 6 can be arbitrarily set. However, considering the optical focus of the high magnification detection system, the thickness of the cover 6 is preferably 2 mm or less.
  (Surface treatment of materials)
  Hereinafter, the surface treatment of the materials of the substrate 2, the separation matrix 5, and the cover 6 will be described. It is desirable that the surface of the substrate 2 is hydrophobic and the contact angle with water is 45 degrees or more. On the other hand, the surface of the groove 3, the surface of the separation matrix 5 (support 4), and the surface of the cover 6 covering the groove 3 are easily contacted with the buffer solution, and the buffer solution of the support column 4 is capillarized. It is preferably hydrophilic so as to facilitate filling the gap spontaneously. Therefore, it is preferable to subject these surfaces to a hydrophilic treatment. Hydrophilization may be performed by any method. Specifically, for example, plasma treatment, plasma polymerization, corona discharge, coating of a hydrophilic chemical substance such as a coating polymer, hydrophilization treatment with a physical fine structure, and the like can be used.
  Examples of the coating polymer include polyhydroxymethyl methacrylate, polyacrylamide, and cellulose polymer. Here, it should be noted that it is necessary to perform a crosslinking treatment so that these coating polymers do not dissolve during electrophoresis. Further, since the electroosmotic flow and electrophoresis can be controlled by the type and concentration of the ionic substance, the same effect can be obtained by adding the ionic substance or the like to the buffer solution.
  Even if the surface of the groove 3, the surface of the separation matrix 5 (support 4), and the surface of the cover 6 covering the groove 3 are hydrophobic as in the surface of the substrate 2, the electrophoresis cell 1 should be used. Is possible. In general, typical elastomers and plastics are hydrophobic without surface treatment. If these are used as they are, the buffer solution cannot be filled in the groove 3 or the separation matrix 5 (gap between the columns 4) by capillary action. Therefore, in this case, it is necessary to perform the deaeration process by immersing the electrophoresis cell 1 in the buffer solution. However, once the groove 3 or the separation matrix 5 is filled with the buffer solution in this way, the effects of the present invention can be sufficiently exhibited thereafter. In addition, if the electrophoresis cell 1 is hydrophobic as a whole, when using an elastomer and a plastic, for example, these adhesiveness will increase. Furthermore, nonspecific adsorption of biomolecules is reduced, and the effect that the electrophoretic cell 1 is hardly contaminated can be obtained.
  (Method for producing electrophoresis cell)
  Hereinafter, a specific manufacturing method of the electrophoresis cell 1 will be described. The electrophoresis cell 1 is manufactured by the following procedure. The materials of the electrophoresis cell 1 are all polymer materials, but these polymer materials are processed by molding. As molding methods for polymer materials, there are known compression molding methods, injection molding methods, extrusion molding methods, blow molding methods, casting molding methods, thin molding methods, and the like. Any method may be used as long as the structure can be formed. In this embodiment, the following two methods are used. That is, one is a method of transferring a fine structure by casting an elastomer using finely processed silicon (Si) as a mold. The other is a method in which a microstructure is transferred by pressing a thermoplastic polymer heated to a temperature higher than the glass transition temperature against a finely processed silicon mold.
  Examples of the mold material include glass, silicon, ceramic, metal, and plastic. Any material can be used as long as a negative pattern of nanometer to submicron level can be finely processed. As a method for producing the mold, a method of producing a mask and processing it by dry etching, or a method of finely processing a resist as it is by lithography can be used. However, in this embodiment, a silicon etching technique is used to improve the peelability. This method can smooth the microstructure surface of the negative pattern, thereby further improving the peelability and the transfer capability of the microstructure.
  (Mold production)
  Hereinafter, with reference to FIGS. 7A to 7G, a method of manufacturing a mold and a method of manufacturing a substrate 2 (hereinafter referred to as “device”) having grooves 3 including a separation matrix 5 using the mold. Will be described more specifically.
  First, as shown in FIG. 7A, a silicon wafer 11 is prepared. Subsequently, as shown in FIG. 7B, a photosensitive organic film 12 is applied to the upper surface of the silicon wafer 11. Next, as shown in FIG. 7C, on the silicon wafer 11 with the photosensitive organic film 12, a mask 14 in which a light transmitting region 14a and a light transmitting region 14b are formed in a predetermined pattern is disposed. To do. Then, a predetermined region of the photosensitive organic film 12 is irradiated with light 13 through the mask 14 and developed using a developer. For example, when a so-called positive photosensitive organic film in which the exposed portion is dissolved is used as the photosensitive organic film 12, the photosensitive organic film 12 is removed at a portion corresponding to the region 14a through which light passes, and the hole 15 is formed. It is formed.
  Next, as shown in FIG. 7D, the silicon wafer 11 is etched. As an etching method, inductive electromagnetic coupling plasma reactive plasma etching which is an existing technique is used. As the reaction gas, a gas mainly composed of fluorocarbon is used. By this etching, the silicon wafer 11 is etched at a portion corresponding to the hole 15 formed in the photosensitive organic film 12, that is, a portion not covered with the photosensitive organic film 12, thereby forming a recess 16. Thereafter, the photosensitive organic film 12 is dissolved using an organic solvent such as acetone. Thereby, as shown in FIG. 7E, a mold 17 made of silicon is obtained.
  Then, a device is manufactured using this mold 17. Specifically, first, as shown in FIG. 7F, a silicone rubber melt 18 is applied to a mold 17 made of silicon. The surface of the mold 17 is subjected to a treatment for maintaining hydrophobicity, for example, by applying a silane coupling agent in advance. By this treatment for maintaining hydrophobicity, the releasability between the silicone rubber and the mold 17 can be improved in the peeling step described later.
  After the silicone rubber solution 18 is solidified at room temperature, the solidified product is peeled from the mold 17. As a result, a device (silicone rubber structure) made of silicone rubber, that is, the substrate 2 provided with the grooves 3 including the separation matrix 5 is obtained. Further, as shown in FIG. 7G, a cover 6 provided with a sample injection port (through hole) at a predetermined position is arranged in the device. As the cover 6, a glass substrate or a plastic substrate can be used. Then, by crimping the cover 6 to the device, the electrophoresis cell 1 in which the cover 6 and the device made of silicone rubber are in close contact with each other is completed. Since the silicone rubber has adhesiveness, the cover 6 made of a glass substrate or a plastic substrate and the device can be sealed simply by pressing the cover 6 to the device, and there is no problem of liquid leakage.
  Thus, the template 17 or the electrophoresis cell 1 can be manufactured by a very simple method. Further, since the mold 17 made of silicon can be used repeatedly, the productivity and production cost of the electrophoresis cell 1 can be improved.
  The substrate 2 provided with the device, that is, the groove 3 including the separation matrix 5 may be manufactured by a method different from the above manufacturing method. For example, a device made of plastic can be obtained by pressing a plastic plate against a press die while applying heat to a press die made of silicon. Thereafter, when the cover 6 is attached to the device, the electrophoresis cell 1 is completed. As described above, the strength of the device or electrophoresis cell 1 using plastic is improved as compared with the case of using silicone rubber.
  Instead of the mold 17 made of silicon, a mold made of a thick photosensitive organic film may be used. In this case, for example, a thick photosensitive organic film may be patterned by light irradiation, and the patterned photosensitive organic film may be used as a template as it is. According to this method, since the etching process of the silicon wafer 11 shown in FIG. 7D can be omitted, the productivity can be further improved.
  (PDMS chip manufacturing method)
  Hereinafter, a process or result of actually manufacturing the electrophoresis cell 1 using PDMS (polydimethylsiloxane), which is a typical silicone rubber, as a device material will be described. In this manufacturing method, a negative pattern mold made of a silicon wafer was formed by a fine processing technique. The mold has a mold surface corresponding to the groove containing the separation matrix. This mold was placed in a plastic petri dish having a diameter of 90 cm, and PDMS was injected from the upper part thereof. PDMS purchased from Toray Dow Corning (trade name “SYLGARD”) was used.
  The silicon wafer (silicon substrate) was surface-treated with a silane coupling agent in advance. After applying a solution of 4% dimethyldichlorosilane in acetonitrile on the surface of the silicon wafer, the silicon wafer was dried at 100 ° C. for 30 minutes. About 40 ml of PDMS was placed in a 100 ml beaker, 4 ml of catalyst was added and mixed well, and then the beaker was placed in a desiccator and deaerated with a vacuum pump for 30 minutes.
  Thereafter, 20 ml of degassed PDMS was placed in a plastic petri dish containing a silicon wafer, and the plastic petri dish was placed in a desiccator and deaerated with a vacuum pump for 30 minutes. After sufficiently expelling air bubbles from the groove containing the separation matrix, air was put into a desiccator and the plastic petri dish was taken out. And degassed PDMS was laminated | stacked by the thickness of about 5-7 mm on the silicon wafer (multilayer). The plastic petri dish on which PDMS was laminated was placed in a constant temperature box of 40 degrees to polymerize PDMS. After more than 48 hours, the plastic petri dish was taken out. Then, the necessary separation matrix portion was cut with a cutter, and the polymerized PDMS was slowly peeled from the mold made of a silicon wafer to obtain a PDMS chip.
  A PDMS chip (width 5 mm, length 20 mm, thickness 5 mm) was covered with a polystyrene plate having the same dimensions (width 5 mm, length 20 mm, thickness 1 mm) as a cover. When the adhesion is poor, oxygen plasma treatment (oxygen pressure 1.0 kPa, excitation power 100 w, treatment time 5 minutes) or ultraviolet treatment (20 w ultraviolet lamp, treatment time 20 minutes, distance 10 cm) is applied to the groove side surface of the PDMS chip. To improve adhesion. Two sample injection holes (holes) having a diameter of 1 mm were formed in the cover along the groove. And the electrode which consists of platinum wires (diameter 0.2mm) was wired so that the front-end | tip vicinity part might contact the buffer solution in a groove | channel directly through a sample injection port (refer FIG. 1). In addition, when forming an electrode by platinum vapor deposition, it is needless to say that the same effect as the case where a platinum wire is used is produced.
  TBE buffer (89 mM Tris-borate, 2 mM EDTA) was used as a buffer for DNA electrophoresis. When the oxygen plasma treatment is performed on the PDMS chip, the surface of the PDMS chip becomes hydrophilic, so that the buffer solution penetrates into the gap between the columns of the separation matrix by capillary action. When such a hydrophilic treatment is not performed, a buffer solution may be supplied from one sample inlet into the groove, and the other sample inlet may be sucked with a syringe to fill the separation matrix with the buffer solution. Alternatively, the PDMS chip may be immersed in a buffer solution, decompressed in a vacuum desiccator, and filled with the buffer solution in the separation matrix. In the latter case, the cover must be carefully placed on the PDMS chip after degassing.
  (Perform electrophoresis)
  Hereinafter, processes and results of actual electrophoresis using the electrophoresis cell according to the present invention will be described. A DNA sample was used as the biomolecule sample. As a DNA sample, λDNA, T4 phage DNA, and ½λDNA obtained by cleaving λDNA with a restriction enzyme Xbo-I about half were used. The base numbers of these DNAs were 48500, 166000, and 24000, respectively. DNA mixture containing 0.05 μg of these DNAs per 1 μl of TBE buffer and 1 μM of YOPRO-I (manufactured by Molecular Probes), Cyber Green (manufactured by Takara Shuzo) or Cyber Gold (manufactured by Takara Shuzo) as a fluorescent staining sample Was used as a sample. 1 μL of this sample was used per electrophoresis. Then, the sample was injected into one of the two sample injection ports formed on the cover, and electrophoresis was performed. As the power source for electrophoresis, 0 to 1000 V, 1 A rated ATTO cross power 1000 was used. With this power source, a voltage of 50 to 100 V / cm was applied to both ends of an electrode made of a platinum wire. The current was 10 mA or less. Since DNA is negatively charged in the TBE buffer, it migrates toward the positive electrode. When the length of the groove was 1 cm, the electrophoresis time was 2 to 5 minutes.
  (Observation of electrophoresis)
  Hereinafter, an example of an electrophoresis observation method will be described. FIG. 8 shows a schematic configuration of an electrophoresis evaluation apparatus for observing or recording electrophoresis of DNA.
  As shown in FIG. 8, the electrophoresis evaluation apparatus includes a fluorescence microscope 20. The fluorescence microscope 20 includes a sample stage 22 on which a sample 21 (electrophoresis cell) is placed, a light source 23 having an excitation filter (not shown), a dichroic mirror 24, and an observation window 25 for observing DNA. A switching mirror 26 for switching the optical path, a photomultiplier tube 27 for converting light into an electrical signal, and a video camera 28 (high-sensitivity CCD camera). The electrophoretic evaluation apparatus includes a VTR 29 (video tape recorder) for recording an image, a personal computer 30, and a display 31.
  In the fluorescence microscope 20, the light L <b> 1 radiated from the light source 23 and having a wavelength adjusted to 420 to 490 nm by the excitation side filter is reflected by the dichroic mirror 24 and then irradiated to the sample 21. As a result, the DNA in the sample emits fluorescence. This fluorescence is adjusted to light L2 having a wavelength of 520 nm by an absorption filter (not shown). The light L2 is split by the dichroic mirror 24 into light L3 that travels toward the observation window 25 and light L4 that travels toward the photomultiplier tube 27 or the video camera 28.
  Thus, by looking through the observation window 25, it is possible to observe how the DNA molecules emitting fluorescence pass through the separation matrix (gap between the columns) by electrophoresis. The light L 4 is input to the photomultiplier tube 27 or the video camera 28 by switching the optical path by the switching mirror 26. The photomultiplier tube 27 converts the light L4 into an electrical signal and outputs it to an external device. The video camera 28, which is a high-sensitivity CCD camera, takes an image of the DNA molecules passing through the separation matrix by electrophoresis, and sends the obtained image to the VTR 29. The VTR 29 records this image. The image recorded in the VTR 29 can be analyzed using the personal computer 30 and can be displayed on the display 31.
  (Relationship between number of bases and movement speed)
  Hereinafter, the relationship between the number of DNA bases and the movement speed (electrophoresis speed) of DNA by electrophoresis will be described.
  FIG. 9 shows the relationship between the number of DNA bases and the DNA transfer speed under the following conditions.
        Groove width: 200 μm
        Groove depth: 10 μm
        Separation matrix length: 10mm
        Prop shape: cylinder,
        Strut diameter: 15 μm
        Gap between struts: approx. 1 μm
        Prop arrangement density: 4000 pieces / mm²
        Voltage: 50V / cm
        Buffer solution: TBE buffer solution
        DNA concentration: 50 ng / μl
        Dye: Cyber Green 1 mM
  As is clear from FIG. 9, the migration speed of DNA by electrophoresis changes significantly depending on the number of DNA bases. Therefore, it can be seen that the separation matrix according to the present invention, which is composed of artificial struts and gaps, has the same effect as the agarose gel or polyacrylamide gel, and can be substituted for these gels.
  (DNA migration model)
  FIG. 10 shows a columnar column 4 and a DNA motion model passing through the gap between the columns 4. As shown in FIG. 10, it is considered that the gap between the pillars 4 as an obstacle plays a role of an entropy barrier against the movement of DNA passing through the gap. That is, when the spherical DNA molecule passes through the gap of the support column 4, it temporarily takes a thermodynamically low-entropy conformation and deforms to pass through the gap. This is considered to affect the movement speed of DNA by electrophoresis.
  (Application example of electrophoresis cell according to the present invention)
  The basic electrophoretic cell according to the present invention has been described in detail with respect to its shape, material, manufacturing method, DNA separation principle, DNA motion model, and the like. A DNA sorting device (biological sample sorting device) using an electrophoresis cell will be described.
  (DNA sorting device 1: application example 1)
  FIG. 11 shows one DNA sorting apparatus to which the electrophoresis cell according to the present invention is applied. As shown in FIG. 11, in this DNA sorting apparatus, the separation matrix 5 is divided into five pillar areas A to E having different arrangement density of pillars or gaps between pillars (pillar gaps). Here, in the pillar areas A, B, C, D, and E, the arrangement density of the support columns increases in this order (the space between the support columns decreases). The DNA sorting apparatus includes one sample injection port 33 with a cathode and five sample recovery ports 34a to 34e provided in the pillar areas A to E with an anode, respectively.
  Thus, in this DNA sorting apparatus, for example, if a sample containing DNA having different molecular weights is supplied to the sample inlet 33, the DNA having a large molecular weight passes through the pillar area (pillar area E side) where the arrangement density of the pillars is large. Since it is difficult to do this, many pillars are gathered in the pillar area (pillar area A side) where the arrangement density of the pillars is small, and collected from the sample collection port in this pillar area. Therefore, the DNA present in the pillar areas A, B, C, D, and E, or the DNA recovered at the sample recovery ports 34a to 34e has a lower molecular weight as the latter. Therefore, the DNA in the sample can be fractionated (fractionated) into five types according to the molecular weight.
  (DNA sorting device 2: application example 2)
  12A and 12B show another DNA sorting apparatus to which the electrophoresis cell according to the present invention is applied. As shown in FIGS. 12A and 12B, in this DNA sorting apparatus, the separation matrix 5 is two-dimensionally separated in the longitudinal direction (X1-X2 direction) and the lateral direction (Y1-Y2 direction) in plan view. It is composed of a large number of prismatic pillars 4 arranged in this manner. In addition, this DNA sorting apparatus includes one sample injection port 35 with a cathode and six sample collection ports 36a to 36f each with an anode.
  The separation matrix 5 is located on the Y1 side in a gap formed between a certain column 4 and the two columns 4 adjacent in the X1-X2 direction on the X2 side and extending in the Y1-Y2 direction. The gap 38 is wide, and the gap 39 located on the Y2 side is narrow. On the other hand, the gaps 40 extending in the X1-X2 direction between the columns 4 adjacent in the Y1-Y2 direction are all wide. Note that a substantially rectangular space 41 is formed at the connection between the end portion on the X2 side of the gap 40 extending in the X1-X2 direction and both the gaps 38, 39 extending in the Y1-Y2 direction.
  Thus, in this DNA sorting apparatus, for example, if a sample containing DNA having different molecular weights is supplied to the sample injection port 35, DNA having a large molecular weight cannot pass through the narrow gap 39. Furthermore, regarding the Y1-Y2 direction (lateral direction), it does not move in the Y1 direction but moves only in the Y2 direction. On the other hand, DNA with a small molecular weight can move in either the Y1 direction or the Y2 direction. However, as described above, since DNA having a large molecular weight almost moves in the Y2 direction, DNA having a small molecular weight has a strong tendency to move in the Y1 direction as indicated by R2, for example. Therefore, in the separation matrix 5, the molecular weight of the DNA recovered at the sample recovery ports 36a to 36f becomes smaller as the latter. Therefore, the DNA in the sample can be fractionated (fractionated) into six types according to the molecular weight.
  (Application to medical diagnosis)
  The electrophoresis cell or DNA sorting device (biological sample sorting device) according to the present invention can be applied to medical diagnosis or gene polymorphism detection. For example, in human genes, there are slight differences (gene polymorphisms) in the nucleotide sequence for each individual. The difference is said to be in the gene regulatory region, the intron region, or the region encoding the protein. The difference between the gene regulatory region and the intron region indicates that the gene expression level varies from individual to individual. In addition, regions encoding proteins and the like indicate that the activity of the enzyme varies depending on the individual. Individual differences are said to be determined by the combination of genetic polymorphisms. For example, there are individual differences in drug metabolism, which are closely related to drug efficacy and side effects. In other words, the gene polymorphism of the drug metabolism gene group possessed by an individual greatly affects these drug efficacy and side effects.
  There are various types of gene polymorphisms. Currently, single nucleotide polymorphisms (SNPs) are said to be the most numerous and easy to analyze in large quantities. SNPs are parts where one gene code (one base) is replaced with another base among individuals, and it is estimated that there are 3 to 10 million human genomes. Therefore, it is expected that genetic polymorphism information of drug metabolism genes will be useful for drug development, effective use of drugs, prevention of side effects, and the like.
  As SNP analysis methods, currently, restriction enzyme methods, SSCP (single-strand conformation polymorphisms) analysis methods, direct sequencing methods, electrochemical detection methods, and the like are known. Among these, those analyzed by electrophoresis are restriction enzyme method, SSCP analysis method, and direct sequencing method. For such rapid separation and detection, an electrophoresis cell using the separation matrix according to the present invention can be applied. By using the electrophoresis cell according to the present invention, SNPs can be rapidly detected with a small amount of sample. Among these, the SSCP method is suitable for the present invention because it amplifies using PCR technology and detects a change in the three-dimensional structure by electrophoresis.
  In this specification, DNA and biomolecules are targeted, but the target is not limited to biomolecules. It can also be applied to separation and detection of environmental chemicals and polymer compounds. Furthermore, it is expected that this separation matrix and its manufacturing method will be useful for separation and detection in microchannels such as chemical microdevices and Lab on achip, which have recently been discussed.
  While the present invention has been described with reference to specific embodiments thereof, it will be apparent to those skilled in the art that numerous other variations and modifications are possible. Therefore, the present invention should not be limited by such embodiments, but should be limited by the appended claims.

Industrial applicability

  As described above, the biomolecule separation cell such as the electrophoresis cell according to the present invention, the manufacturing method thereof, and the DNA sorting device or the biological sample sorting device are particularly useful for detecting or sorting biomolecules such as DNA. It is suitable for use as a medical examination apparatus or diagnostic apparatus.

Claims (12)

A biomolecule separation cell for separating or detecting biomolecules in a liquid sample,
A substrate with grooves formed on the surface;
A separation matrix composed of a plurality of struts arranged in a partial region of the groove and having a narrowest gap between the struts of 0.1 to 5 μm;
A cover that covers the surface of the substrate and the groove and is in close contact with the upper surface of the support;
A buffer filling the groove,
And at least two electrodes for applying a voltage to the buffer in direct contact with the buffer,
A biomolecule separation cell in which the substrate and the separation matrix are formed of a polymer material. The biomolecule separation cell according to claim 1, wherein the biomolecule is separated by electrophoresis. The biomolecule separation cell according to claim 2, wherein a shape of a cross section of the support column is a circle, an oval, a polygon, or a polygon obtained by rounding or cutting corners. The biomolecule separation cell according to claim 2, wherein at least a part of the substrate and the separation matrix is formed of an elastomer or a plastic. The biomolecule separation cell according to claim 4, wherein the elastomer is silicone rubber. The biomolecule separation cell according to claim 4, wherein the plastic is an acrylic resin, a polystyrene resin, or a polycarbonate resin. The biomolecule separation cell according to claim 2, wherein the groove has a width of 20 to 1000 microns, a depth of 1 to 50 µm, and a length of 0.1 to 50 mm. The biomolecule separation cell according to claim 2, wherein the arrangement density of the support columns is 1000 pieces / mm ² or more. A sample inlet for injecting a biomolecule sample into the groove is provided in the cover, and the separation matrix is not in the vicinity of the sample inlet, there is no region or column where the arrangement density of columns is lower than other regions. The biomolecule separation cell according to claim 2, which has a region. Production of a biomolecule separation cell for separating or detecting biomolecules in a liquid sample, comprising a substrate having grooves formed on the surface and a separation matrix comprising a plurality of support columns arranged in a partial region of the grooves. A method,
Forming a mold having a negative pattern corresponding to the groove and the support column by performing fine processing on a silicon substrate, a glass substrate or a polymer substrate;
Pouring a liquid elastomer or plastic, or a monomer or prepolymer as a raw material of the elastomer or plastic into the mold, and solidifying;
A method for producing a biomolecule separation cell, comprising a step of separating a solidified product from a mold to obtain a biomolecule separation cell comprising the substrate and the separation matrix. Production of a biomolecule separation cell for separating or detecting biomolecules in a liquid sample, comprising a substrate having grooves formed on the surface and a separation matrix comprising a plurality of support columns arranged in a partial region of the grooves. A method,
Forming a mold having a negative pattern corresponding to the groove and the column by finely processing a silicon substrate, a glass substrate or a polymer substrate;
After pressing the plastic heated above the glass transition temperature against the mold, the temperature is lowered and cured,
The manufacturing method of the biomolecule separation cell including the process of isolate | separating hardened | cured material from a type | mold and obtaining the biomolecule separation cell provided with the said board | substrate and the said separation matrix. A substrate with grooves formed on the surface;
A separation matrix composed of a plurality of struts arranged in a partial region of the groove and having a narrowest gap between the struts of 0.1 to 5 μm;
A cover that covers the surface of the substrate and the groove and is in close contact with the upper surface of the support;
A buffer filling the groove,
And at least two electrodes for applying a voltage to the buffer in direct contact with the buffer,
The substrate and the separation matrix are formed of a polymer material,
A DNA fractionation device configured to fractionate or fractionate DNA from a liquid sample containing DNA supplied into the groove based on the characteristics and structure of the DNA.

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