JP2009122022A - Biological material sensing chip, device, and method - Google Patents

Biological material sensing chip, device, and method Download PDF

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JP2009122022A
JP2009122022A JP2007297809A JP2007297809A JP2009122022A JP 2009122022 A JP2009122022 A JP 2009122022A JP 2007297809 A JP2007297809 A JP 2007297809A JP 2007297809 A JP2007297809 A JP 2007297809A JP 2009122022 A JP2009122022 A JP 2009122022A
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reservoir
biological material
flow path
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pressure
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Yuji Saito
祐司 斉藤
富美男 ▲高▼城
Fumio Takagi
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Seiko Epson Corp
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Abstract

<P>PROBLEM TO BE SOLVED: To provide a nucleic acid detection device, capable of sensitively detecting with little detection liquid and capable of processing a plurality of analytes at once. <P>SOLUTION: The nucleic acid detection device is provided with a flow path 103 formed with a plurality of probe regions with intervals in a flowing direction of the analytes; reservoirs 104, 105 connected to both the ends of the flow path 103; and gas-liquid separation membranes 106, 107 are provided so as to cover the apertures of the reservoirs 104, 105. The reserver 104 is connected with a pump 14 through the gas-liquid separation membrane 106, the pressurization, pressure reduction are repeated by the pump 14, thereby the analyte in the flow path 103 is made to reciprocally move. <P>COPYRIGHT: (C)2009,JPO&INPIT

Description

本発明は、特定の塩基配列を有する核酸分子などの生体物質を検出するための、生体物質検出チップ、生体物質検出装置、および生体物質検出方法に関するものである。   The present invention relates to a biological material detection chip, a biological material detection device, and a biological material detection method for detecting a biological material such as a nucleic acid molecule having a specific base sequence.

血液や組織細胞などの検体中に、疾患に由来する特定の遺伝子が存在するか否かを検査する手法の一つにＤＮＡマイクロアレイがある。ＤＮＡマイクロアレイは、基板上に固定化されたプローブ遺伝子と検体中の遺伝子とを反応（ハイブリダイゼーション）させることにより、目標の遺伝子の有無を検出する。従来、検体中に含まれる特定の遺伝子の検出精度をあげるため、検体中の特定の遺伝子とプローブ遺伝子との反応効率を高くする工夫がなされている。   One technique for examining whether or not a specific gene derived from a disease is present in a sample such as blood or tissue cell is a DNA microarray. A DNA microarray detects the presence or absence of a target gene by reacting (hybridizing) a probe gene immobilized on a substrate with a gene in a specimen. Conventionally, in order to increase the detection accuracy of a specific gene contained in a sample, a device for increasing the reaction efficiency between the specific gene in the sample and the probe gene has been devised.

例えば、特許文献１には、反応中に検体液の攪拌を行って検体液の分布を改善させることにより、使用する検体液の量を少なくし、反応時間を短縮する方法が開示されている。   For example, Patent Document 1 discloses a method of reducing the amount of sample liquid to be used and reducing the reaction time by improving the distribution of the sample liquid by stirring the sample liquid during the reaction.

特開２００３−５７２３７号公報JP 2003-57237 A

しかし、特許文献１に開示された装置では、スライド上に検体液を分布させているため、プローブの配置位置から離れた位置に分布する検体液は反応に用いられないので、より検体液の利用効率の向上が望まれる。また、一度に多数の検体の処理を行うことが困難であった。   However, in the apparatus disclosed in Patent Document 1, since the sample liquid is distributed on the slide, the sample liquid distributed at a position distant from the arrangement position of the probe is not used for the reaction. Improvement in efficiency is desired. In addition, it has been difficult to process a large number of samples at a time.

そこで、本発明の目的は、より少ない検体液で感度の高い検出が可能で、一度に多くの検体の処理を行うことが可能な生体物質検出チップ、生体物質検出装置、および生体物質検出方法を得ることである。   Therefore, an object of the present invention is to provide a biological material detection chip, a biological material detection device, and a biological material detection method capable of highly sensitive detection with a smaller amount of sample liquid and capable of processing many samples at once. Is to get.

本発明に係る生体物質検出チップは、検体の流れる方向に間隔をおいて形成された複数の反応領域を備えて形成された流路と、
前記流路の一端に接続され、開口部を有する第１のリザーバと、前記流路の他方の端部に接続され、開口部を有する第２のリザーバと、前記第１のリザーバの開口部を覆うように設けられた第１の気液分離膜と、前記第２のリザーバの開口部を覆うように設けられた第２の気液分離膜とを備え、各々の前記反応領域は、前記検体中の特定の生体物質を検出するためのプローブを固定する領域を有するものである。
本発明によれば、第１のリザーバと第２のリザーバの開口部がそれぞれ第１の気液分離膜と第２の気液分離膜で覆われているため、検体液は第１の気液分離膜と第２の気液分離膜から外へ漏れることがなく、圧力制御手段を用いて加圧、減圧することにより流路内で検体液を往復移動させるのに適している。流路内の検体液を往復移動させることにより、少ない検体液を効率よく反応領域上のプローブと反応させることができる。 The biological material detection chip according to the present invention comprises a flow path formed with a plurality of reaction regions formed at intervals in the direction in which the specimen flows,
A first reservoir connected to one end of the flow path and having an opening; a second reservoir connected to the other end of the flow path and having an opening; and an opening of the first reservoir A first gas-liquid separation membrane provided so as to cover and a second gas-liquid separation membrane provided so as to cover the opening of the second reservoir, and each of the reaction regions includes the sample It has the area | region which fixes the probe for detecting the specific biological substance in it.
According to the present invention, since the openings of the first reservoir and the second reservoir are respectively covered with the first gas-liquid separation film and the second gas-liquid separation film, the sample liquid is the first gas-liquid separation. It does not leak out from the separation membrane and the second gas-liquid separation membrane, and is suitable for reciprocating the sample liquid in the flow path by pressurizing and depressurizing using the pressure control means. By reciprocating the sample liquid in the flow path, a small amount of the sample liquid can be efficiently reacted with the probe on the reaction region.

また、前記流路を複数備え、各々の前記流路は、それぞれ別の前記第１のリザーバ及び前記第２のリザーバに接続されていることが望ましい。
これにより、一度に多くの検体の処理を行うことができる。 Further, it is preferable that a plurality of the flow paths are provided, and each of the flow paths is connected to the different first reservoir and second reservoir.
Thereby, many samples can be processed at a time.

本発明に係る生体物質検出装置は、上記の生体物質検出チップを用いて生体物質検出処理を行うための生体物質検出装置であって、前記第１の分離膜を介して前記第１のリザーバと接続され、前記第１のリザーバ内の加圧または減圧を行う圧力制御手段を備えているものである。
本発明によれば、第１のリザーバ内の加圧または減圧を行うことにより、流路内の検体液を往復移動させることができるので、検体液が流路内で攪拌され、少ない検体液でプローブとの反応効率を高めることができる。また、第１のリザーバと第２のリザーバの開口部がそれぞれ第１の気液分離膜と第２の気液分離膜で覆われているため、第１のリザーバ内の加圧、減圧を行っても検体液は第１の気液分離膜と第２の気液分離膜から外へ漏れることがなく、制御がしやすいという利点がある。 A biological material detection device according to the present invention is a biological material detection device for performing a biological material detection process using the biological material detection chip described above, and the first reservoir and the first reservoir through the first separation membrane. The pressure control means is connected and is configured to pressurize or depressurize the first reservoir.
According to the present invention, the sample liquid in the flow path can be reciprocated by pressurizing or depressurizing the first reservoir. The reaction efficiency with the probe can be increased. In addition, since the openings of the first reservoir and the second reservoir are covered with the first gas-liquid separation membrane and the second gas-liquid separation membrane, respectively, pressurization and decompression of the first reservoir are performed. Even in this case, there is an advantage that the sample liquid does not leak out from the first gas-liquid separation membrane and the second gas-liquid separation membrane and is easy to control.

また、複数の前記流路と接続する各々の前記第１のリザーバが、１つの前記圧力制御手段に接続されるようにすることができる。
これにより、複数の流路を１つの圧力制御手段で制御することができるため、装置の小型化やコストの削減が図れる。また、第１のリザーバと第２のリザーバの開口部がそれぞれ第１の気液分離膜と第２の気液分離膜で覆われているため、流路間で検体の移動時間に差があっても、リザーバ内に検体液を留めておくことで、移動時間の差を吸収することができ、全ての流路で攪拌の回数を一定にすることができる。よって、流路によってばらつきのない信頼性の高い検査結果を得ることができる。 Moreover, each said 1st reservoir | reserver connected with the said several flow path can be made to be connected to one said pressure control means.
Thereby, since several flow paths can be controlled by one pressure control means, size reduction of an apparatus and cost reduction can be achieved. In addition, since the openings of the first reservoir and the second reservoir are respectively covered with the first gas-liquid separation membrane and the second gas-liquid separation membrane, there is a difference in the movement time of the specimen between the flow paths. However, by keeping the sample liquid in the reservoir, the difference in the movement time can be absorbed, and the number of agitation can be made constant in all the channels. Therefore, it is possible to obtain a highly reliable test result that does not vary depending on the flow path.

また、前記圧力制御手段は、ポンプと、圧力がｐ１以上になると開く第１の圧力弁と、圧力がｐ２以下になると開く第２の圧力弁を備え、ｐ２＜大気圧＜ｐ１とすることができる。これにより、第１及び第２の圧力弁の開閉を検知してポンプによる加圧、減圧を切り替えるようにすることができ、簡易な処理で、検体液の往復移動を制御することができる。   The pressure control means includes a pump, a first pressure valve that opens when the pressure becomes equal to or higher than p1, and a second pressure valve that opens when the pressure becomes equal to or lower than p2, wherein p2 <atmospheric pressure <p1. it can. Thus, the opening and closing of the first and second pressure valves can be detected to switch between pressurization and decompression by the pump, and the reciprocating movement of the sample liquid can be controlled with a simple process.

また、前記生体物質検出チップの温度調節機構を備えるようにすれば、温度設定が必要な反応処理に利用することができる。   Further, if the temperature adjustment mechanism of the biological material detection chip is provided, it can be used for a reaction process that requires temperature setting.

本発明に係る生体物質検出方法は、前記リザーバに検体液を供給する工程と、前記圧力制御手段によって前記流路内の検体液を往復移動させる工程を備えている。
本発明によれば、流路内の検体液を往復移動させることにより、検体液が流路内で攪拌され、少ない検体液でプローブとの反応効率を高めることができる。また、第１のリザーバと第２のリザーバの開口部がそれぞれ第１の気液分離膜と第２の気液分離膜で覆われているため、第１のリザーバ内の加圧、減圧を行っても検体液は第１の気液分離膜と第２の気液分離膜から外へ漏れることがなく、制御がしやすいという利点がある。 The biological material detection method according to the present invention includes a step of supplying a sample liquid to the reservoir and a step of reciprocating the sample liquid in the flow path by the pressure control means.
According to the present invention, by reciprocating the sample liquid in the flow path, the sample liquid is stirred in the flow path, and the reaction efficiency with the probe can be increased with a small amount of sample liquid. In addition, since the openings of the first reservoir and the second reservoir are covered with the first gas-liquid separation membrane and the second gas-liquid separation membrane, respectively, pressurization and decompression of the first reservoir are performed. Even in this case, there is an advantage that the sample liquid does not leak out from the first gas-liquid separation membrane and the second gas-liquid separation membrane and is easy to control.

以下、本発明の実施の形態について図面を参照して説明する。
実施の形態１．
図１は、本発明の実施の形態１による核酸検出装置（生体物質検出装置）１０の構成を示す模式図である。図に示すように、核酸検出装置１０は、ＤＮＡチップ（検出用チップ）１１、ステージ１２、コントローラ１３、ポンプ（圧力制御手段）１４、圧力弁（圧力制御手段）１５，１６、管１７を備えている。 Hereinafter, embodiments of the present invention will be described with reference to the drawings.
Embodiment 1 FIG.
FIG. 1 is a schematic diagram showing a configuration of a nucleic acid detection apparatus (biological substance detection apparatus) 10 according to Embodiment 1 of the present invention. As shown in the figure, the nucleic acid detection device 10 includes a DNA chip (detection chip) 11, a stage 12, a controller 13, a pump (pressure control means) 14, pressure valves (pressure control means) 15 and 16, and a tube 17. ing.

図に示すように、ＤＮＡチップ１１はステージ１２上に固定されている。ステージ１２にはヒータ（図示せず）が設置されており、コントローラ１３によって所定の温度に設定することができる。また、ポンプ１４によって加圧、減圧を繰り返すことにより、ＤＮＡチップ１１の流路内の検体液を往復移動させることができるように構成されている。ポンプ１４は例えばシリンジポンプやマイクロポンプを用いることができる。また、管１７は、フッ素樹脂、ポリエーテルエーテルケトン（ＰＥＥＫ）樹脂、シリコーン樹脂等からなるキャピラリーチューブを用いることができる。   As shown in the figure, the DNA chip 11 is fixed on a stage 12. A heater (not shown) is installed on the stage 12 and can be set to a predetermined temperature by the controller 13. Further, the sample liquid in the flow path of the DNA chip 11 can be reciprocated by repeating pressurization and decompression by the pump 14. For example, a syringe pump or a micro pump can be used as the pump 14. The tube 17 can be a capillary tube made of a fluororesin, a polyether ether ketone (PEEK) resin, a silicone resin, or the like.

図２は、ＤＮＡチップ１１の上面図、図３は、図２に示すＢ−Ｂ断面図である。図に示すように、ＤＮＡチップ１１は、透明基板１０１，１０２、流路１０３、リザーバ（第１のリザーバ、第２のリザーバ）１０４，１０５、気液分離膜（第１の気液分離膜、第２の気液分離膜）１０６，１０７、Ｏリング１０８〜１１２、蓋１１３，１１４、カバー１１５、ポンプとの接続部１１６を備えている。   2 is a top view of the DNA chip 11, and FIG. 3 is a cross-sectional view taken along line BB shown in FIG. As shown in the figure, the DNA chip 11 includes transparent substrates 101 and 102, a channel 103, reservoirs (first reservoir and second reservoir) 104 and 105, gas-liquid separation membrane (first gas-liquid separation membrane, (Second gas-liquid separation membrane) 106, 107, O-rings 108 to 112, lids 113, 114, cover 115, and pump connection portion 116 are provided.

流路１０３は透明基板１０２に形成されており、リザーバ１０４，１０５は透明基板１０１に形成されている。透明基板１０１，１０２は例えばガラス基板とすることができ、この場合には、流路１０３、リザーバ１０４，１０５は、サンドブラスト法等を用いて形成することができる。透明基板１０１と透明基板１０２は、リザーバ１０４，１０５が流路１０３の両端に接続されるように貼り合わされている。   The flow path 103 is formed on the transparent substrate 102, and the reservoirs 104 and 105 are formed on the transparent substrate 101. The transparent substrates 101 and 102 can be glass substrates, for example. In this case, the flow path 103 and the reservoirs 104 and 105 can be formed using a sandblast method or the like. The transparent substrate 101 and the transparent substrate 102 are bonded so that the reservoirs 104 and 105 are connected to both ends of the flow path 103.

リザーバ１０４，１０５は、流路１０３との接続部の反対側に開口部を有し、それぞれ気液分離膜１０６，１０７によって覆われている。気液分離膜１０６，１０７は、４フッ化エチレン樹脂等で形成された膜であり、気体は透過させるが、液体の透過は遮断する性質を持つ。気液分離膜１０６，１０７は、それぞれ蓋１１３，１１４によって押さえられており、さらに蓋１１３，１１４はカバー１１５で覆われている。蓋１１３，１１４と透明基板１０１，１０２の間にはＯリング１０８，１０９、蓋１１３，１１４とカバー１１５の間にはＯリング１１０，１１１が設けられており、これにより気液分離膜１０６，１０７を透明基板１０１に密着させている。   The reservoirs 104 and 105 have openings on the side opposite to the connection portion with the flow path 103 and are covered with gas-liquid separation films 106 and 107, respectively. The gas-liquid separation membranes 106 and 107 are membranes formed of tetrafluoroethylene resin or the like, and have a property of allowing gas to permeate but blocking liquid permeation. The gas-liquid separation membranes 106 and 107 are pressed by lids 113 and 114, respectively, and the lids 113 and 114 are covered with a cover 115. O-rings 108 and 109 are provided between the lids 113 and 114 and the transparent substrates 101 and 102, and O-rings 110 and 111 are provided between the lids 113 and 114 and the cover 115, whereby the gas-liquid separation membrane 106, 107 is in close contact with the transparent substrate 101.

リザーバ１０４は、気液分離膜１０６、蓋１１３、カバー１１５、Ｏリング１１２、およびポンプとの接続部１１６を介してポンプ１４に接続されている。ポンプ１４と接続部１１６とを繋ぐ管１７には、図１に示すように圧力弁１５，１６が設置されている。圧力弁１５は、管内の圧力が一定値（ｐ１）以上になると開いて管内の空気を外部へ放出し、圧力弁１６は、管内の圧力が一定値（ｐ２）以下になると開いて外気を管内に取り込む動作をする。ポンプ１４の動作は、制御部（図示せず）が検知した圧力弁１５，１６の開閉に基づいて制御される。なお、ｐ２＜大気圧＜ｐ１である。リザーバ１０５は、気液分離膜１０７、蓋１１４、カバー１１５、を介して外部に通じている。   The reservoir 104 is connected to the pump 14 via a gas-liquid separation membrane 106, a lid 113, a cover 115, an O-ring 112, and a connection portion 116 with the pump. As shown in FIG. 1, pressure valves 15 and 16 are installed in the pipe 17 that connects the pump 14 and the connection portion 116. The pressure valve 15 opens when the pressure in the pipe reaches a certain value (p1) or more and discharges the air inside the pipe to the outside, and the pressure valve 16 opens when the pressure inside the pipe becomes a certain value (p2) or less to allow outside air to enter the pipe. The operation to import to. The operation of the pump 14 is controlled based on the opening and closing of the pressure valves 15 and 16 detected by a control unit (not shown). Note that p2 <atmospheric pressure <p1. The reservoir 105 communicates with the outside through the gas-liquid separation membrane 107, the lid 114, and the cover 115.

図２に示すように、実施の形態１では複数の流路１０３が平行に透明基板１０２に形成されており、各々のリザーバ１０４は、ポンプとの接続部１１６を介して１つのポンプ１４に接続されている。   As shown in FIG. 2, in the first embodiment, a plurality of flow paths 103 are formed in parallel on the transparent substrate 102, and each reservoir 104 is connected to one pump 14 via a connection portion 116 with the pump. Has been.

図４は、流路１０３を上から見た図である。流路１０３の形状は、例えば検体が流れる方向（図２に示すＡ方向）に垂直な断面が直径１００μｍの半円とすることができる。図に示すように、流路１０３の内壁には、検体が流れる方向に間隔をおいてプローブが塗布された領域（反応領域）が形成されている。   FIG. 4 is a view of the flow path 103 as viewed from above. The shape of the channel 103 can be, for example, a semicircle having a diameter of 100 μm in a cross section perpendicular to the direction in which the specimen flows (direction A shown in FIG. 2). As shown in the figure, an area (reaction area) where a probe is applied is formed on the inner wall of the flow path 103 at intervals in the direction in which the specimen flows.

プローブには、例えば血液、尿、唾液、髄液のような検体試料に含まれる標的物質（ターゲット）を捕捉し得る物質を用いることができる。例えば、ターゲットがＤＮＡやＲＮＡのような核酸である場合には、プローブとしては、これらの核酸とハイブリダイゼーション（相補的に結合）する核酸やヌクレオチド（オリゴヌクレオチド）等を用いることができる。このような核酸としては、例えばｃＤＮＡやＰＣＲ産物等が用いられる。なお、ターゲットは核酸に限られず、例えば特定のタンパク質であってもよい。この場合には、プローブとしては、このタンパク質を特異的に捕捉（例えば、吸着、結合等）するもの等が用いられる。具体的には、抗原、抗体、レセプター、酵素等のタンパク質、ペプチド（オリゴペプチド）等である。   As the probe, for example, a substance that can capture a target substance (target) contained in a specimen sample such as blood, urine, saliva, or spinal fluid can be used. For example, when the target is a nucleic acid such as DNA or RNA, a nucleic acid or nucleotide (oligonucleotide) that hybridizes (complementarily binds) to these nucleic acids can be used as the probe. As such a nucleic acid, for example, cDNA or PCR product is used. The target is not limited to a nucleic acid, and may be a specific protein, for example. In this case, a probe that specifically captures (eg, adsorbs, binds, etc.) the protein is used as the probe. Specifically, proteins such as antigens, antibodies, receptors, enzymes, peptides (oligopeptides) and the like.

プローブの塗布は、非接触あるいは接触式スポッター等を用いて行うことができる。なお、各々の反応領域には、それぞれ異なる１種類のプローブが固定されている。これにより、１度に複数種類のターゲットの検出が可能である。   The probe can be applied using a non-contact or contact spotter. In addition, one different type of probe is fixed to each reaction region. Thereby, a plurality of types of targets can be detected at a time.

次に、本実施形態による核酸検出装置１０を用いた、ターゲット（核酸）とプローブとのハイブリダイゼーション処理について説明する。   Next, a hybridization process between a target (nucleic acid) and a probe using the nucleic acid detection device 10 according to the present embodiment will be described.

まず、リザーバ１０４又は１０５にピペット等を用いて検体液を供給する。上述したように、流路１０３の形状を検体が流れる方向に垂直な断面が直径１００μｍの半円とすると、流路１０３の体積は０．１μｌ以下となるため、リザーバ１０４，１０５に供給する検体液の量は１μｌ以下でよい。従来の標準的なハイブリダイゼーション処理において用いられる検体液の量は１００μｌ程度であり、本実施形態では、１／１００以下に減らすことが可能となる。   First, the sample liquid is supplied to the reservoir 104 or 105 using a pipette or the like. As described above, when the shape of the flow channel 103 is a semicircle having a diameter of 100 μm in a cross section perpendicular to the direction in which the sample flows, the volume of the flow channel 103 is 0.1 μl or less, so the sample supplied to the reservoirs 104 and 105 The amount of liquid may be 1 μl or less. The amount of the sample solution used in the conventional standard hybridization process is about 100 μl, and in this embodiment, it can be reduced to 1/100 or less.

検体液は、例えば血液、尿、唾液、髄液のような生体サンプルを含む。なお、必要に応じて、ＰＣＲ法を用いて、ターゲットとなる核酸の増幅処理を行っておいてもよいが、上述したように、本実施形態では検体液の量が従来と比較して非常に少量でありハイブリダイゼーションの効率が高いため、増幅を行わなくても検査が可能となり、増幅処理工程を省くことで検査時間を短縮することができる。   The sample fluid includes biological samples such as blood, urine, saliva, and spinal fluid. If necessary, the target nucleic acid may be amplified using the PCR method. However, as described above, in this embodiment, the amount of the sample solution is much higher than in the conventional case. Since the amount is small and the efficiency of hybridization is high, the inspection can be performed without performing amplification, and the inspection time can be shortened by omitting the amplification process.

リザーバ１０４又は１０５に検体液を供給した後、リザーバ１０４，１０５に気液分離膜１０６，１０７を被せ、Ｏリング１０８，１０９、蓋１１３，１１４、Ｏリング１１０，１１１、カバー１１５を取り付けて、気液分離膜１０６，１０７を密着させる。また、ポンプとの接続部１１６をＯリング１１２を介して取り付け、ポンプ１４を接続する。   After supplying the specimen liquid to the reservoir 104 or 105, the gas-liquid separation membranes 106 and 107 are covered with the reservoirs 104 and 105, and O-rings 108 and 109, lids 113 and 114, O-rings 110 and 111, and a cover 115 are attached. The gas-liquid separation membranes 106 and 107 are brought into close contact with each other. Moreover, the connection part 116 with a pump is attached via the O-ring 112, and the pump 14 is connected.

次に、ＤＮＡチップ１１をステージ１２に取り付け、ヒーターの温度を３７℃に設定する。ステージ１２の温度が３７℃に達したら、ポンプ１４を作動させて加圧と減圧を繰り返し、検体液を流路１０３内で往復させる。これにより、流路１０３内で検体液が攪拌され、ターゲットとプローブとの反応効率を上げることができる。   Next, the DNA chip 11 is attached to the stage 12, and the temperature of the heater is set to 37 ° C. When the temperature of the stage 12 reaches 37 ° C., the pump 14 is operated to repeat pressurization and depressurization, and the sample liquid is reciprocated in the flow path 103. Thereby, the sample liquid is agitated in the flow path 103, and the reaction efficiency between the target and the probe can be increased.

初めにリザーバ１０４に検体液を導入した場合には、ポンプ１４で管１７内を加圧していく。すると気液分離膜１０６を通してリザーバ１０４に空気が入り、検体液は流路１０３を通ってリザーバ１０５へ移動する。リザーバ１０５が検体液で満たされた状態になると、検体液は気液分離膜１０７を通ることができないため、検体液の移動が停止する。その後さらに加圧を行うと、管１７内の圧力がｐ１に達し、圧力弁１５が開いて管内の空気が外部へ放出される。圧力弁１５が開いたことが検知されると、今度は逆にポンプ１４で管１７内を減圧していく。すると、今度はリザーバ１０４内から空気が出ていくため、検体液は流路１０３を通ってリザーバ１０４へ移動する。リザーバ１０４が検体液で満たされた状態になると、検体液は気液分離膜１０６を通ることができないため、検体液の移動が停止する。その後さらに減圧を行うと、管１７内の圧力がｐ２に達し、圧力弁１６が開いて外部の空気が管１７内へ流入する。圧力弁１６が開いたことが検知されると、再びポンプ１４で管１７内が加圧される。以上の動作を繰り返すことにより、流路１０３内の検体液を往復移動させることができる。   When the sample liquid is first introduced into the reservoir 104, the inside of the tube 17 is pressurized by the pump 14. Then, air enters the reservoir 104 through the gas-liquid separation membrane 106, and the sample liquid moves to the reservoir 105 through the flow path 103. When the reservoir 105 is filled with the sample liquid, the sample liquid cannot pass through the gas-liquid separation membrane 107, and the movement of the sample liquid is stopped. When further pressurization is performed thereafter, the pressure in the pipe 17 reaches p1, the pressure valve 15 is opened, and the air in the pipe is released to the outside. When it is detected that the pressure valve 15 is opened, the inside of the pipe 17 is depressurized by the pump 14 on the contrary. Then, since air comes out from the reservoir 104 this time, the sample liquid moves to the reservoir 104 through the flow path 103. When the reservoir 104 is filled with the sample liquid, the sample liquid cannot pass through the gas-liquid separation membrane 106, and the movement of the sample liquid is stopped. When the pressure is further reduced thereafter, the pressure in the pipe 17 reaches p2, the pressure valve 16 is opened, and external air flows into the pipe 17. When it is detected that the pressure valve 16 is opened, the inside of the pipe 17 is pressurized again by the pump 14. By repeating the above operation, the sample liquid in the channel 103 can be reciprocated.

ここで、図２に示すように、本実施形態では、複数の流路１０３が１つのポンプ１４に接続されており、ポンプ１４を用いて全ての流路１０３内の検体液の移動を制御している。一般に、各々の流路１０３は、加工によるばらつきなどの理由から、検体液の流れやすさに違いがあり、一方のリザーバから他方のリザーバへの移動時間は流路１０３によって異なる。しかし、本実施形態によれば、リザーバの開口部を気液分離膜で覆うようにしたため、一方のリザーバに検体液が完全に移動すると、それ以上加圧あるいは減圧を行ってもリザーバ内に検体液が留まるため、検体液の移動の速い流路１０３の移動完了後も、遅い流路１０３の移動が完了するまで、加圧あるいは減圧を続けることができる。そして、全ての流路１０３の移動が完了したところで、ポンプ１４の動作を加圧から減圧へ、あるいは減圧から加圧へ移行することができる。この結果、検体液の往復移動の回数が全ての流路１０３で等しくなる。このように、流路１０３毎に別のポンプ１４及び圧力弁１５，１６を用いなくても、攪拌の回数を一定にすることができる。よって、流路１０３の複数の流路間でばらつきのない信頼性の高い検査結果を得ることができる。   Here, as shown in FIG. 2, in this embodiment, a plurality of flow paths 103 are connected to one pump 14, and the movement of the sample liquid in all the flow paths 103 is controlled using the pumps 14. ing. In general, each flow path 103 has a difference in easiness of flow of the sample liquid due to processing variations and the like, and the movement time from one reservoir to the other reservoir differs depending on the flow path 103. However, according to the present embodiment, since the opening of the reservoir is covered with the gas-liquid separation membrane, when the sample liquid completely moves to one of the reservoirs, the sample is stored in the reservoir even if the pressure is further increased or reduced. Since the liquid remains, pressurization or depressurization can be continued until the movement of the slow flow path 103 is completed even after the movement of the flow path 103 where the movement of the specimen liquid is fast is completed. When the movement of all the flow paths 103 is completed, the operation of the pump 14 can be shifted from pressurization to depressurization or from depressurization to pressurization. As a result, the number of reciprocating movements of the sample liquid is equal in all the flow paths 103. In this manner, the number of stirrings can be made constant without using a separate pump 14 and pressure valves 15 and 16 for each flow path 103. Therefore, it is possible to obtain a highly reliable test result with no variation among the plurality of channels 103.

なお、本実施形態によれば、検体液の往復を繰り返しながらの反応時間は３時間以下でよい。従来の標準的なハイブリダイゼーション処理では、１０時間以上の反応時間が必要であり、本実施形態では大幅に時間短縮を図ることができる。また、１つのポンプ１４及び圧力弁１５，１６による制御で複数の流路１０３の制御が行えるため、従来のハイブリダイゼーション処理と比較し、一度に多くの検体の処理を行うことができる。
ハイブリダイゼーション反応が終了したら、ＣＣＤカメラ等を用いて予めターゲット核酸に結合してある蛍光標識剤を検出するなどの方法により、光学測定を行う。 According to this embodiment, the reaction time while repeating the reciprocation of the sample liquid may be 3 hours or less. The conventional standard hybridization process requires a reaction time of 10 hours or more, and in this embodiment, the time can be greatly reduced. In addition, since the plurality of flow paths 103 can be controlled by the control of one pump 14 and the pressure valves 15 and 16, a larger number of samples can be processed at a time than the conventional hybridization process.
When the hybridization reaction is completed, optical measurement is performed by a method such as detecting a fluorescent labeling agent previously bound to the target nucleic acid using a CCD camera or the like.

以上のように、本実施形態によれば、リザーバ１０４内の加圧または減圧を行うことにより、流路１０３内の検体液を往復移動させることができるので、検体液が流路１０３内で攪拌され、少ない検体液でプローブとの反応効率を高めることができる。また、リザーバ１０４，１０５の開口部がそれぞれ気液分離膜１０６，１０７で覆われているため、リザーバ１０４内の加圧、減圧を行っても検体液は気液分離膜１０６，１０７から外へ漏れることがなく、制御がしやすいという利点がある。   As described above, according to the present embodiment, the sample liquid in the flow path 103 can be reciprocated by pressurizing or depressurizing the reservoir 104, so that the sample liquid is stirred in the flow path 103. Thus, the reaction efficiency with the probe can be increased with a small amount of sample liquid. In addition, since the openings of the reservoirs 104 and 105 are covered with the gas-liquid separation membranes 106 and 107, respectively, the sample liquid is released from the gas-liquid separation membranes 106 and 107 even when the reservoir 104 is pressurized and depressurized. There is an advantage that it does not leak and is easy to control.

また、複数の流路１０３と接続する各々のリザーバ１０４が、１つのポンプ１４及び圧力弁１５，１６に接続されるようにしたので、複数の流路１０３を１つのポンプ１４で制御することができるため、装置の小型化やコストの削減が図れる。また、リザーバ１０４，１０５の開口部がそれぞれ気液分離膜１０６，１０７で覆われているため、流路１０３間で検体の移動時間に差があっても、リザーバ内に検体液を留めておくことで、移動時間の差を吸収することができ、全ての流路１０３で攪拌の回数を一定にすることができる。よって、流路１０３によって検体間の感度ばらつきのない信頼性の高い検査結果を得ることができる。   In addition, since each reservoir 104 connected to the plurality of flow paths 103 is connected to one pump 14 and the pressure valves 15 and 16, the plurality of flow paths 103 can be controlled by one pump 14. Therefore, it is possible to reduce the size and cost of the apparatus. In addition, since the openings of the reservoirs 104 and 105 are covered with the gas-liquid separation films 106 and 107, respectively, the specimen liquid is retained in the reservoir even if there is a difference in specimen movement time between the flow paths 103. Thus, the difference in moving time can be absorbed, and the number of times of stirring can be made constant in all the channels 103. Therefore, a highly reliable test result without sensitivity variation between samples can be obtained by the flow path 103.

また、圧力弁１５，１６の開閉を検知してポンプ１４による加圧、減圧を切り替えるようにしたので、簡易な処理で、検体液の往復移動を制御することができる。
なお、圧力弁１５，１６の代わりに、圧力センサと制御回路を設置し、管１７内の圧力がｐ１以上になったらポンプ１４による減圧に切り替え、管１７内の圧力がｐ２以下になったらポンプ１４による加圧に切り替えるように制御してもよい。 In addition, since the opening and closing of the pressure valves 15 and 16 are detected and the pressurization and depressurization are switched by the pump 14, the reciprocating movement of the sample liquid can be controlled with a simple process.
In place of the pressure valves 15 and 16, a pressure sensor and a control circuit are installed. When the pressure in the pipe 17 becomes p1 or higher, the pressure is reduced by the pump 14, and when the pressure in the pipe 17 becomes p2 or lower, the pump is switched. You may control to switch to the pressurization by 14.

なお、本実施形態では、流路１０３を複数設け、全ての流路１０３内の検体液を１つのポンプ１４を用いて往復移動させるようにしたが、流路１０３が１つの場合でも同様に反応処理を行うことができる。
また、本実施形態では、リザーバ１０４のみがポンプ１４に接続されているが、リザーバ１０４，１０５をそれぞれ別のポンプに接続し、両方のポンプを用いて流路１０３内の検体液を往復移動させるようにしてもよい。 In this embodiment, a plurality of flow paths 103 are provided, and the sample liquids in all the flow paths 103 are reciprocated using one pump 14, but the reaction is similarly performed even when there is only one flow path 103. Processing can be performed.
In this embodiment, only the reservoir 104 is connected to the pump 14, but the reservoirs 104 and 105 are connected to separate pumps, and the sample liquid in the flow path 103 is reciprocated using both pumps. You may do it.

本発明の実施の形態１による核酸検出装置の構成を示す模式図である。It is a schematic diagram which shows the structure of the nucleic acid detection apparatus by Embodiment 1 of this invention. 本発明の実施の形態１によるＤＮＡチップの上面図である。1 is a top view of a DNA chip according to Embodiment 1 of the present invention. 図２のＢ−Ｂ断面図である。It is BB sectional drawing of FIG. 本発明の実施の形態１による流路を上から見た図である。It is the figure which looked at the flow path by Embodiment 1 of this invention from the top.

符号の説明Explanation of symbols

１０ 核酸検出装置、１１ ＤＮＡチップ、１２ ステージ、１３ コントローラ
１４ ポンプ、１５，１６ 圧力弁、１７ 管、１０１，１０２ 透明基板、１０３ 流路、１０４，１０５ リザーバ、１０６，１０７ 気液分離膜、１０８〜１１２ Ｏリング、１１３，１１４ 蓋、１１５ カバー、１１６ ポンプとの接続部 DESCRIPTION OF SYMBOLS 10 Nucleic acid detection apparatus, 11 DNA chip, 12 stage, 13 Controller 14 Pump, 15, 16 Pressure valve, 17 Tube, 101, 102 Transparent substrate, 103 Channel, 104, 105 Reservoir, 106, 107 Gas-liquid separation membrane, 108 ~ 112 O-ring, 113, 114 Lid, 115 cover, 116 Connection with pump

Claims (7)

検体の流れる方向に間隔をおいて形成された複数の反応領域を備えて形成された流路と、
前記流路の一端に接続され、開口部を有する第１のリザーバと、
前記流路の他方の端部に接続され、開口部を有する第２のリザーバと、
前記第１のリザーバの開口部を覆うように設けられた第１の気液分離膜と、
前記第２のリザーバの開口部を覆うように設けられた第２の気液分離膜と、を備え、
各々の前記反応領域は、前記検体中の特定の生体物質を検出するためのプローブを固定する領域を有することを特徴とする生体物質検出チップ。 A flow path formed with a plurality of reaction regions formed at intervals in the flow direction of the specimen;
A first reservoir connected to one end of the flow path and having an opening;
A second reservoir connected to the other end of the flow path and having an opening;
A first gas-liquid separation membrane provided to cover the opening of the first reservoir;
A second gas-liquid separation membrane provided to cover the opening of the second reservoir,
Each of the reaction regions has a region for fixing a probe for detecting a specific biological material in the specimen. 前記流路を複数備え、
各々の前記流路は、それぞれ別の前記第１のリザーバ及び前記第２のリザーバに接続されていることを特徴とする請求項１に記載の生体物質検出チップ。 A plurality of the flow paths are provided,
2. The biological material detection chip according to claim 1, wherein each of the flow paths is connected to the first reservoir and the second reservoir separately from each other. 請求項１または請求項２に記載の生体物質検出チップを用いて生体物質検出処理を行うための生体物質検出装置であって、
前記第１の分離膜を介して前記第１のリザーバと接続され、前記第１のリザーバ内の加圧または減圧を行う圧力制御手段を備えていることを特徴とする生体物質検出装置。 A biological material detection device for performing a biological material detection process using the biological material detection chip according to claim 1,
A biological substance detection apparatus comprising pressure control means connected to the first reservoir via the first separation membrane and configured to pressurize or depressurize the first reservoir. 複数の前記流路と接続する各々の前記第１のリザーバが、１つの前記圧力制御手段に接続されていることを特徴とする請求項３に記載の生体物質検出装置。   The biological substance detection device according to claim 3, wherein each of the first reservoirs connected to the plurality of flow paths is connected to one pressure control unit. 前記圧力制御手段は、ポンプと、圧力がｐ１以上になると開く第１の圧力弁と、圧力がｐ２以下になると開く第２の圧力弁を備え、ｐ２＜大気圧＜ｐ１であることを特徴とする請求項３または請求項４に記載の生体物質検出装置。   The pressure control means includes a pump, a first pressure valve that opens when the pressure becomes p1 or more, and a second pressure valve that opens when the pressure becomes p2 or less, and p2 <atmospheric pressure <p1. The biological substance detection device according to claim 3 or claim 4 to be performed. 前記生体物質検出チップの温度調節機構を備えていることを特徴とする請求項３から請求項５のいずれかに記載の生体物質検出装置。   The biological material detection device according to claim 3, further comprising a temperature adjustment mechanism of the biological material detection chip. 請求項３から請求項６のいずれかに記載の生体物質検出装置を用いた生体物質検出方法であって、
前記リザーバに検体液を供給する工程と、
前記圧力制御手段によって前記流路内の検体液を往復移動させる工程を備えていることを特徴とする生体物質検出方法。 A biological material detection method using the biological material detection device according to any one of claims 3 to 6,
Supplying a sample liquid to the reservoir;
A biological substance detection method comprising a step of reciprocating the sample liquid in the flow path by the pressure control means.
JP2007297809A 2007-11-16 2007-11-16 Biological material sensing chip, device, and method Pending JP2009122022A (en)

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JP2011163939A (en) * 2010-02-10 2011-08-25 Sumitomo Bakelite Co Ltd Microchannel device
WO2012126294A1 (en) * 2011-03-22 2012-09-27 博奥生物有限公司 Biological chip hybridization system
JP2018089611A (en) * 2016-12-01 2018-06-14 富士フイルム和光純薬株式会社 Method for mixing solution in microfluidic apparatus, and apparatus for the same

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WO2011027851A1 (en) * 2009-09-07 2011-03-10 コニカミノルタホールディングス株式会社 Liquid feeding system for microchip, sample detection device, and liquid feeding method for liquid feeding system for microchip
US9375713B2 (en) 2009-09-07 2016-06-28 Konica Minolta, Inc. Liquid feeding system for microchip, sample detection device, and liquid feeding method for liquid feeding system for microchip
JP2011163939A (en) * 2010-02-10 2011-08-25 Sumitomo Bakelite Co Ltd Microchannel device
WO2012126294A1 (en) * 2011-03-22 2012-09-27 博奥生物有限公司 Biological chip hybridization system
US9677133B2 (en) 2011-03-22 2017-06-13 Capitalbio Corporation Biological chip hybridization system
JP2018089611A (en) * 2016-12-01 2018-06-14 富士フイルム和光純薬株式会社 Method for mixing solution in microfluidic apparatus, and apparatus for the same
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