JP6656185B2 - Droplet trapping method, measurement cell, and droplet trapping device - Google Patents

Droplet trapping method, measurement cell, and droplet trapping device Download PDF

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JP6656185B2
JP6656185B2 JP2017000683A JP2017000683A JP6656185B2 JP 6656185 B2 JP6656185 B2 JP 6656185B2 JP 2017000683 A JP2017000683 A JP 2017000683A JP 2017000683 A JP2017000683 A JP 2017000683A JP 6656185 B2 JP6656185 B2 JP 6656185B2
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譲 島崎
譲 島崎
淳子 田中
淳子 田中
小原　賢信
賢信 小原
原田　邦男
邦男 原田
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Description

本発明は、生体分子計測方法、特に遺伝子変異の計測方法に関わる液滴のトラップ方法、測定セル、及び液滴トラップ装置に関する。   The present invention relates to a method for measuring a biomolecule, in particular, a method for trapping a droplet, a measurement cell, and a droplet trap device related to a method for measuring a gene mutation.

現在、遺伝子変異を高感度で検知できるデジタルＰＣＲ法が注目されている。デジタルＰＣＲ法の一種である、液滴を用いたデジタルＰＣＲ法（ｄｄＰＣＲ：droplet digital PCR）では、まず、検体中に含まれるＤＮＡを液滴中に単離し、単離したＤＮＡの種類を蛍光標識などの方法で可視化する。その後、流路を流れる液滴に含まれるＤＮＡの種類をフローサイトメーターで判別し、それぞれの個数を積算して、検体中のターゲットＤＮＡの数を定量する。   At present, a digital PCR method capable of detecting a gene mutation with high sensitivity has attracted attention. In a digital PCR method using droplets (ddPCR: droplet digital PCR), which is a kind of digital PCR method, first, DNA contained in a sample is isolated in a droplet, and the type of the isolated DNA is labeled with a fluorescent label. And visualize it. Thereafter, the type of DNA contained in the droplet flowing through the flow path is determined by a flow cytometer, and the number of each is integrated to quantify the number of target DNAs in the sample.

一方、非特許文献１は、複数のウエルを有する液滴ガイド部を内壁に有するマイクロ流路中に液滴を流し、液滴ガイド部に捕捉された液滴群の蛍光を上面から観察することで、液滴中の蛍光色素の有無を判別する技術を開示している。   On the other hand, Non-Patent Document 1 discloses that a droplet is caused to flow through a microchannel having a droplet guide portion having a plurality of wells on an inner wall, and the fluorescence of the droplet group captured by the droplet guide portion is observed from above. Discloses a technique for determining the presence or absence of a fluorescent dye in a droplet.

L. Labanieh et al. Micromachines 2015, 6, 1469-1482.L. Labanieh et al. Micromachines 2015, 6, 1469-1482.

発明者らは、平面状に配列した液滴群からの蛍光を検出するｄｄＰＣＲ法を検討している。マイクロ流路中の内壁に設置された、複数のウエルを有する液滴ガイド部に液滴を捕捉する方法を検討したところ、液滴の捕捉率（投入液滴数に対する捕捉液滴数の割合）が２０％程度と悪く、十分量の液滴を液滴ガイド部に捕捉させるために、多量の液滴を流す必要があった。一般に生体分子計測に使用できる検体量は少なく、検査に供する検体量は少ないことが望ましい。   The inventors are studying a ddPCR method for detecting fluorescence from a group of droplets arranged in a plane. We examined a method of catching droplets in a droplet guide unit with multiple wells installed on the inner wall in the microchannel, and found that the droplet capture rate (the ratio of the number of captured droplets to the number of input droplets) However, the droplet guide portion was not good at about 20%, and it was necessary to flow a large amount of droplets in order to capture a sufficient amount of droplets in the droplet guide portion. In general, the amount of a sample that can be used for biomolecule measurement is small, and the amount of a sample to be used for a test is desirably small.

本発明は、液滴の捕捉率の高い液滴のトラップ方法、測定セル、液滴トラップ装置を提供するものである。   The present invention provides a method, a measurement cell, and a droplet trap device for a droplet having a high droplet capture rate.

本発明による液滴のトラップ方法は、一例として、内部空間の一方の内壁面に複数のウエルを有する液滴ガイド部が設けられ、一方の内壁面に対向する他方の内壁面に浮遊する液滴を捕捉する窪み部を有するトラップ部が設けられた測定セル内に、トラップ部を上に液滴ガイド部を下にした状態で複数の液滴とオイルとを含む流体を流し、トラップ部に複数の液滴をトラップする工程と、測定セルを反転させ、トラップ部にトラップされた複数の液滴を液滴ガイド部の複数のウエルに捕捉させる工程と、を有する。   The droplet trapping method according to the present invention includes, as an example, a droplet guide portion having a plurality of wells provided on one inner wall surface of an internal space, and a droplet floating on the other inner wall surface facing one inner wall surface. A fluid containing a plurality of droplets and oil is flowed in a measurement cell provided with a trap portion having a concave portion for capturing the liquid, with the trap portion being upward and the droplet guide portion being downward, and a plurality of fluids are passed through the trap portion. And a step of inverting the measurement cell and capturing the plurality of droplets trapped in the trap portion in the plurality of wells of the droplet guide portion.

本発明による測定セルは、一例として、筐体の外表面に開口した試料注入口及び排出口と、試料注入口と排出口とを結ぶ筐体の内部に設けられた流路と、流路の途中に設けられ筐体の外表面に平行な方向に広がりを持つ内部空間と、内部空間の一方の内壁面に設けられた複数のウエルを有する液滴ガイド部と、内部空間の他方の内壁面に設けられた浮遊する液滴を捕捉する窪み部を有するトラップ部と、を有する。   The measurement cell according to the present invention includes, as an example, a sample inlet and an outlet opened on the outer surface of the housing, a flow path provided inside the housing connecting the sample inlet and the outlet, An inner space provided in the middle and extending in a direction parallel to the outer surface of the housing, a droplet guide portion having a plurality of wells provided on one inner wall surface of the inner space, and the other inner wall surface of the inner space And a trap portion having a concave portion for catching a floating droplet provided on the substrate.

また、本発明による液滴トラップ装置は、一例として、測定セルに液滴を捕捉させるための装置であって、内部空間の内壁面に複数のウエルを有する液滴ガイド部と浮遊する液滴を捕捉する窪み部を有するトラップ部とが対面して設けられた測定セルを保持し、反転させる機構を有する反転部と、反転部によって反転された測定セルを配置する設置部と、反転部による測定セルの反転タイミングを制御する制御部と、を有する。   In addition, the droplet trap device according to the present invention is, for example, a device for causing a measurement cell to capture a droplet, and a droplet guide portion having a plurality of wells on an inner wall surface of an internal space and a droplet that floats. A reversing unit having a mechanism for holding and reversing a measuring cell provided with a trap unit having a dent part for capturing and reversing, an installation unit for arranging the measuring cell reversed by the reversing unit, and measurement by the reversing unit And a controller for controlling the inversion timing of the cell.

本発明によれば、測定セルにおける液滴の捕捉率を高めることができる。   ADVANTAGE OF THE INVENTION According to this invention, the capture rate of the droplet in a measurement cell can be improved.

上記した以外の、課題、構成及び効果は、以下の実施形態の説明により明らかにされる。   Problems, configurations, and effects other than those described above will be apparent from the following description of the embodiments.

測定セルの一例を示す断面模式図。FIG. 3 is a schematic cross-sectional view illustrating an example of a measurement cell. 測定セルの中央付近を拡大して示した断面模式図。FIG. 2 is a schematic cross-sectional view showing an enlarged vicinity of the center of a measurement cell. 液滴ガイド部の構造例を示す模式図。FIG. 3 is a schematic diagram illustrating a configuration example of a droplet guide unit. 図２ＡのＢ−Ｂ断面図。BB sectional drawing of FIG. 2A. 測定セル中に液滴とオイルとを含む流体を流した状態を示す断面模式図。FIG. 4 is a schematic cross-sectional view showing a state in which a fluid containing droplets and oil has flowed through the measurement cell. 測定セルを反転させた状態を示す断面模式図。FIG. 3 is a schematic cross-sectional view showing a state where a measurement cell is inverted. 液滴が液滴ガイド部に捕捉された状態を示す断面模式図。FIG. 4 is a schematic cross-sectional view illustrating a state in which a droplet is captured by a droplet guide unit. 液滴トラップ装置を模式的に示す図。The figure which shows a droplet trap apparatus typically. 液滴トラップ装置の動作を模式的に示した説明図。FIG. 4 is an explanatory diagram schematically showing the operation of the droplet trap device. 液滴トラップ装置の動作を模式的に示した説明図。FIG. 4 is an explanatory diagram schematically showing the operation of the droplet trap device. 液滴トラップ装置の動作を模式的に示した説明図。FIG. 4 is an explanatory diagram schematically showing the operation of the droplet trap device. 実施例の測定セルと比較例の測定セルの上面図。The top view of the measuring cell of an Example, and the measuring cell of a comparative example. 実施例で使用した測定セルの断面模式図。FIG. 4 is a schematic cross-sectional view of a measurement cell used in the example. 比較例で使用した測定セルの断面模式図。FIG. 4 is a schematic cross-sectional view of a measurement cell used in a comparative example. 実施例と比較例で用いた測定セルの液滴ガイド部の構造を示す平面模式図。FIG. 4 is a schematic plan view showing the structure of a liquid drop guide section of a measurement cell used in Examples and Comparative Examples. 図７ＡのＢ−Ｂ断面模式図。FIG. 7B is a schematic sectional view taken along line BB of FIG. 7A. 測定セルを反転した直後の液滴ガイド部の光学顕微鏡像。An optical microscope image of the droplet guide immediately after the measurement cell is inverted. 振動を６０分印加した後の液滴ガイド部の光学顕微鏡像。An optical microscope image of the droplet guide portion after applying vibration for 60 minutes. 測定セル中の配列液滴の蛍光像を観察した計測システムの概略図。The schematic diagram of the measurement system which observed the fluorescence image of the arrangement droplet in the measurement cell. 測定セル中の配列液滴の蛍光顕微鏡像。Fluorescence microscope image of arrayed droplets in the measurement cell.

はじめに、本発明の一実施形態に係る測定セルについて説明する。なお、以下の各図において共通する構成については、同一の符号を付し、重複した説明を省略する。   First, a measurement cell according to an embodiment of the present invention will be described. Note that the same reference numerals are given to configurations common to the following drawings, and redundant description will be omitted.

図１Ａは、測定セル１の一例を示す断面模式図である。また、図１Ｂは測定セルの中央付近を拡大して示した断面模式図である。   FIG. 1A is a schematic cross-sectional view illustrating an example of the measurement cell 1. FIG. 1B is an enlarged schematic cross-sectional view of the vicinity of the center of the measurement cell.

本実施例の測定セル１は、外形が板状をした筐体８の一方の外表面に試料注入口４と排出口６が開口し、筐体８の内部に試料注入口４と排出口６を結ぶ流路５が形成されている。流路５の途中には、主に筐体８の外表面に平行な方向に広がりを持った内部空間１５が設けられ、その内部空間１５に液滴ガイド部３とトラップ部２が設けられている。液滴ガイド部３とトラップ部２とは、流路５の上面側と下面側に互いに対向するように設けられている。すなわち、流路５の途中に設けられた内部空間１５の一方の内壁面に液滴ガイド部３が設けられ、その内壁面に対面する他方の内壁面にトラップ部２が設けられている。   In the measurement cell 1 of the present embodiment, the sample inlet 4 and the outlet 6 are opened on one outer surface of a case 8 having a plate-like outer shape, and the sample inlet 4 and the outlet 6 Are formed. In the middle of the flow path 5, an internal space 15 is provided, which has an expansion mainly in a direction parallel to the outer surface of the housing 8. I have. The droplet guide section 3 and the trap section 2 are provided on the upper surface side and the lower surface side of the flow path 5 so as to face each other. That is, the droplet guide portion 3 is provided on one inner wall surface of the internal space 15 provided in the middle of the flow path 5, and the trap portion 2 is provided on the other inner wall surface facing the inner wall surface.

トラップ部２は、試料注入口４から注入され流路５を通して流入した浮遊する液滴をまとめて捕捉する窪み部を有する。トラップ部２は、流路面から５μｍ以上窪んだ単数又は複数の領域を有することが好ましい。トラップ部２の窪みの深さが５μｍより浅いと、流路を通して流入した液滴が十分にトラップできず、液滴の捕捉率が低下するため好ましくない。また、トラップ部２の形状は、流路を通して流入した液滴が十分にトラップできれば、特に制限はない。トラップ部２の形状の例として、液滴ガイド部３に対面した領域の一部が一様に窪んだ形状や、一様に窪んだ領域の底面にさらに複数の窪みがある構造などが挙げられる。また、トラップ部２を構成する材料は、測定セル１を反転した際に、トラップした液滴がトラップ部から離れて浮遊すれば特に制限はない。トラップ部２を構成する材料の例として、ガラス、樹脂、金属などが挙げられる。   The trap unit 2 has a recess for collectively capturing floating droplets injected from the sample injection port 4 and flowing through the channel 5. The trap section 2 preferably has one or more regions depressed by 5 μm or more from the flow path surface. If the depth of the depression of the trap portion 2 is less than 5 μm, the droplets flowing through the flow channel cannot be sufficiently trapped, and the capture rate of the droplets is undesirably reduced. The shape of the trap portion 2 is not particularly limited as long as the droplets flowing through the flow channel can be sufficiently trapped. Examples of the shape of the trap portion 2 include a shape in which a part of the region facing the droplet guide portion 3 is uniformly depressed, and a structure in which a plurality of depressions are further provided on the bottom surface of the uniformly depressed region. . Further, there is no particular limitation on the material forming the trap portion 2 as long as the trapped droplets float away from the trap portion when the measurement cell 1 is inverted. Examples of the material forming the trap section 2 include glass, resin, and metal.

液滴ガイド部３は、測定セル１の反転後、トラップ部２から浮遊した液滴を配列させるためのウエル３１を有する。液滴ガイド部３の形状は、トラップ部２から浮遊した液滴を配列できる形状であれば、特に制限はない。液滴ガイド部３の形状の例として、ウエル３１が周期的に配列した形状が挙げられる。   The droplet guide unit 3 has a well 31 for arranging droplets floating from the trap unit 2 after the measurement cell 1 is inverted. The shape of the liquid drop guide 3 is not particularly limited as long as the liquid drop floating from the trap 2 can be arranged. As an example of the shape of the droplet guide portion 3, there is a shape in which the wells 31 are periodically arranged.

図２Ａは、ウエル３１が２次元的に周期的に配列した形状の液滴ガイド部３の構造例を示す模式図であり、図２ＢはそのＢ−Ｂ断面図である。ウエルは、様々な形状を取ることが可能である。ウエル３１の形状の例として、円柱状、楕円柱状、多角柱状（三角柱状、四角柱状、五角柱状、六角柱状など）、四面体状などが挙げられる。また、ウエル３１の周期配列の例として、正方配列や三角配列が考えられ、ウエル３１の密度は、用途に応じて調整することが可能である。   FIG. 2A is a schematic diagram showing a structural example of the droplet guide unit 3 having a shape in which the wells 31 are two-dimensionally and periodically arranged, and FIG. 2B is a cross-sectional view taken along the line BB. The wells can take various shapes. Examples of the shape of the well 31 include a columnar shape, an elliptical columnar shape, a polygonal columnar shape (such as a triangular column, a quadrangular column, a pentagonal column, and a hexagonal column), and a tetrahedron. Further, as an example of the periodic arrangement of the wells 31, a square arrangement or a triangular arrangement can be considered, and the density of the wells 31 can be adjusted according to the application.

特に、ウエル３１の形状が円柱状の場合、液滴ガイド部３は、液滴の配列において良好な効果を発揮する。円筒状のウエルの形状は下記の（１）〜（３）の関係を全て満たすことが望ましい。本実施例で、既存技術と同等の判別速度を実現するためには、液滴の直径は５０μｍ以下であることが望ましく、ウエルの最大径を液滴の直径の０．５倍から１．２倍の範囲として、液滴位置のドリフトを最小限に抑えることが望ましい。また、ウエルの平均深さは、ウエルの最大径の０．０１倍より大きく１倍より小さいことが望ましい。ウエルの平均深さがウエルの平均径の０．０１倍より小さいと、液滴の捕捉が困難となり、好ましくない。また、ウエルの平均深さがウエルの平均径の１倍より大きいと、液滴ガイド部３の加工が困難となり好ましくない。   In particular, when the shape of the well 31 is cylindrical, the droplet guide portion 3 exhibits a good effect on the arrangement of droplets. It is desirable that the shape of the cylindrical well satisfies all of the following relationships (1) to (3). In this embodiment, in order to realize a discrimination speed equivalent to that of the existing technology, the diameter of the droplet is desirably 50 μm or less, and the maximum diameter of the well is set to 0.5 to 1.2 times the diameter of the droplet. It is desirable to minimize the drift of the droplet position as a double range. Further, it is desirable that the average depth of the well be larger than 0.01 times and smaller than 1 time of the maximum diameter of the well. If the average depth of the well is smaller than 0.01 times the average diameter of the well, it becomes difficult to catch the droplet, which is not preferable. On the other hand, if the average depth of the well is larger than one time the average diameter of the well, it becomes difficult to process the droplet guide portion 3, which is not preferable.

Ｄd＜５０μｍ （１）
０．５Ｄd＜Ｄw＜１．２Ｄd （２）
０．０１≦ｄ／Ｄw≦１ （３）
ただし、Ｄd［μｍ］：液滴の直径の平均、Ｄw［μｍ］：ウエルの最大径の平均、ｄ［μｍ］：ウエルの平均深さである。 D d <50 μm (1)
0.5D d <D w <1.2D d (2)
0.01 ≦ d / D w ≦ 1 (3)
Here, D d [μm]: average of the diameter of the droplet, D w [μm]: average of the maximum diameter of the well, and d [μm]: average depth of the well.

試料注入口４は、液滴とオイルとを含む流体を測定セル１中に注入できる構造であれば特に制限はない。また、流路５は、液滴とオイルとを含む流体をトラップ部２に誘導し、過剰のオイルを排出口６まで誘導する構造であれば特に制約はない。また、排出口６は、オイルを測定セル１の外部に排出できる構造であれば特に制限はない。   The sample injection port 4 is not particularly limited as long as it has a structure capable of injecting a fluid containing a droplet and oil into the measurement cell 1. The flow path 5 is not particularly limited as long as it has a structure that guides a fluid containing droplets and oil to the trap portion 2 and guides excess oil to the outlet 6. Further, the outlet 6 is not particularly limited as long as it has a structure capable of discharging the oil to the outside of the measuring cell 1.

試料注入口４と排出口６には、蓋部材を設置して、オイルの蒸発と液滴の流出を防止することが可能である。この場合、試料注入口４と排出口６は、蓋部材を設置して開口部を塞ぐことができる構造となっていることが望ましい。   Lid members can be provided at the sample inlet 4 and the outlet 6 to prevent evaporation of oil and outflow of droplets. In this case, it is desirable that the sample inlet 4 and the outlet 6 have a structure in which a lid member is installed to close the opening.

筐体８は、測定セル１の構造を維持し、液滴を構成する溶液の沸点（１００℃程度）までの耐熱性を有することが望ましい。また、液滴の光学観察をする場合には、液滴ガイド部３と測定セル１の外周とに挟まれた筐体部分９の、波長４５０〜７５０ｎｍにおける光透過率が７０％以上であることが望ましい。透過率が７０％より小さいと、測定セル１の外部から液滴の蛍光を計測することが困難となるため、好ましくない。また、倒立顕微鏡などの顕微鏡を用いて、トラップ部２を通して液滴を光学観察する場合には、トラップ部２と測定セル１の外周とに挟まれた筐体部分１０の、波長４５０〜７５０ｎｍにおける光透過率が７０％以上であることが望ましい。透過率が７０％より小さいと、測定セル１の外部から液滴の蛍光を計測することが困難となるため、好ましくない。   It is desirable that the housing 8 maintain the structure of the measurement cell 1 and have heat resistance up to the boiling point (about 100 ° C.) of the solution forming the droplet. When optical observation of the droplet is performed, the light transmittance at a wavelength of 450 to 750 nm of the housing portion 9 sandwiched between the droplet guide portion 3 and the outer periphery of the measurement cell 1 is 70% or more. Is desirable. If the transmittance is less than 70%, it becomes difficult to measure the fluorescence of the droplet from outside the measurement cell 1, which is not preferable. In addition, when optical observation of liquid droplets is performed through the trap unit 2 using a microscope such as an inverted microscope, the case portion 10 sandwiched between the trap unit 2 and the outer periphery of the measurement cell 1 has a wavelength of 450 to 750 nm. It is desirable that the light transmittance is 70% or more. If the transmittance is less than 70%, it becomes difficult to measure the fluorescence of the droplet from outside the measurement cell 1, which is not preferable.

次に、本発明の一実施形態に係る液滴のトラップ方法について説明する。なお、以下の各図において共通する構成については、同一の符号を付し、重複した説明を省略する。   Next, a droplet trapping method according to one embodiment of the present invention will be described. Note that the same reference numerals are given to configurations common to the following drawings, and redundant description will be omitted.

図３Ａ〜３Ｃは、実施例の測定セルを用いた液滴のトラップ方法を模式的に示す図である。図３Ａは測定セル１中に液滴１１とオイル１２とを含む流体を流した後の状態を示す断面模式図、図３Ｂは測定セル１を反転させた状態を示す断面模式図、図３Ｃは液滴１１が液滴ガイド部３に捕捉された状態を示す断面模式図である。   3A to 3C are diagrams schematically illustrating a droplet trapping method using the measurement cell of the example. 3A is a schematic cross-sectional view showing a state after a fluid containing a droplet 11 and an oil 12 has flowed into the measurement cell 1, FIG. 3B is a schematic cross-sectional view showing a state where the measurement cell 1 is inverted, and FIG. FIG. 4 is a schematic cross-sectional view illustrating a state in which a droplet 11 is captured by a droplet guide unit 3.

まず、試料注入口４から、測定セル１中に液滴１１とオイル１２とを含む流体を流す。流体の流し方は、流体が測定セル中に入る方法であれば特に制限はない。流体の流し方の例として、マイクロピペットを使って注入する方法や、試料注入口４にチューブなどを接続し、ポンプなどを用いて注入する方法などが挙げられる。液滴１１は、オイル１２よりも密度が低く、オイル１２の表面に浮遊するものが好ましい。また、液滴１１は、液滴中の観察対象や、液滴中で観察対象を形成するために必要な分子を含有してもよい。液滴１１が含有してもよい分子の例として、生体関連分子、ＰＣＲ反応に必要な薬剤、蛍光色素、塩などが挙げられる。また、液滴１１は、界面活性剤で安定化されていることが好ましい。界面活性剤の例として、EA surfactant（RainDance社製）や、Pico-SurfTM 1（dolomite社製）などが挙げられる。また、オイル１２は、液滴１１を浮遊させる液体であればよい。オイル１２の例として、フロリナート（３Ｍ製）、Novec（３Ｍ製）などが挙げられる。 First, a fluid containing the droplet 11 and the oil 12 flows into the measurement cell 1 from the sample inlet 4. The flow of the fluid is not particularly limited as long as the fluid enters the measurement cell. Examples of the method of flowing the fluid include a method of injecting using a micropipette, and a method of connecting a tube or the like to the sample inlet 4 and injecting using a pump or the like. The droplet 11 is preferably lower in density than the oil 12 and floats on the surface of the oil 12. Further, the droplet 11 may contain an observation target in the droplet or a molecule necessary for forming the observation target in the droplet. Examples of molecules that may be contained in the droplet 11 include biologically relevant molecules, drugs required for a PCR reaction, fluorescent dyes, salts, and the like. Further, it is preferable that the droplet 11 is stabilized with a surfactant. Examples of the surfactant include EA surfactant (manufactured by RainDance) and Pico-Surf ™ 1 (manufactured by dolomite). Further, the oil 12 may be any liquid that causes the droplets 11 to float. Examples of the oil 12 include Fluorinert (manufactured by 3M), Novec (manufactured by 3M), and the like.

液滴１１とオイル１２とを含む流体を測定セル１に流すと、液滴１１は浮力を受けて、図３Ａのようにトラップ部２に捕捉される。そのため、排出口６から液滴１１が抜け出ることはほとんどない。従って、本実施例の液滴トラップ方法によって、液滴の捕捉率を大幅に向上させることが可能である。   When a fluid containing the droplet 11 and the oil 12 flows through the measurement cell 1, the droplet 11 receives buoyancy and is captured by the trap unit 2 as shown in FIG. 3A. Therefore, the droplet 11 hardly escapes from the outlet 6. Therefore, the droplet trapping method of this embodiment can significantly improve the droplet capture rate.

液滴１１とオイル１２とを含む流体を測定セル１に流した後、試料注入口４と排出口６に蓋部材７を設置して、オイル１２の蒸発と液滴１１の流出を防止してもよい。蓋部材７は、オイル１２の蒸発と液滴１１の流出を防止できる構造と材質であれば、特に制限はない。また、この場合、試料注入口４と排出口６は、蓋部材７を設置できる構造となっていることが望ましい。また、蓋部材７として、粘着テープなどを使用することも可能である。   After the fluid containing the droplets 11 and the oil 12 flows into the measurement cell 1, the lid member 7 is provided at the sample inlet 4 and the outlet 6 to prevent the evaporation of the oil 12 and the outflow of the droplet 11. Is also good. The lid member 7 is not particularly limited as long as it has a structure and a material capable of preventing the evaporation of the oil 12 and the outflow of the droplet 11. In this case, it is desirable that the sample inlet 4 and the outlet 6 have a structure in which the lid member 7 can be installed. Further, an adhesive tape or the like can be used as the lid member 7.

試料注入口４と排出口６を蓋部材７で塞いだ後、液滴ガイド部３がトラップ部２の上に来るように測定セル１を反転させる。このとき液滴１１に浮力が働き、トラップ部２に捕捉されていた液滴１１は、図３Ｂに示すように液滴ガイド部３に誘導される。   After closing the sample inlet 4 and the outlet 6 with the cover member 7, the measuring cell 1 is inverted so that the droplet guide 3 is located above the trap 2. At this time, buoyancy acts on the droplet 11, and the droplet 11 trapped in the trap unit 2 is guided to the droplet guide unit 3 as shown in FIG. 3B.

その後、時間経過とともに、図３Ｃに示すように、液滴１１が液滴ガイド部３のウエルに従って配列する。このプロセスでは、液滴１１は、液滴ガイド部３に沿って移動し、浮力、または、液滴表面の電荷と、液滴ガイド部３の窪みの角に集中する静電気との間に働く相互作用によって窪みに捕捉される。また、測定セル１を反転させた後に、測定セル１に振動を印加して、液滴１１のウエルへの配列時間を短縮することも可能である。振動の印加方法は、液滴１１の配列時間が短縮できる方法であれば、特に制限はない。   Thereafter, as time elapses, the droplets 11 are arranged according to the wells of the droplet guide unit 3, as shown in FIG. 3C. In this process, the droplet 11 moves along the droplet guide 3 and acts between buoyancy or charge on the surface of the droplet and static electricity concentrated on the corners of the depressions of the droplet guide 3. It is trapped in the depression by the action. After the measurement cell 1 is inverted, vibration can be applied to the measurement cell 1 to shorten the time required for arranging the droplets 11 in the wells. The method of applying the vibration is not particularly limited as long as the arrangement time of the droplets 11 can be shortened.

以上に示した液滴のトラップ方法により、高捕捉率で測定セル１中に液滴１１を捕捉し、液滴ガイド部３のウエルに配列させることができる。なお、液滴１１の蛍光強度変化を効率良く計測する必要がある場合には、液滴１１の直径を小さくして、液滴１１を高密度に配列する方がよい。例えば、液滴流路を流れる液滴に含まれるＤＮＡの種類を判別する現行のｄｄＰＣＲ装置では、液滴を１０4個／分程度の速度で判別している。本実施例で同等の判別速度を実現するためには、液滴１１の直径は５０μｍ以下であることが望ましい。 By the droplet trapping method described above, the droplets 11 can be captured in the measurement cell 1 at a high capture rate and arranged in the wells of the droplet guide unit 3. When it is necessary to efficiently measure a change in the fluorescence intensity of the droplet 11, it is better to reduce the diameter of the droplet 11 and arrange the droplets 11 at a high density. For example, in a current ddPCR apparatus that determines the type of DNA contained in a droplet flowing through a droplet channel, droplets are determined at a speed of about 10 4 droplets / minute. In order to realize the same discrimination speed in this embodiment, the diameter of the droplet 11 is desirably 50 μm or less.

次に、本発明の一実施形態に係る液滴トラップ装置について説明する。なお、以下の各図において共通する構成については、同一の符号を付し、重複した説明を省略する。   Next, a droplet trap device according to an embodiment of the present invention will be described. Note that the same reference numerals are given to configurations common to the following drawings, and redundant description will be omitted.

図４は、本実施例の液滴トラップ装置を模式的に示した図である。本実施例の液滴トラップ装置１００は、設置部１０１と、反転部１０２と、制御部１０３とを有する。設置部１０１は、筐体の外表面に平行な方向に広がりを持つ内部空間の一方の内壁面に窪み部を有するトラップ部が設けられ、他方の内壁面に複数のウエルを有する液滴ガイド部が設けられた測定セル１を配置する部位である。反転部１０２は、設置部１０１に配置した測定セルを反転する機構を有する。制御部１０３は、反転部１０２が測定セルを反転するタイミングを制御する。   FIG. 4 is a diagram schematically illustrating the droplet trap device of the present embodiment. The droplet trap device 100 according to the present embodiment includes an installation unit 101, an inversion unit 102, and a control unit 103. The installation section 101 is provided with a trap section having a recess on one inner wall surface of an internal space extending in a direction parallel to the outer surface of the housing, and a droplet guide section having a plurality of wells on the other inner wall surface. Is a portion where the measurement cell 1 provided with is disposed. The reversing unit 102 has a mechanism for reversing the measurement cell arranged in the installation unit 101. The control unit 103 controls the timing at which the inversion unit 102 inverts the measurement cell.

図５Ａ〜５Ｃは、本実施例の液滴トラップ装置１００の動作を模式的に示した説明図である。各図の上方に、液滴トラップ装置１００の内部での測定セル１の状態を拡大して示した。   5A to 5C are explanatory diagrams schematically showing the operation of the droplet trap device 100 of the present embodiment. Above each figure, the state of the measurement cell 1 inside the droplet trap device 100 is shown in an enlarged manner.

測定セル１の流路に液滴１１とオイル１２とを含む流体を流し、図３Ａに示すようにオイル中に浮遊した液滴をトラップ部２に捕捉して、試料注入口４と排出口６を蓋部材７で塞ぐ。この測定セル１のトラップ部２に液滴を捕捉する工程は、測定セル１を液滴トラップ装置１００の反転部１０２に固定した状態で行ってもよいし、別の場所で行ってもよい。別の場所でトラップ部２に液滴を捕捉する工程を行った場合には、トラップ部２が液滴ガイド部３より上方に位置する姿勢を維持したまま、図５Ａに示すように測定セル１を反転部１０２に固定する。   A fluid containing a droplet 11 and an oil 12 is caused to flow through the flow path of the measurement cell 1, and the droplet floating in the oil is captured by the trap unit 2 as shown in FIG. Is closed with the lid member 7. The step of capturing the liquid droplets in the trap unit 2 of the measurement cell 1 may be performed in a state where the measurement cell 1 is fixed to the reversing unit 102 of the droplet trap device 100, or may be performed in another place. When the step of trapping the liquid droplets in the trap unit 2 is performed at another location, the measurement cell 1 is maintained as shown in FIG. Is fixed to the reversing unit 102.

次に、トラップ部が上面になるように測定セル１を保持した反転部１０２は、制御部１０３の命令により反転し、図５Ｂに示すように、設置部１０１に測定セル１を設置する。このとき、測定セル１は液滴ガイド部３が上面になり、液滴ガイド部３がトラップ部２より上方に位置する。反転の方法は、測定セルの上面と下面とが反転できれば特に制限はない。反転の方法の例として、図５Ａに矢印で示すように、円弧を描くようにセル平面に平行な軸を回転させる方法や、セル平面に平行な軸を中心に回転させる方法が挙げられる。測定セル１を設置部１０１に配置した後に、反転部１０２は測定セル１の保持を解除してもよい。また、反転部１０２が測定セル１の保持を解除した後は、図５Ｃに示すように、反転部１０２は、次の操作に備えて、測定セル１を反転させる前の位置に移動してもよい。   Next, the inversion unit 102 holding the measurement cell 1 so that the trap unit is on the upper surface is inverted according to a command from the control unit 103, and the measurement cell 1 is installed in the installation unit 101 as shown in FIG. 5B. At this time, in the measurement cell 1, the droplet guide unit 3 is on the upper surface, and the droplet guide unit 3 is located above the trap unit 2. The method of inversion is not particularly limited as long as the upper surface and the lower surface of the measurement cell can be inverted. Examples of the inversion method include a method of rotating an axis parallel to the cell plane so as to draw an arc, and a method of rotating around an axis parallel to the cell plane, as shown by an arrow in FIG. 5A. After disposing the measurement cell 1 on the installation unit 101, the reversing unit 102 may release the holding of the measurement cell 1. After the reversing unit 102 releases the holding of the measurement cell 1, as shown in FIG. 5C, the reversing unit 102 may move to a position before the reversal of the measurement cell 1 in preparation for the next operation. Good.

反転部１０２によって反転した測定セル１の中では、トラップ部に捕捉されていた液滴が浮上して上方の液滴ガイド部に誘導され、液滴ガイド部のウエルに従って配列する。この配列に要する時間を短縮するために、設置部１０１と一体化した振動部を設置し、測定セル１に振動を印加してもよい。印加する振動の種類や振幅は、液滴の配列時間が短縮できる振動であれば特に制限はない。振動の種類の例として、測定セル１の法線方向に平行な振動や、測定セル１の平面方向に平行な振動が挙げられる。また、振動の周波数は、液滴径やオイルの物性に応じて１〜１００Ｈｚの範囲、より好適には１０〜５０Ｈｚの範囲から選択することが可能である。また、測定セル１中のオイルを、例えば３５〜６０℃の温度範囲で加熱することにより、オイルの粘度を低減して、液滴の配列時間をさらに短縮することが可能である。従って、設置部１０１は、振動印加時に測定セル１を加熱する機構を有していても良い。   In the measurement cell 1 inverted by the inversion unit 102, the droplets trapped in the trap unit float and are guided to the upper droplet guide unit, and are arranged according to the wells of the droplet guide unit. In order to shorten the time required for this arrangement, a vibrating unit integrated with the setting unit 101 may be installed, and vibration may be applied to the measurement cell 1. The type and amplitude of the applied vibration are not particularly limited as long as the vibration can shorten the arrangement time of the droplets. Examples of the types of vibration include vibration parallel to the normal direction of the measurement cell 1 and vibration parallel to the plane direction of the measurement cell 1. The frequency of the vibration can be selected from the range of 1 to 100 Hz, more preferably from the range of 10 to 50 Hz, depending on the droplet diameter and the physical properties of the oil. Further, by heating the oil in the measurement cell 1 in a temperature range of, for example, 35 to 60 ° C., the viscosity of the oil can be reduced, and the arrangement time of the droplets can be further reduced. Therefore, the installation unit 101 may have a mechanism for heating the measurement cell 1 when applying vibration.

［実施例と比較例］
以下、本発明の実施例、比較例を用いて本発明をより詳細に説明するが、本発明の技術的範囲はこれに限定されるものではない。 [Examples and Comparative Examples]
Hereinafter, the present invention will be described in more detail with reference to Examples and Comparative Examples of the present invention, but the technical scope of the present invention is not limited thereto.

図６Ａ〜６Ｃは、下記の実施例と比較例で使用した測定セルの構造を示す模式図である。図６Ａは、実施例の測定セルと比較例の測定セルの上面図である。図６Ｂは実施例１〜３で使用した測定セルの断面模式図、図６Ｃは比較例で使用した測定セルの断面模式図である。実施例１〜３の測定セルと比較例の測定セルの主な違いは、トラップ部２の有無である。実施例の測定セルにはトラップ部があるが、比較例の測定セルにはトラップ部が無い。   6A to 6C are schematic diagrams showing the structure of a measurement cell used in the following examples and comparative examples. FIG. 6A is a top view of the measurement cell of the example and the measurement cell of the comparative example. FIG. 6B is a schematic sectional view of the measurement cell used in Examples 1 to 3, and FIG. 6C is a schematic sectional view of the measurement cell used in Comparative Example. The main difference between the measurement cells of Examples 1 to 3 and the measurement cell of the comparative example is the presence or absence of the trap unit 2. The measuring cell of the example has a trap portion, but the measuring cell of the comparative example has no trap portion.

これらの測定セルは、スライドガラス（松浪硝子工業製S1111）と粘着シート（３Ｍ製9969）を積層して作製した。流路の幅は１ｍｍ、高さは１００μｍとした。実施例で使用した測定セルのトラップ部２の深さは７５μｍとした。液滴ガイド部３は光ナノインプリント法を用いて作製した。液滴ガイド部３を構成する材料として、光架橋性のポリウレタン（ダイセルサイテック社製EB8405）と光架橋剤（新中村化学製A-NPG）の混合物（１：２）を用いた。   These measurement cells were prepared by laminating a slide glass (S1111 manufactured by Matsunami Glass Industry) and an adhesive sheet (9969 manufactured by 3M). The width of the channel was 1 mm, and the height was 100 μm. The depth of the trap part 2 of the measuring cell used in the example was 75 μm. The droplet guide part 3 was produced by using a photo nanoimprint method. A mixture (1: 2) of a photocrosslinkable polyurethane (EB8405 manufactured by Daicel Cytec) and a photocrosslinking agent (A-NPG manufactured by Shin-Nakamura Chemical Co., Ltd.) was used as a material constituting the liquid droplet guide portion 3.

図７Ａ，７Ｂは、実施例の測定セルと比較例の測定セルの液滴ガイド部の構造を示す模式図である。図７Ａは液滴ガイド部の平面模式図、図７ＢはそのＢ−Ｂ断面模式図である。ウエル３１は正方配列しており、ウエル間の最短ピッチは４０μｍであった。個々のウエル３１は、直径が２０μｍ、深さが１μｍであった。ウエルの総数は約５×１０4個である。 FIGS. 7A and 7B are schematic diagrams illustrating the structures of the liquid drop guide portions of the measurement cell of the example and the measurement cell of the comparative example. FIG. 7A is a schematic plan view of the droplet guide section, and FIG. 7B is a schematic cross-sectional view taken along line BB. The wells 31 were arranged in a square, and the shortest pitch between the wells was 40 μm. Each well 31 had a diameter of 20 μm and a depth of 1 μm. The total number of wells is about 5 × 10 4 .

実施例と比較例のいずれにおいても、RainDance社製のRainDrop Systemを用いて作製した液滴（粒径２０μｍ）を用い、オイルとしてRainDance社製のキャリアオイルを用いた。それぞれの実施例、比較例において、オイル上に浮遊した液滴（０．１μｌ）をオイル（１００μｌ）に添加し、混合液を調製した。混合液中においても、液滴はオイル上に浮遊した。   In each of the examples and comparative examples, droplets (particle diameter: 20 μm) prepared by using a RainDrop System manufactured by RainDance were used, and a carrier oil manufactured by RainDance was used as an oil. In each of Examples and Comparative Examples, droplets (0.1 μl) suspended on oil were added to oil (100 μl) to prepare mixed liquids. Even in the mixture, the droplets floated on the oil.

＜実施例１＞
液滴ガイド部３が下面となるように設置した測定セルの試料注入口４から、調製した混合液を全量注入し、測定セルに入りきらないオイルを排出口６から排出した。その後、試料注入口４と排出口６を粘着テープ（日東電工社製ニトフロン粘着テープ903）で塞ぎ、オイルの蒸発と液滴の流出を防止した。その後、液滴ガイド部３が上面となるように測定セルを反転した。１時間放置後に光学顕微鏡で液滴を観察したところ、液滴はほとんどウエルに捕捉されていなかった。一方、一晩放置後、光学顕微鏡を用いて液滴を観察したところ、液滴は、ウエルに従い規則配列していた。また、光学顕微鏡像から液滴ガイド部に存在する液滴の数を求めた結果、４×１０4個の液滴が捕捉されていることが分かった。 <Example 1>
The whole prepared liquid mixture was injected from the sample injection port 4 of the measurement cell in which the droplet guide section 3 was set to the lower surface, and the oil that could not enter the measurement cell was discharged from the discharge port 6. Thereafter, the sample inlet 4 and the outlet 6 were closed with an adhesive tape (Nitoflon adhesive tape 903 manufactured by Nitto Denko Corporation) to prevent oil evaporation and outflow of droplets. After that, the measurement cell was inverted so that the droplet guide section 3 was on the upper surface. When the droplet was observed with an optical microscope after being left for 1 hour, the droplet was hardly captured by the well. On the other hand, after standing overnight, the droplets were observed using an optical microscope. As a result, the droplets were regularly arranged according to the wells. Further, as a result of obtaining the number of droplets existing in the droplet guide portion from the optical microscope image, it was found that 4 × 10 4 droplets were captured.

比較例１では、実施例１と同様の操作を行い、試料注入口４と排出口６を粘着テープで塞ぎ、オイルの蒸発と液滴の流出を防止した。その後、液滴ガイド部３が上面となるように測定セルを保持し、一晩放置した。光学顕微鏡を用いて液滴を観察したところ、液滴は、ウエルに従い規則配列していた。また、光学顕微鏡像から液滴ガイド部に存在する液滴の数を求めた結果、１×１０4個の液滴が捕捉されていることがわかった。
In Comparative Example 1, the same operation as in Example 1 was performed, and the sample inlet 4 and the outlet 6 were closed with an adhesive tape to prevent oil evaporation and outflow of droplets. Thereafter, the measurement cell was held such that the droplet guide unit 3 was on the upper surface, and left overnight. When the droplets were observed using an optical microscope, the droplets were regularly arranged according to the wells. Further, as a result of obtaining the number of droplets existing in the droplet guide portion from the optical microscope image, it was found that 1 × 10 4 droplets were captured.

＜実施例２＞
実施例１と同様の操作を行い、試料注入口４と排出口６を粘着テープで塞ぎ、オイルの蒸発と液滴の流出を防止した。その後、液滴ガイド部が上面となるように測定セルを反転し、ブロックバスシェーカ（アズワン製ブロックバスシェーカMyBL-100CS）を用いて測定セルに振動を印加した。振動の振動数は２５Ｈｚとし、振動幅は２ｍｍとした。図８Ａは測定セルを反転した直後の液滴ガイド部の光学顕微鏡像、図８Ｂは振動を６０分印加した後の液滴ガイド部の光学顕微鏡像である。この図から、振動印加により、ウエルに従い液滴が迅速に配列することが分かった。 <Example 2>
The same operation as in Example 1 was performed, and the sample inlet 4 and the outlet 6 were closed with an adhesive tape to prevent oil evaporation and outflow of droplets. Thereafter, the measurement cell was inverted so that the liquid drop guide portion was on the upper surface, and vibration was applied to the measurement cell using a block bath shaker (My One BL-100CS, a block bath shaker manufactured by As One Corporation). The vibration frequency was 25 Hz, and the vibration width was 2 mm. FIG. 8A is an optical microscope image of the droplet guide section immediately after the measurement cell is inverted, and FIG. 8B is an optical microscope image of the droplet guide section after applying vibration for 60 minutes. From this figure, it was found that the droplets were quickly arranged according to the wells by the application of the vibration.

＜実施例３＞
ＥＧＦＲ遺伝子（０．４μＭ）とＤＮＡインターカレータ（EvaGreen、０．８μＭ）とを含む液滴をRainDance社製のRainDrop Systemを用いて作製した。その後、実施例２と同じ手順に従い、測定セル中で液滴を配列させた。 <Example 3>
Droplets containing the EGFR gene (0.4 μM) and a DNA intercalator (EvaGreen, 0.8 μM) were prepared using the RainDance RainDrop System. Thereafter, the droplets were arranged in the measurement cell according to the same procedure as in Example 2.

次に、配列した液滴の蛍光像を観察した。図９は、測定セル中の配列液滴の蛍光像を観察した計測システムの概略図である。測定セル１は測定セル設置部２０８に設置されている。測定セル設置部２０８は、図４に示した液滴トラップ装置の設置部１０１であってもよい。すなわち、ｄｄＰＣＲ検出装置を構成する計測システムを、液滴トラップ装置と一体化して構成してもよい。   Next, the fluorescent images of the arranged droplets were observed. FIG. 9 is a schematic diagram of a measurement system that observes a fluorescent image of arrayed droplets in a measurement cell. The measurement cell 1 is installed in the measurement cell installation section 208. The measurement cell installation section 208 may be the installation section 101 of the droplet trap device shown in FIG. That is, the measurement system constituting the ddPCR detection device may be integrated with the droplet trap device.

本観察では、光源２０１から出た光をダイクロイックミラー２０２で反射させ、対物レンズ２０３を通して測定セル１を照射した。光照射により液滴ガイド部のウエルに配列した液滴から発せられた蛍光は、対物レンズ２０３、ダイクロイックミラー２０２、結像レンズ２０４、ロングパスフィルター２０５、バンドパスフィルター２０６を通過し、撮像装置２０７の受光素子表面で結像した。この場合、ダイクロイックミラー２０２と対物レンズ２０３は、光源２０１からの出射光を測定セル設置部２０８に配置された測定セル１に光照射する照射光学系を構成する。また、対物レンズ２０３、ダイクロイックミラー２０２、結像レンズ２０４、ロングパスフィルター２０５、バンドパスフィルター２０６は、測定セル１の複数のウエルから発生された蛍光を撮像装置２０７に結像する結像光学系を構成する。図１０は、図９に示した計測システムを用いて測定セル１中の配列液滴の蛍光像を測定した結果を示す図である。図１０から、図９の光学システムを用いて、配列した液滴の蛍光像が観察できることが分かった。   In the main observation, light emitted from the light source 201 was reflected by the dichroic mirror 202, and the measurement cell 1 was irradiated through the objective lens 203. The fluorescent light emitted from the droplets arranged in the wells of the droplet guide portion by light irradiation passes through the objective lens 203, the dichroic mirror 202, the imaging lens 204, the long-pass filter 205, and the band-pass filter 206. An image was formed on the surface of the light receiving element. In this case, the dichroic mirror 202 and the objective lens 203 constitute an irradiation optical system that irradiates the light emitted from the light source 201 to the measurement cell 1 arranged in the measurement cell installation section 208. The objective lens 203, the dichroic mirror 202, the imaging lens 204, the long-pass filter 205, and the band-pass filter 206 form an imaging optical system that images fluorescence generated from a plurality of wells of the measurement cell 1 on the imaging device 207. Constitute. FIG. 10 is a diagram showing the result of measuring the fluorescence image of the arrayed droplets in the measurement cell 1 using the measurement system shown in FIG. From FIG. 10, it was found that a fluorescent image of the arranged droplets can be observed using the optical system of FIG.

実施例１と比較例１の比較から、実施例１の測定セルの液滴ガイド部に存在する液滴の数が、比較例１に比べて４倍程度多いことが分かった。以上から、トラップ部を有する測定セルを用いると、トラップ部の無い測定セルに比較して液滴の捕捉率を大幅に向上できることが分かった。また、実施例１と実施例２の比較から、測定セルに振動を印加することによって、液滴の配列時間が短縮できることが分かった。また、実施例３から、本実施例の測定セル中で配列させた液滴の蛍光像が観察できることが分かった。   From a comparison between Example 1 and Comparative Example 1, it was found that the number of droplets present in the droplet guide portion of the measurement cell of Example 1 was about four times larger than that in Comparative Example 1. From the above, it was found that the use of the measurement cell having the trap portion can significantly improve the droplet capture rate as compared with the measurement cell having no trap portion. In addition, from a comparison between Example 1 and Example 2, it was found that by applying vibration to the measurement cell, the arrangement time of droplets can be reduced. In addition, it was found from Example 3 that a fluorescent image of the droplets arranged in the measurement cell of the present example could be observed.

なお、本発明は上記した実施例に限定されるものではなく、様々な変形例が含まれる。例えば、上記した実施例は本発明を分かりやすく説明するために詳細に説明したものであり、必ずしも説明した全ての構成を備えるものに限定されるものではない。また、ある実施例の構成の一部を他の実施例の構成に置き換えることが可能であり、また、ある実施例の構成に他の実施例の構成を加えることも可能である。また、各実施例の構成の一部について、他の構成の追加・削除・置換をすることが可能である。   Note that the present invention is not limited to the above-described embodiment, and includes various modifications. For example, the above-described embodiments have been described in detail in order to explain the present invention in an easy-to-understand manner, and are not necessarily limited to those having all the configurations described above. Further, a part of the configuration of one embodiment can be replaced with the configuration of another embodiment, and the configuration of one embodiment can be added to the configuration of another embodiment. Also, for a part of the configuration of each embodiment, it is possible to add, delete, or replace another configuration.

１ 測定セル
２ トラップ部
３ 液滴ガイド部
４ 試料注入口
５ 流路
６ 排出口
７ 蓋部材
８ 筐体
１１ 液滴
１２ オイル
３１ ウエル
１００ 液滴トラップ装置
１０１ 設置部
１０２ 反転部
１０３ 制御部
２０１ 光源
２０２ ダイクロイックミラー
２０３ 対物レンズ
２０４ 結像レンズ
２０７ 撮像装置 DESCRIPTION OF SYMBOLS 1 Measurement cell 2 Trap part 3 Droplet guide part 4 Sample injection port 5 Flow path 6 Outlet 7 Cover member 8 Case 11 Droplet 12 Oil 31 Well 100 Droplet trap device 101 Installation part 102 Inversion part 103 Control part 201 Light source 202 Dichroic mirror 203 Objective lens 204 Imaging lens 207 Imaging device

Claims (11)

内部空間の一方の内壁面に複数のウエルを有する液滴ガイド部が設けられ、前記一方の内壁面に対向する他方の内壁面に、浮遊する液滴を捕捉する窪み部を有するトラップ部が設けられた測定セル内に、前記トラップ部を上に前記液滴ガイド部を下にした状態で複数の液滴とオイルとを含む流体を流し、前記トラップ部に前記複数の液滴をトラップする工程と、
前記測定セルを反転させ、前記トラップ部にトラップされた前記複数の液滴を前記液滴ガイド部の前記複数のウエルに捕捉させる工程と、
を有し、
前記トラップ部は、前記液滴ガイド部に対向する領域の略全面を占める液滴のトラップ方法。 A droplet guide portion having a plurality of wells is provided on one inner wall surface of the internal space, and a trap portion having a concave portion for capturing floating droplets is provided on the other inner wall surface facing the one inner wall surface. Flowing a fluid containing a plurality of droplets and oil in a state where the trap portion is set up and the droplet guide portion is set down, and trapping the plurality of droplets in the trap portion. When,
Inverting the measurement cell, and capturing the plurality of droplets trapped in the trap portion in the plurality of wells of the droplet guide portion,
Have a,
A method for trapping a droplet, wherein the trap portion occupies substantially the entire surface of a region facing the droplet guide portion . 前記測定セルを反転した後に前記測定セルを振動させる、請求項１に記載の液滴のトラップ方法。   The method according to claim 1, wherein the measuring cell is vibrated after the measuring cell is inverted. 前記測定セル内に前記流体を流したのち、前記測定セルに前記流体を注入した注入口及び前記測定セルから前記流体が排出された排出口を塞ぐ、請求項１に記載の液滴のトラップ方法。   2. The droplet trapping method according to claim 1, wherein, after flowing the fluid into the measurement cell, an inlet for injecting the fluid into the measurement cell and an outlet from which the fluid is discharged from the measurement cell are closed. . 筐体の外表面に開口した試料注入口及び排出口と、
前記試料注入口と前記排出口とを結ぶ前記筐体の内部に設けられた流路と、
前記流路の途中に設けられ前記筐体の外表面に平行な方向に広がりを持つ内部空間と、
前記内部空間の一方の内壁面に設けられた複数のウエルを有する液滴ガイド部と、
前記内部空間の他方の内壁面に設けられた浮遊する液滴を捕捉する窪み部を有し、前記液滴ガイド部に対向する領域の略全面を占めるトラップ部と、
を有する測定セル。 A sample inlet and an outlet opening on the outer surface of the housing,
A flow path provided inside the housing connecting the sample inlet and the outlet,
An internal space provided in the middle of the flow path and extending in a direction parallel to the outer surface of the housing,
A droplet guide portion having a plurality of wells provided on one inner wall surface of the internal space,
A trap portion which occupies almost the entire have a recess for catching droplets that float provided on the other inner wall surface of the interior space, facing the droplet guide region,
A measurement cell having: Ｄd［μｍ］を前記液滴の直径の平均、Ｄw［μｍ］を前記ウエルの最大径の平均、ｄ［μｍ］を前記ウエルの平均深さとするとき、下記（１）〜（３）の関係を満たす、請求項４に記載の測定セル。
Ｄd＜５０μｍ （１）
０．５Ｄd＜Ｄw＜１．２Ｄd （２）
０．０１≦ｄ／Ｄw≦１ （３） When D d [μm] is the average of the diameter of the droplet, D w [μm] is the average of the maximum diameter of the well, and d [μm] is the average depth of the well, the following (1) to (3) The measuring cell according to claim 4, which satisfies the following relationship:
D d <50 μm (1)
0.5D d <D w <1.2D d (2)
0.01 ≦ d / D w ≦ 1 (3) 前記トラップ部が流路面から５μｍ以上窪んだ単数又は複数の領域を有する、請求項４に記載の測定セル。   The measurement cell according to claim 4, wherein the trap unit has one or more regions depressed by 5 μm or more from a flow path surface. 前記液滴ガイド部と前記測定セルの外周とに挟まれた前記筐体の部分の波長４５０〜７５０ｎｍにおける光透過率が７０％以上である、請求項４に記載の測定セル。   The measurement cell according to claim 4, wherein a light transmittance at a wavelength of 450 to 750 nm of a portion of the housing sandwiched between the droplet guide portion and an outer periphery of the measurement cell is 70% or more. 前記トラップ部と前記測定セルの外周とに挟まれた前記筐体の部分の波長４５０〜７５０ｎｍにおける光透過率が７０％以上である、請求項４に記載の測定セル。   The measurement cell according to claim 4, wherein a light transmittance at a wavelength of 450 to 750 nm of a portion of the housing sandwiched between the trap portion and an outer periphery of the measurement cell is 70% or more. 測定セルに液滴を捕捉させるための装置であって、
内部空間の内壁面に複数のウエルを有する液滴ガイド部と浮遊する液滴を捕捉する窪み部を有するトラップ部とが対面して設けられた測定セルを保持し、反転させる機構を有する反転部と、
前記反転部によって反転された前記測定セルを配置する設置部と、
前記反転部による前記測定セルの反転タイミングを制御する制御部と、
を有し、
前記トラップ部は、前記液滴ガイド部に対向する領域の略全面を占める液滴トラップ装置。 An apparatus for capturing a droplet in a measurement cell,
A reversing unit having a mechanism for holding and reversing a measuring cell provided with a droplet guide unit having a plurality of wells on an inner wall surface of an internal space and a trap unit having a recess for capturing floating droplets When,
An installation unit for arranging the measurement cell inverted by the inversion unit,
A control unit that controls the inversion timing of the measurement cell by the inversion unit,
Have a,
The droplet trap device , wherein the trap unit occupies substantially the entire surface facing the droplet guide unit . 前記測定セルを振動させる振動部が前記設置部に一体化されている、請求項９に記載の液滴トラップ装置。   The droplet trap device according to claim 9, wherein a vibration unit that vibrates the measurement cell is integrated with the installation unit. 前記設置部の上方に、光源、撮像装置、前記光源からの出射光を前記設置部に配置される前記測定セルに光照射する照射光学系、前記測定セルの前記複数のウエルから発生された蛍光を前記撮像装置に結像する結像光学系を含む計測システムを有する、請求項９に記載の液滴トラップ装置。   Above the installation section, a light source, an imaging device, an irradiation optical system that irradiates light emitted from the light source to the measurement cell arranged in the installation section, and fluorescence generated from the plurality of wells of the measurement cell. The droplet trap device according to claim 9, further comprising a measurement system including an imaging optical system that forms an image on the imaging device.
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