JP6120440B2 - Microchamber and liquid mixing method - Google Patents
Microchamber and liquid mixing method Download PDFInfo
- Publication number
- JP6120440B2 JP6120440B2 JP2013095009A JP2013095009A JP6120440B2 JP 6120440 B2 JP6120440 B2 JP 6120440B2 JP 2013095009 A JP2013095009 A JP 2013095009A JP 2013095009 A JP2013095009 A JP 2013095009A JP 6120440 B2 JP6120440 B2 JP 6120440B2
- Authority
- JP
- Japan
- Prior art keywords
- liquid
- microchamber
- pillars
- mixing
- pillar
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired - Fee Related
Links
- 239000007788 liquid Substances 0.000 title claims description 216
- 238000000034 method Methods 0.000 title claims description 31
- 239000012530 fluid Substances 0.000 claims description 25
- 235000012489 doughnuts Nutrition 0.000 claims description 6
- 239000000243 solution Substances 0.000 description 33
- MHMNJMPURVTYEJ-UHFFFAOYSA-N fluorescein-5-isothiocyanate Chemical compound O1C(=O)C2=CC(N=C=S)=CC=C2C21C1=CC=C(O)C=C1OC1=CC(O)=CC=C21 MHMNJMPURVTYEJ-UHFFFAOYSA-N 0.000 description 16
- 239000008363 phosphate buffer Substances 0.000 description 16
- 239000000203 mixture Substances 0.000 description 8
- 238000010586 diagram Methods 0.000 description 7
- 239000000758 substrate Substances 0.000 description 6
- 239000003566 sealing material Substances 0.000 description 5
- 238000002474 experimental method Methods 0.000 description 4
- 238000011084 recovery Methods 0.000 description 4
- 239000000523 sample Substances 0.000 description 4
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 description 3
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 3
- 230000014759 maintenance of location Effects 0.000 description 3
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 3
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 description 2
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 2
- ZMXDDKWLCZADIW-UHFFFAOYSA-N N,N-Dimethylformamide Chemical compound CN(C)C=O ZMXDDKWLCZADIW-UHFFFAOYSA-N 0.000 description 2
- WYURNTSHIVDZCO-UHFFFAOYSA-N Tetrahydrofuran Chemical compound C1CCOC1 WYURNTSHIVDZCO-UHFFFAOYSA-N 0.000 description 2
- 238000006243 chemical reaction Methods 0.000 description 2
- 238000011156 evaluation Methods 0.000 description 2
- 238000002032 lab-on-a-chip Methods 0.000 description 2
- 239000003960 organic solvent Substances 0.000 description 2
- 230000002093 peripheral effect Effects 0.000 description 2
- 238000012545 processing Methods 0.000 description 2
- 230000000717 retained effect Effects 0.000 description 2
- RYHBNJHYFVUHQT-UHFFFAOYSA-N 1,4-Dioxane Chemical compound C1COCCO1 RYHBNJHYFVUHQT-UHFFFAOYSA-N 0.000 description 1
- SECXISVLQFMRJM-UHFFFAOYSA-N N-Methylpyrrolidone Chemical compound CN1CCCC1=O SECXISVLQFMRJM-UHFFFAOYSA-N 0.000 description 1
- 150000001298 alcohols Chemical class 0.000 description 1
- 239000007864 aqueous solution Substances 0.000 description 1
- 230000000994 depressogenic effect Effects 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 230000008020 evaporation Effects 0.000 description 1
- 238000001704 evaporation Methods 0.000 description 1
- 230000005484 gravity Effects 0.000 description 1
- 238000012423 maintenance Methods 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 239000011259 mixed solution Substances 0.000 description 1
- 230000003287 optical effect Effects 0.000 description 1
- 239000013307 optical fiber Substances 0.000 description 1
- 238000007789 sealing Methods 0.000 description 1
- 230000035939 shock Effects 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 238000011282 treatment Methods 0.000 description 1
Landscapes
- Chemical & Material Sciences (AREA)
- General Health & Medical Sciences (AREA)
- Immunology (AREA)
- Micromachines (AREA)
- Pathology (AREA)
- Analytical Chemistry (AREA)
- Physics & Mathematics (AREA)
- Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- Physical Or Chemical Processes And Apparatus (AREA)
- General Physics & Mathematics (AREA)
- Life Sciences & Earth Sciences (AREA)
- Automatic Analysis And Handling Materials Therefor (AREA)
- Microelectronics & Electronic Packaging (AREA)
- Engineering & Computer Science (AREA)
- Computer Hardware Design (AREA)
- Organic Chemistry (AREA)
- Chemical Kinetics & Catalysis (AREA)
Description
本発明は、マイクロチャンバー及び液体の混合方法に関する。 The present invention relates to a microchamber and a liquid mixing method.
μ-TASやLab-on-a-chipと称される一枚の基板上（マイクロチップ）で、２種類以上のサンプルと液体試料を混合させ、簡便・迅速に検知を行う様々な方法が開示されているが、実用化例は少ない。使い捨て用のマイクロチップの場合、機能が低く、反応や混合の制御は外部装置に依る場合が多いが、一方で、繰り返し使用するマイクロチップの場合、高機能であるがマイクロチップ自体が高価、大型、複雑になる問題がある。 Disclosed are various methods that allow simple and quick detection by mixing two or more samples and a liquid sample on a single substrate (microchip) called μ-TAS or Lab-on-a-chip. However, there are few practical examples. In the case of a disposable microchip, the function is low, and control of reaction and mixing often depends on an external device. On the other hand, in the case of a microchip that is used repeatedly, the microchip itself is expensive but large in size. There is a complicated problem.
そこで、複雑な操作や制御を要せずに、マイクロチップ上で２種類以上の液体を簡易に混合できる手法が望まれている。従来技術として、気体導出用流路を混合チャンバーと平行に備えて、流体を内部の流体と加速・接触させる事により簡易に混合する方法（特許文献１）、混合槽の底部の中央からずれた位置に、２種類の液体が流動する微細流路を接続することで効率良く混合させる方法（特許文献２）、深溝な流路構造を設けて流体同士の接触界面を層流下で大きく確保する事で、容易で速やかに混合できる方法(特許文献３)が公開されている。 Therefore, there is a demand for a technique that can easily mix two or more kinds of liquids on a microchip without requiring complicated operations and controls. As a prior art, a method for easily mixing by providing a gas outlet channel parallel to the mixing chamber and accelerating / contacting the fluid with the fluid inside (Patent Document 1), shifted from the center of the bottom of the mixing tank A method of efficiently mixing by connecting a fine flow path in which two kinds of liquids flow at a position (Patent Document 2), and providing a deep flow path structure to ensure a large contact interface between fluids under laminar flow Thus, a method (Patent Document 3) that allows easy and rapid mixing is disclosed.
特許文献１では、混合チャンバー内の流体１に向けて流体２を加速・接触させる方法を公開しており、特別な装置を用いず単純な方法で混合が可能であるが、気体導出部から混合チャンバー内へ加圧する必要があり、また、混合後の流体はチャンバー内に留まるため、混合後に複数の反応や処理を連続して行う事ができない。特許文献２においても、簡易な構成で２種類以上の液体の混合を効率良く行う方法を公開しているが、混合液を次の操作で扱う方法は説明されておらず、連続処理が必要なマイクロチップには馴染まない。また、液がチャンバー内に保持されているときに、マイクロチップを上下や左右に動かすと、チャンバー内に保持されている液体は漏出するリスクが高い。特許文献３では、複数の流体を接触界面が大きくなるように層流を形成できる合流流路デバイスを公開しており、各流体の量的制御の容易性を達成しているが、其々の流体を流し続ける或いは２種類の同量の液体を適切に接触させる必要があり、混合時の操作方法は複雑になる。 Patent Document 1 discloses a method of accelerating / contacting the fluid 2 toward the fluid 1 in the mixing chamber, and mixing is possible by a simple method without using a special device. It is necessary to pressurize the chamber, and the mixed fluid stays in the chamber. Therefore, a plurality of reactions and treatments cannot be performed continuously after mixing. Patent Document 2 also discloses a method for efficiently mixing two or more types of liquids with a simple configuration. However, a method for handling a mixed liquid in the next operation is not described, and continuous processing is necessary. Not familiar with microchips. Further, if the microchip is moved up and down or left and right while the liquid is held in the chamber, the liquid held in the chamber has a high risk of leakage. Patent Document 3 discloses a confluence channel device that can form a laminar flow so that a contact interface of a plurality of fluids becomes large, and achieves ease of quantitative control of each fluid. It is necessary to keep the fluid flowing or to make two kinds of the same amount of liquid properly contact with each other, and the operation method at the time of mixing becomes complicated.
このように、マイクロチップ上で簡易に混合する方法が公開されているが、何れも混合前の液体の保持、扱いやすさ、必要な液量、混合液を次の操作で扱う自由度において制限があり、実用化に向けて課題がある。 As described above, methods for easy mixing on a microchip have been disclosed, but all have limitations on the retention of liquid before mixing, ease of handling, the required amount of liquid, and the degree of freedom to handle the mixed liquid in the next operation. There is a problem for practical application.
本発明は、混合前の液体の保持、扱いやすさ、必要な液量、混合液を次の操作で扱う自由度において制限のない実用的な液体の混合技術を提供することを目的とする。 An object of the present invention is to provide a practical liquid mixing technique that is not limited in terms of retention of liquid before mixing, ease of handling, required amount of liquid, and freedom of handling the mixed liquid in the following operations.
上記課題に鑑みて、本発明者は、マイクロ空間中での液体試料の界面張力と流力（慣性力）を効果的に活用して、液体を簡易な構造のマイクロチャンバーに保持する事により、２種類以上の液体を簡便で効率良く混合できることを見出した。
本発明は、以下のマイクロチャンバー及び液体の混合方法を提供する。
項１． 液体流入口５及び液体流出口６と接続したマイクロチャンバーであって、前記マイクロチャンバーは液体保持部、中空筒状の流体混合部を備え、液体保持部の底面２及び流体混合部の底面３の両方に交わる位置に複数のピラー１を配置する、マイクロチャンバー。
項２． ピラーの中心軸は液体保持部の底面２の略外周上にある項１に記載のマイクロチャンバー。
項３． ピラーが２、３又は４個配置されている、項１又は２に記載のマイクロチャンバー。
項４． ピラーが３個配置されている、項３に記載のマイクロチャンバー。
項５． ピラーの１つが液体流入口５と対向する位置に配置されている、項１〜４のいずれかに記載のマイクロチャンバー。
項６． 隣接するマイクロチャンバーの液体流入口５と液体流出口６とを接続することで、複数のマイクロチャンバーを連結させてなる、項１〜５のいずれかに記載のマイクロチャンバー。
項７． 前記ピラーは円筒状である、項１〜６のいずれかに記載のマイクロチャンバー。
項８． 前記液体混合部はドーナツ状である、項１〜７のいずれかに記載のマイクロチャンバー。
項９． 前記液体保持部は円筒状であり、かつ、液体保持部は液体混合部よりも低い位置にある、項１〜８のいずれかに記載のマイクロチャンバー。
項１０． 項１〜９のいずれかに記載のマイクロチャンバーを用いた液体の混合方法であって、液体保持部に第１の液体を導入する工程、液体流入口５から第２の液体をマイクロチャンバー内に流入し、液体流出口６から第１の液体と第２の液体の混合液体を流出させる工程を含むことを特徴とする液体の混合方法。
In view of the above problems, the present inventor effectively utilizes the interfacial tension and flow force (inertial force) of a liquid sample in a micro space, and holds the liquid in a micro chamber with a simple structure, It has been found that two or more kinds of liquids can be easily and efficiently mixed.
The present invention provides the following microchamber and liquid mixing method.
Item 1. A microchamber connected to the liquid inlet 5 and the liquid outlet 6, the microchamber comprising a liquid holding portion and a hollow cylindrical fluid mixing portion; and a bottom surface 2 of the liquid holding portion and a bottom surface 3 of the fluid mixing portion A microchamber in which a plurality of pillars 1 are arranged at positions where both intersect.
Item 2. Item 2. The microchamber according to Item 1, wherein the pillar has a central axis substantially on the outer periphery of the bottom surface 2 of the liquid holding portion.
Item 3. Item 3. The microchamber according to Item 1 or 2, wherein 2, 3 or 4 pillars are arranged.
Item 4. Item 4. The microchamber according to Item 3, wherein three pillars are arranged.
Item 5. Item 5. The microchamber according to any one of Items 1 to 4, wherein one of the pillars is disposed at a position facing the liquid inlet 5.
Item 6. Item 6. The microchamber according to any one of Items 1 to 5, wherein a plurality of microchambers are connected by connecting the liquid inlet 5 and the liquid outlet 6 of adjacent microchambers.
Item 7. Item 7. The microchamber according to any one of Items 1 to 6, wherein the pillar is cylindrical.
Item 8. Item 8. The microchamber according to any one of Items 1 to 7, wherein the liquid mixing part has a donut shape.
Item 9. Item 9. The microchamber according to any one of Items 1 to 8, wherein the liquid holding unit is cylindrical, and the liquid holding unit is at a lower position than the liquid mixing unit.
Item 10. Item 10. A liquid mixing method using the microchamber according to any one of Items 1 to 9, wherein the step of introducing the first liquid into the liquid holding unit, and the second liquid from the liquid inlet 5 into the microchamber. A liquid mixing method comprising a step of flowing in and flowing out a mixed liquid of the first liquid and the second liquid from the liquid outlet 6.
本発明によれば、マイクロチャンバー内での２種類以上の液体の混合のための外部装置と煩雑な操作を必要とせず、液体保持部に保持される液体自身の界面張力と、マイクロチャンバー内に流動してくる液体の界面張力と流力（慣性力）を活用する事で、２種類以上の液体を自動的に混合する事が可能である。 According to the present invention, an external device for mixing two or more types of liquids in the microchamber and a complicated operation are not required, and the interfacial tension of the liquid itself held in the liquid holding unit, By utilizing the interfacial tension and flow force (inertial force) of the flowing liquid, it is possible to automatically mix two or more types of liquid.
ピラーの配置と窪みをマイクロチャンバー内で効果的に配置する事で、保持されている液体はマイクロチャンバー内に安定して強く留まり、混合後はマイクロチャンバーに留まらず自動的にマイクロ流路へ流動していくので、連続した処理が容易に可能である。 By arranging the pillars and depressions effectively in the microchamber, the retained liquid stays in the microchamber stably and strongly, and after mixing, it automatically flows to the microchannel instead of staying in the microchamber. Therefore, continuous processing is easily possible.
さらに、本発明のマイクロチャンバーは複数個を連結させる事が可能であり、２種類以上の液体を簡便に混合することが出来る。 Furthermore, a plurality of the microchambers of the present invention can be connected, and two or more kinds of liquids can be easily mixed.
１．ピラー
２．液体保持部の底面
３．流体混合部の底面
４．基板
５．液体流入口
６．液体流出口
７．ピラーの高さ
８．液体保持部の高さ
９．流体混合部及び筒状の空間の高さ
１０．ピラーの直径
１１．液体保持部の底面の直径
１２．流体混合部の外径
１３．流体混合部の幅（３の外径から２の半径を減じた長さ）
１４．液体保持部に保持されている液体
１５．液体流出口から流出する空気の方向
１６．液体流入口に流入する空気の方向
１７．液体保持部に第１の液体が充填されている場合のマイクロチャンバー内を流動する空気の方向
１８．第２の液体が最初に流入するマイクロチャンバー
１９．１８のマイクロチャンバーに接続されたマイクロチャンバー
２０．１９のマイクロチャンバーに接続されたマイクロチャンバー
1. Pillar 2. 2. Bottom surface of liquid holding unit 3. bottom surface of fluid mixing section Substrate 5. Liquid inlet 6. 6. Liquid outlet Pillar height8. 8. Liquid holding part height 9. Height of fluid mixing part and cylindrical space Pillar diameter11. 11. Diameter of bottom surface of liquid holding unit 12. outer diameter of fluid mixing section Width of fluid mixing part (length obtained by subtracting radius 2 from outer diameter 3)
14 14. Liquid held in the liquid holding part Direction of air flowing out from liquid outlet 16. Direction of air entering the liquid inlet 17. 17. Direction of air flowing in the micro chamber when the liquid holding portion is filled with the first liquid Microchamber connected to the microchamber of the microchamber 20.19 connected to the microchamber of the microchamber 19.18 into which the second liquid first flows
本発明では、液体試料の界面張力と流力（慣性力）を効果的に活用して、第１の液体を簡易な構造で保持する事により、２種類以上の液体を簡便で効率良く混合できるマイクロチャンバー及び液体の混合方法を提供する。 In the present invention, it is possible to easily and efficiently mix two or more kinds of liquids by effectively utilizing the interfacial tension and flow force (inertial force) of the liquid sample and holding the first liquid with a simple structure. A microchamber and a method for mixing liquids are provided.
本発明のマイクロチャンバーを用いれば、低い位置にある液体保持部に第１の液体を導入し、その後で液体流入口５から第２の液体を流すことで、２つの液体が混合されて液体流出口６から流出する。 If the microchamber of the present invention is used, the first liquid is introduced into the liquid holding portion located at the lower position, and then the second liquid is allowed to flow from the liquid inlet 5, whereby the two liquids are mixed and the liquid flow It flows out from the outlet 6.
液体保持部は、底面２が第１の液体と強い界面張力を有し、第１の液体を保持できる場合には、底面２と底面３は同じ高さでもよい。この場合、液体保持部は底面２から構成される。１つの好ましい実施形態では、液体保持部の底面２は液体混合物の底面３よりも低い位置にあり、この場合、液体保持部は筒状の形状を有し、液体保持部の上に筒状の空間が形成され、その周囲に中空筒状の液体混合部が形成される。液体保持部、液体保持部上の空間（中空筒状の液体混合部の内部の空間）を構成する筒状の形状としては、例えば円柱、楕円柱、三角柱、四角柱、五角柱、六角柱、八角柱などの任意の筒状の形状でよく、角柱状の場合、角の部分は丸くなり、液体が残留しないようになっていることが好ましい。液体混合部は中空部分(内周部分)及び外周部分はいずれも筒状である。液体混合部の外側（基板４側）の筒状の形状と内側の中空の筒状の形状は同一であっても異なっていてもよい。液体混合物の好ましい形状はドーナツ状の形状である。 When the bottom surface 2 has a strong interfacial tension with the first liquid and can hold the first liquid, the bottom surface 2 and the bottom surface 3 may have the same height. In this case, the liquid holding part is composed of the bottom surface 2. In one preferred embodiment, the bottom surface 2 of the liquid holding part is at a lower position than the bottom surface 3 of the liquid mixture, in which case the liquid holding part has a cylindrical shape and is cylindrical on the liquid holding part. A space is formed, and a hollow cylindrical liquid mixing portion is formed around the space. Examples of the cylindrical shape constituting the liquid holding unit and the space above the liquid holding unit (the space inside the hollow cylindrical liquid mixing unit) include, for example, a cylinder, an elliptical column, a triangular column, a quadrangular column, a pentagonal column, a hexagonal column, An arbitrary cylindrical shape such as an octagonal prism may be used. In the case of a prismatic shape, the corner portion is preferably rounded so that no liquid remains. The liquid mixing part has a hollow part (inner peripheral part) and an outer peripheral part both cylindrical. The cylindrical shape on the outer side (substrate 4 side) of the liquid mixing part and the hollow cylindrical shape on the inner side may be the same or different. The preferred shape of the liquid mixture is a donut shape.
液体保持部の第１の液体は、液体流入口５から供給される第２の液体と混合されて液体流出口６から流出する。 The first liquid in the liquid holding unit is mixed with the second liquid supplied from the liquid inlet 5 and flows out from the liquid outlet 6.
マイクロチャンバー内には複数のピラー１が配置され、第２の液体はこのピラーにより流れが乱され、効率的に第１の液体と混合される。ピラーの形状は上記と同様な筒状（角筒状、円筒状、楕円筒状など）が好ましく、円筒状が最も好ましい。 A plurality of pillars 1 are arranged in the microchamber, and the flow of the second liquid is disturbed by the pillars, and is efficiently mixed with the first liquid. The shape of the pillar is preferably a cylindrical shape (such as a rectangular tube shape, a cylindrical shape, or an elliptical cylinder shape) similar to the above, and a cylindrical shape is the most preferable.
マイクロチャンバーの上は、シーリングテープなどのシール材によりシールして、液体の蒸発や液体のマイクロチャンバー外への飛散を防止するのが好ましい。シール材の内面が第２の液体と接触する場合、第２の液体とシール材の界面張力を考慮して第２の液体の供給速度、液量を適切に調節する。第２の液体の供給速度、液量は、液体保持部の形状、液体保持部の内面と第２の液体の界面張力、液体混合部の底面３及び基板４側の側面と第２の液体の界面張力、液体混合部の形状、ピラーの本数と形状などにより影響を受けるため、マイクロチャンバー、第１の液体、第２の液体の組み合わせに応じて適切な値を決めることができる。このような値は、図面に示される実施形態を参考にして当業者であれば容易に決定できる。第１／第２の液体とマイクロチャンバー（液体混合部、液体保持部）、シール材との間の界面張力は、マイクロチャンバーとシール材の親水性／疎水性の程度により決められる。第１と第２の液体は、通常は水/水溶液であるが、水と混和する有機溶媒（エタノールなどの低級アルコール、DMSO、DMF、アセトン、THF、アセトニトリル、ジオキサン、N-メチルピロリドンなど）を含む含水溶液であってもよい。有機溶媒の種類と含量によっても界面張力は変化する。 The top of the microchamber is preferably sealed with a sealing material such as a sealing tape to prevent liquid evaporation and liquid scattering outside the microchamber. When the inner surface of the sealing material is in contact with the second liquid, the supply speed and amount of the second liquid are appropriately adjusted in consideration of the interfacial tension between the second liquid and the sealing material. The supply speed and amount of the second liquid are the shape of the liquid holding unit, the interfacial tension between the inner surface of the liquid holding unit and the second liquid, the bottom surface 3 of the liquid mixing unit and the side surface of the substrate 4 and the second liquid. Since it is influenced by the interfacial tension, the shape of the liquid mixing portion, the number and shape of pillars, etc., an appropriate value can be determined according to the combination of the microchamber, the first liquid, and the second liquid. Such values can be easily determined by those skilled in the art with reference to the embodiments shown in the drawings. The interfacial tension between the first / second liquid, the microchamber (liquid mixing part, liquid holding part), and the sealing material is determined by the degree of hydrophilicity / hydrophobicity of the microchamber and the sealing material. The first and second liquids are usually water / water solutions, but an organic solvent miscible with water (such as ethanol, lower alcohols, DMSO, DMF, acetone, THF, acetonitrile, dioxane, N-methylpyrrolidone, etc.) It may be an aqueous solution containing. The interfacial tension varies depending on the type and content of the organic solvent.
本発明のマイクロチャンバーの好ましい実施形態が図１に示されている。液体保持部の底面２とドーナツ状の流体混合部の底面３の中心及び液体保持部上の筒状の空間の中心は同軸状にあり、底面２は下方に位置する。液体保持部の上面(形状は底面２と同じ)と底面３を合わせると１つの面を形成する。図１では、液体保持部が円筒形、液体混合部はドーナツ状の形状のものが示されているが、これらは筒状の形状であれば任意の他の形状であってもよい。また、底面２は、底面３よりも下方にあるのが好ましいが、底面２と第１の液体の界面張力が十分強ければ、底面２と底面３が同じ高さでもよく、底面２が底面３よりも高い位置にあってもよい。 A preferred embodiment of the microchamber of the present invention is shown in FIG. The center of the bottom surface 2 of the liquid holding unit and the bottom surface 3 of the doughnut-shaped fluid mixing unit and the center of the cylindrical space on the liquid holding unit are coaxial, and the bottom surface 2 is positioned below. When the upper surface (the shape is the same as that of the bottom surface 2) and the bottom surface 3 of the liquid holding part are combined, one surface is formed. In FIG. 1, the liquid holding part has a cylindrical shape and the liquid mixing part has a donut shape, but any other shape may be used as long as it has a cylindrical shape. The bottom surface 2 is preferably located below the bottom surface 3. However, if the interface tension between the bottom surface 2 and the first liquid is sufficiently strong, the bottom surface 2 and the bottom surface 3 may be the same height, and the bottom surface 2 is the bottom surface 3. It may be in a higher position.
３個のピラーは図２に示すように中心が底面２の外周付近にあり、３つのピラーの中心は正三角形の頂点の位置関係にある。また、１つのピラーは液体流入口５に対向する位置にあると、流入口５から流入した第２の液体の流れがピラーにより乱され、それにより流体混合部内の第１の液体とより十分に混合されることになるので好ましい。 As shown in FIG. 2, the centers of the three pillars are in the vicinity of the outer periphery of the bottom surface 2, and the centers of the three pillars are in the positional relationship of the vertices of an equilateral triangle. Also, if one pillar is at a position facing the liquid inlet 5, the flow of the second liquid flowing in from the inlet 5 is disturbed by the pillar, and thereby the first liquid in the fluid mixing section is more fully affected. Since it will be mixed, it is preferable.
ピラー１の形状は筒状であればよく、好ましくは円筒状である。 The pillar 1 may have a cylindrical shape, preferably a cylindrical shape.
なお、底面２は円形、底面３はドーナツ状と記載したが、これはピラーがない状態での形状であり、実際にはピラーが存在するので、ピラーが存在する場合の形状は図２ｄ、図２ｃに記載される形状になる。図２ａはマイクロチャンバーの平面図であり、図２ｂは底面２とピラーの位置関係をわかりやすく示した図である。複数のピラーの中心軸は液体保持部の底面２の略外周上の対称な位置に配置されるのがよく、２つのピラーは直線状、３つのピラーは正三角形の各頂点、４つのピラーは正方形の各頂点、５つのピラーは正５角形の各頂点に配置するのがよい。ピラーの高さ７は、液体保持部の高さ８よりも高く、好ましくは（ピラーの高さ７）＝（液体保持部の高さ８＋流体混合部の高さ９）である。 Although the bottom surface 2 is described as a circle and the bottom surface 3 is described as a donut shape, this is a shape without a pillar, and since there is actually a pillar, the shape when a pillar is present is shown in FIGS. It becomes the shape described in 2c. FIG. 2a is a plan view of the microchamber, and FIG. 2b is a diagram showing the positional relationship between the bottom surface 2 and the pillars in an easy-to-understand manner. The central axes of the plurality of pillars are preferably arranged at symmetrical positions on the substantially outer periphery of the bottom surface 2 of the liquid holding part. Two pillars are linear, three pillars are vertices of equilateral triangles, and four pillars are Each vertex of the square and the five pillars are preferably arranged at each vertex of the regular pentagon. The height 7 of the pillar is higher than the height 8 of the liquid holding part, and preferably (the height 7 of the pillar) = (the height 8 of the liquid holding part + the height 9 of the fluid mixing part).
ピラーの数が多いほど界面張力が強く作用して液体が流動しにくくなる。図６から、ピラーの数が4以上になると混合後（図５-Ｖ）に、メニスカスで液がチャンバー内に残留し易くなるため、回収率が低下し、混合チャンバーとしての性能が低下する。ピラーが3個のときは回収率が比較的良好なため、ピラーの数は3が望ましい。 The greater the number of pillars, the stronger the interfacial tension acts, making it difficult for the liquid to flow. From FIG. 6, when the number of pillars is 4 or more, the liquid tends to remain in the chamber after mixing (FIG. 5-V), so that the recovery rate is lowered and the performance as a mixing chamber is lowered. When the number of pillars is 3, the recovery rate is relatively good, so the number of pillars is preferably 3.
図３は、マイクロチャンバーの液体保持部に第１の液体を充填した状態を示す。図３の矢印１５、１７は空気の流れる方向を示す。 FIG. 3 shows a state in which the liquid holding part of the microchamber is filled with the first liquid. Arrows 15 and 17 in FIG. 3 indicate the direction of air flow.
図４は、マイクロチャンバーを回転させて遠心力を働かせたときに、液体保持部に充填した第１の液体が流動するかを試験した結果を示す。図４に示すようにピラーの数が多いほど液体保持部における第１の液体の流動が抑制されることが明らかになった。液体の流動は、遠心力で行ってもよい。 FIG. 4 shows a result of testing whether the first liquid filled in the liquid holding portion flows when the micro chamber is rotated and centrifugal force is applied. As shown in FIG. 4, it has been clarified that the flow of the first liquid in the liquid holding portion is suppressed as the number of pillars is increased. The liquid may be flowed by centrifugal force.
第１の液体が第２の液体と混合して流動していく過程が図５に示されている。まず、マイクロチャンバーの流入口５から第２の液体が流入してくると（図５−Ｉ）、界面張力によって一番近傍にあるピラーに接触する（図５−II）。次に、他のピラーとも接触してマイクロチャンバー内が混合液によって満たされ（図５−III）、出口の液体流出口６へ混合液が押し出される（図５−IV）。マイクロチャンバー内の混合液は、表面張力によって途切れることなく出口のマイクロ流路へ流動していくため、マイクロチャンバー内に液は残留しない（図５−V）。 A process in which the first liquid is mixed with the second liquid and flows is shown in FIG. First, when the second liquid flows in from the inflow port 5 of the microchamber (FIG. 5-I), it contacts the nearest pillar by the interfacial tension (FIG. 5-II). Next, the microchamber is filled with the mixed liquid in contact with other pillars (FIG. 5-III), and the mixed liquid is pushed out to the liquid outlet 6 at the outlet (FIG. 5-IV). Since the liquid mixture in the microchamber flows to the microchannel at the outlet without interruption due to surface tension, no liquid remains in the microchamber (FIG. 5-V).
マイクロ空間では、液体自身の表面張力や固液界面に働く界面張力が液体の流動に、最も大きく作用する。仮に、重力が支配的に働くと、図1の２の面は窪んでおり、液体は流出しない。保持されている液体は表面張力があるので、図５-Ｖでは液体は流出する。しかし、図７に示されているようピラーとチャンバー壁面の距離が遠くなると、液体は残留し易くなる。図５−IIIから図５−Vの過程では、液体の流出と空気の流出が競合しており、液体の流出が優位になっている。 In the micro space, the surface tension of the liquid itself and the interfacial tension acting on the solid-liquid interface have the greatest effect on the flow of the liquid. If gravity works dominantly, the surface 2 in FIG. 1 is depressed and the liquid does not flow out. Since the retained liquid has surface tension, the liquid flows out in FIG. However, as the distance between the pillar and the chamber wall surface increases as shown in FIG. 7, the liquid tends to remain. In the process from FIG. 5-III to FIG. 5-V, the outflow of liquid and the outflow of air compete with each other, and the outflow of liquid is superior.
図６は、マイクロチャンバーにおける液体の混合状態を蛍光液を用いてピペッティングを用いた混合方法と比較して評価した結果を示す。蛍光測定は、マイクロチャンバーの先端から1mmの箇所に光学プローブ(No.4040(Spot dia. : 0.4 mm、日本板硝子株式会社))を当て、光ファイバー型蛍光検出器（FLE1100B、日本板硝子株式会社）にて蛍光強度を測定して実施した。 FIG. 6 shows the result of evaluating the mixed state of the liquid in the microchamber in comparison with a mixing method using pipetting using a fluorescent solution. Fluorescence measurement is performed by applying an optical probe (No. 4040 (Spot dia .: 0.4 mm, Nippon Sheet Glass Co., Ltd.)) to a location 1 mm from the tip of the microchamber and applying to an optical fiber type fluorescence detector (FLE1100B, Nippon Sheet Glass Co., Ltd.) The fluorescence intensity was measured.
図６の実験の詳細を以下に示す。 Details of the experiment of FIG. 6 are shown below.
＊ピペッティングで混合したとき
40nM FITC溶液10μL
0.1 M リン酸緩衝液(pH7.4)30μL
＊ピラー（半径が0.8mm）が２個のとき
40nM FITC溶液10.6μL
0.1 M リン酸緩衝液(pH7.4)31.8μL
＊ピラー（半径が0.8mm）が３個のとき
マイクロチャンバーに添加した液：40nM FITC溶液9.6μL,
送液した液:0.1 M リン酸緩衝液(pH7.4)28.8μL
＊ピラー（半径が0.8mm）が４個のとき
マイクロチャンバーに添加した液：40nM FITC溶液8.6μL,
送液した液:0.1 M リン酸緩衝液(pH7.4)25.8μL
＊ピラー（半径が0.4mm）が２個のとき
40nM FITC溶液12.1μL
0.1 M リン酸緩衝液(pH7.4)36.3μL
＊ピラー（半径が0.4mm）が３個のとき
マイクロチャンバーに添加した液：40nM FITC溶液11.8μL,
送液した液:0.1 M リン酸緩衝液(pH7.4)35.4μL
＊ピラー（半径が0.4mm）が４個のとき
マイクロチャンバーに添加した液：40nM FITC溶液11.6μL,
送液した液:0.1 M リン酸緩衝液(pH7.4)34.8μL
＊ピラー（半径が0.4mm）が５個のとき
マイクロチャンバーに添加した液：40nM FITC溶液11.3μL,
送液した液:0.1 M リン酸緩衝液(pH7.4)33.9μL
* When mixed by pipetting
10 nL of 40 nM FITC solution
30 μL of 0.1 M phosphate buffer (pH 7.4)
* When there are two pillars (radius 0.8mm)
40nM FITC solution 10.6μL
0.1 M phosphate buffer (pH 7.4) 31.8 μL
* When 3 pillars (radius 0.8mm) were added to the microchamber: 9.6μL of 40nM FITC solution,
Pumped solution: 28.8 μL of 0.1 M phosphate buffer (pH 7.4)
* When 4 pillars (with a radius of 0.8 mm) were added to the microchamber: 8.6 μL of 40 nM FITC solution,
Pumped solution: 25.8 μL of 0.1 M phosphate buffer (pH 7.4)
* When there are two pillars (radius 0.4mm)
40nM FITC solution 12.1μL
0.1 M phosphate buffer (pH 7.4) 36.3 μL
* When 3 pillars (with a radius of 0.4 mm) were added to the microchamber: 11.8 μL of 40 nM FITC solution,
Liquid sent: 0.1 M phosphate buffer (pH 7.4) 35.4 μL
* When 4 pillars (radius is 0.4mm), the solution added to the microchamber: 11.6μL of 40nM FITC solution,
Pumped solution: 34.8 μL of 0.1 M phosphate buffer (pH 7.4)
* When 5 pillars (0.4mm radius) were added to the microchamber: 10.3μL of 40nM FITC solution,
Pumped solution: 0.1 M phosphate buffer (pH 7.4) 33.9 μL
図６の結果から、この実験条件ではピラーの数は２又は３が優れていることが明らかになった。ピラー数がゼロの場合、回収率(蛍光強度)は優位であるが、液体がチャンバー内に保持される力が弱いため（図４）、第１の液体は液体保持部の底面２からすぐに漏出する。実際に使うときになると扱い方（物理的衝撃や温度など）を著しく注意する必要があるので、ピラー数がゼロのチャンバーは実際には殆ど使えない。混合用マイクロチャンバーの性能は、液体が安定に保持される力（漏出されにくい力）と、混合後の回収率の総合評価で判断されるべきであり、この総合評価でピラー数は２〜４個が優れており、ピラー数２〜３個がより優れており、ピラー数３が最も優れている。 From the results shown in FIG. 6, it was found that 2 or 3 pillars are excellent under this experimental condition. When the number of pillars is zero, the recovery rate (fluorescence intensity) is superior, but the force with which the liquid is held in the chamber is weak (FIG. 4), so the first liquid is immediately from the bottom surface 2 of the liquid holding unit. Leak. Since it is necessary to pay special attention to handling (physical shock, temperature, etc.) when actually used, chambers with zero pillars are practically useless. The performance of the mixing microchamber should be judged by a comprehensive evaluation of the force that keeps the liquid stable (force that does not easily leak) and the recovery rate after mixing, and the number of pillars is 2 to 4 in this comprehensive evaluation. The number is excellent, the number of pillars is 2-3, and the number of pillars 3 is the most excellent.
図７は、マイクロチャンバーに蛍光液を保持して、図５に示す工程により混合操作を行い得られた混合液の蛍光強度を、液体保持部の直径１１の長さが4mmのときのドーナツ状の流体混合部の幅１３の長さを変えて得られた結果を示す。ピラーの数は、図６の結果から３個で行った。 FIG. 7 shows the fluorescence intensity of the mixed solution obtained by holding the fluorescent solution in the microchamber and performing the mixing operation according to the process shown in FIG. 5, and the donut shape when the length 11 of the diameter 11 of the liquid holding unit is 4 mm. The result obtained by changing the length of the width 13 of the fluid mixing part is shown. The number of pillars was three from the result of FIG.
図７の実験の詳細を以下に示す。 Details of the experiment of FIG. 7 are shown below.
＊ピペッティングで混合したとき
200nM FITC溶液9.6μL
0.1 M リン酸緩衝液(pH7.4)144μL
＊ピラー（半径が0.8mm）が３個
マイクロチャンバーに添加した液：200nM FITC溶液9.6μL,
送液した液:0.1 M リン酸緩衝液(pH7.4)144μL
* When mixed by pipetting
200nM FITC solution 9.6μL
144 μL of 0.1 M phosphate buffer (pH 7.4)
* Liquid with 3 pillars (0.8mm radius) added to the microchamber: 9.6μL of 200nM FITC solution,
Liquid sent: 144 μL of 0.1 M phosphate buffer (pH 7.4)
図７の結果から液体保持部の直径を100としたときのドーナツ状の流体混合部の幅(13)は25〜80、好ましくは30〜75程度、より好ましくは35〜70程度である。 From the result of FIG. 7, the width (13) of the doughnut-shaped fluid mixing part when the diameter of the liquid holding part is 100 is 25 to 80, preferably about 30 to 75, more preferably about 35 to 70.
図８は、液体流入口５に対してマイクロチャンバーのピラー１の配置が異なったときに得られる、混合後の蛍光強度の比較を示す図である。図８の実験の詳細を以下に示す。 FIG. 8 is a diagram showing a comparison of the fluorescence intensity after mixing, which is obtained when the arrangement of the microchamber pillars 1 is different from the liquid inlet 5. Details of the experiment of FIG. 8 are shown below.
＊ピペッティングで混合したとき
40nM FITC溶液10μL
0.1 M リン酸緩衝液(pH7.4)30μL
＊ピラー（半径が0.8mm）が３個
マイクロチャンバーに添加した液：40nM FITC溶液9.6μL,
送液した液:0.1 M リン酸緩衝液(pH7.4)28.8μL
* When mixed by pipetting
10 nL of 40 nM FITC solution
30 μL of 0.1 M phosphate buffer (pH 7.4)
* Liquid with 3 pillars (radius 0.8mm) added to micro chamber: 9.6μL of 40nM FITC solution,
Pumped solution: 28.8 μL of 0.1 M phosphate buffer (pH 7.4)
図８の結果から、複数のピラーのうちの１つは流入口５と流出口６を結ぶ直線上に配置されるのがよく、特に流入口５に対向する位置にピラーを配置するのが好ましい。 From the result of FIG. 8, one of the plurality of pillars is preferably arranged on a straight line connecting the inflow port 5 and the outflow port 6, and in particular, the pillar is preferably arranged at a position facing the inflow port 5. .
図９は、３つのマイクロチャンバー直列に配置して液体を混合したときの混合後の蛍光強度の比較を示す図である。図９の実験の詳細を以下に示す。 FIG. 9 is a diagram showing a comparison of fluorescence intensities after mixing when liquids are mixed by arranging them in series in three microchambers. Details of the experiment of FIG. 9 are shown below.
＊ピペッティングで混合したとき
40nM FITC溶液10μL
0.1 M リン酸緩衝液(pH7.4)30μL
＊マイクロチャンバーが2個のとき（ピラー（半径が0.8mm）が３個）
マイクロチャンバーに添加した液：40nM FITC溶液9.6μL×2個
送液した液:0.1 M リン酸緩衝液(pH7.4)57.6μL
＊マイクロチャンバーが3個のとき（ピラー（半径が0.8mm）が３個）
マイクロチャンバーに添加した液：40nM FITC溶液9.6μL×3個
送液した液:0.1 M リン酸緩衝液(pH7.4)86.4μL
＊マイクロチャンバーが4個のとき（ピラー（半径が0.8mm）が３個）
マイクロチャンバーに添加した液：40nM FITC溶液9.6μL×4個
送液した液:0.1 M リン酸緩衝液(pH7.4)115.2μL
* When mixed by pipetting
10 nL of 40 nM FITC solution
30 μL of 0.1 M phosphate buffer (pH 7.4)
* When there are two micro chambers (three pillars (radius 0.8mm))
Solution added to the microchamber: 9.6 μL of 40 nM FITC solution × 2 solution supplied: 57.6 μL of 0.1 M phosphate buffer (pH 7.4)
* When there are three micro chambers (three pillars (radius 0.8mm))
Solution added to the micro chamber: 40nM FITC solution 9.6μL x 3 solution supplied: 0.1M phosphate buffer (pH7.4) 86.4μL
* When there are four micro chambers (three pillars (radius 0.8mm))
Solution added to the microchamber: 9.6 μL of 40 nM FITC solution × 4 solution supplied: 115.2 μL of 0.1 M phosphate buffer (pH 7.4)
図９の結果から、マイクロチャンバーを複数個直列に接続しても問題なく液体の混合が行われることが明らかになった。従って、3種以上の液体混合は、マイクロチャンバーを直列に接続することで対応できることが明らかである。 From the results shown in FIG. 9, it has been clarified that even if a plurality of microchambers are connected in series, the liquid can be mixed without any problem. Therefore, it is clear that mixing of three or more liquids can be handled by connecting the microchambers in series.
本発明のマイクロチャンバーはμ-TASやLab-on-a-chipと称されるマイクロチップ上で、２種類以上の液体を混合する製品全般への応用が可能である。 The microchamber of the present invention can be applied to all products that mix two or more liquids on a microchip called μ-TAS or Lab-on-a-chip.
Claims (9)
前記液体保持部は円筒状であり、前記液体保持部は前記中空筒状の流体混合部と同心円状であり、かつ、液体保持部は液体混合部よりも低い位置にある、マイクロチャンバー。 A microchamber connected to the liquid inlet 5 and the liquid outlet 6, the microchamber comprising a liquid holding portion and a hollow cylindrical fluid mixing portion; and a bottom surface 2 of the liquid holding portion and a bottom surface 3 of the fluid mixing portion A plurality of pillars 1 are arranged in a position across both ,
The microchamber , wherein the liquid holding part is cylindrical, the liquid holding part is concentric with the hollow cylindrical fluid mixing part, and the liquid holding part is lower than the liquid mixing part .
液体保持部に第１の液体を導入する工程、液体流入口５から第２の液体をマイクロチャンバー内に流入し、液体流出口６から第１の液体と第２の液体の混合液体を流出させる工程
を含むことを特徴とする液体の混合方法。 A liquid mixing method using the microchamber according to any one of claims 1 to 8 ,
The step of introducing the first liquid into the liquid holding part, the second liquid is introduced into the microchamber from the liquid inlet 5 and the mixed liquid of the first liquid and the second liquid is caused to flow out from the liquid outlet 6. A liquid mixing method comprising a step.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP2013095009A JP6120440B2 (en) | 2013-04-30 | 2013-04-30 | Microchamber and liquid mixing method |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP2013095009A JP6120440B2 (en) | 2013-04-30 | 2013-04-30 | Microchamber and liquid mixing method |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JP2014213299A JP2014213299A (en) | 2014-11-17 |
| JP6120440B2 true JP6120440B2 (en) | 2017-04-26 |
Family
ID=51939587
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP2013095009A Expired - Fee Related JP6120440B2 (en) | 2013-04-30 | 2013-04-30 | Microchamber and liquid mixing method |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JP6120440B2 (en) |
Families Citing this family (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP6554077B2 (en) * | 2016-07-28 | 2019-07-31 | 積水化学工業株式会社 | Microfluidic merging method |
| JP7178012B2 (en) * | 2018-10-29 | 2022-11-25 | 学校法人 中央大学 | Liquid mixing device and liquid mixing method |
| KR102367263B1 (en) * | 2020-07-08 | 2022-02-25 | 연세대학교 산학협력단 | Microfluidic mixer and microfluidic device comprising the same |
| WO2024071987A1 (en) * | 2022-09-30 | 2024-04-04 | 포항공과대학교 산학협력단 | Microfluidic mixing structure and microfluidic mixing apparatus comprising same |
Family Cites Families (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP1542010A4 (en) * | 2002-07-12 | 2007-11-21 | Mitsubishi Chem Corp | Analytical chip, analytical chip unit, analyzing apparatus, method of analysis using the apparatus, and method of producing the analytical chip |
| DE10326607A1 (en) * | 2003-06-13 | 2005-01-05 | Steag Microparts Gmbh | Microstructure, for minimal- and non-invasive diagnostics, analysis and therapy, has base plate whose surface is sub-divided into zones with different capillary characteristics |
| US20040265171A1 (en) * | 2003-06-27 | 2004-12-30 | Pugia Michael J. | Method for uniform application of fluid into a reactive reagent area |
| JP4598646B2 (en) * | 2005-10-18 | 2010-12-15 | 学校法人早稲田大学 | Micro reactor |
| EP1977829A1 (en) * | 2007-03-29 | 2008-10-08 | Roche Diagnostics GmbH | Device for performing multiple analyses in parallel |
| US9044757B2 (en) * | 2011-03-15 | 2015-06-02 | Carclo Technical Plastics Limited | Capillary fluid flow control |
-
2013
- 2013-04-30 JP JP2013095009A patent/JP6120440B2/en not_active Expired - Fee Related
Also Published As
| Publication number | Publication date |
|---|---|
| JP2014213299A (en) | 2014-11-17 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| US11426726B2 (en) | Systems and methods for serial flow emulsion processes | |
| JP6120440B2 (en) | Microchamber and liquid mixing method | |
| CA2471686C (en) | Reducing working fluid dilution in liquid systems | |
| US8821813B2 (en) | Liquid-feeding chip and analysis method | |
| JP4680037B2 (en) | Fluid handling device and fluid handling unit used therefor | |
| Brouzes | Droplet microfluidics for single-cell analysis | |
| ES2863533T3 (en) | Fluidic system for testing | |
| JP2017523435A (en) | Device for separating bubbles from a fluid | |
| JP2018531369A5 (en) | ||
| US20070275426A1 (en) | Disk-like microfluidic structure for generating diffrent concentration fluid mixtures | |
| US20180149582A1 (en) | Channel structure, measurement unit, method of measuring liquid to be measured, and measurement device for liquid to be measured | |
| WO2021156844A1 (en) | Microfluidic device with interface pinning reaction vessels within a flow-through chamber, kit for forming, and use of same | |
| JP4298616B2 (en) | Treatment tool | |
| WO2014045482A1 (en) | Analysis chip, and analysis device | |
| Jing et al. | Novel Inertial Self-Assembly Dynamics and Long-Range Spatial Ordering of Interacting Droplet Ensembles in Confined Microfluidic Flows | |
| JP2010014437A (en) | Fluid handling device | |
| JP2005304480A (en) | Treating device | |
| JP2017053650A (en) | Sample analysis chip, sample analysis method, and sample analysis device |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| A621 | Written request for application examination |
Free format text: JAPANESE INTERMEDIATE CODE: A621 Effective date: 20160216 |
|
| A977 | Report on retrieval |
Free format text: JAPANESE INTERMEDIATE CODE: A971007 Effective date: 20161220 |
|
| A131 | Notification of reasons for refusal |
Free format text: JAPANESE INTERMEDIATE CODE: A131 Effective date: 20170104 |
|
| A521 | Request for written amendment filed |
Free format text: JAPANESE INTERMEDIATE CODE: A523 Effective date: 20170301 |
|
| TRDD | Decision of grant or rejection written | ||
| A01 | Written decision to grant a patent or to grant a registration (utility model) |
Free format text: JAPANESE INTERMEDIATE CODE: A01 Effective date: 20170314 |
|
| A61 | First payment of annual fees (during grant procedure) |
Free format text: JAPANESE INTERMEDIATE CODE: A61 Effective date: 20170327 |
|
| R150 | Certificate of patent or registration of utility model |
Ref document number: 6120440 Country of ref document: JP Free format text: JAPANESE INTERMEDIATE CODE: R150 |
|
| R250 | Receipt of annual fees |
Free format text: JAPANESE INTERMEDIATE CODE: R250 |
|
| R250 | Receipt of annual fees |
Free format text: JAPANESE INTERMEDIATE CODE: R250 |
|
| R250 | Receipt of annual fees |
Free format text: JAPANESE INTERMEDIATE CODE: R250 |
|
| LAPS | Cancellation because of no payment of annual fees |