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WO2018003856A1 - Plaque de micropuits pour former un réseau de gouttelettes et procédé de fabrication d'un réseau de gouttelettes - Google Patents

Plaque de micropuits pour former un réseau de gouttelettes et procédé de fabrication d'un réseau de gouttelettes Download PDF

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Publication number
WO2018003856A1
WO2018003856A1 PCT/JP2017/023752 JP2017023752W WO2018003856A1 WO 2018003856 A1 WO2018003856 A1 WO 2018003856A1 JP 2017023752 W JP2017023752 W JP 2017023752W WO 2018003856 A1 WO2018003856 A1 WO 2018003856A1
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Prior art keywords
wells
oil
microwell plate
well
flow paths
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English (en)
Japanese (ja)
Inventor
浩規 矢菅
則尚 三木
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Keio University
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Keio University
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Priority to JP2018525211A priority Critical patent/JPWO2018003856A1/ja
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    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J19/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B81—MICROSTRUCTURAL TECHNOLOGY
    • B81B—MICROSTRUCTURAL DEVICES OR SYSTEMS, e.g. MICROMECHANICAL DEVICES
    • B81B1/00—Devices without movable or flexible elements, e.g. microcapillary devices
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B81—MICROSTRUCTURAL TECHNOLOGY
    • B81C—PROCESSES OR APPARATUS SPECIALLY ADAPTED FOR THE MANUFACTURE OR TREATMENT OF MICROSTRUCTURAL DEVICES OR SYSTEMS
    • B81C3/00—Assembling of devices or systems from individually processed components
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N35/00—Automatic analysis not limited to methods or materials provided for in any single one of groups G01N1/00 - G01N33/00; Handling materials therefor
    • G01N35/02—Automatic analysis not limited to methods or materials provided for in any single one of groups G01N1/00 - G01N33/00; Handling materials therefor using a plurality of sample containers moved by a conveyor system past one or more treatment or analysis stations
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N37/00—Details not covered by any other group of this subclass

Definitions

  • the present invention relates to a microwell plate for forming a droplet array and a method for manufacturing the droplet array.
  • microdroplets The two-dimensional array of microdroplets is expected to contribute to higher throughput and lower costs for biochemical assays and medical diagnostics.
  • microdroplets can be applied to various applications such as protein crystallization in drug discovery field, evaluation of cell sensitivity to new drugs, digital PCR used for diagnosis of cancer and infectious diseases, and microorganism / cell culture. (Non-Patent Documents 1 to 3 below).
  • Non-Patent Document 1 describes a technique for generating and arraying droplets in a microfluidic device.
  • Non-Patent Document 2 describes a technique for generating a droplet array using a substrate on which a hydrophilic surface is patterned.
  • Non-Patent Document 3 describes a slip chip.
  • the slip chip is a technique for generating droplets by sliding grooves provided on two substrates facing each other.
  • Non-Patent Document 3 Generally, in order to generate a two-dimensional droplet array, pipetting is required (for example, see Non-Patent Document 3). In particular, obtaining a droplet array having a concentration gradient cannot be achieved by any of the above methods, and requires a human pipette operation or a device using a pump.
  • An object of the present invention is to provide a microwell plate for forming a droplet array and a method for manufacturing the droplet array, which can manufacture a two-dimensional droplet array having a concentration gradient without performing precise pumping and pipetting operations.
  • the present invention basically uses a predetermined microwell plate in which microwells (wells) are connected using flow paths, so that if oil is added in a state where water components are present, the oil is added to the wells. It is based on the knowledge that droplets can be generated easily by propagating through the channel and closing the channel.
  • the sample is added in the presence of the water component, and the oil component is added before the concentration of the sample becomes constant. This is based on the knowledge that a two-dimensional droplet array having a concentration gradient can be manufactured.
  • the present invention firstly provides a microwell plate for forming a droplet array.
  • the plate includes a substrate 11, a plurality of wells 13a, 13b, 13c, 13d, and a plurality of flow paths 17a, 17b, 17c.
  • the plurality of wells 13a, 13b, 13c, and 13d are wells formed in a two-dimensional array on the substrate.
  • the microwell plate 15 for forming a droplet array has a plurality of wells, and is a plate for forming droplets in each of the plurality of wells.
  • the plurality of channels 17a, 17b, and 17c connect adjacent ones of the plurality of wells 13a, 13b, 13c, and 13d. This channel is a channel in which a liquid present in a certain well can move to an adjacent well when there is no obstacle in the channel.
  • the microwell plate for forming a droplet array of the present invention has oil passages 19a, 19b, 19c, 19d, and 19e.
  • the oil passage is a portion where oil components existing in the plurality of wells and the plurality of flow paths move. Oil components are added to the plate (well, channel or oil channel) in a state where water components exist in the plurality of wells and the plurality of channels, so that the oil component passes through the oil channel, and the plurality of wells and Propagates through multiple channels.
  • the plurality of flow paths 17a, 17b, and 17c are adjacent to each other by containing the oil components 23a and 23b in the plurality of flow paths 17a, 17b, and 17c in a state where the water component exists in the plurality of wells. To move to wells. In this way, the droplets 21a, 21b, 21c, 21d and the droplet array are formed.
  • This plate may have grooves 23a, 23b, 23c, 23d.
  • This groove is a groove that exists through a plurality of wells and a plurality of flow paths. This groove functions as an oil passage or part of an oil passage.
  • the oil passage of this plate may be a lipophilic portion provided on the lower wall surface or the bottom surface of the plurality of wells and the plurality of passages.
  • This method uses the plate described above.
  • a water component is added to the plurality of wells 13a, 13b, 13c, 13d and the flow paths 17a, 17b, 17c.
  • the oil component is added to any of the plurality of wells 13a, 13b, 13c, 13d and the flow paths 17a, 17b, 17c.
  • the oil component propagates through the continuous oil passages 19a, 19b, 19c, 19d, and 19e existing in the plurality of wells 13a, 13b, 13c, and 13d and the plurality of flow paths 17a, 17b, and 17c.
  • the oil components 23a and 23b are accommodated in the plurality of flow paths 17a, 17b, and 17c in a state where the water component exists in the plurality of wells 13a, 13b, 13c, and 13d.
  • the water component is prevented from moving to the adjacent well, and droplets 21a, 21b, 21c, and 21d containing the water component are formed.
  • the target liquid is added to the plurality of wells 13a, 13b, 13c, after the step of adding the water component and before the step of adding the oil component. 13d and the process of adding to any of the some flow path 17a, 17b, 17c is further included.
  • the step of adding the oil component is preferably performed as follows. That is, the oil component is added before the concentration of the target liquid becomes uniform in the plurality of wells 13a, 13b, 13c, and 13d. Then, in a state where the target liquid has a concentration gradient in the plurality of wells 13a, 13b, 13c, and 13d, the movement to the adjacent wells is hindered. In this way, a two-dimensional droplet array having a concentration gradient can be easily obtained.
  • the present invention can provide a microwell plate for forming a droplet array and a method for manufacturing the droplet array, which can manufacture droplets simply and at low cost by simply adding an oil component while containing a water component.
  • the present invention adds a target liquid while containing a water component, and only adds an oil component before the concentration of the target liquid becomes uniform, without performing a precise pump and pipetting operation.
  • a microwell plate for forming a droplet array and a method for manufacturing the droplet array can be provided.
  • FIG. 1 is a conceptual diagram of a microwell plate for forming a droplet array.
  • FIG. 2 is a diagram illustrating an example of a peripheral portion of one well.
  • FIG. 3 is an example of a top view of the microwell plate.
  • FIG. 3A shows a microwell plate before droplets are formed, and
  • FIG. 3B shows a microwell plate on which droplets are formed.
  • FIG. 4 is a diagram for explaining the oil passage.
  • FIG. 5 is a top view and a cross-sectional view of a well portion having a groove. 5A shows a top view and FIG. 5B shows a cross-sectional view.
  • FIG. 6 is a conceptual diagram for explaining a method of manufacturing a droplet array.
  • FIG. 1 is a conceptual diagram of a microwell plate for forming a droplet array.
  • FIG. 2 is a diagram illustrating an example of a peripheral portion of one well.
  • FIG. 3 is an example of a top view of the microwell plate
  • FIG. 7 is a conceptual diagram showing how wells, flow paths, and droplets are formed after an oil component is added.
  • FIG. 8 is a conceptual diagram showing a manufacturing process of a two-dimensional droplet array having a concentration gradient.
  • FIG. 9 is a conceptual diagram showing an example of a method for manufacturing a microwell plate.
  • FIG. 10 is a photograph replacing a drawing of the manufactured microwell plate.
  • FIG. 10A shows an overall view of the manufactured microwell plate.
  • FIG. 10B shows a partially enlarged view of the manufactured microwell plate.
  • FIG. 11 is a photograph replacing a drawing showing the state of the microwell plate after addition of nomaldecane.
  • FIG. 11 shows the state of two adjacent wells when nomaldecane is added.
  • FIG. 11B shows the state of two adjacent wells 15 seconds after the addition of nomaldecane.
  • FIG. 11C shows the state of two adjacent wells 30 seconds after the addition of nomaldecane.
  • FIG. 12 is a photograph replacing a drawing showing a microwell plate in which droplets are formed.
  • FIG. 13 is a photograph replacing a drawing showing that a droplet having a concentration gradient was formed.
  • FIG. 13A shows a state when nomaldecane is added, and
  • FIG. 13B shows a state when a predetermined time has elapsed and droplets are formed.
  • FIG. 14 is a photograph replacing a drawing showing the obtained droplet.
  • FIG. 15 is a diagram showing the evaluation results of the obtained droplets.
  • FIG. 16 is a graph instead of a drawing showing the ratio of droplets formed.
  • FIG. 17 is a graph instead of a drawing showing the width and height of the groove of the first apparatus in the embodiment and the rate at which droplets are formed.
  • FIG. 18 is a graph instead of a drawing showing the width and height of the groove of the second device in the embodiment and the rate at which droplets are formed.
  • FIG. 19 is a drawing related to Example 9.
  • FIG. 19A is a conceptual diagram showing an array in the embodiment.
  • FIG. 19B is a diagram for explaining the well size.
  • FIG. 19C is a conceptual diagram for explaining the state of the well.
  • FIG. 19D is a table instead of a drawing showing the result of droplet generation by n-decane based on Ag and Ap.
  • FIG. 19A is a conceptual diagram showing an array in the embodiment.
  • FIG. 19B is a diagram for explaining the well size.
  • FIG. 19C is a conceptual diagram for explaining the state of the well.
  • FIG. 19D is a table instead of a drawing showing the result of droplet generation by
  • FIG. 19E is a table instead of a drawing showing the result of droplet generation by n-hexane based on Ag and Ap.
  • FIG. 19F is a table instead of a drawing showing the result of droplet generation by n-hexadecane based on Ag and Ap.
  • FIG. 19G is a photograph replacing a drawing showing the state of the generated droplet.
  • FIG. 19 (H) is a photograph replacing a drawing showing a connected droplet.
  • FIG. 19I is a photograph replacing a drawing showing that n-hexane is linked in a short period of time.
  • FIG. 19 (J) is a photograph replacing a drawing which shows that n-hexadecane has a lower droplet formation rate than other solvents.
  • FIG. 19G is a photograph replacing a drawing showing the state of the generated droplet.
  • FIG. 19 (H) is a photograph replacing a drawing showing a connected droplet.
  • FIG. 19I is a photograph replacing a drawing
  • FIG. 21 is a drawing related to Example 10.
  • FIG. 21 is a drawing related to Example 11.
  • FIG. FIG. 21A is a conceptual diagram showing a state in which fluorescent molecules Rhodamine B, Fluorescein, and n-decane are dropped onto a microwell plate having three reservoirs.
  • FIG. 21B is a photograph replacing a drawing showing a microwell plate having three reservoirs.
  • FIG. 21 (C) is a photograph replacing a drawing which shows a state in which a concentration gradient is generated by allowing to stand for 2 hours after dropping Rhodamine B and fluorescein.
  • FIG. 21D is a photograph replacing a drawing which shows a state in which n-decane was dropped and droplets were generated after the state of FIG.
  • FIG. 21E is a photograph replacing a drawing showing the concentration gradient of rhodamine B.
  • FIG. 21F is a graph replaced with a drawing showing the concentration gradient of rhodamine B.
  • FIG. 21 (G) is a photograph replacing a drawing showing a concentration gradient of fluorescein.
  • FIG. 21 (H) is a graph replaced with a drawing showing the concentration gradient of fluorescein.
  • FIG. 22 is a fluorescence micrograph in place of a drawing showing that sf9 cells are encapsulated in
  • FIG. 1 is a conceptual diagram of a microwell plate for forming a droplet array.
  • FIG. 2 is a diagram illustrating an example of a peripheral portion of one well.
  • FIG. 3 is an example of a top view of the microwell plate.
  • FIG. 3A shows a microwell plate before droplets are formed
  • FIG. 3B shows a microwell plate on which droplets are formed.
  • the microwell plate has a plurality of wells on a substrate.
  • a microwell plate can accommodate various reagents and samples in a well, and is a kind of detection instrument used for reagent evaluation, biological evaluation, and pathological evaluation, for example.
  • the microwell plate is already known as described in, for example, Japanese Patent No. 5848233, Japanese Patent No. 5642551, Japanese Patent No. 5592606, Japanese Patent No. 4996595, and Japanese Patent No. 4496962.
  • An example of the size of the portion where the well exists in the microwell plate may be 3 mm to 50 cm on a side, 3 mm to 20 cm, or 5 mm to 10 cm.
  • the size of this part may be square, rectangular, polygonal, circular or elliptical.
  • the plate includes a substrate 11, a plurality of wells 13a, 13b, 13c, and 13d, and a plurality of flow paths 17a, 17b, and 17c.
  • the material of the substrate is not particularly limited as long as it can form a well.
  • a known material may be adopted as appropriate for the plate.
  • plate materials are metals, resins and ceramics.
  • metals are copper and aluminum.
  • the resin are methacryl, acrylic, polyethylene, polypropylene, polystyrene, polyethylene terephthalate, vinyl chloride, polyamide, polycarbonate, hot melt, phenol, bakelite, melamine, polytetrafluoroethylene, epoxy, and mixed resins thereof.
  • polypropylene, polystyrene, polycarbonate, polytetrafluoroethylene or polyethylene is preferable.
  • a specific example of the material of the substrate is OSTEMER (registered trademark) crystal clear.
  • the material of the substrate may be adjusted in consideration of the shape of the well and the channel, lipophilicity and hydrophilicity.
  • the substrate may be molded integrally with the well and the flow path, or the substrate may be molded separately from the well and the flow path.
  • a part of the surface of the substrate may be the bottom of a well, a channel, or a groove.
  • the surface of the substrate preferably has water repellency. If the surface of the substrate has water repellency, an oil passage can be formed effectively and droplets can be formed efficiently.
  • a substrate using a fluorine-based resin is a polytetrafluoroethylenic substrate.
  • a water repellent layer may be provided on the surface of the substrate, and wells and flow paths may be formed on the substrate.
  • the plurality of wells 13a, 13b, 13c, and 13d are wells formed in a two-dimensional array on the substrate.
  • the number of wells may be appropriate depending on the measurement target.
  • the number of specific wells is preferably 4 or more (2 ⁇ 2 or more) and 10,000 or less, may be 25 or more and 1000 or less, may be 36 or more and 1000 or less, and may be 100 or more and 400
  • the number may be not more than 225, may be 225 or more and 400 or less, or may be 400 or more and 900 or less.
  • Each well may have the same shape or a different shape.
  • the well shape is different, for example, the well size may be different (for example, smaller) for each column. By doing so, the concentration gradient of the sample can be easily provided. Further, the well size may be different (for example, smaller) as the distance from a certain point increases.
  • the shape seen from the top of the well is usually circular. On the other hand, it may be oval or polygonal.
  • the area viewed from the upper surface of the well is 0.01 mm 2 or more and 10 mm 2 or less, 0.01 mm 2 or more and 4 mm 2 or less, 0.05 mm 2 or more and 2 mm 2 or less, 0.1 mm 2 or more and 1.5 mm 2 or less. Good.
  • Examples of the depth of the well are 0.1 mm or more and 1 cm or less, 0.1 mm or more and 5 mm or less, 0.3 mm or more and 2 mm or less, 5 mm or more and 1 mm or less may be sufficient.
  • the cross-sectional shape of the well may be a rectangle (or a rectangle having a groove portion), or may be a shape in which the width becomes narrower (or becomes wider) as it becomes deeper.
  • the plurality of flow paths 17a, 17b, and 17c connect adjacent ones of the plurality of wells 13a, 13b, 13c, and 13d. Since there is no adjacent well at the end portion of the substrate 11, there is no need for a flow path, and there is provided a flow path that assumes that the arrayed well exists at the end portion of the substrate or at the end of the end portion. May be. In that case, the flow path existing at the end of the substrate may be one or plural.
  • the flow path connects adjacent wells.
  • This channel is a channel in which a liquid present in a certain well can move to an adjacent well when there is no obstacle in the channel.
  • An example of the width of the flow path is that the oil component stays as the oil component passes through the flow path. Therefore, the shape of the flow path may be selected in consideration of physical force such as surface tension in the flow path.
  • the shape of the channel is usually rectangular. However, the shape of the upper surface of the flow path may be reduced as it moves away from a certain point. Further, even if the shape of the flow path is rectangular, it may be designed so that the width of the flow path becomes smaller (or larger) as the distance from a certain well increases.
  • the cross-sectional shape of the flow path is also usually rectangular (or a shape having a groove). On the other hand, the shape of the flow path may be a shape in which the width becomes narrower (or becomes wider) as it becomes deeper.
  • the length of the channel is not less than 1000 times and not more than 1/2 of the width of the well (the maximum length of the well when it is assumed that the channel does not exist), and is not less than 1/500 and not more than 3 minutes. 1/200, 1/200 to 1/5, 1/100 to 1/8, or 1/50 to 1/10.
  • the length of the flow path is 0.01 mm or more and 5 mm or less, 0.01 mm or more and 2 mm or less, or 0.01 mm or more and 0.5 mm or less. 05 mm or more and 0.5 mm or less may be sufficient, and 0.05 mm or more and 0.3 mm or less may be sufficient.
  • the width of the channel is not less than 1/100 and not more than 1/2 of the well width (the maximum length of the well when it is assumed that the channel does not exist). It may be 1 or less, 1/20 or more and 1/5 or less, 1/15 or more and 1/8 or less, or 1/11 or more and 1/9 or less.
  • An example of the width of the channel is one tenth of the width of the well.
  • Specific examples of the width of the flow path are 0.01 mm to 10 mm, 0.01 mm to 0.5 mm, 0.03 mm to 0.2 mm, 0.05 mm to 0.00 mm. It may be 1 mm or less.
  • the depth of the flow path may be the same as or different from the depth of the well.
  • the upper surface of the channel may be the same height as the upper surface of the well.
  • the flow path preferably includes an oil path.
  • the bottom surface of the channel and the bottom surface of the well may be at the same position, or the bottom surface of the channel may be at a position lower than the bottom surface of the well.
  • This plate may have grooves 23a, 23b, 23c, 23d.
  • This groove is a groove that exists through a plurality of wells and a plurality of flow paths. This groove functions as an oil passage or part of an oil passage.
  • the cross-sectional shape of the groove may be rectangular, semicircular, or inverted triangle.
  • the oil passages 19a, 19b, 19c, 19d, and 19e are portions where oil components existing in the plurality of wells and the plurality of flow paths move.
  • the oil passages exist in the wells and the flow passages, and may be portions where the physical properties are not different from the portions other than the oil passages, or may be portions where some physical properties are different.
  • the oil path is preferably present in the lower part of the well and the flow path, and the oil component moves through the oil path.
  • the microwell plate of the present invention has the above-described well and channel, and when the oil component is added in a state where the water component is present in the well and the channel, the oil component moves through the well and the channel. However, it has a shape and physical properties that can be propagated.
  • the part where the oil component moves at this time is an oil passage. That is, the microwell plate of the present invention is a microwell plate for a new application. When an oil component is added in a state where a water component is present in the well and the channel, the oil component moves through the well and the channel. The microwell plate is used for propagating and forming droplets thereby.
  • FIG. 4 is a diagram for explaining the oil passage.
  • the oil passage 19a may be a well or a lower portion of the flow path.
  • the oil passage 19a shown in FIG. 4 (A) has the same physical properties as the portions other than the oil passage.
  • the oil passage may be a portion having physical properties different from those of other portions of the well or the flow passage.
  • An example of the oil passage in this case is one in which different surface processing is applied to portions other than the oil passage and the oil passage.
  • a portion other than the oil passage may be etched to make the surface roughness rough.
  • only the oil passage portion may be mirror-finished.
  • An oil passage may be formed by coating the oil passage portion. Any coating may be used as long as it can change the physical properties of the part other than the oil passage and can function as an oil passage.
  • An example of the coating is a water repellent (hydrophobic agent).
  • water repellents examples include waxes (eg, paraffin wax and natural wax), resins (eg, polypropylene resin, polyethylene resin, polyamide resin, and polyamine resin), fluororesins (eg, resins containing fluorine, poly Tetrafluoroethylene (PTFE), polyvinylidene fluoride (PVDF), polyhexafluoropropylene, tetrafluoroethylene-hexafluoropropylene copolymer (FEP) polytetrafluoroethylene, and polychlorotrifluoroethylene), and silicone resin (polymethyl) Hydrogen siloxane, and polydimethylsiloxane). These may be used alone or in combination.
  • waxes eg, paraffin wax and natural wax
  • resins eg, polypropylene resin, polyethylene resin, polyamide resin, and polyamine resin
  • fluororesins eg, resins containing fluorine, poly Tetrafluoroethylene (PTFE), polyvinylid
  • a specific example of the coating is CYTOP (types A to C) manufactured by Asahi Glass.
  • the oil passage may be formed by depositing a metal on the oil passage portion.
  • the oil passage formed in this way may form a layer called a water repellent layer or a hydrophobic layer. Then, when the oil component is added, the affinity to the oil component is higher than that of the water component, so that the oil component tends to stay in the flow path, and the oil component exists on the bottom of the well, and the liquid is contained in the well. Drops are easily formed.
  • the oil passage 19c may be a groove present on the bottom surface of the well (and the bottom surface of the flow path). Since this groove functions as an oil passage, it is preferable that the groove is provided through the well bottom surface and the channel bottom surface.
  • the groove is a portion provided below the bottom surface of the well and having a shape recessed from the bottom surface. In the example shown in FIG. 4C, only the groove portion may function as the oil passage, or the groove portion and the well bottom surface (and the flow path bottom surface) may function as the oil passage. Moreover, the whole groove part does not need to function as an oil path.
  • the substrate surface may be a groove bottom surface, or the substrate surface may be exposed through the groove. When the substrate surface has water repellency, a good oil passage can be formed without coating the well, and droplets can be preferably produced. This eliminates the need for coating or the like, increasing productivity.
  • the oil passage 19d is a groove present on the bottom surface of the well (and the flow path), and may have a surface with physical properties different from those of the well and the flow path surface.
  • the surfaces having different physical properties are as described in the example of FIG. Even in this case, it is not necessary for the oil to move only through the groove portion, and it is sufficient that the oil contacts at least a part of the groove portion and moves.
  • the oil passage 19d is provided not only in the groove existing on the bottom surface of the well (and flow path) but also in the well (and flow path), and has a physical property different from that of other portions. There may be. Also in this case, it is not necessary for oil to move while adhering to the entire oil passage, and it is sufficient that the oil contacts at least part of the oil passage and moves.
  • FIG. 5 is a top view and a cross-sectional view of a well portion having a groove.
  • 5A shows a top view and
  • FIG. 5B shows a cross-sectional view.
  • the depth D D of the groove examples of the depth of the well (at height) D W 25 over one of may be a 1 or less 1 or 2 minutes of the 20 minutes, 10 1 to 1/3, 1/6 to 1/4, or 1/5 may be used.
  • Width W D of the groove may be the same as the width W W of the channel as shown in FIG. 5 (A), may be smaller than the width of the channel.
  • the width of the groove may be not less than 1/3 and not more than 2/3 of the width of the flow path, and may be 1/2.
  • oil components are added to the plate (well, channel or oil channel) in a state where water components exist in a plurality of wells and a plurality of channels, so that the oil component passes through the oil channel. And propagate through a plurality of wells and a plurality of flow paths.
  • the plurality of flow paths 17a, 17b, and 17c are adjacent to each other by containing the oil components 23a and 23b in the plurality of flow paths 17a, 17b, and 17c in a state where the water component exists in the plurality of wells. To move to wells. In this way, droplets 21a, 21b, 21c, 21d and a droplet array are formed.
  • This method is a method of obtaining a droplet array using the plate described above. This method is briefly described as follows. Add oil component with water component in well and flow path. As a result, the oil component propagates through the well and the oil passage of the flow path, while staying in the flow path. As the oil component stays in the flow path in this way, the water component in the well is isolated and droplets are formed.
  • S means a step.
  • FIG. 6 is a conceptual diagram for explaining a method of manufacturing a droplet array.
  • FIG. 6A is a conceptual diagram showing the state of the plate before adding the water component. In this example, a plate having a groove below the well is used.
  • the lower view of FIG. 6A is a schematic drawing of a top view and a cross-sectional view of a certain plate.
  • Water component is added to the plurality of wells 13a, 13b, 13c, 13d and the flow paths 17a, 17b, 17c.
  • the water component may be an aqueous solution or an alcohol solution containing a target sample, or may be a target sample dispersed or a target sample dissolved.
  • the plate is configured such that a plurality of wells can flow liquid through the flow path. Therefore, for example, if a water component is added to a certain well, the water component is transmitted to a plurality of wells through the flow path.
  • FIG. 6B is a conceptual diagram showing a state in which water components are distributed to the well and the flow path.
  • the water component may be added in an amount contained in the entire well, or an amount obtained by dividing the amount that can be accommodated in the well and the flow path by dividing the amount of components added later (for example, oil component). It may be added.
  • the amount of water component added to the whole well is added, the water component in the well rises and forms droplets after the oil component is added. For this reason, the water component may be added in an amount accommodated in the entire well.
  • the oil component is added to any of the plurality of wells 13a, 13b, 13c, 13d and the flow paths 17a, 17b, 17c.
  • the oil component may be added from any one well.
  • an oil component may be added from any one flow path.
  • an oil component addition part for adding an oil component to the plate may be provided, and the oil component may be added from the oil component addition part.
  • the oil component preferably has a higher affinity for the well and the flow path than the water component.
  • examples of the oil component are liquid alkanes having 4 to 20 carbon atoms, silicon oil, vegetable oil, essential oil, and ester (liquid).
  • An example of the liquid alkane is a liquid alkane having 5 to 16 carbon atoms.
  • examples of vegetable oils are corn oil, palm oil and palm shell oil.
  • esters are myristyl myristate, myristyl palmitate, myristyl stearate, myristyl isostearate, myristyl oleate, myristyl behenate, myristyl erucate, cetyl myristate, cetyl palmitate, cetyl stearate, cetyl isostearate, olein Cetyl acid, cetyl behenate, cetyl elcaate, stearyl myristate, stearyl palmitate, stearyl stearate, stearyl isostearate, stearyl oleate, stearyl behenate, stearyl erucate, isostearyl myristate, isostearyl palmitate, stearin Isostearyl acid, Isostearyl isostearate, Isostearyl oleate, Isostearyl behenate, Isostearyl be
  • FIG. 6 (C) is a schematic drawing of how oil components propagate.
  • the oil component exists in the lower part of the well and propagates to the adjacent well through a groove connected to the flow path. Part of the oil component may propagate through the upper surface of the well. That is, since the oil component has a higher affinity (permeability) with the surface of the substrate than the water component, the oil component propagates through the substrate surface (including the well surface and the channel surface).
  • the oil component is accommodated in the plurality of flow paths 17a, 17b, and 17c in a state where the water component exists in the plurality of wells 13a, 13b, 13c, and 13d.
  • the water component is prevented from moving to the adjacent well, and droplets 21a, 21b, 21c, and 21d containing the water component are formed.
  • FIG. 7 is a conceptual diagram showing how wells, flow paths, and droplets are formed after an oil component is added.
  • the oil component passes through the flow path, a part of the oil component remains in the flow path.
  • the water component can flow in the adjacent wells as shown in FIG.
  • FIG. 7B when a sufficient amount of oil component remains in the flow path, as shown in FIG. 7B, the water component is divided in adjacent wells, and droplets are formed in each well.
  • FIG. 7C is a conceptual diagram showing how droplets are formed.
  • the manufacturing method of the above droplet array is as follows: Adding a water component to the plurality of wells 13a, 13b, 13c, 13d and the flow paths 17a, 17b, 17c; A step of adding an oil component to any of the plurality of wells 13a, 13b, 13c, 13d and the flow paths 17a, 17b, 17c after the water component is added; Including The oil component propagates through the continuous oil passages 19a, 19b, 19c, 19d, 19e existing in the plurality of wells 13a, 13b, 13c, 13d and the plurality of flow paths 17a, 17b, 17c, With the water component A present in the plurality of wells 13a, 13b, 13c, 13d, the oil components 23a, 23b are accommodated in the plurality of flow paths 17a, 17b, 17c, Preventing water components from moving to adjacent wells; Forming droplets 21a, 21b, 21c, 21d containing water components; It is a manufacturing method of a droplet array
  • the target liquid is added to the plurality of wells 13a, 13b, 13c, after the step of adding the water component and before the step of adding the oil component. 13d and the process of adding to any of the some flow path 17a, 17b, 17c is further included.
  • the step of adding the oil component is preferably performed as follows. That is, the oil component is added before the concentration of the target liquid becomes uniform in the plurality of wells 13a, 13b, 13c, and 13d. Then, in a state where the target liquid has a concentration gradient in the plurality of wells 13a, 13b, 13c, and 13d, the movement to the adjacent wells is hindered. In this way, a two-dimensional droplet array having a concentration gradient can be easily obtained.
  • FIG. 8 is a conceptual diagram showing a manufacturing process of a two-dimensional droplet array having a concentration gradient.
  • the first target liquid and the second target liquid are separately added to wells existing at opposite corners.
  • the target liquid is not limited to one type but may be a plurality of types. In this case, each well has various concentrations and concentration ratios for a plurality of types of target liquids.
  • the target liquid is a liquid containing the target sample. Examples of target samples include, but are not limited to, various reagents, markers, cells, microorganisms, genes, genetic materials, peptides, proteins, and dyes.
  • a water component is added to the plurality of wells 13a, 13b, 13c, 13d and the flow paths 17a, 17b, 17c.
  • the target liquid is added to any of the plurality of wells 13a, 13b, 13c, 13d and the plurality of flow paths 17a, 17b, 17c.
  • the first target liquid and the second target liquid are separately added to the wells present at the opposite corners. Then, the first target liquid and the second target liquid propagate through the flow path and propagate to the adjacent wells.
  • the target liquid After the target liquid is added and before the concentration of the target liquid becomes uniform in the plurality of wells 13a, 13b, 13c, 13d, the plurality of wells 13a, 13b, 13c, 13d and the flow paths 17a, 17b, Add oil component to any of 17c.
  • the oil component propagates through the continuous oil passages 19a, 19b, 19c, 19d, and 19e existing in the plurality of wells 13a, 13b, 13c, and 13d and the plurality of flow paths 17a, 17b, and 17c.
  • the oil components 23a and 23b are accommodated in the plurality of flow paths 17a, 17b, and 17c in a state where the water component exists in the plurality of wells 13a, 13b, 13c, and 13d.
  • the water component is prevented from moving to the adjacent well, and droplets 21a, 21b, 21c, and 21d containing the water component are formed.
  • FIG. 8B is a conceptual diagram showing that a plurality of droplets having a concentration gradient are formed for two types of target liquids.
  • the target liquid cannot move to the adjacent wells in a state where the target liquid has a concentration gradient in the plurality of wells 13a, 13b, 13c, and 13d.
  • a two-dimensional droplet array having a concentration gradient for the target liquid can be easily obtained.
  • the microwell plate may be manufactured using a known method.
  • a water repellent layer for example, a water repellent or a hydrophobic agent may be added to the well and the solvent evaporated.
  • An example of a method for changing the surface roughness of a part of the substrate, the well and the flow path is to perform the sand blasting process with a part masked.
  • a method for manufacturing a part having physically or scientifically different physical properties is already known.
  • FIG. 9 is a conceptual diagram showing an example of a manufacturing method of a microwell plate.
  • a template serving as a basis for the microwell plate was prepared (FIG. 9A).
  • the mold was made of acrylic resin.
  • the mold may be a metal.
  • a groove is formed in the lower part of the well.
  • a resin as a raw material for the substrate was injected into the mold (FIG. 9B).
  • OSTECC was used.
  • a side glass serving as a substrate was mounted on the resin, and then ultraviolet rays were irradiated to cure the OSTECC resin (FIG. 9C). Then, the resin on the mold was cured.
  • the substrate was peeled off from the mold (FIG. 9D).
  • the substrate was made of OSTECC resin, so it was flexible (flexible) and could be peeled off from the mold.
  • the CYTOP solution was added so as to be in the fourth to sixth minutes of the well (FIG. 9E). With the CYTOP liquid added, the substrate was heated to 100 ° C. to solidify the CYTOP liquid (FIG. 9F). In this way, a water repellent layer (hydrophobic layer) was formed.
  • the microwell plate produced in Example 1 was as follows.
  • FIG. 10 is a photograph replacing the drawing of the manufactured microwell plate.
  • FIG. 10A shows an overall view of the manufactured microwell plate.
  • FIG. 10B shows a partially enlarged view of the manufactured microwell plate. As shown in FIG. 10A, a microwell plate was manufactured.
  • a microwell plate was manufactured in the same manner as in Example 1 except that there was no groove.
  • a microwell plate was manufactured in the same manner as in Example 1 except that the coating layer by CYTOP was not provided.
  • a microwell plate was manufactured in the same manner as in Example 1 except that the coating layer by CYTOP was provided up to the upper surface of the well.
  • FIG. 11 is a photograph replacing a drawing showing the state of the microwell plate after addition of nomaldecane.
  • FIG. 11A shows the state of two adjacent wells when nomaldecane is added.
  • FIG. 11B shows the state of two adjacent wells 15 seconds after the addition of nomaldecane.
  • FIG. 11C shows the state of two adjacent wells 30 seconds after the addition of nomaldecane. As shown in FIG. 11C, it can be seen that droplets were formed in the wells.
  • FIG. 12 is a photograph replacing a drawing showing a microwell plate in which droplets are formed.
  • FIG. 13 is a photograph replacing a drawing showing that a droplet having a concentration gradient was formed.
  • FIG. 13A shows a state when nomaldecane is added
  • FIG. 13B shows a state when a predetermined time has elapsed and droplets are formed. As shown in FIG. 13B, it can be seen that by using this method, droplets were formed in a two-dimensional array with a concentration gradient.
  • a microwell plate having 9 ⁇ 9 (a total of 81) wells manufactured in Example 1 was prepared. After infiltrating the microwell plate with ethanol, the microwell plate was replaced with 0.5% indigo carmine aqueous solution (blue No. 2). Subsequently, approximately 10 ⁇ l of any of alkanes of normal hexane (Hexane: C 6 H 14 ), normal decane (Dekane: C 10 H 22 ), and normal hexadecane (Hexadecan: C 16 H 34 ) was dropped. For each alkane, the same kind of plate was used, and the state of droplet formation was investigated three times. The evaluation was as follows. Droplet: The well is filled with an independent aqueous solution (droplet). Connected droplet: An aqueous solution (droplet) is connected to an adjacent well or the outside. Empty: The well is filled with alkane. Damaged well (Broken well): The well is damaged.
  • FIG. 14 is a photograph replacing a drawing showing the obtained droplet.
  • FIG. 15 is a diagram showing the evaluation results of the obtained droplets. As shown in FIG. 15, the number of droplets was counted, and the ratio with the number of effective wells was taken. The result is shown in FIG. FIG. 16 is a graph instead of a drawing showing the ratio of droplets formed. As shown in FIG. 16, the ratio of droplets formed is 0.60 ⁇ 0.05 (Hexane), 0.76 ⁇ 0.13 (Decane), and 0.77 ⁇ 0.03 (respectively). Hexadecane).
  • FIG. 17 shows the groove width and height of the first apparatus and the ratio of droplets formed.
  • FIG. 18 shows the width and height of the groove of the second apparatus and the rate at which droplets are formed.
  • a groove height (step) of 0 indicates an example in which a groove is not formed and a channel having a width indicated by a parameter value exists.
  • 17 and 18 are graphs of the contents of Table 2.
  • the x-axis indicates the groove (channel) width
  • the y-axis indicates the groove height (step)
  • the z-axis indicates the droplets. Indicates the rate of formation.
  • the groove width is 0.02 mm or more and 0.08 mm or less, and the ratio of droplet formation is high.
  • the droplets since the droplets are sufficiently formed even when the groove width is 0.02 mm, the droplets may be effectively formed even when the groove width is 0.02 or less. It can be said.
  • the heights (steps) of the grooves were 0.2 mm and 0.4 mm. When the width of the film was 0.08 mm or less, droplets were stably formed.
  • the groove heights were 0.2 mm and 0.4 mm, and both showed good droplet formation rates. Therefore, the height of the groove is not limited to 0.2 mm or 0.4 mm, and it is considered that droplets can be formed even when the height of the groove is 0.2 mm or less or 0.4 mm or more. It is done.
  • FIG. 19 (D) shows the result of droplet generation by n-decane based on Ag and Ap.
  • Ag 15
  • droplets could be generated at a rate of 90% or more.
  • FIGS. 19 (G) and 19 (H) show the generated droplet and the connected droplet, respectively.
  • droplets could be generated using n-hexane and n-hexadecane. When N-hexane was used, it was found that the droplet formation rate was higher than when other solvents were used. This is probably because n-hexane has a lower viscosity than other solvents and can penetrate a wide range.
  • n-hexane is a volatile solvent, it was found that it would be linked in a few seconds as shown in FIG. 19 (I). When n-hexadecane was used, a low droplet generation rate was observed compared to other solvents as shown in FIG. 19 (F).
  • the droplets generated in the respective microwell plates are shown in FIGS. Indicates volume.
  • FIGS. 21 (A) and 21 (B) Demonstration of Array with Concentration Gradient by Fluorescence Intensity Measurement
  • a microwell plate having three reservoirs was fabricated.
  • Fluorescent molecules Rhodamine B that emits red fluorescence, fluorescein that emits green fluorescence, and n-decane are dropped into these reservoirs.
  • the mixture was allowed to stand for 2 hours to generate a concentration gradient (FIG. 21 (C)), and then n-decane was dropped to generate droplets (FIG. 21 (D)).
  • Sf9 Cell encapsulation experiment A cell encapsulation experiment called Sf9 was conducted. First, sf9 cells stained with a substance that emits green fluorescence by an intracellular esterase reaction called calcein-AM were prepared. Subsequently, the microwell plate was filled with the medium containing the sf9 cells, and then droplets were generated by dropping n-decane. Observation with a fluorescence microscope confirmed that sf9 cells were encapsulated in the droplets as shown in FIG.
  • the present invention can be used in the field of physics and chemistry equipment industry.
  • the present invention can be used in the development of new drugs, biological experiments, and chemical experiments.

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Abstract

[Le problème] décrit par la présente invention est de fournir : une plaque de micropuits pour former un réseau de gouttelettes, avec lequel il est possible de fabriquer des gouttelettes d'une manière simple et peu coûteuse et avec lequel il est possible de fabriquer un un réseau de gouttelettes bidimensionnel ayant un gradient de concentration sans effectuer une opération précise de pompe et de pipette l'invention concerne également un procédé de fabrication d'un réseau de gouttelettes. [Solution] une plaque de micropuits prédéterminée, dans laquelle des micropuits (puits) sont reliés par des canaux, est utilisée. Un spécimen est ajouté tandis qu'un composant d'eau est présent. Un composant huileux est ajouté avant que la concentration de l'échantillon ne soit uniforme, ce qui permet à l'huile de bloquer les canaux avec un gradient de concentration dans le spécimen ou l'échantillon. On peut ainsi fabriquer un réseau de gouttelettes bidimensionnel ayant un gradient de concentration.
PCT/JP2017/023752 2016-06-28 2017-06-28 Plaque de micropuits pour former un réseau de gouttelettes et procédé de fabrication d'un réseau de gouttelettes Ceased WO2018003856A1 (fr)

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WO2019208795A1 (fr) * 2018-04-26 2019-10-31 国立大学法人東京大学 Procédé de formation d'un gradient de concentration sur une puce de microréacteur et puce de microréacteur
WO2021040021A1 (fr) * 2019-08-30 2021-03-04 公立大学法人大阪 Procédé de détection de substance à détecter et système de détection pour substance à détecter
CN112881729A (zh) * 2021-01-15 2021-06-01 中山大学 一种药物浓度梯度产生和加样装置及其应用
CN116615526A (zh) * 2020-10-06 2023-08-18 国立研究开发法人理化学研究所 靶标物质的检测方法、流体装置及试剂盒

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JP2010110262A (ja) * 2008-11-06 2010-05-20 Hitachi Maxell Ltd ウェルプレートを用いた核酸増幅法
JP2014503832A (ja) * 2011-01-28 2014-02-13 シロアム バイオサイエンシズ，インコーポレイテッド 微小流体アッセイ装置および方法

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JP2006181407A (ja) * 2004-12-27 2006-07-13 Pentax Corp Ｐｄｍｓ製シート
JP2007017155A (ja) * 2005-07-05 2007-01-25 National Institute Of Advanced Industrial & Technology マイクロ流路ビーズアレイデバイス及びその作製方法
JP2010110262A (ja) * 2008-11-06 2010-05-20 Hitachi Maxell Ltd ウェルプレートを用いた核酸増幅法
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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2019208795A1 (fr) * 2018-04-26 2019-10-31 国立大学法人東京大学 Procédé de formation d'un gradient de concentration sur une puce de microréacteur et puce de microréacteur
WO2021040021A1 (fr) * 2019-08-30 2021-03-04 公立大学法人大阪 Procédé de détection de substance à détecter et système de détection pour substance à détecter
CN116615526A (zh) * 2020-10-06 2023-08-18 国立研究开发法人理化学研究所 靶标物质的检测方法、流体装置及试剂盒
CN112881729A (zh) * 2021-01-15 2021-06-01 中山大学 一种药物浓度梯度产生和加样装置及其应用

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