JP2020054248A - Droplets generator - Google Patents

Abstract

To provide a compact droplets generator capable of reducing a dispersion phase which is residual without transmitting a porous film.SOLUTION: A droplets generator disclosed in this specification is a cylindrical droplets generator comprising: a first space which is surrounded by a sidewall and has a first diameter; a second space which is surrounded by a sidewall and has a second diameter smaller than the first diameter; a third space whose diameter is reduced from the first space toward the second space and which is surrounded by a sidewall; and a generating part which is provided so as to contact the sidewall forming the second space and generates the droplets. The second diameter is equal to or shorter than the diameter forming the third space.SELECTED DRAWING: Figure 1

Description

  The disclosure herein relates to a droplet generation device.

  There is known a direct membrane emulsification method in which a liquid is extruded at a constant pressure through a porous membrane to generate a dispersed phase in a continuous phase. When analyzing the concentration of an object contained therein using the emulsion produced by this production method, it is desirable that a large number of emulsions be produced from the viewpoint of reliability in quantitative analysis.

Patent No. 3242776

  However, while the amount of emulsion that can be produced at one time can be increased by increasing the size of the porous membrane, there is a problem that increasing the size of the porous membrane increases the amount of the dispersed phase that does not pass through the membrane and remains. Was.

  Further, if the size of the porous membrane is reduced without changing the amount of the liquid, the shape of the container is undesirably increased in the height direction.

  In view of the above problems, it is an object of the disclosure of the present specification to provide a droplet generation apparatus that is small and capable of reducing the remaining dispersed phase without passing through a porous membrane.

  The present invention is not limited to the above-described object, and it is an operation effect derived from each configuration shown in the embodiment for carrying out the invention described later, and also has an operation effect that cannot be obtained by the conventional technology. It can be positioned as one of the other purposes.

  The droplet generation device disclosed in the present specification is a cylindrical droplet generation device, wherein a first space having a first diameter surrounded by a side wall, and the first diameter surrounded by a side wall. A second space having a smaller second diameter, a third space surrounded by a side wall decreasing in diameter from the first space toward the second space, and the second space. And a generation unit that generates the droplet, which is provided so as to be in contact with the side wall that forms the third space, wherein the second diameter is equal to or less than the diameter that forms the third space. I do.

  According to the disclosure of the present specification, it is possible to provide a droplet generation apparatus that is small and capable of reducing the remaining dispersed phase without permeating the porous membrane.

The figure which shows an example of the external appearance of the droplet generation apparatus which concerns on this embodiment. FIG. 4 is a diagram illustrating an example of a procedure for performing droplet generation in the present embodiment. The figure which shows an example in which the droplet generation apparatus and container which concern on this embodiment were integrated The figure which shows an example of the relationship between the magnitude | size of the production | generation part of the droplet generation apparatus which concerns on this embodiment, and the remaining aqueous phase. The figure which shows an example of the structure of the space inside the droplet generation apparatus which concerns on this embodiment. The figure which shows an example at the time of inclining the droplet generation apparatus which concerns on this embodiment.

  Hereinafter, an embodiment of an image processing apparatus according to the present disclosure will be described with reference to the drawings. However, the scope of the invention is not limited to the illustrated example.

<First embodiment>
The droplet generation device according to the present embodiment is a droplet generation device used for generating an emulsion. Specifically, for example, it can be used in a nucleic acid sample digital PCR (dPCR: digital Polymerase Chain Reaction) method in which a water-in-oil emulsion is used as a reaction field to analyze an analyte such as a compound or a particle contained in a sample. In the digital PCR method, a sample containing a nucleic acid to be analyzed is mixed and diluted with an amplification reagent for amplifying the nucleic acid, a fluorescent reagent for detecting the nucleic acid, and the like. Divided into Each droplet contained in the emulsion may be used as a reaction field in a digital PCR method. Then, PCR is independently performed in each of a large number of reaction fields (droplets), and the nucleic acid is amplified. Nucleic acid is detected by a fluorescent reagent after amplification, and the number of reaction fields where a signal of the fluorescent reagent is detected (the number of positive reaction fields) and the number of reaction fields where no signal is detected after the amplification (number of negative reaction fields) are determined. Thus, the concentration of the nucleic acid in the sample can be estimated.

  Each of the embodiments described below is merely an example for describing a method of generating an emulsion using a droplet generation device, and the disclosure in this specification is not limited to the embodiments.

  FIG. 1 is a diagram illustrating a configuration of a droplet generation device 100 according to the present embodiment.

  FIG. 1A is a plan view of the droplet generation device 100. FIG. FIG. 1B is a cross-sectional view illustrating an A-A cross section of the droplet generation device 100. In FIG. 1A, the thickness of the side wall is omitted.

  Hereinafter, the configuration of the droplet generation device 100 will be described with reference to FIG.

  The droplet generation device 100 generates a droplet used for quantitative analysis of an object included in the droplet. The droplet generation device 100 is made of a transparent material such as glass, polycarbonate resin (PC), cycloolefin polymer resin (COP), acrylic resin (PMMA), polyvinyl chloride resin (PVC), and polyethylene terephthalate, which can be used to check the state of the sample. Resin (PET), polydimethylsiloxane resin (PDMA) and the like are preferable. Further, in the present embodiment, the droplet generation device 100 will be described as a cylindrical device, but the shape is not limited to the above, and may be, for example, a rectangular tube. In the present embodiment, it is assumed that the droplet generation device 100 is used so as to have an opening in the x direction (horizontal direction). However, it may be used in a state where it is inclined by 90 ° in the y direction (vertical direction). In this case, it is considered that the droplet generation device 100 has an opening in the y-direction (vertical direction).

  The first space 101 is a space facing an opening having a first diameter 110 into which a liquid for generating a droplet can be injected, of the space inside the droplet generation device 100. In addition, when the droplet generation device 100 has a rectangular cylindrical shape, the long side may be the first diameter 110.

  The second space 102 is a space facing an opening having a second diameter 120 smaller than the first diameter 110. When the droplet generation device 100 has a rectangular cylindrical shape, the long side may be the second diameter 120.

  The third space 103 is a space between the first space 101 and the second space 102, and has a diameter equal to or smaller than the first diameter 110 and equal to or larger than the second diameter 120, and is a second space from the first space. It is a space surrounded by side walls whose diameter is reduced toward the space. Note that the side wall forming the third space does not necessarily have to be continuously reduced in diameter, and may have a shape in which the diameter is reduced intermittently in a stepped manner.

  The generation unit 104 is provided so as to be in contact with a side wall forming the second space 102. The generation unit 104 has a plurality of holes, and the liquid injected from the opening having the first diameter 110 passes through the plurality of holes to generate an emulsion, that is, a droplet. Hereinafter, the emulsion is referred to as a droplet. The generation unit 104 is a member in which a plurality of holes are two-dimensionally arranged in a film-like member, a porous body of open cells, a mesh in which fibers are arranged vertically and horizontally, and a microstructure in which a through-hole is arranged in a single member. Channels and the like are used. Note that the generation unit 104 in the present embodiment is desirably an emulsified film. Further, in order to stably produce a water-in-oil emulsion, the surface state is preferably water-repellent. The diameter of the hole of the substance used as the generation unit 104 is preferably about 20 [μm], but is not limited thereto.

  The angle 105 indicates an angle formed by the side wall forming the third space with respect to the x plane.

  Next, the overall processing of the droplet generation method according to the present embodiment will be described with reference to FIG.

  In the step of FIG. 2A, the aqueous phase serving as the dispersed phase is filled in the droplet generation device 100 using the aqueous phase injection means 201. When the droplet generated in the process of the present embodiment is used, for example, for analyzing the concentration of an object contained in the droplet, the aqueous phase is composed of, for example, a reaction liquid. The reaction solution contains water, a sample, an amplification reagent, and a fluorescent reagent. The sample referred to here may be the sample itself or a sample that has been subjected to pretreatment or adjustment for analysis, such as purification or concentration, chemical modification or fragmentation of the analyte, and the like. Is also good. The analytes include, for example, nucleic acids, peptides, proteins, enzymes and the like, which may be present together. The analyte may be a molecule, a microparticle, a nanoparticle, or a virus, a bacterium, a cell, or the like, in which at least one of a nucleic acid, a peptide, a protein, and an enzyme is bound or attached by a covalent bond or the like. The configuration of the reaction solution is not limited to the above, and the aqueous phase may not be the reaction solution. As the aqueous phase injecting means 201, a means such as a syringe for discharging a predetermined volume of liquid, a pump for pressurizing or depressurizing the air in the flow path, or a combination of a pump and a valve can be used. That is, the aqueous phase injecting means 201 is realized by any means having a function of injecting an aqueous phase into the droplet generation device 100.

  In addition, since the water phase injected in this step is preferably not split as much as possible, it is preferable that the water phase is injected from the position close to the droplet generation device 100 by the aqueous phase injection means 201.

  Next, the container 202 is filled with an oil phase to be a continuous phase using an oil phase injection means (not shown). The oil phase contains an oil and a surfactant. The oil phase comprises a solvent which is incompatible with and separates from the aqueous phase, and typically comprises an oil such as an aliphatic hydrocarbon or silicone oil. As the oil phase injecting means, a means such as a syringe for discharging a predetermined volume of liquid, a pump for pressurizing or depressurizing the air in the flow path, a combination of a pump and a valve, and the like can be used. That is, the oil phase injection means is realized by any means having a function of injecting oil into the droplet generation device 100.

  The order of the step of injecting the aqueous phase into the droplet generation device 100 and the step of injecting the oil phase into the container 202 may be any order. Specifically, for example, after injecting the oil phase into the container 202, the droplet generation device 100 may be immersed therein, and then the aqueous phase may be injected, or after the aqueous phase is previously injected into the droplet generation device 100, It may be immersed in the container 202 containing the oil phase.

  In this step, it is desirable that the oil phase be injected at least in such an amount that the generating section 104 is immersed in the oil phase. However, the amount of the oil phase injected is not limited to the above.

  Further, although the droplet generation device 100 that generates droplets and the container 202 that holds the generated droplets are separate bodies, a cartridge shape in which the holding unit is integrated as shown in FIG. 3 may be used.

  In the step of FIG. 2B, the driving unit 205 pressurizes the inside of the droplet generation device 100 from the opening to drive the aqueous phase filled in the droplet generation device 100. Then, the aqueous phase filled on the upper surface of the generator 104 passes through the generator 104 to generate droplets.

  The driving means 205 may use an oil phase injecting means (not shown) or an aqueous phase injecting means 201, or may use another means different from these. That is, various means can be used as the driving means as long as the liquid can be driven by applying pressure.

  Further, by attaching a pressure-resistant plug to the driving means 205, it is possible to prevent the pressure from leaking out, but the pressure-resistant plug is not always necessary.

  In this step, as shown in FIG. 4, if the area of the generating unit 104 is large, the amount of the aqueous phase that does not pass through the generating unit 104 and remains therewith increases. However, from the viewpoint of quantitative analysis, it is not preferable to reduce the amount of the aqueous phase held on the generating unit 104, and thus, by reducing the size of the generating unit 104, the length of the narrowed x direction is increased in the y direction. There is a need. On the other hand, if the length is increased only in the y direction, the size of the droplet generation device 100 increases in one direction. Therefore, as shown in FIG. 1B, a shape having a first space 101 larger than the second space 102 in two axes in the x direction and the y direction on the second space 102 facing the generation unit 104, as shown in FIG. Configuration. That is, the first diameter 110 is longer than the second diameter 120 in the x direction, and the height of the first space 101 is higher than the height of the second space 102 in the y direction. Further, it is desirable that the angle formed between the generation unit 104 and the side wall forming the second space 102 is 90 °. A third space 103 formed between the first space 101 and the second space 102 by a side wall continuously reduced in length between the first diameter 110 and the second diameter 120. Is provided. That is, the first diameter 110 is longer than the diameter forming the third space 103, and the second diameter 120 is shorter than the diameter forming the third space 103. According to the above, even if there is a difference in the length in the x direction between the first diameter 110 and the second diameter 120, the droplets can remain because the side wall forming the third space 103 is tapered. Performance can be reduced. Specifically, when the third space 103 is provided as shown in FIG. 5A, even when the aqueous phase has adhered to the side wall, it can be dropped on the generation unit 104. However, when the third space 103 is not provided between the first space 101 and the second space 102 as shown in FIG. 5B, the aqueous phase may drop onto the generation unit 104. It may not be possible to remain.

  Therefore, from the above viewpoint, it is desirable that one communicating space inside the droplet generating apparatus 100 disclosed in the present specification is constituted by at least three spaces. Further, it is desirable that the angle 105 formed by the side wall forming the third space 103 with respect to the x plane (horizontal plane) is 5 ° or more. The shape of one communicating space inside the droplet generation device 100 is not limited to the above, and any configuration may be used as long as the size of the droplet generation device 100 can be reduced in a desired direction while reducing the remaining aqueous phase. For example, when it is desired to make the droplet generation device 200 thinner, the length of the first space 101 in the x direction is made longer and the length of the first space 101 in the y direction is made shorter.

  According to the above, by reducing the area of the generation unit 104 and configuring one communicating space with at least three spaces including a taper, it is possible to reduce the droplet generation apparatus 100 while reducing the remaining liquid. Can be. Also, as shown in FIG. 6, even when the droplet generation device 100 is inclined by reducing the area of the second diameter 120 and the generation unit 104, it is difficult to form a path through which the oil phase flows, so that the droplets can be efficiently discharged. Can be generated.

REFERENCE SIGNS LIST 100 droplet generation device 101 first space 102 second space 103 third space 104 generation unit 110 first diameter 120 second diameter

Claims (12)

A cylindrical droplet generating device,
A first space having a first diameter surrounded by a side wall;
A second space surrounded by a side wall and having a second diameter smaller than the first diameter;
A third space surrounded by a side wall that is reduced in diameter from the first space toward the second space;
A generator configured to generate the droplet, provided to be in contact with a side wall forming the second space;
With
The droplet generating apparatus according to claim 1, wherein the second diameter is equal to or less than a diameter forming the third space.   The apparatus according to claim 1, wherein the third space is a space surrounded by side walls that are continuously reduced in diameter.   The apparatus according to claim 1, wherein the third space is a space surrounded by side walls whose diameter is reduced intermittently.   4. The droplet generation device according to claim 1, wherein the first space faces an opening into which the liquid serving as the droplet can be injected. 5.   5. The droplet generation device according to claim 1, wherein the one communicating space is formed by the three spaces. 6.   The droplet generation device according to any one of claims 1 to 5, wherein an angle formed by the generation unit and a side wall forming the second space is 90 °.   The droplet generation device according to any one of claims 1 to 6, wherein the droplet generation device is used for analyzing an object included in the droplet.   The droplet generation device according to claim 7, wherein the droplet generation device is used in a digital PCR (Polymerase Chain Reaction) method.   9. The droplet generation device according to claim 1, wherein a height of the first space in the vertical direction is higher than a height of the second space. 10.   10. The droplet generation device according to claim 1, wherein the generation unit includes a porous film.   The droplet generation device according to claim 1, wherein an angle formed by a side wall forming the third space with respect to a horizontal plane is 5 ° or more. A droplet generating apparatus including a cylindrical member provided with a generation unit that generates a droplet, and a holding unit that holds the droplet,
The member has a first space surrounded by a side wall and having a first diameter, and a second diameter smaller than the first diameter surrounded by the side wall, and the generation unit contacts the side wall. And a third space surrounded by a side wall whose diameter is reduced from the first space toward the second space, at least inside the second space. Wherein the diameter of the droplet is less than or equal to the diameter forming the third space.

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