Droplet manufacturing apparatus and droplet manufacturing method

Abstract

The present invention provides a droplet manufacturing apparatus and method that can simply and efficiently produce uniform, minute droplets from a completely water-soluble solution. [Solution] A droplet manufacturing apparatus 1 that uses a microfluidic chip 10 to produce droplets of the first aqueous solution in the second aqueous solution from a mixed solution of the first aqueous solution and the second aqueous solution, wherein the microfluidic chip 10 comprises a chip body 11 formed from a water-absorbing synthetic resin and a substrate 12 attached to the lower surface 11a of the chip body 11, and the chip body 11 has an inlet port 31 for introducing the mixed solution of the first aqueous solution and the second aqueous solution, an outlet port 32 for collecting droplets and a microchannel 21 connected to the inlet port 31 and the outlet port 32 and extending between the inlet port 31 and the outlet port 32.

Description

Technical Field

[0001] The present invention relates to a droplet manufacturing apparatus and a droplet manufacturing method for manufacturing droplets using a microfluidic chip.

Background Art

[0002] In recent years, in the fields of biotechnology, drug discovery research, etc., microscale micro droplets (microdroplets) manufactured using microfluidics have been utilized. Droplets are applied to, for example, nanocarriers of a drug delivery system (DDS).

[0003] Non-Patent Document 1 discloses a method for generating droplets using a microfluidics technique and liquid layer two-phase separation. In this method, two types of aqueous solutions are separately introduced into a flow path by pressure. For example, when using an aqueous solution of PEG and an aqueous solution of DEX, the aqueous solution of PEG and the aqueous solution of DEX are introduced into separate micro flow paths, and at the confluence point of the two flow paths, the continuous flow of DEX is torn by the external force of the confluent PEG flow to generate droplets.

[0004] Non-Patent Document 2 discloses a method for creating a liquid layer in a glass capillary by water-soluble liquid layer two-phase separation. A 5% (w / v): 5% (w / v) = PEG: DEX mixed solution is introduced into a glass capillary with an inner diameter of 140 μm, and by sealing both ends of the capillary, droplets of a uniform size rich in DEX are linearly arranged at equal intervals and generated. It is also disclosed that when DNA (salmon sperm DNA) or living cells (red blood cells, epithelial cells NMuNG) are added to the phase separation solution, they are naturally incorporated into the DEX-rich droplets. The above concentration of the PEG: DEX mixed solution is a concentration under phase separation conditions that are likely to cause phase separation.

[0005] Non-patent document 3 discloses a method for reducing droplet size by dehydrating water-oil droplets within a microchannel. In this method, a fluorinated oil FC-40 (oil phase) with 2% surfactant added and a cell-free expression system solution expressing GFP (aqueous phase) are used to generate droplets by passive flow focusing in a microchannel. The water in the generated droplets is then dehydrated through a PDMS membrane, shrinking the droplet size. [Prior art documents] [Non-patent literature]

[0006] [Non-Patent Document 1] “Exploring New Horizons in Liquid Compartmentalization via Microfluidics”, Shauni Keller, Serena P. Teora, Moussa Boujemaa, and Daniela A. Wilson, Biomacromolecules (American Chemical Society), 2021, 22, 5, 1759-1769 [Non-Patent Document 2] “Emergence of uniform linearly-arranged micro-droplets entrapping DNA and living cells through water / water phase-separation”, Mayu Shono, Ritsuki Ito, Fumika Fujita, Hiroki Sakuta & Kenichi Yoshikawa, Scientific Reports, volume 11, Article number: 23570 (2021). [Non-Patent Document 3] “Enhanced transcription rates in membrane-free protocells formed by coacervation of cell lysate”, Ekaterina Sokolova, et al., PNAS, 110 (29) 11692-11697, 2013 [Overview of the project] [Problems that the invention aims to solve]

[0007] When producing droplets using a microchannel that introduces two types of aqueous solutions separately, as disclosed in Non-Patent Document 1 above, a continuous flow of solution is essential. However, droplets produced by two-phase separation of water-soluble liquid layers have the problem of easily fusing together due to the influence of flow. On the other hand, when producing water-oil droplets, as disclosed in Non-Patent Document 3, problems arise with oil contamination and interactions at the oil interface. Furthermore, when producing droplets in a large bulk space, it is not possible to obtain homogeneous droplets. When producing droplets using a microchannel, special equipment such as precise flow control by a pump may be required, which becomes a problem when mass-producing droplets.

[0008] The present invention has been made in view of the above-described circumstances, and its purpose is to provide a droplet manufacturing apparatus and a droplet manufacturing method that can simply and efficiently produce uniform, minute droplets in a completely water-soluble solution. [Means for solving the problem]

[0009] According to one aspect of the present invention, a droplet manufacturing apparatus for producing droplets of a first aqueous solution in a second aqueous solution from a mixed solution of a first aqueous solution and a second aqueous solution using a microfluidic chip, wherein the microfluidic chip comprises a chip body formed from a water-absorbing resin and a substrate attached to the lower surface of the chip body, the chip body having an inlet port for introducing the mixed solution of the first aqueous solution and the second aqueous solution, an outlet port for collecting droplets, and a microchannel connected to the inlet port and the outlet port and extending between the inlet port and the outlet port, the microchannel having a cross-sectional size perpendicular to the direction of extension of the microchannel such that the mixed solution introduced from the inlet port and in contact with the inner surface of the microchannel is dehydrated by the water absorption of the resin, and a plurality of droplets are generated in the microchannel by two-phase separation of the liquid layer. [Brief explanation of the drawing]

[0010] [Figure 1] Figure 1 is a schematic plan view showing the configuration of a droplet manufacturing apparatus according to the first embodiment of the present invention. [Figure 2] Figure 2 is a partial cross-sectional view of the droplet production apparatus shown in Figure 1, viewed from direction AA. [Figure 3] Figure 3 is a cross-sectional view of BB of the droplet production apparatus shown in Figure 1. [Figure 4] Figure 4 is a graph illustrating the regions where the first polymer aqueous solution and the second polymer aqueous solution exist as single phases and as two phases. [Figure 5] Figure 5 is a perspective view showing a schematic configuration of a droplet manufacturing apparatus according to a second embodiment of the present invention. [Figure 6] Figure 6 is a cross-sectional view of the droplet production apparatus shown in Figure 5. [Figure 7] Figure 7 is a fluorescence microscope image of the microchannel showing the results of droplet production according to Example 1. [Figure 8] Figure 8 is a fluorescence microscope image of the microchannel showing the results of droplet production according to Example 2. [Figure 9] Figure 9 is a bright-field microscope image of the microchannel showing the results of droplet production using Comparative Example 1. [Figure 10] Figure 10 is a fluorescence microscope image of the microchannel showing the results of droplet production using Comparative Example 1. [Figure 11] Figure 11 shows fluorescence microscope images of the microchannel, illustrating the time-dependent results of droplet production according to Example 3. [Modes for carrying out the invention]

[0011] -First Embodiment- Hereinafter, a droplet manufacturing apparatus and droplet manufacturing method according to the first embodiment of the present invention will be described in detail with reference to the drawings. In the droplet manufacturing apparatus and droplet manufacturing method according to this embodiment, the droplet manufacturing apparatus according to the first embodiment includes a step of introducing a mixed solution of a first aqueous solution and a second aqueous solution under non-phase separation conditions, thereby generating microscale minute droplets (fine particles) by completely water-soluble two-phase separation of the liquid layer using a microfluidic chip.

[0012] In this specification, a droplet manufacturing apparatus is a device capable of manufacturing droplets of a first aqueous solution in a second aqueous solution from a mixed solution of a first aqueous solution and a second aqueous solution within a microchannel provided by the apparatus. Both the first aqueous solution and the second aqueous solution are oil-free aqueous solutions. Therefore, the mixed solution is also oil-free. The "droplets of the first aqueous solution" manufactured after phase separation in the microchannel are a solution rich in the solute of the first aqueous solution and mainly composed of the solute of the first aqueous solution, but may not have the exact same composition as the first aqueous solution before separation. Similarly, the second aqueous solution after separation is also a solution rich in the solute of the second aqueous solution and mainly composed of the solute of the second aqueous solution, but may not have the exact same composition as the second aqueous solution before separation. The "main component" of the solute as used herein means, for example, 50% by mass or more, preferably 70% by mass or more, and more preferably 90% by mass or more of the solute. Furthermore, the droplets of the first aqueous solution produced may be liquid or may contain a gel. In this specification, the term "droplet" includes both microdroplets and microgels.

[0013] Figure 1 is a schematic plan view showing the configuration of the droplet manufacturing apparatus 1 according to the first embodiment. Figure 2 is a partial cross-sectional view of the droplet manufacturing apparatus 1 shown in Figure 1 as seen from direction AA, and Figure 3 is a cross-sectional view of the droplet manufacturing apparatus 1 shown in Figure 1 as BB. The droplet manufacturing apparatus 1 includes a microfluidic chip 10, which consists of a chip body 11 and a substrate 12 attached to the lower surface 11a of the chip body 11. In Figure 1, arrow X indicates the width direction of the droplet manufacturing apparatus 1, arrow Y indicates the length direction, and arrow Z indicates the height direction (up and down direction).

[0014] The chip body 11 is a plate-shaped member having a certain thickness and is formed from a resin having water absorption. The chip body 11 is not particularly limited as long as it is a resin having water absorption, and examples thereof include silicone resins, and more preferably, polydimethylsiloxane (PDMS). The substrate 12 can be formed from a plate-shaped member having a certain thickness, and as the plate-shaped member, a glass member such as a slide glass or a cover glass can be used. Note that the substrate 12 may also be formed from a resin having water absorption, similar to the chip body 11. PDMS, which is a representative example of a preferred resin, has strong self-adsorption properties, and by simply bringing the lower surface 11a of the chip body 11 formed from PDMS into contact with the upper surface 12a of the substrate 12, the two can be bonded together in an airtight sealed state (non-covalent bond). Alternatively, after performing plasma treatment (for example, oxygen plasma treatment) on the lower surface 11a of the chip body 11 and the upper surface 12a of the substrate 12, these surfaces may be brought into contact to form a permanent bond (covalent bond). By performing surface treatment using plasma treatment, the lower surface 11a of the chip body 11 and the upper surface 12a of the substrate 12 can be firmly bonded together. Hereinafter, although PDMS will be exemplified and described as the chip body 11 of the present invention, the chip body 11 of the present invention is not limited to being made of PDMS.

[0015] The chip body 11 has an inlet port 31 for introducing a mixed solution, an outlet port 32 for collecting droplets, and a flow path portion 20 connected to the inlet port 31 and the outlet port 32 and extending in the longitudinal direction Y between the inlet port 31 and the outlet port 32. The inlet port 31 and the outlet port 32 penetrate the chip body 11 in the height direction Z and are formed, for example, in a columnar shape. The flow path portion 20 is formed by fine grooves provided on the lower surface 11a of the chip body 11. The flow path portion 20 can be formed in the chip body 11 by using, for example, MEMS technology or a microfabrication technology such as a 3D printer. A specific example of the manufacturing method of the microfluidic chip 10 will be described later.

[0016] The flow path section 20 has a plurality of micro flow paths (channels) 21 arranged parallel to each other. Each micro flow path 21 has one end connected to the inlet port 31 and the other end connected to the outlet port 32, and extends in the longitudinal direction Y. FIG. 1 shows an example in which the flow path section 20 has 11 micro flow paths 21. Note that the number of micro flow paths 21 included in the flow path section 20 is not limited to 11. In FIG. 1, a plurality of micro flow paths 21 are schematically shown as being directly connected to the inlet port 31 and the outlet port 32, but the configuration of the connection portions between the flow path section 20 and the inlet port 31 and the outlet port 32 is not limited to that schematically shown in FIG. 1.

[0017] For example, the inlet port 31 and the flow path section 20 may be configured to be connected by a branch flow path having a symmetric branching structure so that the mixed solution injected from the inlet port 31 is evenly distributed to the plurality of micro flow paths 21 (not shown). The branch flow path has a tree structure in which the branches spread symmetrically. By connecting using the branch flow path, the flow velocities (flow rates) of the mixed solution introduced into the plurality of micro flow paths 21 after branching can be made substantially the same. Therefore, it is advantageous when introducing the mixed solution into the plurality of micro flow paths 21 under the same conditions, such as when it is desired to introduce the mixed solution into the plurality of micro flow paths 21 while controlling the flow velocity (flow rate). Alternatively, the inlet port 31 and the outlet port 32 may be configured to be connected one by one to each of the plurality of micro flow paths 21 (not shown). In this case, since there is no need to provide a branch flow path, the microfluidic chip 10 can be easily fabricated.

[0018] As shown in Figure 2, the top and both sides of the microchannel 21 are defined by the chip body 11, and the bottom surface of the microchannel 21 is defined by the substrate 12. The inner surface (also called the wall surface) of the microchannel 21 may be configured so that the materials constituting the chip body 11 and the substrate 12 are exposed and in contact with the mixed solution. Alternatively, the inner surface of the microchannel 21 may be covered with a polymer coating agent. As shown in Figure 3, the inner circumferential surfaces of the inlet port 31 and the outlet port 32 are defined by the chip body 11, and the bottom surface is defined by the substrate 12. The tops of the inlet port 31 and the outlet port 32 are open. The inlet port 31 has a diameter D1, and the outlet port 32 has a diameter D2 which is smaller than the diameter D1. Here, an example is shown where the diameter D1 of the inlet port 31 is larger than the diameter D2 of the outlet port 32, but the relative sizes of the inlet port 31 and the outlet port 32 are not limited to this. The illustrated inlet port 31 is formed in a substantially cylindrical shape with a constant diameter D1 along the Z direction, but the diameter D1 does not have to be constant as long as liquid can flow in. Also, the shape of the inlet port 31 when viewed from above in the Z direction does not have to be circular; it may be rectangular or elliptical. Similarly, the diameter D2 of the outlet port 32 may vary along the Z direction, and its shape may be rectangular or elliptical.

[0019] The microchannel 21 has a length L1 between the inlet port 31 and the outlet port 32 along the length direction (extension direction) Y. The cross-section of the microchannel 21 perpendicular to the length direction Y is formed as a rectangle with a height H1 and a width W1, as shown in Figure 2. There is a distance T1 between adjacent microchannels 21. That is, a wall with a thickness T1 is provided between the microchannels 21 in the width direction X. The size of the cross-section of the microchannel 21, i.e., the height H1 and width W1, is appropriately determined according to the desired droplet size. For example, the height H1 and width W1 can be set in the range of about 3 μm to about 30 μm, and preferably in the range of about 5 μm to about 20 μm. For the purpose of producing droplets with a shape close to a perfect sphere, it is preferable to set the height H1 and width W1 to the same value.

[0020] Although Figure 2 shows an example where the cross-section of the microchannel 21 is rectangular, the shape of the cross-section of the microchannel 21 is not limited to this. The cross-section of the microchannel 21 can be a polygon including a triangle, pentagon, trapezoid, rhombus, circle, ellipse, etc. The cross-section of the microchannel 21 is selected appropriately considering the dehydration effect of the mixed solution by the microchannel 21, the ease of manufacturing the microchannel 21, etc.

[0021] Furthermore, the length L1 of the microchannel 21 and the distance T1 between multiple parallel microchannels 21, i.e., the thickness of the wall between the microchannels, affect the dehydration effect of the mixed solution by the microchannels 21, so they are appropriately determined according to the concentration and type of the mixed solution.

[0022] Next, a method for manufacturing the microfluidic chip 10 will be described. As mentioned above, the flow channel portion 20 of the chip body 11 can be formed by utilizing MEMS technology or microfabrication technology such as a 3D printer. Below, an example of a method for manufacturing the chip body 11 using a 3D printer will be described. However, the method for manufacturing the microfluidic chip 10 is not limited to a specific method; any method that can realize the above structure is acceptable.

[0023] First, the channel section 20 having the microchannels 21 is drawn using 3D CAD software. When drawing, the design of the microchannels 21 can be created so that the convex and concave surfaces are reversed in order to create a mold.

[0024] Next, a mold is created on the substrate using the design of the created microchannel 21. A 3D printer can be used to create the mold. 3D printer resin is dropped onto the substrate, and the microchannel designed using CAD software is fabricated by the 3D printer. After fabrication, the mold is developed and washed to create the mold on the substrate.

[0025] An uncured, fluid-like water-absorbing resin is poured into a mold and cured. If the resin is PDMS, heat curing is preferable. After the cured resin and mold are slowly cooled to room temperature, the resin is peeled off the mold. The size of the peeled resin is adjusted to fit on the substrate 12, and the parts corresponding to the inlet port 31 and outlet port 32 are punched out, for example, using a drilling device, to form the chip body 11. Plasma treatment is applied to the lower surface 11a of the chip body 11 and the upper surface 12a of the substrate 12, and these surfaces are brought into contact to firmly bond the chip body 11 and the substrate 12. Furthermore, by applying plasma treatment to the chip body 11 and the substrate 12, the effect of hydrophilization of the inner surface of the microchannel 21 is also obtained. In other words, plasma treatment makes it possible to simultaneously obtain both the effect of strong bonding through covalent bonding between the chip body 11 and the substrate 12 and the effect of hydrophilization. This is how the microfluidic chip 10 is manufactured.

[0026] Next, a droplet manufacturing method according to a first embodiment of the present invention will be described. The droplet manufacturing method includes the step of introducing a mixed solution of a first aqueous solution and a second aqueous solution into a microchannel of a droplet manufacturing apparatus according to the first embodiment under non-phase separation conditions.

[0027] The droplet production method according to the present invention is a method for producing multiple droplets of the first aqueous solution having substantially uniform diameters in the second aqueous solution from a mixed solution of the first aqueous solution and the second aqueous solution. The conditions and preparation method of the mixed solution will be described in detail below.

[0028] The first aqueous solution is an aqueous solution in which the first solute is dissolved in water. The second aqueous solution is an aqueous solution in which the second solute is dissolved in water. Both the first and second aqueous solutions should be in a fluid state that can be introduced into the microchannel of the droplet production apparatus. The first and second solutes are different substances, and both should be compounds that dissolve in water. Both the first and second solutes may be one type or two or more types. Furthermore, both the first and second solutes may contain a main component and one or more other water-soluble molecules or granules other than the main component, and should be an aqueous solution that causes phase separation by dehydration. The main component is, for example, a substance that accounts for 50% or more, preferably 70% or more by mass, and more preferably 90% or more by mass, of both the first and second solutes. The combination of the main components of the first solute and the main components of the second solute may be any combination that dissolves in water to form an soluble two-phase system (ATPS). Such combinations are known to those skilled in the art and are disclosed, for example, in Aqueous two-phase system (ATPS): an overview and advances in its applications, M. Iqbal, et al., Biological Procedures Online, 18:18(2016). DOI 10.1186 / s12575-016-0048-8. Examples of combinations of the main components of the first solute and the main components of the second solute include, but are not limited to, polymer compounds and polymer compounds, polymer compounds and salts, ionic liquids and short-chain alcohols, alcohols and salts, etc. Other substances constituting the first and second solutes may include water-soluble metal ions, low molecular weight compounds, biomolecules (nucleic acids, amino acids, proteins, antibodies, enzymes, cells, organs), viruses, or virus-like particles. In particular, the first solute may include a substance that is active in the body, such as a drug, and a first polymer compound that can serve as a carrier for it. Hereinafter, the explanation will be given using examples where the main components of both the first and second solutes are polymer compounds, and these will be referred to as the first polymer compound and the second polymer compound, respectively.However, the main components of the first and second solutes of the present invention are not limited to polymer compounds.

[0029] The first and second polymer compounds are not particularly limited as long as they are water-soluble polymer compounds, but can be selected from, for example, biocompatible polymers. When the manufactured droplets are used in a drug delivery system (DDS), these molecules are useful because they decompose over time in the body and do not harm the body. Examples of biocompatible polymers include, but are not limited to, polyethylene glycol, dextran, gelatin, collagen, albumin, polyglycolic acid, poly(lactic acid + glycolic acid), poly(acrylic acid), polyvinyl alcohol, poly(γ-glutamic acid), carboxymethylcellulose, polydioxanone, trimethylene carbonate, hyaluronic acid, chitosan, and alginic acid.

[0030] In order for the first polymer aqueous solution to form droplets in the second polymer aqueous solution, the first and second polymer compounds can be selected based on prior experiments, for example, based on disclosures in literature such as M. Iqbal, et al., to suit the intended use of the droplets. Polymer compounds generally have large molecular weights, so the effect of mixing entropy between different polymers is small (when the degree of polymerization is n, the mixing entropy is about 1 / n or less). Therefore, even if the chemical structures are similar, phase separation is likely to occur, and by inducing such a phase separation phenomenon due to polymer effects in a microchannel, droplets of uniform size will be arranged. Furthermore, even when using polymer compounds composed of monomers with the same chemical structure, it is necessary to consider the non-phase separation conditions described later, keeping in mind that the concentration that causes phase separation changes depending on the molecular weight (degree of polymerization). If a coating is provided on the inner surface of the microchannel 21, and a hydrophobic substance is used for the coating, then if a more hydrophilic aqueous solution is used as the first aqueous solution, droplets of the first aqueous solution will spontaneously form. Conversely, when a hydrophilic substance is used as the coating, if a more hydrophobic aqueous solution is used as the first aqueous solution, droplets of the first aqueous solution will spontaneously form. Alternatively, a polymer aqueous solution containing the same polymer compound as the coating on the inner surface of the microchannel 21 can be used as the second aqueous solution. In this case, the first aqueous solution can be arbitrary. Alternatively, even without providing a coating, the inner surface of the microchannel 21 can be made hydrophilic by plasma treatment, and the first and second polymer compounds can be selected so that droplets of a more hydrophobic aqueous solution are formed.

[0031] Specific combinations of the first polymer compound and the second polymer compound include, but are not limited to, the following. In the following enumeration, two polymers that can be combined are indicated by a hyphen, but which one corresponds to the first polymer compound that forms the droplet is determined by the hydrophilicity of each polymer compound, as described above, or can be appropriately controlled by a person skilled in the art through the coating of the microchannel 21. Dextran-PEG, Gelatin-PEG, Dextran-Methylcellulose, Dextran-[Sucrose-Epichlorohydrin copolymer], [Sucrose-Epichlorohydrin copolymer]-PEG, [DEAE-Dextran]-PEG, [Na-Dextran sulfate]-PVA, PEG-PPG, PEG-PVP, PEG-PAA, PEG-PAM, PEG-PVME, PEG-[HP-Starch], PEG-PES, PEG-[PEG-PPG copolymer], PEG-Pullulan, PEG-PVA, PEG-PPGDME, PEG-Maltodextrin, PEG-Carboxylmethyldextran, Dextran-PVP, Dextran-[PEG-PPG copolymer], Dextran-PVA, Dextran-PPG, Dextran-HEC, Dextran-[HP-Starch], Dextran sulfate-PEG, Dextran sulfate-PSS, Dextran sulfate-[DEAE-Dextran], PPG-PVA, PPG-[sucrose-epichlorohydrin copolymer], PPG-PEGME, PPG-PEGDME, [PEG-PPG copolymer]-[sucrose-epichlorohydrin copolymer], [HP-Starch]-[sucrose-epichlorohydrin copolymer], HP-Starch-[PEG-PPG copolymer], PVP-PAM, agarose-PEG, agarose-PAA, agarose-dextran, agarose-[sucrose-epichlorohydrin copolymer].

[0032] In the above combinations, the definitions of the abbreviations are as follows: PEG: Polyethylene Glycol PEGME: Polyethylene Glycol Methyl Ether PEGDME: Polyethylene Glycol Dimethyl Ether PPG: Polypropylene Glycol PPGDME: Polypropylene Glycol Dimethyl Ether PVP: Polyvinylpyrrolidone PVA: Polyvinyl Alcohol PVME: Poly(N-vinyl methyl ether) PES: Polyether Sulfones HP-Starch: Hydroxypropyl Starch PAA: Polyacrylic Acid PAM: Polyacrylamide PSS: Polystyrene Sulfonate Dextran sulfate: Dextran sulfate DEAE-Dextran: Diethylaminoethyl dextran Maltodextrin: Maltodextrin (malt sugar dextrin) Pullulan: A water-soluble polysaccharide produced by fungi. HEC: Hydroxyethylcellulose Furthermore, the PEG-PPG copolymer may be, for example, UCON®. The sucrose-epichlorohydrin copolymer may be, for example, Ficol®, a highly branched polymer.

[0033] Examples of combinations that produce phase separation using solutes other than polymers as the main component include, but are not limited to, potassium phosphate and PEG, and ammonium sulfate and PEG.

[0034] The first polymer aqueous solution and the second polymer aqueous solution are introduced into the microchannel of the droplet production apparatus as a mixed solution under non-phase separation conditions. Here, the phase separation of the two polymer aqueous solutions will be explained with reference to Figure 4. In general, water / aqueous phase separation systems undergo phase separation at high concentrations and remain single-phase at low concentrations. Figure 4 is a graph in which the vertical axis represents the concentration of the first polymer aqueous solution and the horizontal axis represents the concentration of the second polymer aqueous solution, with "○" plotting the concentration conditions that cause phase separation. In the graph, "□" represents the conditions under which phase separation occurs in solutions with concentrations on the horizontal and vertical axes, and the values ​​on the horizontal and vertical axes at the intersection (small circle) of the straight line passing through it and the dashed line (phase separation curve) represent the concentrations of each component after phase separation. In the present invention, it is desirable that the concentrations of the first polymer aqueous solution and the second polymer aqueous solution in the mixed solution are near and below the curves in Figure 4. Here, "nearby" refers to a concentration lower than the concentration at which phase separation occurs, where water is not removed, and where phase separation does not occur or is not visible even after a long period of time (e.g., 1 hour). This is determined by the properties of the polymer solute and its combination. For example, with dextran and PEG, this means a concentration about 10% to 50% lower than the concentration on the phase separation curve (for example, if the concentration on the phase separation curve is 5%, then 4.5% to 2.5%), preferably about 20% to 40% lower (for example, if the concentration on the phase separation curve is 5%, then 4.0% to 3.0%), i.e., a condition with a high water content. Even under non-phase separation conditions, if the concentration is set near the phase separation curve, the stirring effect from injection into the microchannel will cause a mixed state as an initial condition, and phase separation will progress over time, generating droplets. Even in the case of a homogeneous phase (single phase) far from the phase separation curve, it is possible to induce a phase separation state by promoting water absorption from the walls of the channel, and thus generate droplets. Distance from the phase separation curve is an effective control parameter for adjusting the size and uniformity of the droplets generated.

[0035] A curve representing the phase separation of two different polymer aqueous solutions can be created by those skilled in the art, and the concentration conditions of the mixed solution in the present invention can be determined based on such a curve and by conducting appropriate preliminary experiments. For the creation of phase separation curves, the following documents can be consulted, but are not limited thereto. 1.Albertsson, P.-A. (1986). Partitioning of Cell Particles and Macromolecules. John Wiley & Sons. 2.Zaslavsky, Boris (1995). Aqueous Two-Phase Partitioning: Physical Chemistry and Bioanalytical Applications. Marcel Dekker Inc. ISBN: 978-0-8247-9461-3 3.Bakhshi, Hamid; Mobalegholeslam, Poorya (2017). "Phase equilibria calculations of electrolyte solutions containing water- polymer- salt using a new thermodynamic model, applicable in aqueous two phase systems". Fluid Phase Equilibria. 434: 222-232. DOI:10.1016 / j.fluid.2016.11.033. 4.Hamta, Afshin; Dehghani, Mohammad Reza; Gholami, Mahsa (2017). "experimental data on aqueous two-phase system containing PEG-6000 and Na2CO3at T = (293.15, 303.15 and 313.15) K''. Journal of Molecular Liquids. 241: 144-149. DOI: 10.1016 / j.molliq.2017.05.149. Therefore, even if polymers have the same name (including common or comprehensive names) but differ in molecular weight and molecular structure, the precise non-phase separation conditions can be determined by observing the solution state at the test tube level and creating a phase separation curve. Examples of polymers with the same name but different structures include carbohydrates such as starch, which have linear and branched structures, and structural isomers with different degrees and positions of branching. Copolymers have sequence isomers such as alternating copolymers, random copolymers, and block copolymers. Similarly, even when solutes other than polymers are present, the precise non-phase separation conditions can be determined. Furthermore, even when the main component of the solute is not a polymer, a curve representing phase separation can be created using the same approach, and the conditions of a mixed solution under non-phase separation conditions close to phase separation can be determined.

[0036] Next, the method for preparing the mixed solution will be described. The first polymer aqueous solution can be prepared by completely dissolving the first solute in an aqueous solvent. Similarly, the second polymer aqueous solution can be prepared by completely dissolving the second solute in an aqueous solvent. The aqueous solvent can contain water and is determined appropriately depending on the purpose. It may be a buffer solution or culture medium that satisfies the specified salt concentration and pH conditions, but it must be a solvent that does not affect phase separation. Both the first and second polymer aqueous solutions can be prepared by any method, for example, by inversion mixing. The mixed solution can be prepared by mixing and stirring the first and second polymer aqueous solutions. For example, stirring the first and second polymer aqueous solutions can be done using a vortex mixer or the like. Alternatively, the mixed solution can also be prepared by completely dissolving the first and second solutes in an aqueous solvent after mixing them. A similar mixed solution can be obtained by either preparation method.

[0037] The first polymer aqueous solution, the second polymer aqueous solution, and the mixed solution can be prepared at any temperature suitable for the purpose, as long as it does not affect the phase separation conditions, ensures the fluidity of the mixed solution, and does not affect biomolecules contained in the solute. For example, by preparing the solutions at room temperature, or at higher or lower temperatures, and injecting them into a microfluidic channel, it is possible to adjust stability and elasticity by utilizing sol-gel transitions, etc. It should be noted that the polymer aqueous solution is just one example of an aqueous solution that undergoes phase separation, and the mixed solution of the present invention is not limited to polymer aqueous solutions.

[0038] The operation of introducing the mixed solution into the microchannel of the droplet manufacturing apparatus can be carried out, for example, by injecting the mixed solution into the inlet port 31 shown in Figures 1 and 3 using a pipette or the like. By injecting the mixed solution into the inlet port 31, the mixed solution can be filled into one or more microchannels 21 by capillary action. However, the introduction of the mixed solution into the microchannels 21 is not limited to capillary action; it can also be carried out by connecting a pump such as a syringe pump to the inlet port 31 to pressurize it from the outside, or by connecting a suction device to the outlet port 32 to apply negative pressure from the outside. Furthermore, the temperature conditions during introduction are not particularly limited; it can be carried out at room temperature, or at a temperature higher or lower than room temperature. If necessary, the mixed solution and the droplet manufacturing apparatus can each be maintained at the desired temperature conditions before carrying out the introduction operation.

[0039] The mixed solution introduced into the microchannel 21 can be allowed to flow within the microchannel 21 and collected from the outlet port 32. The rate at which the mixed solution flows within the microchannel 21 can be controlled as appropriate by pressurization or suction. The flow rate within the microchannel 21 should be sufficient to hold the mixed solution within the microchannel 21 for a sufficient amount of time to produce droplets. This time can be determined, for example, through prior experiments.

[0040] The mixed solution introduced into the microchannel 21 can also be left to stand in the microchannel 21 for a predetermined period of time. When the mixed solution is left to stand in the microchannel 21, the mixed solution can also be injected into the outlet port 32.

[0041] The mixed solution introduced into the microchannel 21 is in contact with the inner surface of the microchannel 21, specifically the top and both sides, and is gradually dehydrated by the water absorption of the PDMS forming the chip body 11. If the substrate 12 is also made of PDMS, the mixed solution is also dehydrated from the bottom surface of the microchannel 21. Although PDMS has only slight water absorption, in the microchannel 21, the surface area in contact with the inner surface of the microchannel 21, i.e., the specific surface area, becomes large relative to the volume of the solution, thus achieving a dehydrating effect on the solution. When the mixed solution under non-phase separation conditions is dehydrated by the inner surface of the microchannel 21, the solute concentration increases, and it transitions to phase separation conditions. Then, phase separation of the mixed solution begins, and spherical droplets of the first aqueous solution are self-constructed. As a result, spherical droplets corresponding to the size of the microchannel 21 (height H1 and width W1) are formed within the microchannel 21.

[0042] The droplet manufacturing apparatus 1 and droplet manufacturing method according to this embodiment, as described above, can provide the following effects.

[0043] (1) The droplet manufacturing apparatus 1 is configured to manufacture droplets using a microfluidic chip 10. The microfluidic chip 10 comprises a chip body 11 formed from a water-absorbing synthetic resin and a substrate 12 attached to the lower surface 11a of the chip body 11. The chip body 11 has an inlet port 31 for introducing a mixed solution of a first aqueous solution and a second aqueous solution, an outlet port 32 for collecting droplets, and a microchannel 21 connected to the inlet port 31 and the outlet port 32 and extending between the inlet port 31 and the outlet port 32. The microchannel 21 is designed with a cross-sectional size perpendicular to the extension direction Y of the microchannel 21 so that the mixed solution introduced from the inlet port 31 and in contact with the inner surface of the microchannel 21 is dehydrated by the water absorption of the synthetic resin, and multiple droplets are generated in the microchannel 21 by two-phase separation of the liquid layer. By configuring the apparatus to dehydrate the mixed solution using the water absorption of the chip body 11 itself that forms the microchannel 21, the configuration of the droplet manufacturing apparatus 1 can be simplified. The size (diameter) of the droplets produced by the droplet manufacturing apparatus 1 corresponds to the size of the cross-section of the microchannel 21. Therefore, the size of the droplets can be controlled by designing the size of the cross-section of the microchannel 21.

[0044] (2) The cross-sectional size of the microchannel 21 is substantially constant along the entire length L1 of the microchannel 21. This simplifies the configuration of the microchannel 21 and, consequently, the configuration of the droplet production apparatus 1.

[0045] (3) The width W1 and height H1 of the cross-section of the microchannel 21 can be set to the desired dimensions of the droplet to be manufactured. For example, the width W1 and height H1 can be set within the range of 3 μm to 30 μm, respectively. This makes it possible to manufacture droplets of a minute size that matches the size of the cross-section of the microchannel 21.

[0046] (4) The cross-sectional shape of the microchannel 21 is polygonal. This makes it possible to increase the specific surface area of ​​the inner surface of the microchannel 21 that comes into contact with the mixed solution (the surface area in contact with the inner surface of the microchannel 21 relative to the volume of the solution), thereby improving the dehydration effect of the solution.

[0047] (5) Multiple microchannels 21 are arranged parallel to each other between the inlet port 31 and the outlet port 32. This makes it possible to mass-produce droplets by using multiple microchannels 21.

[0048] One application of this embodiment is the production of solidified, or gelled, droplets within the microchannel 21. In this specification, a solidified droplet refers to a droplet that can maintain its shape even after flowing out of the microchannel 21 into a space without particle size restrictions, such as the outlet port 32.

[0049] In the first application embodiment, the first solute can be prepared to include a compound that hardens when irradiated with light of a predetermined wavelength. Examples of such compounds include water-soluble ultraviolet-curable resins. With the mixed solution standing in the microchannel 21, the mixed solution can be irradiated with light of a predetermined wavelength from the substrate 12 side. This makes it possible to produce droplets or microgels of the first aqueous solution that have solidified in the microchannel 21, which can then be recovered and used by an appropriate method from the outlet port 32.

[0050] In a second application embodiment, the first solute can be prepared to contain a compound that is liquid or fluid at a first temperature and solidifies when heated or cooled to a second temperature. Examples of such compounds include substances that undergo a sol-gel transition, such as starch, agar, pectin, and methylcellulose. Before introduction into the microchannel 21, the mixed solution and apparatus can be heated to a temperature at which the gelatin does not solidify. After phase separation in the microchannel 21, the droplets can be left to stand in the microchannel until they cool to room temperature, or they can be cooled from the outside. This allows the gelatin solidified in the microchannel 21 to be recovered and used in its solidified state from the outlet port 32. Therefore, for example, if the first solute contains gelatin and a pharmaceutical compound, droplets consisting of gelatin with a predetermined particle size and containing the pharmaceutical compound can be produced. Such droplets may be promising as carriers in DDS.

[0051] -Second Embodiment- The droplet manufacturing apparatus according to the second embodiment of the present invention will be described below. The basic configuration of the droplet manufacturing apparatus according to the second embodiment is the same as that of the first embodiment described above. The differences from the first embodiment will be mainly described below.

[0052] Figure 5 shows a perspective view illustrating the schematic configuration of the droplet manufacturing apparatus 1A according to the second embodiment, and Figure 6 shows a cross-sectional view of the droplet manufacturing apparatus 1A shown in Figure 5. Figures 5 and 6 show an example in which the flow channel section 20 has a single microchannel 21 extending between the inlet port 31 and the outlet port 32.

[0053] The droplet production apparatus 1A according to the second embodiment further includes a dehydration mechanism 40 for promoting the dehydration of the mixed solution introduced into the microchannel 21. The dehydration mechanism 40 has dehydration channels 41 and 42 arranged adjacent to each other on both sides of the microchannel 21, dehydration inlet ports 43 and 44 for introducing a dehydrating substance (described later) into the dehydration channels 41 and 42, respectively, and dehydration outlet ports 45 and 46 for accumulating the dehydrating substance discharged from the dehydration channels 41 and 42. The dehydration channels 41 and 42 and the dehydration inlet ports 43 and 44 are connected via an introduction channel 47, and the dehydration channels 41 and 42 and the dehydration outlet ports 45 and 46 are connected via an discharge channel 48. The dewatering channels 41, 42, the introduction channel 47, and the discharge channel 48 are formed by fine grooves provided on the lower surface 11a of the chip body 11, and the dewatering inlet ports 43, 44 and the dewatering outlet ports 45, 46 penetrate the chip body 11 in the height direction Z and are formed, for example, in a cylindrical shape.

[0054] The dehydration channels 41 and 42 each extend along the microchannel 21. The length L2 of the dehydration channels 41 and 42 in the longitudinal direction Y is shorter than the length L1 of the microchannel 21. The cross-section of the dehydration channels 41 and 42 perpendicular to the longitudinal direction Y is formed as a rectangle with height H2 and width W2, as shown in Figure 5. The distance T2 between the microchannel 21 and the dehydration channels 41 and 42 is. That is, a partition wall 49 with a thickness T2 is provided between the microchannel 21 and the dehydration channels 41 and 42 in the width direction X. The partition wall 49 is made of PDMS, which has water absorption properties, and plays the role of a permeable membrane, as will be described later.

[0055] The size of each cross-section of the dehydration channels 41 and 42 is formed to be larger than the size of the cross-section of the microchannel 21. The height H2 and width W2 of the cross-sections of the dehydration channels 41 and 42 are appropriately determined considering the dehydration effect of the mixed solution in the microchannel 21. For example, the height H2 and width W2 can be set within the range of approximately 80 μm to approximately 150 μm, respectively. However, the size of each cross-section of the dehydration channels 41 and 42 is not limited to those described above, and can be made smaller than the size of the cross-section of the microchannel 21, as long as the dehydration effect of the mixed solution in the microchannel 21 can be achieved. However, since the dehydration effect is enhanced by increasing the surface area of ​​the inner surface of the dehydration channels 41 and 42, it is preferable that the size of each cross-section of the dehydration channels 41 and 42 be larger than the size of the cross-section of the microchannel 21.

[0056] The manufacturing method for the microfluidic chip 10 equipped with a dehydration mechanism 40 is basically the same as that of the first embodiment described above. That is, a single phase-separation microchannel 21 and dehydration channels 41 and 42 arranged on both sides of the microchannel 21 are drawn using 3D CAD software. Using the created design of the microchannel 21 and dehydration channels 41 and 42, a mold is fabricated on a glass substrate using a microscale 3D printer. A PDMS solution is poured into the mold and heat-cured, then the PDMS is peeled off to adjust the size, and the parts corresponding to the inlet port 31 and outlet port 32, and the parts corresponding to the dehydration inlet ports 43 and 44 and dehydration outlet ports 45 and 46 are punched out. The microfluidic chip 10 is fabricated by joining the chip body 11 formed in this way to a glass slide 12.

[0057] Although Figure 6 shows an example where the cross-sections of the dehydration channels 41 and 42 are rectangular, the shape of the cross-sections of the dehydration channels 41 and 42 is not limited to this. The cross-sections of the dehydration channels 41 and 42 can be, for example, polygons including triangles, pentagons, trapezoids, and rhombuses, as well as circular and elliptical shapes. The cross-sections of the dehydration channels 41 and 42 are selected appropriately considering the dehydration effect of the mixed solution in the microchannel 21, the ease of manufacturing the microfluidic chip 10, etc. The length L2 of the dehydration channels 41 and 42, and the distance T2 between the microchannel 21 and the dehydration channels 41 and 42, affect the dehydration effect of the mixed solution in the microchannel 21, so they are appropriately determined according to the concentration and type of the mixed solution, etc.

[0058] The introduction channel 47 is a channel for guiding the dewatering material injected into the dewatering inlet ports 43 and 44 to the dewatering channels 41 and 42, and is connected such that the angle between the introduction channel 47 and the dewatering channels 41 and 42 is acute. The discharge channel 48 is a channel for guiding the dewatering material discharged from the dewatering channels 41 and 42 to the dewatering outlet ports 45 and 46, and is connected such that the angle between the discharge channel 48 and the dewatering channels 41 and 42 is acute. The cross-sections of the introduction channel 47 and the discharge channel 48 are formed to be the same shape as those of the dewatering channels 41 and 42.

[0059] The dehydrating substance injected into the dehydration inlet ports 43 and 44 is introduced into the dehydration channels 41 and 42 via the introduction channel 47 by capillary action. The partition wall 49 between the microchannel 21 and the dehydration channels 41 and 42 functions as a permeable membrane, allowing water from the mixed solution in the microchannel 21 to permeate (move) into the dehydration channels 41 and 42. In this way, the dehydration mechanism 40 is configured to promote the dehydration of the mixed solution introduced into the microchannel 21 by providing dehydration channels 41 and 42 adjacent to the microchannel 21 and making the partition wall 49 function as a permeable membrane.

[0060] The configuration of the dewatering mechanism 40 is not limited to those shown in Figures 5 and 6 and described above, and various modifications are possible as long as the mixed solution in the microchannel 21 can be properly dewatered. For example, in the above-described embodiment, a pair of dewatering channels 41 and 42 are arranged on both sides in the width direction X of the microchannel 21, but instead, the dewatering channel may be provided on only one side of the microchannel 21.

[0061] Next, a droplet production method according to a second embodiment of the present invention will be described. The droplet production method includes the steps of introducing a mixed solution of a first aqueous solution and a second aqueous solution into a microchannel of a droplet production apparatus according to the second embodiment under non-phase separation conditions, and introducing a dehydrating substance into a dehydration channel of the droplet production apparatus according to the second embodiment.

[0062] The step of introducing the mixed solution into the microchannel may be the same as in the first embodiment. Therefore, the preparation conditions and preparation operation of the mixed solution may be the same as in the first embodiment, and the operation of introducing the mixed solution into the microchannel may also be the same as in the first embodiment. In addition, the droplet production apparatus according to the second embodiment has a large dehydration effect due to the provision of a dehydration channel, so it is possible to produce droplets using a mixed solution that is in non-phase separation conditions away from the phase separation curve. The mixed solution in non-phase separation conditions away from the phase separation curve may have an even lower concentration than the concentration in non-phase separation conditions near the phase separation curve described above. For example, in the case of a mixed solution of dextran and PEG, the concentration of the mixed solution in non-phase separation conditions away from the phase separation curve is about 40% to 80% (if the concentration on the phase separation curve is 5% by mass, then 3% to 1% by mass), preferably 40% (if the concentration on the phase separation curve is 5% by mass, then 3% by mass) lower than the concentration on the phase separation curve, meaning that it is in a condition with a high water content.

[0063] The dehydrating substance used in the second embodiment may be any substance that absorbs water in the mixed solution, and may be a liquid or a solid. If the dehydrating substance is a liquid, a liquid with a higher solute concentration than the mixed solution and capable of drawing water from the mixed solution through the septum can be used. For example, an aqueous sodium chloride solution with a higher osmotic pressure than the mixed solution can be used, but it is not limited to a specific salt solution. The dehydrating substance may also be a solid substance such as a water-absorbing polymer, and water in the mixed solution can be absorbed by filling the dehydration channel with the solid substance.

[0064] The introduction of the dehydrating substance into the dehydration channel may be performed before the introduction of the mixed solution into the microchannel, substantially simultaneously with the introduction, or after the introduction. For the purpose of using the introduction of the dehydrating substance as a trigger for droplet production and generating droplets at a predetermined time, it is preferable to introduce the dehydrating substance into the dehydration channel after the introduction of the mixed solution into the microchannel. The dehydrating substance may be flowed through the dehydration channel or left to stand in the dehydration channel. The method of flowing the dehydrating substance through the dehydration channel at a predetermined rate and the method of leaving it to stand can be carried out in the same manner as the method of flowing the mixed solution through the microchannel at a predetermined rate and the method of leaving it to stand, as described in the first embodiment.

[0065] In addition to the effects and advantages of the first embodiment described above, the droplet manufacturing apparatus 1A and droplet manufacturing method according to the second embodiment described above can also provide the following effects and advantages.

[0066] (1) The chip body 11 is positioned adjacent to the microchannel 21 and further has dehydration channels 41 and 42 through which a dehydrating substance flows to promote dehydration of the mixed solution. In addition to the water absorption of the chip body 11 itself that forms the microchannel 21, the provision of dehydration channels 41 and 42 enables effective dehydration from the mixed solution. Furthermore, the timing of dehydration from the mixed solution in the microchannel 21 can be adjusted by the timing of introducing the dehydrating substance into the dehydration channels 41 and 42, thereby controlling the timing of droplet formation in the microchannel 21. [Examples]

[0067] The present invention will be described in more detail with reference to examples. However, the present invention is not limited to the following examples.

[0068] [Example 1] Droplet production using a 4%:4%=PEG:DEX mixed solution (non-phase separation conditions) (Manufacturing of a droplet production device) The droplet production apparatus shown in Figures 1-3 was manufactured using PDMS. Hereafter, the apparatus used in this embodiment will also be referred to as the "PDMS device." To produce the PDMS device, a mold was first created, and then a PDMS sheet with microchannels was fabricated on the mold using a PDMS molding method. Subsequently, the PDMS sheet was attached to a glass substrate to complete the PDMS device used in this experiment. A detailed explanation follows below.

[0069] (Mold fabrication for PDMS devices (1): CAD design) First, the microchannel design was drawn on a PC using 3D CAD software Rhinocerous3D (version 7, TLM, Inc.) so that the concave and convex surfaces were reversed. Each microchannel had dimensions based on Figure 2: W1=20μm, H1=20μm, and L1=30mm. A structure was designed in which 11 of these microchannels were arranged in parallel at intervals of T1=20μm.

[0070] (Mold preparation for PDMS devices (2): Glass substrate preparation) Next, the manufacturing process for the PDMS device was carried out in a yellow cleanroom facility. Using the created design, a microscale 3D printer (Quantum X Bio, Nanoscribe GmbH&Co.KG) employing two-photon polymerization was used to fabricate a mold on a glass substrate. A methacrylated glass slide (thickness: 1.0~1.2 mm, 76 × 26 mm, Matsunami Glass Industry Co., Ltd. S1112) was used as the glass substrate. The glass slide substrate was cleaned by immersing it in acetone (reagent grade, Kishida Chemical), isopropyl alcohol (reagent grade, Kishida Chemical), and ultrapure water, respectively, and ultrasonically cleaning for 10 minutes, followed by air drying with nitrogen gas. After cleaning, the substrate was treated with oxygen plasma for 15 seconds using an RF / DC sputtering apparatus (SC-708, Sanyu Electronics) to introduce hydroxyl groups to the surface. The substrate was immersed in a 2% (vol / vol) solution of TMSPMA (3-(trimethoxysilyl)propyl methacrylate, Sigma-Aldrich) prepared in isopropyl alcohol (same as above) and left to stand at room temperature for 2 hours. After the methacrylate reaction, the substrate was removed and thoroughly rinsed with acetone (same as above), isopropyl alcohol (same as above), and ultrapure water to remove any unreacted TMSPMA. Immediately after washing, the substrate was air-dried using a nitrogen gas blower. It was stored in a desiccator until use, and the storage (use) period was within 2 days after substrate preparation.

[0071] (Mold fabrication for PDMS devices (3): Mold fabrication using a microscale 3D printer) On a substrate with a methacrylate-treated glass slide surface, 500 μL of negative-type two-photon polymerization resin IP-S (Nanoscribe GmbH&Co.KG) was dropped, and a microchannel designed in CAD was fabricated using a microscale 3D printer. Fabrication was performed using a femtosecond laser (wavelength 780 nm) with a laser output of 115 mW and a laser scanning speed of 250 mm / s. After fabrication, the substrate was developed by immersion in propylene glycol monomethyl ether acetate (≧99.5%, Sigma-Aldrich) for 10 minutes, and then washed by immersion in isopropyl alcohol (≧99.9%, Sigma-Aldrich) for 1 minute. Immediately after washing, the substrate was air-dried using a nitrogen gas blower.

[0072] (PDMS molding and attachment to glass substrate, completed) For molding, a solution of PDMS (polydimethylsiloxane, Toray Dow Corning) was prepared. The PDMS substrate solution and the PDMS catalyst solution were mixed in a weight ratio of 10:1. After mixing, the mixture was degassed in a desiccator for 30 minutes to remove air bubbles. The degassed PDMS mixture was poured into the mold (the slide glass created above), and degassed again in a desiccator for 30 minutes to remove air bubbles. The mold was placed in a 70°C oven for 2 hours to heat-cur the PDMS. The cured PDMS and mold were slowly cooled to room temperature, and the cured PDMS was carefully peeled off the mold. The peeled PDMS was cut with a utility knife to a size that could be placed on the slide glass, and then an inlet port (3 mm in diameter) and an outlet port (1.5 mm in diameter) were punched out using a biopsy trephine (Kai Industries).

[0073] (assembly) Both the molded PDMS and the cleaned glass slide surface were treated with oxygen plasma for 15 seconds using an RF / DC sputtering apparatus (same as above). For cleaning the glass slide substrate, the glass slide (same as above) was immersed in acetone (reagent grade, Kishida Chemical), isopropyl alcohol (reagent grade, Kishida Chemical), and ultrapure water respectively, and ultrasonically cleaned for 10 minutes, after which the substrate was air-dried with nitrogen gas. The oxygen plasma-treated PDMS and the glass surface were immediately brought into contact at room temperature and left to stand for 10 minutes to permanently bond, thereby manufacturing the PDMS device of Example 1.

[0074] (Mixed solution preparation) For the two-phase separation solution, an aqueous two-phase system consisting of polyethylene glycol (hereinafter referred to as PEG) (MW=7300-9300, Fujifilm) and dextran (hereinafter referred to as DEX) (MW=180,000-210,000, Fujifilm) was used. Each was mixed in ultrapure water to a concentration of 8% (wt / vol), and then mixed by inversion using a rotator at room temperature for 24 hours to ensure complete dissolution. The prepared 8% (wt / vol) PEG and DEX solutions were mixed in a volume ratio of 1:1 using a vortex mixer, and the phase separation near the binomial line was observed. Non The composition was set to PEG:DEX = 4% (wt / vol):4% (wt / vol), corresponding to the phase separation state. To visualize the DEX phase using a fluorescence microscope, 0.0125% (wt / vol) fluorescein isothiocyanate-bound DEX (hereinafter referred to as FITC-DEX) (average MW = 250,000, Sigma-Aldrich) was added. Here, the DEX phase refers to a droplet of the first aqueous solution that is rich in DEX.

[0075] (Solution introduction into PDMS device and observation) After fixing the PDMS device prepared using the method described above onto the stage of an inverted fluorescence microscope (TE-2000, Nikon), it was confirmed that there was no dust or other foreign matter in the inlet and outlet ports. The PEG:DEX = 4% (wt / vol):4% (wt / vol) solution containing the above-mentioned FITC-DEX was vigorously mixed for 20 seconds using a vortex mixer. Immediately after mixing, 20 μL of the solution was carefully injected into the inlet port. The injection was done slowly to avoid introducing air bubbles. The solution was allowed to stand until it was naturally drawn into the microchannels within the PDMS device by capillary action, completely filling the microchannels and reaching the outlet port. Immediately thereafter, the solution was injected into the outlet port, taking care not to introduce air bubbles. The amount of liquid injected into the outlet port was adjusted as needed while observing under the microscope to minimize flow during microscopic observation. Furthermore, the introduction of solution into the microfluidic channel is not limited to capillary action; it can also be performed by external pressurization (inlet port) or aspiration (outlet port) using a pump (syringe pump, etc.). Observation of bright-field and fluorescence images with a microscope was performed using a 20x objective lens (Plan Fluor ELWD, NA=0.45, Nikon) and an EM-CCD digital camera (C9100-13, Hamamatsu Photonics), and video frames were recorded using the recording software AQUACOSMOS (Hamamatsu Photonics) on a PC (video is not shown in the examples). All observations were performed at room temperature.

[0076] Figure 7 is a fluorescence microscope image of the microchannel, where the white areas represent the DEX phase containing FITC. Multiple nearly spherical, uniform droplets with a diameter of approximately 20 μm, corresponding to the width and height of the microchannel, were formed and aligned within the microchannel. Initially, the mixed solution was at a concentration for non-phase separation conditions. However, upon introduction into the microchannel, dehydration and absorption occurred due to the inner wall surface of the PDMS that defines the microchannel, causing the mixed solution to reach a phase separation concentration, leading to phase separation and droplet formation.

[0077] [Example 2] Droplet production using a 3.4%:4.5% PEG:gelatin mixed solution (non-phase separation conditions) Droplet production was carried out using a mixed solution under non-phase separation conditions, with the same droplet separation apparatus as in Example 1.

[0078] (Coating of microfluidic channels) The inner walls of the microchannels of the PDMS device, similar to that in Example 1, were coated with poly(D-lysine)-graft-poly(ethylene glycol) (hereinafter referred to as PDL-g-PEG). The preparation method for PDL-g-PEG is as follows: 100 μL of 0.5 M Borate Buffer (pH 8.5±0.2, Polyscience Inc.) was added to a 1.0 mg / mL aqueous solution of poly(D-lysine) (A-003-E, Sigma-Aldrich) and mixed. Then, 70 μg of N-hydroxylsuccinimide-functionalized poly(ethylene glycol) derivative (SUNBRIGHT ME-020AS, NOF Corporation) was added and stirred with a vortex mixer until completely dissolved. The solution was then allowed to stand at room temperature in the dark for 60 minutes. The resulting solution was dispensed into 100 μL portions and stored at -20°C until use. The coating was performed immediately after the permanent bonding of the PDMS and the glass slide as described in Example 1. Specifically, the microchannel was filled with a 100 μg / mL PDL-g-PEG solution and incubated at room temperature for 60 minutes. Then, the unreacted PDL-g-PEG was washed out by passing 1 mL of ultrapure water through the microchannel, and the device was allowed to air dry. The PDMS device with the PEG-coated microchannel was stored in a desiccator and used within 3 days of preparation.

[0079] (Mixed solution preparation) For the two-phase separation solution, an aqueous two-phase system consisting of polyethylene glycol (hereinafter referred to as PEG) (average MW = 20000, Fujifilm) and gelatin (derived from bovine bone, 071-06291, lot number: LEQ1590, Fujifilm) was used. PEG was added to ultrapure water at 65°C to a concentration of 3.4% (wt / vol) and stirred with a vortex mixer until completely dissolved. Subsequently, gelatin was added to the 3.4% (wt / vol) PEG solution at 65°C to a concentration of 4.5% (wt / vol) and stirred with a vortex mixer until completely dissolved. This solution was incubated at 65°C until it was introduced into the PDMS device.

[0080] (Solution introduction into PDMS device and observation) A PDMS device having a microchannel coated with PEG using the method described above was incubated with the above-mentioned PEG:gelatin = 3.4% (wt / vol):4.5% (wt / vol) solution until it reached 65°C. While the PDMS device was kept warm, the PEG:gelatin solution was vigorously mixed for 20 seconds using a vortex mixer. Immediately after mixing, 20 μL of the solution was carefully injected into the inlet port. The injection was done slowly to avoid introducing air bubbles. The solution was naturally drawn into the microchannel within the PDMS device by capillary action from the inlet port until the microchannel was completely filled and the solution reached the outlet port. Immediately thereafter, the solution was injected into the outlet port, taking care not to introduce air bubbles. The device filled with the PEG:gelatin solution was slowly cooled to room temperature and allowed to stand for 2 hours to initiate phase separation and gel the gelatin-rich phase. The microchannel was observed in the same manner as in Example 1.

[0081] Figure 8 is a bright-field microscope image of the microchannel. A uniform arrangement of droplets composed of gelatin of the same size as the channel (i.e., approximately 20 μm) was observed.

[0082] [Comparative Example 1] Droplet production using a 5%:5% PEG:DEX mixed solution (phase separation conditions) Using a droplet separation apparatus similar to that in Example 1, we attempted to produce droplets using a mixed solution under phase separation conditions.

[0083] (Preparation of a mixed solution under phase separation conditions) As a mixed solution, an aqueous two-phase system consisting of PEG (MW=7300-9300, Fujifilm) and DEX (MW=180,000-210,000, Fujifilm) was used. Each was mixed in ultrapure water to a concentration of 10% (wt / vol), and the mixture was inverted and mixed at room temperature for 24 hours using a rotator to ensure complete dissolution. The prepared 10% (wt / vol) aqueous solutions of PEG and DEX were mixed in a volume ratio of 1:1 using a vortex mixer to obtain a composition of PEG:DEX = 5% (wt / vol):5% (wt / vol), which corresponds to the phase separation state near the binomial line of phase separation. This composition was a mixed solution under phase separation conditions. To visualize the DEX phase using a fluorescence microscope, 0.0125% (wt / vol) of FITC-DEX (average MW=250,000, Sigma-Aldrich) was added.

[0084] The introduction and observation of the mixed solution into the PDMS device was performed in the same manner as in Example 1. Figure 9 is a bright-field microscope image of the microchannel. Figure 10 is a fluorescence microscope image of the microchannel, where the white areas represent the DEX-rich phase containing FITC. Figures 9 and 10 show images of different locations in the microchannel. Non-spherical or heterogeneous droplet formation was observed in both Figures 9 and 10.

[0085] [Example 3] Droplet production using a 3%:3%=PEG:DEX mixed solution (non-phase separation conditions) and a droplet separation apparatus equipped with a dehydration channel. The droplet separation device shown in Figures 5 and 6 was manufactured using PDMS. The manufacturing process for the droplet separation device was the same as in Example 1, except that the CAD design process involved designing the microchannel for introducing the mixed solution and the dehydration channel for introducing the dehydration solution, as described below. The microchannel had a width W1=20μm, a height H1=20μm, and a length L1=28mm, based on Figures 2 and 6. Two dehydration channels were placed on either side of the microchannel in the x-direction, separated by a distance of T1=20μm, for a total of two channels. Based on Figure 6, the width W2=100μm and height H2=20μm were set, and the length of the region where the microchannel and dehydration channel ran parallel was 20mm.

[0086] In the subsequent steps of glass substrate preparation, mold fabrication using a 3D printer, attachment to the glass substrate, and assembly, the droplet separation device of Example 3 was manufactured in the same manner as in Example 1, except that the inlet port of the phase separation microchannel and all ports of the dehydration microchannel were punched out with a 3 mm diameter biopsy trephine, and the outlet port of the phase separation microchannel was punched out with a 1.5 mm diameter biopsy trephine.

[0087] The preparation of the mixed solution was carried out in the same manner as in Example 1, except that PEG and DEX were each mixed in ultrapure water to a concentration of 6% (wt / vol), resulting in a final mixed solution with a composition of PEG:DEX = 3% (wt / vol):3% (wt / vol). The introduction of the mixed solution into the microfluidic channel and observation were carried out in the same manner as in Example 1.

[0088] Next, after confirming by observation that no phase separation had occurred in the microchannel, 20 μL of 5 M NaCl aqueous solution (31334-51, Nacalai Tesque) was carefully injected into one port of the dehydration channel to initiate phase separation. The injection was done slowly to avoid introducing air bubbles. The solution was naturally drawn from one port to the other by capillary action and filled. The observation method and observation temperature conditions were the same as in Example 1. Figure 11 is a fluorescence microscope image of the microchannel, where the white areas are the DEX phase containing FITC. The dehydration channel was invisible because no fluorescent substance was present. (b) is an image taken when the NaCl aqueous solution was fully filled, with t=0. (c) is a fluorescence microscope image taken at t=30 seconds, (d) at t=60 seconds, and (a) at t=-300 seconds when the mixed solution had fully filled the microchannel. These results confirmed that as dehydration progresses, uniformly sized droplets with a shape close to a perfect sphere are formed. [Explanation of symbols]

[0089] 1,1A droplet production equipment 10 Microfluidic Chips 11 Chip body, 11a Bottom surface 12 boards 20 Flow channel section, 21 Microchannel 31 Inlet port, 32 Exit port 40 Dehydration mechanism, 41, 42 Dehydration channels

Claims

1. A droplet manufacturing apparatus that uses a microfluidic chip to produce droplets of the first aqueous solution in the second aqueous solution from a mixed solution of the first aqueous solution and the second aqueous solution, The aforementioned microfluidic chip is A chip body formed from a water-absorbing resin, A substrate attached to the lower surface of the chip body and Equipped with, The chip body is An inlet port for introducing the aforementioned mixed solution, An outlet port for collecting droplets, A microchannel connected to the inlet port and the outlet port, extending between the inlet port and the outlet port, It has, A droplet manufacturing apparatus in which the size of the cross-section perpendicular to the extending direction of the microchannel is designed such that the mixed solution introduced from the inlet port and in contact with the inner surface of the microchannel is dehydrated by the water absorption of the resin, and multiple droplets are generated in the microchannel by two-phase separation of the liquid layer.

2. The droplet manufacturing apparatus according to claim 1, wherein the size of the cross-section of the microchannel is substantially constant over the entire length of the microchannel.

3. The droplet manufacturing apparatus according to claim 2, wherein the width and height of the cross-section of the microchannel are set within the range of 3 μm to 30 μm, respectively.

4. The droplet manufacturing apparatus according to claim 1, wherein the cross-sectional shape of the microchannel is polygonal.

5. The droplet manufacturing apparatus according to claim 1, wherein a plurality of the microchannels are arranged parallel to each other between the inlet port and the outlet port.

6. The droplet manufacturing apparatus according to claim 1, wherein the chip body further comprises a dehydration channel disposed adjacent to the microchannel, into which a dehydrating substance is introduced to promote dehydration of the mixed solution.

7. A method for producing droplets of a first aqueous solution in a second aqueous solution, comprising the step of introducing a mixed solution of the first aqueous solution and the second aqueous solution into the microchannel of the droplet production apparatus described in claim 1 under non-phase separation conditions.

8. A method for producing droplets according to claim 7, comprising the step of introducing a dehydrating substance near the microchannel, thereby promoting the absorption of water from the mixed solution from the inner surface of the microchannel.

9. A method for producing droplets of a first aqueous solution in a second aqueous solution, comprising the steps of introducing a mixed solution of the first aqueous solution and the second aqueous solution into the microchannel of the droplet production apparatus described in claim 6 under non-phase separation conditions, A step of introducing a dehydrating substance into the dehydration channel described in claim 6. A method for producing droplets, including the method described above.

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