Method and device for generating emulsion in microfluidic chip

Abstract

To provide an improved method and device for generating an emulsion, the method for separately supplying a continuous phase liquid and a dispersed phase liquid to a micro-channel chip.SOLUTION: In a micro-channel chip 10, dispersed phase liquid holding units 102, 103 are connected with an emulsion forming unit 120 through a dispersed phase liquid channel 117; a continuous phase liquid holding unit 101 is connected with the emulsion forming unit 120; the emulsion forming unit 120 is connected with an exhaust port 150 through an emulsion channel 130. A method includes, with an external liquid sending driving force, in the emulsion forming unit 120, generating an emulsion including droplets formed of the dispersed phase liquid and a continuous phase formed of the continuous phase liquid, and causing the emulsion to enter the emulsion channel 130. A surface of a wall surface of a dispersed phase liquid channel 117 is formed of an inorganic material and / or an organic material not containing a fluorine compound and not treated with a fluorine compound.SELECTED DRAWING: Figure 1

Description

[Technical field]

[0001] The present invention relates to a method and device for producing an emulsion in a microchannel chip. [Background technology]

[0002] The microdroplet method, which divides a reaction solution into microcompartments and performs reactions independently, is known as a technique for dividing a reaction solution into microcompartments and performing reactions independently. This method is expected to be applied to, for example, the production of micro- and nano-particles, and in particular, it is used for digital measurement to measure the presence or absence of a target molecule by the presence or absence of a signal by dividing a target molecule into microcompartments using a microfluidic device and performing a reaction in the microdroplet, thereby performing absolute quantification of the number of target molecules.

[0003] Microdroplet techniques generally use emulsions that consist of a continuous phase, such as oil, and droplets of an aqueous solution dispersed in the continuous phase.

[0004] Non-Patent Document 1 discloses a centrifugal step droplet generation method. The document describes that the inlet of the device is filled with oil, and the oil is sent to a droplet collection chamber by centrifugation, and then a sample solution is introduced from the same inlet and droplets are generated by centrifugation.

[0005] In contrast to such droplet generation methods, a method is known in which a dispersed phase liquid such as a reaction liquid and a continuous phase liquid such as an oil are supplied to a microchannel chip via separate supply parts and then merged within the chip to generate an emulsion.

[0006] US Patent No. 5,999,633 discloses such a system and method for generating droplets suitable for droplet assays. The document describes transporting the generated droplets to an outlet area consisting of a pipette tip or a droplet well. The document also describes an air trap that substantially separates the sample and oil until application of a fluid driving force (such as negative or positive pressure).

[0007] In addition, in recent years, droplet array measurement, in which droplets are aligned in a monolayer in a detection area to easily measure signals, has been attracting attention as a method for simplifying and speeding up the device.

[0008] Patent Documents 2 and 3 disclose a microchannel chip having a channel for forming droplets and a droplet holder for holding droplets. Patent Document 2 describes forming droplets by joining two or more reaction liquids together and then contacting them with an immiscible liquid that is not miscible with the reaction liquids. Patent Document 3 describes forming droplets by contacting the dispersed phase and continuous phase flowing in from a dispersed phase inlet and a continuous phase inlet in a droplet generating section via a channel.

[0009] Non-Patent Document 2 describes a method for generating droplets on a chip and an apparatus for doing so. The method described in this document includes an operation of filling a droplet array part with oil (filling operation) before liquid transfer. [Prior art documents] [Patent documents]

[0010] [Patent Document 1] European Patent No. 2550528 [Patent Document 2] JP 2019-170363 A [Patent Document 3] JP 2020-169911 A [Non-patent literature]

[0011] [Non-Patent Document 1] Centrifugal step emulsification applied for absolute quantification of nucleic acids by digital droplet RPA, Lab Chip, 2015, 15, 2759-2766 [Non-Patent Document 2] 1-Million droplet array with wide-field fluorescence imaging for digital PCR, Lab on a Chip, 2011, 11, 3838-3845 Summary of the Invention [Problem to be solved by the invention]

[0012] The method of supplying the continuous phase liquid and the dispersed phase liquid to respective supply parts provided separately on the microchannel chip and passing them through the respective channels from the supply parts to join at the emulsion forming part has an advantage of being able to increase the droplet generation speed, etc. However, when the continuous phase liquid moves through the dispersed phase liquid channel, such as when the channel in the microchannel chip is filled with the continuous phase liquid in advance, the stability of the liquid transfer and the uniformity of the droplets may become insufficient.

[0013] An object of the present invention is to provide an improved method for producing an emulsion in a method in which a continuous phase liquid and a dispersed phase liquid are supplied separately to a microchannel chip, and to provide an apparatus that can be used in such a method. [Means for solving the problem]

[0014] The above-mentioned problems of the present invention can be solved by the following aspects of the present invention: <Aspect 1> A method for producing an emulsion by supplying a dispersed phase liquid and a continuous phase liquid to a microchannel chip, comprising the steps of: the microchannel chip has a dispersed phase liquid holding section, a dispersed phase liquid flow path, a continuous phase liquid holding section, a continuous phase liquid flow path, an emulsion forming section, an emulsion flow path, and an outlet; the dispersed phase liquid holding unit is connected to the emulsion forming unit via the dispersed phase liquid flow path, the continuous phase liquid holding unit is connected to the emulsion forming unit via the continuous phase liquid flow path, the emulsion forming section is connected to the outlet through the emulsion flow path, Supplying a dispersed phase liquid to the dispersed phase liquid holding unit; supplying a continuous phase liquid to the continuous phase liquid holding section; and generating an emulsion including droplets composed of the dispersed phase liquid and a continuous phase composed of the continuous phase liquid in the emulsion forming section by an external liquid sending driving force, and causing the emulsion thus generated to enter the emulsion flow path; Including, The surface of the wall of the dispersed phase liquid flow path is made of an inorganic material and / or an organic material that does not contain a fluorine compound and is not treated with a fluorine compound. method. <Aspect 2> The method according to aspect 1, wherein the surface of the wall of the dispersed phase liquid holding portion is made of an inorganic material and / or an organic material that does not contain a fluorine compound and is not treated with a fluorine compound. <Aspect 3> 3. The method of claim 1 or 2, wherein the continuous phase liquid is a fluorinated dispersion medium. <Aspect 4> The method according to any one of aspects 1 to 3, wherein the continuous phase liquid comprises a fluorosurfactant. <Aspect 5> The method according to any one of aspects 1 to 4, wherein the dispersed phase liquid is an aqueous solution. <Aspect 6> The method according to any one of aspects 1 to 5, wherein the inorganic material and / or organic material that does not contain a fluorine compound and is not treated with a fluorine compound is at least one selected from polydimethylsiloxane (PDMS), glass, cyclic olefin copolymer (COC), cyclic olefin polymer (COP), and polymethyl methacrylate (PMMA). <Aspect 7> 7. The method according to any one of aspects 1 to 6, wherein the walls of the dispersed phase liquid flow channels have surfaces treated with a non-fluorine-based surface treatment agent. <Aspect 8> 8. The method according to any one of aspects 1 to 7, wherein the external liquid delivery driving force is a negative pressure applied to the outlet. <Aspect 9> The dispersed phase liquid holding portion includes a first dispersed phase liquid holding portion and a second dispersed phase liquid holding portion, The dispersed phase liquid flow path includes a first dispersed phase liquid flow path connected to the first dispersed phase liquid holding portion, a second dispersed phase liquid flow path connected to the second dispersed phase liquid holding portion, and a dispersed phase liquid junction portion; the first dispersed phase liquid flow path and the second dispersed phase liquid flow path are each connected to the emulsion forming section via a dispersed phase liquid junction section; 9. The method according to any one of aspects 1 to 8, characterized in that <Aspect 10> the microchannel chip further has an emulsion-holding channel, the emulsion forming section is connected to the emulsion holding flow path via the emulsion flow path, the emulsion holding channel is connected to the outlet, and applying a negative pressure to the outlet, in the emulsion forming section, to generate an emulsion containing droplets composed of the dispersed phase liquid and a continuous phase composed of the continuous phase liquid, and transporting the emulsion thus generated through the emulsion flow path to the emulsion holding flow path filled with gas. The method according to any one of aspects 1 to 9. <Aspect 11> A microchannel chip having a dispersed phase liquid holder, a dispersed phase liquid flow path, a continuous phase liquid holder, a continuous phase liquid flow path, an emulsion forming section, an emulsion flow path, and an outlet, the dispersed phase liquid holding unit is connected to the emulsion forming unit via the dispersed phase liquid flow path, the continuous phase liquid holding unit is connected to the emulsion forming unit via the continuous phase liquid flow path, the emulsion forming section is connected to the outlet through the emulsion flow path, and a dispersed phase liquid is supplied to the dispersed phase liquid holding section, a continuous phase liquid is supplied to the continuous phase liquid holding section, and an external liquid sending driving force is applied to the micro-channel chip, whereby an emulsion containing droplets constituted of the dispersed phase liquid and a continuous phase constituted of the continuous phase liquid is generated in the emulsion forming section, and the emulsion thus generated enters the emulsion channel; The surface of the wall of the dispersed phase liquid flow path is made of an inorganic material and / or an organic material that does not contain a fluorine compound and is not treated with a fluorine compound. Microfluidic chip. Effect of the Invention

[0015] According to the present invention, it is possible to provide an improved method for producing an emulsion in a method in which a continuous phase liquid and a dispersed phase liquid are supplied separately to a microchannel chip, and an apparatus that can be used in such a method. [Brief description of the drawings]

[0016] [Figure 1] FIG. 1 is a schematic plan view of one embodiment of a microchannel chip according to the method of the present disclosure. [Diagram 2] FIG. 2 is a cross-sectional schematic diagram showing one embodiment of a dispersed phase liquid retaining portion according to the present disclosure. [Diagram 3] FIG. 3 is a schematic plan view of another embodiment of a microchannel chip according to the method of the present disclosure. [Figure 4] FIG. 4 is a photograph showing emulsion formation in one embodiment of the method of the present disclosure. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0017] <Method of producing emulsion> The method according to the present disclosure is a method for generating an emulsion by supplying a dispersed phase liquid and a continuous phase liquid to a microchannel chip, comprising the steps of: the microchannel chip has a dispersed phase liquid holding section, a dispersed phase liquid flow path, a continuous phase liquid holding section, a continuous phase liquid flow path, an emulsion forming section, an emulsion flow path, and an outlet; the dispersed phase liquid holding section is connected to the emulsion forming section via a dispersed phase liquid flow path, the continuous phase liquid holding section is connected to the emulsion forming section via a continuous phase liquid flow path; the emulsion forming section is connected to the outlet through an emulsion flow path, Supplying a dispersed phase liquid to a dispersed phase liquid holding unit; Supplying a continuous phase liquid to a continuous phase liquid holding portion; and generating an emulsion including droplets composed of a dispersed phase liquid and a continuous phase composed of a continuous phase liquid in an emulsion forming section by an external liquid sending driving force, and causing the emulsion thus generated to enter an emulsion flow path; Including, The surface of the wall of the dispersed phase liquid flow path is characterized in that it is made of an inorganic material and / or an organic material that does not contain a fluorine compound and is not treated with a fluorine compound.

[0018] As a method for generating an emulsion in a microchannel chip, a method can be used in which a continuous phase liquid and a dispersed phase liquid are supplied to respective supply parts provided separately on the microchannel chip and merged in the chip to generate an emulsion. This method has an advantage over a method in which a continuous phase liquid and a dispersed phase liquid are supplied from a single identical holding part (for example, the method of Non-Patent Document 1), in that the droplet generation speed per emulsion forming part can be increased.

[0019] However, in such a method, if the continuous phase liquid enters the dispersed phase liquid flow path, the stability of the liquid delivery and the uniformity of the droplets may become insufficient during the production of the emulsion.

[0020] Without intending to be limited by theory, it is believed that in the above case, the continuous phase liquid remains in the dispersed phase liquid flow path, which may result in undesirable effects on the stability of the liquid transfer and the formation of an emulsion. In particular, when a liquid having a relatively low surface tension and viscosity, such as a fluorinated dispersion medium, is used as the continuous phase liquid, the continuous phase liquid is likely to remain in the flow path, and this tendency is particularly pronounced.

[0021] In particular, when using a microchannel chip including a dispersed phase liquid junction that can mix two or more dispersed phase liquids in a channel, the continuous phase liquid filled in the dispersed phase liquid channel, dispersed phase liquid junction, and dispersed phase liquid holding section may remain in the channel during droplet generation, hindering uniform mixing in the droplets due to laminarization of the dispersed phase liquid at the dispersed phase liquid junction. The hindrance of uniform mixing due to the remaining continuous phase liquid is thought to be caused by the continuous phase liquid forming a thin layer between the channel wall surface and the dispersed phase liquid, and therefore it is thought that the thin layer of the continuous phase liquid can be easily removed by reducing the stability of the continuous phase liquid-dispersed phase liquid interface without adding a surfactant, but this is not preferable because it may have a contradictory effect on the generation and retention of stable droplets. In addition, in order to suppress mixing of the dispersed phase liquids before emulsion formation, an example has been shown in which emulsion formation is performed while maintaining the laminar flow state at the dispersed phase liquid junction. However, even in a dispersed phase liquid junction having a structure that promotes stirring (micromixer structure), it is thought that the behavior of the interface between the dispersed phase liquids becomes unstable, preventing uniform mixing within the droplets.

[0022] In the present invention, since the surface of the wall of the dispersed phase liquid flow path is made of an inorganic material and / or an organic material that does not contain a fluorine compound and is not treated with a fluorine compound, the affinity of the continuous phase liquid made of an oil mainly composed of fluorocarbon to the wall of the dispersed phase liquid flow path is reduced. Therefore, according to the present invention, it is possible to suppress the entry of the continuous phase liquid into the dispersed phase liquid flow path before the start of liquid transfer by application of an external liquid transfer driving force and / or the residue of the continuous phase liquid in the dispersed phase liquid flow path during liquid transfer.

[0023] The present invention will be specifically described using the microchannel chip shown in Fig. 1. Note that the microchannel chip in Fig. 1 is a schematic diagram of an exemplary embodiment, and the method according to the present invention is not limited to this embodiment and can be carried out in microchannel chips having various aspects. Fig. 1 is a schematic diagram for ease of understanding and is not drawn to scale.

[0024] The microchannel chip of FIG. 1 has a planar configuration, that is, the generation and transportation of the emulsion are substantially performed within one plane. In FIG. 1, the direction W indicates the width direction, and the direction L indicates the length direction. The direction perpendicular to W and L is the vertical direction. The microchannel chip 10 of FIG. 1 has a first dispersed phase liquid holding section 102, a second dispersed phase liquid holding section 103, a dispersed phase liquid flow path 117, a continuous phase liquid holding section 101, a continuous phase liquid flow path 111, an emulsion forming section 120, an emulsion flow path 130, and an outlet 150. The dispersed phase liquid holding sections 102 and 103 are connected to the emulsion forming section 120 via the dispersed phase liquid flow path 117, and the continuous phase liquid holding section 101 is connected to the emulsion forming section 120 via the continuous phase liquid flow path 111. 1, the dispersed phase liquid flow path 117 has a first dispersed phase liquid flow path 114, a second dispersed phase liquid flow path 115, and a dispersed phase liquid junction 116, and the continuous phase liquid flow path 111 has a first continuous phase liquid flow path 112 and a second continuous phase liquid flow path 113. The emulsion forming section 120 is connected to the discharge port 150 via the emulsion flow path 130.

[0025] When supplying the continuous phase liquid and the dispersed phase liquid to such a microchannel chip, the dispersed phase liquid flow channel may be filled with the continuous phase liquid in advance. Specifically, for example, before supplying the dispersed phase liquid, the continuous phase liquid may be moved to the dispersed phase liquid holding section filled with gas through the continuous phase liquid flow channel filled with gas, the emulsion forming section, and the dispersed phase liquid flow channel.

[0026] In this way, for example, clogging of the flow path caused by the dispersed phase liquid passing through the dispersed phase liquid flow path filled with gas can be suppressed. However, in this case, the continuous phase liquid may remain in the dispersed phase liquid flow path.

[0027] In addition, when supplying the continuous phase liquid and the dispersed phase liquid to the microchannel chip, the dispersed phase liquid can be moved to the inlet to the emulsion forming section through the dispersed phase liquid flow path filled with gas before the continuous phase liquid reaches the emulsion forming section. In this way, the intrusion of the continuous phase liquid into the dispersed phase liquid flow path can be suppressed. However, even if the dispersed phase liquid flow path is filled with the dispersed phase liquid in this way, the continuous phase liquid may intrude into the dispersed phase liquid flow path.

[0028] In the present invention, the wall surface of the dispersed phase liquid flow path 117 is made of an inorganic material and / or an organic material that does not contain a fluorine compound and is not treated with a fluorine compound. This makes it possible to suppress the continuous phase liquid from entering the dispersed phase liquid flow path and / or to efficiently remove the continuous phase liquid remaining in the dispersed phase liquid flow path, thereby suppressing or avoiding destabilization of liquid delivery and droplet generation caused by the remaining continuous phase liquid.

[0029] 1 is configured to move the dispersed phase liquid and / or the continuous phase liquid from each holding portion through each flow channel to the emulsion forming portion 120 by, for example, capillary force and / or liquid surface pressure difference. Therefore, the dispersed phase liquid and / or the continuous phase liquid supplied to the holding portion moves toward the emulsion forming portion 120.

[0030] As described above, the continuous phase liquid supplied to the continuous phase liquid holding section can be moved to the dispersed phase liquid flow path and the dispersed phase liquid holding section through the emulsion forming section filled with gas. In this case, the continuous phase liquid can be moved to the dispersed phase liquid holding section and then the dispersed phase liquid can be supplied to bring them into contact with each other. It is preferable to shorten the time from when the continuous phase liquid reaches the dispersed phase liquid holding section until the dispersed phase liquid is supplied to the dispersed phase liquid holding section and / or the time from when the dispersed phase liquid is supplied to the dispersed phase liquid holding section until the liquid transfer is started by the external liquid transfer driving force, thereby reducing the amount of continuous phase liquid flowing back to the holding section and reducing non-uniform droplets generated when the dispersed phase liquid and the continuous phase liquid contact each other in the dispersed phase liquid holding section. From the viewpoint of the residual continuous phase liquid in the dispersed phase liquid holding section and the dispersed phase liquid flow path after the liquid transfer starts, it is particularly preferable to reduce the amount of continuous phase liquid flowing back to the holding section. In this embodiment, it is preferable to prevent the continuous phase liquid from excessively filling the emulsion holding flow path before the liquid transfer (before the application of the external liquid transfer driving force).

[0031] By using the capillary force and / or the liquid level difference to move the dispersed phase liquid and the continuous phase liquid, the movement of the dispersed phase liquid and the continuous phase liquid can be easily controlled without requiring any additional device. For example, by appropriately setting the flow channel structure of the microchannel chip (characteristics of the flow channel surface, pressure loss of the flow channel, etc.), a desired capillary force can be obtained. In addition, by adjusting the amount of liquid (particularly the liquid level height) supplied to each phase liquid holding section, a desired liquid level differential pressure can be obtained. In particular, the flow channel resistance in the emulsion forming section and its upstream is sufficiently small so that the dispersed phase liquid and the continuous phase liquid can move without an external liquid delivery driving force.

[0032] In one embodiment of the present disclosure, the resistance of the emulsion forming section and its upstream channel is sufficiently small so that the dispersed phase liquid and the continuous phase liquid can come into contact with each other without an external liquid delivery driving force, so that, for example, when the dispersed phase liquid is moved to the emulsion forming section through a dispersed phase liquid channel filled with gas before the contact, the time until the dispersed phase liquid supplied to the dispersed phase liquid holding section (of an empty microchannel chip) comes into contact with the continuous phase liquid is within 5 minutes. This time is preferably within 3 minutes, more preferably within 1 minute. In this case, the time required for the generation (and optional retention) of the emulsion can be further shortened.

[0033] In another embodiment of the present disclosure, the resistance of the emulsion forming section and its upstream flow path is sufficiently small so that the dispersed phase liquid and the continuous phase liquid can contact each other without an external liquid delivery driving force, so that, for example, when the continuous phase liquid is moved to the dispersed phase liquid holding section through the continuous phase liquid flow path filled with gas, the emulsion forming section, and the dispersed phase liquid flow path before the contact, the time from when the continuous phase liquid is supplied to the continuous phase liquid holding section (of the empty microchannel chip) to when the continuous phase liquid comes into contact with the dispersed phase liquid in the dispersed phase liquid holding section is within 1 minute. This time is preferably within 30 seconds, more preferably within 15 seconds. In this case, the time required for the generation (and optional retention) of the emulsion can be further shortened.

[0034] For the above movement (of the dispersed phase liquid and / or the continuous phase liquid, particularly the dispersed phase liquid), pressure applied from the outside may be used. In particular, negative pressure is applied to the outlet, and thus the dispersed phase liquid is moved to the emulsion forming section through the dispersed phase liquid flow path filled with gas. In general, since the dispersed phase liquid has a low affinity for the surface of the flow path, it is preferable to increase the moving speed of the dispersed phase liquid. By using negative pressure as one of the means, the device can be simplified. For the pressure applied from the outside, the following description regarding the external liquid sending driving force can be referred to.

[0035] When controlling the above-mentioned pre-contact movement by applying positive pressure to each holding part, in order to control the minute amounts of each phase liquid in the flow path, it is necessary to control the positive pressure source to a small pressure and / or control the application time of the positive pressure to an extremely short time, which may reduce the reproducibility of the device.

[0036] On the other hand, for example, when negative pressure is applied to the outlet when the dispersed phase liquid moves through the dispersed phase liquid flow path before the continuous phase liquid is supplied, the continuous phase liquid flow path and the continuous phase liquid holder are not blocked by liquid, so the flow path from the outlet to the continuous phase liquid holder is open to the outside air and a negative pressure smaller than the negative pressure of the negative pressure supply source is applied to the dispersed phase liquid. This is preferable in terms of precisely controlling the movement of minute amounts of dispersed phase liquid within the flow path, as the applied pressure can be easily controlled to be small, making it easier to ensure reproducibility as an apparatus.

[0037] Furthermore, when negative pressure is applied to the outlet when the continuous phase liquid moves through the continuous phase liquid flow path before the dispersed phase liquid is supplied, since the continuous phase liquid generally has a low surface tension and / or viscosity, the movement using the external liquid delivery driving force requires more precise control, which is more preferable. Note that the control of the movement is important because it is directly linked to the suppression of the generation of bubbles due to the blockage of the flow path. In addition, in the above case, since the pressure of the negative pressure supply source is released to the outside air and gradually decreases, it is more preferable to have a mechanism that can maintain the pressure.

[0038] In the present invention, an emulsion containing droplets composed of a dispersed phase liquid and a continuous phase composed of a continuous phase liquid is generated in the emulsion forming unit 120 by an external liquid-sending driving force, and the emulsion thus generated is caused to enter the emulsion flow channel 130. Negative pressure or positive pressure can be used as the external liquid-sending driving force. Negative pressure can be applied, for example, by a suction device connected to the outlet 150 of the microchannel chip 10. Positive pressure can be applied, for example, by a pressure application device connected to the dispersed phase liquid holding units 102 and 103 and the continuous phase liquid holding unit 101.

[0039] The negative pressure can be applied, for example, by a suction device connected to the outlet 150 of the microchannel chip 10. Moreover, the positive pressure can be applied, for example, by a pressure application device connected to the dispersed phase liquid holding units 102 and 103 and the continuous phase liquid holding unit 101.

[0040] <Wall surface of dispersed phase liquid flow path> As described above, in the method according to the present disclosure, the wall surfaces of the dispersed phase liquid flow paths are made of inorganic and / or organic materials that do not contain or are not treated with a fluorine compound.

[0041] In addition to the dispersed phase liquid flow path, the wall surface of the dispersed phase liquid retaining portion may be made of an inorganic material and / or an organic material that does not contain a fluorine compound and is not treated with a fluorine compound. In this case, the continuous phase liquid can be prevented from remaining in the dispersed phase liquid retaining portion, which is particularly preferable.

[0042] Preferably, the inorganic material and / or organic material that does not contain a fluorine compound and is not treated with a fluorine compound is at least one selected from polydimethylsiloxane (PDMS), glass, cyclic olefin copolymer (COC), cyclic olefin polymer (COP), and polymethyl methacrylate (PMMA). These materials are highly transparent, and therefore are also preferred from the viewpoint of ease of quality control in the manufacture of microchannel chips. In addition, they are particularly preferred when optical signal detection is performed within the microchannel chip.

[0043] These materials, in particular, have a relatively low affinity for the continuous phase liquid composed of a fluorine-based dispersion medium, so that the continuous phase liquid can be prevented from entering the dispersed phase liquid flow path before the liquid is fed by application of an external liquid feeding driving force, and even if the continuous phase liquid remains on the surface of the dispersed phase liquid flow path, the residual continuous phase liquid is relatively quickly removed by the dispersed phase liquid passing through the dispersed phase liquid flow path. In addition, these materials are also preferable in that they are suitable as materials for forming the substrate or flow path constituting the microchannel chip in terms of rigidity, processability, leak resistance, etc. Furthermore, these materials have a relatively low affinity for the dispersed phase liquid, which is generally an aqueous solution, so that good droplet generation is ensured.

[0044] For example, when a microchannel chip is fabricated by bonding a polymer sheet made of PDMS having a channel structure to a cover glass as a substrate, when the microchannel chip is in use, the surfaces of the side walls and upper wall (ceiling) of the dispersed phase liquid channel are made of PDMS, and the lower wall (floor) of the dispersed phase liquid channel is made of glass.

[0045] Here, in the case where the flow path is formed of a surface (such as glass) having a relatively high affinity for the dispersed phase liquid, when the dispersed phase liquid flow path is filled with the continuous phase liquid and the liquid transfer is started, the dispersed phase liquid wets the wall surface early, the flow path is blocked by the dispersed phase liquid, and a large amount of the continuous phase liquid remains (particularly, the continuous phase liquid remains in the dispersed phase liquid flow path in a droplet-like shape). However, when a surface with a high affinity and a surface with a low affinity coexist when viewed from the cross section of the flow path, the wall surface with a high affinity is wetted by the dispersed phase liquid early, and the cross-sectional shape of the dispersed phase liquid during the liquid transfer is easily stabilized in terms of surface free energy, while the wall surface with a low affinity is difficult to wet, so it is not blocked early by the dispersed phase liquid, and it is considered that the amount of the residual continuous phase liquid can be reduced.

[0046] Therefore, in a particularly preferred embodiment of the present disclosure, particularly with respect to the dispersed phase liquid flow path, (i) At least half of the flow channel wall surface in the flow channel cross section has a contact angle with water of 90 degrees or more, and / or (ii) the bottom surface or the top surface of the flow channel is at least one selected from glass, cyclic olefin copolymer (COC), cyclic olefin polymer (COP), and polymethyl methacrylate (PMMA), and the other wall surfaces of the flow channel (i.e., the bottom surface or the top surface of the flow channel, and the side walls, which are not glass, COC, COP, or PMMA) have a contact angle of 90 degrees or more with water; Here, it is particularly preferable that the flow channel wall surface having a contact angle of 90 degrees or more with water is formed from PDMS.

[0047] (Non-fluorine based surface treatment agents) In one embodiment of the present disclosure, the walls of the dispersed phase liquid channels are treated with a non-fluorine based surface treatment agent.

[0048] In order to stabilize emulsion formation, it is generally preferable to use a channel wall surface that has a high affinity for the continuous phase liquid and a low affinity for the dispersed phase liquid, but when using a fluorine-based dispersion medium as the continuous phase liquid, in order to suppress the continuous phase liquid from remaining in the dispersed phase liquid channel or flowing back during liquid transfer, it is preferable to use a channel wall surface that does not contain a fluorine compound, i.e., a channel wall surface that has a relatively low affinity for the continuous phase liquid, so that emulsion formation is difficult to stabilize. Therefore, in order to make the surface less compatible with the dispersed phase liquid, it is preferable to perform a surface treatment that is not based on fluorine. Examples of the flow path surface treated with a non-fluorine-based surface treatment agent include deposition of a non-fluorine-based surface treatment agent (which generally has a reactive group on the surface) such as silane coupling agents (preferably so-called long-chain spacer type silane coupling agents having 5 or more carbon atoms and no cationic or anionic terminal group) onto the substrate, surface treatments such as wet and dry treatments, surface coatings containing non-fluorine-containing hydrophobic resins such as silicone rubber (e.g., PDMS), polyethylene, and polypropylene, and application of paraffin-based wax.

[0049] In contrast, fluorine-based surface treatments include deposition, wet, and dry treatments of fluorine-based surface treatment agents (generally with reactive groups on the surface), such as silane coupling agents such as 1H,1H,2H,2H-perfluorodecyltrichlorosilane (FDTS) and trifluoropropyltrimethoxysilane, modification of the surface properties of the substrate by compounding with a fluorine-based surfactant, which eliminates the need for post-molding surface treatment, direct fluorination of the substrate with fluorine gas, and surface coatings (or direct use as a substrate) containing fluororesins such as polytetrafluoroethylene (PTFE), perfluoroalkoxyethylene (PFA), ethylene tetrafluoroethylene (ETFE), tetrafluoroethylene-hexafluoropropylene copolymer (FEP), and polyvinylidene fluoride (PVDF). Compared to the flow channel surfaces treated with these fluorine compounds, non-fluorine-based surface treatments have a lower affinity for the continuous phase liquid consisting of a fluorine-based dispersion medium. Therefore, by treating the wall surfaces of the dispersed phase liquid flow paths with a surface treatment agent that is not based on fluorine, it may be possible to more effectively suppress the ingress of continuous phase liquid into the dispersed phase liquid flow paths and / or the residue of continuous phase liquid on the wall surfaces of the dispersed phase liquid flow paths.

[0050] The fluorine compound may be present on the surface of the wall of the dispersed phase liquid flow channel (and / or the dispersed phase liquid holding portion) in an amount unintentionally mixed in during manufacturing. It is difficult to precisely determine the amount of fluorine compound that must be present on the flow channel surface to cause the continuous phase liquid to remain or flow back, but it is believed that the amount of fluorine compound unintentionally mixed in during manufacturing will not make a difference.

[0051] Each of the components constituting the invention according to the present disclosure and the embodiments thereof will be described in more detail below.

[0052] <Emulsion> The emulsions produced by the methods of the present disclosure are disperse solutions that contain droplets made up of the dispersed phase liquid and a continuous phase made up of the continuous phase liquid. In an emulsion, the droplets made up of the dispersed phase liquid are dispersed in the continuous phase made up of the continuous phase liquid.

[0053] (dispersed phase liquid) The dispersed phase liquid is the liquid that makes up the droplets contained in the emulsion.

[0054] The dispersed phase liquid is preferably an aqueous solution. The dispersed phase liquid may optionally contain a surfactant, an organic solvent, a thickener, serum, an enzyme, etc. The dispersed phase liquid may be a reaction liquid, for example, a liquid containing a sample to be detected in the detection process described below, a liquid containing a detection reagent, or a mixture thereof.

[0055] (Continuous phase liquid) The continuous phase liquid is the liquid that constitutes the continuous phase contained in the emulsion.

[0056] The continuous phase liquid is preferably an immiscible liquid with the dispersed phase liquid, for example, if the dispersed phase liquid is an aqueous solution, the continuous phase liquid may be an oil, in which case a water-in-oil (W / O) emulsion is formed.

[0057] When the continuous phase liquid is an oil, the oil may include silicone oil, mineral oil, fluorinated dispersion medium, vegetable oil, or a combination thereof.

[0058] For the purpose of improving the thermal stability of the droplets, additives such as surfactants can be added to the continuous phase liquid. It is preferable that these additives do not inhibit the detection reaction in the droplets. Examples of surfactants include non-ionic surfactants such as PLURONIC (registered trademark) and TETRONIC (registered trademark), which are block copolymers of polyethylene glycol and polypropylene glycol, as well as Tween, Span, and Zonyl (registered trademark). When a fluorine-based dispersion medium is used as the continuous phase liquid, it is preferable to use a fluorine-based surfactant, such as a block copolymer of perfluoropolyether and polyethylene glycol.

[0059] (Fluorocarbon) In one preferred embodiment of the present disclosure, the continuous phase liquid is a fluorinated dispersion medium, in particular a fluorocarbon-based oil.

[0060] Since fluorocarbon-based oils exhibit a relatively low affinity for surfaces composed of inorganic and / or organic materials that do not contain fluorine compounds and are not treated with fluorine compounds, when used in the present invention, the ingress of continuous phase liquid into the dispersed phase liquid flow paths and / or the residue of continuous phase liquid in the dispersed phase liquid flow paths can be further effectively reduced or avoided.

[0061] In addition, when a fluorine-based dispersion medium, particularly the above-mentioned fluorocarbon, is used as the continuous phase liquid, droplets can be generated particularly stably and quickly. In addition, since it has a characteristic of being extremely poorly compatible with polar solvents and non-polar solvents, it is possible to suppress the problem (crosstalk, contamination) that the components of the droplets in the emulsion move to other droplets through the continuous phase liquid. In addition, when a liquid with low surface tension or viscosity is selected such as a hydrocarbon-based dispersion medium or silicone oil, the risk of flammability and other dangerous substances generally increases, but a fluorine-based dispersion medium is characterized by its high safety, so that it is used as a fire extinguisher and a cooling medium.

[0062] Fluorocarbon-based oils contain or consist essentially of fluorocarbons. Fluorocarbon-based oils can contain, for example, 50% or more, 75% or more, 80% or more, 90% or more, or 95% or more fluorocarbons by weight.

[0063] Fluorocarbons include, for example, perfluorohexane, hexafluorobenzene, perfluoromethylcyclohexane, perfluorooctane, and perfluorotripentylamine.

[0064] Commercially available fluorocarbons include FC-3283 (Fluorinert (trade name) manufactured by 3M Corporation), FC-40 (Fluorinert (trade name) manufactured by 3M Corporation), and HFE-7500 (Fluorinert (trade name) manufactured by 3M Corporation). TM Novec TM Highly functional liquid (manufactured by 3M).

[0065] (droplet) The droplets contained in an emulsion are composed of the dispersed phase liquid, for example, by encapsulation of the dispersed phase liquid through contact with the continuous phase liquid.

[0066] The droplets contain, for example, a sample to be detected. In the droplets, a target substance contained in the sample is reacted with a reagent, and the sample can be analyzed via a detectable signal (e.g., a fluorescent signal) indicating the presence or absence and / or the extent of the reaction. The reaction may be, for example, a chemical reaction, a binding reaction, a phenotypic change, or a combination thereof.

[0067] The volume of the droplet is preferably large enough to hold approximately one target substance (for example, one molecule). Specifically, the average volume is preferably 0.00001 nL or more, 0.0001 nL or more, 0.001 nL or more, 0.01 nL or more, 0.1 nL or more, or 1 nL or more, and / or 100 nL or less, 50 nL or less, or 10 nL or less. From the viewpoint of uniformly reacting the target molecules in the droplet, it is preferable that the volume of the droplet to be formed is highly monodisperse. Specifically, the monodisperse here means that the coefficient of variation (CV) of the droplet volume is 20% or less, 10% or less, 5% or less, 2% or less, or 1% or less. In the following description, the droplets are treated as spherical for ease of understanding, but the same can be considered even if the droplets are non-spherical due to the flow channel structure or the surrounding flow.

[0068] The droplets preferably have a thermal stability sufficient to maintain their shape at least under the reaction temperature conditions of the target substance.Specifically, in the detection process, when nucleic acid amplification is performed by the TRC method, the droplets preferably have a thermal stability sufficient to maintain their shape under temperature conditions of 40°C to 48°C, and when nucleic acid amplification is performed by the PCR method, the droplets preferably have a thermal stability sufficient to maintain their shape under temperature conditions of 50°C to 100°C.

[0069] <Microfluidic Chip> The micro-channel chip of the present disclosure has a dispersed phase liquid holding section, a dispersed phase liquid flow path, a continuous phase liquid holding section, a continuous phase liquid flow path, an emulsion forming section, an emulsion flow path, and an outlet. The dispersed phase liquid holding section is connected to the emulsion forming section via the dispersed phase liquid flow path, the continuous phase liquid holding section is connected to the emulsion forming section via the continuous phase liquid flow path, and the emulsion forming section is connected to the outlet via the emulsion flow path. The micro-channel chip may further have an emulsion holding flow path.

[0070] The dispersed phase liquid holding section, dispersed phase liquid flow path, continuous phase liquid holding section, continuous phase liquid flow path, emulsion forming section, emulsion flow path, (optional emulsion holding flow path), and outlet are fluidly connected to each other and collectively form a single flow path structure.

[0071] In particular, this flow path structure is capable of being connected to the outside atmosphere (in particular the outside atmosphere) only via the dispersed phase liquid holding portion, the continuous phase liquid holding portion, and the outlet.

[0072] The microchannel chip according to the present disclosure has, for example, a substrate and an upper structure disposed on the substrate. Preferably, the upper structure has a channel structure, i.e., a dispersed phase liquid holder, a dispersed phase liquid channel, a continuous phase liquid holder, a continuous phase liquid channel, an emulsion forming section, an emulsion channel, (optionally an emulsion holding channel), and an outlet. The substrate may be made of glass. The upper structure may be made of resin. The microchannel chip can be fabricated, for example, by bonding a resin upper structure to a glass substrate constituting the bottom of the microchannel chip.

[0073] The size (width, depth, etc.) of the flow channel constituting the microchannel chip can be appropriately determined in consideration of the volume of the target droplets, and in particular, in consideration of the reaction form of the target substance. For example, when the target substance is a nucleic acid such as DNA or RNA, and the reaction of the target substance is a digital amplification reaction of the nucleic acid (amplification reaction in single molecule units), it is necessary to prepare droplets on the order of pL or nL, so that the width and depth of the flow channel around the emulsion forming part are preferably in the range of 0.1 μm to 1000 μm, particularly 1 μm to 300 μm, respectively.

[0074] Microchannel chips can be fabricated by combining techniques that use molds, such as molding or embossing, which can accurately and easily fabricate channel structures, or techniques that are commonly used by those skilled in the art, such as photolithography, soft photolithography, wet etching, dry etching, nanoimprinting, laser processing, direct electron beam drawing, additive manufacturing (AM), and machining.

[0075] Materials used to fabricate microchannel chips include polymer materials such as PDMS (polydimethylsiloxane) and acrylic, metal materials such as stainless steel, glass, silicone, ceramics, etc. Among these, polymer materials allow the flow channel itself to be fabricated inexpensively and are easy to make in a disposable form. Therefore, it is preferable to use polymer materials at least in part.

[0076] (Dispersed phase liquid holding section) The dispersed phase liquid holding portion is a portion that holds the dispersed phase liquid that is a material for producing an emulsion. The dispersed phase liquid holding portion may be, for example, a hole and / or a well that extends vertically when the microchannel chip is in use, and the dispersed phase liquid can be supplied and held in the hole and / or well. The dispersed phase liquid holding portion may be, for example, composed of a hole and / or a well with a diameter of 0.1 mm to 20 mm. When the dispersed phase liquid holding portion is composed of a hole and a well, the well extending vertically can be connected to the dispersed phase liquid flow channel via the hole extending vertically.

[0077] FIG. 2 is a cross-sectional schematic diagram showing one embodiment of a dispersed phase liquid holding portion according to the present disclosure. FIG. 2 is a schematic diagram for ease of understanding and is not drawn to scale. In FIG. 2, H is the height direction, and L is the length direction. The dispersed phase liquid holding portion 200 shown in FIG. 2 has a well 21 and a hole 23. The well 21 is connected to the dispersed phase liquid flow path 115 via the hole 23. The well 21 and the hole 23 extend in the height direction (here, the vertical direction). The reference numeral 25 in FIG. 2 indicates an extension portion which is a joint between the bottom surface of the well and the hole. In the embodiment of FIG. 2, the bottom surface of the well 21 extends perpendicular to the extension direction of the hole 23 (particularly the side wall of the hole 23 in the vicinity of the extension portion 25).

[0078] With respect to the dispersed phase liquid holder, it is preferable to carry out further optimization as follows.

[0079] The driving force during liquid transfer is mainly liquid transfer pressure (positive pressure, negative pressure) and / or liquid level differential pressure (hydrostatic pressure) and / or capillary force. Of these, the capillary force is determined by the difference in surface tension between the gas-liquid interface (and solid-liquid interface) downstream of the flow path (generally, the emulsion-holding flow path) during liquid transfer and the gas-liquid interface (and solid-liquid interface) in each holding part. In other words, since the liquid transfer speed in the flow path changes depending on the shape of the gas-liquid interface in each holding part (wetting behavior on the wall surface), the shape of each holding part is an important factor for realizing stable liquid transfer, particularly in the emulsion filling method in which liquid transfer (emulsion generation and retention) is performed by controlling the gas-liquid interface in the flow path. In particular, in a typical emulsion generation chip, stable emulsion generation can be achieved by making at least the flow channel wall surface of the emulsion formation section a surface with low affinity to the dispersed phase liquid (a hydrophobic surface if the dispersed phase liquid is an aqueous liquid), so in the case where surface treatment within the flow channel or a separate substrate is not combined from the viewpoint of chip manufacturing costs, the wall surface of the dispersed phase liquid holding section also generally has a surface with low affinity to the dispersed phase liquid. In this case, the change in the surface tension of the gas-liquid interface accompanying the change in the interface shape in the dispersed phase liquid holding section increases, which has a greater impact on liquid transfer.

[0080] From the viewpoint of reducing the change in the capillary force as described above, the holes and / or wells are preferably substantially free of discontinuous shapes on the wall surface and extend vertically. In addition, when the remaining amount of the dispersed phase liquid in the dispersed phase liquid holding portion decreases, the gas-liquid interface substantially comes into contact with the bottom surface of the well of the holding portion, and the interface shape is likely to change. In order to reduce this effect, it is preferable that the diameter of the holes and / or wells directly fluidically connected to the flow path is small, for example, 5 mm or less, more preferably 2 mm or less, and particularly preferably 1 mm or less. On the other hand, the amount of dispersed phase liquid held can be increased by expanding the diameter from the holes or wells directly fluidically connected to the dispersed phase liquid flow path, but in this case, if the gas-liquid interface is present near the expansion portion, the gas-liquid interface shape is likely to change. Therefore, in one preferred embodiment, the shape of the expansion portion can be adjusted, and / or the height of the holes or wells directly fluidically connected to the flow path to the expansion portion can be reduced, for example, to 3 mm or less, more preferably 1 mm or less, and particularly preferably 0.5 mm or less. Furthermore, when the amount of dispersed phase liquid supplied is small and / or the external surface of the chip around the dispersed phase liquid holding portion has low affinity for the dispersed phase liquid, and the external liquid delivery driving force is the application of negative pressure to the outlet, it is preferable to configure the chip with only a hole and supply the dispersed phase liquid to the hole so that it forms a hemispherical shape, because this minimizes the contact area with the chip wall surface and suppresses changes in the shape of the gas-liquid interface even when the remaining amount of dispersed phase liquid becomes small.

[0081] When positive pressure is used as the external liquid delivery driving force, it is preferable that the dispersed phase liquid holding portion is suitable for connection to a positive pressure source. In this case, it is preferable that the dispersed phase liquid holding portion has resistance to the applied pressure.

[0082] (Supply of dispersed phase liquid) The method according to the present disclosure includes providing a dispersed phase liquid to a dispersed phase liquid holding portion.

[0083] In order to supply the dispersed phase liquid to the dispersed phase liquid holding section, a separate container (phase liquid holding container) may be used to hold the dispersed phase liquid. From the viewpoint of preventing the liquid from leaking during storage and operation, such a container is preferably completely or variably sealed while holding the dispersed phase liquid.

[0084] The supply of the dispersed phase liquid (and / or the supply of the continuous phase liquid) can be carried out by a dispensing means. The use of a dispensing means is preferable in that it can suppress the remaining amount of the dispersed phase liquid and can suppress the measurement time and / or the contamination between the reagents (dispersed phase liquid).

[0085] For example, each phase liquid can be dropped into each holding part using a dispensing means, or each phase liquid can be introduced along the wall surface of each holding part. This is particularly advantageous when the liquid is fed under negative pressure. In the conventional supply method, particularly in the method of simultaneously feeding liquid and feeding pressure to each holding part by fluidly connecting (sealedly connecting) a tube or manifold to each holding part, it is not easy to accurately control the progress of the dispersed phase liquid and the continuous phase liquid due to unexpected pressure fluctuations at the time of connection and the time required for the pressure to stabilize, and it is difficult to contact the dispersed phase liquid and the continuous phase liquid without blocking the flow path. In contrast, when a dispensing means is used and the liquid is fed under negative pressure, there is no pressure fluctuation when connecting a phase liquid supply device and a pressure source to each holding part, so it is possible to more accurately control the movement of each phase liquid and the contact between the dispersed phase liquid and the continuous phase liquid before the application of negative pressure, and as a result, the generation of bubbles can be more effectively suppressed.

[0086] The dispensing means is preferably one that does not cause pressure fluctuations in the holding parts. The dispensing means may be, for example, a pipette. Preferably, the dispensing means (particularly, the liquid outlet constituting the dispensing means) is not fluidically connected (sealed connected) to each holding part, but is spatially separated.

[0087] For example, the dispensing means may be a mechanism including a pump, an actuator, and a pipette. It is preferable that the pump sucks up each phase liquid held in a separate container, moves the tip of the pipette to each holding part by the actuator, and then pushes each phase liquid into each holding part by the pump. In addition, it is preferable that a part of the pipette etc. that each phase liquid comes into contact with is removable and can be replaced after each use, since this suppresses contamination. Furthermore, it is preferable to use the pump of the dispensing means in combination as a liquid sending means, since this simplifies the device configuration. In addition, when repeated use is intended, the phase liquid may be added by a dispensing means including a dispensing means including a disposable pipette, or a common line may be used to add the phase liquid from the phase liquid holding container to the microchannel chip. In the latter case, in order to suppress contamination of the dispersed phase liquid with the continuous phase liquid, it is preferable to include a step of cleaning the shared line to the connection part to the microchannel chip. In addition, as a dispensing means that does not include a pipette, a method of adding (dripping) each liquid directly to the holding part from a container that holds the phase liquid by an external force is also preferable (for example, a means for pushing out the liquid in the container by breaking the thermocompression-bonded part of the container by pressure, etc.). Furthermore, for example, when performing a TRC reaction or a PCR reaction, a dispensing means may be used in combination as a purification means or a preparation means for an aqueous solution sample.

[0088] (Dispersed phase liquid flow path) The dispersed phase liquid flow path connects the dispersed phase liquid holding section and the emulsion forming section. The dispersed phase liquid flow path is configured so that the dispersed phase liquid passes therethrough. It is also possible to assume that in addition to the dispersed phase liquid, the continuous phase liquid passes through the dispersed phase liquid flow path.

[0089] The dimensions of the dispersed phase liquid flow path can be appropriately set depending on the type and characteristics of the dispersed phase liquid used. The dispersed phase liquid flow path can have a width of, for example, 10 to 500 μm or 50 to 200 μm, and a length of 1 mm to 500 mm or 10 to 200 mm. The dispersed phase liquid flow path can have a flow path height of 1 to 200 μm or 10 to 100 μm. The dispersed phase liquid flow path may be bent at one or more locations, and may have a serpentine shape.

[0090] The microchannel chip according to the present disclosure can have two or more dispersed phase liquid holders and two or more dispersed phase liquid channels corresponding thereto.

[0091] In particular, the dispersed phase liquid holding section includes a first dispersed phase liquid holding section and a second dispersed phase liquid holding section, the dispersed phase liquid flow path includes a first dispersed phase liquid flow path connected to the first dispersed phase liquid holding section, a second dispersed phase liquid flow path connected to the second dispersed phase liquid holding section, and a dispersed phase liquid junction section, and the first dispersed phase liquid flow path and the second dispersed phase liquid flow path are each connected to the emulsion forming section via the dispersed phase liquid junction section.

[0092] It may be preferable to use two dispersed phase liquids, a sample solution containing a substance to be detected and a reagent solution containing a detection reagent, as the dispersed phase liquid. That is, a reaction liquid containing a sample for analysis and a reaction liquid containing a detection reagent can be separately supplied to a microchannel chip so that the two liquids are not mixed until just before droplets are generated. This is preferable because the timing of the reaction start can be better controlled. However, in this case, due to the continuous phase liquid remaining in the dispersed phase liquid flow path (especially the dispersed phase liquid junction), an uneven mixing ratio of the two dispersed phase liquids may occur. This leads to a decrease in the accuracy of the detection reaction.

[0093] In contrast, according to the present invention, it is possible to suppress or avoid the continuous phase liquid from entering the dispersed phase liquid flow path (particularly the dispersed phase liquid junction) and / or the continuous phase liquid from remaining in the dispersed phase liquid flow path (particularly the dispersed phase liquid junction), thereby ensuring uniformity of the mixing ratio when multiple reaction liquids are mixed within a microchannel chip.

[0094] From the viewpoint of further suppressing the continuous phase liquid from remaining in the dispersed phase liquid flow path, the flow path and / or flow rate can be set so that the pressure drop resistance of the dispersed phase liquid flow path is relatively large. For this purpose, for example, the length of the dispersed phase liquid flow path can be increased, the width and / or height (cross-sectional area of ​​the flow path) can be decreased, or the flow rate can be set to be high. In this case, the pressure acting on the dispersed phase liquid present near the wall surface of the flow path can be relatively increased, so that it is considered that the continuous phase liquid remaining in the dispersed phase liquid flow path can be more effectively reduced.

[0095] In addition, when the cross section of the dispersed phase liquid flow path (including the dispersed phase liquid junction) is a typical rectangle, the continuous phase liquid is likely to remain at the four corners of the flow path cross section. Therefore, it is preferable to make the flow path width larger than the flow path height, for example, 2 times, 3 times, 5 times, or 10 times. In this case, the amount of residual continuous phase liquid relative to the flow rate of dispersed phase liquid can be made relatively small. In addition, it is also preferable to use a flow path cross section with fewer corners (such as a circle) from the viewpoint of suppressing the residual continuous phase liquid.

[0096] (Continuous phase liquid holding section) The continuous phase liquid holding portion is a portion that holds the continuous phase liquid that is a material for producing an emulsion. The structure of the continuous phase liquid holding portion is not particularly limited as long as it can hold the continuous phase liquid. The continuous phase liquid holding portion may be a hole or well, for example, a hole or well extending in a vertical direction, and the continuous phase liquid can be supplied and held in this hole or well. The continuous phase liquid holding portion may be, for example, a hole or well having a diameter of 0.1 mm to 20 mm.

[0097] In addition, since the continuous phase liquid generally uses a liquid with low surface tension and viscosity, the amount of change in surface tension accompanying a change in the interface shape in the continuous phase liquid holding section is small. Therefore, the shape of the continuous phase liquid holding section does not have a significant effect on the liquid transfer. In addition, in the present invention, for example, when an emulsion is held and a detection reaction or the like is performed, a sufficient amount of continuous phase liquid is supplied so that the continuous phase liquid in the continuous phase liquid holding section does not run out, so that the remaining amount of continuous phase liquid in the holding section does not decrease during liquid transfer, and the interface shape is likely to change. Therefore, again, the shape of the continuous phase liquid holding section is unlikely to have a significant effect on the liquid transfer.

[0098] When positive pressure is used as the external liquid delivery driving force, the continuous phase liquid holder is preferably suitable for connection to a positive pressure source. In this case, it is preferable that the continuous phase liquid holder has resistance to the applied pressure.

[0099] (Continuous phase liquid supply) The supply of the continuous phase liquid can be performed using a separate container and / or a dispensing means, as described above with respect to the supply of the dispersed phase liquid. For example, the continuous phase liquid can be dropped into the holding section using a dispensing means, or the continuous phase liquid can be introduced along the wall surface of the holding section. For details of the separate container and the dispensing means, please refer to the above description regarding the supply of the dispersed phase liquid.

[0100] (Continuous phase liquid flow path) The continuous phase liquid flow path connects the continuous phase liquid holding section and the emulsion forming section, and is configured so that the continuous phase liquid passes therethrough.

[0101] The dimensions of the continuous phase liquid flow path can be appropriately set depending on the type and characteristics of the continuous phase liquid used. The continuous phase liquid flow path can have a width of, for example, 10 to 500 μm or 50 to 200 μm, and a length of 1 mm to 500 mm or 10 to 200 mm. The continuous phase liquid flow path can have a flow path height of 1 to 200 μm or 10 to 100 μm. The continuous phase liquid flow path may be bent at one or more locations, and may have at least a partially serpentine shape.

[0102] The micro-channel chip may have two or more continuous phase liquid channels. In particular, the micro-channel chip according to the present disclosure has a first continuous phase liquid channel and a second continuous phase liquid channel, each of which connects the continuous phase liquid holding section and the emulsion forming section.

[0103] Referring to the exemplary embodiment of FIG. 1, the continuous phase liquid flow path 111 is composed of two flow paths (a first continuous phase liquid flow path 112 and a second continuous phase liquid flow path 113). These two flow paths 112, 113 face each other in the emulsion forming section 120, and are substantially perpendicular to the dispersed phase liquid flow path 117 (more precisely, the dispersed phase liquid junction 116) connected to the emulsion forming section 120. In the embodiment of FIG. 1, the first continuous phase liquid flow path 112 and the second continuous phase liquid flow path 113 have substantially the same structure and flow path length, so that the speed of the continuous phase liquid moving through each flow path is substantially the same. In addition, when it is desired to suppress the mixing of the dispersed phase liquids before the emulsion is generated as described above, it is preferable that the length of the flow path connecting the downstream part of the dispersed phase liquid junction 116 and the emulsion forming section 120 is relatively short (for example, 3 mm or less, more preferably 0.5 mm or less), and it is preferable that the dispersed phase liquids are kept in a laminar flow state separately within the flow path.

[0104] (emulsion forming part) The emulsion forming section is configured to generate an emulsion. The emulsion forming section is supplied with a dispersed phase liquid and a continuous phase liquid via a dispersed phase liquid flow path and a continuous phase liquid flow path, respectively. The emulsion forming section is also connected to the emulsion flow path, and the emulsion generated in the emulsion forming section is sent to the emulsion flow path.

[0105] The emulsion former may have one or more openings that open into the dispersed phase liquid flow path and one or more openings that open into the continuous phase liquid flow path, and may have one or more openings that open into the emulsion flow path.

[0106] The emulsion forming section will be described with reference to the exemplary embodiment of FIG. 1. In the emulsion forming section 120 of FIG. 1, the continuous phase liquid flow path 111 consisting of two flow paths 112 and 113 and the dispersed phase liquid flow path 117 (more precisely, the dispersed phase liquid junction 116) are substantially perpendicular to each other. During the application of an external liquid delivery driving force, the continuous phase liquid flows into the emulsion forming section 120 from two directions substantially opposite to each other, and the dispersed phase liquid flows into the emulsion forming section 120 in a direction substantially perpendicular to the inflow direction of the continuous phase liquid. As a result, in the emulsion forming section 120, droplets dispersed in the continuous phase (i.e., emulsion) are generated. The emulsion thus generated passes through the emulsion flow path 130 and enters the emulsion holding flow path 140 filled with gas.

[0107] The emulsion forming section may appropriately use a flow path utilizing a general droplet generation method such as T-junction, Flow-Focus, co-flow, or step-emulsification. In order to generate the emulsion quickly, a plurality of emulsion forming sections may be arranged in parallel. In addition, the emulsion forming section may be provided with a meandering flow path for stirring the droplets in the emulsion.

[0108] (Emulsion flow path) The emulsion flow path is connected to the emulsion forming section, and can optionally connect the emulsion forming section and the emulsion holding flow path. The emulsion flow path connects the emulsion forming section and the outlet (or the emulsion holding flow path). The portion of the emulsion flow path adjacent to the emulsion forming section is preferably arranged so as to face the dispersed phase liquid flow path (particularly the dispersed phase liquid junction). FIG. 1 shows an emulsion flow path of such an embodiment. Also, in FIG. 1, the portion of the emulsion flow path adjacent to the emulsion forming section is substantially perpendicular to the inlet portion of the continuous phase liquid flow path to the emulsion forming section. In the case of FIG. 1, when an emulsion is generated, the dispersed phase liquid flowing from the dispersed phase liquid flow path to the emulsion forming section becomes droplets, and enters the emulsion flow path without changing the flow angle.

[0109] The emulsion flow path may have an expanded width and / or height downstream (toward the outlet) of the portion adjacent to the emulsion formation portion, and / or may be serpentine. Such an embodiment is preferred because it can promote agitation in the droplet.

[0110] (Emulsion holding channel)

[0111] In one embodiment according to the present disclosure, the microchannel chip further comprises an emulsion holding channel.

[0112] In this embodiment, the emulsion forming section is connected to the emulsion holding flow path via the emulsion flow path, and the emulsion holding flow path is connected to the outlet. By applying negative pressure to the outlet, an emulsion containing droplets composed of the dispersed phase liquid and a continuous phase composed of the continuous phase liquid is generated in the emulsion forming section, and the emulsion thus generated is transported to the emulsion holding flow path via the emulsion flow path.

[0113] This embodiment will be specifically described with reference to the microchannel chip shown in Figure 3. Figure 3 is a schematic diagram for ease of understanding and is not necessarily drawn to scale. The microchannel chip in Figure 3 is similar to that in Figure 1, except that it has an emulsion-holding channel and the arrangement of the outlet is different. Only the differences will be described below.

[0114] 3 further includes an emulsion holding channel 140. The emulsion forming section 120 is connected to the emulsion holding channel 140 via an emulsion channel 130, and the emulsion holding channel 140 is connected to an outlet 150.

[0115] The emulsion produced in the emulsion forming section is transported via the emulsion flow path 130 to the emulsion holding flow path 140 .

[0116] When an emulsion is generated and held using a microchannel chip having an emulsion holding channel, if the continuous phase liquid remains in the dispersed phase liquid channel (particularly the wall surface of the dispersed phase liquid channel), the generation of bubbles in the emulsion holding channel may increase. This is particularly because, when the dispersed phase liquid holding section becomes empty, the remaining continuous phase liquid acts as a lubricant, so to speak, and gas (particularly air) enters the droplet holding channel from the surrounding atmosphere through the emptied dispersed phase liquid holding section. The entry of gas through the emptied dispersed phase liquid holding section can be prevented by stopping the liquid transfer before the dispersed phase liquid holding section becomes empty, but in this case, unused dispersed phase liquid remains in the dispersed phase liquid holding section, resulting in waste. In particular, when a relatively expensive reaction liquid for genetic testing or a reaction liquid containing a valuable sample is used as the dispersed phase liquid, there is a risk of an increase in costs.

[0117] According to the present invention, it is possible to suppress or avoid the continuous phase liquid from entering the dispersed phase liquid flow path and / or the continuous phase liquid from remaining in the dispersed phase liquid flow path. Therefore, by carrying out the present invention in a microchannel chip having an emulsion holding flow path, it is possible to generate and hold an emulsion having particularly good stability.

[0118] In the method of the present disclosure, the emulsion (droplets + continuous phase) generated in the emulsion formation section can be transported to an emulsion holding channel filled with gas. For example, referring to FIG. 3, the emulsion generated in the emulsion formation section 120 can be transported via the emulsion channel 130 to the emulsion holding channel 140 filled with gas ("emulsion filling method"). Note that the emulsion holding channel 140 does not need to be completely filled with gas when the emulsion is transported, and may be partially filled with, for example, continuous phase liquid.

[0119] In such an emulsion filling method, the emulsion generated in the emulsion formation section moves through the emulsion holding flow channel filled with gas (particularly toward the outlet) and fills the emulsion holding flow channel. That is, the "gas-liquid interface" formed by the gas filling the emulsion holding flow channel and the emulsion moves downstream of the emulsion holding flow channel (particularly toward the outlet). The emulsion filling method is preferable because it can omit the preparation step of filling the flow channel of the microchannel chip with a continuous phase liquid in advance and the device for this purpose.

[0120] The width and length of the emulsion-holding flow channel can be appropriately set according to the volume and number of droplets to be held. For example, it may be a wide, simple flow channel with approximately the same width and length, a single continuous long flow channel arranged in a serpentine or spiral shape, or branched straight flow channels arranged in parallel.

[0121] In the present invention, the cross section of the emulsion holding flow channel is preferably circular, semicircular, elliptical, convex, concave, rectangular or trapezoidal, since the center of the droplets easily flows along the center of the flow channel.

[0122] In the emulsion filling method, emulsions are generally generated and held simultaneously by controlling the movement of the gas-liquid interface in the emulsion-holding channel, so it is desirable that the channel cross-sectional shape is constant and straight without bending so that the shape of the gas-liquid interface during liquid transfer is maintained (the change in shape is small even while moving). However, if the channel width is extremely large relative to the channel height in order to increase the number of droplets to be detected, it may be difficult to stably manufacture a chip having the intended channel structure, and / or the bottom and / or top of the channel may be deformed, and the height of the channel region far from the side of the channel may change due to the liquid transfer pressure or the fixing pressure to the chip, which may adversely affect the measurement (roof collapse). As a countermeasure, for example, the ratio of the channel width to the channel height is preferably 100 or less, more preferably 50 or less, 25 or less, and particularly preferably 10 or less (when there are no pillars as described below). Alternatively, a pillar is provided in the center of the flow channel, and the ratio of the distance between the pillars and / or the distance between the pillar and the side of the flow channel to the flow channel height is, for example, 100 or less, more preferably 50 or less, 25 or less, and particularly preferably 10 or less. On the other hand, when performing a batch detection process using an image sensor or the like, it is preferable that the emulsion-holding flow channel is densely packed on a horizontal surface (for example, in a shape close to a square or circle) because the number of droplets detected can be increased. Therefore, continuous single long flow channels with the same flow channel cross-sectional shape may be arranged in a meandering or spiral shape, or branched straight flow channels may be arranged in parallel. In this case, the interface shape is likely to change during liquid transfer due to the presence of a bend, but the effect can be reduced by adjusting the cross-sectional shape at the bend.

[0123] In one embodiment, it is intended to carry out a detection process on the held emulsion, i.e., the emulsion held in the emulsion holding channel can be optionally subjected to a detection process, which will be described later.

[0124] Preferably, the emulsion-holding flow channel is configured so that most or all of the emulsion held therein does not come into contact with the atmosphere (particularly the atmosphere) external to the micro-channel chip. Also, preferably, among the droplets held in the emulsion-holding flow channel, the droplets that are the subject of the detection process do not come into contact with the atmosphere (particularly the atmosphere).

[0125] This can be achieved, for example, by setting the length of the emulsion holding flow channel to be relatively long so that the emulsion can come into contact with the outside atmosphere only at the downstream end of the emulsion holding flow channel.

[0126] It is particularly preferable that the emulsion held in the emulsion holding flow channel is sealed against the external atmosphere (particularly the external air).

[0127] Preferably, the emulsion holding flow path is suitable for carrying out a detection process on the emulsion held in the emulsion holding flow path.

[0128] Preferably, the emulsion holding flow path is configured so that the detection process can be performed on the emulsion that is not exposed to the outside atmosphere. More specifically, for example, a structure that isolates the emulsion from the outside atmosphere is present between the held emulsion and the detection means. This structure is made of, for example, a material that transmits light. In this case, among the emulsions held in the emulsion holding flow path, the emulsion that is not the target of the detection process, for example, the emulsion located at the discharge side end of the emulsion holding flow path, may be exposed to the outside atmosphere.

[0129] Preferably, the flow channel volume of the emulsion holding flow channel is equal to or larger than the total volume of droplets produced in the emulsion forming section (particularly equal to or larger than the total volume of droplets to be detected in the detection process), and / or the flow channel volume of the emulsion holding flow channel is equal to or larger than 1 μL, equal to or larger than 5 μL, or equal to or larger than 10 μL. Such an emulsion holding flow channel allows the detection process to be carried out efficiently. The upper limit of the flow channel volume of the emulsion holding flow channel may be, for example, equal to or smaller than 1000 μL.

[0130] Preferably, the emulsion-holding flow channel has a flow channel volume capable of holding 500 or more, 1000 or more, 2500 or more, 5000 or more, or 10000 or more, and / or 100000 or less, 80000 or less, 60000 or less, or 40000 or less, of droplets having an average volume of 0.1 nL to 10 nL, particularly 0.3 to 3 nL. Detection processing can be performed on these droplets held in the emulsion-holding flow channel.

[0131] The average volume of droplets can be calculated by acquiring bright-field images using an image acquisition device such as a digital camera, and calculating the average volume of droplets for N=10 or more in the acquired images based on the following formula.

[0132] The volume of a spherical droplet and the volume of a disk-shaped droplet (V drop and V disk [nL]) are expressed by the following formulas (1) and (2), respectively. drop , D disk are the diameters of a spherical droplet and a disk-shaped droplet, respectively, when the droplets held in the emulsion holding channel are observed from above in the normal use state of the microchannel chip. Also, in formula (2), h is the channel height of the emulsion holding channel.

[0133]

number

[0134]

number

[0135] Preferably, the droplets in the emulsion to be detected do not overlap with each other, and in particular form a single layer extending in a plane perpendicular to the detection direction, relative to the detection means (such as a camera) used in the detection process. In this case, most of the droplets to be detected can be easily observed from one detection direction, thereby further improving the detection accuracy.

[0136] Particularly preferably, the "channel height" of the emulsion-holding channel is adjusted so that, when the microchannel chip is in use, the droplets held in the emulsion-holding channel do not overlap each other in the vertical direction (i.e., a single droplet layer is formed). When a detection process is performed in such an emulsion-holding channel, the detection accuracy is further improved. Note that the "channel height" usually refers to the length of the channel in the vertical direction (perpendicular direction) when the microchannel chip is in use.

[0137] Preferably, the height of the emulsion holding channel has a dimension corresponding to the diameter of the droplets to be detected in the detection process, and for example, the channel height is preferably 1 / 10 to 10 times, 1 / 4 to 4 times, or 1 / 2 to 2 times the diameter of the droplets. The height of the emulsion holding channel may be 1 / 4 times or less the width of the channel. The width of the channel is usually the length in the horizontal direction perpendicular to the length of the channel when the microchannel chip is in use. The diameter of the droplets can be measured in the width direction of the channel.

[0138] In the emulsion filling method, the emulsion is filled in the emulsion holding channel depending on the flow rate ratio of the continuous phase liquid to the dispersed phase liquid in the emulsion formation, so that, for example, when the flow rate ratio of the continuous phase liquid to the dispersed phase liquid is increased to stabilize the emulsion formation, it is preferable to design the channel height to be larger than usual in order to pack the droplets densely in the horizontal direction. For example, when the flow rate ratio of the continuous phase liquid to the dispersed phase liquid is 8 to 12, the height of the emulsion holding channel can be 2 to 4 times the diameter of the droplets.

[0139] (Stopping the liquid supply) In order to retain the emulsion in the emulsion holding flow channel, for example, when a desired amount of emulsion is filled in the emulsion holding flow channel, the liquid supply is stopped and the outlet is optionally closed. When negative pressure is used for liquid supply, the application of negative pressure can also be stopped by opening the outlet (at least partially) to the outside atmosphere. When carrying out the above-mentioned emulsion filling method, the liquid supply can be stopped after the gas that filled the emulsion holding flow channel is completely replaced with the emulsion, but it is preferable to stop the liquid supply before the gas that filled the emulsion holding flow channel is completely replaced with the emulsion. In other words, the liquid supply is stopped when a gas-emulsion interface exists in the flow channel or in the outlet.

[0140] When stopping the liquid transfer, it may be possible to suppress backflow at the gas-liquid interface (gas-emulsion interface) by gradually opening the outlet to the outside atmosphere. For example, a relatively low-pressure tank is provided separately between the two solenoid valves, and the outlet is opened to this tank before opening to the outside atmosphere, allowing for gradual opening. Liquid transfer can also be stopped by adjusting the opening speed of the solenoid valve so that the degree of opening increases in stages.

[0141] Regarding the stopping of liquid flow, it is preferable to increase the flow rate ratio of the continuous phase liquid or reduce the flow channel volume of the emulsion-holding flow channel so as to increase the minimum distance between the gas-emulsion interface and all of the droplets. This makes it difficult for droplets in the emulsion to flow out to the outlet and also suppresses the adverse effects of the gas-emulsion interface on nearby droplets (aggregation and coalescence of droplets, and inhibition of intra-droplet reactions and signal detection).

[0142] Regarding the stopping of the liquid transfer and the subsequent retention of the emulsion, when a highly fluid continuous phase liquid is used, the gas-emulsion interface may flow downstream due to capillary force and / or liquid level differential pressure even if an external liquid transfer driving force is not applied. In addition, when a highly volatile continuous phase liquid is used and / or the emulsion is heated for a reaction within the droplets, the continuous phase liquid may evaporate and the amount of continuous phase liquid in the flow path may decrease, and / or the continuous phase liquid remaining in the continuous phase liquid holding section may flow into the flow path to replenish the evaporated amount of continuous phase liquid, causing the droplets in the emulsion holding flow path to flow even after the liquid transfer is stopped. Such a flow of droplets after the liquid transfer is stopped may cause the droplets in the emulsion holding flow path to flow out toward the outlet or emulsion flow path, so it is desirable to take measures against this. For this reason, the flow of droplets caused by the capillary force and / or liquid level differential pressure, and the evaporation of the continuous phase liquid may be suppressed by sealing each holding section and the outlet and controlling the pressure, and / or using a material with low gas permeability as the flow path substrate. Incidentally, when a dispensing means is provided, such a sealing operation must be carried out after the dispensing operation or after the liquid transfer is stopped, which is not preferable from the viewpoint of automating the device.

[0143] (Exhaust port) The microchannel chip has an outlet that is connected to the emulsion channel or the emulsion-holding channel. As described below, the outlet can also function as a negative pressure source connection portion for applying negative pressure to the microchannel chip.

[0144] When negative pressure is used for the delivery of the liquid, it is preferred that the outlet is adapted to be connected to a negative pressure source, in which case it is preferred that the outlet is resistant to the applied pressure.

[0145] When the microchannel chip has an emulsion-holding channel and negative pressure is used for liquid delivery, the outlet is usually located downstream of the emulsion-holding channel, and by applying negative pressure to the outlet, the entire length or most of the emulsion-holding channel can be filled with the generated emulsion.

[0146] (Liquid transfer conditions) In the present invention, since the flow channel wall surface has a low affinity for the continuous phase liquid, it is preferable to ensure the stability of emulsion generation and retention by keeping the emulsion generation speed relatively low. Specifically, for example, the emulsion generation speed is preferably 1000 particles / second or less, 500 particles / second or less, and particularly preferably 300 particles / second or less.

[0147] On the other hand, from the viewpoint of rapid measurement, a faster droplet generation speed is preferable. For example, the droplet generation speed is preferably 50 droplets / second or more, 100 droplets / second or more, and particularly preferably 200 droplets / second or more.

[0148] In addition, in order to reduce the amount of residual continuous phase liquid in the dispersed phase liquid flow path, it is preferable to reduce the length, width, and / or height of the flow path to reduce the flow path volume of the dispersed phase liquid flow path. However, regarding the width and height of the flow path, there is a lower limit in consideration of ease of manufacture and / or stability of emulsion production, so that the flow path pressure drop resistance tends to be small as a result. However, when the upper limit of the above-mentioned emulsion production rate is also taken into consideration, the liquid delivery pressure becomes smaller and the reproducibility of the liquid delivery tends to decrease. Therefore, it is preferable to design the flow path pressure drop resistance so that the liquid delivery pressure is, for example, 1 kPa or more, 1.5 kPa or more, 3 kPa or more, and particularly preferably 5 kPa or more.

[0149] (External liquid delivery driving force) The external liquid delivery driving force provides a driving force for generating an emulsion from the dispersed phase liquid and the continuous phase liquid in the emulsion forming section. In addition, when the microchannel chip has an emulsion holding channel, the external liquid delivery driving force provides a driving force for transporting the generated emulsion to the emulsion holding channel. The external liquid delivery driving force may be application of negative pressure to the outlet, or application of positive pressure to the dispersed phase liquid holding section and the continuous phase liquid holding section. From the viewpoint of simplicity of the device, it is preferable to keep each holding section at normal pressure while applying negative pressure to the outlet, and to keep the outlet at normal pressure while applying positive pressure to the dispersed phase liquid holding section and the continuous phase liquid holding section. However, in order to stabilize the liquid delivery under negative and / or positive pressure and / or to also serve as a sealing operation for holding the emulsion, pressure control to an emergency pressure state may be performed.

[0150] When negative pressure is applied to the outlet, the dispersed phase liquid holding section and the continuous phase liquid holding section can be open to the external atmosphere (particularly the external atmosphere). Similarly, when positive pressure is applied to the dispersed phase liquid holding section and the continuous phase liquid holding section, the outlet can be open to the external atmosphere (particularly the external atmosphere).

[0151] When negative pressure is applied, for example, a pressure tank or a syringe pump can be used to aspirate fluid (eg, gas or continuous phase liquid) in a channel of a microchannel chip through an outlet.

[0152] When a pressure tank is used as a negative pressure source, the volume of the pressure tank is preferably larger than the total volume of the flow channel from the outlet to the pressure tank and the volume of the flow channel of the microchannel chip. The pressure tank may be designed to be open to the outside atmosphere (particularly the outside atmosphere).

[0153] When applying positive pressure, for example, pressure can be applied to the fluid (gas, dispersed phase liquid, and / or continuous phase liquid) in the flow channel of the microchannel chip via the dispersed phase liquid holder and the continuous phase liquid holder using a pressure application means.

[0154] In addition, a monitoring means for monitoring the pressure value of the applied negative or positive pressure can be used to check the liquid transfer state, for example, to check whether an emulsion is being generated without any problems.

[0155] (Fluid delivery by negative pressure) In one embodiment of the present disclosure, negative pressure is used as the external liquid delivery driving force, that is, in one embodiment of the present disclosure, negative pressure is applied to the outlet to generate emulsion in the emulsion forming section, and the emulsion thus generated is transported through the emulsion flow path to the emulsion holding flow path filled with gas.

[0156] Negative pressure liquid delivery is preferable because it requires simpler equipment than positive pressure liquid delivery.

[0157] In addition, in the case of positive pressure liquid transfer, particularly when applying positive pressure to each holding part via gas, it is necessary to add the dispersed phase liquid and the continuous phase liquid to each holding part and then seal-connect the liquid transfer means and each holding part, but unnecessary external pressure is likely to be applied during connection. In particular, when a highly flexible substrate such as silicone rubber is used, the microchannel chip may deform during connection, and the cross section of the channel may be deformed. In addition, by using a liquid with low viscosity and surface tension as the continuous phase liquid, stable and rapid droplet generation using the shear force in the emulsion forming part is possible, but liquids with the above physical properties tend to leak when the sealed state is incomplete, making it difficult to apply appropriate pressure. In addition, by introducing the continuous phase liquid and / or the dispersed phase liquid after the sealed connection, it is possible to suppress the problems of liquid transfer and liquid leakage due to unnecessary external pressure during the above-mentioned connection, but this is an example of a means for simultaneously performing the above-mentioned liquid introduction and pressure liquid transfer, and the reproducibility of the operation of contacting the dispersed phase liquid and the continuous phase liquid is likely to decrease.

[0158] In contrast, negative pressure liquid transfer has the advantage that the liquid transfer means can be connected before the dispersed phase liquid and the continuous phase liquid are added, so that the pressure at the time of connection is not applied to the dispersed phase liquid and the continuous phase liquid, and also has the advantage that it is possible to avoid the need to seal the continuous phase liquid holding section that holds the continuous phase liquid, which has low viscosity and surface tension.

[0159] Furthermore, when using a microchannel chip with low flow channel pressure drop resistance, the method of applying positive pressure to each holding section may result in the dispersed phase liquid or continuous phase liquid unintentionally entering the flow channel due to unnecessary pressure when connecting to the liquid delivery means.

[0160] In contrast, the method of applying negative pressure to the outlet has the advantage that unnecessary pressure is not applied because the outlet can be connected to the liquid delivery means before the dispersed phase liquid and the continuous phase liquid are added.

[0161] Thus, negative pressure liquid transfer has many advantages. However, on the other hand, when negative pressure is applied, it is not as easy to remove the continuous phase liquid remaining on the wall surface of the dispersed phase liquid flow path as compared with the case of positive pressure. That is, in negative pressure liquid transfer, pressure is applied to the dispersed phase liquid through the continuous phase liquid, so that the continuous phase liquid remaining in the dispersed phase liquid flow path is likely to cause instability of the laminar flow of the dispersed phase liquid.

[0162] According to the present invention, it is possible to suppress or avoid the continuous phase liquid from entering the dispersed phase liquid flow path and / or the continuous phase liquid from remaining in the dispersed phase liquid flow path, so that a good emulsion can be produced and maintained even when negative pressure liquid transfer is used.

[0163] In one embodiment of the method according to the present disclosure, a negative pressure control means is fluidly connected to the outlet.

[0164] In particular, the negative pressure control means is composed of a negative pressure source, a connection part, and a valve, the negative pressure source is controlled to a constant negative pressure, and the valve is disposed between the negative pressure source and the connection part. The negative pressure control means can be connected to the exhaust port via the connection part. According to this aspect, negative pressure can be applied instantly by opening the valve, so that the timing of application of negative pressure can be controlled more accurately.

[0165] There is no particular limitation on the specific embodiment of the valve. From the viewpoint of preventing backflow when liquid transfer is stopped, it is preferable that the valve can suppress pressure fluctuations in the flow channel when opening and closing, for example, that the opening and closing operation is relatively slow. The valve may be, for example, a three-way valve, and may be capable of connecting the microchannel chip to either a negative pressure source (for example, a pressure tank) or the external atmosphere (particularly the external atmosphere).

[0166] In particular, a negative pressure control means is fluidly connected to the outlet before the dispersed phase liquid and the continuous phase liquid come into contact, in which case negative pressure can be applied quickly after the dispersed phase liquid and the continuous phase liquid come into contact, which may suppress early droplet generation and further improve droplet uniformity.

[0167] (Liquid delivery in emulsion filling method) In the emulsion filling method, it is generally preferable to stop the liquid transfer before the emulsion reaches the outlet (i.e., before the gas-liquid interface reaches the outlet) because this reduces the loss of detected droplets and increases the number of detected droplets. In addition, the flow rate ratio of the continuous phase liquid to the dispersed phase liquid during the liquid transfer may be increased so that only the continuous phase liquid in the emulsion fills the outlet, but it is difficult to fully fill the outlet with the continuous phase liquid.

[0168] For these reasons, in the emulsion filling method, when the liquid supply is stopped, the gas-liquid interface often exists within the flow channel or near the flow channel opening of the outlet, and the sudden pressure fluctuation when the liquid supply is stopped (air flowing into the outlet in the case of negative pressure liquid supply) causes the gas-liquid interface to flow back inside the flow channel, which is likely to have an adverse effect on emulsion retention (droplets flowing out of the emulsion-retaining flow channel, or droplets coagulate and coalesce near the gas-liquid interface).

[0169] In addition, in the case of negative pressure liquid delivery, air flows into the outlet when liquid delivery is stopped, which makes it easier for pressure to be applied to the gas-liquid interface, and if a highly flexible material such as silicone rubber (PDMS) is used for the substrate, or if the substrate is extremely thin (about 1mm or less for COC), the flow path is more likely to deform during negative pressure liquid delivery, and the force that restores this deformation when liquid delivery is stopped makes it more likely for backflow to occur. (In other words, in the center of the flow path far from the side wall of the flow path, the top and bottom surfaces of the flow path bend in a direction that reduces the flow path cross-sectional area (roof collapse). As mentioned above, it is preferable for the width of the emulsion-holding flow path to be large relative to the flow path height, so the impact is thought to be particularly large.)

[0170] In order to suppress such backflow when liquid supply is stopped, it is preferable to reduce the liquid supply pressure. For example, the pressure applied to the microchannel chip by the external liquid supply driving force can be 30 kPa or less, 10 kPa or less, and particularly preferably 5 kPa or less. The pressure applied to the microchannel chip by the external liquid supply driving force is, in particular, a negative pressure applied to the outlet, or a positive pressure applied to the dispersed phase liquid holding portion and the continuous phase liquid holding portion.

[0171] On the other hand, for example, when the present invention is used for rapid digital measurement, the droplet generation speed calculated from the liquid delivery speed and the ratio of the flow speed of the continuous phase liquid to the dispersed phase liquid is preferably high. For example, the droplet generation speed in the emulsion forming section is preferably 5 droplets / second or more, 20 droplets / second or more, 50 droplets / second or more, 100 droplets / second or more, and particularly preferably 200 droplets / second or more.

[0172] When it is desired to reduce the liquid delivery pressure and increase the droplet generation rate, it is desirable to adjust the flow path pressure drop resistance value (= liquid delivery pressure / liquid delivery rate) to be small. The flow path pressure drop resistance value depends on the flow path structure, the surface physical properties of the flow path wall surface, the physical properties of each phase liquid, the pressure control method of the liquid delivery means, etc., so the above parameters can be appropriately adjusted so that the liquid delivery rate and / or the droplet generation rate and liquid delivery pressure during liquid delivery become appropriate values.

[0173] (Installation of microfluidic chip) Microchannel chips are generally installed horizontally in the vertical direction (top-to-bottom direction), but they may be installed with an intentional inclination in a certain direction from the viewpoint of emulsion retention, etc. For example, when the continuous phase liquid has a higher specific gravity than the dispersed phase liquid (e.g., when a fluorine-based dispersant is used as the continuous phase liquid and an aqueous solution is used as the dispersed phase liquid), the droplets have buoyancy due to the difference in specific gravity, so the microchannel chip may be installed with an intentional inclination so that the droplets are less likely to flow out of the emulsion-holding channel.

[0174] <Detection process> A detection process can be performed on droplets in an emulsion produced according to the method of the present disclosure. The detection process can include, for example, a reaction of a target substance in the droplet and detection of the reaction (e.g., detection of a reaction product). The detection process can be performed on droplets in an emulsion held in an emulsion holding flow path.

[0175] Target substances (particularly target molecules) include nucleic acids, proteins, peptides, enzymes, cells, bacteria, spores, viruses, organelles, macromolecular assemblies, drug candidates, lipids, carbohydrates, metabolites, or any combination thereof.

[0176] The reaction of the target substance is not particularly limited. Examples of the reaction of the target substance include enzyme reactions, more specifically, enzyme reactions using kinases, nucleases, nucleotide cyclases, nucleotide ligases, nucleotide phosphodiesterases, polymerases (DNA or RNA), prenyltransferases, pyrophospatases, reporter enzymes, reverse transcriptases, topoisomerases, and the like. When the target molecule is a nucleic acid such as DNA or RNA, and the reaction of the target molecule is an amplification reaction of the nucleic acid, examples of the reaction include reactions capable of isothermal amplification of nucleic acids, such as the LAMP method, the NASBA method, the TMA method, and the TRC method. In addition, in the case of one-step RT-PCR, it is preferable to prepare droplets at a temperature suitable for the reverse transcription reaction in terms of the reaction efficiency and reaction time of the reverse transcription reaction. In addition, it is also possible to detect cDNA, which is a product of the reverse transcription reaction, by the cycling probe method.

[0177] When carrying out a reaction, it is preferable to mix two or more types of reaction liquids upstream of the emulsion forming section (for example, the dispersed phase liquid merging section) and generate droplets using this mixture. In the present invention, the reaction liquid refers to a solution containing at least a part of the target substance and the components necessary for reacting the target substance. It is sufficient that all the components necessary for the reaction of the target substance are prepared by mixing all the reaction liquids, and the target substance is contained in one of the reaction liquids. There is no problem even if three or more types of reaction liquids are used.

[0178] For example, when the target substance is a nucleic acid (DNA, RNA) containing a specific sequence, and the reaction of the target substance is a reaction to amplify the specific sequence, the components contained in the reaction solution include a primer containing a sequence homologous to a part of the specific sequence, a primer containing a sequence complementary to a part of the specific sequence, a detection probe containing a sequence homologous or complementary to a part of the specific sequence, a polymerase, a nucleotide, salts, and a buffer solution component. Note that it is preferable that the composition of the reaction solution is designed so that the target molecule, reaction substrate, enzyme, etc. do not decompose, change, or undergo non-specific reactions, and glycerol, a surfactant, etc. may be further added in consideration of the behavior in the device.

[0179] <Other methods>

[0180] (Detection means) To detect the reaction, for example, a detection means capable of detecting the product of the reaction can be used.

[0181] The detection method can be appropriately selected according to the reaction product, and can be detected using known methods such as optical, X-ray, MALDI (matrix-assisted laser desorption / ionization), FCS (fluorescence correlation spectroscopy), FP (fluorescence polarization) / FCS, fluorometry, colorimetry, chemiluminescence, bioluminescence, scattering, surface plasmon resonance, electrochemistry, electrophoresis, laser, mass spectrometry, Raman spectroscopy, FLIPR (Molecular Devices), etc. When detecting using transmitted light, it is preferable to prepare a microchannel chip from a material that transmits light, since the reaction product can be detected by simply placing the microchannel chip on an optical detector without moving the droplets in the chip.

[0182] The detection means (detector) used to detect the reaction products can be an imaging sensor and optionally its components for recording and measuring the reaction of the target substance. One example of the detection is a camera or imaging device with suitable illumination and resolution to spatially resolve the individual signals to be detected. The camera or imaging device can be any known one, for example, the camera can be any common semiconductor image sensor including a charge-coupled device (CCD), a charge injection device (CID), a photodiode array (PDA) or a complementary metal oxide semiconductor (CMOS). Detection can also be improved by using polarization of the excitation / emitted light. For example, when detecting droplets that emit fluorescent signals, the detection area can be photographed all at once by an optical unit with a large field of view, allowing rapid and high-throughput signal detection.

[0183] (Temperature control means) The temperature control means has a role of maintaining the liquid in the microchannel chip at a temperature suitable for the reaction of the target substance. The temperature control means does not necessarily have to be flat as long as it can be in close proximity (preferably in close contact) with the microchannel chip.

[0184] Of the temperature control means, at least a portion close to (preferably in close contact with) the microchannel chip is preferably made of a metal material with high thermal conductivity. When the microchannel chip is fabricated by bonding a substrate and a superstructure, it is preferable to reduce the thickness of the substrate and / or superstructure in contact with the temperature control means in order to more efficiently conduct heat to the channels provided in the microchannel chip. The temperature control means is sufficient to at least control the temperature of the emulsion-holding channel, which is the reaction field of the target substance, but it is preferable to control the temperature of the phase liquid holding section and the channel in order to suppress nonspecific reactions of the target molecules. As a specific example, when the reaction of the target substance is a nucleic acid amplification reaction, the temperature control means controls the temperature in each holding section and channel so that it is higher than the reaction temperature of the target substance in the emulsion-holding channel, thereby reducing nonspecific annealing between primers / probes. Furthermore, when the bottom surface of the micro-channel chip is heated to the reaction temperature by a temperature control means, and the top surface substrate of the micro-channel chip is made of a light-transmitting material and transmitted light detection is performed from the top surface, this is preferable because it enables easy evaluation of the position of the empty micro-channel chip before supply of each phase liquid and / or the flow channel structure and / or dust inside and outside the chip, evaluation of the behavior inside the flow channel when supplying each phase liquid and / or the behavior of emulsion generation during liquid transfer, and improvement of the upper limit of quantification of digital detection using the signal detection results of the emulsion during the reaction to be performed on the device.

[0185] The present invention will be described in more detail below with reference to examples, but the present invention is not limited to these descriptions. EXAMPLES

[0186] The microchannel chip used in the examples is described below.

[0187] <Fabrication of microfluidic chip> A microfluidic chip was fabricated using photolithography and soft lithography techniques. The specific steps are as follows.

[0188] (1) Photoresist SU-8 3050 (Microchem) was dropped onto a 4-inch bare silicon wafer (Filtech), and then a thin photoresist film was formed using a spin coater (MIKASA).

[0189] (2) Using a mask aligner (Ushio Inc.) and a chrome mask on which the flow channel pattern of the microfluidic chip was formed, the flow channel pattern was formed on a photoresist film. The flow channel pattern was then developed using SU-8 Developer (Microchem Inc.) to create a mold for the flow channels that make up the microfluidic chip.

[0190] (3) To suppress adsorption onto SU-8, a surface treatment was performed by vapor deposition using trichloro(1H,1H,2H,2H-perfluoro-octyl)silane (Thermo Fisher Scientific).

[0191] (4) A mixture of uncured siloxane monomer and polymerization initiator (weight ratio 10:1) prepared using a Sylgard Silicone Elastomer Kit (Dow Corning Toray Co., Ltd.) was poured into the mold treated in (3) above, and heated at 80°C for 2 hours to produce a polymer (PDMS) substrate onto which the shape of the flow path was transferred.

[0192] (5) The obtained polymer substrate was carefully peeled off from the mold and shaped with a cutter, and then a dispersed phase liquid holding portion, a continuous phase liquid holding portion, and an outlet were formed using a puncher.

[0193] (6) The polymer substrate with the holding part and the outlet formed and the cover glass (Matsunami Glass Co., Ltd.) were surface-treated with an oxygen plasma generator (Meiwa Force Systems Co., Ltd.), and then the patterned surface of the PDMS substrate was bonded to the cover glass. The fabricated chip was stored in a desiccator.

[0194] The fabricated microchannel chip was 34 cm long and 75 cm wide, and had a φ4 mm hole for the dispersed phase liquid holding area, a φ8 mm hole for the continuous phase liquid holding area, and a φ1.5 mm hole for the outlet.

[0195] (Flow path structure) The microchannel chip had two dispersed phase liquid holders, a dispersed phase liquid flow path (having a first dispersed phase liquid flow path, a second dispersed phase liquid flow path, and a dispersed phase liquid junction), a continuous phase liquid holder, two continuous phase liquid flow paths, an emulsion forming section, an emulsion flow path, an emulsion holding flow path, and an outlet. The two dispersed phase liquid holders were connected to the emulsion forming section via the first or second dispersed phase liquid flow path, the continuous phase liquid holder was connected to the emulsion forming section via the two continuous phase liquid flow paths, the emulsion forming section was connected to the emulsion holding flow path via the emulsion flow path, and the emulsion holding flow path was connected to the outlet.

[0196] The microchannel chips used in the examples had one of the following detailed structures (No. 1 or No. 2).

[0197] (No.1) The first dispersed phase liquid flow path and the second dispersed phase liquid flow path are meandering flow paths with a height of 80 μm, a width of 100 μm, and a length of 30 mm, and join at a dispersed phase liquid junction, narrowed to a flow path width of 100 μm, and join at an emulsion formation section. The two continuous phase liquid flow paths are straight flow paths with two bends, each with a height of 80 μm, a width of 100 μm, and a length of 30 mm, and in the emulsion formation section, the dispersed phase liquid junction and the two continuous phase liquid flow paths cross at an angle of 90 degrees, and the reaction liquid and the immiscible liquid (oil) join to form droplets. The emulsion flow path is a straight flow path with a width of 80 μm and a length of 100 μm near the emulsion formation section, a width of 200 μm and a length of 680 μm downstream, and a stirring flow path with a width of 200 μm and a length of 11.5 mm including a meandering formed by a circular arc curve with a radius of 275 μm downstream, which is connected to the emulsion holding flow path. The emulsion holding flow path is a meandering flow path with a flow height of 130 μm, a width of 2 mm, and a length of 350 mm, and is directly connected to the outlet via an outlet communication flow path with a width of 2 mm and a length of 10 mm. The volume of the emulsion holding flow path was 91 μL.

[0198] (No.2) The first dispersed phase liquid flow path and the second dispersed phase liquid flow path are meandering flow paths with a height of 100 μm, a width of 200 μm, and a length of 4200 μm. They join at a dispersed phase liquid junction, narrow to a flow path width of 100 μm, and join at an emulsion forming section. The two continuous phase liquid flow paths are straight flow paths with two bends, each with a height of 100 μm, a width of 280 μm, and a length of 26 mm. In the emulsion forming section, the dispersed phase liquid junction and the two continuous phase liquid flow paths cross at an angle of 90 degrees. In the emulsion forming section, the dispersed phase liquid junction and the two continuous phase liquid flow paths cross at an angle of 90 degrees. In the emulsion forming section, the reaction liquid and the immiscible liquid (oil) join to form droplets. The emulsion flow path is a straight flow path with a width of 80 μm and a length of 100 μm near the emulsion formation section, a width of 200 μm and a length of 680 μm downstream, and a stirring flow path with a width of 200 μm and a length of 11.5 mm including a meandering formed by a circular arc curve with a radius of 275 μm downstream, which is connected to the emulsion holding flow path. The emulsion holding flow path is a meandering flow path with a flow height of 130 μm, a width of 2 mm, and a length of 350 mm, and is directly connected to the outlet via an outlet communication flow path with a width of 2 mm and a length of 10 mm. The volume of the emulsion holding flow path was 91 μL.

[0199] Example 1 (Emulsion Creation and Maintenance) Using the above microchannel chip (No. 1), emulsions were produced and maintained according to the present invention, as detailed in the following (1) to (6).

[0200] (dispersed phase liquid) (1) As the dispersed phase liquid to be introduced into the dispersed phase liquid holding section, two types of aqueous solutions with the following compositions ("initiation liquid" and "reaction liquid") were prepared. The aqueous solutions with the following compositions mimic the composition of the reaction initiation liquid when using the TRC reaction, which is one of the nucleic acid amplification reactions.

[0201] (starting solution) 36.8mM Magnesium chloride 180.0mM Potassium Chloride 0.2% (w / v) Tween 20 18.0% (v / v) DMSO 5.0% (v / v) glycerol

[0202] (Reaction solution) 0.2% (w / v) Tween 20 300nM trehalose 5.0% (v / v) glycerol

[0203] (2) A glass heater (Blast) heated to 46°C, the TRC reaction temperature, was placed on an inverted microscope IX71 (Olympus), and the microfluidic chip was placed on top of it and fixed with tape.

[0204] (3) As a liquid delivery means, we used a device that can control the pressure in a 200 mL tank to -1 to -10 kPa, consisting of a peristaltic pump (Takasago Kogyo), an electromagnetic valve (Takasago Kogyo), and a pressure sensor (Keyence). This tank was connected to the outlet of the microfluidic chip with a PTFE tube (Nichias), and a pressure difference (negative pressure) was applied to the chip by releasing the pressure in the tank.

[0205] (4) 200 μL of Droplet Generator Oil for EvaGreen (Biorad, hereinafter also referred to simply as oil) was dropped into the continuous phase liquid holding part using a pipette (Gilson). 30 seconds after the oil was dropped, 20 μL of the reaction solution and the starting solution were dropped into the two dispersed phase liquid holding parts using a 2.0 to 20.0 μL pipette, respectively, and the continuous phase liquid and the dispersed phase liquid were brought into contact with each other in the dispersed phase liquid holding part. The above pipette was calibrated to a maximum allowable error of ±0.06 μL.

[0206] (5) 20 seconds after the reaction liquid was dropped, the pressure in the tank connected to the outlet was adjusted to -5 kPa in advance, and negative pressure was applied to start droplet generation and retention. Note that each phase liquid retention part was open to atmospheric pressure during the transfer.

[0207] (Evaluation of the time until the flow of the reaction solution and the starting solution stabilizes) Using a digital CMOS camera (ORCA-FLASH (product name), Hamamatsu Photonics), the state of emulsion generation was captured as a bright field image, and the time from when the reaction liquid and the starting liquid came into contact at the dispersed phase liquid merging channel until a stable phase liquid flow (uniformly mixed flow) was formed was measured (N=4). The results are shown in Table 1 below.

[0208] Figure 4 is a photograph showing the state of emulsion production in Example 1. As can be seen in Figure 4, the reaction liquid and the initiation liquid form a stable phase liquid flow at the dispersed phase liquid junction. Also, Figure 4 shows that a thin layer of continuous phase liquid exists between the reaction liquid and the initiation liquid.

[0209] Comparative Example 1 An emulsion was produced in the same manner as in Example 1, except that the channel surface of the microchannel chip (No. 1) was subjected to a fluorine treatment using a long-chain fluorocarbon silanizing agent (fluorine compound) as described below.

[0210] (Fluorine treatment) In the manufacturing process of the microchannel chip described above, after bonding the PDMS substrate and the cover glass, ethanol containing 2% Trichloro(1H,1H,2H,2H-perfluoro-octyl)silane (Thermo Fisher Scientific) was introduced into the channel and left for 30 minutes to modify the surface of the channel wall. After that, the inside of the channel was washed with ethanol, heated at 120 degrees for 3 minutes to evaporate the ethanol, and air-dried overnight to produce a microchannel chip. The fabricated chip was stored in a desiccator.

[0211] (Evaluation of time until flow stabilization of reaction solution and starting solution) In Comparative Example 1, the time from when the reaction liquid and the starting liquid came into contact with each other in the dispersed phase liquid joining passage until they became a stable phase liquid flow (uniformly mixed flow) was measured (N=4) in the same manner as in Example 1. The results are shown in Table 1 below.

[0212] [Table 1]

[0213] As seen in Table 1, in Example 1 in which the wall surface of the dispersed phase liquid flow path was made of PDMS and glass, the time until the reaction liquid and the initiation liquid merged to form a stable phase liquid flow (homogeneous mixed flow) was significantly shorter than in Comparative Example 1 in which the wall surface of the dispersed phase liquid flow path was treated with a fluorine compound.

[0214] Example 2 An emulsion was generated and maintained in the same manner as in Example 1 above, except that the dispersed phase liquid and continuous phase liquid were supplied and delivered using the above microchannel chip (No. 2) as described below.

[0215] (Supply of dispersed phase liquid and continuous phase liquid) In Example 2, 20 μL of the reaction liquid and the initiation liquid were dropped into two dispersed phase liquid holding parts using a pipette. After 40 seconds, 200 μL of oil was dropped into the continuous phase liquid holding part. Before the continuous phase liquid reached the emulsion forming part, the dispersed phase liquid (a mixture of the reaction liquid and the initiation liquid) passed through the dispersed phase liquid flow path filled with gas and reached the inlet to the emulsion forming part.

[0216] (liquid delivery) In Example 2, 30 seconds after the oil was dripped, the pressure inside the tank of the liquid delivery device connected to the outlet was adjusted to -5 kPa in advance, and a pressure difference (negative pressure) was applied to start droplet generation and droplet retention. Note that each retention part during the liquid transfer was open to atmospheric pressure.

[0217] (evaluation) For Example 2, the presence or absence of intrusion of the continuous phase liquid into the dispersed phase liquid flow paths was evaluated.

[0218] Specifically, a digital CMOS camera (ORCA-FLASH (product name), Hamamatsu Photonics) was used to obtain bright-field images of the flow paths when the dispersed phase liquid and the continuous phase liquid were in contact before the application of negative pressure, and the images were evaluated. As a result, in Example 2, no intrusion of the continuous phase liquid into the dispersed phase liquid flow paths was observed.

[0219] Comparative Example 2 An emulsion was generated and maintained in the same manner as in Example 2, except that the channel surface of the microchannel chip was subjected to a fluorine treatment using a long-chain fluorocarbon silanizing agent (fluorine compound) as described below.

[0220] (Fluorine treatment) In the manufacturing process of the microchannel chip described above, after bonding the PDMS substrate and the cover glass, ethanol containing 2% Trichloro(1H,1H,2H,2H-perfluoro-octyl)silane (Thermo Fisher Scientific) was introduced into the channel and left for 30 minutes to modify the surface of the channel wall. After that, the inside of the channel was washed with ethanol, heated at 120 degrees for 3 minutes to evaporate the ethanol, and air-dried overnight to produce a microchannel chip. The fabricated chip was stored in a desiccator.

[0221] (evaluation) The presence or absence of intrusion of the continuous phase liquid into the dispersed phase liquid flow path was evaluated in the same manner as in Example 2. In Comparative Example 2, the continuous phase liquid that reached the emulsion formation section was observed to intrude into the dispersed phase liquid flow path (more precisely, the dispersed phase liquid junction section).

[0222] As described above, inflow of the continuous phase liquid into the dispersed phase liquid flow paths was not observed in Example 2, in which the wall surfaces of the dispersed phase liquid flow paths were made of PDMS and glass. On the other hand, in Comparative Example 2, in which the wall surfaces of the dispersed phase liquid flow paths were treated with a fluorine compound, intrusion of the continuous phase liquid into the dispersed phase liquid flow paths was observed, even though the dispersed phase liquid flow paths were filled with the dispersed phase liquid. [Explanation of symbols]

[0223] 10, 20 Microfluidic chip 101 Continuous phase liquid holding section 102 First dispersed phase liquid holding section 103 Second dispersed phase liquid holding section 111 Continuous phase liquid flow path 112 First continuous phase liquid flow path 113 Second continuous phase liquid flow path 114 First dispersed phase liquid flow path 115 Second dispersed phase liquid flow path 116 Dispersed phase liquid confluence section 117 Dispersed phase liquid flow path 120 Emulsion forming section 130 Emulsion channel 140 Emulsion holding channel 150 Outlet 200 Dispersed phase liquid holding section 21 wells 23 Hole 25 Expansion Area W Width direction L Length direction H Height direction

Claims

1. A method for producing an emulsion by supplying a dispersed phase liquid and a continuous phase liquid to a microchannel chip, comprising the steps of: the microchannel chip has a dispersed phase liquid holding section, a dispersed phase liquid flow path, a continuous phase liquid holding section, a continuous phase liquid flow path, an emulsion forming section, an emulsion flow path, an outlet, and an emulsion holding flow path; the dispersed phase liquid holding unit is connected to the emulsion forming unit via the dispersed phase liquid flow path, the continuous phase liquid holding unit is connected to the emulsion forming unit via the continuous phase liquid flow path, the emulsion forming section is connected to the emulsion holding flow path via the emulsion flow path, the emulsion holding flow path is connected to the outlet, Supplying a dispersed phase liquid to the dispersed phase liquid holding unit; supplying a continuous phase liquid to the continuous phase liquid holding section; and applying a negative pressure to the outlet, in the emulsion forming section, to generate an emulsion containing droplets composed of the dispersed phase liquid and a continuous phase composed of the continuous phase liquid, and transporting the emulsion thus generated through the emulsion flow path to the emulsion holding flow path filled with gas; Including, The surface of the wall of the dispersed phase liquid flow path is made of an inorganic material and / or an organic material that does not contain a fluorine compound and is not treated with a fluorine compound; and The continuous phase liquid is a fluorine-based dispersion medium; Characterized by: method.

2. The method according to claim 1, characterized in that the surface of the wall of the dispersed phase liquid retaining portion is made of an inorganic material and / or an organic material that does not contain a fluorine compound and is not treated with a fluorine compound.

3. 3. The method of claim 1 or 2, wherein the continuous phase liquid comprises a fluorosurfactant.

4. The method according to any one of claims 1 to 3, characterized in that the dispersed phase liquid is an aqueous solution.

5. The method according to any one of claims 1 to 4, characterized in that the inorganic material and / or organic material that does not contain a fluorine compound and is not treated with a fluorine compound is at least one selected from polydimethylsiloxane (PDMS), glass, cyclic olefin copolymer (COC), cyclic olefin polymer (COP), and polymethyl methacrylate (PMMA).

6. The method according to any one of claims 1 to 5, characterized in that the surface of the wall of the dispersed phase liquid flow channel is treated with a non-fluorine-based surface treatment agent.

7. The dispersed phase liquid holding portion includes a first dispersed phase liquid holding portion and a second dispersed phase liquid holding portion, The dispersed phase liquid flow path includes a first dispersed phase liquid flow path connected to the first dispersed phase liquid holding portion, a second dispersed phase liquid flow path connected to the second dispersed phase liquid holding portion, and a dispersed phase liquid junction portion; the first dispersed phase liquid flow path and the second dispersed phase liquid flow path are each connected to the emulsion forming section via a dispersed phase liquid junction section; The method according to any one of claims 1 to 6, characterized in that

8. A microchannel chip having a dispersed phase liquid holding section, a dispersed phase liquid flow path, a continuous phase liquid holding section, a continuous phase liquid flow path, an emulsion forming section, an emulsion flow path, an outlet, and an emulsion holding flow path, the dispersed phase liquid holding unit is connected to the emulsion forming unit via the dispersed phase liquid flow path, the continuous phase liquid holding unit is connected to the emulsion forming unit via the continuous phase liquid flow path, the emulsion forming section is connected to the emulsion holding flow path via the emulsion flow path, the emulsion holding flow path is connected to the outlet, and, a dispersed phase liquid is supplied to the dispersed phase liquid holding section, a continuous phase liquid is supplied to the continuous phase liquid holding section, and a negative pressure is applied to the outlet, whereby an emulsion containing droplets composed of the dispersed phase liquid and a continuous phase composed of the continuous phase liquid is generated in the emulsion forming section, and the emulsion thus generated is transported through the emulsion flow path to the emulsion holding flow path filled with gas, The surface of the wall of the dispersed phase liquid flow path is made of an inorganic material and / or an organic material that does not contain a fluorine compound and is not treated with a fluorine compound; and The continuous phase liquid is a fluorine-based dispersion medium. Microfluidic chip.

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