Method and apparatus for generating an emulsion in a microchannel chip

Abstract

To provide an improved emulsion generation method and device, 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 the application of 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. Before the continuous phase liquid reaches the emulsion forming unit 120, the dispersed phase liquid is passed through a dispersed phase liquid channel 117 filled with gas and moved to an inflow part of the emulsion forming unit 120.SELECTED DRAWING: Figure 1

Description

Technical Field

[0001] The present invention relates to a method and an apparatus for generating an emulsion in a microchannel chip. In particular, the present invention relates to an apparatus and a method capable of performing droplet array measurement more efficiently, simply, and rapidly.

Background Art

[0002] As a technique for fractionating a reaction solution into minute compartments and performing reactions independently, a microdroplet method of fractionating a reaction solution into microdroplets is known. This technique is expected to be applied, for example, to the production of micro- and nanoparticles. In particular, by using a microfluidic device to fractionate target molecules into minute compartments on a single-molecule basis and performing reactions within the microdroplets, the presence or absence of target molecules is measured by the presence or absence of signals, and digital measurement for absolute quantification of the number of target molecules is utilized.

[0003] In the microdroplet method, generally, an emulsion composed of a continuous phase such as oil and droplets of an aqueous solution dispersed in this continuous phase is used.

[0004] Non-Patent Document 1 discloses a centrifugal step droplet generation method. This document describes filling oil into the injection port of the apparatus, sending this oil to the droplet collection chamber by centrifugation, and then introducing a sample solution from the same injection port to generate droplets by centrifugation.

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

[0006] Patent Document 1 discloses such a system and method for generating droplets suitable for droplet assays. The document describes transporting the generated droplets to an outlet region consisting of a pipette tip or a droplet well. The document also describes a bubble trap (air trap), and it is described that the sample and the oil are substantially separated by this bubble trap until the application of a fluid driving force (such as negative pressure or positive pressure).

[0007] Regarding the micro-droplet method, in recent years, from the perspective of simplifying and accelerating the device, droplet array measurement that simply measures signals by aligning droplets in a single layer in the detection region has attracted attention.

[0008] Patent Documents 2 and 3 disclose a microchannel chip having a flow path for forming droplets and a droplet holding portion for holding droplets. Patent Document 2 describes forming droplets by bringing a non-miscible liquid that does not mix with the reaction liquid into contact after merging two or more reaction liquids. Patent Document 3 describes droplet formation by bringing the dispersed phase and the continuous phase flowing in from the dispersed phase inflow portion and the continuous phase inflow portion into contact in the droplet generation portion through the flow path.

[0009] Non-Patent Document 2 describes a method for generating droplets on a chip and an apparatus therefor. The method described in the document includes an operation (filling operation) of filling the droplet array portion with oil before liquid feeding.

Prior Art Documents

Patent Documents

[0010]

Patent Document 1

Patent Document 2

Patent Document 3

Non-Patent Documents

[0011]

Non-Patent Document 1

Non-Patent Document 2

Summary of the Invention

Problems to be Solved by the Invention

[0012] A method of supplying a continuous phase liquid and a dispersed phase liquid to respective supply parts separately prepared on a microchannel chip and merging them in an emulsion forming part through respective channels from the supply parts has advantages such as being able to increase the droplet generation rate, while problems may occur in the flow rate or mixing of the dispersed phase liquid.

[0013] An object of the present invention is to provide an improved emulsion generation method in a method of separately supplying a continuous phase liquid and a dispersed phase liquid to a microchannel chip, and to provide an apparatus that can be used in such a method.

Means for Solving the Problems

[0014] The above problems according to the present invention can be solved by the following aspects according to the present invention. <Aspect 1> A method of supplying a dispersed phase liquid and a continuous phase liquid to a microchannel chip to generate an emulsion, wherein the microchannel chip has a dispersed phase liquid holding part, a dispersed phase liquid flow path, a continuous phase liquid holding part, a continuous phase liquid flow path, an emulsion forming part, an emulsion flow path, and a discharge port, The dispersed-phase liquid holding part is connected to the emulsion forming part through the dispersed-phase liquid flow path, The continuous-phase liquid holding part is connected to the emulsion forming part through the continuous-phase liquid flow path, The emulsion forming part is connected to the discharge port through the emulsion flow path, Supplying a dispersed-phase liquid to the dispersed-phase liquid holding part, Supplying a continuous-phase liquid to the continuous-phase liquid holding part, and By applying an external liquid feeding driving force, an emulsion including droplets composed of the dispersed-phase liquid and a continuous phase composed of the continuous-phase liquid is generated in the emulsion forming part, and the emulsion thus generated is made to enter the emulsion flow path, including Before the continuous-phase liquid reaches the emulsion forming part, moving the dispersed-phase liquid through the dispersed-phase liquid flow path filled with gas to the inflow part to the emulsion forming part A method characterized by the above. <Aspect 2> The method according to aspect 1, characterized in that, before applying the external liquid feeding driving force, the continuous-phase liquid is moved to the emulsion forming part. <Aspect 3> The method according to aspect 1 or 2, characterized in that the dispersed-phase liquid and / or the continuous-phase liquid is moved by capillary force and / or liquid surface differential pressure. <Aspect 4> Applying a negative pressure to the discharge port, thereby moving the dispersed-phase liquid through the dispersed-phase liquid flow path filled with gas to the inflow part to the emulsion forming part, the method according to any one of aspects 1 to 3. <Aspect 5> The inflow part has an entry suppression structure, This entry suppression structure can suppress the entry of the dispersed-phase liquid into the emulsion forming part before applying the external liquid feeding driving force, A method according to any one of aspects 1 to 4, characterized by the above. <Aspect 6> The method according to any one of Aspects 1 to 5, characterized in that the external liquid feeding driving force is a negative pressure applied to the discharge port. <Aspect 7> The dispersed-phase liquid holding part includes a first dispersed-phase liquid holding part and a second dispersed-phase liquid holding part. The dispersed-phase liquid flow path includes a first dispersed-phase liquid flow path connected to the first dispersed-phase liquid holding part, a second dispersed-phase liquid flow path connected to the second dispersed-phase liquid holding part, and a dispersed-phase liquid confluence part. The first dispersed-phase liquid flow path and the second dispersed-phase liquid flow path are each connected to the emulsion forming part via a dispersed-phase liquid confluence part. The method according to any one of Aspects 1 to 6. <Aspect 8> The microchannel chip further has an emulsion holding flow path. The emulsion forming part is connected to the emulsion holding flow path via the emulsion flow path, and The emulsion holding flow path is connected to the discharge port. By applying a negative pressure to the discharge port, 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 part, and the emulsion thus generated is transported to the emulsion holding flow path filled with gas via the emulsion flow path. The method according to any one of Aspects 1 to 7. <Aspect 9> A microchannel chip having a dispersed-phase liquid holding part, a dispersed-phase liquid flow path, a continuous-phase liquid holding part, a continuous-phase liquid flow path, an emulsion forming part, an emulsion flow path, and a discharge port, The dispersed-phase liquid holding part is connected to the emulsion forming part via the dispersed-phase liquid flow path. The continuous-phase liquid holding part is connected to the emulsion forming part via the continuous-phase liquid flow path. The emulsion forming part is connected to the discharge port via the emulsion flow path, and By supplying a dispersed-phase liquid to the dispersed-phase liquid holding part, supplying a continuous-phase liquid to the continuous-phase liquid holding part, and applying an external liquid-feeding driving force to the microchannel chip, 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 part, and the emulsion thus generated is configured to enter the emulsion flow channel. Here, an inflow part of the dispersed-phase liquid flow channel into the emulsion forming part has an entry suppression structure, and this entry suppression structure can suppress the entry of the dispersed-phase liquid into the emulsion forming part before the application of the external liquid-feeding driving force. Microchannel chip.

Advantages of the Invention

[0015] According to the present invention, in a method of separately supplying a continuous-phase liquid and a dispersed-phase liquid to a microchannel chip, an improved emulsion generation method can be provided.

Brief Description of the Drawings

[0016]

Figure 1

Figure 2

Figure 3

Figure 4

Embodiments for Carrying Out the Invention

[0017] ≪Method for Generating an 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, 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 a discharge port, 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, The emulsion forming section is connected to the discharge port via the emulsion flow path, Supplying a dispersed-phase liquid to the dispersed-phase liquid holding section, Supplying a continuous-phase liquid to the continuous-phase liquid holding section, and By applying an external liquid feeding driving force, an emulsion including liquid 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 caused to enter the emulsion flow path, including Before the continuous-phase liquid reaches the emulsion forming section, the dispersed-phase liquid is moved to the inflow section to the emulsion forming section through the dispersed-phase liquid flow path filled with gas.

[0018] As a method for generating an emulsion with a microchannel chip, a method can be used in which the continuous-phase liquid and the dispersed-phase liquid are supplied to respective supply sections separately prepared on the microchannel chip, and are caused to merge in the emulsion forming section through the respective flow paths to generate an emulsion. This method has advantages such as being able to increase the droplet generation rate per emulsion forming section as compared with a method of supplying the continuous-phase liquid and the dispersed-phase liquid from one and the same holding section (for example, the method of Non-Patent Document 1).

[0019] On the other hand, in the method using separate supply sections, when the continuous-phase liquid enters the dispersed-phase liquid flow path, the flow of the dispersed-phase liquid in the dispersed-phase liquid flow path tends to become unstable, and thereby the flow rate of the dispersed-phase liquid tends to become unstable.

[0020] In particular, when two types of dispersed-phase liquids are mixed in a microchannel chip, such destabilization of the flow rates of the dispersed-phase liquids leads to a non-uniform mixing ratio of the two dispersed-phase liquids. This can result in a decrease in the accuracy of the detection reaction.

[0021] Although not intended to be limited by theory, it is considered that one of the factors is that the continuous-phase liquid remains on the wall surface of the dispersed-phase liquid flow path. In particular, when a liquid with relatively low surface tension and viscosity, such as a fluorine-based dispersion medium, is used as the continuous-phase liquid, it is considered that the continuous-phase liquid can quickly flow by capillary action or the like and easily enter the dispersed-phase liquid flow path, and the continuous-phase liquid easily remains in the flow path. Further, when using a microchannel chip including a dispersed-phase liquid confluence part where two or more dispersed-phase liquids can be mixed in the flow path, the continuous-phase liquid filled in the dispersed-phase liquid flow path, the dispersed-phase liquid confluence part, and the dispersed-phase liquid holding part remains in the flow path even during the generation of droplets, and it is considered that it inhibits the uniform mixing in the droplets due to the laminar flow of the dispersed-phase liquid in the dispersed-phase liquid confluence part. The inhibition of uniform mixing by the remaining continuous-phase liquid is considered to be caused by the continuous-phase liquid forming a thin layer between the flow path wall surface and the dispersed-phase liquid. Therefore, it is considered 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. However, this may show an effect contrary to the generation and retention of stable droplets, so it is not preferable. In addition, for the purpose of suppressing the mixing of the dispersed-phase liquids before emulsion formation, an example of performing emulsion formation while maintaining the laminar flow state of the dispersed-phase liquid confluence part has been shown. However, for example, even in a dispersed-phase liquid confluence part having a structure (micromixer structure) that promotes stirring, it is considered that the behavior of the interface between the dispersed-phase liquids becomes unstable and inhibits the uniform mixing in the droplets.

[0022] In the present invention, before the continuous-phase liquid reaches the emulsion formation part, the dispersed-phase liquid is moved through the dispersed-phase liquid flow path filled with gas to the inflow part to the emulsion formation part. According to the method of the present invention, since the dispersed-phase liquid advances in the dispersed-phase liquid flow path and blocks the dispersed-phase liquid flow path, the continuous-phase liquid cannot enter the dispersed-phase liquid flow path. As a result, it is considered that the problem of the continuous-phase liquid flowing backward and remaining in the dispersed-phase liquid flow path can be suppressed or prevented.

[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 implemented with various microchannel chips. FIG. 1 is a schematic diagram for easy understanding and is not to scale.

[0024] The microchannel chip in FIG. 1 has a planar configuration, that is, the generation and transportation of the emulsion are substantially performed in one plane. The direction W in FIG. 1 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 in FIG. 1 includes a first dispersed-phase liquid holding portion 102, a second dispersed-phase liquid holding portion 103, a dispersed-phase liquid flow path 117, a continuous-phase liquid holding portion 101, a continuous-phase liquid flow path 111, an emulsion forming portion 120, an emulsion flow path 130, and a discharge port 150. The dispersed-phase liquid holding portions 102 and 103 are connected to the emulsion forming portion 120 via the dispersed-phase liquid flow path 117, and the continuous-phase liquid holding portion 101 is connected to the emulsion forming portion 120 via the continuous-phase liquid flow path 111. In the embodiment according to FIG. 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 confluence portion 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 portion 120 is connected to the discharge port 150 via the emulsion flow path 130.

[0025] In the method according to the present disclosure, a dispersed-phase liquid such as a water-soluble reaction solution containing a substance to be detected is supplied to the dispersed-phase liquid holding portions 102 and 103, and a continuous-phase liquid such as oil is supplied to the continuous-phase liquid holding portion 101. At this point, each flow path of the microchannel chip 10 is filled with gas (particularly air) (that is, a "hollow" microchannel chip is used). In the method according to the present invention, since the flow paths of the microchannel chip are not pre-filled with the continuous-phase liquid and the dispersed solution and the continuous-phase liquid are supplied to the chip, the preparation process can be omitted.

[0026] The term "empty microchannel chip" refers to a state in which the inside of the channel is filled with gas (especially air), and it is preferable that there is no liquid (for example, a dispersed-phase liquid, a continuous-phase liquid, etc.) in the entire channel, that is, the entire channel is filled with gas. Note that even if liquid remains or is generated in the channel of the microchannel chip due to surface treatment or condensation of water in the air, this is not the case as long as at least a part of the channel is not blocked by the liquid. In particular, it is preferable that the dispersed-phase liquid channel, the continuous-phase liquid channel, and the emulsion formation part are not blocked.

[0027] The microchannel chip 10 in FIG. 1 is configured such that, for example, the dispersed-phase liquid and the continuous-phase liquid can be moved from their respective holding parts through their respective channels to the emulsion formation part 120 by capillary force and / or liquid level differential pressure. Therefore, the dispersed-phase liquid and the continuous-phase liquid supplied to each of the holding parts 101, 102, 103 move in the direction (downstream) of the emulsion formation part 120. Note that, for this movement (especially the movement of the dispersed-phase liquid), a pressure applied from the outside (especially a negative pressure applied to the discharge port) may be used.

[0028] According to the present invention, before the continuous-phase liquid reaches the emulsion formation part 120, the dispersed-phase liquid is moved through the dispersed-phase liquid channel 117 filled with gas to the inflow part 118 to the emulsion formation part 120. The inflow part 118 is a part of the dispersed-phase liquid channel (especially the dispersed-phase liquid confluence part) adjacent to the emulsion formation part 120. According to the method of the present invention, since the dispersed-phase liquid advances in the dispersed-phase liquid channel 117 and blocks the dispersed-phase liquid channel 117, the continuous-phase liquid cannot enter the dispersed-phase liquid channel 117. As a result, the problem that the continuous-phase liquid flows backward into the dispersed-phase liquid channel 117 and remains can be suppressed.

[0029] 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 an emulsion forming section 120 by an external liquid-feeding driving force, and the emulsion thus generated is caused to enter an emulsion flow path 130. As the external liquid-feeding driving force, a negative pressure or a positive pressure can be used. The application of the negative pressure can be performed, for example, by a suction device connected to an outlet 150 of a microchannel chip 10. Also, the application of the positive pressure can be performed, for example, by a pressure applying device connected to dispersed-phase liquid holding sections 102 and 103 and a continuous-phase liquid holding section 101.

[0030] <Movement> As described above, in the present invention, before the continuous-phase liquid reaches the emulsion forming section, the dispersed-phase liquid is moved through a dispersed-phase liquid flow path filled with a gas to an inflow section to the emulsion forming section. In one embodiment according to the present disclosure, the emulsion forming section and the flow path resistance upstream thereof are sufficiently small such that the dispersed-phase liquid can move even without an external liquid-feeding driving force.

[0031] Preferably, immediately after the dispersed-phase liquid reaches the inflow section to the emulsion forming section (particularly, before the dispersed-phase liquid enters the emulsion forming section), the continuous-phase liquid is caused to enter the emulsion forming section. In this case, it is possible to prevent the emulsion forming section from being blocked by the dispersed-phase liquid and the generation of associated bubbles.

[0032] Note that even after the dispersed-phase liquid enters the emulsion forming section, by causing the continuous-phase liquid to enter the emulsion forming section before the dispersed-phase liquid fills and blocks the emulsion forming section, it is possible to prevent the flow path from being blocked.

[0033] Also, particularly preferably, before applying the external liquid-feeding driving force, the continuous-phase liquid is moved to the emulsion forming section. By moving the continuous-phase liquid to the emulsion forming section before applying the external liquid-feeding driving force to start liquid feeding, it is possible to more effectively suppress the generation of bubbles (flow path blockage) caused by the gas present in the continuous-phase liquid flow path.

[0034] The movement of such a dispersed-phase liquid and / or continuous-phase liquid can be carried out, for example, by capillary force and / or liquid level difference. According to this aspect, the movement of the dispersed-phase liquid and the continuous-phase liquid can be easily controlled without the need for additional devices. For example, by appropriately setting the flow path structure (such as the characteristics of the flow path surface and the pressure loss of the flow path) of the micro flow path chip, a desired capillary force can be obtained. Also, for example, by adjusting the amount of liquid supplied to each phase liquid holding portion (particularly the liquid level height), a desired liquid level differential pressure can be obtained.

[0035] Capillary force, also called capillary action, is a force that acts in the direction of invading a flow path generated by the surface tension difference between the gas-liquid interface in a largely open holding portion and the gas-liquid interface in a flow path having a smaller cross-section. Therefore, capillary force affects not only the characteristics of each flow path but also the structure of the holding portion. Particularly in the emulsion filling method, in order to control the movement of the gas-liquid interface of the emulsion, capillary force has a great influence even during liquid feeding (droplet generation) and during droplet holding after the liquid feeding stops.

[0036] Also, the liquid level differential pressure, also called hydrostatic pressure, generally refers to the pressure generated by gravity in a stationary liquid, that is, the pressure depending on the supply amount (weight) of each phase liquid to each holding portion.

[0037] In one embodiment according to the present disclosure, the emulsion forming portion and the flow path resistance upstream thereof are sufficiently small so that the dispersed-phase liquid and the continuous-phase liquid can come into contact even without an external liquid feeding driving force. As a result, for example, from the time when the dispersed-phase liquid is supplied to the dispersed-phase liquid holding portion (of the micro flow path chip in an empty state), the time until the dispersed-phase liquid comes into contact with the continuous-phase liquid is within 5 minutes. This time is preferably within 3 minutes, and more preferably within 1 minute. In this case, the time required for emulsion generation (and optionally holding) can be further shortened.

[0038] For the above-described movement, pressure applied from the outside may be used. In particular, a negative pressure may be applied to the discharge port, and in this way, the dispersed-phase liquid can be moved through the dispersed-phase liquid flow path filled with gas to the emulsion formation section (particularly the inlet section to the emulsion formation section). Generally, since the dispersed-phase liquid has low affinity for the flow path surface, it is preferable to increase the movement speed of the dispersed-phase liquid. By using negative pressure as one of the means, the apparatus can be simplified. Regarding the pressure applied from the outside, reference can be made to the following description regarding the external liquid feeding driving force.

[0039] Particularly preferably, after the state where the dispersed-phase liquid fills the dispersed-phase liquid flow path and the continuous-phase liquid fills the continuous-phase liquid flow path and the emulsion formation section, the external liquid feeding driving force is applied.

[0040] When controlling the movement before the above contact by applying a positive pressure to each holding section, in order to control a minute amount 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 positive pressure application time to an extremely short time. Therefore, there is a possibility that the reproducibility of the apparatus may decrease.

[0041] On the other hand, for example, when a negative pressure is applied to the discharge port when the dispersed-phase liquid moves in the dispersed-phase liquid flow path before supplying the continuous-phase liquid, since the continuous-phase liquid flow path and the continuous-phase liquid holding section are not blocked by liquid, the flow path from the discharge port to the continuous-phase liquid holding section is open to the outside air, and a negative pressure smaller than the negative pressure of the negative pressure source is applied to the dispersed-phase liquid. Therefore, it is easy to control the applied pressure to be small for accurately controlling the movement of a minute amount of the dispersed-phase liquid in the flow path, and it is preferable because it is easy to ensure the reproducibility of the apparatus.

[0042] (Entry suppression structure) In a preferred embodiment according to the present disclosure, the inlet section has an entry suppression structure, and this entry suppression structure can suppress the entry of the dispersed-phase liquid into the emulsion formation section before applying the external liquid feeding driving force. Note that the "inlet section" is a portion adjacent to the emulsion formation section in the dispersed-phase liquid flow path (particularly the dispersed-phase liquid confluence section).

[0043] By retaining the dispersed - phase liquid at the inflow portion to the emulsion - forming section by the entry - inhibiting structure, it becomes easier to control the timing of arrival of the dispersed - phase liquid and the continuous - phase liquid, and it is possible to more effectively suppress the occurrence of channel blockage and bubbles.

[0044] The specific design of the entry - inhibiting structure is not particularly limited as long as it exhibits the desired function, that is, the function of suppressing the entry of the dispersed - phase liquid into the emulsion - forming section before the application of an external liquid - feeding driving force. The specific design of the entry - inhibiting structure can be appropriately determined according to the types of the dispersed - phase liquid and the continuous - phase liquid used, the amounts of the supplied dispersed - phase liquid and the continuous - phase liquid, the channel structure of the micro - flow - path chip other than the emulsion - forming section (such as the characteristics of the channel surface, the pressure loss of the channel, etc.).

[0045] Preferably, the entry - inhibiting structure and other channels are configured such that the dispersed - phase liquid flowing in the dispersed - phase liquid channel filled with gas and moving toward the emulsion - forming section by capillary force and / or liquid - level differential pressure (and / or negative pressure) stops at the inflow portion by the entry - inhibiting structure and does not enter the inside of the emulsion - forming section beyond the inflow portion unless an external liquid - feeding driving force is applied.

[0046] The entry - inhibiting structure may be, for example, a structure in which the channel cross - section (channel cross - sectional area), particularly the channel height, rapidly increases in the direction from the inflow portion toward the emulsion - forming section. By adopting such a structure, a local force that inhibits further movement of the dispersed - phase liquid can be generated. This local force is more likely to occur as the affinity of the channel wall surface of the entry - inhibiting structure for the dispersed - phase liquid is lower. For example, when the dispersed - phase liquid is an aqueous solution, the contact angle of the aqueous solution with the wall surface of the dispersed - phase liquid channel is preferably 30 degrees or more, more preferably 45 degrees or more, and particularly preferably 60 degrees or more. When different materials are used for the upper structure and the base material, for example, when PDMS is used as the upper structure and glass is used as the base material, the affinity of the wall surface for the dispersed - phase liquid within the channel cross - section of the entry - inhibiting structure may be partially low, but it is also possible to design it so that the entry - inhibiting structure functions well.

[0047] From the perspective of manufacturing the microchannel chip, generally, since the flow channel structure is arranged such that the liquid flows horizontally with respect to the vertical direction (up-down direction), the inflow angle to the emulsion forming section is substantially zero (or 180°) in many cases on the upper surface and the bottom surface of the flow channel. Particularly in such cases, it is preferable that the upper surface (and / or the bottom surface) of the dispersed phase liquid flow channel and the emulsion forming section do not exist on the same plane, and in the direction from the inflow section to the emulsion forming section, the cross-section of the flow channel (the cross-sectional area of the flow channel) increases, that is, the height of the flow channel rapidly increases (so-called "step" structure). For example, it can be implemented by the bottom surface (floor portion) inside the emulsion forming section being at a lower position than the bottom surface inside the inflow section connected thereto, or by the upper surface (ceiling portion) inside the emulsion forming section being at a higher position than the upper surface inside the inflow section connected thereto, or by both of these. Here, the "bottom surface" and "upper surface" can be defined based on the up-down direction (particularly the vertical direction) in the normal use state of the microchannel chip. Also, in the step structure, it is more preferable that the upper surface of the dispersed phase liquid flow channel and the upper surface of the emulsion forming section (and / or the bottom surface of the dispersed phase liquid flow channel and the bottom surface of the emulsion forming section) are connected in a more angular (discontinuous and acute angle) manner rather than being smoothly connected, because it becomes more difficult for the dispersed phase liquid to enter the emulsion forming section.

[0048] FIG. 2 is a schematic cross-sectional view showing one embodiment of the entry suppression structure according to the present disclosure. FIG. 2 is a schematic view for easy understanding and is not to scale. H in FIG. 2 indicates the height direction, and L indicates the length direction. The entry suppression structure 200 in FIG. 2 has a so-called "step" structure. Specifically, the upper surface of the flow channel of the emulsion forming section 120 and the upper surface of the inflow section 118 of the dispersed phase liquid flow channel are not on the same plane, and in the direction from the inflow section 118 to the emulsion forming section 120, the height of the flow channel rapidly increases.

[0049] In the entry suppression structure 200 of FIG. 2, the upper surface of the flow path of the dispersed phase liquid flow path and the upper surface of the flow path of the emulsion forming section 120 are discontinuously connected, and the bottom surface of the flow path of the dispersed phase liquid flow path and the bottom surface of the flow path of the emulsion forming section are discontinuously connected (in particular, in FIG. 2, the wall surface has a corner). However, either the upper surface or the bottom surface of the flow path may be discontinuously connected, and in particular, only the upper surface is discontinuously connected.

[0050] In the entry suppression structure 200 of FIG. 2, the upper surface of the flow path of the emulsion forming section 120 and the upper surface of the inflow section 118 of the dispersed phase liquid flow path are connected by a connection section 220a that constitutes the wall surface of the emulsion forming section 120. Similarly, the bottom surface is also connected by a connection section 220b. The connection section 220a is arranged at an angle α1 of 90° with respect to the upper surface of the flow path of the inflow section 118 of the dispersed phase liquid flow path, and the connection section 220b is arranged at an angle α2 of 90° with respect to the bottom surface of the flow path of the inflow section 118 of the dispersed phase liquid flow path. The angle between the connection section and the upper surface or the bottom surface of the inflow section may be 45 to 135°, 60 to 120°, 75 to 105°, 85° to 95°, or substantially 90°.

[0051] When assuming that the roundness of the discontinuous portion (the portion indicated by reference numeral 230a or 230b in FIG. 2) on the wall surface is a circle, the so-called corner R is preferably, for example, 0.3 mm or less, 0.15 mm or less, 0.5 mm or less, 0.3 mm or less, 0.1 mm or less, 0.05 mm or less, 0.03 mm or less, 0.01 mm or less, 0.005 mm or less, 0.003 mm or less, or 0.001 mm or less.

[0052] In the above, for convenience, the existence of the bottom surface and the upper surface of the flow path is defined, but the cross-sectional shape of the flow path may be circular, semi-circular, elliptical, convex, concave, rectangular, or trapezoidal.

[0053] In particular, in the portion adjacent to the inflow portion, the emulsion forming portion can have a channel height that is 1.1 times or more, 1.2 times or more, 1.3 times or more, 1.5 times or more, 2 times or more, 3 times or more, 5 times or more, or 10 times or more that of the inflow portion. Particularly preferably, in the portion adjacent to the inflow portion, the emulsion forming portion is 5 times or less, 3 times or less, 2 times or less, or 1.5 times or less that of the inflow portion. In this case, it is possible to suppress the excessive increase in the flow rate ratio of the continuous phase liquid to the dispersed phase liquid and the decrease in the droplet generation rate during negative pressure liquid feeding. Here, the channel height can be measured in the vertical direction in the normal use state of the microchannel chip and can be measured as the distance from the bottom surface to the upper surface inside the channel.

[0054] When the dispersed phase liquid enters until it contacts the channel side surface of the emulsion forming portion formed by the inflow portion into the emulsion channel and the continuous phase liquid channel, the emulsion forming portion is immediately blocked by the dispersed phase liquid. Therefore, the distance between the channel side surface near the inflow portion from the dispersed phase liquid channel and the channel side surface near the inflow portion into the emulsion channel is preferably greater, for example, 1 time, 1.2 times, 1.5 times, 2 times, 3 times, 5 times, 10 times the channel height of the emulsion forming portion. However, since this distance is related to the stability of emulsion generation, it is particularly preferable to adjust it according to the embodiment.

[0055] (Wall surface of the dispersed phase liquid channel) From the viewpoint of shortening the measurement time, it is desirable to shorten the time (arrival time) for the dispersed phase liquid to reach the emulsion forming portion through the dispersed phase liquid channel. However, from the viewpoint of the stability of droplet generation, a channel surface with low affinity for the dispersed phase liquid is desirable. Therefore, when using a liquid with high surface tension and / or viscosity as the dispersed phase liquid, this arrival time may be extremely delayed. In this case, it is possible to add a surfactant or the like to the dispersed phase liquid and adjust the affinity of the channel surface for the dispersed phase liquid.

[0056] Hereinafter, each component constituting the invention according to the present disclosure and its embodiments will be described in more detail.

[0057] <Emulsion> The emulsion produced by the method according to the present disclosure is a dispersion solution and includes droplets composed of a dispersed phase liquid and a continuous phase composed of a continuous phase liquid. In the emulsion, the droplets composed of the dispersed phase liquid are dispersed in the continuous phase composed of the continuous phase liquid.

[0058] (Dispersed phase liquid) The dispersed phase liquid is a liquid that constitutes the droplets contained in the emulsion.

[0059] The dispersed phase liquid may be, for example, an aqueous solution. The dispersed phase liquid may optionally contain a surfactant, an organic solvent, a thickening agent, serum, an enzyme, etc. The dispersed phase liquid may be a reaction solution, for example, a liquid containing a sample to be detected in the detection process described later, a liquid containing a detection reagent, or a mixture thereof.

[0060] (Continuous phase liquid) The continuous phase liquid is a liquid that constitutes the continuous phase contained in the emulsion.

[0061] The continuous phase liquid is preferably an immiscible liquid that is immiscible with the dispersed phase liquid. For example, when the dispersed phase liquid is an aqueous solution, the continuous phase liquid may be oil, and in this case, a water-in-oil (W / O) type emulsion is formed.

[0062] When the continuous phase liquid is oil, examples of the oil include silicone oil, mineral oil, fluorinated dispersion media, vegetable oil, or a combination thereof.

[0063] Examples of the fluorinated dispersion media include fluorocarbons, particularly perfluorohexane, hexafluorobenzene, perfluoromethylcyclohexane, perfluorooctane, and perfluorotripentylamine.

[0064] Examples of commercially available fluorocarbons include FC-3283 (manufactured by 3M under the trade name Fluorinert), FC-40 (manufactured by 3M under the trade name Fluorinert), and HFE-7500 (a high-performance liquid manufactured by 3M). TM Novec TM high-performance liquid, manufactured by 3M).

[0065] When a fluorinated dispersion medium, particularly the above-mentioned fluorocarbons, is used as the continuous-phase liquid, particularly stable and rapid droplet generation becomes possible. In addition, since it has extremely low compatibility with polar solvents and non-polar solvents, problems such as the components of the droplets in the emulsion moving to other droplets through the continuous-phase liquid (crosstalk, contamination) can be suppressed. Also, when selecting a liquid with low surface tension and viscosity using a hydrocarbon-based dispersion medium or silicone oil, generally the risk as a dangerous substance such as flammability increases, but the fluorinated dispersion medium is characterized by high safety as it is used as a fire extinguishing agent or a cooling medium.

[0066] For the purpose of the thermal stability of the droplets, etc., additives such as surfactants can also be added to the continuous-phase liquid. These additives are preferably those that do not inhibit the detection reaction in the droplets. Examples of surfactants include non-ionic surfactants such as PLURONIC (registered trademark), TETRONIC (registered trademark), which are block copolymers of polyethylene glycol and polypropylene glycol, Tween, Span, Zonyl (registered trademark), etc. When using a fluorinated dispersion medium as the continuous-phase liquid, it is preferable to use a fluorinated surfactant, for example, block copolymers of perfluoropolyether and polyethylene glycol, etc.

[0067] (Droplets) The droplets contained in the emulsion are composed of the dispersed-phase liquid. The droplets are formed, for example, by encapsulating the dispersed-phase liquid through contact with the continuous-phase liquid.

[0068] The droplet contains, for example, a sample to be detected. In the droplet, the target substance contained in the sample is reacted with a reagent, and the sample can be analyzed via a detectable signal (e.g., a fluorescence signal) indicating the presence and / or degree of the reaction. This reaction may be, for example, a chemical reaction, a binding reaction, a phenotypic change, or a combination thereof.

[0069] The volume of the droplet preferably has a volume capable of holding approximately one (e.g., one molecule) of the target substance. Specifically, it is preferable that the average volume is 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 performing the reaction of the target molecule in the droplet, the volume of the formed droplet preferably has a high monodispersity. The monodispersity referred to here specifically 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, for the sake of clarity, the droplet is treated as spherical, but the same consideration may apply even if the droplet is non-spherical due to the flow path structure or the surrounding flow.

[0070] The droplet preferably has a thermal stability sufficient to maintain its shape at least under the reaction temperature conditions of the target substance. As a specific example, in the detection process, when nucleic acid amplification is performed by the TRC method, the droplet preferably has a thermal stability sufficient to maintain its shape under the temperature conditions of 40°C to 48°C, and when nucleic acid amplification is performed by the PCR method, the droplet preferably has a thermal stability sufficient to maintain its shape under the temperature conditions of 50°C to 100°C.

[0071] <Microfluidic chip> The microchannel chip of the present disclosure has a dispersed-phase liquid holding part, a dispersed-phase liquid flow path, a continuous-phase liquid holding part, a continuous-phase liquid flow path, an emulsion forming part, an emulsion flow path, and a discharge port. The dispersed-phase liquid holding part is connected to the emulsion forming part via the dispersed-phase liquid flow path, the continuous-phase liquid holding part is connected to the emulsion forming part via the continuous-phase liquid flow path, and the emulsion forming part is connected to the discharge port via the emulsion flow path. Note that the microchannel chip can further have an emulsion holding flow path.

[0072] The dispersed-phase liquid holding part, the dispersed-phase liquid flow path, the continuous-phase liquid holding part, the continuous-phase liquid flow path, the emulsion forming part, the emulsion flow path, (optional emulsion holding flow path), and the discharge port are fluidly connected to each other to form a single flow path structure as a whole.

[0073] In particular, this flow path structure can be connected to the external atmosphere (in particular, the external air) only through the dispersed-phase liquid holding part, the continuous-phase liquid holding part, and the discharge port.

[0074] The microchannel chip according to the present disclosure has, for example, a base material and an upper structure disposed on the base material. Preferably, the upper structure has a flow path structure, that is, a dispersed-phase liquid holding part, a dispersed-phase liquid flow path, a continuous-phase liquid holding part, a continuous-phase liquid flow path, an emulsion forming part, an emulsion flow path, (optionally an emulsion holding flow path), and a discharge port. The base material can be made of glass. The upper structure can be made of resin. The microchannel chip can be produced, for example, by bonding a resin upper structure and a glass base material constituting the bottom of the microchannel chip.

[0075] The size (such as width and depth) of the flow channels constituting the microfluidic chip can be appropriately determined in consideration of the volume of the target droplets, etc., and in particular, can be appropriately determined 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 (amplification reaction at the single molecule unit) of the nucleic acid, it is necessary to produce droplets in the pL order or nL order. Therefore, it is preferable that the width and depth of the flow channels around the emulsion formation part are in the ranges of 0.1 μm to 1000 μm, particularly 1 μm to 300 μm, respectively.

[0076] The microfluidic chip can be fabricated by combining techniques using a mold such as molding or embossing that can accurately and easily fabricate the flow channel structure, or techniques commonly used by those skilled in the art, such as photolithography, soft photolithography, wet etching, dry etching, nanoimprinting, laser processing, electron beam direct writing, additive manufacturing (AM), and machining.

[0077] Examples of the materials used for fabricating the microfluidic chip include polymer materials such as PDMS (polydimethylsiloxane) and acrylic, metal materials such as stainless steel, glass, silicone, and ceramics. Among these, polymer materials can fabricate the flow channels themselves at low cost and are easily disposable. Therefore, it is preferable to use polymer materials at least partially.

[0078] (Dispersed phase liquid holding part) The dispersed-phase liquid holding part is a part that holds the dispersed-phase liquid which is a material for generating an emulsion. The dispersed-phase liquid holding part may be, for example, a hole and / or a well extending in the vertical direction in the usage state of the microchannel chip, and the dispersed-phase liquid can be supplied and held in this hole and / or well. The dispersed-phase liquid holding part may be composed of, for example, holes and / or wells with a diameter of 0.1 mm to 20 mm. When the dispersed-phase liquid holding part is composed of a hole and a well, the well extending in the vertical direction can be connected to the dispersed-phase liquid flow path through the hole extending in the vertical direction.

[0079] FIG. 3 is a schematic cross-sectional view showing one embodiment of the dispersed-phase liquid holding part according to the present disclosure. FIG. 3 is a schematic view for easy understanding and is not to scale. H in FIG. 3 is the height direction, and L is the length direction. The dispersed-phase liquid holding part 300 shown in FIG. 3 has a well 31 and a hole 33. The well 31 is connected to the dispersed-phase liquid flow path 115 through the hole 33. The well 31 and the hole 33 extend in the height direction (here, the vertical direction). Reference numeral 35 in FIG. 3 indicates an extended part which is the connection point between the bottom surface of the well and the hole. In the embodiment of FIG. 3, the bottom surface of the well 31 extends perpendicular to the extending direction of the hole 33 (particularly, the side wall of the hole 33 in the vicinity of the extended part 35).

[0080] Regarding the dispersed-phase liquid holding part, it is preferable to perform further optimization according to the following.

[0081] As the driving force during liquid delivery, mainly, liquid delivery pressure (positive pressure, negative pressure) and / or liquid level differential pressure (hydrostatic pressure) and / or capillary force (capillary force) act. Among these, the capillary force is determined by the difference in surface tension between the gas-liquid interface (and solid-liquid interface) of the downstream of the flow path during liquid delivery (especially the emulsion holding flow path) and the gas-liquid interface (and solid-liquid interface) within each holding part. That is, since the liquid delivery speed in the flow path and the like change depending on the shape of the gas-liquid interface (wetting behavior on the wall surface) in each holding part, particularly in the emulsion filling method that performs liquid delivery (emulsion generation and holding) by controlling the gas-liquid interface in the flow path, the shape of each holding part is an important factor for realizing stable liquid delivery. In particular, in a general emulsion generation chip, stable emulsion generation can be achieved by making at least the flow path wall surface of the emulsion forming part a surface with low affinity for the dispersed phase liquid (a hydrophobic surface if the dispersed phase liquid is an aqueous liquid). Therefore, from the perspective of chip manufacturing cost, when the inner surface treatment of the flow path or a separate substrate is not combined, the wall surface of the dispersed phase liquid holding part generally also has a surface with low affinity for the dispersed phase liquid. At this time, since the amount of change in the surface tension of the gas-liquid interface accompanying the change in the interface shape in the dispersed phase liquid holding part becomes large, the influence on liquid delivery becomes greater.

[0082] From the viewpoint of reducing the change in capillary force as described above, holes and / or wells that substantially have no discontinuous shape on the wall surface and extend in the vertical direction are preferable. Further, when the remaining amount of the dispersed-phase liquid in the dispersed-phase liquid holding portion decreases, the gas-liquid interface substantially contacts the bottom surface of the well in the holding portion and the interface shape is likely to change. Therefore, in order to reduce the influence, it is preferable that the diameter of the hole and / or well that is in direct fluid connection with the flow path is smaller, for example, 5 mm or less, more preferably 2 mm or less, and particularly preferably 1 mm or less. On the other hand, the holding amount of the dispersed-phase liquid can also be increased by expanding the diameter from the hole or well that is in direct fluid connection with the dispersed-phase liquid flow path. In this case, however, when the gas-liquid interface exists near the expansion site, the gas-liquid interface shape is likely to change. Therefore, as one preferable aspect, the shape of the expansion site can be adjusted, and / or the height to the expansion site of the hole or well that is in direct fluid connection with the flow path can be made smaller, for example, 3 mm or less, more preferably 1 mm or less, and particularly preferably 0.5 mm or less. Further, when the supply amount of the dispersed-phase liquid is small and / or the outer surface of the chip around the dispersed-phase liquid holding portion has low affinity for the dispersed-phase liquid and the external liquid supply driving force is negative pressure applied to the discharge port, in the configuration of only the hole, when the dispersed-phase liquid is supplied to the hole so as to form a hemispherical surface, the contact area with the chip wall surface is minimized, and even when the remaining amount of the dispersed-phase liquid decreases, the change in the shape of the gas-liquid interface can be suppressed, which is preferable.

[0083] Note that when a positive pressure is used as the external liquid supply 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.

[0084] (Supply of Dispersed-Phase Liquid) The method according to the present disclosure includes supplying a dispersed-phase liquid to a dispersed-phase liquid holding portion.

[0085] The supply of the dispersed-phase liquid can be performed by dispensing means.

[0086] In order to supply the dispersed-phase liquid to the dispersed-phase liquid holding section, a separate container (phase liquid holding container) prepared separately for holding the dispersed-phase liquid can also be used. From the viewpoint of preventing the outflow of the liquid during storage and operation, it is preferable that such a container is completely or variably sealed while holding the dispersed-phase liquid.

[0087] Also, the supply of the dispersed-phase liquid (and / or the supply of the continuous-phase liquid) can be performed by dispensing means. The use of the dispensing means is preferable in that it can suppress the remaining amount of the dispersed-phase liquid and suppress the contamination between the measurement time and / or the reagents (dispersed-phase liquid).

[0088] For example, each phase liquid can be dropped into each holding section using the dispensing means, or each phase liquid can be introduced along the wall surface of each holding section. This is particularly advantageous when the liquid feeding is performed under negative pressure. In the conventional supply method, particularly the method of fluidly connecting (sealing connection) a tube or a manifold to each holding section to simultaneously perform the liquid introduction and the liquid feeding pressure to each holding section, due to the unexpected pressure fluctuations during connection and the time required for pressure stabilization, it is not easy to accurately control the progress of the dispersed-phase liquid and the continuous-phase liquid, and it has been difficult to bring the dispersed-phase liquid and the continuous-phase liquid into contact without clogging the flow path. On the other hand, when using the dispensing means and performing the liquid feeding under negative pressure, the pressure fluctuations when connecting the phase liquid supply device and the pressure source to each holding section are eliminated, so that the movement of each phase liquid before the application of negative pressure and the contact between the dispersed-phase liquid and the continuous-phase liquid can be controlled more accurately. As a result, the generation of bubbles can be more effectively suppressed.

[0089] The dispensing means preferably does not cause pressure fluctuations in the holding section. The dispensing means may be, for example, a pipette. Preferably, the dispensing means (particularly, the liquid discharge port constituting the dispensing means) is not fluidly connected (sealed connection) to each holding section and is spatially separated.

[0090] For example, the dispensing means may be a mechanism including a pump, an actuator, and a pipette. It is preferable that each phase liquid held in a separate container is sucked up by the pump, the tip of the pipette is moved to each holding part by the actuator, and then each phase liquid is extruded into each holding part by the pump. In addition, it is preferable that a part such as a pipette in contact with each phase liquid is removable and can be replaced for each use, because contamination can be suppressed. Furthermore, it is preferable to use the pump of the dispensing means also as a liquid feeding means, because the device configuration can be simplified. Also, when repeated use is intended, the phase liquid may be added by the dispensing means including a disposable pipette, or the addition may be performed from the phase liquid holding container to the microchannel chip using a common line. In the latter case, it is preferable to include a step of cleaning the common line up to the connection part to the microchannel chip in order to suppress contamination of the dispersed phase liquid in the continuous phase liquid. Also, as a dispensing means not including a pipette, a method of directly adding (dropping) each liquid from the container holding the phase liquid to the holding part by an external force is also preferable (for example, means for breaking the thermocompression-bonded part of the container by pressure to extrude the liquid in the container). Also, for example, when performing a TRC reaction or a PCR reaction, the dispensing means may be used in combination as a purification means or a preparation means for an aqueous solution sample.

[0091] (Dispersed phase liquid flow path) The dispersed phase liquid flow path connects the dispersed phase liquid holding part and the emulsion forming part. The dispersed phase liquid flow path is configured such that the dispersed phase liquid passes through it. It can also be assumed that, in addition to the dispersed phase liquid, the continuous phase liquid passes through the dispersed phase liquid flow path.

[0092] The dimensions of the dispersed phase liquid flow path can be appropriately set according to the type and characteristics of the dispersed phase liquid to be used. The dispersed phase liquid flow path can have, for example, a width of 10 to 500 μm, or 50 to 200 μm, and can have a length of 1 mm to 500 mm, or 10 to 200 mm. Also, 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 bend at one or more locations and may have a meandering shape.

[0093] In order for the dispersed phase liquid to quickly reach the emulsion forming section, it is preferable that the length of the dispersed phase liquid flow path is short. Since the above arrival time is also an important factor such as the flow path cross-sectional area and / or the flow path volume, and the flow path pressure loss resistance, it is preferable to appropriately adjust according to the embodiment.

[0094] In the present invention, after the dispersed phase liquid that has reached the emulsion forming section comes into contact with the continuous phase liquid, it is preferable that the continuous phase liquid does not flow backward through the dispersed phase liquid flow path until the start of liquid feeding. However, even when the above-mentioned backflow occurs, the remaining of the continuous phase liquid can be suppressed by the presence of the dispersed phase liquid that has already been filled. In addition, it is also possible to reduce the influence of the backflow by shortening the time from the above contact to the start of liquid feeding. For example, it is preferable to have means capable of applying a negative pressure driving force to the discharge port before the contact. Therefore, the measures for suppressing the backflow shown below are preferable countermeasures, and the present invention is not limited by those measures.

[0095] The above-mentioned backflow phenomenon mainly depends on: 1. the force of the continuous-phase liquid flowing backward while forming a thin layer of the continuous-phase liquid between the wall surface of the dispersed-phase liquid flow path and the dispersed-phase liquid due to the surface tension and / or viscosity of the continuous-phase liquid being lower than that of the dispersed-phase liquid; 2. the liquid level differential pressure (vector component to the inflow part of the dispersed-phase liquid flow path in the emulsion formation part) depending on the supply amount of the dispersed-phase liquid to the dispersed-phase liquid holding part in the emulsion formation part; 3. the liquid level differential pressure (the same vector component as 3) depending on the supply amount of the continuous-phase liquid to the continuous-phase liquid holding part; 4. the shear stress on the dispersed-phase liquid caused by the flow of the continuous-phase liquid from the emulsion formation part to the emulsion flow path. If the forces of 2 and 4 are greater than those of 1 and 3, the above-mentioned backflow phenomenon is less likely to occur, which is preferable. Since 1 is a value depending on the physical properties of each phase liquid and the flow path wall surface, it is difficult to prevent the formation of the above-mentioned thin layer. However, by increasing the cross-sectional area of the flow path, the volume of the dispersed-phase liquid relative to the contact area of the thin layer and the dispersed-phase liquid can be increased, and as a result, the backflow of the dispersed-phase liquid near the center of the flow path cross-section due to the shear stress caused by the backflow of the thin layer can be suppressed. Also, by increasing the cross-sectional area of the dispersed-phase liquid flow path and shortening the flow path length to reduce the flow path pressure loss resistance, the force of 2 is more likely to be transmitted to the emulsion formation part, which is preferable. However, in the case of positive pressure liquid feeding to each holding part, if the flow path pressure loss resistance of the dispersed-phase liquid flow path at the start of liquid feeding is smaller than the total flow path pressure loss resistance in the downstream direction of the emulsion formation part, the continuous-phase liquid will be fed (flow backward) not only in the direction of the emulsion flow path but also in the direction of the dispersed-phase liquid flow path in the emulsion formation part, making it difficult to generate an emulsion. Therefore, it is advisable to appropriately set the flow path pressure loss resistance of the dispersed-phase liquid flow path according to the embodiment. In addition, by reducing the cross-sectional area of the continuous-phase liquid flow path and increasing the flow path length to increase the flow path pressure loss resistance, the force of 3 is less likely to be transmitted to the emulsion formation part, which is preferable. However, in order to adjust the flow rate ratio of the continuous-phase liquid and the dispersed-phase liquid in emulsion generation, the flow path pressure loss resistance ratio between the dispersed-phase liquid flow path and the continuous-phase liquid flow path is important, so it can be appropriately set according to the embodiment. Also, the force of 3 also depends on the outflow cross-sectional area on the emulsion flow path side and the inflow cross-sectional area on the dispersed-phase liquid flow path side in the emulsion formation part (it is easier to flow to the side with a larger cross-sectional area). Since it is important for the stability of emulsion generation, it can be appropriately set according to the embodiment.If the continuous phase liquid can smoothly move through the emulsion formation section, the emulsion flow path (and optionally the emulsion holding flow path), it is a force that continuously occurs depending on the moving speed of the continuous phase liquid during that time, and as long as no external force is applied (as long as the liquid moves depending on the liquid level differential pressure and capillary force), the backflow is continuously suppressed. In summary, in order to suppress the above-mentioned backflow, it is preferable to reduce the flow path pressure loss resistance by increasing the flow path cross-sectional area of the dispersed phase liquid flow path and decreasing the flow path length.

[0096] The microchannel chip according to the present disclosure can have two or more dispersed phase liquid holding portions and two or more dispersed phase liquid flow paths corresponding to them respectively.

[0097] In particular, the microchannel chip according to the present disclosure has a first dispersed phase liquid holding portion and a second dispersed phase liquid holding portion, and 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 confluence portion. The first dispersed phase liquid flow path and the second dispersed phase liquid flow path are each connected to the emulsion formation section via the dispersed phase liquid confluence portion.

[0098] By using two or more dispersed phase liquid holding portions, for example, a reaction liquid containing an analysis sample and a reaction liquid containing a detection reagent can be separately supplied to the microchannel chip so that they do not mix until immediately before droplet generation. This is preferable because the timing of the start of the reaction can be controlled better.

[0099] On the other hand, when two types of dispersed phase liquids are supplied to the emulsion formation section using two dispersed phase liquid flow paths, the remaining continuous phase liquid in the dispersed phase liquid flow path (especially the dispersed phase liquid confluence portion) leads to a non-uniform mixing ratio of the two dispersed phase liquids. This can cause a decrease in the accuracy of the detection reaction. According to the method of the present invention, the remaining continuous phase liquid in the dispersed phase liquid flow path can be avoided or suppressed, so the uniformity of the mixing ratio can be ensured when mixing a plurality of reaction liquids in the chip.

[0100] In a microchannel chip having a dispersed-phase liquid confluence section, when moving the dispersed-phase liquid through a dispersed-phase liquid flow path filled with gas to an emulsion formation section (or an inlet section to the emulsion formation section), it is preferable to merge the first dispersed-phase liquid and the second dispersed-phase liquid respectively supplied to the first dispersed-phase liquid holding section and the second dispersed-phase liquid holding section at the dispersed-phase liquid confluence section.

[0101] If the flow path is blocked by bubbles at the dispersed-phase liquid confluence section, there is a risk that the generation of the emulsion may not be possible. Especially when the liquid is sent under negative pressure, it is generally not easy to eliminate the flow path blockage by bubbles due to the properties of the dispersed-phase liquid which is water-soluble. By merging the first dispersed-phase liquid and the second dispersed-phase liquid respectively supplied to the first dispersed-phase liquid holding section and the second dispersed-phase liquid holding section at the dispersed-phase liquid confluence section, it becomes possible to avoid such problems.

[0102] (Continuous-phase liquid holding section) The continuous-phase liquid holding section is a part that holds the continuous-phase liquid which is the material for generating the emulsion. The structure of the continuous-phase liquid holding section is not particularly limited as long as it can hold the continuous-phase liquid. The continuous-phase liquid holding section may be a hole or a well, for example, a hole or a well extending in the vertical direction, and the continuous-phase liquid can be supplied into and held in this hole or well. The continuous-phase liquid holding section may be, for example, a hole or a well with a diameter of 1 mm to 20 mm.

[0103] In addition, since generally a liquid with low surface tension and viscosity is used as the continuous-phase liquid, the amount of change in surface tension accompanying the 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 great influence on the liquid delivery. In addition, in the present invention, for example, when holding an emulsion and performing a detection reaction, in order to supply a sufficient amount of the continuous-phase liquid so that the continuous-phase liquid in the continuous-phase liquid holding section does not run out, the remaining amount of the continuous-phase liquid in the holding section does not decrease during liquid delivery and the interface shape does not easily change. Therefore, still, the shape of the continuous-phase liquid holding section is not likely to have a great influence on the liquid delivery.

[0104] When using positive pressure as the external liquid supply driving force, it is preferable that the continuous phase liquid holding part is suitable for connection to the positive pressure source. In this case, it is preferable that the continuous phase liquid holding part has resistance to the applied pressure.

[0105] (Supply of continuous phase liquid) The method according to the present disclosure includes supplying a continuous phase liquid to the continuous phase liquid holding part.

[0106] The supply of the continuous phase liquid can be performed using a separate container and / or by dispensing means, in the same manner as described above for the supply of the dispersed phase liquid. For example, the phase liquid can be dropped into the holding part using the dispensing means, or the phase liquid can be introduced along the wall surface of the holding part. For details of the separate container and the dispensing means, reference can be made to the above description regarding the supply of the dispersed phase liquid.

[0107] (Continuous phase liquid flow path) The continuous phase liquid flow path connects the continuous phase liquid holding part and the emulsion forming part. The continuous phase liquid flow path is configured such that the continuous phase liquid passes through it.

[0108] The dimensions of the continuous phase liquid flow path can be appropriately set according to the type and characteristics of the continuous phase liquid to be used. The continuous phase liquid flow path can have a width of, for example, 10 to 500 μm, or 50 to 200 μm, and can have a length of 1 mm to 500 mm, or 10 to 200 mm. Also, 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 bend at one or more locations and may have a meandering shape at least partially.

[0109] The microchannel chip can have two or more continuous phase liquid flow paths. In particular, the microchannel chip according to the present disclosure has a first continuous phase liquid flow path and a second continuous phase liquid flow path, and these flow paths connect the continuous phase liquid holding part and the emulsion forming part, respectively.

[0110] Referring to the exemplary embodiment of FIG. 1, the continuous phase liquid flow path 111 is composed of two flow paths (the first continuous phase liquid flow path 112 and the second continuous phase liquid flow path 113). These two flow paths 112 and 113 are arranged to face each other in the emulsion forming section 120, and are substantially orthogonal to the dispersed phase liquid flow path 117 (more precisely, the dispersed phase liquid confluence section 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 velocities of the continuous phase liquid moving through the respective flow paths are substantially the same. Further, when it is desired to suppress the mixing of the dispersed phase liquids before emulsion generation as described above, the length of the flow path connecting the downstream portion of the dispersed phase liquid confluence section 116 and the emulsion forming section 120 is preferably relatively short (for example, 3 mm or less, more preferably 0.5 mm or less), and it is preferable that the dispersed phase liquid maintains a separate laminar flow state within the flow path.

[0111] (Emulsion forming section) The emulsion forming section is configured to generate an emulsion. The emulsion forming section receives the supply of the dispersed phase liquid and the continuous phase liquid through the dispersed phase liquid flow path and the continuous phase liquid flow path, respectively. Further, the emulsion forming section is connected to the emulsion flow path, and the emulsion generated in the emulsion forming section is sent to the emulsion flow path.

[0112] The emulsion forming section can have one or more openings opening to the dispersed phase liquid flow path and one or more openings opening to the continuous phase liquid flow path. Further, the emulsion forming section can have one or more openings opening to the emulsion flow path.

[0113] Referring to the exemplary embodiment of FIG. 1, the emulsion forming section will be described. In the emulsion forming section 120 of FIG. 1, a continuous phase liquid flow path 111 composed of two flow paths 112 and 113 and a dispersed phase liquid flow path 117 (more precisely, a dispersed phase liquid confluence section 116) are substantially orthogonal. During the application of an external liquid feeding driving force, the continuous phase liquid flows into the emulsion forming section 120 from two substantially opposite directions to each other, and the dispersed phase liquid flows into the emulsion forming section 120 in a direction substantially orthogonal to the inflow direction of the continuous phase liquid. As a result, droplets (i.e., an emulsion) dispersed in the continuous phase are generated in the emulsion forming section 120. The emulsion thus generated enters the emulsion flow path 130.

[0114] The emulsion forming section can appropriately use flow paths that utilize common droplet generation methods such as T-janction, Flow-Focus, co-flow, step-emulsification, etc. In order to generate an emulsion quickly, a plurality of emulsion forming sections may be arranged in parallel. Also, it may be provided with a meandering flow path or the like for stirring the droplets in the emulsion.

[0115] (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 discharge port (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 (especially the dispersed phase liquid confluence section). FIG. 1 shows an emulsion flow path in such a manner. Also, in FIG. 1, the portion of the emulsion flow path adjacent to the emulsion forming section is substantially orthogonal to the inflow portion of the continuous phase liquid flow path to the emulsion forming section. In the case of FIG. 1, when the emulsion is generated, the dispersed phase liquid flowing into the emulsion forming section from the dispersed phase liquid flow path becomes droplets and enters the emulsion flow path without changing the flow angle as it is.

[0116] The emulsion flow path can have a flow path with an expanded width and / or height and / or can be serpentine on the downstream side (in the direction of the discharge port) of the portion adjacent to the emulsion forming portion. According to such an aspect, stirring in the droplets can be promoted, which is preferable.

[0117] (Emulsion holding flow path) In yet another embodiment of the method according to the present disclosure, the microfluidic chip further has an emulsion holding flow path, the emulsion forming portion is connected to the emulsion holding flow path via the emulsion flow path, and the emulsion holding flow path is connected to the discharge port. By applying an external liquid feeding driving force, an emulsion including droplets composed of the dispersed phase liquid and a continuous phase composed of the continuous phase liquid is generated in the emulsion forming portion, and the emulsion thus generated is transported to the emulsion holding flow path via the emulsion flow path.

[0118] With reference to the microfluidic chip shown in FIG. 4, this embodiment will be specifically described. FIG. 4 is a schematic diagram for easy understanding and is not to scale. The microfluidic chip of FIG. 4 has a structure similar to that of FIG. 1 except that it has an emulsion holding flow path and the arrangement of the discharge port is different. Only the differences will be described below.

[0119] The microfluidic chip 20 of FIG. 4 further has an emulsion holding flow path 140. The emulsion forming portion 120 is connected to the emulsion holding flow path 140 via the emulsion flow path 130, and the emulsion holding flow path 140 is connected to the discharge port 150.

[0120] The emulsion generated in the emulsion forming portion 120 is transported to the emulsion holding flow path 140 via the emulsion flow path 130.

[0121] When generating and holding an emulsion using a microchannel chip having an emulsion holding channel, if the continuous phase liquid remains in the dispersed phase liquid channel (particularly on the wall surface of the dispersed phase liquid channel), the generation of bubbles in the emulsion holding channel may increase. This is presumably because when the dispersed phase liquid holding portion becomes empty, the remaining continuous phase liquid acts as a lubricant, and through the emptied dispersed phase liquid holding portion, gas (particularly air) enters the droplet holding channel from the surrounding atmosphere (particularly the surrounding air). The entry of gas through the emptied dispersed phase liquid holding portion can be prevented by stopping the liquid feeding before the dispersed phase liquid holding portion becomes empty. However, in this case, unused dispersed phase liquid remains in the dispersed phase liquid holding portion, resulting in waste. Particularly, when using a reaction solution for gene testing, which is relatively expensive as the dispersed phase liquid, there is a risk of increasing costs. Particularly, when using a reaction solution for gene testing that detects a low-concentration nucleic acid as the target substance as the dispersed phase liquid, the above waste may cause a decrease in detection sensitivity and measurement reproducibility.

[0122] According to the present invention, the entry of the continuous phase liquid into the dispersed phase liquid channel and / or the remaining of the continuous phase liquid in the dispersed phase liquid channel can be suppressed or avoided. Therefore, by implementing the present invention using a microchannel chip having an emulsion holding channel, it becomes possible to realize the generation and holding of an emulsion with simple operation and good stability.

[0123] In the method of the present disclosure, the emulsion (droplets + continuous phase) generated in the emulsion forming portion can be transported to the emulsion holding channel filled with gas. For example, referring to FIG. 4, the emulsion generated in the emulsion forming portion 120 can be transported through the emulsion channel 130 to the emulsion holding channel 140 filled with gas (the "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 the continuous phase liquid, for example.

[0124] In such an emulsion filling method, the emulsion generated in the emulsion forming section moves through the emulsion holding flow path filled with gas (especially towards the discharge port) to fill the emulsion holding flow path. That is, the "gas-liquid interface" formed by the gas filling the emulsion holding flow path and the emulsion moves towards the downstream (especially the discharge port) of the emulsion holding flow path. The emulsion filling method is preferable because it can omit the preparation process of pre-filling the flow path of the microchannel chip with the continuous phase liquid and the device therefor.

[0125] The width and length of the emulsion holding flow path can be appropriately set according to the volume and number of the droplets to be held. For example, it may be a wide and simple flow path with the width and length being substantially equal, or a continuous single long flow path may be arranged in a meandering or spiral shape, or branched straight flow paths may be arranged in parallel.

[0126] In the present invention, the cross-section of the emulsion holding flow path is preferably circular, semi-circular, elliptical, convex, concave, rectangular, or trapezoidal because the center of the droplet can easily flow along the center of the flow path.

[0127] In the emulsion filling method, generally, in order to simultaneously generate and hold an emulsion by controlling the movement of the gas-liquid interface in the emulsion holding flow path, it is desirable that the cross-sectional shape of the flow path is a straight flow path with a constant cross-sectional shape and no bending so that the shape of the gas-liquid interface during liquid feeding is maintained (with little change in its shape even while moving). However, if the flow path width is extremely increased with respect to the flow path height in order to increase the number of detected droplets, it is difficult to stably manufacture a chip having the intended flow path structure, and / or the bottom surface and / or the upper surface of the flow path may be deformed, and the height of the flow path region far from the side surface of the flow path may change due to the liquid feeding pressure, the fixing pressure to the chip, etc., which may have an adverse effect on the measurement (roof collapse). As a countermeasure, for example, the ratio of the flow path width to the flow path height is preferably 100 or less, more preferably 50 or less, 25 or less, and particularly preferably 10 or less (when there is no pillar described below). Alternatively, by providing a pillar (pillar) in the center of the flow path, the ratio of the distance between the pillars and / or the distance between the pillar and the side surface of the flow path to the flow path height is, for example, preferably 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 paths are densely packaged in a horizontal plane (for example, in a shape close to a square or a circle) because the number of detected droplets can be increased. Therefore, a single long continuous flow path having the same cross-sectional shape may be arranged in a meandering shape or a spiral shape, or branched straight flow paths may be arranged in parallel. In this case, since there are bent portions, the interface shape is likely to change during liquid feeding, but the influence can be reduced by adjusting the cross-sectional shape at the bent portions.

[0128] In one embodiment, it is intended to perform a detection process on the held emulsion. That is, the detection process described below can be optionally performed on the emulsion held in the emulsion holding flow path.

[0129] Preferably, the emulsion holding channel is configured such that most or all of the held emulsion does not come into contact with the external atmosphere (particularly the external air) of the microchannel chip. Preferably, among the droplets held in the emulsion holding channel, the droplets to be subjected to the detection process do not come into contact with the external atmosphere (particularly the external air). Particularly preferably, the emulsion held in the emulsion holding channel is sealed against the external atmosphere (particularly the external air). This can be achieved, for example, by setting the channel length of the emulsion holding channel to be relatively long so that the emulsion can come into contact with the external atmosphere (particularly the external air) only at the downstream end of the emulsion holding channel.

[0130] Preferably, the emulsion holding channel is suitable for performing a detection process on the emulsion held in the emulsion holding channel.

[0131] Preferably, the emulsion holding channel is configured to be able to perform a detection process on an emulsion that is not open to the external air. For example, there is a structure between the held emulsion and the detection means that isolates the emulsion from the external air. This structure is made of, for example, a material that transmits light. In this case, among the emulsions held in the emulsion holding channel, emulsions that are not the subject of the detection process, for example, emulsions located at the discharge-side end of the emulsion holding channel, may be open to the external air.

[0132] Preferably, the channel volume of the emulsion holding channel is equal to or greater than the total volume of the droplets generated in the emulsion forming section (particularly the droplets to be detected in the detection process), and / or the channel volume of the emulsion holding channel is 1 μL or more, 5 μL or more, or 10 μL or more. According to such an emulsion holding channel, the detection process can be efficiently performed. The upper limit of the channel volume of the emulsion holding channel may be, for example, 1000 μL or less.

[0133] Preferably, the emulsion holding channel has a channel volume capable of holding droplets of an average volume of 0.1 nL to 10 nL, particularly 0.3 nL to 3 nL, of 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. Detection processing can be performed on these droplets held in the emulsion holding channel.

[0134] Note that the average volume of the droplets can be calculated based on the following for N = 10 or more droplets in the acquired bright-field image using an image acquisition device such as a digital camera.

[0135] The volume (V drop of each spherical or disk-shaped droplet is represented by the following formulas (1) and (2). In the following formulas (1) and (2), D disk and D drop are the diameters of the spherical droplet and the disk-shaped droplet, respectively, when observing the droplets held in the emulsion holding channel 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. disk

[0136]

Number

[0137]

Number

[0138] Further, preferably, the droplets in the emulsion to be detected do not overlap with each other with respect to the detection means (such as a camera) used in the detection process, and in particular, a single layer spreading in a plane orthogonal to the detection direction is formed. In this case, since most of the droplets to be detected in the detection process can be easily observed from one detection direction, the detection accuracy can be further improved.

[0139] Particularly preferably, the "channel height" of the emulsion holding channel is adjusted so that the droplets held in the emulsion holding channel do not overlap with each other in the vertical direction (i.e., a single droplet layer is formed) in the state of use of the microchannel chip. When the detection process is performed with such an emulsion holding channel, the detection accuracy is further improved. The "channel height" is usually the length of the channel in the vertical direction (vertical direction) in the state of use of the microchannel chip.

[0140] Preferably, the channel height of the emulsion holding channel preferably has a dimension corresponding to the diameter of the droplets to be detected in the detection process. For example, it preferably has a channel height of 1 / 10 to 10 times, 1 / 4 to 4 times, or 1 / 2 to 2 times the diameter of the droplets. Further, the channel height of the emulsion holding channel may be 1 / 4 times or less of the channel width. The width of the channel is usually the length in the horizontal direction orthogonal to the length direction of the channel in the state of use of the microchannel chip. The diameter of the droplets can be measured in the width direction of the channel.

[0141] In the emulsion filling method, since the emulsion holding channel is filled with the emulsion depending on the flow rate ratio of the continuous phase liquid and the dispersed phase liquid in the emulsion forming section, for example, in order to stabilize the emulsion generation, when the flow rate ratio of the continuous phase liquid to the dispersed phase liquid is increased, it is preferable to design the channel height larger than usual to densely package the droplets 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.

[0142] (Stopping the liquid delivery) In order to hold the emulsion in the emulsion holding flow path, for example, when the emulsion holding flow path is filled with a desired amount of emulsion, stop the liquid delivery and optionally close the discharge port. When negative pressure is used for liquid delivery, the application of negative pressure can also be stopped by (at least partially) opening the discharge port to the external atmosphere. When performing the emulsion filling method, the liquid delivery can be stopped after the gas filling the emulsion holding flow path has been completely replaced by the emulsion, but preferably, the liquid delivery is stopped while the gas filling the emulsion holding flow path has not been completely replaced by the emulsion. In other words, stop the liquid delivery with a gas-emulsion interface existing in the flow path or the discharge port.

[0143] When stopping the liquid delivery, in some cases, backflow of the gas-liquid interface (gas-emulsion interface) can be suppressed by gradually opening the discharge port to the external atmosphere. For example, a relatively low-pressure tank can be separately provided between two valves (especially solenoid valves), and the discharge port can be opened to this tank before opening to the external atmosphere to achieve a gradual opening. Also, the liquid delivery can be stopped by adjusting the opening speed of the solenoid valve so that the degree of opening increases step by step.

[0144] Regarding the stopping of liquid delivery, it is preferable to adjust so that the minimum distance between the gas-emulsion interface and all the droplets increases by increasing the flow rate ratio of the continuous phase liquid or decreasing the flow path volume of the emulsion holding flow path. This is because it is difficult for the droplets in the emulsion to flow out of the discharge port, and adverse effects (coagulation and coalescence of droplets, in-droplet reactions, and inhibition of signal detection) on the nearby droplets caused by the gas-emulsion interface can be suppressed.

[0145] Also, regarding the stop of liquid feeding and the subsequent holding of the emulsion, when using a highly fluid continuous phase liquid, even without an externally applied liquid feeding driving force, the gas-emulsion interface may flow downstream due to capillary force and / or liquid level differential pressure. In addition, when using a highly volatile continuous phase liquid and / or when heating the emulsion due to in-drop reaction or the like, the continuous phase liquid evaporates and the continuous phase liquid in the flow path decreases, and / or the continuous phase liquid remaining in the continuous phase liquid holding portion flows into the flow path to replenish the evaporated continuous phase liquid, and the droplets in the emulsion holding flow path may also flow after the liquid feeding stops. Such flow of droplets after the liquid feeding stops is a factor for the droplets in the emulsion holding flow path to flow out in the direction of the discharge port or the emulsion flow path direction, so it is desirable to take countermeasures. For this purpose, by sealing each holding portion and the discharge port and performing pressure control, and / or by using a material with low gas permeability as the flow path substrate, the flow of droplets caused by capillary force and / or liquid level differential pressure and the evaporation of the continuous phase liquid can be suppressed. Note that when having a dispensing means, since such a sealing operation needs to be performed after the dispensing operation or after the liquid feeding stops, it is not preferable from the viewpoint of the automation of the apparatus.

[0146] (Discharge port) The microchannel chip has a discharge port. This discharge port is connected to the emulsion flow path or optionally to the emulsion holding flow path. The discharge port can also function as a negative pressure source connection portion for applying a negative pressure to the microchannel chip.

[0147] When using negative pressure for liquid feeding, it is preferably configured such that the discharge port is suitable for connection to a negative pressure source. In this case, it is preferable that the discharge port has resistance to the applied pressure.

[0148] When the microchannel chip has an emulsion holding flow path and uses negative pressure for liquid feeding, usually, the discharge port is located on the downstream side of the emulsion holding flow path. By applying a negative pressure to this discharge port, the entire length or most of the emulsion holding flow path can be filled with the generated emulsion.

[0149] (Liquid feeding) The external liquid feeding 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. Further, when the microchannel chip has an emulsion holding channel, the external liquid feeding driving force provides a driving force for transporting the generated emulsion to the emulsion holding channel. The external liquid feeding driving force may be the application of negative pressure to the discharge port or the application of positive pressure to the dispersed phase liquid holding section and the continuous phase liquid holding section. Note that, from the viewpoint of device simplicity, it is preferable to keep each holding section at normal pressure while applying negative pressure to the discharge port, and to keep the discharge port at normal pressure while applying positive pressure to the dispersed phase liquid holding section and the continuous phase liquid holding section. However, for stabilizing the liquid feeding of negative pressure and / or positive pressure, and / or for also serving as a sealing operation for holding the emulsion, the pressure may be controlled to a non-normal pressure state.

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

[0151] When applying negative pressure, for example, a pressure tank or a syringe pump can be used to suck the fluid (such as gas or continuous phase liquid) in the flow path of the microchannel chip through the discharge port.

[0152] When using a pressure tank as the negative pressure source, it is preferable that the volume of the pressure tank is larger than the total volume of the flow path from the discharge port to the pressure tank and the volume of the flow path of the microchannel chip. The pressure tank can also be designed to be openable to the external atmosphere (particularly the external air). Further, for example, it is preferable to control the pressure in the pressure tank with a pump. Also, a pressure sensor may be provided so that the pressure value in the pressure tank can be monitored.

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

[0154] In addition, by using monitoring means for monitoring the pressure value of the applied negative pressure or positive pressure, it is possible to confirm the liquid feeding state, for example, to confirm whether an emulsion is being generated without problems.

[0155] (Liquid feeding by negative pressure) In one embodiment according to the present disclosure, a negative pressure is used as an external liquid feeding driving force. That is, in one embodiment according to the present disclosure, by applying a negative pressure to the discharge port, an emulsion is generated in the emulsion forming portion, and the emulsion thus generated is transported to the emulsion flow path and optionally the emulsion holding flow path.

[0156] Negative pressure liquid feeding is preferable because the required apparatus can be simplified as compared with positive pressure liquid feeding.

[0157] Further, in positive pressure liquid feeding, particularly when applying a positive pressure to each holding portion through gas, it is necessary to add the dispersed phase liquid and the continuous phase liquid to each holding portion and then make a sealed connection between the liquid feeding means and each holding portion, but an unnecessary external pressure is likely to be applied during the connection. In particular, when using a highly flexible substrate such as silicone rubber, the microchannel chip may be deformed during connection, and the cross-section of the flow path may be deformed. Further, by using a liquid having low viscosity and surface tension as the continuous phase liquid, stable and rapid droplet generation using the shearing force in the emulsion forming portion becomes possible, but a liquid having the above physical properties is likely to leak when the sealed state is incomplete, and it becomes difficult to apply an appropriate pressure. Further, 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 feeding and liquid leakage due to the unnecessary external pressure during the above connection, but this is an example of a means for simultaneously performing the above-described liquid introduction and pressure liquid feeding, and the reproducibility of the operation of bringing the dispersed phase liquid and the continuous phase liquid into contact is likely to decrease.

[0158] On the other hand, in the case of negative pressure liquid feeding, since the liquid feeding means can be connected before adding the dispersed phase liquid and the continuous phase liquid, it has the advantage that the pressure at the time of connection is not applied to the dispersed phase liquid and the continuous phase liquid. It also has the advantage of avoiding the need to seal the continuous phase liquid holding part that holds the continuous phase liquid with low viscosity and surface tension.

[0159] Furthermore, in the present invention, in order to shorten the time for the dispersed phase liquid to reach the emulsion forming part through the dispersed phase liquid flow path, it is often preferable to shorten the length of the dispersed phase liquid flow path. Therefore, correspondingly, it is preferable to use a microchannel chip with a small pressure loss resistance for the entire flow path. However, in the method of applying a positive pressure to each holding part, there is a risk that the dispersed phase liquid or the continuous phase liquid may inadvertently enter the flow path due to unnecessary pressure when connecting to the liquid feeding means.

[0160] On the other hand, the method of applying a negative pressure to the discharge port has the advantage that the discharge port and the liquid feeding means can be connected before adding the dispersed phase liquid and the continuous phase liquid, so that no unnecessary pressure is applied.

[0161] Thus, negative pressure liquid feeding has many advantages. However, on the other hand, when applying negative pressure, since pressure is not directly applied to the dispersed phase liquid to be fed, it is not easy to remove the continuous phase liquid remaining on the wall surface of the dispersed phase liquid flow path. As described above, the continuous phase liquid remaining in the dispersed phase liquid flow path causes instability in droplet generation and the like.

[0162] On the contrary, according to the present invention, since the remaining of the continuous phase liquid in the dispersed phase liquid flow path can be substantially avoided, even when negative pressure is used as the external liquid feeding driving force, it is possible to generate a stable emulsion.

[0163] In one embodiment of the method according to the present disclosure, a negative pressure control means is fluidly connected to the discharge port. 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 this valve is disposed between the negative pressure source and the connection part. The negative pressure control means can be connected to the discharge port via the connection part. According to this embodiment, the negative pressure can be instantaneously applied by opening the valve.

[0164] There is no particular limitation on the specific embodiment of the valve. From the viewpoint of preventing backflow when the liquid feeding is stopped, those that can suppress pressure fluctuations in the flow path when the valve opens and closes, for example, those with a relatively slow opening and closing operation, are preferable. The valve may be, for example, a three-way valve, and may be configured to be able to connect the microchannel chip to either a negative pressure source (for example, a pressure tank) or the external atmosphere (particularly the external air).

[0165] In particular, before the dispersed-phase liquid and the continuous-phase liquid come into contact, the negative pressure control means is fluidly connected to the discharge port. In this case, since the negative pressure can be rapidly applied after the contact between the dispersed-phase liquid and the continuous-phase liquid, it may be possible to suppress the generation of early droplets and further improve the uniformity of the droplets.

[0166] (Liquid feeding in the emulsion filling method) In the emulsion filling method, generally, it is preferable to stop the liquid feeding before the emulsion reaches the discharge port (that is, before the gas-liquid interface reaches the discharge port) because it can reduce the loss of detected droplets and increase the number of detected droplets. Also, the flow rate ratio of the continuous-phase liquid to the dispersed-phase liquid during liquid feeding may be increased so that only the continuous-phase liquid in the emulsion fills the discharge port, but it is difficult to sufficiently fill the discharge port with the continuous-phase liquid.

[0167] For these reasons, in the emulsion filling method, when the liquid feeding is stopped, the gas-liquid interface often exists in the flow path or near the flow path opening of the discharge port. Due to the sudden pressure fluctuation when the liquid feeding is stopped (in the case of negative pressure liquid feeding, air flows into the discharge port), the gas-liquid interface flows backward in the flow path, and it is likely to have an adverse effect on the emulsion retention (droplets flow out of the emulsion retention flow path, or droplets aggregate and coalesce near the gas-liquid interface).

[0168] In addition, in the case of negative pressure liquid feeding, since air flows into the discharge port when the liquid feeding is stopped, the pressure is likely to be applied to the gas-liquid interface. When a highly flexible material such as silicon rubber (PDMS) is used for the substrate material, or the thickness of the substrate is extremely thin (about 1 mm or less in the case of COC), the flow path is likely to deform during negative pressure liquid feeding, and the force to restore the deformation when the liquid feeding is stopped makes the reverse flow more likely to occur. (That is, the upper and lower surfaces of the flow path in the central part of the flow path far from the wall surface of the flow path side deflect in the direction of reducing the flow path cross-sectional area (roof collapse). Since the emulsion retention flow path is preferably wider than the flow path height as described above, it is considered to have a particularly large impact.

[0169] To suppress such reverse flow when the liquid feeding is stopped, it is preferable to reduce the liquid feeding pressure. For example, the pressure applied to the microchannel chip by the external liquid feeding 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 feeding driving force is, in particular, the pressure of the negative pressure applied to the discharge port, or the pressure of the positive pressure applied to the dispersed phase liquid holding part and the continuous phase liquid holding part.

[0170] On the other hand, for example, when the present invention is used for rapid digital measurement, it is preferable that the droplet generation speed calculated from the ratio of the liquid feeding speed to the flow rate of the continuous phase liquid with respect to the dispersed phase liquid is high. For example, it is preferable that the droplet generation speed in the emulsion forming part is 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.

[0171] When it is desired to reduce the liquid feeding pressure and increase the droplet generation rate, it is desirable to adjust so that the flow path pressure loss resistance value (= liquid feeding pressure / liquid feeding rate) becomes small. Since the flow path pressure loss 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 feeding means, etc., the above parameters may be appropriately adjusted so that the liquid feeding rate and / or the droplet generation rate during liquid feeding and the liquid feeding pressure become appropriate values.

[0172] (Installation of Microchannel Chip) The microchannel chip is generally installed horizontally in the vertical direction (up and down direction), but from the viewpoint of holding the emulsion, etc., it may be installed with an intentional inclination in a certain direction. For example, when the continuous phase liquid has a greater specific gravity than the dispersed phase liquid (e.g., using a fluorine-based dispersant as the continuous phase liquid and an aqueous solution as the dispersed phase liquid), since the droplets have buoyancy due to the specific gravity difference, the microchannel chip may be installed with an intentional inclination so that the droplets do not easily flow out from the emulsion holding flow path.

[0173] <Detection Process> Detection processing can be performed on the droplets in the emulsion generated according to the method according to the present disclosure. The detection processing includes, for example, the reaction of the target substance in the droplet and the detection of the reaction (for example, the detection of the reaction product). The detection processing can be performed on the droplets in the emulsion held in the emulsion holding flow path.

[0174] Examples of the target substance (particularly the target molecule) include nucleic acids, proteins, peptides, enzymes, cells, bacteria, spores, viruses, organelles, polymer assemblies, drug candidates, lipids, carbohydrates, metabolites, or any combination thereof.

[0175] 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, pyrophosphatases, reporter enzymes, reverse transcriptases, topoisomerases, etc. can be exemplified. 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, reactions capable of isothermal amplification of nucleic acids such as the LAMP method, NASBA method, TMA method, and TRC method can be mentioned. In the case of one-step RT-PCR, it is preferable to create droplets at a temperature suitable for the reverse transcription reaction in terms of the reaction efficiency and reaction time of the reverse transcription reaction. It is also possible to detect cDNA, which is a product of the reverse transcription reaction, by the cycling probe method.

[0176] When performing the reaction, it is preferable to mix two or more reaction solutions upstream of the emulsion formation part (for example, the dispersed phase liquid confluence part) and generate droplets using this mixture. In the present invention, the reaction solution 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 solutions, and the target substance may be contained in any of the reaction solutions. There is no problem even if there are three or more reaction solutions.

[0177] 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 for amplifying 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, polymerase, nucleotides, salts, and buffer components. It is preferable that the composition is devised so that the target molecule, reaction substrate, enzyme, etc. do not decompose, deteriorate, or cause non-specific reactions in the reaction solution, and glycerol, surfactant, etc. may be further added in consideration of the behavior in the apparatus.

[0178] <Other means> (Detection means) For the detection of the reaction, for example, detection means capable of detecting the product resulting from the reaction can be used.

[0179] The detection method can appropriately select an appropriate method according to the reaction product. For example, optical, X-ray, MALDI (Matrix-Assisted Laser Desorption / Ionization), FCS (Fluorescence Correlation Spectroscopy), FP (Fluorescence Polarization) / FCS, fluorescence method, colorimetric analysis, chemiluminescence, bioluminescence, scattering, surface plasmon resonance, electrochemical method, electrophoresis, laser, mass spectrometry, Raman spectroscopy, FLIPR (Molecular Devices), etc. can be used for detection. When detecting using transmitted light, if a microchannel chip is fabricated with a material that transmits light, it is preferable in that the reaction product can be detected only by placing the microchannel chip on an optical detector without moving the droplets in the chip.

[0180] As the detection means (detector) used for detecting the reaction product, an imaging sensor for recording / measuring the reaction of the target substance and optionally its components can be used. As an example of detection, a camera or imaging device having appropriate illumination and resolution for spatially decomposing individual signals to be detected can be mentioned. As the camera or imaging device, known ones can be used. For example, the camera can use any ordinary 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). Also, it can be improved by using the polarization of the excited / emitted light during detection. For example, when detecting droplets that emit a fluorescent signal, by collectively photographing the detection area with an optical unit having a large field of view, rapid and high-throughput signal detection can be performed.

[0181] (Temperature control means) The temperature control means has the role of keeping the liquid in the microchannel chip at a temperature suitable for the reaction of the target substance. The temperature control means may have a shape that can be in proximity (preferably in close contact) with the microchannel chip, and does not necessarily have to be flat.

[0182] Among the temperature control means, at least the portion in proximity (preferably in close contact) with the microchannel chip is preferably made of a metal material with high thermal conductivity. In addition, when the microchannel chip is fabricated by bonding a substrate and an upper structure, reducing the thickness of the substrate and / or the upper structure in contact with the temperature control means is preferable because heat conduction to the channels provided in the microchannel chip can be performed more efficiently. The temperature control means only needs to be able to control the temperature of at least the emulsion holding channel, which is the reaction field of the target substance. However, if it can also control the temperature of the phase liquid holding part and the channels, it is preferable in that it can suppress the non-specific reaction of the target molecule. As a specific example, when the reaction of the target substance is a nucleic acid amplification reaction, by controlling the temperature of each holding part and channel with the temperature control means so that it is higher than the reaction temperature of the target substance in the emulsion holding channel, non-specific annealing between primers / probes can be reduced. Also, when the bottom surface of the microchannel chip is heated to the reaction temperature by the temperature control means and the upper surface substrate of the microchannel chip is made of a material that transmits light and transmission light detection is performed from the upper surface, it is preferable because it can easily improve the quantitative upper limit of digital detection on the device by using the position of the empty microchannel chip before supplying each phase liquid and / or the evaluation of the channel structure and / or dust inside and outside the chip, the behavior in the channel when supplying each phase liquid and / or the behavior of emulsion generation during liquid feeding, and the signal detection result of the emulsion during the reaction.

[0183] Hereinafter, the present invention will be described in more detail using examples. The present invention is not limited to these descriptions.

Examples

[0184] The microchannel chip used in the present invention will be described below.

[0185] ≪Fabrication of Microchannel Chip≫ A microchannel chip was fabricated using photolithography and soft lithography techniques. The specific procedure is shown below.

[0186] (1) After dropping photoresist SU-8 3050 (Microchem) onto a 4-inch bare silicon wafer (Filtec), a photoresist thin film was formed using a spin coater (MIKASA).

[0187] (2) Using a mask aligner (USHIO Electric) and a chrome mask forming the channel pattern of the microchannel chip, the channel pattern was formed on the photoresist film. Then, the channel pattern was developed using SU-8 Developer (Microchem) to fabricate a mold for the channels constituting the microchannel chip.

[0188] (3) To suppress adsorption to SU-8, vapor deposition surface treatment was performed with Trichloro(1H,1H,2H,2H-perfluoro-octyl)silane (Thermo Fisher Scientific).

[0189] (4) A mixture of an uncured siloxane monomer and a polymerization initiator (weight ratio 10:1) prepared using SYLGARD SILICONE ELASTOMER KIT (Toray Dow Corning) was poured into the mold processed in (3) above, and heated at 80 °C for 2 hours to fabricate a polymer (PDMS) substrate with the channel shape transferred.

[0190] (5) The obtained polymer substrate was carefully peeled from the mold, shaped with a cutter, and then the dispersed phase liquid holding part, continuous phase liquid holding part, and discharge port were formed using a punch.

[0191] (6) After surface treatment of the polymer substrate with the holding part and discharge port formed and the cover glass (Matsunami Glass) using an oxygen plasma generator (Maywa Focus), the patterned surface of the PDMS substrate and the cover glass were bonded together. The fabricated chip was stored in a desiccator.

[0192] The fabricated microchannel chip has dimensions of 34 cm in length and 75 cm in width. As the dispersed-phase liquid holding part, it has a hole with a diameter of φ4 mm, as the continuous-phase liquid holding part, it has a hole with a diameter of φ8 mm, and as the discharge port, it has a hole with a diameter of φ1.5 mm, respectively.

[0193] (Flow channel structure) The microchannel chip had two dispersed-phase liquid holding parts, a dispersed-phase liquid flow channel (having a first dispersed-phase liquid flow channel, a second dispersed-phase liquid flow channel, and a dispersed-phase liquid confluence part), a continuous-phase liquid holding part, two continuous-phase liquid flow channels, an emulsion formation part, an emulsion flow channel, an emulsion holding flow channel, and a discharge port. The two dispersed-phase liquid holding parts were connected to the emulsion formation part via the first or second dispersed-phase liquid flow channel, the continuous-phase liquid holding part was connected to the emulsion formation part via the two continuous-phase liquid flow channels, the emulsion formation part was connected to the emulsion holding flow channel via the emulsion flow channel, and the emulsion holding flow channel was connected to the discharge port.

[0194] The microchannel chip used in the example had a structure of any one of the following (No.1 or No.2).

[0195] (No.1) Regarding Microchannel Chip No.1, the first dispersed-phase liquid flow path and the second dispersed-phase liquid flow path are flow paths with a height of 80 μm, a width of 100 μm, and a length of 30 mm including meandering. They merge at the dispersed-phase liquid confluence section, narrow down to a flow path width of 100 μm, and then merge into the emulsion formation section. The two continuous-phase liquid flow paths are each straight flow paths with a height of 80 μm, a width of 100 μm, and a length of 30 mm, each having two bending sections. In the emulsion formation section, the dispersed-phase liquid confluence section and the two continuous-phase liquid flow paths intersect at a 90-degree angle in a cross shape, where the reaction liquid and the immiscible liquid (oil) merge to form droplets. The emulsion flow path is a straight flow path with a width of 80 μm × length of 100 μm in the part close to the emulsion formation section, a width of 200 μm × length of 680 μm in its downstream part, and further downstream, it is a stirring flow path with a width of 200 μm × length of 11.5 mm including a meandering composed of an arc curve with an R of 275 μm, which is connected to the emulsion holding flow path. The emulsion holding flow path is a meandering flow path with a flow path height of 130 μm, a width of 2 mm, and a length of 350 mm, and is directly connected to the discharge port through a discharge port 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.

[0196] (No.2) Regarding Microchannel Chip No.2, the first dispersed-phase liquid flow path and the second dispersed-phase liquid flow path are flow paths with a height of 80 μm, a width of 200 μm, and a length of 5000 μm. They merge at the dispersed-phase liquid confluence section, narrow down to a flow path width of 100 μm, and then merge into the emulsion formation section. The two continuous-phase liquid flow paths are each straight flow paths with a height of 80 μm, a width of 280 μm, and a length of 26 mm, each having two bent sections. In the emulsion formation section, the dispersed-phase liquid confluence section and the two continuous-phase liquid flow paths intersect at a 90-degree angle in a cross shape. In the emulsion formation section, the reaction liquid and the immiscible liquid (oil) merge to form droplets. The emulsion flow path is a straight flow path with a width of 80 μm × a length of 100 μm in the part close to the emulsion formation section, a straight flow path with a width of 200 μm × a length of 680 μm in its downstream part, and further downstream, it is a stirring flow path with a width of 200 μm × a length of 11.5 mm including a meander composed of an arc curve with an R of 275 μm, which is connected to the emulsion holding flow path. The emulsion holding flow path is a meandering flow path with a flow path height of 130 μm, a width of 2 mm, and a length of 350 mm, and is directly connected to the discharge port through a discharge port 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.

[0197] (No.3) Regarding the microchannel chip No.3, the dispersed-phase liquid flow path is the same as that of No.2. The two continuous-phase liquid flow paths are straight flow paths with a height of 130 μm, a width of 280 μm, and a length of 33 mm, each having two bent portions. In the emulsion formation section, the dispersed-phase liquid confluence section and the two continuous-phase liquid flow paths intersect at a 90-degree angle in a cross shape. In the emulsion formation section, the reaction liquid and the immiscible liquid (oil) merge to form droplets. The height of the dispersed-phase liquid flow path is 80 μm up to the inflow section to the emulsion formation section, and the flow path height of the emulsion formation section is the same as that of the continuous-phase liquid flow path and the downstream flow path of the emulsion formation section, which is 130 μm. Therefore, the inflow section has a stepped structure (inflow suppression structure) with a step of 50 μm in height (the emulsion formation section has a flow path height approximately 1.6 times that of the inflow section). Note that the mold was fabricated such that the step shape is approximately a right angle. The emulsion flow path is a straight flow path with a width of 80 μm × a length of 100 μm in the portion close to the emulsion formation section, a width of 200 μm × a length of 680 μm in its downstream portion, and a stirring flow path with a width of 200 μm × a length of 11.5 mm including a meandering shape composed of an arc curve with an R of 275 μm in its further downstream portion, and is connected to the emulsion holding flow path. The emulsion holding flow path is a meandering flow path with a flow path height of 130 μm, a width of 2 mm, and a length of 350 mm, and is directly connected to the discharge port through a discharge port 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] ≪Example 1≫ (Generation and retention of emulsion) Using the above microchannel chip (No.2), the generation and retention of emulsion were carried out according to the present invention. The details are shown in (1) to (6) below.

[0199] (Dispersed-phase liquid) (1) As the dispersed-phase liquid introduced into the dispersed-phase liquid holding section, aqueous solutions ("starting liquid" and "reaction liquid") with the following two types of compositions were prepared. Note that the aqueous solutions with the following compositions mimic the composition of the reaction starting liquid when using the TRC reaction, which is one of the nucleic acid amplification reactions.

[0200] (Starting liquid) 36.8 mM magnesium chloride 180.0 mM potassium chloride 0.2% (w / v) Tween 20 18.0% (v / v) DMSO 5.0% (v / v) glycerol

[0201] (Reaction solution) 0.2% (w / v) Tween 20 300 nM trehalose 5.0% (v / v) glycerol

[0202] (2) A glass heater (manufactured by Blast) heated at 46 °C, which is the TRC reaction temperature, was installed on an inverted microscope IX71 (manufactured by Olympus), and a microchannel chip was placed on it and fixed with tape.

[0203] (3) As a liquid delivery means, a device composed of a peristaltic pump (manufactured by Takasago Industries), a solenoid valve (manufactured by Takasago Industries), and a pressure sensor (manufactured by Keyence) that can control the pressure in a 200 mL tank to -1 to -10 kPa was used. This tank and the outlet of the microchannel chip were connected with a PTFE tube (manufactured by Nitto Seiko), and by releasing the pressure in the tank, a pressure difference (negative pressure) was applied to the chip.

[0204] (4) 20 μL each of the reaction solution and the starting solution were dropped into the two dispersed phase liquid holding parts using a pipetteman. 40 seconds after dropping the reaction solution and the starting solution, 200 μL of oil (Droplet Generator oil for EvaGreen (manufactured by Biorad)) as the continuous phase liquid was dropped. Note that the pipetteman used above was calibrated with a maximum allowable error of ±0.06 μL. Before the continuous phase liquid reached the emulsion forming part, the dispersed phase liquid (a mixed liquid of the reaction solution and the starting solution) passed through the dispersed phase liquid flow path filled with gas and reached the inflow part to the emulsion forming part.

[0205] (5) Twenty seconds after the oil was dropped, with the internal pressure of the tank of the liquid feeding device connected to the discharge port in advance adjusted to -5 kPa, a pressure difference (negative pressure) was applied to start droplet generation and droplet capture. Each holding part during liquid feeding was in a state of being open to the atmospheric pressure. Before the application of the negative pressure, the continuous phase liquid had moved to the emulsion formation part.

[0206] (6) After the interface between the oil and air (the interface between the emulsion and air) reached the downstream end of the emulsion holding flow path (about 150 seconds after the application of the negative pressure), the negative pressure applied to the discharge port was released to normal pressure, and the liquid feeding was stopped.

[0207] (Evaluation of mixing ratio) The mixing ratio of the two dispersed phase liquids (starting liquid and reaction liquid) in Example 1 was evaluated as follows.

[0208] After the liquid feeding was stopped, the amounts of the reaction liquid and the starting liquid remaining in the dispersed phase liquid holding part were measured with a pipetteman of 0.2 to 10.0 μL respectively. This remaining amount was measured in 0.2 μL increments. When the liquid in the dispersed phase liquid holding part dried up and air invaded the pipette, the set liquid amount of the pipetteman was taken as the maximum value of the remaining amount. The value obtained by subtracting 0.2 μL from the maximum value was taken as the minimum value. The remaining amount was taken as its median value (the value obtained by subtracting 0.1 μL from the maximum value), and its standard deviation was assumed to be ±0.1 μL. Considering that the pipetteman used was calibrated with a maximum allowable error of ±0.03 μL, the standard deviation of the remaining amount measurement by this measurement was ±0.11 μL. In addition, since the standard deviation when adding 20 μL of the reaction liquid and the starting liquid was ±0.06 μL, the standard deviation δa of the measurement of the total liquid feeding amount of the reaction liquid and the starting liquid (= the difference between the added liquid amount and the remaining amount of each liquid) was ±(0.11 2 ×2 + 0.06 2 ×2) 0.5 = ±0.18 μL. Also, the relative error δb of the measurement of the difference in the liquid feeding amounts of the reaction liquid and the starting liquid (= the difference between the remaining amounts of each, absolute value) was ±(0.11 2 ×2) 0.5 ×2 = ±0.32 μL.

[0209] In this experiment, it was assumed that the ideal mixing ratio c was reaction solution: starting solution = 1:1, and the error in the mixing ratio for each experiment was defined as (the difference b in the liquid feeding amounts of the reaction solution and the starting solution) / (the total liquid feeding amount a of the reaction solution and the starting solution), and the error in the mixing ratio was measured. The relative error δc in the measurement of the above mixing ratio error was calculated from δc = (δa / a + δb / b)×c.

[0210] The average error in the mixing ratio according to Example 1 (number of trials = 10 times) was 1.0%, and the maximum value of the error in the mixing ratio was 2.4%. The results are shown in Table 1 below.

[0211] (Evaluation of the time until air intrusion into the emulsion holding flow path) Emulsion generation and retention were performed in the same manner as above, except that the supply amount of the dispersed phase liquid (starting solution and reaction solution respectively) was set to 10 μL, and then the inflow of air into the emulsion holding flow path through the dispersed phase liquid holding part was evaluated.

[0212] The time from when the reaction solution or the starting solution was depleted and air began to intrude into the chip flow path until the air intruded into the emulsion holding flow path as bubbles was measured. For three trials (N = 3), the times from the depletion of the dispersed phase liquid to the intrusion of bubbles were 16 seconds, 19 seconds, and 17 seconds respectively, and the average value was 17.3 seconds. The results are shown in Table 1 below.

[0213] ≪Comparative Example 1≫ (Emulsion generation and retention) Emulsion generation and retention were performed in the same manner as in Example 1, except that the supply and liquid feeding of the dispersed phase liquid and the continuous phase liquid were performed as follows using the above microchannel chip (No. 1).

[0214] 200 μL of oil was dropped into the continuous phase liquid holding part using a pipetteman (Gilson). 40 seconds after the oil was dropped, the reaction solution and the starting solution were dropped into the dispersed phase liquid holding part in 20 μL portions each using a pipetteman with a volume of 2.0 to 20.0 μL. Before dropping the dispersed phase liquid, the continuous phase liquid had reached the dispersed phase liquid holding part through the dispersed phase liquid flow path.

[0215] Twenty seconds after the dropping of the reaction solution, with the internal pressure of the tank of the liquid feeding device connected to the discharge port in advance adjusted to -5 kPa, a negative pressure was applied to start droplet generation and retention. Each holding part during liquid feeding was in a state of being open to the atmospheric pressure.

[0216] (Evaluation of mixing ratio) The mixing ratio was evaluated in the same manner as in Example 1.

[0217] The average error of the mixing ratio according to Comparative Example 1 (number of trials = 10 times) was 6.7%, and the maximum value of the error of the mixing ratio was 15.0%. The results are shown in Table 1 below.

[0218] (Evaluation of the time until air intrusion into the emulsion holding flow path) Emulsion generation and retention were carried out in the same manner as above, except that the supply amount of the dispersed phase liquid (starting liquid and reaction liquid respectively) was set to 10 μL. Then, the inflow of air into the emulsion holding flow path through the dispersed phase liquid holding part was evaluated.

[0219] The time from when the reaction liquid or the starting liquid was depleted and air began to intrude into the chip flow path until air intruded into the emulsion holding flow path as bubbles was measured. For three trials (N = 3), the times from the depletion of the dispersed phase liquid sound to the intrusion of bubbles were 3 seconds, 2 seconds, and 3 seconds respectively, and the average value was 2.3 seconds. The results are shown in Table 1 below.

[0220]

Table 1

[0221] As shown in Table 1, when the dispersed-phase liquid reached the inlet part to the emulsion forming part through the dispersed-phase liquid flow path in a state where the dispersed-phase liquid was filled with gas before the continuous-phase liquid reached the emulsion forming part (Example 1), compared with the case where the continuous-phase liquid reached the emulsion forming part before the dispersed-phase liquid reached the inlet part of the emulsion forming part (Comparative Example 1), the mixing ratio of the two dispersed-phase liquids was stable, and the intrusion of air after the dispersed-phase liquid was depleted in the dispersed-phase liquid holding part was suppressed.

[0222] In particular, in the case of Example 1, it was confirmed that the air intrusion speed was about 10 times slower than that in the case of Comparative Example 1. The above intrusion speed varies greatly depending on the liquid feeding pressure, flow path structure, physical properties of each liquid, physical properties of the flow path wall surface, etc. On the other hand, when using a dispersed-phase liquid with a high surface tension such as an aqueous solution and a continuous-phase liquid with a low surface tension and viscosity used for general droplet generation, the speed at which air intrudes by displacing the dispersed-phase liquid after the dispersed-phase liquid is depleted is slower than the speed at which air intrudes by displacing the continuous-phase liquid. Therefore, generally, by introducing the dispersed-phase liquid first and allowing it to reach the inlet part to the emulsion forming part, the intrusion of air can be suppressed, and as a result, it is considered possible to minimize sample loss.

[0223] ≪Reference Example 1≫ (Evaluation of Suppression of Flow Path Blockage by the Ingress Suppression Structure) The same operations as in Example 1 were performed up to (1) to (4) described in the above “(Generation and Retention of Emulsion)”, except that oil was not dropped, and the time from when the dispersed-phase liquid reached the emulsion forming part until the emulsion forming part was blocked by the dispersed-phase liquid was measured (N = 3). As a result, the measured times were 21 seconds, 28 seconds, and 29 seconds (average 26 seconds). The results are shown in Table 2 below.

[0224] ≪Reference Example 2≫ Except for using the above chip No. 3, in the same manner as in Reference Example 1, the time from when the dispersed-phase liquid reached the emulsion formation section until the emulsion formation section was blocked by the dispersed-phase liquid in the flow path was measured (N = 3). As a result, the measured times were 77 seconds, 86 seconds, and 102 seconds (average 88 seconds). The results are shown in Table 2 below.

[0225]

Table 2

[0226] As can be seen from Table 2, it can be understood that by providing an entry suppression structure (step structure), the blockage of the flow path is particularly effectively suppressed. By retaining the dispersed-phase liquid in the inflow section to the emulsion formation section by the entry suppression structure, the blockage of the flow path and the generation of bubbles can be more effectively suppressed.

Explanation of Reference Signs

[0227] 10, 20 microchannel chips 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 118 inflow section to the emulsion formation section 120 emulsion formation section 130 emulsion flow path 140 emulsion holding flow path 150 discharge port 200 entry suppression structure 220a, 220b connection parts 230a, 230b discontinuous parts, corner parts on the wall surface α1, α2 angles 300 dispersed-phase liquid holding section 31 wells 33 hole parts 35 expansion parts W width direction L length direction H height direction

Claims

1. A method for generating an emulsion by supplying a dispersed-phase liquid and a continuous-phase liquid to a microchannel chip, comprising: the microchannel chip having a dispersed-phase liquid holding portion, a dispersed-phase liquid flow path, a continuous-phase liquid holding portion, a continuous-phase liquid flow path, an emulsion forming portion, an emulsion flow path, and a discharge port; the dispersed-phase liquid holding portion being connected to the emulsion forming portion via the dispersed-phase liquid flow path; the continuous-phase liquid holding portion being connected to the emulsion forming portion via the continuous-phase liquid flow path; the emulsion forming portion being connected to the discharge port via the emulsion flow path; supplying a dispersed-phase liquid to the dispersed-phase liquid holding portion; supplying a continuous-phase liquid to the continuous-phase liquid holding portion; and applying an external liquid feeding driving force to generate, in the emulsion forming portion, an emulsion containing droplets composed of the dispersed-phase liquid and a continuous phase composed of the continuous-phase liquid, and causing the emulsion thus generated to enter the emulsion flow path; including: before the continuous-phase liquid reaches the emulsion forming portion, moving the dispersed-phase liquid through the dispersed-phase liquid flow path filled with gas to the inflow portion to the emulsion forming portion; and before applying the external liquid feeding driving force, moving the continuous-phase liquid to the emulsion forming portion. A method characterized by the above.

2. A method for generating an emulsion by supplying a dispersed-phase liquid and a continuous-phase liquid to a microchannel chip, comprising: the microchannel chip having a dispersed-phase liquid holding portion, a dispersed-phase liquid flow path, a continuous-phase liquid holding portion, a continuous-phase liquid flow path, an emulsion forming portion, an emulsion flow path, and a discharge port; the dispersed-phase liquid holding portion being connected to the emulsion forming portion via the dispersed-phase liquid flow path; the continuous-phase liquid holding portion being connected to the emulsion forming portion via the continuous-phase liquid flow path; the emulsion forming portion being connected to the discharge port via the emulsion flow path; supplying a dispersed-phase liquid to the dispersed-phase liquid holding portion; supplying a continuous-phase liquid to the continuous-phase liquid holding portion; and applying an external liquid feeding driving force to generate, in the emulsion forming portion, an emulsion containing droplets composed of the dispersed-phase liquid and a continuous phase composed of the continuous-phase liquid, and causing the emulsion thus generated to enter the emulsion flow path; including: Before the continuous phase liquid reaches the emulsion forming section, moving the dispersed phase liquid through the dispersed phase liquid flow path filled with gas to the inflow section to the emulsion forming section, and moving the dispersed phase liquid and / or the continuous phase liquid by capillary force and / or liquid level differential pressure A method characterized by the above. **Claim 3**: A method for generating an emulsion by supplying a dispersed phase liquid and a continuous phase liquid to a microchannel chip, wherein 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 a discharge port, the dispersed phase liquid holding section is connected to the emulsion forming section through the dispersed phase liquid flow path, the continuous phase liquid holding section is connected to the emulsion forming section through the continuous phase liquid flow path, the emulsion forming section is connected to the discharge port through the emulsion flow path, supplying a dispersed phase liquid to the dispersed phase liquid holding section, supplying a continuous phase liquid to the continuous phase liquid holding section, and applying an external liquid feeding driving force to generate an emulsion including liquid droplets composed of the dispersed phase liquid and a continuous phase composed of the continuous phase liquid in the emulsion forming section, and causing the emulsion thus generated to enter the emulsion flow path, including before the continuous phase liquid reaches the emulsion forming section, moving the dispersed phase liquid through the dispersed phase liquid flow path filled with gas to the inflow section to the emulsion forming section, and applying a negative pressure to the discharge port, thereby moving the dispersed phase liquid through the dispersed phase liquid flow path filled with gas to the inflow section to the emulsion forming section A method characterized by the above. **Claim 4**: A method for generating an emulsion by supplying a dispersed phase liquid and a continuous phase liquid to a microchannel chip, wherein 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 a discharge port, the dispersed phase liquid holding section is connected to the emulsion forming section through the dispersed phase liquid flow path, the continuous phase liquid holding section is connected to the emulsion forming section through the continuous phase liquid flow path, the emulsion forming section is connected to the discharge port through the emulsion flow path, Supplying a dispersed-phase liquid to the dispersed-phase liquid holding part, Supplying a continuous-phase liquid to the continuous-phase liquid holding part, and By applying an external liquid-feeding driving force, generating an emulsion containing droplets composed of the dispersed-phase liquid and a continuous phase composed of the continuous-phase liquid in the emulsion forming part, and causing the emulsion thus generated to enter the emulsion flow path, including Before the continuous-phase liquid reaches the emulsion forming part, moving the dispersed-phase liquid through the dispersed-phase liquid flow path filled with gas to the inflow part to the emulsion forming part, and the inflow part has an entry suppression structure, and this entry suppression structure can suppress the entry of the dispersed-phase liquid into the emulsion forming part before the application of the external liquid-feeding driving force, A method characterized by the above.

5. A method for generating an emulsion by supplying a dispersed-phase liquid and a continuous-phase liquid to a microchannel chip, the microchannel chip has a dispersed-phase liquid holding part, a dispersed-phase liquid flow path, a continuous-phase liquid holding part, a continuous-phase liquid flow path, an emulsion forming part, an emulsion flow path, and a discharge port, the dispersed-phase liquid holding part is connected to the emulsion forming part through the dispersed-phase liquid flow path, the continuous-phase liquid holding part is connected to the emulsion forming part through the continuous-phase liquid flow path, the emulsion forming part is connected to the discharge port through the emulsion flow path, Supplying a dispersed-phase liquid to the dispersed-phase liquid holding part, Supplying a continuous-phase liquid to the continuous-phase liquid holding part, and By applying an external liquid-feeding driving force, generating an emulsion containing droplets composed of the dispersed-phase liquid and a continuous phase composed of the continuous-phase liquid in the emulsion forming part, and causing the emulsion thus generated to enter the emulsion flow path, including Before the continuous-phase liquid reaches the emulsion forming part, moving the dispersed-phase liquid through the dispersed-phase liquid flow path filled with gas to the inflow part to the emulsion forming part, and the external liquid-feeding driving force is a negative pressure applied to the discharge port A method characterized by the above.

6. the dispersed-phase liquid holding part includes a first dispersed-phase liquid holding part and a second dispersed-phase liquid holding part, 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 confluence portion. The first dispersed-phase liquid flow path and the second dispersed-phase liquid flow path are each connected to the emulsion forming portion via the dispersed-phase liquid confluence portion. The method according to any one of claims 1 to 5. **Claim 7**: A method for generating an emulsion by supplying a dispersed-phase liquid and a continuous-phase liquid to a microchannel chip, wherein the microchannel chip has a dispersed-phase liquid holding portion, a dispersed-phase liquid flow path, a continuous-phase liquid holding portion, a continuous-phase liquid flow path, an emulsion forming portion, an emulsion flow path, an emulsion holding flow path, and a discharge port, the dispersed-phase liquid holding portion is connected to the emulsion forming portion via the dispersed-phase liquid flow path, the continuous-phase liquid holding portion is connected to the emulsion forming portion via the continuous-phase liquid flow path, the emulsion forming portion is connected to the emulsion holding flow path via the emulsion flow path, and the emulsion holding flow path is connected to the discharge port, supplying a dispersed-phase liquid to the dispersed-phase liquid holding portion, supplying a continuous-phase liquid to the continuous-phase liquid holding portion, and applying a negative pressure to the discharge port to generate, in the emulsion forming portion, an emulsion including liquid droplets composed of the dispersed-phase liquid and a continuous phase composed of the continuous-phase liquid, and transporting the emulsion thus generated to the emulsion holding flow path filled with gas via the emulsion flow path, including moving the dispersed-phase liquid through the dispersed-phase liquid flow path filled with gas to the inflow portion to the emulsion forming portion before the continuous-phase liquid reaches the emulsion forming portion. A method characterized by the above. **Claim 8** A microchannel chip having a dispersed-phase liquid holding portion, a dispersed-phase liquid flow path, a continuous-phase liquid holding portion, a continuous-phase liquid flow path, an emulsion forming portion, an emulsion flow path, and a discharge port, wherein the dispersed-phase liquid holding portion is connected to the emulsion forming portion via the dispersed-phase liquid flow path, the continuous-phase liquid holding portion is connected to the emulsion forming portion via the continuous-phase liquid flow path, the emulsion forming portion is connected to the discharge port via the emulsion flow path, and By supplying a dispersed phase liquid to the dispersed phase liquid holding section, supplying a continuous phase liquid to the continuous phase liquid holding section, and applying an external liquid feeding driving force to the microchannel chip, 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 configured to enter the emulsion flow path. Here, an inflow section of the dispersed phase liquid flow path to the emulsion forming section has an entry suppression structure, and this entry suppression structure is characterized in that it can suppress the dispersed phase liquid from entering the emulsion forming section before the application of the external liquid feeding driving force. Microchannel chip.

Images