Microchannel chip and use thereof

Abstract

To provide a micro-channel chip improved in the retention of an emulsion and to provide a method for using the same.SOLUTION: A micro-channel chip for filling an emulsion has a dispersion phase liquid holding portion, a dispersion phase liquid channel, a continuous phase liquid holding portion, a continuous phase liquid channel, an emulsion forming portion, an emulsion channel, an emulsion holding channel, and a discharge port; wherein the dispersion phase liquid holding portion is connected to the emulsion forming portion via the dispersion phase liquid channel; wherein the continuous phase liquid holding portion is connected to the emulsion forming portion via the continuous phase liquid channel; wherein the emulsion forming portion is connected to the emulsion holding channel via the emulsion channel; wherein the emulsion holding channel is connected to the discharge port; wherein a terminal part on a discharge port side of the emulsion holding channel constitutes a bubble trap stop valve portion.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] The present invention relates to a microchannel chip and use thereof. In particular, the present invention relates to a microchannel chip for more efficiently, conveniently, and quickly performing droplet array measurement, and use thereof. [Background technology]

[0002] The microdroplet method, in which a reaction solution is fractionated into microcompartments and reacted independently, is known. This method is expected to be applied to, for example, the production of micro- and nanoparticles. In particular, by using a microfluidic device to microcompartmentalize target molecules at the single molecule level and carrying out reactions within the microdroplets, the presence or absence of target molecules can be measured by the presence or absence of a signal, and this is used for digital measurement to perform absolute quantification of the number of target molecules.

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

[0004] U.S. Patent No. 6,277,663 discloses a system and method for generating droplets suitable for droplet assays. The method described in this document transports the generated droplets to an outlet region consisting of a pipette tip or droplet well. The document also describes an air trap that substantially separates the sample and oil until the application of a fluid driving force.

[0005] Non-Patent Document 1 discloses a centrifugal step droplet generation method, which describes filling an inlet of a device with oil, sending the oil to a droplet collection chamber by centrifugation, and then introducing a sample solution through the same inlet and generating droplets by centrifugation.

[0006] In recent years, droplet array measurement, in which droplets are aligned in a single layer in a detection area to easily measure signals, has attracted attention from the perspective of simplifying and speeding up the device used for such microdroplet methods.

[0007] Patent Documents 2 and 3 disclose microchannel chips having channels for forming droplets and droplet holders for holding droplets. Document 2 describes forming droplets by converging two or more reaction solutions and then contacting them with an immiscible liquid that is not miscible with the reaction solutions. Document 3 describes forming droplets by contacting the dispersion layer and continuous layer that flow in from a dispersion layer inlet and a continuous layer inlet through channels in a droplet generation unit.

[0008] Non-Patent Document 2 describes a method for generating droplets on a chip and an apparatus for doing so. The method described in this document includes an operation of filling a droplet array section with oil (filling operation) before liquid transfer. [Prior art documents] [Patent documents]

[0009] [Patent Document 1] European Patent No. 2550528 [Patent Document 2] Japanese Patent Application Publication No. 2019-170363 [Patent Document 3] Japanese Patent Application Publication No. 2020-169911 [Non-patent literature]

[0010] [Non-Patent Document 1] Centrifugal step emulsification applied for absolute quantification of nucleic acids by digital droplet RPA, Lab Chip, 2015, 15, 2759-2766 [Non-patent document 2] 1-Million droplet array with wide-field fluorescence imaging for digital PCR, Lab on a Chip, 2011, 11, 3838-3845 Summary of the Invention [Problem to be solved by the invention]

[0011] A microchannel chip having an emulsion-holding channel can be used when generating and holding an emulsion for performing a detection process on droplets, etc. By holding the generated emulsion in the emulsion-holding channel within the same microchannel chip, a simple method suitable for automation can be provided.

[0012] When an emulsion is produced in such a microchannel chip, the emulsion produced in the emulsion formation unit can be transported to an emulsion-holding channel filled with gas. The transported emulsion replaces the gas filling the emulsion-holding channel and is held there. This method is referred to as the "emulsion filling method" in this disclosure.

[0013] The emulsion filling method is extremely advantageous in automating the droplet array method because it can eliminate the preparatory operation of filling the microchannel chip with the continuous phase liquid in advance, the accompanying operation of discharging the excess continuous phase liquid, and the devices required for these operations. However, when the emulsion filling method is applied to a microchannel chip having an emulsion-retaining channel, the emulsion retention may be insufficient.

[0014] An object of the present invention is to provide a microchannel chip with improved emulsion (particularly droplet) retention, and to provide a method for using the same. [Means for solving the problem]

[0015] The above-mentioned problems of the present invention can be solved by the following aspects of the present invention. <Aspect 1> the dispersion liquid holding section, the dispersion liquid flow path, the continuous phase liquid holding section, the continuous phase liquid flow path, the emulsion forming section, the emulsion flow path, the emulsion holding flow path, and the discharge port; the dispersed phase liquid holding unit is connected to the emulsion forming unit via the dispersed phase liquid flow path, the continuous phase liquid holding unit is connected to the emulsion forming unit via the continuous phase liquid flow path, the emulsion forming section is connected to the emulsion holding flow path via the emulsion flow path, the emulsion holding channel is connected to the outlet, an end portion of the emulsion holding flow path on the discharge port side constitutes an air bubble trap stop valve portion; Microfluidic chip for emulsion filling. <Aspect 2> 2. The micro-channel chip according to aspect 1, wherein the bubble trap stop valve portion has a constriction portion. <Aspect 3> 3. The micro-channel chip according to aspect 2, wherein the constricted portion is constricted in the vertical direction. <Aspect 4> 4. The micro-channel chip according to aspect 3, wherein the constricted portion is constricted in both the vertical and horizontal directions. <Aspect 5> 5. The micro-channel chip according to aspect 3 or 4, wherein the narrowed portion has a step structure, thereby narrowing in the vertical direction. <Aspect 6> 6. The micro-channel chip according to any one of aspects 2 to 5, wherein the bubble trap stop valve section has an expansion section disposed downstream of the narrowing section, and an additional narrowing section disposed downstream of the expansion section. <Aspect 7> 7. The micro-channel chip according to aspect 6, wherein the additional constriction section is constricted in the vertical direction. <Aspect 8> 8. The micro-channel chip according to aspect 7, wherein the additional constriction section is constricted in both the vertical and horizontal directions. <Aspect 9> 9. The micro-channel chip according to aspect 7 or 8, wherein the additional constriction section has a step structure, thereby constricting in the vertical direction. <Aspect 10> A method for generating and maintaining an emulsion by supplying a dispersed phase liquid and a continuous phase liquid to the micro-channel chip according to any one of Aspects 1 to 9, comprising: 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 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 unit by an external liquid feeding driving force, and transporting the emulsion thus generated through the emulsion flow path to the emulsion holding flow path filled with gas; Including, the external liquid-transport driving force is stopped before the emulsion completely fills the emulsion-holding channel. method. <Aspect 11> 11. The method of claim 10, further comprising adjusting an amount of the continuous phase liquid supplied to the continuous phase liquid holding section, such that the continuous phase liquid is present in the continuous phase liquid holding section for at least a certain period of time after the external liquid-feeding driving force is stopped. <Aspect 12> 12. The method of claim 10 or 11, wherein the external fluid delivery driving force is negative pressure applied to the outlet. <Aspect 13> 13. The method according to any one of aspects 10 to 12, wherein at least one of the outlet and each phase liquid holding section is not sealed while the droplets are held in the emulsion holding channel after the external liquid delivery driving force is stopped. [Effects of the Invention]

[0016] According to the present invention, it is possible to provide a microchannel chip with improved emulsion (particularly droplet) retention, and a method for using the same.

[0017] In particular, the microchannel chip of the present invention can hold droplets well even when each holding section and outlet is open to atmospheric pressure. [Brief explanation of the drawings]

[0018] [Figure 1] FIG. 1 is a schematic plan view of one embodiment of a microchannel chip according to the present disclosure. [Figure 2] FIG. 2 is a top schematic view of one embodiment of a bubble trap stop valve portion according to the present disclosure. [Figure 3] 3 is a schematic diagram of the bubble trap stop valve portion of FIG. 2 in a cross section perpendicular to a horizontal plane. [Figure 4] FIG. 4 is a cross-sectional schematic diagram showing one embodiment of a dispersed phase liquid retaining portion according to the present disclosure. [Figure 5] FIG. 5 is a photograph of the bubble trap stop valve part according to Example 1. [Figure 6] FIG. 6 is a photograph showing the state of the detection process for droplets held in the emulsion holding channel according to Example 1. [Figure 7] FIG. 7 is an enlarged view of a portion of the photograph of FIG. DETAILED DESCRIPTION OF THE INVENTION

[0019] <Microfluidic chip for emulsion filling> A microchannel chip according to the present disclosure is used for filling an emulsion, and includes a dispersed phase liquid holding section, a dispersed phase liquid flow path, a continuous phase liquid holding section, a continuous phase liquid flow path, an emulsion forming section, an emulsion flow path, an emulsion holding flow path, and an outlet; the dispersed phase liquid holding section is connected to the emulsion forming section via a dispersed phase liquid flow path, the continuous phase liquid holding section is connected to the emulsion forming section via a continuous phase liquid flow path; the emulsion forming section is connected to the emulsion holding flow path via an emulsion flow path, an emulsion holding channel connected to the outlet; The end portion of the emulsion holding flow path on the discharge port side constitutes the bubble trap stop valve portion.

[0020] When an emulsion filling method is performed in a microchannel chip having an emulsion-holding channel, the emulsion may not be retained sufficiently.

[0021] Specifically, in the emulsion filling method, the emulsion advances through an emulsion-holding channel filled with gas, causing the gas-liquid interface between the gas and the emulsion to move within the emulsion-holding channel. In this case, after the liquid transfer is stopped (after the external liquid transfer driving force is stopped), the interface between the held emulsion and the gas advances toward the outlet due to the liquid level differential pressure (particularly due to the continuous phase liquid held in the continuous phase liquid holding section) and the capillary force at the interface, which could cause droplets in the emulsion to flow out from the outlet.

[0022] Furthermore, under conditions where the continuous phase liquid is likely to volatilize (evaporate) due to heating or the like, for example, when a volatile continuous phase liquid is used or the emulsion needs to be heated, and / or when the vaporized continuous phase liquid is likely to leak into the external atmosphere, for example, when the holders are not sealed or a highly gas-permeable chip substrate (such as PDMS) is used, the continuous phase of the emulsion will evaporate, causing the interface between the emulsion and the gas to move in a direction away from the outlet, which could cause droplets in the emulsion to flow out toward the holders and / or cause the droplets to aggregate and coalesce at the gas-liquid interface.

[0023] In other words, in order to improve the emulsion retention in the emulsion holding channel, it is considered important to control the position of this gas-liquid interface and stably retain the droplets in the emulsion holding channel. However, with conventional technology, it has not been easy to control the position of this gas-liquid interface.

[0024] In contrast, in the present invention, the emulsion holding flow path has a bubble trap stop valve section at the end on the discharge port side. According to the present invention, when the gas-liquid interface progressing through the emulsion holding flow path approaches the end on the discharge port side, bubbles are formed in the bubble trap stop valve section, causing the continuous phase liquid in the emulsion to flow out toward the discharge port, but the formed bubbles are retained in the bubble trap stop valve section (particularly before the narrowed section), making it difficult for the droplets to pass through the thin layer of continuous phase liquid formed by the bubbles and the flow path wall surface (acting like a size exclusion filter), so that only the droplets can be selectively retained within the emulsion holding flow path, and as a result, it is possible to prevent the droplets in the emulsion from flowing out and being wasted. In addition, even if the vapor pressure inside the bubbles increases and / or the amount of continuous phase liquid in the emulsion decreases due to the volatilization (evaporation) of the continuous phase liquid, as long as there is a force that causes the continuous phase liquid in the emulsion to flow out toward the outlet, the gas-liquid interface where the bubbles and droplets are in contact can be prevented from moving away from the outlet (the bubbles can expand). Therefore, even under conditions such as heating that make it easy for the continuous phase liquid to volatilize (evaporate) and / or conditions that make it easy for the vaporized continuous phase liquid to leak into the external atmosphere, it is possible to prevent the outflow of droplets and the aggregation / coalescence of droplets at the gas-liquid interface.

[0025] The present invention will be specifically described with reference to the drawings. Note that the drawings show exemplary embodiments, and the method according to the present invention is not limited to these embodiments. Figure 1 is a schematic diagram for ease of understanding and is not drawn to scale.

[0026] The microchannel chip shown schematically in FIG. 1 has a planar configuration, i.e., emulsion generation, transport, and retention occur substantially within a single plane. In FIG. 1, direction W indicates the width direction, and 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 holder 102, a second dispersed phase liquid holder 103, a dispersed phase liquid flow path (comprising a first dispersed phase liquid flow path 114, a second dispersed phase liquid flow path 115, and a dispersed phase liquid junction 116), a continuous phase liquid holder 101, a continuous phase liquid flow path 111, an emulsion formation section 120, an emulsion flow path 130, an emulsion retention flow path 140, and an outlet 150. The dispersed phase liquid holding units 102 and 103 are connected to the emulsion forming unit 120 via dispersed phase liquid channels, and the continuous phase liquid holding unit 101 is connected to the emulsion forming unit 120 via a continuous phase liquid channel 111. In the embodiment shown in Figure 1, the continuous phase liquid channel 111 has a first continuous phase liquid channel 112 and a second continuous phase liquid channel 113. The emulsion forming unit 120 is connected to an emulsion holding channel 140 via an emulsion channel 130, and the emulsion holding channel 140 is connected to an outlet 150.

[0027] According to the present invention, the end portion of the emulsion holding channel 140 on the outlet side constitutes the bubble trap stop valve portion 160 .

[0028] When generating and holding an emulsion, for example, an emulsion containing droplets made of a dispersed phase liquid and a continuous phase made of a continuous phase liquid is generated in the emulsion forming section 120 by an external liquid supply driving force, and the emulsion thus generated is transported via the emulsion flow path 130 to the emulsion holding flow path 140 filled with gas.

[0029] In this case, the gas-liquid interface between the gas filling the emulsion holding flow path 140 and the emulsion moves through the emulsion holding flow path 140 toward the outlet 150. When this gas-liquid interface reaches the bubble trap stop valve portion 160, bubbles are formed due to the structure of the bubble trap stop valve portion 160.

[0030] That is, when the gas-liquid interface progressing within the emulsion holding flow path 140 approaches the terminal end on the outlet 150 side, bubbles are formed in the bubble trap stop valve portion 160, thereby automatically stopping or suppressing the progression and backflow of the gas-liquid interface. Note that, for example, the application of the external liquid-transfer driving force can be stopped before (particularly just before or when) the gas-liquid interface reaches the bubble trap stop valve portion.

[0031] The function of the bubble trap stop valve section is to prevent the outflow of droplets while allowing some outflow of the continuous phase (continuous phase liquid). That is, the continuous phase in the emulsion passes through the bubble trap stop valve section and gradually flows out to the outlet due to capillary force and liquid surface differential pressure, but the droplets in the emulsion (dispersed phase liquid) have difficulty passing through the thin layer of continuous phase liquid that exists between the bubbles formed in the bubble trap stop valve section and the wall surface of the flow path, so their outflow to the outlet is suppressed.

[0032] Capillary force, also known as capillary force, is a force that acts in the direction of intrusion into the channel, generated by the difference in surface tension between the gas-liquid interface in the wide-open holding section and the gas-liquid interface in the channel with a smaller cross section. Therefore, capillary force is affected not only by the characteristics of each channel but also by the structure of the holding section. In particular, in the emulsion filling method, capillary force has a significant effect during liquid transfer (droplet generation) and while the droplets are being held after the liquid transfer has stopped, as it controls the movement of the emulsion's gas-liquid interface.

[0033] The liquid level differential pressure is also known as hydrostatic pressure, and generally refers to the pressure generated by gravity in a stationary liquid, i.e., the pressure that depends on the amount (weight) of each phase liquid supplied to each holding section.

[0034] <Emulsion filling method> The microchannel chip according to the present disclosure is used to generate and hold emulsions by an emulsion filling method. In the emulsion filling method, emulsions (droplets + continuous phase) generated in an emulsion formation unit are transported to an emulsion holding channel filled with gas. The emulsion transported to the emulsion holding channel is held there and can optionally be subjected to a detection process.

[0035] In the emulsion filling method, it is preferable that the entire channel is free of liquid (e.g., dispersed phase liquid, continuous phase liquid, etc.), that is, the entire channel is filled with gas. This does not apply 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, but it is preferable that at least a part of the channel is not clogged with liquid, and it is particularly preferable that the dispersed phase liquid channel, continuous phase liquid channel, and emulsion formation section are not clogged.

[0036] <Air bubble trap stop valve> The microchannel chip according to the present disclosure has a bubble trap stop valve section, which is configured by the end portion of the emulsion holding channel on the outlet side.

[0037] The structure of the bubble trap stop valve section is not particularly limited as long as it can perform the above-mentioned function, that is, as long as bubbles are formed due to the structure of the bubble trap stop valve section when the gas-liquid interface moving through the emulsion holding flow path toward the discharge port reaches the bubble trap stop valve section, thereby suppressing or preventing the outflow of emulsion (particularly droplets) through the discharge port. Preferably, the bubble trap stop valve section has a narrowed section and, optionally, an additional narrowed section.

[0038] (Stenosis) In one embodiment of the bubble trap stop valve portion according to the present disclosure, the bubble trap stop valve portion has a constriction.

[0039] The "narrowed portion" is a portion of the bubble trap stop valve where the flow path is narrowed. That is, the emulsion-holding flow path is narrowed at the narrowed portion. The narrowed portion is configured to generate bubbles by being blocked by the continuous phase liquid before the air completely escapes the narrowed portion as the air-liquid interface reaches the narrowed portion, and is configured to be able to suppress the movement of emulsion (especially droplets) from the emulsion-holding flow path to the outlet.

[0040] More specifically, the "narrowed portion" is a portion of the bubble trap stop valve where the cross-sectional area of the flow path is reduced. In particular, the cross-sectional area of the emulsion holding flow path is abruptly reduced at the narrowed portion.

[0041] For example, the width and / or height of the channel can be reduced at the narrowed portion. The width of the channel is the length in the horizontal direction perpendicular to the length of the channel when the microchannel chip is in normal use. The height of the channel is the length in the vertical direction (direction of gravity) when the microchannel chip is in normal use.

[0042] When the upper surface (ceiling) of the inside of the flow channel at the constriction is lower than the upper surface of the inside of the flow channel adjacent to the constriction (i.e., when the constriction is constricted in the vertical direction (height direction)), the buoyancy of the bubbles can prevent the bubbles from passing through the constriction (note that as long as the bubbles are prevented from passing through the constriction, there is no problem if only some of the bubbles continue to flow out). In this case, it is possible to particularly effectively prevent the outflow of bubbles formed upstream of the constriction (toward the emulsion formation section). Note that "high" and "low" in this case are based on the vertical direction (direction of gravity) in the normal usage state of the microchannel chip.

[0043] In particular, the narrowed portion is narrowed in both the vertical and horizontal directions, which is particularly preferable since this further improves the stability of bubble formation and retention.

[0044] In particular, the narrowing in the vertical direction is achieved by a step structure. That is, the narrowed portion has a step structure, which narrows the narrowed portion in the vertical direction. The step structure is preferable because the sudden change in vertical height is discontinuous and acute, making it difficult for bubbles to flow out toward the outlet. Furthermore, from the viewpoint of stability in bubble formation and retention, it is particularly preferable that the narrowed portion further has a structure that narrows the narrowed portion in the horizontal direction in addition to the step structure.

[0045] Furthermore, a continuous phase liquid that easily wets the surface of the flow channel, i.e., a continuous phase liquid with low surface tension and / or viscosity, is preferred because it easily forms bubbles. Note that in technologies aimed at emulsion filling, a continuous phase liquid having the above liquid properties is generally used to generate and maintain the emulsion.

[0046] (Upstream of the narrowed area) In the flow channel upstream of the constriction, bubbles are formed / maintained near the constriction, and therefore bubbles with a shape that reflects the flow channel structure can be formed and maintained. In particular, the cross-sectional shape of the flow channel is related to the cross-sectional shape of the bubble and the shape of the thin layer between the flow channel wall and the bubble, so it is preferable to adjust the cross-sectional shape of the flow channel to an appropriate shape depending on the size of the droplet, etc.

[0047] (transition area) A "transition region" in which the cross-sectional area of the flow path gradually decreases can be provided between the narrowed portion and the flow path adjacent to the narrowed portion (particularly the upstream portion of the narrowed portion). Providing such a transition region may improve bubble retention.

[0048] For example, when viewed from above in a normal use state of the microchannel chip, the wall surfaces on both sides of the transition region form an angle α (α in Figure 2), and this angle α can form an angle of 30° to 150°, 45° to 125°, or 60° to 100°.

[0049] The manner in which the cross-sectional area of the channel is reduced in the transition region is not particularly limited. For example, when the microchannel chip is viewed from above in a normal use state, the wall surface of the transition region may be linear or curved, and the channel width may be reduced in this manner.

[0050] (Additional stenosis) In another embodiment of the bubble trap stop valve portion according to the present disclosure, the bubble trap stop valve portion has an additional constriction portion downstream of the constriction portion, where "downstream" means the downstream side when assuming the direction from the emulsion holding channel to the outlet, i.e., the outlet side.

[0051] Similar to the above-described narrowing portion, the "additional narrowing portion" is a portion of the bubble trap stop valve section where the flow path is narrowed, and is configured to hold bubbles upstream of the portion so as to suppress the movement of emulsion (particularly droplets) from the emulsion-holding flow path to the outlet. Similar to the narrowing portion, the "additional narrowing portion" is, for example, a portion in the bubble trap stop valve section where the flow path cross-sectional area is reduced (particularly abruptly). For the structure of the additional narrowing portion, please refer to the above description of the narrowing portion.

[0052] When the bubble trap stop valve portion has a narrowed portion and an additional narrowed portion (especially when the expansion portion described below exists between them), bubbles can be generated and maintained reproducibly in the bubble trap stop valve portion, and the bubble trap stop valve portion can function even better.

[0053] (extension) The bubble trap stop valve portion may have an expansion portion between the narrowed portion and the additional narrowed portion. The expansion portion is configured to be suitable for retaining bubbles, and has, for example, a larger flow channel cross section (and / or flow channel width) than the narrowed portion and the additional narrowed portion. Preferably, the expansion portion also has a larger flow channel height than the narrowed portion and the additional narrowed portion.

[0054] (Dimensions of each part of the bubble trap stop valve) For example, when the emulsion holding flow path has a width of 500 μm to 5 mm or 1 mm to 3 mm and a height of 50 μm to 300 μm or 100 μm to 200 μm, the "narrowed portion" and optional "additional narrowed portion" of the bubble trap stop valve portion can have a width of 10 μm to 300 μm or 50 μm to 150 μm, a height of 20 μm to 200 μm or 50 μm to 100 μm, and a length of 100 μm to 800 μm or 200 μm to 600 μm, and the optional "expanded portion" can have a width of 100 μm to 600 μm or 200 μm to 450 μm, a height of 50 μm to 300 μm or 100 μm to 200 μm, and a length of 300 μm to 1000 μm or 400 μm to 800 μm.

[0055] In addition, the cross-sectional area of the emulsion holding flow path (upstream portion 168 of the bubble valve portion) is sufficiently small (especially 2 mm 2 or less), it is possible to ensure a sufficient linear velocity of the flow of the continuous phase liquid from the continuous phase liquid holding section (which replenishes the continuous phase liquid), and therefore it may be possible to suppress the movement of the gas-liquid interface in a direction away from the discharge port (it may be possible to suppress the expansion of the bubbles).

[0056] In addition, the cross-sectional area of the emulsion holding flow path (upstream portion 168 of the bubble valve portion) is sufficiently large (especially 0.05 mm 2 In the above cases, a sufficient cross-sectional area for the bubbles can be ensured, and the linear velocity of the continuous phase liquid in the outflow direction is relatively reduced, so that the outflow of bubbles can be effectively suppressed. In order to suppress the outflow of bubbles, it is particularly preferable that the bubble valve has an expanding portion and an additional narrowing portion toward the downstream.

[0057] One exemplary embodiment of a bubble trap stop valve portion according to the present disclosure will now be described with reference to the schematic diagram of the exemplary embodiment in Figure 2. Figure 2 (and Figure 3 below) are schematic diagrams for ease of understanding and are not drawn to scale.

[0058] The bubble trap stop valve section 160 in Fig. 2 is configured by the terminal portion on the outlet side of the emulsion holding flow path 140 (see Fig. 1), and has a narrowed section 162, an additional narrowed section 164, and an expanded section 166 arranged therebetween. Note that the narrowed section 162 has a transition region 170 between it and the adjacent flow path (the upstream portion 168 of the bubble trap stop valve section). The bubble trap stop valve section in Fig. 2 has its flow path filled with the continuous phase of the emulsion, and has two bubbles A.

[0059] 2, the narrowed section 162 and the additional narrowed section 164 have flow path cross-sectional areas that are significantly smaller than the upstream section 168 of the bubble trap stop valve section. For example, in the narrowed section 162 and the additional narrowed section 164, the flow path cross-sectional areas are reduced to 1 / 4 to 1 / 400 times that of the upstream section 168. Also, in the narrowed section 162 and the additional narrowed section 164, the flow path width is reduced to 1 / 3 to 1 / 100 times that of the upstream section 168, and the flow path height is reduced to 3 / 4 to 1 / 10 times that of the upstream section 168.

[0060] The bubble trap stop valve unit 160 of FIG. 2 has an expansion section 166 between the narrowed section 162 and the additional narrowed section 164. The expansion section 166 has a larger flow path cross-sectional area than the narrowed section 162 and the additional narrowed section 164, and is suitable for retaining bubbles. For example, in the expansion section 166, the flow path cross-sectional area is increased by 4 to 100 times compared to the narrowed section 162 and the additional narrowed section 164. Also, for example, in the expansion section 166, the flow path width is increased by 2 to 25 times compared to the narrowed section 162 and the additional narrowed section 164, and the flow path height is increased by 4 / 3 to 10 times compared to the upstream section 168. Between the expansion section 166 and the narrowed section 162 and the additional narrowed section 164, respectively, there are preferably transition regions in which the flow path cross-sectional area gradually decreases.

[0061] In the bubble trap stop valve section 160 in Figure 2, the continuous phase (continuous phase liquid) in the emulsion passes through the bubble trap stop valve section due to capillary force and / or liquid level differential pressure and gradually flows out to the outlet, but the droplets in the emulsion (dispersed phase liquid) cannot pass through the thin layer of continuous phase liquid that exists between the bubbles A formed in the bubble trap stop valve section and the flow path wall surface, and so do not flow out to the outlet (droplets are present above in Figure 2, although not shown in Figure 2).

[0062] Figure 3 is a schematic cross-sectional view perpendicular to the horizontal plane of the bubble trap stop valve unit in Figure 2. H in Figure 3 indicates the height direction. As can be seen in Figure 3, the upper surfaces (ceiling portions) of the inside of the flow paths of the narrowed portion 162 and the additional narrowed portion 164 are lower than the upper surfaces of the inside of the flow paths adjacent to them. As described above, in this case, the buoyancy of the bubbles can be prevented from causing the bubbles to pass through the narrowed portion (and the additional narrowed portion), further improving the function of the bubble trap stop valve unit.

[0063] (Flow of continuous phase liquid in emulsion) As described above, the bubble trap stop valve portion has the effect of preventing the outflow of droplets while allowing some outflow of the continuous phase (continuous phase liquid).

[0064] Here, the bubbles formed in the bubble trap stop valve portion tend to expand over time due to evaporation of the continuous phase liquid, etc. Such bubble expansion can sometimes cause the gas-liquid interface to flow back upstream of the emulsion holding channel (towards the emulsion formation section), particularly under conditions such as heating. On the other hand, bubble expansion can also be desirable for suppressing the outflow of bubbles to the outlet of the bubble trap stop valve portion and for maintaining a thin layer of bubbles on the channel wall surface. Therefore, even in embodiments in which bubbles expand, it is preferable to be able to suppress the gas-liquid interface from flowing back upstream of the emulsion holding channel (towards the emulsion formation section).

[0065] Such backflow at the gas-liquid interface can be suppressed by maintaining the outflow of the continuous phase liquid in the emulsion toward the outlet. For example, in an embodiment in which a bubble trap stop valve is not provided and the emulsion continues to flow toward the outlet even after the liquid feed is stopped (after the external liquid feed driving force becomes zero), adding a bubble trap stop valve allows the continuous phase liquid in the emulsion to continue flowing toward the outlet through the bubble trap stop valve, thereby suppressing backflow at the gas-liquid interface. However, unlike the backflow at the air-emulsion interface in the absence of a bubble trap stop valve, backflow at the bubble-emulsion interface in the bubble trap stop valve is affected by the expansion of bubbles due to vapor pressure, and therefore, it is preferable to appropriately adjust the amount of backflow. For example, when the retained emulsion is heated (e.g., when adjusting the reaction temperature), the vapor pressure of the continuous phase liquid increases, which makes it easier for bubbles in the bubble trap stop valve to expand. Therefore, it may be preferable to perform the reaction under conditions that make it easier for the continuous phase liquid to flow toward the outlet.

[0066] (pressure loss resistance of flow path) Such backflow at the gas-liquid interface can be suppressed or avoided, for example, by adjusting the channel characteristics of the microchannel chip (e.g., the pressure drop resistance of the channel.) For example, it is preferable to reduce the pressure drop resistance of the channel from the continuous phase liquid holder to the bubble trap stop valve so that continuous phase liquid lost due to evaporation can be easily replenished from the continuous phase liquid holder.

[0067] Below, each component constituting the invention according to the present disclosure and its embodiments will be described in more detail.

[0068] <Microfluidic chip> The microchannel chip of the present disclosure has a dispersed phase liquid holder, a dispersed phase liquid flow path, a continuous phase liquid holder, a continuous phase liquid flow path, an emulsion forming section, an emulsion flow path, an emulsion holding flow path, and an outlet. The dispersed phase liquid holder is connected to the emulsion forming section via the dispersed phase liquid flow path, the continuous phase liquid holder is connected to the emulsion forming section via the continuous phase liquid flow path, the emulsion forming section is connected to the emulsion holding flow path via the emulsion flow path, and the emulsion holding flow path is connected to the outlet. An end portion of the emulsion holding flow path on the outlet side constitutes a bubble trap stop valve unit.

[0069] The dispersed phase liquid holding section, dispersed phase liquid flow path, continuous phase liquid holding section, continuous phase liquid flow path, emulsion forming section, emulsion flow path, emulsion holding flow path (having a bubble trap stop valve section), and discharge port are fluidly connected to each other and form a single flow path structure as a whole.

[0070] In particular, this flow path structure is capable of being connected to the external atmosphere (particularly the external air) only via the dispersed phase liquid holding portion, the continuous phase liquid holding portion, and the outlet.

[0071] The microchannel chip according to the present disclosure has, for example, a substrate and an upper structure disposed on the substrate. Preferably, the upper structure has a channel structure, i.e., a dispersed phase liquid holder, a dispersed phase liquid flow path, a continuous phase liquid holder, a continuous phase liquid flow path, an emulsion forming section, an emulsion flow path, an emulsion holding flow path, and an outlet. The substrate may be made of glass. The upper structure may be made of resin. The microchannel chip can be fabricated, for example, by bonding a resin upper structure to a glass substrate that constitutes the bottom of the microchannel chip.

[0072] The size (width, depth, etc.) of the channels constituting the microchannel chip can be appropriately determined taking into consideration the volume of the desired droplets, and in particular the reaction mode of the target substance. For example, when the target substance is a nucleic acid such as DNA or RNA and the reaction of the target substance is a digital amplification reaction of the nucleic acid (amplification reaction at the single molecule level), droplets on the order of pL or nL must be produced, and therefore the width and depth of the channel around the emulsion-forming portion are preferably in the range of 0.1 μm to 1000 μm, particularly 1 μm to 300 μm.

[0073] It is preferable that the channels and each device constituting the microchannel chip have a channel wall surface with low affinity for the dispersed phase liquid. The microchannel chip may be fabricated using a material with low affinity for the dispersed phase liquid, or the portions corresponding to the channel wall surface may be surface-treated with a material with low affinity for the dispersed phase liquid. For example, the microchannel chip may be fabricated using a polymer material such as PDMS (polydimethylsiloxane), acrylic, cycloolefin polymer, or PTFE (polytetrafluoroethylene), and the channel wall surface may be surface-treated with a hydrocarbon-based silanizing agent, a fluorocarbon-based silanizing agent, or the like.

[0074] The wall surfaces of the dispersed phase liquid flow paths can also be made of inorganic and / or organic materials that do not contain or are not treated with fluorine compounds. In this case, the stability of emulsion production can be further improved by suppressing the amount of continuous phase liquid remaining in the dispersed phase liquid flow paths.

[0075] Microchannel chips can be fabricated by combining techniques that use molds, such as molding or embossing, which enable accurate and easy fabrication of channel structures, or techniques commonly used by those skilled in the art, such as photolithography, soft photolithography, wet etching, dry etching, nanoimprinting, laser processing, direct electron beam writing, additive manufacturing (AM), and machining.

[0076] Materials used to fabricate microchannel chips include polymer materials such as PDMS (polydimethylsiloxane) and acrylic, metal materials such as stainless steel, glass, silicone, and ceramics. Among these, polymer materials allow the channel itself to be fabricated inexpensively and are easily made disposable. Therefore, it is preferable to use polymer materials at least in part.

[0077] (Dispersed phase liquid holding section) The dispersed phase liquid holding section is a section that holds the dispersed phase liquid, which is a material for producing an emulsion. The dispersed phase liquid holding section may be, for example, a hole and / or well that extends vertically when the microchannel chip is in use, and the dispersed phase liquid can be supplied to and held in the hole and / or well. The dispersed phase liquid holding section may be composed of, for example, a hole and / or well with a diameter of 0.1 mm to 20 mm. When the dispersed phase liquid holding section is composed of a hole and a well, the well that extends vertically can be connected to the dispersed phase liquid flow path via the hole that also extends vertically.

[0078] FIG. 4 is a cross-sectional schematic diagram showing one embodiment of a dispersed phase liquid holding section according to the present disclosure. FIG. 4 is a schematic diagram for ease of understanding and is not drawn to scale. In FIG. 4, H represents the height direction, and L represents the length direction. The dispersed phase liquid holding section 400 shown in FIG. 4 has a well 41 and a hole 43. The well 41 is connected to the dispersed phase liquid flow path 115 via the hole 43. The well 41 and the hole 43 extend in the height direction (here, the vertical direction). Reference numeral 45 in FIG. 4 indicates an extension portion, which is a joint between the bottom surface of the well and the hole. In the embodiment of FIG. 4, the bottom surface of the well 41 extends perpendicular to the extension direction of the hole 43 (particularly the side wall of the hole 43 near the extension portion 45).

[0079] It is preferable to further optimize the dispersed phase liquid holder as follows.

[0080] The driving forces during liquid transfer are mainly liquid transfer pressure (positive pressure, negative pressure) and / or liquid level differential pressure (hydrostatic pressure) and / or capillary force. Of these, capillary force is determined by the difference in surface tension between the gas-liquid interface (and solid-liquid interface) downstream of the flow channel (generally, the emulsion-holding flow channel) during liquid transfer and the gas-liquid interface (and solid-liquid interface) within each holding section. In other words, the liquid transfer speed within the flow channel changes depending on the shape of the gas-liquid interface (wetting behavior on the wall surface) in each holding section. Therefore, in emulsion filling methods, in which liquid transfer (emulsion generation and retention) is performed by controlling the gas-liquid interface within the flow channel, the shape of each holding section is an important factor for achieving stable liquid transfer. In particular, in a typical emulsion generation chip, stable emulsion generation is possible by making at least the wall surface of the emulsion-forming section a surface with low affinity for the dispersed phase liquid (a hydrophobic surface if the dispersed phase liquid is an aqueous liquid). Therefore, if the inner surface of the channel is not treated or a separate substrate is not used, from the perspective of chip manufacturing costs, the wall surface of the dispersed phase liquid holding section also generally has a surface with low affinity for the dispersed phase liquid. In this case, the change in the interfacial shape in the dispersed phase liquid holding section increases the amount of change in surface tension at the gas-liquid interface, which has a greater impact on liquid transfer.

[0081] From the viewpoint of reducing the change in capillary force as described above, holes and / or wells that are substantially free of discontinuities on the wall surface and extend vertically are preferred. Furthermore, when the remaining amount of dispersed phase liquid in the dispersed phase liquid holding section decreases, the gas-liquid interface substantially contacts the bottom surface of the well in the holding section, which tends to change the shape of the interface. To reduce this effect, the diameter of the holes and / or wells directly fluidically connected to the flow path is preferably small, for example, 5 mm or less, more preferably 2 mm or less, and particularly preferably 1 mm or less. On the other hand, the amount of dispersed phase liquid retained can be increased by expanding the diameter of the holes or wells directly fluidically connected to the dispersed phase liquid flow path. However, in this case, if the gas-liquid interface is located near the expanded region, the shape of the gas-liquid interface is likely to change. Therefore, in one preferred embodiment, the shape of the expanded region can be adjusted, and / or the height of the holes or wells directly fluidically connected to the flow path to the expanded region can be reduced, for example, to 3 mm or less, more preferably 1 mm or less, and particularly preferably 0.5 mm or less. Furthermore, when the supply amount of the dispersed phase liquid is small and / or the external surface of the chip around the dispersed phase liquid holding portion has low affinity for the dispersed phase liquid, and the external liquid delivery driving force is the application of negative pressure to the outlet, it is preferable to use a configuration with only a hole and supply the dispersed phase liquid to the hole so that it forms a hemispherical shape, because this minimizes the contact area with the chip wall surface and prevents changes in the shape of the gas-liquid interface even when the remaining amount of dispersed phase liquid becomes small.

[0082] When positive pressure is used as the external liquid transport driving force as described below, the dispersed phase liquid holder is preferably suitable for connection to a positive pressure source, and in this case, it is preferable that the dispersed phase liquid holder has resistance to the applied pressure.

[0083] (Dispersed phase liquid flow path) The dispersed phase liquid flow path connects the dispersed phase liquid holding section and the emulsion forming section. The dispersed phase liquid flow path is configured so that the dispersed phase liquid passes through it. It is also possible to assume that in addition to the dispersed phase liquid, the continuous phase liquid passes through the dispersed phase liquid flow path.

[0084] The dimensions of the dispersed phase liquid flow path can be appropriately set depending on the type and properties of the dispersed phase liquid used. The dispersed phase liquid flow path can have a width of, for example, 10 to 500 μm or 50 to 200 μm, and a length of 1 mm to 500 mm or 10 to 200 mm. The dispersed phase liquid flow path can also have a flow path height of 1 to 200 μm or 10 to 100 μm. The dispersed phase liquid flow path may be bent at one or more locations, or may have a serpentine shape.

[0085] The microchannel chip according to the present disclosure can have two or more dispersed phase liquid holders and two or more dispersed phase liquid channels corresponding thereto.

[0086] In particular, the microchannel chip according to the present disclosure has a first dispersed phase liquid holder and a second dispersed phase liquid holder, and the dispersed phase liquid flow path includes a first dispersed phase liquid flow path connected to the first dispersed phase liquid holder, a second dispersed phase liquid flow path connected to the second dispersed phase liquid holder, and a dispersed phase liquid junction. The first dispersed phase liquid flow path and the second dispersed phase liquid flow path are each connected to an emulsion formation part via the dispersed phase liquid junction.

[0087] By using two or more dispersed phase liquid holders, for example, a reaction liquid containing an analytical sample and a reaction liquid containing a detection reagent can be supplied separately to the microchannel chip, and the two can be prevented from mixing until just before droplets are generated, which is preferable because it allows for better control of the timing of reaction initiation.

[0088] (Continuous phase liquid holding section) The continuous phase liquid holding section is a section that holds the continuous phase liquid that is the material for producing the emulsion.

[0089] The structure of the continuous phase liquid holding portion is not particularly limited as long as it can hold the continuous phase liquid. The continuous phase liquid holding portion may be a hole or well, for example, a hole or well extending in the vertical direction, and the continuous phase liquid can be supplied to and held in this hole or well. The continuous phase liquid holding portion may be, for example, a hole or well with a diameter of 0.1 mm to 20 mm.

[0090] In addition, since the continuous phase liquid generally has low surface tension and viscosity, the change in surface tension associated with a change in the interfacial shape in the continuous phase liquid holding section is small. Therefore, the shape of the continuous phase liquid holding section does not significantly affect the liquid transfer. In addition, in the present invention, when, for example, an emulsion is held and a detection reaction or the like is performed, a sufficient amount of continuous phase liquid is supplied so that the continuous phase liquid in the continuous phase liquid holding section does not run out. This prevents the remaining amount of continuous phase liquid in the holding section from becoming small during liquid transfer, making the interfacial shape prone to change. Therefore, the shape of the continuous phase liquid holding section is unlikely to significantly affect the liquid transfer.

[0091] When positive pressure is used as the external liquid transport driving force, the continuous phase liquid holder is preferably suitable for connection to a pressure source, and in this case, it is preferable that the continuous phase liquid holder has resistance to the applied pressure.

[0092] (Continuous phase liquid flow path) The continuous phase liquid flow path connects the continuous phase liquid holding section and the emulsion forming section, and is configured so that the continuous phase liquid passes through it.

[0093] The dimensions of the continuous phase liquid flow path can be appropriately set depending on the type and properties of the continuous phase liquid used. The continuous phase liquid flow path can have a width of, for example, 10 to 500 μm or 50 to 200 μm, and a length of 1 mm to 500 mm or 10 to 200 mm. The continuous phase liquid flow path can also 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 at least a partially serpentine shape.

[0094] The micro-channel chip can have two or more continuous phase liquid channels. In particular, the micro-channel chip according to the present disclosure has a first continuous phase liquid channel and a second continuous phase liquid channel, which respectively connect the continuous phase liquid holding section and the emulsion forming section.

[0095] 1 , the continuous-phase liquid flow path 111 is composed of two flow paths (a first continuous-phase liquid flow path 112 and a second continuous-phase liquid flow path 113). These two flow paths 112 and 113 face each other in the emulsion-forming section 120 and are substantially perpendicular to the dispersed-phase liquid flow path (more precisely, the dispersed-phase liquid junction 116) connected to the emulsion-forming section 120. In the embodiment of FIG. 1 , the first continuous-phase liquid flow path 112 and the second continuous-phase liquid flow path 113 have substantially the same structure and flow path length, thereby allowing the continuous-phase liquids moving through the respective flow paths to travel at substantially the same speed. Furthermore, in order to suppress mixing of the dispersed-phase liquids before emulsion formation as described above, the length of the flow path connecting the downstream side of the dispersed-phase liquid junction 116 and the emulsion-forming section 120 is preferably relatively short (e.g., 3 mm or less, more preferably 0.5 mm or less), and the dispersed-phase liquids preferably maintain separate laminar flow states within the flow paths.

[0096] (emulsion forming part) The emulsion forming unit is configured to generate an emulsion. The emulsion forming unit receives a dispersed phase liquid and a continuous phase liquid via a dispersed phase liquid flow path and a continuous phase liquid flow path, respectively. The emulsion forming unit is also connected to the emulsion flow path, and the emulsion generated in the emulsion forming unit is sent to the emulsion flow path.

[0097] The emulsion-forming section may have one or more openings that open to the dispersed phase liquid flow path and one or more openings that open to the continuous phase liquid flow path, and may also have one or more openings that open to the emulsion flow path.

[0098] The emulsion-forming section will be described with reference to the exemplary embodiment of FIG. 1. In the emulsion-forming section 120 of FIG. 1, a continuous-phase liquid flow path 111, consisting of two flow paths 112 and 113, and a dispersed-phase liquid flow path (more precisely, a dispersed-phase liquid junction 116) are substantially perpendicular to each other. During application of an external liquid-transport driving force, the continuous-phase liquid flows into the emulsion-forming section 120 from two substantially opposite directions, and the dispersed-phase liquid flows into the emulsion-forming section 120 in a direction substantially perpendicular to the inflow direction of the continuous-phase liquid. As a result, droplets dispersed in the continuous phase (i.e., an emulsion) are generated in the emulsion-forming section 120. The emulsion thus generated passes through the emulsion flow path 130 and enters the emulsion-holding flow path 140, which is filled with gas.

[0099] The emulsion-forming unit can be a flow path that utilizes a common droplet generation method, such as T-junction, flow-focus, co-flow, or step-emulsification. To rapidly generate emulsion, multiple emulsion-forming units may be arranged in parallel. Furthermore, the unit may be equipped with a serpentine flow path to agitate the droplets in the emulsion.

[0100] (Emulsion flow path) The emulsion flow path connects the emulsion formation section and the emulsion holding flow path. The portion of the emulsion flow path adjacent to the emulsion formation section is preferably arranged to face the dispersed phase liquid flow path (particularly the dispersed phase liquid junction). Figure 1 shows an emulsion flow path of such an embodiment. In Figure 1, the portion of the emulsion flow path adjacent to the emulsion formation section is substantially perpendicular to the inlet portion of the continuous phase liquid flow path to the emulsion formation section. In the case of Figure 1, when an emulsion is generated, the dispersed phase liquid flowing from the dispersed phase liquid flow path into the emulsion formation section becomes droplets and enters the emulsion flow path without changing its flow angle.

[0101] The emulsion flow path may have an expanded width and / or height downstream (toward the outlet) of the emulsion-forming section, and / or may be serpentine, which is preferred because it can promote agitation within the droplets.

[0102] (Emulsion holding channel) The emulsion holding flow path is connected to the emulsion forming part via an emulsion flow path and has the function of holding the emulsion produced in the emulsion forming part. The emulsion holding flow path is also connected to a discharge port.

[0103] The width and length of the emulsion-holding channel can be set appropriately according to the volume and number of droplets to be held. For example, it may be a wide, simple channel with approximately the same width and length, or a single, continuous, long channel arranged in a serpentine or spiral pattern, or branched, linear channels arranged in parallel.

[0104] In the present invention, the cross section of the emulsion holding channel is preferably circular, semicircular, elliptical, convex, concave, rectangular or trapezoidal, since the centers of the droplets tend to flow along the center of the channel.

[0105] In emulsion filling, emulsion generation and retention are generally achieved by controlling the movement of the gas-liquid interface within the emulsion-holding channel. Therefore, a straight, unbent channel with a constant cross-sectional shape is desirable to maintain the shape of the gas-liquid interface during flow (i.e., the shape changes little even as the flow moves). However, excessively increasing the channel width relative to the channel height to increase the number of detectable droplets can make it difficult to consistently manufacture chips with the intended channel structure. Furthermore, the bottom and / or top surfaces of the channel can deform, potentially changing the height of the channel region far from the sides due to factors such as the flow pressure or the pressure applied to the chip, adversely affecting measurements (roof collapse). To address this issue, for example, the ratio of channel width to channel height is preferably 100 or less, more preferably 50 or less, 25 or less, and particularly preferably 10 or less (when no pillars are present, as described below). Alternatively, pillars can be provided in the center of the channel, so that the ratio of the spacing between the pillars and / or the spacing between the pillars and the side of the channel to the channel height is, for example, 100 or less, more preferably 50 or less, 25 or less, and particularly preferably 10 or less. On the other hand, when performing batch detection processing using an image sensor or the like, it is preferable for the emulsion-holding channel to be densely packed on a horizontal surface (e.g., in a shape close to a square or circle) because this increases the number of droplets detected. Therefore, continuous single long channels with the same channel cross-sectional shape can be arranged in a serpentine or spiral pattern, or branched linear channels can be arranged in parallel. In this case, the presence of bends makes it easy for the interface shape to change during liquid transfer, but this effect can be reduced by adjusting the cross-sectional shape of the bends.

[0106] In one embodiment, it is intended that a detection process be performed on the retained emulsion, i.e., the emulsion retained in the emulsion-retaining channel can be optionally subjected to the detection process described below.

[0107] Preferably, the emulsion holding channel is configured so that most or all of the emulsion held therein does not come into contact with the atmosphere outside the micro-channel chip (particularly the external atmosphere). Preferably, of the droplets held in the emulsion holding channel, the droplets that are the subject of the detection process do not come into contact with the atmosphere outside the micro-channel chip (particularly the external atmosphere).

[0108] Preferably, the emulsion holding channel is suitable for carrying out a detection process on the emulsion held in the emulsion holding channel.

[0109] Preferably, the emulsion-holding flow path is configured so that the detection process can be performed on emulsions that are not exposed to the outside atmosphere. More specifically, for example, a structure that isolates the emulsion from the outside atmosphere is present between the held emulsion and the detection means. This structure is made, for example, of a light-transmitting material. In this case, it should be noted that among the emulsions held in the emulsion-holding flow path, emulsions that are not subject to the detection process, for example, emulsions located at the discharge end of the emulsion-holding flow path, may be exposed to the outside atmosphere.

[0110] Preferably, the flow channel volume of the emulsion holding flow channel is equal to or greater than the total volume of droplets generated in the emulsion forming section (particularly droplets to be detected in the detection process), and / or the flow channel volume of the emulsion holding flow channel is equal to or greater than 1 μL, 5 μL, or 10 μL. Such an emulsion holding flow channel allows the detection process to be carried out efficiently. The upper limit of the flow channel volume of the emulsion holding flow channel may be, for example, 1000 μL or less.

[0111] Preferably, the emulsion-holding channel has a channel volume capable of holding 500 or more, 1000 or more, 2500 or more, 5000 or more, or 10000 or more, and / or 100000 or less, 80000 or less, 60000 or less, or 40000 or less droplets with an average volume of 0.1 nL to 10 nL, particularly 0.3 nL to 3 nL. Detection processing can be performed on these droplets held in the emulsion-holding channel.

[0112] The average volume of droplets can be calculated by acquiring bright-field images using an image acquisition device such as a digital camera, and calculating the average volume of droplets in the acquired images based on the following formula:

[0113] The volume of a spherical droplet and the volume of a disk-shaped droplet (V drop and V disk [nL]) are expressed by the following formulas (1) and (2), respectively. drop , D disk are the diameters of spherical and disk-shaped droplets, respectively, when droplets held in the emulsion holding channel are observed from above under normal use of the microchannel chip. Also, in equation (2), h is the channel height of the emulsion holding channel.

[0114]

number

[0115]

number

[0116] Particularly preferably, the "channel height" of the emulsion-holding channel is adjusted so that droplets held in the emulsion-holding channel do not overlap each other in the vertical direction (i.e., a single droplet layer is formed) when the microchannel chip is in use. When a detection process is performed using such an emulsion-holding channel, detection accuracy is further improved. Note that the "channel height" usually refers to the length of the channel in the vertical direction (perpendicular direction) when the microchannel chip is in use.

[0117] Preferably, the height of the emulsion-holding channel has a dimension corresponding to the diameter of the droplets to be detected in the detection process, and for example, the channel height is preferably 1 / 10 to 10 times, 1 / 4 to 4 times, or 1 / 2 to 2 times the diameter of the droplets. The height of the emulsion-holding channel may be 1 / 4 times or less the width of the channel. The width of the channel is usually the length in the horizontal direction perpendicular to the length of the channel when the microchannel chip is in use. The diameter of the droplets can be measured in the width direction of the channel.

[0118] In the emulsion filling method, the amount of emulsion filling the emulsion-holding channel depends on the flow rate ratio of the continuous phase liquid to the dispersed phase liquid in the emulsion formation section. Therefore, for example, if the flow rate ratio of the continuous phase liquid to the dispersed phase liquid is increased to stabilize emulsion production, it is preferable to design the channel height to be larger than usual in order to pack the droplets tightly in the horizontal direction. For example, if 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 made 2 to 4 times the diameter of the droplets.

[0119] (Exhaust port) The microchannel chip has an outlet connected to the emulsion-holding channel. As will be described later, the outlet can also function as a negative pressure source connection for applying negative pressure to the microchannel chip.

[0120] When negative pressure is used for the delivery of the liquid, the outlet is preferably adapted to be connected to a negative pressure source, and in this case, the outlet is preferably resistant to the applied pressure.

[0121] The outlet is located downstream of the bubble trap stop valve portion.

[0122] <Using microfluidic chips> The present disclosure further includes the use of a microchannel chip according to the present disclosure.

[0123] One embodiment of the present disclosure is a method for generating and maintaining an emulsion by supplying a dispersed phase liquid and a continuous phase liquid to a microchannel chip according to the present disclosure, comprising the steps of: Supplying a dispersed phase liquid to a dispersed phase liquid holding section; Supplying a continuous phase liquid to a continuous phase liquid holding section; and generating an emulsion containing droplets composed of a dispersed phase liquid and a continuous phase composed of the continuous phase liquid in an emulsion forming section by an external liquid feeding driving force, and transporting the emulsion thus generated through an emulsion flow path to an emulsion holding flow path filled with a gas; Including, stopping the external liquid delivery driving force before the emulsion completely fills the emulsion holding channel; It is characterized by:

[0124] According to this method, the external liquid-transfer driving force is stopped before the emulsion holding flow path is completely filled with the emulsion, thereby ensuring bubble formation in the bubble trap stop valve portion. In particular, the external liquid-transfer driving force can be stopped before the emulsion holding flow path is completely filled with the emulsion and before the emulsion reaches the bubble trap stop valve portion (particularly, just before or when it reaches the bubble trap stop valve portion).

[0125] Furthermore, the method according to the present disclosure uses a microchannel chip having a bubble trap stop valve, so that droplets in the emulsion can be stably held in the emulsion holding channel without having to seal the channel of the microchannel chip after the external liquid delivery driving force is stopped.

[0126] Preferably, after the external liquid delivery driving force is stopped, while droplets are held in the emulsion holding channel, at least one of the outlet, the dispersed phase liquid holding section, and the continuous phase liquid holding section is not sealed, and in particular is substantially open to the atmosphere.

[0127] Furthermore, particularly after the external liquid delivery driving force is stopped, while a detection process is being performed on the droplets held in the emulsion holding flow path (more particularly, while a reaction is being carried out in the droplets and / or while signal detection is being carried out), at least one of the outlet, the dispersed phase liquid holding section, and the continuous phase liquid holding section is not sealed, and particularly is substantially open to the atmosphere.

[0128] (Supply of dispersed phase liquid) The method according to the present disclosure includes providing a dispersed phase liquid to a dispersed phase liquid holding portion.

[0129] To supply the dispersed phase liquid to the dispersed phase liquid holder, a separate container (phase liquid holder) may be used. From the viewpoint of preventing the liquid from leaking during storage and operation, such a container is preferably completely or variably sealed while holding the dispersed phase liquid.

[0130] The supply of the dispersed phase liquid (and / or the continuous phase liquid) can be carried out by a dispensing means. The use of a dispensing means is preferred in that it can reduce the amount of residual dispersed phase liquid and reduce the measurement time and / or contamination between reagents (dispersed phase liquids).

[0131] For example, a dispensing means can be used to drop each phase liquid into each holding section or to introduce each phase liquid along the wall surface of each holding section. This is particularly advantageous when liquid delivery is performed under negative pressure. Conventional supply methods, particularly those in which tubes or manifolds are fluidly connected (sealed) to each holding section to simultaneously introduce liquid into each holding section and apply liquid delivery pressure, make it difficult to accurately control the progress of the dispersed phase liquid and the continuous phase liquid due to unexpected pressure fluctuations during connection and the time required for the pressure to stabilize. This makes it difficult to bring the dispersed phase liquid and the continuous phase liquid into contact without blocking the flow path. In contrast, when a dispensing means is used and liquid delivery is performed under negative pressure, pressure fluctuations that occur when connecting a phase liquid supply device and a pressure source to each holding section are eliminated. This makes it possible to more accurately control the movement of each phase liquid and the contact between the dispersed phase liquid and the continuous phase liquid before applying negative pressure, thereby more effectively suppressing the generation of bubbles.

[0132] The dispensing means is preferably one that does not cause pressure fluctuations in the holding parts. The dispensing means may be, for example, a pipette. Preferably, the dispensing means (particularly, the liquid outlet constituting the dispensing means) is not fluidically connected (sealedly connected) to each holding part, but is spatially separated.

[0133] For example, the dispensing means may be a mechanism including a pump, an actuator, and a pipette. Preferably, the pump sucks up each phase liquid held in a separate container, moves the tip of the pipette to each holding section using the actuator, and then pushes each phase liquid into each holding section using the pump. In addition, it is preferable that parts of the pipettes or other devices that come into contact with each phase liquid are removable and replaceable after each use, since this helps prevent contamination. Furthermore, it is preferable to use the pump of the dispensing means as a liquid delivery means, since this simplifies the device configuration. Furthermore, if repeated use is intended, the phase liquids may be added using a dispensing means including a disposable pipette, or they may be added from a 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 shared line leading to the connection to the microchannel chip to prevent contamination of the dispersed phase liquid with the continuous phase liquid. Alternatively, a dispensing means that does not include a pipette may be used, in which each liquid is added (dropped) directly from a container holding the phase liquid to a holding section using an external force (e.g., a means of forcing the liquid out of the container by rupturing a thermocompression-bonded portion of the container using pressure). Furthermore, when carrying out a TRC reaction or a PCR reaction, for example, a dispensing means may be used in combination as a means for purifying or preparing an aqueous solution sample.

[0134] (Continuous phase liquid supply) The method according to the present disclosure includes providing a continuous phase liquid to a continuous phase liquid holding portion.

[0135] The supply of the continuous phase liquid can be carried out using a separate container and / or a dispensing means, as described above with respect to the supply of the dispersed phase liquid. For example, the continuous phase liquid can be dropped into the holding section using a dispensing means, or the continuous phase liquid can be introduced along the wall surface of the holding section. For details about the separate container and the dispensing means, please refer to the above description regarding the supply of the dispersed phase liquid.

[0136] (Maintaining the volume of the continuous phase liquid) As described above, in the present invention, the continuous phase liquid in the emulsion continues to flow out to the outlet through the bubble trap stop valve, thereby suppressing backflow at the gas-liquid interface. On the other hand, as described above, backflow at the bubble-emulsion interface at the bubble trap stop valve is different from backflow at the air-emulsion interface when there is no bubble trap stop valve, and is therefore affected by the expansion of bubbles due to vapor pressure, so it is preferable to adjust it appropriately.

[0137] In one embodiment of the present disclosure, the amount of continuous phase liquid supplied to the continuous phase liquid holding section is adjusted so that the continuous phase liquid remains in the continuous phase liquid holding section for at least a certain period of time after the external liquid delivery driving force is stopped.

[0138] Although there is no intention to be limited by theory, in this case, it is believed that backflow of the gas-liquid interface in the emulsion holding channel is suppressed or avoided by balancing the expansion pressure of the bubbles formed in the bubble trap stop valve portion with the capillary force and / or liquid level differential pressure acting on these bubbles via the continuous phase liquid held in the continuous phase liquid holding portion. Note that, under conditions where vaporized continuous phase liquid is likely to leak, it is preferable to design the system so that the decrease in continuous phase liquid from the channel is compensated for by the continuous phase liquid from the continuous phase liquid holding portion.

[0139] Here, the "certain time" can be set appropriately depending on the use of the emulsion held in the microchannel chip, and in particular, can be set depending on the detection process to be performed on the held emulsion. Specifically, for example, when a nucleic acid amplification reaction and fluorescence detection are performed in the held droplets, the "certain time" can be set to a time corresponding to the sum of the time for the amplification reaction and the time for fluorescence detection (for example, 10 minutes to 2 hours).

[0140] (liquid transfer) The external liquid delivery driving force provides a driving force for producing an emulsion from the dispersed phase liquid and the continuous phase liquid in the emulsion formation section. The external liquid delivery driving force also provides a driving force for transporting the produced emulsion to the emulsion holding channel. The external liquid delivery driving force may be the application of negative pressure to the outlet or positive pressure to the dispersed phase liquid holding section and the continuous phase liquid holding section. From the standpoint of simplicity of the device, it is preferable to maintain each holding section at normal pressure while applying negative pressure to the outlet, and to maintain the outlet at normal pressure while applying positive pressure to the dispersed phase liquid holding section and the continuous phase liquid holding section. However, the pressure may be controlled to an emergency pressure state to stabilize the liquid delivery under negative and / or positive pressure and / or to double as a sealing operation for holding the emulsion.

[0141] When negative pressure is applied to the discharge port, the dispersed phase liquid holding portion and the continuous phase liquid holding portion can be open to the external atmosphere (particularly the external atmosphere). Similarly, when positive pressure is applied to the dispersed phase liquid holding portion and the continuous phase liquid holding portion, the discharge port can be open to the external atmosphere (particularly the external atmosphere).

[0142] When negative pressure is applied, for example, a pressure tank or a syringe pump can be used to suck fluid (for example, gas or continuous phase liquid) in the channel of the microchannel chip through the outlet.

[0143] When a pressure tank is used as a negative pressure source, the volume of the pressure tank is preferably larger than the sum of the volume of the channel from the outlet to the pressure tank and the volume of the channel in the microchannel chip. The pressure tank may also be designed to be open to the outside atmosphere (particularly the outside air).

[0144] When applying positive pressure, for example, pressure can be applied to the fluid (gas, dispersed phase liquid, and / or continuous phase liquid) in the channel of the microchannel chip via the dispersed phase liquid holder and the continuous phase liquid holder using a pressure application means.

[0145] In addition, by using a monitoring means for monitoring the pressure value of the applied negative or positive pressure, it is possible to check the liquid transfer state, for example, whether an emulsion is being generated without any problems.

[0146] (Fluid delivery by negative pressure) In one embodiment of the present disclosure, negative pressure is used as the external liquid transfer driving force, i.e., in one embodiment of the present disclosure, an emulsion is generated in the emulsion forming section by applying negative pressure to the outlet, and the generated emulsion is transported through the emulsion flow path to the emulsion holding flow path filled with gas.

[0147] Negative pressure liquid transfer is preferable because it can simplify the required equipment compared to positive pressure liquid transfer.

[0148] Furthermore, when using positive pressure liquid transfer, particularly when applying positive pressure to each holding section via gas, it is necessary to add the dispersed phase liquid and the continuous phase liquid to each holding section before sealingly connecting the liquid transfer means to each holding section. However, this can easily result in unnecessary external pressure during connection. In particular, when using a highly flexible substrate such as silicone rubber, the microchannel chip may deform during connection, potentially deforming the channel cross section. Furthermore, using a liquid with low viscosity and surface tension as the continuous phase liquid enables stable and rapid droplet generation using shear forces in the emulsion formation section. However, liquids with these physical properties are prone to leakage if the sealing is incomplete, making it difficult to apply appropriate pressure. Furthermore, introducing the continuous phase liquid and / or the dispersed phase liquid after sealing and connecting the sections can reduce the liquid transfer and leakage problems caused by unnecessary external pressure during connection. However, this is only an example of a method for simultaneously introducing the liquid and pressure transferring the liquid, and it can easily reduce the reproducibility of the operation of contacting the dispersed phase liquid and the continuous phase liquid.

[0149] In contrast, negative pressure liquid transfer has the advantage that the liquid transfer means can be connected before the dispersed phase liquid and the continuous phase liquid are added, so that pressure at the time of connection is not applied to the dispersed phase liquid and the continuous phase liquid, and also has the advantage that it is possible to avoid the need to seal the continuous phase liquid holding section that holds the continuous phase liquid, which has low viscosity and surface tension.

[0150] Furthermore, when using a microchannel chip with low channel pressure drop resistance, applying positive pressure to each holding section may result in the dispersed phase liquid or continuous phase liquid unintentionally entering the channel due to unnecessary pressure when connecting to the liquid delivery means.

[0151] In contrast, the method of applying negative pressure to the outlet has the advantage that unnecessary pressure is not applied because the outlet can be connected to the liquid delivery means before the dispersed phase liquid and the continuous phase liquid are added.

[0152] In one embodiment of the method according to the present disclosure, a negative pressure control means is fluidly connected to the outlet, the negative pressure control means being composed of a negative pressure source, a connection part, and a valve, the negative pressure source being controlled to a constant negative pressure, and the valve being disposed between the negative pressure source and the connection part. The negative pressure control means can be connected to the outlet via the connection part.

[0153] According to this aspect, the negative pressure can be applied or stopped instantaneously by opening or closing the valve, which allows for more accurate control of the timing of applying or stopping the negative pressure. In particular, according to this aspect, when the negative pressure is stopped before the emulsion holding flow path is completely filled with emulsion, the timing of stopping the negative pressure can be more accurately controlled. Therefore, according to this aspect, bubble formation in the bubble trap stop valve can be more reliably performed.

[0154] There are no particular limitations on the specific form of the valve. From the viewpoint of preventing backflow when liquid transfer is stopped, it is preferable that the valve be capable of suppressing pressure fluctuations in the flow channel when opening and closing, for example, one with a relatively slow opening and closing operation. The valve may be, for example, a three-way valve, and may be capable of connecting the microchannel chip to either a negative pressure source (e.g., a pressure tank) or the external atmosphere (particularly the external atmosphere).

[0155] In one embodiment of the method according to the present disclosure, a negative pressure control means is fluidly connected to the outlet before the dispersed phase liquid and the continuous phase liquid come into contact, which may allow negative pressure to be applied quickly after the dispersed phase liquid and the continuous phase liquid come into contact, thereby suppressing premature droplet generation and further improving droplet uniformity.

[0156] (Liquid transfer in emulsion filling method) In the emulsion filling method, when the liquid supply is stopped, the gas-liquid interface is often present within the flow channel or near the flow channel opening of the outlet, and the sudden pressure fluctuation when the liquid supply is stopped (air flowing into the outlet in the case of negative pressure liquid supply) causes the gas-liquid interface to flow back inside the flow channel, which is likely to have an adverse effect on emulsion retention (droplets flowing out of the emulsion-retaining flow channel, or droplets agglomerating and coalescing near the gas-liquid interface).

[0157] Additionally, in the case of negative pressure liquid transfer, air flows into the outlet when liquid transfer is stopped, which makes it easier for pressure to be applied to the gas-liquid interface, and if a highly flexible material such as silicone rubber (PDMS) is used for the substrate, or if the substrate is extremely thin (about 1 mm or less for COC), the flow path is more likely to deform during negative pressure liquid transfer, and the force that restores this deformation when liquid transfer is stopped makes it more likely for backflow to occur. (In other words, in the center of the flow path far from the side walls of the flow path, the top and bottom surfaces bend in a direction that reduces the cross-sectional area of the flow path (roof collapse). As mentioned above, it is preferable for the width of the emulsion-holding flow path to be large relative to the height of the flow path, so this is thought to have a particularly large impact.)

[0158] In order to suppress such backflow when liquid supply is stopped, it is preferable to reduce the liquid supply pressure. For example, the pressure applied to the microchannel chip by the external liquid supply driving force can be 30 kPa or less, 10 kPa or less, and particularly preferably 5 kPa or less. The pressure applied to the microchannel chip by the external liquid supply driving force is, in particular, a negative pressure applied to the outlet or a positive pressure applied to the dispersed phase liquid holder and the continuous phase liquid holder.

[0159] On the other hand, when the present invention is used for rapid digital measurement, for example, the droplet generation rate calculated from the liquid delivery rate and the ratio of the flow rate of the continuous phase liquid to the dispersed phase liquid is preferably high. For example, the droplet generation rate in the emulsion formation section is preferably 5 droplets / second or more, 20 droplets / second or more, 50 droplets / second or more, 100 droplets / second or more, and particularly preferably 200 droplets / second or more.

[0160] To reduce the liquid delivery pressure and increase the droplet generation rate, it is desirable to adjust the flow path pressure drop resistance (= liquid delivery pressure / liquid delivery rate) to a small value. The flow path pressure drop resistance depends on the flow path structure, the surface physical properties of the flow path wall, the physical properties of each phase liquid, the pressure control method of the liquid delivery means, etc. Therefore, the above parameters can be appropriately adjusted so that the liquid delivery rate and / or droplet generation rate during liquid delivery and the liquid delivery pressure are appropriate.

[0161] (Installation of microfluidic chip) Microchannel chips are generally installed horizontally in the vertical direction (top-to-bottom direction), but they may be installed with an intentional tilt in a certain direction from the perspective of emulsion retention, etc. For example, when the continuous phase liquid has a higher specific gravity than the dispersed phase liquid (e.g., when a fluorinated dispersant is used as the continuous phase liquid and an aqueous solution is used as the dispersed phase liquid), the droplets have buoyancy due to the difference in specific gravity, so the microchannel chip may be installed with an intentional tilt to make it difficult for the droplets to flow out of the emulsion-holding channel.

[0162] Furthermore, when the continuous phase liquid has a smaller specific gravity than the dispersed phase liquid, a force acts to cause bubbles to rise vertically and droplets to fall vertically, so the above-mentioned inclined installation is particularly preferable because it is possible to control the gas-liquid interface in the bubble trap stop valve portion while making it difficult for the gas-liquid interface to come into contact with the droplets.

[0163] <Emulsion> The emulsions produced by the methods of the present disclosure are disperse solutions that contain droplets made up of a dispersed phase liquid and a continuous phase made up of a continuous phase liquid. In the emulsion, the droplets made up of the dispersed phase liquid are dispersed in the continuous phase made up of the continuous phase liquid.

[0164] (dispersed phase liquid) The dispersed phase liquid is the liquid that makes up the droplets contained in the emulsion.

[0165] The dispersed phase liquid is, for example, an aqueous solution. The dispersed phase liquid may optionally contain a surfactant, an organic solvent, a thickener, serum, an enzyme, etc. The dispersed phase liquid may be a reaction liquid, such as a liquid containing a sample to be detected in the detection process described below, a liquid containing a detection reagent, or a mixture thereof.

[0166] (Continuous phase liquid) The continuous phase liquid is the liquid that constitutes the continuous phase contained in the emulsion.

[0167] The continuous phase liquid is preferably an immiscible liquid that is not miscible with the dispersed phase liquid. For example, if the dispersed phase liquid is an aqueous solution, the continuous phase liquid may be an oil, in which case a water-in-oil (W / O) emulsion is formed.

[0168] When the continuous phase liquid is an oil, the oil may be a silicone oil, a mineral oil, a fluorinated dispersion medium, a vegetable oil, or a combination thereof.

[0169] Fluorine-based dispersion media include fluorocarbons, particularly perfluorohexane, hexafluorobenzene, perfluoromethylcyclohexane, perfluorooctane, and perfluorotripentylamine.

[0170] Commercially available fluorocarbons include FC-3283 (Fluorinert (trade name) manufactured by 3M Corporation), FC-40 (Fluorinert (trade name) manufactured by 3M Corporation), and HFE-7500 (Fluorinert (trade name) manufactured by 3M Corporation). TM Novec TM High-performance liquid (manufactured by 3M).

[0171] When a fluorine-based dispersion medium, especially the fluorocarbons mentioned above, is used as the continuous phase liquid, droplet generation is particularly stable and rapid. Furthermore, because of its extremely low compatibility with polar and nonpolar solvents, it can suppress the problem of droplet components in the emulsion migrating to other droplets via the continuous phase liquid (crosstalk, contamination). Furthermore, when using hydrocarbon-based dispersion mediums or silicone oils with low surface tension and viscosity, there is generally an increased risk of flammability and other hazards. However, fluorine-based dispersion mediums are characterized by their high safety, making them suitable for use as fire extinguishers and coolants.

[0172] Additives such as surfactants can be added to the continuous phase liquid for the purpose of improving the thermal stability of the droplets. It is preferable that these additives do not inhibit the detection reaction in the droplets. Examples of surfactants include nonionic surfactants such as PLURONIC (registered trademark) and TETRONIC (registered trademark), which are block copolymers of polyethylene glycol and polypropylene glycol, as well as Tween, Span, and Zonyl (registered trademark). When a fluorine-based dispersion medium is used as the continuous phase liquid, it is preferable to use a fluorine-based surfactant, such as a block copolymer of perfluoropolyether and polyethylene glycol.

[0173] (droplet) The droplets contained in an emulsion are composed of the dispersed phase liquid, for example, by encapsulation of the dispersed phase liquid through contact with the continuous phase liquid.

[0174] The droplets contain, for example, a sample to be detected. A target substance contained in the sample is reacted with a reagent in the droplet, and the sample can be analyzed via a detectable signal (e.g., a fluorescent signal) indicating the presence or absence and / or extent of the reaction. This reaction may be, for example, a chemical reaction, a binding reaction, a phenotypic change, or a combination thereof.

[0175] The volume of the droplets is preferably large enough to hold approximately one target substance (e.g., one molecule). Specifically, the average volume is preferably 0.00001 nL or more, 0.0001 nL or more, 0.001 nL or more, 0.01 nL or more, 0.1 nL or more, 0.5 nL or more, or 1 nL or more, and / or 100 nL or less, 50 nL or less, or 10 nL or less. To ensure uniform reaction of the target molecules within the droplets, the droplets preferably have a highly monodisperse volume. Specifically, monodisperse refers to a droplet volume coefficient of variation (CV) of 20% or less, 10% or less, 5% or less, 2% or less, or 1% or less. For ease of explanation, droplets will be treated as spherical below; however, droplets that are non-spherical due to the flow channel structure or surrounding flow may also be considered.

[0176] The droplets preferably have sufficient thermal stability to maintain their shape at least under the reaction temperature conditions of the target substance. Specifically, in the detection process, the droplets preferably have sufficient thermal stability to maintain their shape under temperatures of 40°C to 48°C when nucleic acid amplification is performed by the TRC method, and under temperatures of 50°C to 100°C when nucleic acid amplification is performed by the PCR method.

[0177] <Detection process> A detection process can be performed on the droplets in the emulsion. The detection process includes, for example, a reaction of a target substance in the droplet and detection of the reaction (e.g., detection of a reaction product). The detection process can be performed on the droplets in the emulsion held in the emulsion holding channel.

[0178] Target substances (particularly target molecules) include nucleic acids, proteins, peptides, enzymes, cells, bacteria, spores, viruses, organelles, macromolecular assemblies, drug candidates, lipids, carbohydrates, metabolites, or any combination thereof.

[0179] The reaction of the target substance is not particularly limited. Examples of the reaction of the target substance include enzymatic reactions, more specifically, enzymatic reactions using kinases, nucleases, nucleotide cyclases, nucleotide ligases, nucleotide phosphodiesterases, polymerases (DNA or RNA), prenyltransferases, pyrophospatases, reporter enzymes, reverse transcriptases, topoisomerases, etc. When the target molecule is a nucleic acid such as DNA or RNA and the reaction of the target molecule is an amplification reaction of the nucleic acid, examples of the reaction include reactions capable of isothermal amplification of nucleic acids, such as the LAMP method, NASBA method, TMA method, and TRC method. Furthermore, in the case of one-step RT-PCR, it is preferable to prepare droplets at a temperature suitable for the reverse transcription reaction in terms of the reaction efficiency and reaction time of the reverse transcription reaction. Furthermore, it is also possible to detect cDNA, the product of the reverse transcription reaction, using a cycling probe method.

[0180] When carrying out a reaction, it is preferable to mix two or more reaction liquids upstream of the emulsion formation section (for example, at the dispersed phase liquid confluence section) and use this mixture to generate droplets. In the present invention, the reaction liquid refers to a solution containing at least a portion of the target substance and the components necessary for reacting the target substance. All components necessary for the reaction of the target substance need to be present when all reaction liquids are mixed, and the target substance need only be contained in one of the reaction liquids. There is no problem if three or more reaction liquids are used.

[0181] For example, when the target substance is a nucleic acid (DNA, RNA) containing a specific sequence and the reaction of the target substance is a reaction to amplify the specific sequence, the components contained in the reaction solution include a primer containing a sequence homologous to a portion of the specific sequence, a primer containing a sequence complementary to a portion of the specific sequence, a detection probe containing a sequence homologous or complementary to a portion of the specific sequence, a polymerase, nucleotides, salts, and buffer components. Note that the composition of the reaction solution is preferably designed so that the target molecule, reaction substrate, enzyme, etc. do not decompose, deteriorate, or undergo nonspecific reactions, and glycerol, surfactants, etc. may be further added in consideration of their behavior in the device.

[0182] <Other methods> (Detection means) To detect the reaction, for example, a detection means capable of detecting the reaction product can be used.

[0183] The detection method can be appropriately selected depending on the reaction product, and can be detected using known methods such as optical, X-ray, MALDI (matrix-assisted laser desorption / ionization), FCS (fluorescence correlation spectroscopy), FP (fluorescence polarization) / FCS, fluorometry, colorimetric analysis, chemiluminescence, bioluminescence, scattering, surface plasmon resonance, electrochemistry, electrophoresis, laser, mass spectrometry, Raman spectroscopy, FLIPR (Molecular Devices), etc. When detection is performed using transmitted light, it is preferable to fabricate a microchannel chip from a light-transmitting material, since the reaction product can be detected simply by placing the microchannel chip on an optical detector without moving the droplets within the chip.

[0184] The detection means (detector) used to detect reaction products can be an imaging sensor and optional components for recording and measuring the reaction of the target substance. One example of a detection method is a camera or imaging device with appropriate illumination and resolution to spatially resolve the individual signals to be detected. Known cameras or imaging devices can be used. For example, the camera can be any common semiconductor image sensor, including a charge-coupled device (CCD), a charge injection device (CID), a photodiode array (PDA), or a complementary metal-oxide semiconductor (CMOS). Detection can also be improved by using polarization of the excitation / emitted light. For example, when detecting droplets emitting fluorescent signals, rapid, high-throughput signal detection can be achieved by simultaneously capturing images of the detection area using an optical unit with a large field of view.

[0185] (Temperature control means) The temperature control means has the role of maintaining the liquid in the microchannel chip at a temperature suitable for the reaction of the target substance. The temperature control means does not necessarily have to be flat, as long as it can be in close proximity (preferably in close contact) with the microchannel chip.

[0186] At least the portion of the temperature control means that is in close proximity (preferably in close contact) with the microchannel chip is preferably made of a metal material with high thermal conductivity. When the microchannel chip is fabricated by bonding a substrate and a superstructure, reducing the thickness of the substrate and / or superstructure in contact with the temperature control means is preferable in terms of more efficient heat conduction to the channels provided in the microchannel chip. The temperature control means is sufficient to control the temperature of at least the emulsion-holding channel, which is the reaction site for the target substance. However, it is also preferable to be able to control the temperature of the phase liquid supply unit and the channel, in order to suppress nonspecific reactions of the target molecule. Specifically, when the reaction of the target substance is a nucleic acid amplification reaction, the temperature in each holder and channel can be controlled by the temperature control means so that it is higher than the reaction temperature of the target substance in the emulsion-holding channel, thereby reducing nonspecific annealing between primers and probes. Furthermore, when the bottom surface of the micro-channel chip is heated to the reaction temperature by a temperature control means, and the top surface substrate of the micro-channel chip is made of a light-transmitting material and transmitted light is detected from the top surface, this is preferable because it allows for easy evaluation of the position of the empty micro-channel chip before supplying each phase liquid and / or the channel structure and / or dust inside and outside the chip, evaluation of the behavior inside the channel when supplying each phase liquid and / or the behavior of emulsion generation during liquid delivery, and improvement of the upper limit of quantitation in digital detection using the signal detection results of the emulsion during the reaction.

[0187] The present invention will now be described in more detail with reference to examples. [Example]

[0188] The microchannel chip used in the examples is described below.

[0189] <Fabrication of microfluidic chip>

[0190] A microfluidic chip was fabricated using photolithography and soft lithography techniques. The specific procedures are as follows.

[0191] (1) Photoresist SU-8 3050 (Microchem) was dropped onto a 4-inch bare silicon wafer (Filtech), and then a thin photoresist film was formed using a spin coater (MIKASA).

[0192] (2) Using a mask aligner (Ushio Inc.) and a chrome mask with the channel pattern of the microchannel chip formed on it, the channel pattern was formed on a photoresist film, and then the channel pattern was developed using SU-8 Developer (Microchem Inc.) to create a mold for the channel that constitutes the microchannel chip.

[0193] (3) To suppress adsorption onto SU-8, a vapor deposition surface treatment was performed using trichloro(1H,1H,2H,2H-perfluoro-octyl)silane (Thermo Fisher Scientific).

[0194] (4) A mixture of uncured siloxane monomer and polymerization initiator (weight ratio 10:1) prepared using a SYLGARD SILICONE ELASTOMER KIT (Dow Corning Toray Co., Ltd.) was poured into the mold treated in (3) above, and heated at 80°C for 2 hours to produce a polymer (PDMS) substrate with the shape of the flow channel transferred thereto.

[0195] (5) The obtained polymer substrate was carefully peeled off from the mold and shaped with a cutter, and then a dispersed phase liquid holding portion, a continuous phase liquid holding portion, and an outlet were formed using a puncher.

[0196] (6) The polymer substrate with the holding section and outlet formed and the cover glass (Matsunami Glass Co., Ltd.) were surface treated with an oxygen plasma generator (Meiwafosis Co., Ltd.), and then the patterned surface of the PDMS substrate was bonded to the cover glass. The fabricated chip was stored in a desiccator.

[0197] The fabricated microchannel chip was 34 cm long and 75 cm wide, and had a φ4 mm hole for the dispersed phase liquid holding portion, a φ8 mm hole for the continuous phase liquid holding portion, and a φ1.5 mm hole for the outlet.

[0198] (flow path structure) The microchannel chip had two dispersed phase liquid holders, a dispersed phase liquid flow path (having a first dispersed phase liquid flow path, a second dispersed phase liquid flow path, and a dispersed phase liquid junction), a continuous phase liquid holder, two continuous phase liquid flow paths, an emulsion-forming section, an emulsion flow path, an emulsion-holding flow path, and an outlet. The two dispersed phase liquid holders were connected to the emulsion-forming section via the first or second dispersed phase liquid flow path, the continuous phase liquid holder was connected to the emulsion-forming section via the two continuous phase liquid flow paths, the emulsion-forming section was connected to the emulsion-holding flow path via the emulsion flow path, and the emulsion-holding flow path was connected to the outlet. The end portion of the emulsion-holding flow path on the outlet side constituted a bubble trap stop valve.

[0199] More specifically, the first dispersed phase liquid flow path and the second dispersed phase liquid flow path are meandering paths with a height of 100 μm, a width of 200 μm, and a length of 4200 μm. They join at a dispersed phase liquid confluence, narrow to a width of 100 μm, and then join at an emulsion formation section. The two continuous phase liquid flow paths are straight paths with two bends, each measuring 100 μm in height, 280 μm in width, and 26 mm in length. In the emulsion formation section, the dispersed phase liquid confluence and the two continuous phase liquid flow paths cross at a 90-degree angle. In the emulsion formation section, the reaction liquid and the immiscible liquid (oil) join to form droplets.

[0200] The emulsion flow path is a flow path that directly intersects with the emulsion formation section and is 80 μm wide x 100 μm long, and downstream of that is a straight flow path with a width of 200 μm x length of 680 μm.Further downstream of that is a stirring flow path with a width of 200 μm x length of 11.5 mm, including a meandering path composed of an arc curve with a radius of 275 μm, and is connected to the emulsion holding flow path.

[0201] The emulsion-holding channel was a serpentine channel with a channel height of 130 μm, a width of 2 mm, and a length of 350 mm, and its outlet end formed a bubble trap stop valve. The volume of the emulsion-holding channel was 91 μL.

[0202] The bubble trap stop valve section includes a 400 μm long flow path narrowed to a width of 100 μm and a height of 80 μm, a 600 μm long flow path downstream of which is expanded to a width of 300 μm and a height of 130 μm, and a further 400 μm long flow path downstream of which is narrowed to a width of 100 μm and a height of 80 μm, and is connected to the outlet-connected flow path.

[0203] The outlet-connected flow path is a flow path with a width of 2 mm and a length of 10 mm, and is directly connected to the outlet.

[0204] Example 1 Using the above-mentioned microfluidic chip, emulsions were generated and held, and the droplets held in the emulsion-holding channels were subjected to a detection process. Specifically, digital isothermal nucleic acid amplification of hepatitis C virus (HCV) RNA was performed. Details are given below.

[0205] (Reaction solution) HCV standard RNA (SEQ ID NO: 1) was prepared by in vitro transcription from the plasmid into which the HCV gene was inserted. 4 The solution was diluted with water for injection to a concentration of 2 μL per copy, and used as an RNA sample.

[0206] A 10 μL aqueous solution containing the following composition was prepared and used as a reaction solution containing standard RNA. The molecular beacon probe (SEQ ID NO: 6) had six bases of oligonucleotides attached to the 5'- and 3'-ends of the homologous strand of the standard RNA (only one base overlaps on the 5'-end), which are capable of forming a stem-loop structure when not forming a complementary double-stranded strand with the standard RNA. Furthermore, FAM was attached to the 5'-end and Iowa Black FQ (manufactured by IDT) to the 3'-end. 132mM Tris-HCl buffer (pH8.36) 5.0% (v / v) glycerol 0.66mM each dATP, dCTP, dGTP, dTTP 4.0mM each ATP, CTP, GTP, TTP 6.6mM ITP 193.2mM trehalose 100 nM molecular beacon probe (containing a portion of the homologous strand of standard RNA [from positions 107 to 123 of SEQ ID NO: 1]: SEQ ID NO: 6) 2.0 μM first primer (SEQ ID NO: 2) 2.0 μM second primer (SEQ ID NO: 3) 8.5U AMV reverse transcriptase 94U T7 RNA polymerase 50nM fluorescein 10 4 Copy standard RNA

[0207] The first primer (SEQ ID NO: 2) is an oligonucleotide consisting of a partial sequence of the complementary strand of the standard RNA (specifically, from positions 125 to 145 of SEQ ID NO: 1; SEQ ID NO: 4) with a T7 promoter sequence (SEQ ID NO: 5) added to the 5' end of that sequence. The second primer (SEQ ID NO: 3) is an oligonucleotide consisting of a partial sequence of the homologous strand of the standard RNA (specifically, from positions 1 to 16 of SEQ ID NO: 1). Fluorescein was added at a concentration that did not inhibit the TRC reaction in order to improve the S / N ratio of negative droplets.

[0208] (starting solution) An aqueous solution containing the following composition was prepared and used as a starting solution. 36.8mM magnesium chloride 180.0mM potassium chloride 0.2% (w / v) Tween 20 18.0% (v / v) DMSO 5.0% (v / v) glycerol 100 nM Molecular Beacon Probe (SEQ ID NO: 6) 50nM fluorescein

[0209] (Emulsion generation and maintenance) The microfluidic chip was fixed on a thermal cycler (Master cycler nexus flat eco, Eppendorf) and heated to 46°C.

[0210] As a liquid delivery means, a device was used that was capable of controlling the pressure in a 200 mL tank to -1 to -10 kPa, consisting of a peristaltic pump (Takasago Kogyo), a solenoid valve (Takasago Kogyo), and a pressure sensor (Keyence Corporation).The tank and the outlet 90 of the microchannel chip 100 were connected with a PTFE tube (Nichias Corporation), and a pressure difference was applied by releasing the pressure in the tank.

[0211] Using a pipette, 20 μL of each of the reaction solution and the starting solution was dropped into the two dispersed phase liquid holders. After 40 seconds, 200 μL of oil (Droplet Generator oil for EvaGreen (Biorad)) was dropped into the continuous phase liquid holder.

[0212] Thirty seconds after the oil was dripped, the pressure inside the tank of the liquid delivery device connected to the outlet was adjusted to -5 kPa, and a pressure difference (negative pressure) was applied to start droplet generation and droplet retention. Note that each retention part was open to atmospheric pressure.

[0213] When the oil-air interface (emulsion-air interface) reached the bubble trap stop valve (approximately 150 seconds later), the connection between the pressure tank and the outlet was closed, and at the same time the connection between the outlet and the outside atmosphere was opened, thereby releasing the negative pressure applied to the outlet to normal pressure and stopping the liquid transfer. Air bubbles formed at the bubble trap stop valve (see Figure 5), which held the droplets in the emulsion holding channel.

[0214] (Detection process) After the liquid supply was stopped, the mixture was left standing for 20 minutes while being heated at 46°C, to complete the TRC reaction.

[0215] After the TRC reaction, the microfluidic chip was subjected to a detection process. Detection was performed using an X-Cite 110 LED as the light source for fluorescence imaging and a Cooling Camera System (Pacific Image Electronics, Taiwan) as the CCD camera for image capture. The detection results are shown in Figures 6 and 7.

[0216] As can be seen from FIGS. 6 and 7, the droplets were well held in the emulsion holding channel during the detection process, and the detection process was carried out satisfactorily. [Explanation of symbols]

[0217] 10 Microfluidic Chip 101 Continuous phase liquid holding section 102 First dispersed phase liquid holding section 103 Second dispersed phase liquid holding section 111 Continuous phase liquid flow path 112 First continuous phase liquid flow path 113 Second continuous phase liquid flow path 114 First dispersed phase liquid flow path 115 Second dispersed phase liquid flow path 116 Dispersed phase liquid confluence section 120 Emulsion forming section 130 Emulsion channel 140 Emulsion holding channel 150 Outlet 160 Bubble trap stop valve part 162 Stenosis 164 Additional stenosis 166 Extension 168 Upstream part 170 Transition area 400 Dispersed phase liquid holding section 41 wells 43 Hole 45 Expansion Area A. Air bubbles W width direction L lengthwise H Height direction α angle between sidewalls in the transition region

Claims

1. the dispersion liquid holding section, the dispersion liquid flow path, the continuous phase liquid holding section, the continuous phase liquid flow path, the emulsion forming section, the emulsion flow path, the emulsion holding flow path, and the discharge port; the dispersed phase liquid holding unit is connected to the emulsion forming unit via the dispersed phase liquid flow path, the continuous phase liquid holding unit is connected to the emulsion forming unit via the continuous phase liquid flow path, the emulsion forming section is connected to the emulsion holding flow path via the emulsion flow path, the emulsion holding channel is connected to the outlet, an end portion of the emulsion holding flow path on the discharge port side constitutes an air bubble trap stop valve portion; Microfluidic chip for emulsion filling.

2. 2. The micro-channel chip according to claim 1, wherein the bubble trap stop valve portion has a narrowed portion.

3. 3. The microchannel chip according to claim 2, wherein the narrowed portion is narrowed in the vertical direction.

4. 4. The microchannel chip according to claim 3, wherein the narrowed portion is narrowed in both the vertical and horizontal directions.

5. 5. The micro-channel chip according to claim 3, wherein the narrowed portion has a step structure, thereby narrowing in the vertical direction.

6. 6. The micro-channel chip according to claim 2, wherein the bubble trap stop valve section has an expansion section disposed downstream of the narrowed section, and an additional narrowed section disposed downstream of the expansion section.

7. 7. The micro-channel chip according to claim 6, wherein the additional constriction section is constricted in the vertical direction.

8. 8. The micro-channel chip according to claim 7, wherein the additional constriction section is constricted in both the vertical and horizontal directions.

9. 9. The micro-channel chip according to claim 7, wherein the additional constriction section has a step structure to constrict in the vertical direction.

10. A method for generating and maintaining an emulsion by supplying a dispersed phase liquid and a continuous phase liquid to the microchannel chip according to any one of claims 1 to 9, comprising: 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 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 unit by an external liquid feeding driving force, and transporting the emulsion thus generated through the emulsion flow path to the emulsion holding flow path filled with gas; Including, when the gas-liquid interface between the gas filling the emulsion holding flow path and the emulsion proceeding through the emulsion holding flow path reaches the bubble trap stop valve section, bubbles are formed in the bubble trap stop valve section, thereby suppressing or avoiding the outflow of the droplets through the outlet; and the external liquid-transport driving force is stopped before the emulsion completely fills the emulsion-holding channel. method.

11. 11. The method of claim 10, wherein the amount of the continuous phase liquid supplied to the continuous phase liquid holding section is adjusted so that the continuous phase liquid is present in the continuous phase liquid holding section for at least a certain period of time after the external liquid delivery driving force is stopped.

12. 12. The method of claim 10 or 11, wherein the external fluid delivery driving force is a negative pressure applied to the outlet.

13. The method according to any one of claims 10 to 12, wherein at least one of the outlet and each phase liquid holding section is not sealed while the droplets are held in the emulsion holding channel after the external liquid sending driving force is stopped.

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