Microchannel chip for charging emulsion

Abstract

To provide a microchannel chip for charging emulsion which can remove air bubbles present in emulsion in an emulsion holding channel.SOLUTION: In a microchannel chip for charging emulsion of the present invention, at least a part of emulsion flows into an emulsion storage part 142 and / or is guided to an emulsion guiding part 144 when emulsion flows from an emulsion supply channel 130 into an emulsion holding channel 140 while substituting gas inside the emulsion holding channel, by which a gas-liquid interface between emulsion and gas inside the emulsion holding channel is temporarily maintained in the vicinity of an emulsion flow inlet.SELECTED DRAWING: Figure 11

Description

[Technical field]

[0001] The present invention relates to a microchannel chip for filling an emulsion, which generates an emulsion and holds the generated emulsion for measurement. In particular, the present invention relates to a microchannel chip for filling an emulsion, which enables droplet array measurement to be performed more efficiently, simply, and quickly. [Background technology]

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

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

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

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

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

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

[0008] Patent Documents 2 and 3 disclose a microchannel chip having a channel for forming droplets and a droplet holder for holding droplets. Patent Document 2 describes that droplets are formed by joining two or more reaction solutions together and then contacting them with an immiscible liquid that is not miscible with the reaction solutions.

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

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

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

[0012] In the conventional droplet array method, it was necessary to fill the channels of the microchannel chip with a continuous phase liquid in advance as a preparatory process before supplying a reaction solution for gene detection to the microchannel chip. Specifically, the conventional droplet array method required the steps of filling the liquid delivery line with the continuous phase liquid, filling the chip with the continuous phase liquid, and removing excess continuous phase liquid (continuous phase liquid filling method). Such preparatory operations before measurement were complicated with many steps, and also made it difficult to automate the device.

[0013] In contrast, the emulsion filling method, in which a continuous liquid phase and a dispersed liquid phase are supplied to a flow channel of a gas-filled microchannel chip, an emulsion is formed in an emulsion forming section, and the formed emulsion is then filled into the emulsion holding section flow channel while displacing the gas, does not require the above preparatory operations.

[0014] However, in the emulsion filling method, because the continuous and dispersed liquid phases are supplied to the gas-filled flow channel of the microchannel chip to form an emulsion, and the emulsion thus formed is then filled into the emulsion holding section flow channel, the emulsion flowing into the emulsion holding section flow channel may contain air bubbles, particularly near the tip of the emulsion. Specifically, for example, such air bubbles may be formed in the emulsion forming section where the dispersed phase liquid and the continuous phase liquid join to form the emulsion, or in a pillar structure for trapping dust in the continuous phase liquid flow channel.

[0015] Such bubbles tend to remain in the emulsion-holding flow path, and can grow large due to the volatility of the continuous phase liquid, which can be a factor that impedes good analysis.

[0016] In contrast, the present invention has an object to remove air bubbles present in the emulsion in the emulsion holding flow path. [Means for solving the problem]

[0017] <Aspect 1> dispersed phase liquid holding section, A dispersed phase liquid flow path connected to the dispersed phase liquid holding portion; A continuous phase liquid holder; A continuous phase liquid flow path connected to the continuous phase liquid holding portion; an emulsion forming section connected to the dispersed phase liquid flow path and the continuous phase liquid flow path; an emulsion supply flow path connected to the emulsion forming section; and an emulsion holding channel connected to the emulsion supply channel; A microchannel chip for filling an emulsion, comprising: when a dispersed phase liquid is supplied to the dispersed phase liquid holding section, a continuous phase liquid is supplied to the continuous phase liquid holding section, and an external liquid sending driving force is applied to the micro-channel chip, an emulsion containing droplets constituted of the dispersed phase liquid and a continuous phase constituted of the continuous phase liquid is generated in the emulsion forming section, and the emulsion enters the emulsion holding channel via the emulsion supply channel and a single emulsion inlet, (i) the emulsion holding flow channel has an emulsion reservoir in a region where the expansion angle with respect to the direction in which the emulsion flows from the emulsion supply flow channel through the emulsion inlet into the emulsion holding flow channel is 60° or more, and / or in a region upstream of the emulsion inlet, As a result, when the emulsion flows from the emulsion supply flow path into the emulsion holding flow path while replacing the gas in the emulsion holding flow path, at least a portion of the emulsion flows into the emulsion storage section, thereby temporarily maintaining a gas-liquid interface between the emulsion and the gas in the emulsion holding flow path in the vicinity of the emulsion inlet, and / or (ii) the emulsion holding flow path has an emulsion guide portion in the vicinity of the emulsion inlet, As a result, when the emulsion flows from the emulsion supply flow path into the emulsion holding flow path while replacing the gas in the emulsion holding flow path, at least a part of the emulsion is guided to the emulsion guide section, so that a gas-liquid interface between the emulsion and the gas in the emulsion holding flow path is temporarily maintained in the vicinity of the emulsion inlet. Microfluidic chip for emulsion filling. <Aspect 2> The microchannel chip according to aspect 1, which satisfies the above (i). <Aspect 3> 3. The micro-channel chip according to aspect 2, wherein the emulsion holding channel has the emulsion reservoir in a region where the expansion angle is 60° or more. <Aspect 4> 4. The micro-channel chip according to aspect 3, wherein the emulsion holding channel has the emulsion reservoirs on the right and left sides with respect to a flow direction of the emulsion in the emulsion supply channel. <Aspect 5> 3. The micro-channel chip according to aspect 2, wherein the emulsion holding channel has the emulsion reservoir in a region upstream of the emulsion inlet. Aspect 6 6. The microchannel chip according to aspect 5, wherein an angle between a direction in which the emulsion flows from the emulsion supply channel through the emulsion inlet into the emulsion holding channel and a flow direction of the emulsion holding channel in the vicinity of the emulsion inlet is 30° or more. Aspect 7 The microchannel chip according to aspect 1, which satisfies the above (ii). <Aspect 8> 8. The microchannel chip according to aspect 7, wherein the emulsion guide portion has a stepped structure. <Aspect 9> The microchannel chip according to aspect 1, which satisfies both of the above (i) and (ii). Aspect 10 10. The microchannel chip according to aspect 9, wherein the emulsion guide portion guides at least a portion of the emulsion toward the emulsion reservoir portion. <Aspect 11> 2. The microchannel chip according to aspect 1, wherein the width of the emulsion holding channel is four times or more the width of the emulsion inlet. Effect of the Invention

[0018] According to the present invention, air bubbles that may be present in the emulsion can be removed in the emulsion holding flow path. [Brief description of the drawings]

[0019] [Figure 1]FIG. 1 is a schematic diagram showing the top surface (top center of the drawing), front cross section (bottom center of the drawing), and side cross section (right side of the drawing) of an emulsion holding channel of a microchannel chip according to a first configuration example of the first aspect of the present invention. [Diagram 2] FIG. 2 is a conceptual top view showing a process in which an emulsion flows into an emulsion holding channel of a micro-channel chip according to a first configuration example of the first aspect of the present invention. [Diagram 3] FIG. 3 is a schematic diagram showing the top surface (top center of the drawing), front cross section (bottom center of the drawing), and side cross section (right side of the drawing) of an emulsion holding channel of a microchannel chip according to a second configuration example of the first aspect of the present invention. [Figure 4] FIG. 4 is a conceptual top view showing a process in which an emulsion flows into an emulsion holding channel of a micro-channel chip according to a second configuration example of the first aspect of the present invention. [Diagram 5] FIG. 5 is a schematic diagram showing the top surface (top center of the drawing), front cross section (bottom center of the drawing), and side cross section (right side of the drawing) of an emulsion holding channel of a microchannel chip according to a second configuration example of the first aspect of the present invention. [Figure 6] FIG. 6 is a conceptual top view showing a process in which an emulsion flows into an emulsion holding channel of a micro-channel chip according to a second configuration example of the first aspect of the present invention. [Figure 7] FIG. 7 is a schematic diagram showing the top surface (top center of the drawing), front cross section (bottom center of the drawing), and side cross section (right side of the drawing) of an emulsion holding channel of a microchannel chip according to a third configuration example of the first aspect of the present invention. [Figure 8] FIG. 8 is a conceptual top view showing a process in which an emulsion flows into an emulsion holding channel of a micro-channel chip according to a third configuration example of the first aspect of the present invention. [Figure 9] FIG. 9 is a schematic diagram showing the top surface (upper center of the drawing), a front cross section (lower center of the drawing), and a side cross section (right side of the drawing) of an emulsion holding channel of a micro-channel chip according to the second embodiment of the present invention. [Figure 10] FIG. 10 is a conceptual top view showing a process in which an emulsion flows into an emulsion holding channel of a micro-channel chip according to a second embodiment of the present invention. [Figure 11] FIG. 11 is a schematic diagram showing the top surface (upper center of the drawing), front cross section (lower center of the drawing), and side cross section (right side of the drawing) of an emulsion holding channel of a micro-channel chip according to the third embodiment of the present invention. [Figure 12] FIG. 12 is a conceptual top view showing a process in which an emulsion flows into an emulsion holding channel of a micro-channel chip according to a third embodiment of the present invention. [Figure 13] FIG. 13 is a series of top view photographs showing the process of emulsion flowing into an emulsion holding channel of a micro-channel chip according to a third embodiment of the present invention. [Figure 14] FIG. 14 is a schematic top view of a microchannel chip according to one embodiment of the prior art. [Figure 15] FIG. 15 is a schematic diagram showing the top surface (upper center of the drawing), a front cross section (lower center of the drawing), and a side cross section (right side of the drawing) of an emulsion holding channel of a micro-channel chip according to one embodiment of the prior art. [Figure 16] FIG. 16 is a conceptual top view showing a process in which an emulsion flows into an emulsion holding channel of a micro-channel chip according to one embodiment of the prior art. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0020] The microchannel chip for filling an emulsion according to the present invention comprises: dispersed phase liquid holding section, a dispersed phase liquid flow path connected to the dispersed phase liquid holding portion; A continuous phase liquid holder; a continuous phase liquid flow path connected to the continuous phase liquid holding portion; an emulsion former connected to the dispersed phase liquid flow path and the continuous phase liquid flow path; An emulsion supply channel connected to the emulsion forming section; and An emulsion holding channel connected to the emulsion supply channel It has.

[0021] In the emulsion-filling microchannel chip of the present invention, when a dispersed phase liquid is supplied to the dispersed phase liquid holding section, a continuous phase liquid is supplied to the continuous phase liquid holding section, and an external liquid delivery driving force is applied to the microchannel chip, an emulsion containing droplets composed of the dispersed phase liquid and a continuous phase composed of the continuous phase liquid is generated in the emulsion formation section, and the emulsion enters the emulsion holding channel via the emulsion supply channel and the single emulsion inlet.

[0022] According to the present invention, by having the emulsion supply flow channel and the emulsion holding flow channel have structures as shown in the following first to third aspects, the gas-liquid interface between the emulsion and the gas in the emulsion holding flow channel is maintained in the vicinity of the emulsion inlet of the emulsion holding flow channel, and air bubbles present in the emulsion can be caused to collide with this gas-liquid interface and removed. Note that, in the present invention, the "emulsion inlet" means the point where the emulsion supply flow channel is connected to the emulsion holding flow channel, and in the case where the width of the emulsion holding flow channel expands in the liquid delivery direction, it means the point where this expansion starts.

[0023] In the emulsion-filling microchannel chip according to the present invention, the width of the emulsion holding channel can be determined relative to the width of the emulsion inlet, and for example, the width of the emulsion holding channel may be 4 times or more, 5 times or more, 6 times or more, 7 times or more, 8 times or more, 9 times or more, or 10 times or more the width of the emulsion inlet, and may be 50 times or less, 40 times or less, 30 times or less, 20 times or less, or 10 times or less. Specifically, in one embodiment, the width of the emulsion holding channel may be 2.0 mm, and the width of the emulsion inlet may be 0.1 mm.

[0024] The width of the emulsion-holding flow channel can be defined as the width, i.e., the distance between both sides of the emulsion-holding flow channel, when the width of the emulsion-holding flow channel is approximately constant at a position away from the emulsion inlet, or as the average width of the emulsion-holding flow channel, i.e., the average distance between both sides of the emulsion-holding flow channel, when the width of the emulsion-holding flow channel is not approximately constant at a position away from the emulsion inlet.

[0025] In this emulsion-filling microchannel chip according to the present invention, the height of the emulsion-holding channel can be determined relative to the height of the emulsion inlet, and for example, the height of the emulsion-holding channel may be 1.1 times or more, 1.2 times or more, 1.3 times or more, 1.4 times or more, 1.5 times or more, or 2.0 times or more the width of the emulsion inlet, and may be 5.0 times or less, 4.0 times or less, 3.0 times or less, or 2.0 times or less. Specifically, in one embodiment, the height of the emulsion-holding channel may be 0.2 mm, and the height of the emulsion inlet may be 0.1 mm.

[0026] The height of the emulsion-holding flow channel can be defined as the height, i.e., the distance between the top and bottom surfaces of the emulsion-holding flow channel, when the height of the emulsion-holding flow channel is approximately constant at a position away from the emulsion inlet, or as the average height of the emulsion-holding flow channel, i.e., the average distance between the top and bottom surfaces of the emulsion-holding flow channel, when the height of the emulsion-holding flow channel is not approximately constant at a position away from the emulsion inlet.

[0027] First Aspect In a first embodiment, the emulsion supply channel and the emulsion holding channel have the following structure: (i) the emulsion holding flow path has an emulsion reservoir in a region where the expansion angle with respect to the direction in which the emulsion flows from the emulsion supply flow path through the emulsion inlet into the emulsion holding flow path is 60° or more and / or in a region upstream of the emulsion inlet, As a result, when the emulsion flows from the emulsion supply flow path into the emulsion holding flow path while replacing the gas in the emulsion holding flow path, at least a portion of the emulsion flows into the emulsion storage section, thereby temporarily maintaining the gas-liquid interface between the emulsion and the gas in the emulsion holding flow path in the vicinity of the emulsion inlet.

[0028] That is, the emulsion reservoir makes it possible to temporarily maintain the gas-liquid interface between the emulsion and the gas in the emulsion holding flow channel in the vicinity of the emulsion inlet, compared to a case in which the emulsion reservoir is not provided.

[0029] In addition, with regard to the present invention, the "emulsion storage section" means a section into which at least a portion of the emulsion flows when the emulsion flows from the emulsion supply flow path through the emulsion inlet into the emulsion holding flow path while replacing the gas in the emulsion holding flow path, thereby temporarily maintaining the gas-liquid interface between the emulsion and the gas in the emulsion holding flow path in the vicinity of the emulsion inlet.

[0030] The emulsion reservoir may have any size. Specifically, the size of the emulsion reservoir may be determined as desired depending on the intended effect of removing air bubbles and the effect of retaining droplets uniformly, densely, and in a single layer in the emulsion holding flow path.

[0031] The distance in the liquid sending direction from the emulsion inlet to the emulsion reservoir is preferably relatively small, and may be, for example, 5 times or less, 4 times or less, 3 times or less, 2 times or less, or 0.5 times or less the width of the emulsion inlet. It is particularly preferable that the emulsion inlet and the emulsion reservoir are adjacent to each other.

[0032] (First configuration example) In a first configuration example of the first aspect, the emulsion holding channel has an emulsion reservoir in a region where the expansion angle with respect to the direction in which the emulsion flows from the emulsion supply channel into the emulsion holding channel via the emulsion inlet is 60° or more.

[0033] In the present invention, the "region with an expansion angle of 60° or more" may be present on either side of the emulsion supply channel. For example, the "region with an expansion angle of 60° or more" may be present on one or more of the right, left, upper, and lower sides of the emulsion supply channel, particularly on one or both of the right and left sides of the emulsion supply channel. That is, in the present invention, the "expansion angle" may be different or the same on the right, left, upper, and lower sides of the emulsion supply channel, particularly on the right and left sides of the emulsion supply channel. In the present invention, the upper and lower sides of the emulsion supply channel refer to the upper and lower sides, respectively, when the emulsion-filled microchannel chip is in use. In the present invention, the right and left sides of the emulsion supply channel refer to the right and left sides, respectively, with respect to the flow direction of the emulsion in the emulsion supply channel when the emulsion-filled microchannel chip is in use.

[0034] In addition, when the emulsion is filled in the region with an upward expansion angle of 60° or more, gravity acts from the top to the bottom, so the emulsion is preferentially filled in the bottom unless the upper side has an extremely higher affinity for the emulsion and / or continuous phase liquid than the lower side. In other words, when the emulsion is filled in the region with an upward expansion angle of 60° or more, the gas-liquid interface is likely to separate from the vicinity of the inlet, so the region with an upward expansion angle of 60° or more is relatively unlikely to function as a storage section. Therefore, it is preferable that the emulsion holding flow path is expanded on the right side, left side, and lower side of the emulsion supply flow path, particularly on the right side and left side of the emulsion supply flow path, thereby forming an emulsion storage section.

[0035] Specifically, in a first configuration example of the first aspect, the emulsion supply flow path and the emulsion holding flow path can have a structure as shown in Figures 1 and 2, a structure as shown in Figures 3 and 4, or a structure as shown in Figures 5 and 6.

[0036] In the embodiment shown in these figures, the emulsion holding flow path 140 can have an emulsion storage section 142 in a region where the expansion angle (α1, α2) relative to the direction in which the emulsion flows from the emulsion supply flow path 130 into the emulsion holding flow path 140 (white arrow) is 60° or more.

[0037] As described above, in the present invention, the "region with an expansion angle of 60° or more" may be present on either side of the emulsion supply flow channel. Thus, in the embodiment shown in Figs. 1 to 6, the emulsion holding flow channel 140 has a symmetrical shape on the right and left sides of the emulsion supply flow channel, but the emulsion holding flow channel 140 may have an asymmetrical shape on the right and left sides of the emulsion supply flow channel. Thus, the emulsion holding flow channel 140 may have an emulsion storage section in a region with an expansion angle (α1, α2) of 60° or more on only one of the right and left sides of the emulsion supply flow channel, or may have an emulsion storage section in a region with an expansion angle (α1, α2) of 60° or more on both the right and left sides of the emulsion supply flow channel.

[0038] This region having an emulsion reservoir may be a region in which the expansion angle (α1, α2) is 65° or more, 70° or more, 80° or more, 85° or more, 90° or more, 95° or more, 100° or more, 105° or more, 110° or more, 115° or more, or 120° or more, and may be a region in which the expansion angle (α) is 180° or less, 170° or less, 160° or less, 150° or less, 140° or less, 130° or less, 120° or less, 110° or less, 100° or less, 90° or less, 80° or less, or 70° or less.

[0039] In this case, when the emulsion 210 flows from the emulsion supply flow path 130 into the emulsion holding flow path 140 while replacing the gas in the emulsion holding flow path 140, at least a portion of the emulsion 210 flows into the emulsion storage section 142, whereby the gas-liquid interface between the emulsion 210 and the gas in the emulsion holding flow path 140 is temporarily maintained in the vicinity of the emulsion inlet.

[0040] In addition, in conventional emulsion holding flow paths, the above-mentioned expansion angles are relatively small; for example, the expansion angles on the right and left sides of the emulsion supply flow path are typically 45° or less (i.e., the sum of the expansion angles on the right and left sides of the emulsion supply flow path is 90° or less), and therefore the emulsion storage section referred to in the present invention was not formed.

[0041] For reference, the embodiment shown in Figures 1 and 2 will be described in detail. In this embodiment, the emulsion holding flow path 140 expands at an expansion angle of 90° on both the right and left sides of the emulsion supply flow path 130 relative to the inflow direction of the emulsion, and therefore the range of expansion angles from 60° to 90° functions as the emulsion storage section 142.

[0042] 3 and 4 will be described in more detail. In this embodiment, the emulsion holding flow channel 140 expands at an expansion angle of 110° with respect to the inflow direction of the emulsion on both the right and left sides of the emulsion supply flow channel 130, and therefore the range of expansion angles from 60° to 110° functions as the emulsion storage section 142. In this embodiment, the range of expansion angles of 90° or more in the emulsion storage section 142 can also be said to exist in the region upstream of the emulsion inlet.

[0043] 5 and 6, in this embodiment, the emulsion holding flow path 140 has partition walls 140a extending along both the right and left sides of the emulsion supply flow path 130, and therefore the range in which the expansion angle is greater than 60° functions as the emulsion storage section 142. In this embodiment, it can also be said that the range in which the expansion angle is greater than 90° exists in the region upstream of the emulsion inlet.

[0044] (Second configuration example) In a second configuration example of the first aspect, the emulsion holding flow path has an emulsion reservoir in a region upstream of the emulsion inlet.

[0045] Specifically, in the second configuration example of the first aspect, the emulsion supply flow channel and the emulsion holding flow channel can have structures as shown in FIGS.

[0046] In the embodiment shown in these figures, the emulsion holding channel 140 can have an emulsion reservoir 142 in a region upstream of the emulsion inlet (i.e., the point where the emulsion flows from the emulsion supply channel 130 into the emulsion holding channel 140).

[0047] In this case, when the emulsion 210 flows from the emulsion supply flow path 130 into the emulsion holding flow path 140 while replacing the gas in the emulsion holding flow path 140, at least a portion of the emulsion 210 flows into the emulsion storage section 142, whereby the gas-liquid interface between the emulsion 210 and the gas in the emulsion holding flow path 140 is temporarily maintained in the vicinity of the emulsion inlet.

[0048] In order to make the emulsion holding flow channel have an emulsion reservoir in a region upstream of the emulsion inlet, the direction in which the emulsion 210 flows from the emulsion supply flow channel 130 into the emulsion holding flow channel 140 via the emulsion inlet can be made to differ from the liquid sending direction of the emulsion holding flow channel 140 in the vicinity of the emulsion inlet. Specifically, the angle β between the direction in which the emulsion 210 flows from the emulsion supply flow channel 130 into the emulsion holding flow channel 140 and the flow direction of the emulsion holding flow channel 140 in the vicinity of the emulsion inlet is preferably 30° or more. These angles β may be 30° or more, 40° or more, 50° or more, 60° or more, 70° or more, 80° or more, 90° or more, 100° or more, or 120° or more, or may be 170° or less, 160° or less, 150° or less, 140° or less, 130° or less, 120° or less, 100° or less, 90° or less, 80° or less, 70° or less, 60° or less, 50° or less, or 40° or less. In order to promote the inflow of the emulsion 210 from the emulsion supply channel 130 to the emulsion reservoir 142, the angle β may be 90° or less, 80° or less, 70° or less, 60° or less, 50° or less, or 40° or less.

[0049] Second Aspect In a second embodiment, the emulsion supply channel and the emulsion holding channel have the following structure: (ii) the emulsion holding flow path has an emulsion guide portion in the vicinity of the emulsion inlet; As a result, when the emulsion flows from the emulsion supply flow path into the emulsion holding flow path while replacing the gas in the emulsion holding flow path, at least a portion of the emulsion is guided to the emulsion guiding section, so that the gas-liquid interface between the emulsion and the gas in the emulsion holding flow path is temporarily maintained in the vicinity of the emulsion inlet. In other words, the emulsion guiding section makes it possible to temporarily maintain the gas-liquid interface between the emulsion and the gas in the emulsion holding flow path in the vicinity of the emulsion inlet, compared to the case where there is no emulsion guiding section.

[0050] When the emulsion supply flow path and the emulsion holding flow path have a structure as shown in this second aspect, specifically, for example, when the emulsion supply flow path and the emulsion holding flow path have a structure as shown in Figures 9 and 10, when the emulsion 210 flows from the emulsion supply flow path 130 to the emulsion holding flow path 140 while replacing the gas in the emulsion holding flow path 140, at least a portion of the emulsion 210 is guided to the emulsion guide section 144, and the gas-liquid interface between the emulsion 210 and the gas in the emulsion holding flow path 140 is temporarily maintained in the vicinity of the emulsion inlet.

[0051] Here, the emulsion guiding section 144 of the emulsion holding flow channel 140 may be formed with a step structure. That is, the emulsion guiding section 144 is formed as a section having a lower height than other sections of the emulsion holding flow channel 140, and is particularly formed so that the height increases sharply at the transition point from the emulsion guiding section 144 to other sections of the emulsion holding flow channel 140, whereby the emulsion 210 can be guided to the emulsion guiding section 144 by the action of the surface tension of the emulsion.

[0052] Furthermore, the emulsion guiding portion 144 of the emulsion holding flow path 140 may be formed due to a difference in wettability with respect to the emulsion. That is, the emulsion guiding portion 144 is formed as a portion having a higher wettability with respect to the emulsion than other portions of the emulsion holding flow path 140, and this allows the emulsion 210 to be guided to the emulsion guiding portion 144.

[0053] The width and height areas of the emulsion guiding part may be set to any size depending on the intended effect of removing air bubbles. Specifically, the width and height areas of the emulsion guiding part may be determined arbitrarily depending on the intended effect of removing air bubbles and the effect of retaining droplets uniformly, densely, and in a single layer in the emulsion holding flow path.

[0054] In the emulsion holding flow path, it is preferable that the boundary between the induction part and the other part (for example, the step structure in Figs. 9 and 10) is present near the opening of the emulsion supply flow path, since the air bubbles flowing in are more likely to come into contact with the air-liquid interface held near the boundary. The shortest distance between the boundary and the opening of the emulsion supply flow path may be 3 times or less, 2 times or less, 1.5 times or less, 1 times or less, 1 / 2 times or less, 1 / 3 times or less, 1 / 4 times or less, 1 / 5 times or less, 1 / 7 times or less, 1 / 10 times or less, 1 / 15 times or less, 1 / 20 times or less, or 1 / 30 times or less of the height of the emulsion holding flow path and / or the emulsion supply flow path, or 1 / 100 times or more, 1 / 70 times or more, or 1 / 50 times or more. It may be 1 / 30 times or more, 1 / 20 times or more, 1 / 15 times or more, 1 / 10 times or more, 1 / 7 times or more, or 1 / 5 times or more.

[0055] The emulsion guided to the emulsion guide section is preferably prevented from flowing out too far from the boundary, so that the gas-liquid interface near the opening of the emulsion supply flow path can be maintained for a long time. In particular, it is preferable that the emulsion guide section is present up to the vicinity of the side wall surface of the emulsion holding flow path, because this reduces the substantial boundary and allows the gas-liquid interface to be stably maintained so as not to flow out too far from the boundary.

[0056] Third Aspect In the third embodiment, the emulsion supply channel and the emulsion holding channel have the features of both the first and second embodiments.

[0057] 11 and 12, when the emulsion 210 flows from the emulsion supply flow path 130 into the emulsion holding flow path 140 while replacing the gas in the emulsion holding flow path 140, at least a portion of the emulsion 210 is guided to the emulsion inducing section 144 and flows into the emulsion storage section, whereby the gas-liquid interface between the emulsion 210 and the gas in the emulsion holding flow path 140 is temporarily maintained in the vicinity of the emulsion inlet of the emulsion holding flow path 140. In other words, the emulsion inducing section and the emulsion storage section make it possible to temporarily maintain the gas-liquid interface between the emulsion and the gas in the emulsion holding flow path in the vicinity of the emulsion inlet, compared to a case in which there is no emulsion inducing section and / or emulsion storage section.

[0058] In order to efficiently guide the emulsion from the emulsion induction section near the opening of the emulsion supply flow path to the emulsion storage section while maintaining the gas-liquid interface near the opening of the emulsion supply flow path, it is preferable that the emulsion induction section and the emulsion storage section are connected, and it is particularly preferable that they are connected along the side wall surface of the emulsion holding flow path.

[0059] Summary of the first to third aspects In these first to third aspects, even if the emulsion 210 entering the emulsion holding flow path 140 from the emulsion supply flow path 130 contains air bubbles 220, the air bubbles 220 can be removed by colliding with the air-liquid interface maintained in the vicinity of the emulsion inlet of the emulsion holding flow path, that is, by being united with the gas in the emulsion holding flow path 140.

[0060] <<When there is no structure as shown in the first to third embodiments>> In contrast to this, if there is no structure as shown in the first to third embodiments, the air bubbles present in the emulsion cannot be removed, and therefore the air bubbles may remain in the emulsion-holding flow path and impede the measurement.

[0061] Specifically, for example, when the emulsion supply flow path and the emulsion holding flow path have the structures as shown in Figures 15 and 16, that is, when the emulsion holding flow path 140 has neither an emulsion storage section 142 nor an emulsion induction section 144, if the emulsion 210 entering the emulsion holding flow path 140 from the emulsion supply flow path 130 holds air bubbles 220, the air bubbles that were in the emulsion supply flow path 130 will enter the emulsion holding flow path 140 as they are and be maintained in the emulsion 210.

[0062] <Emulsion, configuration of microfluidic chip, detection process using microfluidic chip, etc.> The following describes the emulsion used in combination with the micro-channel chip of the present invention, the configuration of the micro-channel chip other than the emulsion reservoir and emulsion inducer, and detection processing using the micro-channel chip.

[0063] <Emulsion> An emulsion is a disperse solution that contains droplets made up of a dispersed phase liquid and a continuous phase made up of a continuous phase liquid. In an emulsion, droplets made up of the dispersed phase liquid are dispersed in a continuous phase made up of the continuous phase liquid.

[0064] (dispersed phase liquid) The dispersed phase liquid is the liquid that makes up the droplets contained in the emulsion.

[0065] 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, for example, a liquid containing a sample to be detected in the detection process described below, a liquid containing a detection reagent, or a mixture thereof.

[0066] (Continuous phase liquid) The continuous phase liquid is the liquid that constitutes the continuous phase contained in the emulsion.

[0067] The continuous phase liquid is preferably an immiscible liquid with the dispersed phase liquid, for example, if the dispersed phase liquid is an aqueous solution, the continuous phase liquid may be an oil, in which case a water-in-oil (W / O) emulsion is formed.

[0068] When the continuous phase liquid is an oil, the oil may include silicone oil, mineral oil, fluorinated dispersion medium, vegetable oil, or a combination thereof.

[0069] Fluorine-based dispersion media include fluorocarbons, particularly perfluorohexane, hexafluorobenzene, perfluoromethylcyclohexane, perfluorooctane, and perfluorotripentylamine.

[0070] Commercially available fluorocarbons include FC-3283 (Fluorinert (trade name) manufactured by 3M Corporation), FC-40 (Fluorinert (trade name) manufactured by 3M Corporation), and HFE-7500 (Fluorinert (trade name) manufactured by 3M Corporation). TM Novec TM Highly functional liquid (manufactured by 3M).

[0071] When a fluorine-based dispersion medium, particularly the above-mentioned fluorocarbon, is used as the continuous phase liquid, droplets can be generated particularly stably and quickly. In addition, since it has the characteristic of being extremely poorly compatible with polar and non-polar solvents, it is possible to suppress the problem (crosstalk, contamination) that the components of the droplets in the emulsion move to other droplets through the continuous phase liquid. In addition, when a liquid with low surface tension or viscosity is selected such as a hydrocarbon-based dispersion medium or silicone oil, the risk of flammability and other hazards generally increases, but a fluorine-based dispersion medium is characterized by its high safety, so that it is used as a fire extinguisher and a cooling medium.

[0072] When the continuous phase liquid contains oil (particularly a fluorine-based dispersion medium), the oil (particularly a fluorine-based dispersion medium) may be 50.0 to 99.9 mass % or even 80 to 99.0 mass % relative to the continuous phase liquid.

[0073] For the purpose of improving the thermal stability of the droplets, additives such as surfactants can be added to the continuous phase liquid. It is preferable that these additives do not inhibit the detection reaction in the droplets. Examples of surfactants include non-ionic surfactants such as PLURONIC (registered trademark) and TETRONIC (registered trademark), which are block copolymers of polyethylene glycol and polypropylene glycol, as well as Tween (registered trademark), Span (registered trademark), 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, particularly a fluorocarbon-based surfactant, such as a block copolymer of perfluoropolyether and polyethylene glycol.

[0074] When the continuous phase liquid contains a surfactant, the surfactant may be present in an amount of 1 to 20% by weight, or even 2 to 10% by weight, based on the continuous phase liquid.

[0075] (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.

[0076] The droplets contain, for example, a sample to be detected. In the droplets, a target substance contained in the sample is reacted with a reagent, and the sample can be analyzed via a detectable signal (e.g., a fluorescent signal) indicating the presence or absence and / or the extent of the reaction. The reaction may be, for example, a chemical reaction, a binding reaction, a phenotypic change, or a combination thereof.

[0077] The volume of the droplet is preferably large enough to hold approximately one target substance (target molecule) (for example, one molecule). Specifically, the average volume is preferably 0.00001 nL or more, 0.0001 nL or more, 0.001 nL or more, 0.01 nL or more, 0.1 nL or more, 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. From the viewpoint of uniformly reacting the target substance in the droplet, it is preferable that the volume of the droplet to be formed is highly monodisperse. Specifically, the monodisperse here means that the coefficient of variation (CV) of the droplet volume is 20% or less, 10% or less, 5% or less, 2% or less, or 1% or less. In the following description, the droplet is treated as a sphere for ease of understanding, but the same can be considered even if the droplet is non-spherical due to the flow channel structure or the surrounding flow.

[0078] The droplets preferably have a thermal stability sufficient to maintain their shape at least under the reaction temperature conditions of the target substance.Specifically, in the detection process, when nucleic acid amplification is performed by the TRC method, the droplets preferably have a thermal stability sufficient to maintain their shape under temperature conditions of 40°C to 48°C, and when nucleic acid amplification is performed by the PCR method, the droplets preferably have a thermal stability sufficient to maintain their shape under temperature conditions of 50°C to 100°C.

[0079] <Microfluidic Chip> The microchannel chip for filling an emulsion of the present invention comprises: dispersed phase liquid holding section, a dispersed phase liquid flow path connected to the dispersed phase liquid holding portion; A continuous phase liquid holder; a continuous phase liquid flow path connected to the continuous phase liquid holding portion; an emulsion former connected to the dispersed phase liquid flow path and the continuous phase liquid flow path; An emulsion supply channel connected to the emulsion forming section; and An emulsion holding channel connected to the emulsion supply channel These are fluidly connected to each other and form a single flow path structure as a whole.

[0080] The microchannel chip of the present invention is a microchannel chip for filling with an emulsion, which is used in an emulsion filling method.

[0081] In the "emulsion filling method," the emulsion (droplets + continuous phase) generated in the emulsion formation unit is transported to an emulsion holding flow path filled with gas. In the emulsion filling method, the emulsion generated in the emulsion formation unit moves through the emulsion holding flow path filled with gas toward the discharge outlet, filling the emulsion holding flow path. In other words, the "gas-liquid interface" formed by the gas filling the emulsion holding flow path and the emulsion moves downstream of the emulsion holding flow path (or toward the discharge outlet).

[0082] A specific description will be given with reference to a conventional microchannel chip shown in FIG.

[0083] The micro-channel chip 10 in Fig. 14 has a planar configuration, i.e., the generation, transportation, and retention of the emulsion are substantially performed within one plane. The micro-channel chip 10 in Fig. 14 has a first dispersed phase liquid holding section 102, a second dispersed phase liquid holding section 103, a dispersed phase liquid flow path (comprised of a first dispersed phase liquid flow path 114, a second dispersed phase liquid flow path 115, and a dispersed phase liquid junction section 116), a continuous phase liquid holding section 101, a continuous phase liquid flow path 111, an emulsion forming section 120, an emulsion supply flow path 130, an emulsion holding flow path 140, and an outlet 150. The dispersed phase liquid holding sections 102 and 103 are connected to the emulsion forming section 120 via the first dispersed phase liquid flow path 114 and the second dispersed phase liquid flow path 115, respectively, and the continuous phase liquid holding section 101 is connected to 120 via the continuous phase liquid flow path 111. 14, the continuous phase liquid flow path 111 is composed of a first continuous phase liquid flow path 112 and a second continuous phase liquid flow path 113. The emulsion forming section 120 is connected to an emulsion holding flow path 140 via an emulsion supply flow path 130, and the emulsion holding flow path 140 is connected to a discharge port 150.

[0084] In the emulsion filling method, a dispersed phase liquid such as an aqueous reaction liquid containing a substance to be detected is supplied to dispersed phase liquid holding portions 102 and 103, and a continuous phase liquid such as oil is supplied to continuous phase liquid holding portion 101. At this point, each flow path of micro-channel chip 10 is filled with gas (particularly air) (i.e., an "empty" micro-channel chip is used).

[0085] An empty microchannel chip refers to a state in which the inside of the channel is filled with gas (particularly air), and preferably means a state in which there is no liquid (e.g., dispersed phase liquid, continuous phase liquid, etc.) in the entire channel, that is, a state in which 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 in particular, it is preferable that the dispersed phase liquid channel, continuous phase liquid channel, and emulsion formation section are not clogged.

[0086] In using the microchannel chip of the present invention, the emulsion-holding channel is (at least partially) filled with gas before application of an external liquid-transport driving force (particularly negative pressure). This method differs from the conventional method of filling the entire channel of the microchannel chip with a continuous phase liquid in advance, and can omit the preparatory steps of filling with the continuous phase liquid and removing excess continuous phase liquid, which have been required in the conventional method.

[0087] In one embodiment of the microchannel chip of the present invention, the liquids (dispersed phase liquid and continuous phase liquid) supplied to the dispersed phase liquid holding section and the continuous phase liquid holding section can be moved to the emulsion forming section by capillary force and / or liquid level differential pressure. In this case, the movement of the pre-filling liquid and the dispersed phase liquid and the continuous phase liquid can be easily controlled without the need for an additional device.

[0088] For example, a desired capillary force can be obtained by appropriately setting the channel structure of the microchannel chip (channel surface characteristics, channel pressure loss, etc.). Also, a desired liquid level differential pressure can be obtained by adjusting the amount of liquid (particularly the liquid level height) supplied to each phase liquid holding section.

[0089] Capillary force is a force that acts in the direction of penetrating into the flow path, which is generated by the difference in surface tension between the gas-liquid interface in the wide-open holding part and the gas-liquid interface in the flow path with a smaller cross section. Therefore, the capillary force is affected not only by the characteristics of each flow path but also by the structure of the holding part, and in particular, in the emulsion filling method, the capillary force has a large effect during the liquid transfer (droplet generation) and while the droplets are being held after the liquid transfer has stopped, in order to control the movement of the emulsion's gas-liquid interface.

[0090] The liquid level differential pressure is also known as hydrostatic pressure, and generally refers to the pressure generated by gravity in a stationary liquid, that is, the pressure that depends on the amount (weight) of each phase liquid supplied to each holding section.

[0091] For example, by applying an external liquid delivery driving force (negative pressure) to the outlet, an emulsion containing droplets composed of a dispersed phase liquid and a continuous phase composed of a continuous phase liquid can be generated in the emulsion formation section, and the emulsion thus generated can be transported via an emulsion supply flow path to an emulsion holding flow path filled with gas.

[0092] The microchannel chip according to the present invention 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 holding section, a dispersed phase liquid channel, a continuous phase liquid holding section, a continuous phase liquid channel, an emulsion forming section, an emulsion supply channel, and an emulsion holding channel. The substrate may be made of glass. The upper structure may be made of resin. The microchannel chip can be fabricated, for example, by bonding a resin upper structure to a glass substrate constituting the bottom of the microchannel chip.

[0093] The size (width, depth, etc.) of the flow channel constituting the microchannel chip can be appropriately determined in consideration of the volume of the target droplet, etc., and in particular, in consideration of the reaction form of the target substance. For example, in the microdroplet method described below, when the target substance is a nucleic acid such as DNA or RNA, and the reaction of the target substance is a digital amplification reaction of the nucleic acid (amplification reaction in single molecule units), it is necessary to prepare droplets on the order of pL or nL, so that the width and depth of the flow channel around the emulsion formation part are preferably in the range of 0.1 μm to 1000 μm, particularly 1 μm to 300 μm, respectively.

[0094] Microchannel chips can be fabricated by combining techniques that use molds, such as molding or embossing, which can accurately and easily fabricate channel structures, or techniques that are commonly used by those skilled in the art, such as photolithography, soft photolithography, wet etching, dry etching, nanoimprinting, laser processing, direct electron beam drawing, additive manufacturing (AM), and machining.

[0095] Materials used to fabricate microchannel chips include polymer materials such as PDMS (polydimethylsiloxane) and acrylic, metal materials such as stainless steel, glass, silicone, ceramics, etc. Among these, polymer materials allow the flow channel itself to be fabricated inexpensively and are easy to make in a disposable form. Therefore, it is preferable to use polymer materials at least in part.

[0096] In addition, the flow channels constituting the micro-channel chip are preferably made to have flow channel walls with low affinity to liquids (particularly the dispersed phase liquid described later). The micro-channel chip may be fabricated using a material with low affinity to liquids (particularly the dispersed phase liquid described later), and the portions corresponding to the flow channel walls may be surface-treated with a material with low affinity to liquids (particularly the dispersed phase liquid described later). For example, the micro-channel chip may be fabricated using a polymer material such as PDMS (polydimethylsiloxane), acrylic, cycloolefin polymer, or PTFE (polytetrafluoroethylene), and the flow channel walls may be surface-treated with a hydrocarbon-based silanizing agent, a fluorocarbon-based silanizing agent, or the like.

[0097] (Dispersed phase liquid holding section) The dispersed phase liquid holding section is a section that holds the dispersed phase liquid that is a material for producing an emulsion. The dispersed phase liquid holding section may be an inlet, and liquid can be introduced into the micro-channel chip through the dispersed phase liquid holding section. The dispersed phase liquid holding section may be, for example, a hole and / or a well that extends vertically when the micro-channel chip is in use, and the dispersed phase liquid can be supplied and held in the hole and / or well. The dispersed phase liquid holding section may be, for example, composed of a hole and / or a well with a diameter of 0.1 mm to 20 mm. When the dispersed phase liquid holding section is composed of a hole and a well, the well extending vertically can be connected to the dispersed phase liquid flow channel through the hole extending vertically.

[0098] (Supply of dispersed phase liquid) The dispersed phase liquid can be supplied to the dispersed phase liquid holding portion. In one embodiment, the dispersed phase liquid is supplied after the pre-fill liquid has traveled to the dispersed phase liquid holding portion.

[0099] In order to supply the dispersed phase liquid to the dispersed phase liquid holding section, a separate container (phase liquid holding container) may be used to hold the dispersed phase liquid. From the viewpoint of preventing the liquid from leaking during storage and operation, such a container is preferably completely or variably sealed while holding the dispersed phase liquid.

[0100] The supply of the dispersed phase liquid (and / or the supply of the continuous phase liquid) can be carried out by a dispensing means. The use of a dispensing means is preferable in that it can suppress the remaining amount of the dispersed phase liquid and suppress the measurement time and / or the contamination between the reagents (dispersed phase liquid).

[0101] For example, each phase liquid can be dropped into each holding part using a dispensing means, or each phase liquid can be introduced along the wall surface of each holding part. This is particularly advantageous when the liquid is delivered under negative pressure. In conventional supply methods, particularly in methods in which a tube or manifold is fluidly connected (sealed connected) to each holding part to simultaneously introduce liquid into each holding part and apply liquid delivery pressure, droplets may be generated early before the start of liquid delivery due to unexpected pressure fluctuations at the time of connection and the time required for the pressure to stabilize. In contrast, when a dispensing means is used and the liquid is delivered under negative pressure, there is no pressure fluctuation when a phase liquid supply device and a pressure source are connected to each holding part, so that early droplet generation before the start of liquid delivery can be suppressed.

[0102] The dispensing means is preferably one that does not cause pressure fluctuations in the holding parts. The dispensing means may be, for example, a pipette. Preferably, the dispensing means (particularly, the liquid outlet constituting the dispensing means) is not fluidically connected (sealed connected) to each holding part, but is spatially separated.

[0103] For example, the dispensing means may be a mechanism including a pump, an actuator, and a pipette. It is preferable to use a pump to suck up each phase liquid held in a separate container, move the tip of the pipette to each holding part by the actuator, and then use a pump to push each phase liquid into each holding part. In addition, it is preferable that a part of the pipette or the like that comes into contact with each phase liquid is removable and can be replaced after each use, since this can suppress contamination. Furthermore, it is preferable to use the pump of the dispensing means as a liquid sending means, since this can simplify the device configuration. In addition, when repeated use is intended, the phase liquid may be added by a dispensing means including a disposable pipette, or a common line may be used to add the phase liquid from the phase liquid holding container to the microchannel chip. In the latter case, it is preferable to include a step of washing the shared line up to the connection part to the microchannel chip in order to suppress contamination of the dispersed phase liquid with the continuous phase liquid. As a dispensing means not including a pipette, a method of adding (dripping) each liquid directly from a container holding the phase liquid to the holding part by external force is also preferable (for example, a means of breaking a thermocompression-bonded part of the container by pressure to push out the liquid inside the container, etc.). In addition, when performing a TRC reaction or a PCR reaction, for example, a dispensing means may be used in combination as a purification means or a preparation means for an aqueous solution sample.

[0104] (Dispersed phase liquid flow path) The dispersed phase liquid flow path connects the dispersed phase liquid holding section and the emulsion forming section. The dispersed phase liquid flow path is configured so that the dispersed phase liquid passes therethrough.

[0105] The dimensions of the dispersed phase liquid flow path can be appropriately set depending on the type and characteristics of the dispersed phase liquid used. The dispersed phase liquid flow path can have a width of, for example, 10 to 500 μm or 50 to 200 μm, and a length of 1 mm to 500 mm or 10 to 200 mm. The dispersed phase liquid flow path can have a flow path height of 1 to 200 μm or 10 to 100 μm. The dispersed phase liquid flow path may be bent at one or more locations, and may have a serpentine shape.

[0106] (Continuous phase liquid holding section) The continuous phase liquid holding portion is a portion that holds the continuous phase liquid for generating / holding an emulsion. The continuous phase liquid holding portion may be an inlet, and liquid can be introduced into the microchannel chip through the continuous phase liquid holding portion. The structure of the continuous phase liquid holding portion is not particularly limited as long as it can hold the continuous phase liquid. The continuous phase liquid holding portion may be a hole or well, for example, a hole or well extending in a vertical direction, and the continuous phase liquid can be supplied into and held in this hole or well. The continuous phase liquid holding portion may be, for example, a hole or well having a diameter of 0.1 mm to 20 mm.

[0107] In addition, since the continuous phase liquid generally uses a liquid with low surface tension and viscosity, the amount of change in surface tension accompanying a change in the interface shape in the continuous phase liquid holding section is small. Therefore, the shape of the continuous phase liquid holding section does not have a significant effect on the liquid transfer. In addition, in the present invention, for example, when an emulsion is held and a detection reaction or the like is performed, a sufficient amount of continuous phase liquid is supplied so that the continuous phase liquid in the continuous phase liquid holding section does not run out, so that the remaining amount of continuous phase liquid in the holding section does not decrease during liquid transfer, and the interface shape is likely to change. Therefore, again, the shape of the continuous phase liquid holding section is unlikely to have a significant effect on the liquid transfer.

[0108] (Continuous phase liquid flow path) The continuous phase liquid flow path connects the continuous phase liquid holding section and the emulsion forming section, and is configured so that the continuous phase liquid passes therethrough.

[0109] The dimensions of the continuous phase liquid flow path can be appropriately set depending on the type and characteristics of the continuous phase liquid used. The continuous phase liquid flow path can have a width of, for example, 10 to 500 μm or 50 to 200 μm, and a length of 1 mm to 500 mm or 10 to 200 mm. The continuous phase liquid flow path can have a flow path height of 1 to 200 μm or 10 to 100 μm. The continuous phase liquid flow path may be bent at one or more locations, and may have at least a partially serpentine shape.

[0110] The micro-channel chip may have two or more continuous phase liquid flow paths. In particular, the micro-channel chip according to the present invention has a first continuous phase liquid flow path and a second continuous phase liquid flow path, and these flow paths connect the continuous phase liquid holding section and the emulsion forming section, respectively.

[0111] Referring to the exemplary embodiment of FIG. 14, 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 junction where the dispersed phase liquid flow paths 114 and 115 connected to the emulsion forming section 120 join. In the embodiment of FIG. 14, the first continuous phase liquid flow path 112 and the second continuous phase liquid flow path 113 have substantially the same structure and flow path length, so that the speed of the continuous phase liquid moving through each flow path is substantially the same. In addition, when it is desired to suppress the mixing of the dispersed phase liquids before the emulsion is generated as described above, it is preferable that the length of the flow path connecting the downstream part of the dispersed phase liquid junction and the emulsion forming section 120 is relatively short (for example, 3 mm or less, more preferably 0.5 mm or less), and it is preferable that the dispersed phase liquids maintain a laminar flow state separately within the flow path.

[0112] (emulsion forming part) The emulsion forming section (also referred to as "emulsion forming section") is configured to generate droplets. The emulsion forming section is supplied with a dispersed phase liquid and a continuous phase liquid via a dispersed phase liquid flow path and a continuous phase liquid flow path, respectively. The emulsion forming section is also connected to an emulsion supply flow path, and the emulsion generated in the emulsion forming section is sent to the emulsion supply flow path.

[0113] The emulsion former may have one or more openings that open into the dispersed phase liquid flow path and one or more openings that open into the continuous phase liquid flow path, and may have one or more openings that open into the emulsion feed flow path.

[0114] The emulsion forming section will be described with reference to the exemplary embodiment of FIG. 14. In the emulsion forming section 120 of FIG. 14, the continuous phase liquid flow path 111 consisting of two flow paths 112 and 113 and the dispersed phase liquid junction where the two dispersed phase liquid flow paths 114 and 115 join are substantially perpendicular to each other. During the application of negative pressure, the continuous phase liquid flows into the emulsion forming section 120 from two directions substantially opposite to each other, and the dispersed phase liquid flows into the emulsion forming section 120 in a direction substantially perpendicular to the inflow direction of the continuous phase liquid. As a result, droplets dispersed in the continuous phase (i.e., emulsion) are generated in the emulsion forming section 120. The emulsion generated in this way enters the emulsion holding flow path 140 filled with gas through the emulsion supply flow path 130.

[0115] The emulsion forming section may appropriately use a flow path utilizing a general droplet generation method such as T-junction, Flow-Focus, co-flow, or step-emulsification. In order to generate the emulsion quickly, a plurality of emulsion forming sections may be arranged in parallel. In addition, the emulsion forming section may be provided with a meandering flow path for stirring the droplets in the emulsion.

[0116] (Emulsion supply channel) The emulsion supply flow path is a flow path through which the generated emulsion (droplets and continuous phase) is transported. In one embodiment of the present invention, the emulsion supply flow path connects the emulsion formation section and the emulsion holding flow path. The portion of the emulsion supply flow path adjacent to the emulsion formation section is preferably arranged so as to face the dispersed phase liquid flow path (particularly the dispersed phase liquid junction section). FIG. 14 shows an emulsion supply flow path of such an embodiment. In addition, in FIG. 14, the portion of the emulsion supply flow path adjacent to the emulsion formation section is substantially perpendicular to the inlet section of the continuous phase liquid flow path to the emulsion formation section. In the case of FIG. 14, when an emulsion is generated, the dispersed phase liquid flowing from the dispersed phase liquid flow path to the emulsion formation section becomes droplets, and enters the emulsion supply flow path without changing the flow angle.

[0117] The emulsion supply channel may have an expanded width and / or height downstream (toward the outlet) of the portion adjacent to the emulsion formation section, and / or may be serpentine. Such an embodiment is preferred because it can promote agitation in the droplets.

[0118] (Emulsion holding channel) In one embodiment of the present invention, the microchannel chip has an emulsion holding channel (also referred to as "emulsion holding channel"). The emulsion holding channel is connected to the emulsion forming section via an emulsion supply channel, and has a function of holding the emulsion generated in the emulsion forming section. In addition, the emulsion holding channel is optionally connected to an outlet via an outlet-connected channel.

[0119] The width and length of the emulsion-holding flow channel can be appropriately set according to the volume and number of droplets to be held. For example, it may be a wide, simple flow channel with approximately the same width and length, a single continuous long flow channel arranged in a serpentine or spiral shape, or branched straight flow channels arranged in parallel.

[0120] In the present invention, the cross section of the emulsion holding flow channel is preferably circular, semicircular, elliptical, convex, concave, rectangular or trapezoidal, since the center of the droplets easily flows along the center of the flow channel.

[0121] In the emulsion filling method, emulsions are generally generated and held simultaneously by controlling the movement of the gas-liquid interface in the emulsion-holding channel, so it is desirable that the channel cross-sectional shape is constant and straight without bending so that the shape of the gas-liquid interface during liquid transfer is maintained (the change in shape is small even while moving). However, if the channel width is extremely large relative to the channel height in order to increase the number of droplets to be detected, it may be difficult to stably manufacture a chip having the intended channel structure, and / or the bottom and / or top of the channel may be deformed, and the height of the channel region far from the side of the channel may change due to the liquid transfer pressure or the fixing pressure to the chip, which may adversely affect the measurement (roof collapse). As a countermeasure, for example, the ratio of the channel width to the channel height is preferably 100 or less, more preferably 50 or less, 25 or less, and particularly preferably 10 or less (when there are no pillars as described below). Alternatively, a pillar is provided in the center of the flow channel, and the ratio of the distance between the pillars and / or the distance between the pillar and the side of the flow channel to the flow channel height is, for example, 100 or less, more preferably 50 or less, 25 or less, and particularly preferably 10 or less. On the other hand, when performing a batch detection process using an image sensor or the like, it is preferable that the emulsion-holding flow channel is densely packed on a horizontal surface (for example, in a shape close to a square or circle) because the number of droplets detected can be increased. Therefore, continuous single long flow channels with the same flow channel cross-sectional shape may be arranged in a meandering or spiral shape, or branched straight flow channels may be arranged in parallel. In this case, the interface shape is likely to change during liquid transfer due to the presence of a bend, but the effect can be reduced by adjusting the cross-sectional shape at the bend.

[0122] In one embodiment, it is intended to carry out a detection process on the held emulsion, i.e., the emulsion held in the emulsion holding channel can be optionally subjected to a detection process, which will be described later.

[0123] Preferably, the emulsion-holding flow channel is configured so that most or all of the emulsion held therein does not come into contact with the atmosphere (particularly the external atmosphere) external to the micro-channel chip. Preferably, among the droplets held in the emulsion-holding flow channel, the droplets that are the subject of the detection process do not come into contact with the atmosphere (particularly the external atmosphere). For this purpose, for example, the length of the emulsion-holding flow channel can be set relatively long so that the emulsion can come into contact with the atmosphere (particularly the external atmosphere) only at the downstream end of the emulsion-holding flow channel.

[0124] Preferably, the emulsion held in the emulsion holding flow channel is sealed against the outside atmosphere (particularly the outside air).

[0125] Preferably, the emulsion holding flow path is suitable for carrying out a detection process on the emulsion held in the emulsion holding flow path.

[0126] Preferably, the emulsion holding flow path is configured so that the detection process can be performed on the emulsion that is not exposed to the outside atmosphere. More specifically, for example, a structure that isolates the emulsion from the outside atmosphere is present between the held emulsion and the detection means. This structure is made of, for example, a material that transmits light. In this case, among the emulsions held in the emulsion holding flow path, the emulsion that is not the target of the detection process, for example, the emulsion located at the discharge side end of the emulsion holding flow path, may be exposed to the outside atmosphere.

[0127] Preferably, the flow channel volume of the emulsion holding flow channel is equal to or larger than the total volume of droplets produced in the emulsion forming section (particularly equal to or larger than the total volume of droplets to be detected in the detection process), and / or the flow channel volume of the emulsion holding flow channel is equal to or larger than 1 μL, equal to or larger than 5 μL, or equal to or larger than 10 μL. Such an emulsion holding flow channel allows the detection process to be carried out efficiently. The upper limit of the flow channel volume of the emulsion holding flow channel may be, for example, equal to or smaller than 1000 μL.

[0128] Preferably, the emulsion-holding flow channel has a flow channel volume capable of holding 500 or more, 1000 or more, 2500 or more, 5000 or more, or 10000 or more, and / or 100000 or less, 80000 or less, 60000 or less, or 40000 or less, of droplets having an average volume of 0.1 nL to 10 nL, particularly 0.3 to 3 nL. Detection processing can be performed on these droplets held in the emulsion-holding flow channel.

[0129] The average volume of droplets can be calculated by acquiring bright-field images using an image acquisition device such as a digital camera, and calculating the average volume of droplets for N=10 or more in the acquired images based on the following formula.

[0130] The volume of a spherical droplet and the volume of a disk-shaped droplet (V drop and V disk [nL]) are expressed by the following formulas (1) and (2), respectively. drop , D disk are the diameters of a spherical droplet and a disk-shaped droplet, respectively, when the droplets held in the emulsion holding channel are observed from above in the normal use state of the microchannel chip. Also, in formula (2), h is the channel height of the emulsion holding channel.

[0131]

number

[0132]

number

[0133] Preferably, the droplets in the emulsion to be detected by the detection means (such as a camera) do not overlap each other, and in particular form a monolayer in a plane perpendicular to the detection direction, which can further improve the detection accuracy.

[0134] Particularly preferably, the "channel height" of the emulsion-holding channel is adjusted so that, when the microchannel chip is in use, the droplets held in the emulsion-holding channel do not overlap each other in the vertical direction (i.e., a single droplet layer is formed). When a detection process is performed in such an emulsion-holding channel, the detection accuracy is further improved. Note that the "channel height" usually refers to the length of the channel in the vertical direction (perpendicular direction) when the microchannel chip is in use.

[0135] 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.

[0136] In the emulsion filling method, the emulsion is filled in the emulsion holding channel depending on the flow rate ratio of the continuous phase liquid to the dispersed phase liquid in the emulsion formation, so that, for example, when the flow rate ratio of the continuous phase liquid to the dispersed phase liquid is increased to stabilize the emulsion formation, it is preferable to design the channel height to be larger than usual in order to pack the droplets densely in the horizontal direction. For example, when the flow rate ratio of the continuous phase liquid to the dispersed phase liquid is 8 to 12, the height of the emulsion holding channel can be 2 to 4 times the diameter of the droplets.

[0137] (Stopping the liquid supply) In order to retain the emulsion in the emulsion-holding flow channel, for example, when a desired amount of emulsion is filled in the emulsion-holding flow channel, the liquid supply is stopped and the outlet is optionally closed. The application of negative pressure can also be stopped by (at least partially) opening the outlet to the outside atmosphere. The liquid supply can be stopped after the gas that filled the emulsion-holding flow channel has been completely replaced with the emulsion, but it is preferable to stop the liquid supply before the gas that filled the emulsion-holding flow channel has been completely replaced with the emulsion. In other words, the liquid supply is stopped when a gas-emulsion interface exists in the flow channel or in the outlet.

[0138] (Exhaust port) The outlet connects to an emulsion supply channel or, optionally, to an emulsion holding channel, and can also function as a negative pressure source connection for applying negative pressure to the microchannel chip.

[0139] Preferably, the outlet is adapted to be suitable for connection to a negative pressure source, more preferably, the outlet is resistant to the applied pressure.

[0140] Typically, the outlet is located downstream of the emulsion supply channel or, optionally, downstream of the emulsion holding channel, where application of negative pressure to the outlet can fill the entire length or most of the emulsion holding channel with the generated emulsion.

[0141] (liquid delivery) In one embodiment of the present invention, an emulsion containing droplets of the dispersed phase liquid and a continuous phase of the continuous phase liquid is generated in the emulsion forming section by applying negative pressure to the outlet, and the emulsion thus generated is transported to the emulsion supply channel. In an embodiment having an emulsion holding channel, the emulsion thus generated is transported via the emulsion supply channel to the emulsion holding channel, which is an optional component.

[0142] While the liquid is being fed by applying negative pressure to the outlet, the dispersed phase liquid holding section and the continuous phase liquid holding section can be open to the outside atmosphere (particularly the outside atmosphere).

[0143] From the viewpoint of simplicity of the device, it is preferable to keep each holding section at normal pressure while applying negative pressure to the outlet to transfer the liquid, but the pressure may be controlled to an emergency pressure state in order to stabilize the transfer of the liquid under negative pressure and / or to also serve as a sealing operation for retaining the emulsion.

[0144] With regard to application of negative pressure, for example, a pressure tank or syringe pump can be used to draw fluid (gas or continuous phase liquid) through the outlet.

[0145] When a pressure tank is used as the negative pressure source, the volume of the pressure tank is preferably larger than the total volume of the flow path from the outlet to the pressure tank and the volume of the flow path of the micro-channel chip. The pressure tank can also be designed so as to be open to the external atmosphere (external air). It is also preferable that the pressure in the pressure tank is controlled by, for example, a pump. A pressure sensor may also be provided so that the pressure value in the pressure tank can be monitored.

[0146] By using a monitoring means for monitoring the pressure value of the applied negative pressure, it is possible to check the liquid transfer state, for example, whether an emulsion is being formed without any problems.

[0147] In one embodiment of the present invention, a negative pressure control means is fluidly connected to the outlet, and the negative pressure control means is composed of a negative pressure source, a connection part, and a valve, the negative pressure source is controlled to a constant negative pressure, and the valve is disposed between the negative pressure source and the connection part. The negative pressure control means can be connected to the outlet via the connection part. According to this embodiment, negative pressure can be applied instantly by opening the valve, so that the timing of application of negative pressure can be controlled more accurately.

[0148] There is no particular limitation on the specific embodiment of the valve. From the viewpoint of preventing backflow when liquid transfer is stopped, it is preferable that the valve can suppress pressure fluctuations in the flow channel when opening and closing, for example, that the opening and closing operation is relatively slow. The valve may be, for example, a three-way valve, and may be capable of connecting the microchannel chip to either a negative pressure source (for example, a pressure tank) or the external atmosphere (particularly the external atmosphere).

[0149] (Installation of microfluidic chip) Microchannel chips are generally installed horizontally in the vertical direction (top-to-bottom direction), but they may be installed with an intentional inclination in a certain direction from the viewpoint of emulsion retention, etc. For example, when the continuous phase liquid has a higher specific gravity than the dispersed phase liquid (e.g., when a fluorine-based dispersant is used as the continuous phase liquid and an aqueous solution is used as the dispersed phase liquid), the droplets have buoyancy due to the difference in specific gravity, so the microchannel chip may be installed with an intentional inclination so that the droplets are less likely to flow out of the emulsion-holding channel.

[0150] <Detection process> A detection process can be performed on the liquid in the microchannel chip, particularly on droplets in the emulsion. The detection process includes, for example, a reaction of a target substance in the liquid (particularly droplets) and detection of the reaction (for example, detection of a reaction product). In an embodiment having an emulsion-holding channel, the detection process is usually performed on droplets in the emulsion held in the emulsion-holding channel.

[0151] 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.

[0152] The reaction of the target substance is not particularly limited. Examples of the reaction of the target substance include enzyme reactions, more specifically, enzyme reactions using kinases, nucleases, nucleotide cyclases, nucleotide ligases, nucleotide phosphodiesterases, (DNA or RNA) polymerases, prenyltransferases, pyrophospatases, reporter enzymes, reverse transcriptases, topoisomerases, and the like. When the target molecule is a nucleic acid such as DNA or RNA, and the reaction of the target molecule is an amplification reaction of the nucleic acid, examples of the reaction include reactions capable of isothermal amplification of nucleic acids, such as the LAMP method, the NASBA method, the TMA method, and the TRC method. In addition, in the case of one-step RT-PCR, it is preferable to prepare droplets at a temperature suitable for the reverse transcription reaction in terms of the reaction efficiency and reaction time of the reverse transcription reaction. In addition, it is also possible to detect cDNA, which is a product of the reverse transcription reaction, by the cycling probe method.

[0153] When carrying out a reaction, it is preferable to mix two or more types of reaction liquids upstream of the emulsion forming section (for example, the dispersed phase liquid merging section) and generate droplets using this mixture. In the present invention, the reaction liquid refers to a solution containing at least a part of the target substance and the components necessary for reacting the target substance. It is sufficient that all the components necessary for the reaction of the target substance are prepared by mixing all the reaction liquids, and the target substance is contained in one of the reaction liquids. There is no problem even if three or more types of reaction liquids are used.

[0154] 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 this specific sequence, the components contained in the reaction solution include a primer containing a sequence homologous to a part of the specific sequence, a primer containing a sequence complementary to a part of the specific sequence, a detection probe containing a sequence homologous or complementary to a part of the specific sequence, a polymerase, a nucleotide, salts, and a buffer component. Note that it is preferable that the composition of the reaction solution is designed so that the target molecule, reaction substrate, enzyme, etc. do not decompose, change in quality, or cause a non-specific reaction, and glycerol, a surfactant, etc. may be further added in consideration of the behavior in the device.

[0155] (Detection means) To detect the reaction, for example, a detection means capable of detecting the product of the reaction can be used.

[0156] The detection method can be appropriately selected according to the reaction product, and can be detected using known methods such as optical, X-ray, MALDI (matrix-assisted laser desorption / ionization), FCS (fluorescence correlation spectroscopy), FP (fluorescence polarization) / FCS, fluorometry, colorimetry, chemiluminescence, bioluminescence, scattering, surface plasmon resonance, electrochemistry, electrophoresis, laser, mass spectrometry, Raman spectroscopy, FLIPR (Molecular Devices), etc. When detecting using transmitted light, it is preferable to prepare a microchannel chip from a material that transmits light, since the reaction product can be detected by simply placing the microchannel chip on an optical detector without moving the droplets in the chip.

[0157] The detection means (detector) used to detect the reaction products can be an imaging sensor and optionally its components for recording and measuring the reaction of the target substance. One example of the detection is a camera or imaging device with suitable illumination and resolution to spatially resolve the individual signals to be detected. The camera or imaging device can be any known one, for example, the camera can be any common semiconductor image sensor including a charge-coupled device (CCD), a charge injection device (CID), a photodiode array (PDA) or a complementary metal oxide semiconductor (CMOS). Detection can also be improved by using polarization of the excited / emitted light. For example, when detecting droplets that emit fluorescent signals, the detection area can be photographed all at once by an optical unit with a large field of view, allowing rapid and high-throughput signal detection.

[0158] <others> (Temperature control means) The temperature control means has a role of maintaining the liquid in the microchannel chip at a temperature suitable for the reaction of the target substance. The temperature control means does not necessarily have to be flat as long as it can be in close proximity (preferably in close contact) with the microchannel chip.

[0159] Of the temperature control means, at least a portion close to (preferably in close contact with) the microchannel chip is preferably made of a metal material with high thermal conductivity. When the microchannel chip is manufactured by bonding a substrate and a superstructure, it is preferable to reduce the thickness of the substrate and / or superstructure in contact with the temperature control means, since this allows for more efficient heat conduction to the channels provided in the microchannel chip. The temperature control means is required to at least control the temperature of the emulsion-holding channel, which is the reaction field of the target substance, but it is preferable to control the temperature of the phase liquid supply unit and the channel as well, since this can suppress nonspecific reactions of the target molecules. As a specific example, when the reaction of the target substance is a nucleic acid amplification reaction, the temperature control means controls the temperature in each holding unit and channel so that it is higher than the reaction temperature of the target substance in the emulsion-holding channel, thereby reducing nonspecific annealing between primers / probes. Furthermore, when the bottom surface of the micro-channel chip is heated to the reaction temperature by a temperature control means, and the top surface substrate of the micro-channel chip is made of a light-transmitting material and transmitted light detection is performed from the top surface, this is preferable because it enables easy evaluation of the position of the empty micro-channel chip before supply of each phase liquid and / or the flow channel structure and / or dust inside and outside the chip, evaluation of the behavior inside the flow channel when supplying each phase liquid and / or the behavior of emulsion generation during liquid transfer, and improvement of the upper limit of quantification of digital detection using the signal detection results of the emulsion during the reaction to be performed on the device. EXAMPLES

[0160] The present invention will be described in more detail below with reference to examples, but the present invention is not limited to these descriptions.

[0161] Example 1 <Fabrication of microfluidic chip> A microfluidic chip was fabricated using photolithography and soft lithography techniques. The specific steps are as follows.

[0162] (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).

[0163] (2) Using a mask aligner (Ushio Inc.) and a chrome mask on which the flow channel pattern of the microfluidic chip was formed, the flow channel pattern was formed on a photoresist film. The flow channel pattern was then developed using SU-8 Developer (Microchem Inc.) to create a mold for the flow channels that make up the microfluidic chip.

[0164] (3) To suppress adsorption onto SU-8, a surface treatment was performed by vapor deposition using trichloro(1H,1H,2H,2H-perfluoro-octyl)silane (Thermo Fisher Scientific).

[0165] (4) A mixture of uncured siloxane monomer and polymerization initiator (weight ratio 10:1) prepared using a Sylgard Silicone Elastomer Kit (Dow Corning Toray Co., Ltd.) was poured into the mold treated in (3) above, and heated at 80°C for 2 hours to produce a polymer (PDMS) substrate onto which the shape of the flow path was transferred.

[0166] (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.

[0167] (6) The polymer substrate with the holding part and the outlet formed and the cover glass (Matsunami Glass Co., Ltd.) were surface-treated with an oxygen plasma generator (Meiwa Force Systems Co., Ltd.), and then the patterned surface of the PDMS substrate was bonded to the cover glass. The fabricated chip was stored in a desiccator.

[0168] The fabricated microchannel chip was 34 mm long and 75 mm wide, and had a φ4 mm hole as the dispersed phase liquid holding area, a φ8 mm hole as the continuous phase liquid holding area, and a φ1.5 mm hole as the outlet.

[0169] (Flow path structure) The microchannel chip used in the examples 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 supply 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 supply flow path, and the emulsion holding flow path was connected to the outlet.

[0170] Therefore, the microchannel chip used in the examples had the channel structure shown in FIG. 14, except that the emulsion supply channel and emulsion holding channel had the structures shown in FIGS.

[0171] The details of the channel structure of the microchannel chip are as follows.

[0172] The first dispersed phase liquid flow path and the second dispersed phase liquid flow path are meandering flow paths with a height of 80 μm, a width of 100 μm and a length of 30 mm. They join at a dispersed phase liquid junction, where the flow path width is narrowed to 80 μm, and then join the emulsion formation section.

[0173] Each of the two continuous phase liquid flow paths is a straight flow path with two bends, measuring 80 μm in height, 100 μm in width, and 30 mm in length. In the emulsion formation section, the dispersed phase liquid junction and the two continuous phase liquid flow paths cross at an angle of 90 degrees, allowing the reaction liquid and immiscible liquid (oil) to join together and form droplets.

[0174] The emulsion supply channel is a straight channel with height 80μm x width 80μm x length 100μm in the part close to the emulsion formation part, height 80μm x width 200μm x length 680μm in the downstream part, and further downstream is a stirring channel with width 200μm x length 11.5mm including a meandering composed of an arc curve with height 80μm and radius 275μm, which is connected to the emulsion holding channel. The angle between the liquid sending direction of the emulsion supply channel and the liquid sending direction of the emulsion holding channel is 90°.

[0175] The emulsion-holding channel is a serpentine channel with a channel height of 200 μm, a width of 2 mm, and a length of 350 mm, and is directly connected to the outlet via an outlet-connected channel with a width of 2 mm and a length of 10 mm. The volume of the emulsion-holding channel was 91 μL. The emulsion-holding channel forms an expansion angle of 90° in both the left and right directions with respect to the emulsion supply channel.

[0176] The emulsion reservoir has a flow channel height of 200 μm or 80 μm (emulsion guide section), and is 1.0 mm in width and height. 2 A part of the emulsion reservoir forms a part of the emulsion guiding section, and the ceiling surface of the region with a flow channel height of 80 μm (emulsion guiding section) and the ceiling surface of the region with a flow channel height of 200 μm form a step structure at a substantially right angle.

[0177] The emulsion guide section has a flow channel height of 80 μm and a width and height of 1.4 mm. 2 The emulsion holding flow channel has an area of ​​about 100 μm, and a step structure is formed at a substantially right angle to the ceiling surface of an area of ​​the emulsion holding flow channel with a flow channel height of 200 μm. In order to facilitate the maintenance of an air-liquid interface near the opening of the emulsion supply flow channel, the above-mentioned step structure is located 30 μm away from the emulsion supply flow channel opening in the liquid sending direction of the emulsion supply flow channel, and an induction region formed by this step structure and the side wall surface of the extended flow channel in the opposite direction to the liquid sending direction of the emulsion holding flow channel has a flow channel height of 80 μm, a width of 30 μm and a length of 100 μm, and is connected to the emulsion induction section of the emulsion storage section.

[0178] (Evaluation of air bubble removal) The microchannel chip according to the above embodiment was used to examine the bubble removal capability.

[0179] Specifically, when the flow channels of the microchannel chip are filled with gas and continuous phase liquid is supplied to the continuous phase liquid holding section, there is a high probability that air bubbles will be generated during the process in which the branching structure of the continuous phase liquid flow channels is filled with the continuous phase liquid, and during the process in which the two continuous phase liquid flow channels join at the emulsion forming section, causing the tip of the emulsion supplied to the emulsion supply flow channel to contain air bubbles. Then, the emulsion is supplied from the emulsion supply flow channel to the emulsion holding flow channel without any external driving force. Figures 13(A) to 13(E) show a series of photographs taken from the top of the microchannel chip at this time.

[0180] Specifically, a digital CMOS camera (ORCA-FLASH (product name), Hamamatsu Photonics) was used to capture bright-field images of the emulsion-holding channel being filled with the emulsion. Figure 13 shows time-lapse images, with time passing in the order of Figure 13(A) to Figure 13(E).

[0181] 13(A) shows the emulsion supply flow path 130 and the emulsion holding flow path 140 in a state where they are filled with gas. Here, this emulsion holding flow path 140 has an emulsion reservoir section 142 upstream of the emulsion inlet, and an emulsion guide section 144 located in the vicinity of the emulsion inlet.

[0182] 13(B) shows a state in which an emulsion 210 having droplets 211 and bubbles 220 flows from the emulsion supply flow path 130 into the emulsion holding flow path 140. Here, it is shown that the emulsion 210 flows along the emulsion guide portion 144 toward the emulsion reservoir portion 142, which is upstream of the emulsion inlet.

[0183] FIG. 13(C) shows a state in which air bubbles present in the emulsion collide with the air-liquid interface near the emulsion inlet of the emulsion holding channel 140 and are removed.

[0184] 13(D) and (E) show the state in which emulsion containing no air bubbles fills the emulsion holding channel 140. FIG.

[0185] As described above, in the embodiment, the air bubbles present at the tip of the emulsion could be removed by colliding with the air-liquid interface near the emulsion inlet of the emulsion holding flow path. [Explanation of symbols]

[0186] 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 120 Emulsion forming section 130 Emulsion supply channel 140 Emulsion holding channel 142 Emulsion storage section 144 Emulsion Derivative 150 Outlet 210 Emulsion 211 Emulsion Droplets 220 Bubbles W: Width direction of the microfluidic chip L: Length direction of the microfluidic chip U h Upstream direction of emulsion holding channel D h Downstream of the emulsion holding channel W h Width direction of emulsion holding channel H hHeight direction of emulsion holding channel

Claims

1. dispersed phase liquid holding section, A dispersed phase liquid flow path connected to the dispersed phase liquid holding portion; A continuous phase liquid holder; A continuous phase liquid flow path connected to the continuous phase liquid holding portion; an emulsion forming section connected to the dispersed phase liquid flow path and the continuous phase liquid flow path; an emulsion supply flow path connected to the emulsion forming section; and an emulsion holding channel connected to the emulsion supply channel; A microchannel chip for filling an emulsion, comprising: when a dispersed phase liquid is supplied to the dispersed phase liquid holding section, a continuous phase liquid is supplied to the continuous phase liquid holding section, and an external liquid sending driving force is applied to the micro-channel chip, an emulsion containing droplets constituted of the dispersed phase liquid and a continuous phase constituted of the continuous phase liquid is generated in the emulsion forming section, and the emulsion enters the emulsion holding channel via the emulsion supply channel and a single emulsion inlet, (i) the emulsion holding flow path has an emulsion reservoir in a region where the expansion angle with respect to the direction in which the emulsion flows from the emulsion supply flow path through the emulsion inlet into the emulsion holding flow path is 60° or more, and / or in a region upstream of the emulsion inlet, As a result, when the emulsion flows from the emulsion supply flow path into the emulsion holding flow path while replacing the gas in the emulsion holding flow path, at least a portion of the emulsion flows into the emulsion storage section, thereby temporarily maintaining a gas-liquid interface between the emulsion and the gas in the emulsion holding flow path in the vicinity of the emulsion inlet, and / or (ii) the emulsion holding flow path has an emulsion guide portion in the vicinity of the emulsion inlet, As a result, when the emulsion flows from the emulsion supply flow path into the emulsion holding flow path while replacing the gas in the emulsion holding flow path, at least a part of the emulsion is guided to the emulsion guide section, so that a gas-liquid interface between the emulsion and the gas in the emulsion holding flow path is temporarily maintained in the vicinity of the emulsion inlet. Microfluidic chip for emulsion filling.

2. The microchannel chip according to claim 1 , which satisfies the condition (i).

3. The micro-channel chip according to claim 2 , wherein the emulsion holding channel has the emulsion reservoir in a region where the expansion angle is 60° or more.

4. The micro-channel chip according to claim 3 , wherein the emulsion holding channel has the emulsion reservoirs on the right and left sides with respect to a flow direction of the emulsion in the emulsion supply channel.

5. The micro-channel chip according to claim 2 , wherein the emulsion holding channel has the emulsion reservoir in a region upstream of the emulsion inlet.

6. 6. The microchannel chip according to claim 5, wherein an angle between a direction in which the emulsion flows from the emulsion supply channel through the emulsion inlet into the emulsion holding channel and a flow direction of the emulsion holding channel in the vicinity of the emulsion inlet is 30° or more.

7. The microchannel chip according to claim 1 , which satisfies the condition (ii).

8. The micro-channel chip according to claim 7 , wherein the emulsion guide portion has a stepped structure.

9. The microchannel chip according to claim 1 , which satisfies both of (i) and (ii).

10. The micro-channel chip according to claim 9 , wherein the emulsion guide portion guides at least a portion of the emulsion toward the emulsion reservoir portion.

11. 2. The microchannel chip according to claim 1, wherein the width of the emulsion holding channel is at least four times the width of the emulsion inlet.

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