JP2024038752A - microchannel chip - Google Patents

Abstract

The present invention provides a microchannel chip capable of improved droplet generation, particularly improved droplet generation and droplet retention. [Solution] A dispersed phase liquid holding section, a dispersed phase liquid flow path connected to the dispersed phase liquid holding section, a continuous phase liquid holding section, and a first continuous phase liquid flow connected to the continuous phase liquid holding section. A microchannel chip having a channel 212, a second continuous phase liquid flow path 213, an emulsion forming section 220, and an emulsion flow path 230 connected to the emulsion forming section, the microchannel chip having a dispersed phase liquid flow path. a channel is connected to the emulsion forming section between the first continuous phase liquid channel and the second continuous phase liquid channel, and the flow direction of the first continuous phase liquid channel into the emulsion forming section; and the inflow direction of the second continuous phase liquid flow path into the emulsion forming section form an angle α of 0° or more and less than 180°. [Selection diagram] Figure 2

Description

The present invention relates to an apparatus for producing an emulsion in a microchannel chip. In particular, the present invention relates to an apparatus that can perform droplet array measurements more efficiently, simply, and quickly.

A microdroplet method is known as a technique for fractionating a reaction solution into microcompartments and conducting reactions independently. This method is expected to be applied, for example, to the production of micro/nanoparticles, and in particular, it uses a microfluidic device to divide the target molecule into microcompartments in units of molecules and conduct reactions within microdroplets. It is used in digital measurements to measure the presence or absence of target molecules based on the presence or absence of signals, and to perform absolute quantification of the number of target molecules.

The microdroplet method generally uses an emulsion consisting of a continuous phase such as oil and droplets of an aqueous solution dispersed in the continuous phase.

Non-Patent Document 1 discloses a centrifugal step droplet generation method. This document describes that after filling the inlet of the device with oil and sending this oil to a droplet collection chamber by centrifugation, a sample solution is introduced from the same inlet and droplets are generated by centrifugation. are doing.

For such droplet generation methods, a dispersed phase liquid such as a reaction liquid and a continuous phase liquid such as oil are supplied to a microchannel chip through separate supply sections, and are combined in the chip to form an emulsion. There are known methods for performing this generation.

US Pat. No. 5,090,502 discloses such a system and method for producing droplets suitable for droplet assays. The document describes transporting the generated droplets to an exit area consisting of a pipette tip or a droplet well. The document also describes an air bubble trap that essentially keeps the sample and oil together until the application of a fluid motive force (such as negative or positive pressure). It states that it will be separated.

Regarding the microdroplet method, in recent years, droplet array measurement, in which droplets are arranged in a single layer in a detection region to easily measure signals, has been attracting attention from the viewpoint of simplifying and speeding up the apparatus.

Patent Documents 2 and 3 disclose microchannel chips having a flow path for forming droplets and a droplet holding section for holding the droplets. Patent Document 2 describes that two or more reaction liquids are brought together and then an immiscible liquid that is immiscible with the reaction liquid is brought into contact with the liquid to form droplets. Patent Document 3 describes that the dispersed phase and the continuous phase that have flowed in from the dispersed phase inflow section and the continuous phase inflow section are made into droplets by being brought into contact with each other in a droplet generation section via a flow path.

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

Non-Patent Document 3 describes droplet generation by the Flow Focusing method. The microfluidic device described in this document has a central channel for a liquid such as water, two outer channels for oil etc., and an orifice section, and has two liquid phases (i.e. water and oil, etc.) is adapted to flow through small orifices located downstream of the central channel and the two outer channels. The pressure and viscous stress caused by the two liquids (oil) flowing in the two outer channels forces the liquid (water) flowing in the central channel into the narrow channel (orifice section), thereby causing the orifice section to Droplets are generated within or downstream thereof.

European Patent No. 2550528 JP 2019-170363 Publication JP2020-169911A

Centrifugal step emulsification applied for absolute quantification of nuclear acids by digital droplet RPA, Lab Chip, 20 15, 15, 2759-2766 1-Million droplet array with wide-field fluorescence imaging for digital PCR, Lab on a Chip, 2011, 11, 3838-3845 Formation of dispersions using "flow focusing" in microchannels Appl. Phys. Lett. , Vol. 82, No. 3,20 January 2003

When generating droplets in a microchannel chip, two continuous phase liquids are sent through two separate continuous phase liquid channels, and a dispersed phase liquid is delivered to an emulsion forming part where these two continuous phase liquids meet. By feeding the dispersed phase liquid, the dispersed phase liquid can be formed into droplets (flow-focusing method, etc.).

In an emulsion forming section (also referred to as a "droplet generating section") where an emulsion containing droplets is generated, blockage of a flow path or generation of air bubbles may occur. In particular, when a continuous phase liquid passes through an empty channel (particularly a channel filled with gas), as in the emulsion filling method described below, bubbles are likely to occur and droplet generation becomes unstable. There was a risk that More specifically, for example, if there is a difference in the timing at which continuous phase liquids traveling through two gas-filled continuous phase liquid channels reach the emulsion forming section, the continuous phase liquid may arrive at the emulsion forming section first. The other continuous phase liquid flow path was blocked by the continuous phase liquid, gas remained between the continuous phase liquids, and this gas was further divided to generate bubbles.

The bubbles generated in the channel cause a problem of obstructing the progress of the liquid in the channel, and may also interfere with the detection reaction to droplets in the microchannel chip. Specifically, for example, a method in which droplets are generated and held on one microchannel chip is advantageous from the viewpoint of simple and rapid measurement, but it is relatively difficult to remove air bubbles, and However, the detection reaction may be inhibited by air bubbles remaining in the emulsion holding channel, which is the droplet holding section.

The present invention aims to provide a microchannel chip capable of improved droplet generation, particularly improved droplet generation and droplet retention.

<Aspect 1>
dispersed phase liquid holding section,
a dispersed phase liquid flow path connected to the dispersed phase liquid holding section;
continuous phase liquid holding section,
a first continuous phase liquid flow path and a second continuous phase liquid flow path connected to the continuous phase liquid holding section;
an emulsion forming section, and
It has an emulsion flow path connected to the emulsion forming section,
The dispersed phase liquid flow path, the first continuous phase liquid flow path, and the second continuous phase liquid flow path are connected to the emulsion forming section,
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 feeding driving force is applied to the microchannel chip, the emulsion forming section An emulsion including droplets made of the dispersed phase liquid and a continuous phase made of the continuous phase liquid is produced, and the emulsion enters the emulsion flow path.
A microchannel chip,
The dispersed phase liquid flow path is connected to the emulsion forming section between the first continuous phase liquid flow path and the second continuous phase liquid flow path, and The inflow direction of the first continuous phase liquid flow path and the inflow direction of the second continuous phase liquid flow path into the emulsion forming section form an angle α of 0° or more and less than 180°.
Microfluidic chip.
<Aspect 2>
The emulsion forming part includes a phase liquid collection part and an orifice part connected to this,
The emulsion flow path is connected to the emulsion forming section via the orifice section,
The inflow direction of the first continuous phase liquid flow path into the emulsion forming section and the flow path direction of the orifice section form an angle γ1, and
The inflow direction of the second continuous phase liquid flow path into the emulsion forming part and the flow path direction of the orifice part form an angle γ2,
The angle γ1 and the angle γ2 are both 0° or more and less than 90°,
The microchannel chip according to aspect 1.
<Aspect 3>
The inflow direction of the first continuous phase liquid flow path into the emulsion forming section and the inflow direction of the dispersed phase liquid flow path into the emulsion forming section form an angle β1 of 0° or more and less than 90°. and
The inflow direction of the second continuous phase liquid flow path into the emulsion forming section and the inflow direction of the dispersed phase liquid flow path into the emulsion forming section form an angle β2 of 0° or more and less than 90°. ing,
The microchannel chip according to aspect 1 or 2.
<Aspect 4>
The emulsion forming section includes a phase liquid collection section and an orifice section connected thereto,
A connecting portion connecting the side wall surface of the first continuous phase liquid flow path and the side wall surface of the orifice portion, and/or the side wall surface of the second continuous phase liquid flow path and the side wall of the orifice portion. The connection part that connects the part wall surface is rounded,
The microchannel chip according to any one of aspects 1 to 3.
<Aspect 5>
Before applying the external liquid feeding driving force, the first continuous phase liquid channel filled with gas and the second continuous phase liquid channel filled with gas are transferred from the continuous phase liquid holding section to the first continuous phase liquid channel filled with gas. 5. The microchannel chip according to any one of aspects 1 to 4, for use in a method comprising filling a continuous phase liquid through the emulsion forming section filled with gas.
<Aspect 6>
The microchannel chip according to any one of aspects 1 to 5, further comprising an emulsion holding channel.
<Aspect 7>
The microchannel chip according to any one of aspects 1 to 6, for use in an emulsion filling method.

According to the present invention, it is possible to provide a microchannel chip capable of improved droplet generation, particularly improved droplet generation and droplet retention.

FIG. 1 is a schematic plan view of one embodiment of a microchannel chip according to the present disclosure. FIG. 2 is a schematic plan view showing a channel structure near an emulsion forming part of a microchannel chip according to one embodiment of the present disclosure. FIG. 3 is the same embodiment as the embodiment of FIG. 2, and is a schematic plan view for explaining the relationship between two continuous phase liquid channels and an orifice section. FIG. 4 is the same embodiment as the embodiment of FIG. 2, and is a schematic plan view for explaining the relationship between two continuous phase liquid channels and a dispersed phase liquid channel. FIG. 5 is a schematic plan view showing a channel structure near an emulsion forming part in a microchannel chip according to another embodiment of the present disclosure. FIG. 6a is a schematic plan view showing the channel structure near the emulsion forming part of the microchannel chip according to Example 1. FIG. 6b is a photograph showing how droplets are generated in Example 1. FIG. 7 is a schematic plan view showing the channel structure near the emulsion forming part of the microchannel chip according to Comparative Example 1. FIG. 8a is a schematic plan view showing the channel structure near the emulsion forming part of the microchannel chip according to Example 2. FIG. 8b is a photograph showing how droplets are generated in Example 2. FIG. 9 is a schematic plan view showing the channel structure near the emulsion forming part of the microchannel chip according to Comparative Example 2. FIG. 10 is a schematic plan view showing the channel structure near the emulsion forming part of the microchannel chip according to Example 3. FIG. 11 is a schematic plan view showing the channel structure near the emulsion forming part of the microchannel chip according to Comparative Example 3.

The microchannel chip according to the present disclosure includes:
dispersed phase liquid holding section,
a dispersed phase liquid flow path connected to the dispersed phase liquid holding section;
continuous phase liquid holding section,
a first continuous phase liquid flow path and a second continuous phase liquid flow path connected to the continuous phase liquid holding section;
an emulsion forming section, and
It has an emulsion flow path connected to the emulsion forming part,
The dispersed phase liquid flow path, the first continuous phase liquid flow path, and the second continuous phase liquid flow path are connected to the emulsion forming section,
When the dispersed phase liquid is supplied to the dispersed phase liquid holding section, the continuous phase liquid is supplied to the continuous phase liquid holding section, and an external liquid feeding driving force is applied to the microchannel chip, the dispersed phase is An emulsion is produced that includes droplets made up of a liquid and a continuous phase made up of a continuous phase liquid, and the emulsion enters an emulsion flow path.
A microchannel chip,
The dispersed phase liquid flow path is connected to the emulsion forming section between the first continuous phase liquid flow path and the second continuous phase liquid flow path, and the first continuous phase liquid flow path is connected to the emulsion forming section. The inflow direction of the channel and the inflow direction of the second continuous phase liquid channel into the emulsion forming section form an angle α of 0° or more and less than 180°.

For droplet generation using a microfluidic chip, two continuous phase liquids flow into the emulsion formation section through two separate continuous phase liquid channels that are empty (particularly filled with gas). , droplets can be generated by contacting the dispersed phase liquid. However, it has been found that when the two continuous phase liquids enter the emulsion forming section, the flow path may be blocked and bubbles may be generated. Such channel blockage and generation of bubbles often occur due to timing shifts in the arrival of the two continuous phase liquids to the emulsion forming section. If the timing of the arrival of the two continuous phase liquids to the emulsion forming part is different, gas will be left behind in the continuous phase liquid flow path and flow path blockage will easily occur, and the gas will be separated in the flow path and bubbles will form. is more likely to occur.

In contrast, in the present invention, the inflow direction of the first continuous phase liquid flow path into the emulsion forming section and the inflow direction of the second continuous phase liquid flow path into the emulsion forming section are 0° or more and 180°. forming an angle α less than °. According to such a configuration, even if there is a difference in the timing at which the two continuous phase liquids reach the emulsion forming section, blockage of the flow path and generation of bubbles can be suppressed.

Although not intended to be limited by theory, according to the configuration of the present invention, even if the timing of the two continuous phase liquids reaching the emulsion forming part is different, the collision angle of the continuous phase liquid with respect to the gas in the flow path is relaxed. Therefore, the possibility of gas being separated by the continuous phase liquid is reduced, and as a result, it is thought that the generation of bubbles is suppressed.

The present invention has a particularly advantageous effect when the dispersed phase liquid reaches the emulsion forming section before the continuous phase liquid.

In other words, when supplying a dispersed phase liquid and a continuous phase liquid to a microchannel chip filled with gas, the dispersed phase liquid is first filled up to the inlet of the emulsion forming part, and then the continuous phase liquid is supplied to the chip. can do. According to such an embodiment, it is possible to suppress the continuous phase liquid from entering the dispersed phase liquid flow path, so that the stability of droplet generation can be improved. However, in such an embodiment, pressure is applied from the dispersed phase liquid present at the inlet to the emulsion forming part to the gas present between the two continuous phase liquids, and as a result, bubbles are generated. There is a possibility that the possibility of On the other hand, according to the present invention, even in such a case, the collision angle of the continuous phase liquid with respect to the gas can be relaxed, so that the generation of bubbles can be suppressed.

In addition, in the microchannel chip according to the present invention, before applying an external liquid feeding driving force, from the continuous phase liquid holding section, the first continuous phase liquid channel filled with gas and the first continuous phase liquid channel filled with gas are This is particularly advantageous when the continuous phase liquid is used in a method in which the continuous phase liquid passes through the second continuous phase liquid flow path and fills the emulsion forming section filled with gas.

Furthermore, the present invention has particularly advantageous effects when used in a microchannel chip for "emulsion filling method." In the "emulsion filling method," an emulsion (droplets + continuous phase) generated in an emulsion forming section is transported to a droplet holding section (emulsion holding channel) filled with gas. More specifically, in the "emulsion filling method," droplets generated in the emulsion forming section move toward the discharge port in an emulsion holding channel filled with gas, and the emulsion holding flow is Fill the tract. That is, the "gas-liquid interface" formed by the emulsion and the gas filling the emulsion holding channel moves toward the downstream (or outlet) of the emulsion holding channel.

In the "emulsion filling method," the liquid moves through a channel filled with gas, so there is a relatively high possibility that bubbles will occur, and the bubbles that are generated will hold the emulsion in the emulsion holding channel. There is a relatively high risk that the stability of the droplets and the detection reaction to the droplets will be inhibited. According to the present invention, since the generation of bubbles can be effectively suppressed, droplet stability and better detection reaction in the emulsion filling method can be realized.

The present invention will be exemplarily explained below using the drawings. Note that the drawings are exemplary schematic diagrams for explaining the present invention, are not to scale, and do not limit the present invention. Unless otherwise specified, in the figures, L represents the length direction and W represents the width direction.

The present invention will be specifically explained using the microchannel chip shown in FIG. Note that the microchannel chip in FIG. 1 is a schematic diagram of an exemplary embodiment, and the present invention is not limited to this embodiment and can be implemented with various microchannel chips.

The microchannel chip 10 of FIG. 1 has a planar configuration, ie, emulsion generation, transport, and retention occur substantially within one plane. Direction W in FIG. 1 indicates the width direction, and direction L indicates the length direction. The direction perpendicular to W and L is the vertical direction. The microchannel chip 10 in FIG. A dispersed phase liquid flow path (consisting of a dispersed phase liquid confluence section), a continuous phase liquid holding section 101, a continuous phase liquid flow path 111, an emulsion forming section 120, an emulsion flow path 130, an emulsion holding flow path 140, and a discharge port. It has 150. Note that in the present invention, the emulsion holding channel, which is a droplet holding section, is an optional component. The dispersed phase liquid holding sections 102 and 103 are connected to the emulsion forming section 120 via a first dispersed phase liquid flow path 114 and a second dispersed phase liquid flow path 115, respectively, and the continuous phase liquid holding section 101 is , are connected to the emulsion forming section 120 via a continuous phase liquid flow path 111. In the embodiment according to FIG. 1, the continuous phase liquid flow path 111 includes a first continuous phase liquid flow path 112 and a second continuous phase liquid flow path 113. Emulsion forming section 120 is connected to emulsion holding channel 140 via emulsion channel 130, and emulsion holding channel 140 is connected to discharge port 150.

When generating droplets using the microchannel chip shown in FIG. A continuous phase liquid such as oil is supplied to the continuous phase liquid holding unit 101 (which is an inlet for the continuous phase liquid). At this point, each channel of the microchannel chip 10 is filled with gas (particularly air) (ie, an "empty" microchannel chip is used).

Note that an empty microchannel chip refers to a state in which the channel is filled with gas (especially air), and there is no liquid (e.g., dispersed phase liquid, continuous phase liquid, etc.) in the entire channel. The preferred state is that the entire channel is filled with gas. Note that this does not apply even if liquid remains or occurs in the channel of the microchannel chip due to surface treatment or condensation of water in the air, but at least part of the channel is not blocked by liquid. It is preferable that the dispersed phase liquid flow path, continuous phase liquid flow path, and emulsion forming part are not blocked.

The emulsion holding channel may be (at least partially) filled with gas prior to application of an external liquid delivery driving force (eg positive or negative pressure, in particular negative pressure). This differs from the conventional method of pre-filling the entire channel of a microchannel chip with a continuous phase liquid, and requires preparations such as filling the continuous phase liquid and removing excess continuous phase liquid. This has the advantage that a step can be omitted.

The microchannel chip 10 of FIG. 1 allows the dispersed phase liquid and/or continuous phase liquid supplied to the holding part (inlet) to move to the emulsion forming part by capillary force and/or liquid level differential pressure. It is configured. According to this aspect, the movement of the dispersed phase liquid and the continuous phase liquid can be easily controlled without requiring any additional equipment. For example, a desired capillary force can be obtained by appropriately setting the channel structure (characteristics of the channel surface, pressure loss in the channel, etc.) of the microchannel chip. Furthermore, a desired liquid level differential pressure can be obtained, for example, by adjusting the amount of liquid (particularly the liquid level height) supplied to each phase liquid holding section.

Capillary force is, in particular, a force that is also called capillary force and is generated by the difference in surface tension between the gas-liquid interface in a wide open holding part and the gas-liquid interface in a channel with a smaller cross section. It is a force that acts on Therefore, capillary force is affected not only by the characteristics of each channel but also by the structure of the holding part. In particular, in the emulsion filling method, in order to control the movement of the emulsion's gas-liquid interface, it is necessary to Capillary force also has a large influence during the subsequent droplet retention.

In addition, the liquid level differential pressure is a force also called 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 part. .

When generating droplets, for example, by applying an external liquid feeding driving force (particularly negative pressure) to the discharge port 150, the droplets composed of the dispersed phase liquid and the continuous phase are An emulsion including a continuous phase composed of a liquid can be generated, and the emulsion thus generated can be transported via an emulsion channel 130 to an emulsion holding channel 140 filled with gas. .

(Structure near emulsion forming part)
FIG. 2 is a schematic plan view showing a channel structure near an emulsion forming part of a microchannel chip according to one embodiment of the present disclosure.

In the microchannel chip of FIG. 2, a dispersed phase liquid channel 216 is connected to an emulsion forming section 220 between a first continuous phase liquid channel 212 and a second continuous phase liquid channel 213. . The first continuous phase liquid flow path 212 and the second continuous phase liquid flow path 213 may flow into a common continuous phase liquid holding section, as illustrated in FIG. 1, for example. The dispersed phase liquid channel 216 may be, for example, a channel that connects a dispersed phase liquid merging section where two dispersed phase liquids meet and an emulsion forming section.

For example, by applying negative pressure as an external liquid feeding driving force to the downstream side of the emulsion flow path 230, the continuous phase liquid from the first continuous phase liquid flow path 212 and the second continuous phase liquid flow path 213 can be , and the dispersed phase liquid from the dispersed phase liquid channel 216, an emulsion including droplets and a continuous phase can be generated in the emulsion forming section 220.

The emulsion forming part 220 may have a phase liquid collecting part and an orifice part connected thereto. This embodiment will be described with reference to FIG. FIG. 3 is a schematic illustration of the same embodiment as shown in FIG. 2, including a more detailed description of the emulsion former. In FIG. 3, a phase liquid collecting section 222 and an orifice section 224 that constitute the emulsion forming section 220 are shown.

The phase liquid collection section 222 is a space within the flow path, in which the first continuous phase liquid flow path 212, the second continuous phase liquid flow path 213, the dispersed phase liquid flow path 216, and the orifice section 224 are connected. , are directly connected to the phase liquid collecting section 222. The phase liquid collection section 222 in FIG. 3 has no obvious sidewalls and is defined by the bottom and top surfaces of the channels and the connections of each channel.

The orifice portion 224 is a flow path directly connected to the phase liquid collection portion 222, and connects the phase liquid collection portion 222 and the emulsion flow path 230 to each other. The orifice section 224 is arranged to face the dispersed phase liquid channel 216.

At the orifice section 224, droplets can be generated. For example, in the Flow Focus method, droplets are generated within or near an orifice. Explaining the aspect of FIG. 3, under the external liquid feeding driving force applied to the downstream side of the emulsion flow path 230, two continuous phase liquids and a dispersed phase liquid flowing from opposite directions are combined into a phase liquid aggregate. 222. Then, the dispersed phase liquid sandwiched between the two continuous phase liquids flowing in from opposite directions passes through the orifice part 224 in a forced manner, thereby generating droplets in or near the orifice part. . The generated droplets (emulsion) proceed to the emulsion channel 230.

The emulsion channel 230 is a channel through which droplets flow, and is connected to the phase liquid collecting section 222 of the emulsion forming section via the orifice section 224. In the embodiment of FIG. 3, emulsion channel 230 has the same shape as orifice portion 224. That is, orifice portion 224 and emulsion channel 230 form one channel that is linear and has a substantially constant channel size. Such an embodiment is preferable from the viewpoint of stable droplet generation.

(Flow path direction of two continuous phase liquid flow paths)
The invention will be described with reference to the exemplary embodiment of FIG. In the embodiment of FIG. 2, the inflow direction of the first continuous phase liquid channel 212 into the emulsion forming section 220 (particularly the phase liquid collecting section) and the inflow direction of the first continuous phase liquid channel 212 into the emulsion forming section 220 (particularly the phase liquid collecting section) are shown. The inflow direction of the continuous phase liquid flow path 213 of No. 2 forms an angle α. This angle α is greater than or equal to 0° and less than 180°.

The "inflow direction" of a flow channel means the flow direction of the flow channel (especially the overall direction of movement of the liquid flowing in the flow channel) at the part connecting to the emulsion forming section (especially the phase liquid gathering section). do.

In the conventional manner for generating droplets in microchannel chips, the two continuous phase liquids were facing each other, ie, the angle between the two continuous phase liquid channels was 180°. In such cases, channel blockage and bubble generation occur when the two continuous phase liquids enter the emulsion forming section through an empty (particularly gas-filled) continuous phase liquid channel. There was a case. Such channel blockage and generation of bubbles often occur due to timing shifts in the arrival of the two continuous phase liquids to the emulsion forming section.

On the other hand, when the two continuous phase liquids form an angle α of 0° or more and less than 180° as shown in Figure 2, there is a difference in the timing at which the two continuous phase liquids reach the emulsion forming part. Even if there is, clogging of the flow path and generation of bubbles can be suppressed. Although not intended to be limited by theory, according to such a configuration, the gas existing between the two continuous phase liquids and the continuous phase liquid entering the emulsion forming part (particularly the phase liquid collecting part) Since the collision angle between the two is relaxed, the possibility of the gas in the flow path being separated by the continuous phase liquid is reduced, and as a result, it is thought that the generation of bubbles is suppressed.

The angle α is more than 0°, 15° or more, 30° or more, 40° or more, 45° or more, 50° or more, 60° or more, 70° or more, 75° or more, 80° or more, or 85° or more. and/or may be 170° or less, 160° or less, 150° or less, 140° or less, 130° or less, 120° or less, 110° or less, or 100° or less. Preferably, angle α ranges from 75° to 105°, from 85° to 95°, or from 89° to 91°, in particular approximately a right angle. When the angle α is within these ranges, it is possible to particularly effectively suppress the generation of bubbles and to produce particularly good droplets. When the angle α is relatively large (for example, when α is 30° or more or 40° or more), the two continuous phase liquid channels sufficiently oppose each other in the emulsion forming part, so that droplet generation is further enhanced. It may be stabilized.

The first continuous phase liquid channel and the second continuous phase liquid channel can each have a width of 50 μm to 2000 μm at the channel portion connected to the emulsion forming section. This width is more preferably 100 μm or more, 200 μm or more, more than 200 μm, 250 μm or more, or 300 μm or more, and/or 1500 μm or less, 1200 μm or less, 1000 μm or less, 900 μm or less, 800 μm or less, 700 μm or less, 600 μm or less , 500 μm or less, or 400 μm or less.

The width of the channel can be determined by measuring the distance between the side wall surfaces in a direction horizontally orthogonal to the channel direction when the microchannel chip is in use.

The first continuous phase liquid channel and the second continuous phase liquid channel can each have a height of 50 to 1000 μm at the channel portion connected to the emulsion forming section. This height is more preferably 75 μm or more, or 100 μm or more, and/or 750 μm or less, 500 μm or less, 250 μm or less, or 200 μm or less.

The height of the channel can be determined by measuring the length of the channel in the vertical direction when the microchannel chip is in use.

(Flow path direction of continuous phase liquid flow path and flow path direction of orifice part)

In the embodiment of FIG. 3, the inflow direction of the first continuous phase liquid flow path 212 into the emulsion forming section (particularly the phase liquid collection section 222) and the flow path direction (outflow direction) of the orifice section 224 are at an angle γ1. is formed,
The inflow direction of the second continuous phase liquid flow path 213 into the emulsion forming part (particularly the phase liquid collection part 222) and the flow path direction (outflow direction) of the orifice part 224 form an angle γ2,
The angle γ1 and the angle γ2 are both 0° or more and less than 90°.

When the angle γ1 and/or the angle γ2 are within the above range, the generation of bubbles can be effectively suppressed.

That is, when the angle γ1 and/or the angle γ2 is within the above range, the angle between the continuous phase liquid flow path and the orifice section becomes gentle, so that the phase liquid advances to the phase liquid gathering section of the emulsion forming section. Even if the timing of the two continuous phase liquids coming in is shifted and gas is left behind in the continuous phase liquid flow path, the gas existing between the two continuous phase liquids will flow relatively smoothly into the orifice. can flow to the department. This reduces the possibility that the gas existing between the two continuous phase liquids will be separated, so it is thought that the generation of bubbles can be suppressed.

The flow path direction of the orifice section is usually the direction in which liquid (particularly droplets) flows in the orifice section. The angle γ1 and the angle γ2 may be the same or different. Preferably, angle γ1 and angle γ2 are substantially the same as each other.

The angle γ1 and the angle γ2 may be respectively greater than 0°, 5° or more, 10° or more, 20°C or more, 30° or more, or 40° or more, and/or 80° or less, 70° or less, It may be 60° or less, or 50° or less.

(Flow path direction of continuous phase liquid flow path and flow path direction of dispersed phase liquid flow path)
FIG. 4 is an embodiment corresponding to the embodiment of FIG. 2, and is a schematic plan view for explaining the relationship between two continuous phase liquid channels and a dispersed phase liquid channel.

In the embodiment of FIG. 4, the inflow direction of the first continuous phase liquid flow path 212 into the emulsion forming part (particularly the phase liquid collecting part 222) and the inflow direction of the dispersed phase into the emulsion forming part (particularly the phase liquid collecting part 222) The inflow direction of the liquid flow path 216 forms an angle β1,
The inflow direction of the second continuous phase liquid channel 213 into the emulsion forming section (especially the phase liquid collecting section 222) and the inflow direction of the dispersed phase liquid channel 216 into the emulsion forming section (especially the phase liquid collecting section 222) the direction forms an angle β2, and
The angle β1 and the angle β2 are both 0° or more and less than 90°.

The angle β1 and the angle β2 may be the same or different. Preferably, angle β1 and angle β2 are substantially the same as each other.

Angle β1 and angle β2 may be greater than 0°, greater than or equal to 10°, greater than or equal to 20°, greater than or equal to 30°, or greater than or equal to 40°, and/or less than or equal to 85°, less than or equal to 80°, less than or equal to 75°, respectively. It may be 70° or less, 60° or less, or 50° or less. When the angle β1 and/or the angle β2 are relatively large (for example, when β1 and/or β2 are 15° or more or 20° or more), the two continuous phase liquid channels sufficiently oppose each other in the emulsion forming part. This may further stabilize droplet formation.

<Orifice part>
As described above, the orifice section is a flow path directly connected to the phase liquid collecting section of the emulsion forming section.

The channel shape of the orifice section can be adapted to the desired size of droplets generated using the microchannel chip. Specifically, the desired droplet volume is, for example, 0.1 pL to 50 nL (spherical diameter 3 μm to 1000 μm), more preferably 5 nL or less (spherical diameter ~200 μm), particularly preferably 1 nL or less (spherical diameter ~100 μm). The width of the orifice (particularly the width of the opening in the phase liquid collecting section) is particularly preferably ≦100 μm or less. However, the shapes of droplets during generation vary widely, such as disk shapes and plugs (rectangles), and factors such as the flow rate ratio of continuous phase liquid and dispersed phase liquid are also greatly involved, so the droplet size and orifice size must be strictly determined. It is not necessarily necessary to match.

From the viewpoint of droplet generation, the width, height, and length of the orifice are important, and it is desirable that the orifice portion has dimensions close to the droplet size desired to be generated. In particular, in the microdroplet method using a microchannel, it is necessary to obtain an appropriate droplet size (droplet volume) according to the application, and in the flow-focus method, the opening size of the orifice part is adjusted according to the droplet size. Often needs to be relatively small.

In one preferred embodiment of the present disclosure, the typical diameter of the monodisperse droplets in the generated emulsion is configured to be less than five times the minimum cross-sectional area of the orifice.

Note that the "representative diameter" is a diameter value that can be calculated back from the average volume of the actually measured monodisperse droplets in the emulsion, assuming that the monodisperse droplets in the emulsion are spheres.

The orifice portion can have a channel width of 10 μm to 200 μm. The channel width may be 20 μm or more, 30 μm or more, 40 μm or more, or 50 μm or more, and/or 150 μm or less, 120 μm or less, or 100 μm or less. Particularly preferably, the channel width of the orifice portion is 100 μm or less.

The orifice portion can have a channel height of 10 μm to 200 μm. This height may be 20 μm or more, 30 μm or more, 40 μm or more, or 50 μm or more, and/or 150 μm or less, 120 μm or less, or 100 μm or less.

The length of the orifice portion (flow path length) is not particularly limited. From the viewpoint of the stability of droplet generation, it is important to note that the orifice and part of the emulsion flow path connected to it form one straight flow path, as shown in Figure 3. preferable. The length of this straight channel is preferably 10 μm or more, 50 μm or more, or even 100 μm or more.

<Ceiling surface of the emulsion forming section and the channel connected thereto>
(Ceiling surface)
In one embodiment according to the present disclosure,
The emulsion forming part has a phase liquid gathering part and an orifice part,
The first continuous phase liquid flow path, the second continuous phase liquid flow path, the dispersed phase liquid flow path, and the orifice portion are directly connected to the phase liquid collection portion,
The ceiling surface of the first continuous phase liquid flow path, the ceiling surface of the second continuous phase liquid flow path, the ceiling surface of the dispersed phase liquid flow path, and the ceiling surface of the orifice portion are connected to the phase liquid collecting portion. It exists on the same plane as the ceiling surface of the phase liquid collecting part.

This embodiment will be described with reference to FIG. 3. The channels connected to the phase liquid collecting section 222, namely, the first continuous phase liquid channel 212, the second continuous phase liquid channel 213, and the dispersed phase liquid flow Regarding the channel 216 and the orifice section 224, the ceiling surfaces forming the respective flow channels exist on the same plane as the ceiling surface of the phase liquid collection section.

According to this aspect, the generation of bubbles (in particular, the generation of bubbles due to contact between continuous phase liquids flowing through an empty flow path) and the flow path blockage caused by this can be particularly effectively suppressed or avoided.

Although it is not intended to be limited by theory, bubbles tend to be located toward the ceiling of the channel due to buoyancy, so if there is a difference (especially a step) in the height of the ceiling of the emulsion forming part and the channel connected to it. In this case, there is a risk that air bubbles may remain in a flow path portion (for example, a phase liquid gathering portion) that has a relatively high ceiling surface.

On the other hand, if the phase liquid collection part of the emulsion forming part and the ceiling surface of the flow path connected thereto are on the same plane (i.e., there is no step), air bubbles due to the difference in the height of the ceiling surface may occur. It is thought that even if air bubbles are generated in the flow path, the air bubbles can be easily discharged from the emulsion forming part because the amount of remaining air is reduced.

(bottom)
In the above embodiment regarding the ceiling surface of the channel, more preferably the bottom surface of the channel also exists on the same plane. That is, at the portion where the bottom surface of the first continuous phase liquid flow path, the bottom surface of the second continuous phase liquid flow path, the bottom surface of the dispersed phase liquid flow path, and the bottom surface of the orifice portion connect to the phase liquid collecting portion. , exists on the same plane as the bottom surface of the phase liquid collecting part.

According to this aspect, the orifice portion can be kept small according to the droplet size.

Particularly preferably, the height of the flow path in the orifice portion is the same as the height of the flow path in the phase liquid collecting portion.

(Separation of opening of continuous phase liquid flow path)
In one embodiment according to the present disclosure,
The first continuous phase liquid flow path and the second continuous phase liquid flow path are connected through a first opening and a second opening provided in the wall surface of the emulsion forming section (particularly the phase liquid gathering section), respectively. , connected to the emulsion forming part,
The first opening and the second opening are spaced apart from each other by a distance greater than the width of the dispersed phase liquid channel (and/or the width of the orifice portion) at the portion connected to the emulsion forming portion.

The phase liquid collection part of the emulsion forming part can be defined as a space surrounded by walls that constitute a flow path. In other words, when considering the microchannel chip in use, the phase liquid collection part of the emulsion forming part is formed by horizontal walls extending in the horizontal direction (particularly the ceiling and floor surfaces) and a plurality of side walls. It can be an enclosed space.

The "horizontal wall surface" specifically refers to a wall surface that mainly extends in the horizontal direction among the wall surfaces that constitute the flow path. Horizontal walls are in particular the ceiling and floor of the channel. In particular, the "side wall surface" is a wall surface that mainly extends in the horizontal and vertical directions among the wall surfaces that constitute the flow path.

In the embodiment of FIG. 3, the phase liquid collecting section of the emulsion forming section does not have a defined side wall surface. That is, in the embodiment shown in FIG. 3, the phase liquid collection part of the emulsion forming part is connected to the ceiling surface and floor surface, which are the horizontal walls of the flow channel, to the side wall surface of the continuous phase liquid flow channel, and to the dispersed phase liquid flow. and a connection between a side wall of the continuous phase liquid flow path and a side wall of the orifice section.

On the other hand, for example, the side wall surface of the continuous phase liquid flow path and the side wall surface of the dispersed phase liquid flow path can be connected to each other via a connecting wall surface. By using such a connecting wall surface, the first opening of the first continuous phase liquid channel and the second opening of the second continuous phase liquid channel are connected to the emulsion forming section for dispersion. They can be separated from each other by a distance greater than the width of the phase liquid flow path (and/or the width of the orifice portion). In this case, the connecting wall surface extends in the horizontal and vertical directions and constitutes a side wall surface of the phase liquid collection section of the emulsion forming section.

When the first opening and the second opening are separated from each other by a distance greater than the width of the dispersed phase liquid flow path (and/or the width of the orifice portion) at the portion connecting to the emulsion forming portion, the flow Pathway blockage is effectively suppressed, and generation of bubbles in the emulsion forming part can be further effectively suppressed. Note that the first opening and the second opening are, for example, 1.1 times or 1.3 times the width of the dispersed phase liquid flow path (and/or the width of the orifice portion) at the portion connected to the emulsion forming portion. , 1.5 times, 2.0 times, or 3.0 times.

(Width of continuous phase liquid flow path)
In another embodiment of the present disclosure,
Channel cross-sectional area S ₁ of the first continuous phase liquid flow path at the part connected to the emulsion forming part (particularly the phase liquid collecting part), and/or the part connected to the emulsion forming part (particularly the phase liquid collecting part) The flow path cross-sectional area S ₂ of the second continuous phase liquid flow path in is 2.0 times or more the flow path cross-sectional area S _a of the orifice portion.

According to such an embodiment, the stress exerted on the gas existing between the two continuous phase liquids can be reduced, so that the generation of bubbles in the emulsion forming part can be more effectively suppressed.

(Dispersed phase liquid flow path)
The dispersed phase liquid channel is connected to the emulsion forming section between the first continuous phase liquid channel and the second continuous phase liquid channel. That is, when the microchannel chip is in use, the dispersed phase liquid channel is located between the first continuous phase liquid channel and the second continuous phase liquid channel in the horizontal direction, and emulsion formation is prevented. It is distributed to the department.

The dispersed phase liquid channel can have a width of 10 μm to 300 μm at the channel portion connected to the emulsion forming section. This width may be 20 μm or more, 30 μm or more, 40 μm or more, or 50 μm or more, and/or 250 μm or less, 200 μm or less, 150 μm or less, 120 μm or less, or 100 μm or less.

The dispersed phase liquid channel can have a height of 10 μm to 300 μm at the channel portion connected to the emulsion forming section. This height may be 20 μm or more, 30 μm or more, 40 μm or more, or 50 μm or more, and/or 250 μm or less, 200 μm or less, 150 μm or less, 120 μm or less, or 100 μm or less.

(emulsion flow path)
The emulsion channel is a channel into which droplets flow.
The emulsion channel can have a width of 10 μm to 300 μm at the channel portion connected to the emulsion forming part (particularly the orifice part). This width may be 20 μm or more, 30 μm or more, 40 μm or more, or 50 μm or more, and/or 250 μm or less, 200 μm or less, 150 μm or less, 120 μm or less, or 100 μm or less.

The emulsion channel can have a channel height of 10 μm to 300 μm at the channel portion connected to the emulsion forming part (particularly the orifice part). This height may be 20 μm or more, 30 μm or more, 40 μm or more, or 50 μm or more, and/or 250 μm or less, 200 μm or less, 150 μm or less, 120 μm or less, or 100 μm or less.

(Wall connection)
In one embodiment of the present invention, the connection between the side wall surface of the continuous phase liquid flow path and the side wall surface of the orifice portion is rounded. In this case, it may be possible to more effectively suppress the generation of bubbles.

FIG. 5 shows an example of a flow path structure according to such an aspect. In the embodiment of FIG. 5, the connecting portion 57a between the side wall surface of the first continuous phase liquid flow path 512 and the side wall surface of the orifice portion 524 is rounded, and the second continuous phase liquid flow path 513 A connecting portion 57b between the side wall surface of the orifice portion 524 and the side wall surface of the orifice portion 524 is rounded. Emulsion channel 530 connects to orifice section 524 and together forms a straight channel.

Without intending to be limited by theory, if the wall connection is not rounded but forms a corner, the gas between the two continuous phase liquids entering the emulsion formation will press against this corner. This increases the risk of the film becoming separated and creating bubbles. On the other hand, if the connection part is rounded, the separation pressure when gas is pressed against this connection part is reduced, so the generation of air bubbles can be suppressed more effectively. Conceivable.

In particular, when the radius of the corner formed at the connection between the side wall surface of the continuous phase liquid flow path and the side wall surface of the orifice section is assumed to be a circle, the so-called corner R is 0.1 mm or more. It may be. This angle R may further be greater than 0.1 mm, greater than or equal to 0.3 mm, greater than or equal to 0.5 mm, or greater than or equal to 1.0 mm. The upper limit of the angle R is not particularly limited, but may be, for example, 10.0 mm or less, or 5.0 mm or less.

(Dimensions of emulsion forming part)
The phase liquid collecting portion of the emulsion forming portion can have a length of 100 μm to 2000 μm in the width direction of the phase liquid collecting portion of the emulsion forming portion. This length (width) may be 200 μm or more, 300 μm or more, 400 μm or more, or 500 μm or more, and/or 1500 μm or less, 1200 μm or less, 1000 μm or less, or 900 μm or less.

The phase liquid collecting part of the emulsion forming part can have a length of 100 μm to 2000 μm in the length direction of the phase liquid collecting part of the emulsion forming part. This length may be 200 μm or more, 300 μm or more, 400 μm or more, or 500 μm or more, and/or 1500 μm or less, 1200 μm or less, 1000 μm or less, or 900 μm or less.

"The length direction of the phase liquid collecting part of the emulsion forming part (the length direction of the phase liquid collecting part)" can be defined as the flow path direction of the dispersed phase liquid flow path at the part connected to the emulsion forming part. . In addition, "the width direction of the phase liquid collecting part of the emulsion forming part (the width direction of the phase liquid collecting part)" is the horizontal direction with respect to the flow path direction of the dispersed phase liquid flow path in the part connected to the emulsion forming part. It can be defined as orthogonal directions.

<Emulsion>
The emulsion produced by the method of the present disclosure is a dispersed solution and includes 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 a dispersed phase liquid are dispersed in a continuous phase made up of a continuous phase liquid.

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

The dispersed phase liquid is, for example, an aqueous solution. The dispersed phase liquid can optionally contain surfactants, organic solvents, thickeners, serum, enzymes, and the like. The dispersed phase liquid may be a reaction liquid, for example, a liquid containing a sample to be detected in a detection process described below, a liquid containing a detection reagent, or a mixture thereof.

(continuous phase liquid)
The continuous phase liquid is a liquid that constitutes the continuous phase contained in the emulsion.

Preferably, the continuous phase liquid is an immiscible liquid that is immiscible 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) type emulsion is formed.

When the continuous phase liquid is an oil, the oil includes silicone oil, mineral oil, fluorinated dispersion medium, vegetable oil, or combinations thereof.

Fluorinated dispersion media include fluorocarbons, particularly perfluorohexane, hexafluorobenzene, perfluoromethylcyclohexane, perfluorooctane, and perfluorotripentylamine.

Commercially available fluorocarbons include FC-3283 (Florinat (trade name) manufactured by 3M Company), FC-40 (Florinat (trade name) manufactured by 3M Company), and HFE-7500 (3M ^TM Novec ^TM High Performance Liquid, (manufactured by 3M).

When a fluorinated dispersion medium, particularly the above-mentioned fluorocarbon, is used as the continuous phase liquid, particularly stable and rapid droplet formation is possible. In addition, because it has extremely low compatibility with polar and non-polar solvents, there is a problem of components of droplets in the emulsion moving to other droplets via the continuous phase liquid (crosstalk, contamination). ) can be suppressed. In addition, when selecting a liquid with low surface tension or viscosity such as a hydrocarbon dispersion medium or silicone oil, the risk of being a hazardous substance such as flammability generally increases, but fluorine dispersion medium It is characterized by its high safety so that it can be used as a

When the continuous phase liquid contains oil (especially fluorinated dispersion medium), the oil (especially fluorinated dispersion medium) may be 50.0 to 99.9% by mass of the continuous phase liquid, or even 80 to 99.0% by mass.

Note that additives such as surfactants can also be added to the continuous phase liquid for the purpose of thermal stability of the droplets. It is preferable that these additives do not inhibit the detection reaction in the droplet. Examples of surfactants include nonionic surfactants such as PLURONIC (registered trademark) and TETRONIC (registered trademark), which are block copolymers of polyethylene glycol and polypropylene glycol, Tween (registered trademark), Span, Zonyl (registered trademark), etc. Can be mentioned. When a fluorinated dispersion medium is used as the continuous phase liquid, it is preferable to use a fluorinated surfactant, particularly a fluorocarbon surfactant, such as a block copolymer of perfluoropolyether and polyethylene glycol. .

If the continuous phase liquid includes a surfactant, the surfactant may be from 1 to 20% by weight, or even from 2 to 10% by weight, based on the continuous phase liquid.

(droplet)
The droplets contained in the emulsion are composed of dispersed phase liquid. Droplets are formed, for example, by encapsulation of a dispersed phase liquid through contact with a continuous phase liquid.

The droplet contains, for example, a sample to be detected. The target substance contained in the sample and the reagent are reacted in the droplet, and the sample is analyzed via a detectable signal (e.g., a fluorescent signal) that indicates the presence or absence of the reaction and/or the extent of the reaction. It can be carried out. This reaction may be, for example, a chemical reaction, a binding reaction, a phenotypic change, or a combination thereof.

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 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, It is preferably 50 nL or less, or 10 nL or less. Note that, from the viewpoint of uniformly reacting the target substance within the droplet, it is preferable that the volume of the droplet to be formed is highly monodisperse. Monodispersity here specifically means that the coefficient of variation (CV) of droplet volume is 20% or less, 10% or less, 5% or less, 2% or less, or 1% or less. Note that, in the following, in order to make the explanation easier to understand, the droplets are treated as spherical, but the same idea may be applied even if the droplets are non-spherical due to the channel structure or the surrounding flow.

Preferably, the droplet has sufficient thermal stability to maintain the shape of the droplet at least under the reaction temperature conditions of the target substance. As a specific example, in the detection process, when performing nucleic acid amplification by the TRC method, under a temperature condition of 40 ° C. to 48 ° C., and when performing nucleic acid amplification by the PCR method, under a temperature condition of 50 ° C. to 100 ° C. Preferably, each droplet has sufficient thermal stability to maintain its shape.

<Microchannel chip>
The microchannel chip of the present disclosure includes 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 channel, and an outlet. The dispersed phase liquid holding section is connected to the emulsion forming section via the dispersed phase liquid channel, and the continuous phase liquid holding section is connected to the emulsion forming section via the continuous phase liquid channel, An emulsion channel connects to the outlet.

In one embodiment according to the present disclosure,
The microchannel chip further includes an emulsion holding channel,
The emulsion forming section is connected to the emulsion holding channel via the emulsion channel,
An emulsion holding channel connects to the outlet.

The dispersed phase liquid holding portion, the dispersed phase liquid flow path, the continuous phase liquid holding portion, the continuous phase liquid flow path, the emulsion forming portion, the emulsion flow path, the optional emulsion holding flow path, and the outlet are fluidly connected to each other. , forming one channel structure as a whole.

In particular, the flow path structure is such that it can be connected to the external atmosphere (particularly the external atmosphere) only via the dispersed phase liquid holding part, the continuous phase liquid holding part, and the outlet.

The microchannel chip according to the present disclosure includes, for example, a base material and an upper structure disposed on the base material. Preferably, the upper structure has a channel structure, that is, 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 channel, and an optional emulsion holding section. It has a flow path and a discharge port. The substrate may be made of glass. The upper structure may be made of resin. The microchannel chip can be produced, for example, by bonding together a resin upper structure and a glass base material that constitutes the bottom of the microchannel chip.

The size (width, depth, etc.) of the channels that make up the microchannel chip can be determined as appropriate, taking into account the volume of the desired droplet, etc., and in particular, the reaction form of the target substance. and can be determined accordingly. For example, if the target substance is a nucleic acid such as DNA or RNA, and the reaction with the target substance is a digital amplification reaction (amplification reaction in units of one molecule) of the nucleic acid, droplets of pL order or nL order are prepared. Therefore, the width and depth of the channel around the emulsion forming part are preferably in the range of 0.1 μm to 1000 μm, particularly 1 μm to 300 μm.

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

Examples of materials used for manufacturing the microchannel chip include polymer materials such as PDMS (polydimethylsiloxane) and acrylic, metal materials such as stainless steel, glass, silicone, and ceramics. Among these, polymer materials allow the flow path itself to be produced at low cost and are easily made into a disposable form. Therefore, it is preferred to use polymeric materials at least in part.

Note that it is preferable that the channels constituting the microchannel chip have channel walls that have a low affinity for at least the dispersed phase liquid. A microchannel chip may be fabricated using a material that has a low affinity for the dispersed phase liquid, or the portion corresponding to the channel wall may be surface-treated with a material that has a low affinity for the dispersed phase liquid. good. For example, microchannel chips may be fabricated using polymer materials such as PDMS (polydimethylsiloxane), acrylic, cycloolefin polymer, and PTFE (polytetrafluoroethylene), and hydrocarbon-based silanizing agents, fluorocarbons, etc. The surface of the channel wall surface may be treated with a silanizing agent or the like.

(Dispersed phase liquid holding section)
The dispersed phase liquid holding section is a part that holds a dispersed phase liquid that becomes a material for producing an emulsion. The dispersed phase liquid holding section may be an introduction port, and the liquid can be introduced into the microchannel chip via the dispersed phase liquid holding section. The dispersed phase liquid holding section may be, for example, a hole and/or a well that extends in the vertical direction when the microchannel chip is in use, and the dispersed phase liquid is supplied into the hole and/or well. and can be maintained. The dispersed phase liquid holding section may be composed of holes and/or wells having a diameter of 0.1 mm to 20 mm, for example. When the dispersed phase liquid holding section is composed of a hole and a well, the well extending in the vertical direction can be connected to the dispersed phase liquid flow path via the hole extending in the vertical direction.

(Supply of dispersed phase liquid)
A dispersed phase liquid can be supplied to the dispersed phase liquid holding section.

In order to supply the dispersed phase liquid to the dispersed phase liquid holding section, a separate container (phase liquid holding container) prepared separately for holding the dispersed phase liquid can also be used. From the viewpoint of preventing liquid from flowing out during storage and operation, such a container is preferably completely or variably sealed while holding the dispersed phase liquid.

Further, the supply of the dispersed phase liquid (and/or the supply of the continuous phase liquid) can be performed by a dispensing means. The use of a dispensing means is preferable in that it is possible to suppress the remaining amount of the dispersed phase liquid and to suppress the measurement time and/or contamination between reagents (dispersed phase liquid).

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 delivery is carried out under negative pressure. Conventional supply methods, particularly methods in which tubes or manifolds are fluidically connected (sealed connection) to each holding part to simultaneously introduce liquid into each holding part and supply liquid pressure, prevent unexpected pressure fluctuations and pressure fluctuations at the time of connection. Due to the time required for stabilization, droplets were sometimes generated early before the start of liquid feeding. On the other hand, when a dispensing means is used and the liquid is delivered under negative pressure, there is no pressure fluctuation when connecting the phase liquid supply device and the pressure source to each holding part, so the liquid is sent under negative pressure. Early droplet formation before initiation can be suppressed.

Preferably, the dispensing means does not cause pressure fluctuations in the holding section. The dispensing means may be, for example, a pipette. Preferably, the dispensing means (particularly, the liquid discharge ports constituting the dispensing means) are not fluidly connected (hermetically connected) to each holding section, but are spatially separated from each other.

For example, the dispensing means may be a mechanism including a pump, an actuator, and a pipette, in which the pump sucks up each phase liquid held in a separate container, the pipette tip is moved to each holding part by the actuator, and then the pipette tip is moved to each holding part by the pump. It is preferable to perform an operation of pushing out each phase liquid to the holding part. In addition, it is preferable that a part of the pipette or the like that has come into contact with each phase liquid is removable and can be replaced each time it is used, since contamination can be suppressed. Furthermore, it is preferable to use a pump as a dispensing means in combination as a liquid feeding means because the device configuration can be simplified. In addition, if repeated use is intended, the phase liquid may be added by a dispensing means including a disposable pipette, or the phase liquid may be added from the phase liquid holding container to the microchannel chip using a common line. Also good. In the latter case, in order to suppress contamination of the dispersed phase liquid into the continuous phase liquid, it is preferable to include a step of cleaning the shared line up to the connection part to the microchannel chip. In addition, as a dispensing means that does not involve a pipette, it is also preferable to use an external force to add (drop) each liquid directly from the container holding the phase liquid to the holding part (for example, by breaking the thermocompression-bonded part of the container by pressure). (e.g. means for forcing out liquid in a container). Further, for example, when performing a TRC reaction or a PCR reaction, a dispensing means may be used in combination as a means for purifying or preparing an aqueous solution sample.

(Dispersed phase liquid flow path)
The dispersed phase liquid channel connects the dispersed phase liquid holding section and the emulsion forming section. The dispersed phase liquid channel is configured such that the dispersed phase liquid passes therethrough.

The dimensions of the dispersed phase liquid channel can be appropriately set depending on the type and characteristics of the dispersed phase liquid used. The dispersed phase liquid flow path may be curved at one or more locations and may have a serpentine shape.

Depending on the method of supplying liquid to the microchannel chip, at the start of liquid feeding, the dispersed phase liquid displaces the continuous phase liquid present in the dispersed phase liquid channel and the dispersed phase liquid holding section and flows toward the emulsion forming section. . At this time, it is preferable that the continuous phase liquid is efficiently discharged from the dispersed phase liquid flow path and that the amount of continuous phase liquid remaining is reduced. The following measures are shown, but the present invention is not limited thereto: First, the cross-sectional shape of the channel preferably has a structure with few discontinuous surfaces (corners). For example, it may be circular, oval, or semicircular. It is preferable that the channel surface of the dispersed phase liquid channel has a low affinity for the continuous phase liquid. Further, in order to reduce the residual amount, it is preferable that the length of the dispersed phase liquid channel is short and the cross-sectional area of the channel is small.

The microchannel chip according to the present disclosure can have two or more dispersed phase liquid holding sections and two or more dispersed phase liquid channels corresponding thereto.

In particular, the microchannel chip according to the present disclosure has a first dispersed phase liquid holding section and a second dispersed phase liquid holding section, and the dispersed phase liquid channel is connected to the first dispersed phase liquid holding section. a first dispersed phase liquid flow path connected to a second dispersed phase liquid holding section, and a dispersed phase liquid confluence section. The first dispersed phase liquid flow path and the second dispersed phase liquid flow path are each connected to the emulsion forming section via the dispersed phase liquid confluence section.

By using two or more dispersed phase liquid holding units, for example, a reaction liquid containing an analysis sample and a reaction liquid containing a detection reagent can be separately supplied to a microchannel chip, and droplets can be formed. It is possible to prevent the two from mixing until immediately before generation. This is preferred as it allows better control of the timing of reaction initiation.

(Continuous phase liquid holding section)
The continuous phase liquid holding section is a part that holds a continuous phase liquid for producing/holding an emulsion. The continuous phase liquid holding section may be an introduction port, and the liquid can be introduced into the microchannel chip via the continuous phase liquid holding section. The structure of the continuous phase liquid holding section is not particularly limited as long as it can hold the continuous phase liquid. The continuous phase liquid holding portion may be a hole or a well, for example a vertically extending hole or well, into which a continuous phase liquid is supplied and retained. is now possible. The continuous phase liquid holding portion may be, for example, a hole or well with a diameter of 0.1 mm to 20 mm.

Note that since the continuous phase liquid generally uses a liquid with low surface tension and low viscosity, the amount of change in surface tension due to the change in the interface shape in the continuous phase liquid holding portion is small. Therefore, the shape of the continuous phase liquid holding section does not have a large effect on liquid feeding. In addition, in the present invention, for example, when holding an emulsion and performing a detection reaction, in order to supply a sufficient amount of continuous phase liquid so that the continuous phase liquid in the continuous phase liquid holding unit is not depleted, the holding unit is There is no situation where the remaining amount of the continuous phase liquid becomes small and the interface shape is likely to change. Therefore, the shape of the continuous phase liquid holding section is unlikely to have a large effect on liquid feeding.

(Continuous phase liquid flow path)
The continuous phase liquid channel connects the continuous phase liquid holding section and the emulsion forming section. The continuous phase liquid flow path is configured such that the continuous phase liquid passes therethrough.

The dimensions of the continuous phase liquid channel can be appropriately set depending on the type and characteristics of the continuous phase liquid used. The continuous phase liquid flow path may be curved at one or more locations and may have an at least partially serpentine shape.

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

Referring to the exemplary embodiment of FIG. 1, continuous phase liquid channel 111 is comprised of two channels (first continuous phase liquid channel 112 and second continuous phase liquid channel 113). These two channels 112 and 113 are at least partially opposed to each other in the emulsion forming section 120, and are connected to the emulsion forming section 120 in a dispersed phase liquid channel (more precisely, The flow path direction is substantially symmetrical with respect to the dispersed phase liquid confluence section). In the embodiment of FIG. 1, the first continuous phase liquid flow path 112 and the second continuous phase liquid flow path 113 have substantially the same structure and flow path length, such that the respective flow paths The velocities of the continuous phase liquids moving through the two channels are substantially the same. Further, when it is desired to suppress mixing of dispersed phase liquids before emulsion generation, it is preferable that the length of the flow path connecting the downstream part of the dispersed phase liquid confluence part and the emulsion forming part 120 is relatively short (for example, 3 mm). (more preferably 0.5 mm or less), the dispersed phase liquid preferably maintains a laminar flow state separately within the flow path.

(Emulsion forming part)
The emulsion forming section is configured to generate an emulsion. The emulsion forming section receives supply of the dispersed phase liquid and the continuous phase liquid via the dispersed phase liquid channel and the continuous phase liquid channel, respectively. Further, the emulsion forming section is connected to the emulsion channel, and the emulsion generated in the emulsion forming section is sent to the emulsion channel.

The emulsion forming section can have one or more openings that open to the dispersed phase liquid flow path and one or more openings that open to the continuous phase liquid flow path. The emulsion forming section can also have one or more openings that open into the emulsion flow path.

The emulsion forming section will now be described with reference to the exemplary embodiment of FIG. In the emulsion forming section 120 of FIG. 1, a continuous phase liquid channel 111 composed of two channels 112 and 113 and a dispersed phase liquid channel (more precisely, a dispersed phase liquid confluence section) intersect with each other. There is. During application of an external liquid delivery driving force (particularly negative pressure), two continuous phase liquids flow into the emulsion forming section 120 and a dispersed phase liquid flows into the emulsion forming section 120. As a result, in the emulsion forming section 120, droplets (ie, an emulsion) dispersed in the continuous phase are generated. The emulsion thus produced passes through the emulsion channel 130 and enters the emulsion holding channel 140 filled with gas.

The emulsion forming section can appropriately use a flow path using the principles of general droplet generation methods such as T-junction, flow-focus, co-flow, and step-emulsification. In order to quickly generate an emulsion, a plurality of emulsion forming units may be arranged in parallel. Further, a meandering channel or the like may be provided for stirring the droplets in the emulsion.

(emulsion flow path)
The emulsion channel is a channel through which the generated emulsion is transported. In one aspect according to the present disclosure, the emulsion channel connects the emulsion forming section and the emulsion holding channel. The part of the emulsion channel that connects to the emulsion forming section is preferably arranged to face the dispersed phase liquid channel (particularly the dispersed phase liquid confluence section). FIG. 1 shows an emulsion flow path in such an embodiment. Further, in FIG. 1, the inflow portions of the two continuous phase liquid flow paths into the emulsion formation portion are substantially symmetrical with respect to the portion of the emulsion flow path that connects to the emulsion formation portion. In the case of Figure 1, when an emulsion is generated, the dispersed phase liquid flowing into the emulsion forming part from the dispersed phase liquid flow path becomes droplets and enters the emulsion flow path without changing the flow angle. .

The emulsion channel may have an expanded width and/or height downstream of the part connecting to the emulsion forming part (in the direction of the outlet) and/or may be meandering. can. Such an embodiment is preferable because stirring in the droplet can be promoted.

(Emulsion holding channel)
In one embodiment according to the present disclosure, the microchannel chip has an emulsion holding channel that is a droplet holding section. The emulsion holding channel is connected to the emulsion forming section via the emulsion channel, and has a function of holding the emulsion produced in the emulsion forming section. Further, the emulsion holding channel is optionally connected to the discharge port via the discharge port communication channel.

The width and length of the emulsion holding channel can be set appropriately according to the volume and number of droplets to be held, and for example, it may be a wide and simple channel with approximately the same width and length. A single continuous long flow path may be arranged in a meandering or spiral shape, or branched straight flow paths may be arranged in parallel.

In the present invention, the cross section of the emulsion holding channel is preferably circular, semicircular, elliptical, convex, concave, rectangular, or trapezoidal because the center of the droplet tends to flow along the center of the channel. .

In the emulsion filling method, generally, emulsion generation and retention are performed simultaneously by controlling the movement of the gas-liquid interface in the emulsion holding channel, so the shape of the gas-liquid interface is maintained during liquid feeding (even though it moves). It is desirable that the cross-sectional shape of the channel is constant and that the channel is a straight channel without bending so that the change in shape is small. However, if the channel width is made extremely large relative to the channel height in order to increase the number of detected droplets, it becomes difficult to stably manufacture a chip with the intended channel structure, and/or the flow The bottom and/or top surface of the channel may be deformed, and the height of the channel area far from the channel side may change due to liquid delivery pressure or fixing pressure to the chip, which may adversely affect 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, particularly preferably 10 or less (if there is no pillar described below). Alternatively, by providing a pillar in the center of the flow path, the ratio of the distance between the pillars and/or the distance between the pillar and the side surface of the flow path to the height of the flow path is, for example, 100 or less, more preferably 50 or less, It is preferably 25 or less, particularly preferably 10 or less. On the other hand, when performing batch detection processing using an image sensor or the like, it is preferable that the emulsion holding channels are densely packaged on a horizontal plane (for example, in a shape close to a square or circle) because the number of detected droplets can be increased. Therefore, continuous single long channels having the same cross-sectional shape may be arranged in a meandering or spiral shape, or branched straight channels may be arranged in parallel. In this case, since there is a bend, the interface shape is likely to change during liquid feeding, but this effect can be reduced by adjusting the cross-sectional shape at the bend.

In one embodiment, it is provided that the retained emulsion is subjected to a detection process. That is, the detection process described below can be optionally performed on the emulsion held in the emulsion holding channel.

Preferably, the emulsion holding channel is configured such that most or all of the held emulsion does not come into contact with the external atmosphere (particularly the external atmosphere) of the microchannel chip. Preferably, among the droplets held in the emulsion holding channel, the droplets that are the object of the detection process do not come into contact with the external atmosphere (particularly the external atmosphere). For this purpose, for example, the length of the emulsion holding channel is set relatively long so that the emulsion can come into contact with the external atmosphere (in particular the external atmosphere) only at the downstream end of the emulsion holding channel. be able to.

Preferably, the emulsion held in the emulsion holding channel is sealed against the external atmosphere (particularly the external atmosphere).

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

Preferably, the emulsion holding channel is configured such that detection processing can be performed on emulsions that are not exposed to the outside atmosphere. More specifically, for example, there is a structure between the retained emulsion and the detection means that isolates the emulsion from the external atmosphere. This structure is made, for example, of a material that is transparent to light. In this case, among the emulsions held in the emulsion holding channel, emulsions that are not subject to detection processing, such as emulsions located at the discharge end of the emulsion holding channel, may be exposed to the outside atmosphere. good.

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

Preferably, the emulsion holding channel holds 500 or more, 1000 or more, 2500 or more, 5000 or more, or 10000 or more droplets with an average volume of 0.1 nL to 10 nL, particularly 0.3 to 3 nL. It has a channel volume that can hold at least 100,000 pieces, and/or 100,000 pieces or less, 80,000 pieces or less, 60,000 pieces or less, or 40,000 pieces or less. Detection processing can be performed on these droplets held in the emulsion holding channel.

Note that the average volume of droplets can be calculated based on the following by acquiring a bright field image using an image acquisition device such as a digital camera, and regarding droplets with N=10 or more in the acquired image.

The volume of a spherical droplet and the volume of a disk-shaped droplet (V _drop and V _disk [nL], respectively) are expressed by the following equations (1) and (2), respectively. In addition, D _drop and D _disk in the following formulas (1) and (2) respectively represent the case where the droplet held in the emulsion holding channel is observed from above in the normal usage state of the microchannel chip. , the diameter of a spherical droplet, and the diameter of a disc-shaped droplet. Moreover, in formula (2), h is the channel height of the emulsion holding channel.

Preferably, for the detection means (such as a camera) used in the detection process, the droplets in the emulsion to be detected do not overlap each other, and in particular, droplets in the emulsion to be detected do not overlap each other, and in particular, droplets in the emulsion that are to be detected do not overlap one another in a plane orthogonal to the detection direction. forming layers. In this case, detection accuracy can be further improved.

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

Preferably, the channel height of the emulsion holding channel has a dimension corresponding to the diameter of the droplet to be detected in the detection process, for example, 1/10 to 10 times the diameter of the droplet. , 1/4 to 4 times, or 1/2 to 2 times the flow path height. Further, the height of the emulsion holding channel may be equal to or less than 1/4 times the width of the channel. Note that the width of the channel is usually the length in the horizontal direction orthogonal to the length direction of the channel when the microchannel chip is in use. The diameter of the droplet can be measured across the width of the channel.

In addition, in the emulsion filling method, the emulsion is filled into the emulsion holding channel depending on the flow rate ratio of the continuous phase liquid and the dispersed phase liquid during emulsion formation. When the flow rate ratio of the phase liquid is increased, it is preferable to design the channel height to be larger than usual in order to package the droplets closely in the horizontal direction. For example, if the flow rate ratio of continuous phase liquid to dispersed phase liquid is 8 to 12, the height of the emulsion holding channel can be 2 to 4 times the droplet diameter.

(Stopping liquid feeding)
In order to hold the emulsion in the emulsion holding channel, for example, when a desired amount of emulsion is filled in the emulsion holding channel, liquid feeding is stopped and the discharge port is optionally closed. The application of negative pressure can also be stopped by opening the outlet (at least partially) to the outside atmosphere. The liquid feeding can be stopped after the gas filling the emulsion holding channel is completely replaced by the emulsion, but preferably, the gas filling the emulsion holding channel is completely replaced by the emulsion. Stop the liquid supply while the machine is not running. In other words, the liquid feeding is stopped in a state where the gas-emulsion interface exists within the channel or the outlet.

(Vent)
The microchannel chip has an outlet. This outlet connects to an emulsion channel or an optional emulsion holding channel. The outlet may also function as a negative pressure source connection for applying negative pressure to the microchannel chip.

Preferably, the outlet is configured to be suitable for connection to a source of negative pressure. In this case, it is more preferable that the outlet has resistance to the applied pressure.

Typically, the outlet is located downstream of the emulsion flow path or downstream of the optional emulsion retention flow path. If the outlet is located downstream of the emulsion holding channel, by applying negative pressure to the outlet, the entire length or most of the emulsion holding channel can be filled with the produced emulsion.

(liquid delivery)
By applying an external liquid feeding driving force to the microchannel chip, for example, by applying negative pressure to the outlet, droplets composed of the dispersed phase liquid and continuous phase composed of the continuous phase liquid are generated in the emulsion forming section. The emulsion thus produced can be transported to an emulsion flow path. In embodiments having an emulsion holding channel, the emulsion thus produced can be transported via the emulsion channel to the optional emulsion holding channel.

The dispersed phase liquid holding section and the continuous phase liquid holding section can be open to the external atmosphere (particularly the external atmosphere) while the liquid is being fed by applying a negative pressure to the outlet.

Note that while it is preferable to keep each holding part at normal pressure while applying negative pressure to the discharge port and transferring liquid, it is preferable from the viewpoint of device simplicity. The pressure may be controlled to an emergency pressure state in order to make the emulsion evaporate and/or also serve as a sealing operation for retaining the emulsion.

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

When using a pressure tank as a negative pressure source, the volume of the pressure tank is preferably larger than the sum of the volume of the flow path from the outlet to the pressure tank and the volume of the flow path of the microchannel chip. The pressure tank can also be designed to be open to the outside atmosphere. Further, it is preferable that the pressure inside the pressure tank is controlled by, for example, a pump. Additionally, a pressure sensor may be provided to monitor the pressure value within the pressure tank.

Using monitoring means for monitoring the pressure value of the applied negative pressure, it is possible to check the liquid delivery status, for example to check whether the emulsion is being produced without problems.

In one embodiment of the method according to the present disclosure, a negative pressure control means is fluidly connected to the outlet, the negative pressure control means is comprised of a negative pressure source, a connection and a valve, and the negative pressure source is , is controlled to a constant negative pressure, and this valve is arranged between the negative pressure source and the connection part. Via the connection, negative pressure control means can be connected to the outlet. According to this aspect, negative pressure can be instantaneously applied by opening the valve, making it possible to more accurately control the timing of application of negative pressure.

There are no particular restrictions on the specific form of the valve. From the viewpoint of preventing backflow when liquid feeding is stopped, it is preferable to use a valve that can suppress pressure fluctuations in the flow path when opening and closing the valve, for example, one that opens and closes relatively slowly. The valve may be, for example, a three-way valve, allowing the microchannel chip to be connected either to a source of negative pressure (for example a pressure tank) or to an external atmosphere (in particular to the external atmosphere).

(Installation of microchannel chip)
The microchannel chip is generally installed horizontally in the vertical direction (vertical direction), but from the viewpoint of retaining the emulsion, etc., it may be installed with an intentional inclination in a certain direction. For example, when the continuous phase liquid has a higher specific gravity than the dispersed phase liquid (e.g., a fluorinated dispersant is used as the continuous phase liquid and an aqueous solution is used as the dispersed phase liquid), the droplets have buoyancy due to the difference in specific gravity, so the emulsion retains flow. The microchannel chip may be installed with an intentional slope so that droplets are less likely to flow out of the channel.

<Detection processing>
A detection process can be performed on droplets in an emulsion produced according to the methods of the present disclosure. The detection process includes, for example, a reaction of a target substance in a droplet and detection of the reaction (for example, detection of a reaction product). In embodiments having an emulsion holding channel, the detection process is usually performed on droplets in the emulsion held in the emulsion holding channel.

Target substances (particularly target molecules) can be nucleic acids, proteins, peptides, enzymes, cells, bacteria, spores, viruses, organelles, macromolecular assemblies, drug candidates, lipids, carbohydrates, metabolites, or any combination thereof. Can be mentioned.

The reaction of the target substance is not particularly limited. Target substance reactions include enzyme reactions, more specifically, kinases, nucleases, nucleotide cyclase, nucleotide ligases, nucleotide phosphodiesterases, (DNA or RNA) polymerases, prenyltransferases, pyrophospatases, reporter enzymes, reverse transcriptases. An example is an enzymatic reaction using topoisomerase or 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 include reactions capable of isothermal amplification of the nucleic acid such as LAMP method, NASBA method, TMA method, and TRC method. Furthermore, in the case of one-step RT-PCR, it is preferable to produce droplets at a temperature suitable for reverse transcription reaction in terms of reaction efficiency and reaction time of reverse transcription reaction. It is also possible to detect cDNA, which is a product of reverse transcription reaction, by the cycling probe method.

When performing a reaction, it is preferable to mix two or more reaction liquids upstream of the emulsion forming section (for example, at the dispersed phase liquid confluence section) and use this mixture to generate droplets. In the present invention, the reaction solution refers to a solution containing at least a portion of the target substance and the components necessary for reacting the target substance. It is only necessary that all the components necessary for the reaction of the target substance are prepared by mixing all the reaction liquids, and the target substance only needs to be contained in any one of the reaction liquids. There is no problem even if three or more types of reaction liquids are used.

For example, if the target substance is a nucleic acid (DNA, RNA) containing a specific sequence, and the reaction of the target substance is a reaction that amplifies this specific sequence, the components contained in the reaction solution include a primer containing a sequence complementary to a part of a specific sequence, a detection probe containing a sequence homologous or complementary to a part of a specific sequence, polymerase, nucleotides, salts, and buffer components. can be given. It is preferable that the composition of the reaction solution is designed to prevent decomposition, denaturation, and non-specific reactions of the target molecules, reaction substrates, enzymes, etc. Considering their behavior within the apparatus, glycerol, A surfactant or the like may be further added.

<Other means>
(Detection means)
For detection of the reaction, for example, a detection means capable of detecting a product of the reaction can be used.

An appropriate detection method can be selected depending on the reaction product, such as optical, X-ray, MALDI (matrix-assisted laser desorption ionization), FCS (fluorescence correlation spectroscopy), FP ( Fluorescence polarization)/FCS, fluorescence method, colorimetry, chemiluminescence, bioluminescence, scattering, surface plasmon resonance, electrochemical method, electrophoresis, laser, mass spectrometry, Raman spectroscopy, FLIPR (Molecular Devices), etc. It can be detected using the following method. In addition, when detecting using transmitted light, if the microchannel chip is made of a material that transmits light, droplets within the chip can be moved simply by placing the microchannel chip on an optical detector. This is preferable in that the reaction product can be detected without any trace.

As the detection means (detector) used to detect the reaction product, an imaging sensor for recording and measuring the reaction of the target substance and optionally its components can be used. An example of detection is a camera or imaging device with adequate illumination and resolution to spatially resolve the individual signals to be detected. Any known camera or imaging device can be used; for example, the camera may be a charge coupled device (CCD), a charge injection device (CID), a photodiode array (PDA), or a complementary metal oxide semiconductor (CMOS). ) can be used. Detection can also be improved by using the polarization of the excited/emitted light. For example, when detecting droplets that emit fluorescent signals, rapid and high-throughput signal detection can be achieved by photographing the detection area all at once using an optical unit with a large field of view.

(Temperature control means)
The temperature control means has the role of keeping the liquid within the microchannel chip at a temperature suitable for reaction of the target substance. The temperature control means may have any shape as long as it can be brought close to (preferably in close contact with) the microchannel chip, and does not necessarily have to be in the shape of a flat plate.

It is preferable that at least a portion of the temperature control means that is close to (preferably in close contact with) the microchannel chip is made of a metal material with high thermal conductivity. Note that when a microchannel chip is fabricated by bonding a base material and an upper structure, reducing the thickness of the base material and/or the upper structure in contact with the temperature control means will reduce the thickness of the microchannel chip. This is preferable because heat can be conducted more efficiently to the flow path. It is sufficient that the temperature control means can at least control the temperature of the emulsion holding channel, which is the reaction field of the target substance, but if the temperature of the phase liquid supply section and the flow channel can also be controlled, non-specific reactions of the target molecules can be suppressed. preferable. As a specific example, when the reaction of the target substance is a nucleic acid amplification reaction, the temperature in each holding section and channel can be adjusted using a temperature control means so that it is higher than the reaction temperature of the target substance in the emulsion holding channel. , non-specific annealing between primers/probes can be reduced. In addition, when the bottom surface of the microchannel chip is heated to the reaction temperature by a temperature control means, and the top substrate of the microchannel chip is made of a material that transmits light, and transmitted light is detected from the top surface, it is necessary to Evaluation of the position of an empty microchannel chip and/or channel structure and/or dust inside and outside the chip, behavior within the channel when supplying each phase liquid, and/or behavior of emulsion generation during liquid feeding. This method is preferable because it is possible to easily improve the upper limit of quantification in digital detection using the signal detection result of the emulsion during the reaction.

In the following, the present invention will be explained in more detail using examples. The present invention is not limited to these descriptions.

≪Example 1≫
≪Preparation of microchannel chip≫
A microchannel chip was fabricated using photolithography and soft lithography techniques. The specific steps are shown below.

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

(2) After forming a channel pattern on the photoresist film using a mask aligner (Ushio Inc.) and a chrome mask on which the channel pattern of the microchannel chip was formed, the SU-8 Developer (Microchem Inc.) was used to form a channel pattern on the photoresist film. ) was used to develop the channel pattern, thereby creating a mold for the channel that constitutes the microchannel chip.

(3) In order to suppress adsorption to SU-8, vapor deposition surface treatment was performed using Trichloro (1H, 1H, 2H, 2H-perfluoro-octyl) silane (Thermo Fisher Scientific).

(4) A mixture of uncured siloxane monomer and polymerization initiator (weight ratio 10:1) prepared using SYLGARD SILICONE ELASTOMER KIT (Toray Dow Corning) was added to the mold treated in (3) above. A polymer (PDMS) substrate on which the shape of the flow path was transferred was prepared by pouring and heating at 80° C. for 2 hours.

(5) The obtained polymer substrate was carefully peeled off from the mold, and after molding with a cutter, a dispersed phase liquid holding part, a continuous phase liquid holding part, and a discharge port were formed using a puncher.

(6) After surface treatment of the polymer substrate and the cover glass (Matsunami Glass Co., Ltd.) on which the holding part and the discharge port were formed using an oxygen plasma generator (Meiwaforsys Co., Ltd.), the patterned surface of the PDMS substrate and the cover glass were bonded together. The prepared chip was stored in a desiccator.

The fabricated microchannel chip has a size of 34 mm in length x 75 mm in width, and has a φ4 mm hole as a dispersed phase liquid holding part, a φ8 mm hole as a continuous phase liquid holding part, and a φ1.5 mm hole as a discharge port. Each had a hole.

(Flow path structure)
The microchannel chip includes one dispersed phase liquid holding section, a dispersed phase liquid channel, a continuous phase liquid holding section, two continuous phase liquid channels, an emulsion forming section, an emulsion channel, an emulsion holding channel, and an outlet. It had The dispersed phase liquid holding section is connected to the emulsion forming section via a dispersed phase liquid channel, and the continuous phase liquid holding section is connected to the emulsion forming section via two continuous phase liquid channels. The emulsion forming section was connected to the emulsion holding channel via the emulsion channel, and the emulsion holding channel was connected to the discharge port.

In the microchannel chip used in Example 1, the dispersed phase liquid channel is a meandering channel with a height of 80 μm, a width of 100 μm, and a length of 8 mm, and merges into the emulsion forming section. In the emulsion forming section, the dispersed phase liquid confluence section and two continuous phase liquid channels intersect, and the reaction liquid and the immiscible liquid (oil) merge to form droplets. .

FIG. 6a shows a schematic plan view of the channel structure near the emulsion forming part in the microchannel chip according to Example 1.

As seen in FIG. 6a, a first continuous phase liquid channel 612 and a second continuous phase liquid channel 613 connect to the emulsion forming section, and both channels connect to the emulsion forming section. The channel width at the site was 200 μm. A dispersed phase liquid channel 616 with a channel width of 100 μm is connected to the emulsion forming section between the first continuous phase liquid channel 612 and the second continuous phase liquid channel 613. The inflow direction of the first continuous phase liquid channel 612 into the emulsion forming section and the inflow direction of the second continuous phase liquid channel 613 into the emulsion forming section formed an angle α of 90°.

Furthermore, in the microchannel chip according to Example 1, the inflow direction of the first continuous phase liquid channel 612 into the emulsion forming section and the inflow direction of the dispersed phase liquid channel 616 into the emulsion forming section are 45 The inflow direction of the second continuous phase liquid flow path 613 into the emulsion forming section and the inflow direction of the dispersed phase liquid flow path 616 into the emulsion forming section form an angle β1 of 45°. An angle β2 was formed. The first continuous phase liquid flow path 612 and the second continuous phase liquid flow path 613 were arranged symmetrically with respect to the dispersed phase liquid flow path 616.

Furthermore, in the microchannel chip according to Example 1, the inflow direction of the first continuous phase liquid channel 612 into the emulsion forming section and the channel direction (outflow direction) of the orifice section 624 are at an angle of 45°. γ1, and the inflow direction of the second continuous phase liquid flow path 613 into the emulsion forming portion and the flow path direction of the orifice portion 624 formed an angle γ2 of 45°.

In Example 1, the channel size of the orifice portion was 80 μm in height and 80 μm in width.

The emulsion channel was a straight channel with a width of 80 μm and a length of 1000 μm in a portion close to the emulsion forming part, a straight channel with a width of 200 μm and a length of 680 μm in the downstream portion thereof, and a circular arc curve with a radius of 275 μm in the downstream portion. This is a stirring channel with a width of 200 μm and a length of 11.5 mm including meandering, and is connected to the emulsion holding channel.

The emulsion holding channel is a meandering channel with a channel height of 130 μm, a width of 2 mm, and a length of 350 mm, and is directly connected to the discharge port via a discharge port communication channel with a width of 2 mm and a length of 10 mm. The volume of the emulsion holding channel was 91 μL.

(Generation of droplets)
Using the above microchannel chip according to Example 1, droplets were generated as described in (0) to (4) below.

(0) An aqueous solution ("starting solution") having the following composition was prepared as a dispersed phase liquid to be introduced into the dispersed phase liquid holding section. Note that the aqueous solution having the following composition imitates the composition of a reaction starting solution when using the TRC reaction, which is one of the nucleic acid amplification reactions.
(starting solution)
36.8mM Magnesium Chloride 180.0mM Potassium Chloride 0.2% (w/v) Tween 20 (“Tween” is a registered trademark)
18.0% (v/v) DMSO
5.0% (v/v) Glycerol (1) The pressure inside the 200 mL tank, which is composed of a peristaltic pump (Takasago Industries), a solenoid valve (Takasago Industries), and a pressure sensor (Keyence Corporation) as a liquid feeding means. A device capable of controlling the pressure from -1 to -10 kPa was used. A pressure difference (negative pressure) is applied to the chip by connecting this tank and the outlet of the microchannel chip with a PTFE tube (Nichias Corporation) and releasing the pressure inside the tank.
(2) 20 μL of the above-mentioned starting liquid as a dispersed phase liquid was dropped into the dispersed phase liquid holding section. At this time, the channels of the microchannel chip were filled with gas. The dropped dispersed phase liquid passed through the dispersed phase liquid channel filled with gas and proceeded to the opening of the emulsion forming section.
(3) 30 seconds after dropping the dispersed phase liquid, add Droplet Generator oil for EvaGreen (Biorad Co., hereinafter simply oil) as a continuous phase liquid to the continuous phase liquid holding unit using a pipetman (Gilson). 200 μL of (also referred to as ) was added dropwise. The continuous phase liquid was divided into two continuous phase liquid channels, proceeded to the emulsion forming section, and merged at the emulsion forming section.

(4) 20 seconds after oil dripping, droplet generation was started by applying a pressure difference (negative pressure) while adjusting the tank pressure of the liquid feeding device connected to the discharge port to -5 kPa in advance. . Note that each holding section was kept open to atmospheric pressure while the liquid was being fed.

(Droplet generation evaluation)
The formation of droplets in the emulsion forming section was photographed using a digital CMOS camera (ORCA-FLASH (trade name), Hamamatsu Photonics). As seen in Figure 6b, good droplet formation was observed in Example 1.

(Bubble generation evaluation)
In Example 1, bubble generation was evaluated. That is, the above-mentioned droplet generation was performed multiple times, and the generation of bubbles when the two continuous phase liquids entered the emulsion forming section was evaluated.

Specifically, we used a digital CMOS camera (ORCA-FLASH (product name), Hamamatsu Photonics) to observe the continuous phase liquid entering the emulsion forming section through two continuous phase liquid channels filled with gas. The timing difference (delay time) in which the two continuous phase liquids each reach the emulsion forming part, the moving speed of the continuous phase liquid-air interface in the flow path, and the occurrence of bubbles. I checked to see if it existed. The results are shown in Table 1 below.

≪Comparative example 1≫

In Comparative Example 1, bubble generation as the two continuous phase liquids progressed was evaluated in the same manner as in Example 1, except that the angles of the two continuous phase liquid channels were different.

As seen in the schematic diagram of FIG. 7, in the microchannel chip of Comparative Example 1, the first continuous phase liquid channel 712 and the second continuous phase liquid channel 713 form an angle α of 180°. Both had a channel width of 200 μm at the portion connected to the emulsion forming section. Moreover, angle β1 and angle β2, and angle γ1 and angle γ2 were all 90°.

The results are shown in Table 2 below.

A summary of the results of Example 1 and Comparative Example 1 is shown in Table 3 below.

As seen in Tables 1 to 3 (particularly Table 3), in Example 1, the timing difference (delay time) between the two continuous phase liquids reaching the emulsion forming part was within the range of 50 ms to 69 ms. No bubbles were observed in this case. On the other hand, in Comparative Example 1, generation of bubbles was observed when the delay time was within the same range.

≪Example 2 and Comparative Example 2≫
In Example 2 and Comparative Example 2, droplets were generated in the same manner as in Example and Comparative Example 1, respectively, except that the channel width was changed to 400 μm.

Schematic plan views of the vicinity of the emulsion forming portion of the microchannel chips used in Example 2 and Comparative Example 2 are shown in FIGS. 8a and 9, respectively.

(Droplet generation evaluation)
The appearance of droplets being generated in the emulsion forming section of Example 2 was photographed using a digital CMOS camera (ORCA-FLASH (trade name), Hamamatsu Photonics Co., Ltd.). As seen in Figure 8b, good droplet formation was observed in Example 2.

(Bubble generation evaluation)
In the same manner as in Example 1, the generation of bubbles as the continuous phase liquid progressed through the two continuous phase liquid channels filled with gas was evaluated. The results of bubble generation evaluation for Example 2 and Comparative Example 2 are shown in Tables 4 and 5 below, respectively. A summary of these results is shown in Table 6 below.

As seen in Tables 4 to 6 (particularly Table 6), in Example 2, the timing difference (delay time) between the two continuous phase liquids reaching the emulsion forming part was within the range of 100 ms to 199 ms. No bubbles were observed in this case. On the other hand, in Comparative Example 2, generation of bubbles was observed when the delay time was within the same range.

In particular, in Example 2, no bubbles were observed even though the timing difference (delay time) between the two continuous phase liquids reaching the emulsion forming part was 194.7 ms, while in Comparative Example 2, Generation of bubbles was observed when the timing difference (delay time) between the two continuous phase liquids reaching the emulsion forming section was 121.8 ms.

≪Example 3 and Comparative Example 3≫
In Example 3 and Comparative Example 3, droplet generation and bubble generation were evaluated in the same manner as in Example 1 and Comparative Example 1, respectively, except that the channel width was changed to 800 μm.

Schematic plan views of the vicinity of the emulsion forming portion of the microchannel chips used in Example 3 and Comparative Example 3 are shown in FIGS. 10 and 11, respectively.

(Droplet generation evaluation)
The appearance of droplets being generated in the emulsion forming section of Example 3 was photographed using a digital CMOS camera (ORCA-FLASH (trade name), Hamamatsu Photonics Co., Ltd.). In Example 3, good droplet formation was observed in the emulsion forming part.

(Bubble generation evaluation)
The generation of bubbles caused by the progression of two continuous phase liquids through a continuous phase liquid channel filled with gas was evaluated in the same manner as in Example 1. The results of Example 3 and Comparative Example 3 are shown in Table 7 and Table 8 below, respectively. A summary of these results is shown in Table 9 below.

As seen in Tables 7 to 9 (particularly Table 9), in Example 3, the timing difference (delay time) between the two continuous phase liquids reaching the emulsion forming part was within the range of 300 ms to 599 ms. No bubbles were observed in this case. On the other hand, in Comparative Example 3, generation of bubbles was observed when the delay time was within the same range.

In particular, in Example 3, no bubbles were observed even though the timing difference (delay time) between the two continuous phase liquids reaching the emulsion forming section was 517.2 ms. On the other hand, in Comparative Example 3, the generation of bubbles was observed when the timing difference (delay time) between the two continuous phase liquids reaching the emulsion forming part was 317.3 ms.

<About evaluation results>
From the above results, when the angle α of the two continuous phase liquid flow paths satisfies the conditions according to the present disclosure (Examples 1 to 3), when the angle α is 180° (Comparative Examples 1 to 3), In comparison, it was found that the generation of bubbles was suppressed.

Further, from the above results, it was found that when the angle α of the two continuous phase liquid channels satisfies the conditions according to the present disclosure, good droplet generation can be performed.

10 Microchannel 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 channel 115 Second dispersed phase liquid channel 120 Emulsion forming section (droplet generating section)
130 Emulsion channel 140 Droplet holding section (emulsion holding channel)
150 outlet

W Width direction L Length direction

212, 512 First continuous phase liquid channel 213, 513 Second continuous phase liquid channel 216, 516 Dispersed phase liquid channel 220 Emulsion forming section (droplet generating section)
222 Phase liquid collecting section 224, 524 Orifice section 230, 530 Emulsion channel 57a, 57b Connection section between the side wall surface of the continuous phase liquid channel and the side wall surface of the orifice section

α, β1, β2, γ1, γ2 angle

612, 712, 812, 912, 1012, 1112 First continuous phase liquid flow path 613, 713, 813, 913, 1013, 1113 Second continuous phase liquid flow path 616, 716, 816, 916, 1016, 1116 Dispersion Phase liquid channel channel 624, 724, 824, 924, 1024, 1124 Orifice part

Claims (7)

dispersed phase liquid holding section,
a dispersed phase liquid flow path connected to the dispersed phase liquid holding section;
continuous phase liquid holding section,
a first continuous phase liquid flow path and a second continuous phase liquid flow path connected to the continuous phase liquid holding section;
an emulsion forming section, and
It has an emulsion flow path connected to the emulsion forming section,
The dispersed phase liquid flow path, the first continuous phase liquid flow path, and the second continuous phase liquid flow path are connected to the emulsion forming section,
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 feeding driving force is applied to the microchannel chip, the emulsion forming section an emulsion including droplets composed of the dispersed phase liquid and a continuous phase composed of the continuous phase liquid is produced, and the emulsion enters the emulsion flow path.
A microchannel chip,
The dispersed phase liquid flow path is connected to the emulsion forming section between the first continuous phase liquid flow path and the second continuous phase liquid flow path, and The inflow direction of the first continuous phase liquid flow path and the inflow direction of the second continuous phase liquid flow path into the emulsion forming section form an angle α of 0° or more and less than 180°.
Microfluidic chip. The emulsion forming section includes a phase liquid collecting section and an orifice section connected thereto,
The emulsion flow path is connected to the emulsion forming section via the orifice section,
The inflow direction of the first continuous phase liquid flow path into the emulsion forming section and the flow path direction of the orifice section form an angle γ1, and
The inflow direction of the second continuous phase liquid flow path into the emulsion forming section and the flow path direction of the orifice section form an angle γ2,
The angle γ1 and the angle γ2 are both 0° or more and less than 90°,
The microchannel chip according to claim 1. The inflow direction of the first continuous phase liquid flow path into the emulsion forming section and the inflow direction of the dispersed phase liquid flow path into the emulsion forming section form an angle β1 of 0° or more and less than 90°. and
The inflow direction of the second continuous phase liquid flow path into the emulsion forming section and the inflow direction of the dispersed phase liquid flow path into the emulsion forming section form an angle β2 of 0° or more and less than 90°. ing,
The microchannel chip according to claim 1 or 2. The emulsion forming section includes a phase liquid collection section and an orifice section connected thereto,
A connecting portion connecting the side wall surface of the first continuous phase liquid flow path and the side wall surface of the orifice portion, and/or the side wall surface of the second continuous phase liquid flow path and the side wall of the orifice portion. The connection part that connects the part wall surface is rounded,
The microchannel chip according to claim 1 or 2. Before applying the external liquid feeding driving force, the first continuous phase liquid channel filled with gas and the second continuous phase liquid channel filled with gas are transferred from the continuous phase liquid holding section to the first continuous phase liquid channel filled with gas. 3. The microchannel chip according to claim 1, for use in a method including filling a continuous phase liquid through the emulsion forming section filled with gas. The microchannel chip according to claim 1 or 2, further comprising an emulsion holding channel. The microchannel chip according to claim 1 or 2, for use in an emulsion filling method.