JP7585960B2 - Method for generating emulsions in microfluidic chips - Google Patents

Description

The present invention relates to a method for generating an emulsion in a microfluidic chip. In particular, the present invention relates to a method for performing droplet array measurement more efficiently, simply, and quickly.

The microdroplet method, in which a reaction solution is fractionated into microdroplets, is known as a technique for dividing a reaction solution into microcompartments and carrying out reactions independently. This method is expected to be applied to, for example, the production of micro- and nano-particles, and is particularly used in digital measurements in which target molecules are divided into microcompartments at the single molecule level using a microfluidic device, reactions are carried out within the microdroplets, and the presence or absence of target molecules is measured by the presence or absence of a signal, resulting in absolute quantification of the number of target molecules.

The microdroplet method generally uses an emulsion that consists 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. The document describes how the inlet of the device is filled with oil, and the oil is sent to a droplet collection chamber by centrifugation. After that, a sample solution is introduced from the same inlet, and droplets are generated by centrifugation.

As an alternative to this type of droplet generation method, a method is known in which a dispersed phase liquid such as a reaction liquid and a continuous phase liquid such as an oil are supplied to a microchannel chip via separate supply parts and then merged within the chip to generate an emulsion.

US Patent No. 5,399,633 discloses such a system and method for generating droplets. The document describes transporting the generated droplets to an outlet region consisting of a pipette tip or a droplet well. The document also describes an air trap that substantially separates the sample and the oil until application of a fluid driving force (such as negative or positive pressure), which then forms an emulsion having droplets of the sample and a continuous phase of the oil.

In addition, in recent years, droplet array measurement, which aligns droplets in a single layer in a detection area to easily measure signals, has been attracting attention as a method for simplifying and speeding up the device.

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

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

European Patent No. 2550528 JP 2019-170363 A JP 2020-169911 A

Centrifugal step emulsification applied for absolute quantification of nuclear acids by digital droplet RPA, Lab Chip, 2 015, 15, 2759-2766 1-Million droplet array with wide-field fluorescence imaging for digital PCR, Lab on a Chip, 2011, 11, 3838-3845

Conventional methods sometimes resulted in insufficient stability in the formation of emulsions.

The problem of instability in emulsion production occurs when the size of droplets becomes non-uniform due to the unintended formation of an emulsion. Therefore, in order to solve the problem of instability in emulsion production, it was previously thought that it was essential to use a bubble trap as in Patent Document 1.

However, it was found that when a bubble trap is used, the structure of the flow path becomes complicated, and the bubbles used for the bubble trap remain in the emulsion, which can cause problems in the flow of the emulsion in the flow path and in evaluations using the emulsion.

When generating emulsions using an emulsion channel chip, negative pressure liquid transfer is preferable from the perspective of automation, as it requires simpler equipment and processes than positive pressure liquid transfer.

On the other hand, when negative pressure is used, it may not be easy to remove air bubbles that have formed in the flow path. More specifically, when positive pressure is used, pressure can be applied directly to both the continuous phase liquid and the dispersed phase liquid, whereas when negative pressure is used, it is not easy to apply pressure directly to the dispersed phase liquid, so it may be relatively difficult to remove air bubbles that have formed in the dispersed solution, for example. When gas and liquid are mixed in the flow path, it takes time to stabilize the flow rate, which may result in a loss of the sample to be detected.

Therefore, the present invention aims to provide an improved emulsion generation method that stably forms an emulsion without using a bubble trap in a method in which a continuous phase liquid and a dispersed phase liquid are supplied separately to a microchannel chip and transported under negative pressure.

The above-mentioned problems of the present invention can be solved by the following aspects of the present invention.
<Aspect 1>
A method for producing an emulsion by supplying a dispersed phase liquid and a continuous phase liquid to a microchannel chip, comprising the steps of:
the microchannel chip has a dispersed phase liquid holding section, a dispersed phase liquid flow path, a continuous phase liquid holding section, a continuous phase liquid flow path, an emulsion forming section, an emulsion flow path, and an outlet;
the dispersed phase liquid holding unit is connected to the emulsion forming unit via the dispersed phase liquid flow path,
the continuous phase liquid holding unit is connected to the emulsion forming unit via the continuous phase liquid flow path,
the emulsion forming section is connected to the outlet through the emulsion flow path,
Supplying a dispersed phase liquid to the dispersed phase liquid holding unit;
Supplying a continuous phase liquid to the continuous phase liquid holding section; and
generating an emulsion including droplets composed of the dispersed phase liquid and a continuous phase composed of the continuous phase liquid in the emulsion forming section by applying a negative pressure to the outlet, and allowing the emulsion thus generated to enter the emulsion flow path;
Including,
contacting the dispersed phase liquid with the continuous phase liquid at or upstream of the emulsion formation section without using a bubble trap prior to the application of the negative pressure; and
administering said application of negative pressure within 30 seconds after said contact;
A method comprising:
<Aspect 2>
A negative pressure control means is fluidly connected to the outlet;
The negative pressure control means comprises a negative pressure source, a connection and a valve;
The negative pressure source is controlled to a constant negative pressure,
the connection is for fluidly connecting the negative pressure control means to the outlet, and the valve is disposed between the negative pressure source and the connection.
The method according to aspect 1.
<Aspect 3>
3. The method of claim 2, wherein the negative pressure control means is fluidly connected to the outlet prior to the contacting of the dispersed phase liquid and the continuous phase liquid.
<Aspect 4>
4. The method according to any one of the preceding aspects, characterized in that the application of negative pressure is stopped by connecting the outlet to the outside atmosphere.
<Aspect 5>
A method according to any one of aspects 1 to 4, wherein the flow resistance of the emulsion formation section and the flow path upstream thereof is sufficiently small so that the dispersed phase liquid and the continuous phase liquid can come into contact with each other without an external liquid-transport driving force.
<Aspect 6>
Prior to said contacting,
The dispersed phase liquid is moved to the emulsion forming section through the dispersed phase liquid flow path filled with gas, and
A method according to any one of the preceding aspects, characterized in that the continuous phase liquid is moved to the emulsion forming section through the continuous phase liquid flow path in a gas-filled state.
<Aspect 7>
7. The method of claim 6, wherein the dispersed phase liquid and/or the continuous phase liquid are moved by capillary forces and/or liquid level differential pressure.
<Aspect 8>
A method according to claim 6 or 7, characterized in that a negative pressure is applied to the outlet, thereby moving the dispersed phase liquid through the dispersed phase liquid flow path filled with gas to the emulsion forming section prior to the contacting.
<Aspect 9>
A method according to any one of aspects 1 to 5, characterized in that, prior to the contacting, the continuous phase liquid is moved through the continuous phase liquid flow path, the emulsion forming section, and the dispersed phase liquid flow path, all of which are filled with gas, to the dispersed phase liquid holding section.
<Aspect 10>
10. The method of claim 9, wherein the continuous phase liquid is moved by capillary forces and/or liquid head pressure differentials.
<Aspect 11>
the microchannel chip further has an emulsion-holding channel,
the emulsion forming section is connected to the emulsion holding flow path via the emulsion flow path, and the emulsion holding flow path is connected to the discharge port,
applying a negative pressure to the outlet, in the emulsion forming section, to generate an emulsion containing droplets composed of the dispersed phase liquid and a continuous phase composed of the continuous phase liquid, and transporting the emulsion thus generated through the emulsion flow path to the emulsion holding flow path filled with gas;
11. The method of any one of aspects 1 to 10, comprising:
<Aspect 12>
12. The method of claim 11, wherein the magnitude of the negative pressure applied to the outlet is 30 kPa or less.
<Aspect 13>
13. The method according to claim 11 or 12, wherein a production speed of the emulsion in the emulsion forming section is 5 droplets/second or more.

The present invention provides an improved emulsion generation method that stably forms an emulsion without using a bubble trap in a method for supplying a continuous phase liquid and a dispersed phase liquid separately to a microchannel chip.

FIG. 1 is a schematic plan view of one embodiment of a microchannel chip according to the method of the present disclosure. FIG. 2 is a schematic plan view of another embodiment of a microchannel chip according to the method of the present disclosure. FIG. 3 is a cross-sectional schematic diagram showing one embodiment of a dispersed phase liquid retaining portion according to the present disclosure.

<Method of producing emulsion>
The method according to the present disclosure is a method for generating an emulsion by supplying a dispersed phase liquid and a continuous phase liquid to a microchannel chip, comprising the steps of:
the microchannel chip has a dispersed phase liquid holding section, a dispersed phase liquid flow path, a continuous phase liquid holding section, a continuous phase liquid flow path, an emulsion forming section, an emulsion flow path, and an outlet;
the dispersed phase liquid holding section is connected to the emulsion forming section via a dispersed phase liquid flow path,
the continuous phase liquid holding section is connected to the emulsion forming section via a continuous phase liquid flow path;
the emulsion forming section is connected to the outlet through an emulsion flow path,
Supplying a dispersed phase liquid to a dispersed phase liquid holding unit;
Supplying a continuous phase liquid to a continuous phase liquid holding section; and
generating an emulsion including droplets composed of a dispersed phase liquid and a continuous phase composed of a continuous phase liquid in an emulsion forming section by applying a negative pressure to an outlet, and allowing the emulsion thus generated to enter the emulsion flow path;
Including,
contacting the dispersed phase liquid with the continuous phase liquid at or upstream of the emulsion formation section without using a bubble trap prior to the application of negative pressure; and
The application of negative pressure is performed within 30 seconds of contact.

In the method disclosed herein, the dispersed phase liquid and the continuous phase liquid are brought into contact without using a bubble trap before applying negative pressure, thereby suppressing the generation of bubbles.

In addition, in the present invention, after the dispersed phase liquid and the continuous phase liquid are brought into contact with each other, an external liquid-transport driving force is applied within a certain period of time to start the liquid transport, thereby ensuring the uniformity of the droplets and solving the problem of instability in emulsion formation, even without using a bubble trap. Therefore, according to the present invention, it is possible to reduce the loss of sample solution, etc., and realize highly accurate detection reactions, etc.

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

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

According to the method of the present disclosure, a dispersed phase liquid such as an aqueous reaction solution containing a substance to be detected is supplied to the dispersed phase liquid holding units 102 and 103, and a continuous phase liquid such as oil is supplied to the continuous phase liquid holding unit 101. At this point, each channel of the microchannel chip 10 can be filled with a gas (particularly air) (i.e., an "empty" microchannel chip can be used). In this case, the dispersed solution and the continuous phase liquid are supplied to the chip without previously filling the channels of the microchannel chip with the continuous phase liquid, so that the preparation process can be omitted.

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

The microchannel chip 10 in FIG. 1 is configured so that the dispersed phase liquid and the continuous phase liquid can be moved from their respective holding parts through their respective flow paths to the emulsion forming part 120 by capillary force and/or liquid level differential pressure. Therefore, the dispersed phase liquid and the continuous phase liquid supplied to each holding part 101, 102, 103 move toward the emulsion forming part 120 (downstream) by capillary force and/or liquid level differential pressure. Note that for this movement (particularly the movement of the dispersed phase liquid), pressure applied from the outside (particularly negative pressure applied to the outlet) may be used.

According to the present invention, before starting liquid transfer by applying an external liquid transfer driving force (negative pressure) to the microchannel chip, the dispersed phase liquid and the continuous phase liquid are brought into contact with each other upstream of the emulsion formation section without using a bubble trap. According to the method disclosed herein, it is possible to reduce the generation of bubbles, thereby shortening the time required for early stabilization of liquid transfer and reducing the loss of reaction liquids that may contain valuable samples.

Furthermore, in the method of the present invention, after the dispersed phase liquid and the continuous phase liquid come into contact as described above, negative pressure is applied through the outlet 150 within a certain period of time (within 30 seconds) to generate an emulsion in the emulsion forming section 120, which contains droplets composed of the dispersed phase liquid and a continuous phase composed of the continuous phase liquid. According to the method of the present invention, premature droplet generation due to contact between the dispersed phase liquid and the continuous phase liquid can be minimized, thereby ensuring the uniformity of the droplets.

The time until negative pressure is applied after the above-mentioned contact between the dispersed phase liquid and the continuous phase liquid can be set according to the structure and characteristics of the microchannel chip used and the required uniformity of the size of the droplets to be generated, but good droplet uniformity can be ensured if it is within 30 seconds. Particularly preferably, negative pressure is applied within 20 seconds, 15 seconds, 10 seconds, 5 seconds, 2 seconds, or 1 second after the above-mentioned contact.

Negative pressure can be applied, for example, by a suction device connected to the outlet 150 of the microchannel chip 10.

＜Application of negative pressure＞

In one preferred embodiment of the method according to the present disclosure, a negative pressure control means is fluidly connected to the outlet, and the negative pressure control means is composed of a negative pressure source, a connection part, and a valve, the negative pressure source is controlled to a constant negative pressure, and the valve is disposed between the negative pressure source and the connection part. The negative pressure control means can be connected to the outlet via the connection part. According to this embodiment, negative pressure can be applied instantly by opening the valve, so that the timing of application of negative pressure can be more precisely controlled, particularly after contact of the phase liquids.

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

In particular, a negative pressure control means is fluidly connected to the outlet prior to contact of the dispersed phase liquid and the continuous phase liquid.

In an embodiment in which the negative pressure control means is fluidly connected to the outlet before the contact of the dispersed phase liquid with the continuous phase liquid, negative pressure can be applied immediately after the continuous phase liquid and the dispersed phase liquid come into contact. This is particularly preferred because it can suppress unintended early droplet generation and further improve the uniformity of the droplets.

Also, in particular by (at least partially) connecting the exhaust outlet to the external atmosphere (in particular the external atmosphere), the application of the negative pressure is stopped.

When negative pressure is applied to the outlet, the dispersed phase liquid holding portion and the continuous phase liquid holding portion of the microchannel chip can be open to the outside atmosphere.

<Emulsion holding flow path>
In yet another embodiment of the present disclosure, the microchannel chip has an emulsion-holding channel.

More specifically, the microchannel chip further has an emulsion holding channel, the emulsion forming section is connected to this emulsion holding channel via the emulsion channel, and this emulsion holding channel is connected to an outlet. By applying negative pressure to the outlet, an emulsion is generated in the emulsion forming section, and the emulsion thus generated is transported to the emulsion holding channel via the emulsion channel.

This embodiment will be specifically described with reference to the microchannel chip shown in FIG. 2. FIG. 2 is a schematic diagram for ease of understanding and is not drawn to scale. The microchannel chip 20 in FIG. 2 is similar to that in FIG. 1, except that it has an emulsion-holding channel and the placement of the outlet is different. Only these differences will be described below.

The microchannel chip 20 in FIG. 2 has an emulsion holding channel 140. The emulsion forming section 120 is connected to the emulsion holding channel 140 via the emulsion channel 130, and the emulsion holding channel 140 is connected to the outlet 150.

The emulsion produced in the emulsion forming section 120 is transported to the emulsion holding flow path 140 via the emulsion flow path 130.

When a conventional air bubble trap is applied to a microchannel chip having such an emulsion-holding channel, air bubbles may get mixed into the emulsion-holding channel, causing a decrease in the uniformity of the emulsion, and may cause, for example, droplets to coalesce. In particular, when the emulsion held in the emulsion-holding channel for detection processing or the like is configured not to come into contact with the external atmosphere (particularly the external air), it is not easy to remove air bubbles once they have formed.

In addition, when generating and holding an emulsion using a microchannel chip with an emulsion holding channel, it is difficult to selectively remove only the droplets that are generated before the liquid is sent due to early contact, and such droplets also end up being held in the emulsion channel. This causes non-uniformity in the held droplets.

Furthermore, when liquid is delivered under negative pressure, it is not as easy to eliminate air bubbles that occur in the flow channels of the microchannel chip as it is when liquid is delivered under positive pressure.

In contrast, in the method disclosed herein, the dispersed phase liquid and the continuous phase liquid are brought into contact with each other at the emulsion forming section or upstream thereof without using a bubble trap before applying negative pressure, and negative pressure is applied within 30 seconds after this contact, which makes it possible to suppress the generation of bubbles and to suppress droplets that do not have the desired characteristics (particularly size) that are caused by early contact. Therefore, the present invention is particularly useful in microchannel chips having emulsion-holding channels.

In the method of the present disclosure, the emulsion (droplets + continuous phase) generated in the emulsion forming section can be transported to an emulsion holding channel filled with gas. For example, referring to FIG. 2, the emulsion generated in the emulsion forming section 120 can be transported via the emulsion channel 130 to the emulsion holding channel 140 filled with gas ("emulsion filling method"). Note that the emulsion holding channel 140 does not need to be completely filled with gas when the emulsion is transported, and may be partially filled with, for example, continuous phase liquid.

In this type of emulsion filling method, the emulsion generated in the emulsion forming section moves through the emulsion-holding flow path, which is filled with gas (particularly toward the outlet), filling the emulsion-holding flow path. In other words, the "gas-liquid interface" formed by the gas and emulsion filling the emulsion-holding flow path moves downstream of the emulsion-holding flow path (particularly toward the outlet). The emulsion filling method is preferable because it can omit the preparation step of filling the flow path of the microchannel chip with a continuous phase liquid in advance and the device for this purpose.

When the above-mentioned emulsion filling method is performed in a microchannel chip having an emulsion-holding channel, it is preferable from the viewpoint of preventing droplets from flowing out that the gas-liquid interface moving through the emulsion-holding channel is present within the channel (particularly within the channel near the outlet) when droplet generation is stopped. In addition, it is preferable to perform liquid transfer by negative pressure, since this simplifies the connection between the microchannel chip and an external liquid transfer drive source. However, when the emulsion filling method is performed by negative pressure, backflow of the gas-liquid interface (progression toward the emulsion formation part) may become a problem. Note that in the conventional method of filling the entire channel with a continuous phase liquid before liquid transfer, or in the conventional method of recovering droplets in a droplet well, the effect of pressure fluctuation due to the inflow of air when the negative pressure is stopped is mitigated or avoided by the continuous phase liquid or emulsion filling the outlet, so such a problem does not occur.

From the viewpoint of preventing backflow of the gas-liquid interface proceeding through such an emulsion-holding flow path, it is preferable to gradually change the pressure when stopping the liquid transfer, or to reduce the liquid transfer pressure.

<Contact>
As described above, in the method of the present disclosure, the dispersed phase liquid and the continuous phase liquid are brought into contact with each other at the emulsion forming section or upstream thereof without using a bubble trap before applying negative pressure. In this disclosure, the term "contact" means contact between the dispersed phase liquid and the continuous phase liquid, and particularly means contact between the dispersed phase liquid and the continuous phase liquid in a stage prior to the production of an emulsion, unless otherwise specified. In addition, the "bubble trap" is a structure for generating bubbles that inhibit contact between the continuous phase liquid and the dispersed phase liquid separately supplied to the chip, and this structure is provided, for example, in a dispersed phase liquid holding flow path and is configured to promote the generation of bubbles in the flow path.

The above-mentioned contact can be achieved by adjusting the timing of supply of each phase liquid (dispersed phase liquid and continuous phase liquid, respectively) to each holding section (dispersed phase liquid holding section and continuous phase liquid holding section, respectively), and the movement speed of each phase liquid in the flow path. The movement speed of each phase liquid in each flow path can be adjusted, for example, by appropriately selecting the amount of each phase liquid supplied to each holding section, the characteristics of the dispersed phase liquid flow path and the continuous phase liquid flow path (characteristics of the flow path surface, pressure loss in the flow path, etc.), and the liquid properties of each phase liquid. The movement speed can be adjusted mainly by capillary force and liquid level differential pressure.

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

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

Ideally, from the viewpoint of reproducibility for each measurement, it is preferable to control the timing of supply and the movement speed of each of the above-mentioned phase liquids by the physical properties and supply amount of each phase liquid, the capillary force that depends on the characteristics of each flow path, and/or the liquid level differential pressure. However, in reality, unintended pressure may be applied to each phase liquid due to the discharge pressure during supply and convection within each holding part. Therefore, it is preferable to supply the continuous phase liquid and/or the dispersed phase liquid by dropping using a tip using a dispensing means such as a pipette described below.

Note that "upstream" refers to the "upstream" when considering the overall flow direction of the liquid in the microchannel chip. Specifically, in the microchannel chip, the phase liquid holding section side is the upstream side, and the outlet side is the downstream side.

(Contact at the emulsion forming part)
In one embodiment of the present disclosure, prior to contact, the dispersed phase liquid is moved through a dispersed phase liquid flow path filled with gas to the emulsion-forming section, and the continuous phase liquid is moved through a continuous phase liquid flow path filled with gas to the emulsion-forming section.

The method of supplying the dispersed phase liquid and the continuous phase liquid to the chip without filling the flow path with the continuous phase liquid in advance is preferable because it eliminates the preparation step, but air bubbles may be generated depending on the timing of arrival of each phase liquid at the emulsion forming section. For example, if the dispersed phase liquid reaches the emulsion forming section before the continuous phase liquid and fills the emulsion forming section, there is a risk that gas (especially air bubbles) will remain between the continuous phase liquid that moves toward the emulsion forming section and the dispersed phase liquid that fills the emulsion forming section (for example, in the continuous phase liquid flow path).

In contrast, according to the above embodiment in which the dispersed phase liquid is moved to the emulsion forming section through the dispersed phase liquid flow path filled with gas, and the continuous phase liquid is moved to the emulsion forming section through the continuous phase liquid flow path filled with gas, the continuous phase liquid and the dispersed phase liquid come into contact with each other in the emulsion forming section after the continuous phase liquid flow path and the dispersed phase liquid flow path are filled completely, respectively. According to this embodiment, it is possible to suppress or prevent gas from remaining in the phase liquid flow path, and as a result, it is possible to more effectively suppress the generation of bubbles.

Particularly preferably, the dispersed phase liquid is moved to an inlet to the emulsion forming section through a dispersed phase liquid flow path filled with gas, and the continuous phase liquid is moved to the emulsion forming section through a continuous phase liquid flow path filled with gas. The inlet is a portion of the dispersed phase liquid flow path adjacent to the emulsion forming section.

In this case, the continuous phase liquid is allowed to enter the emulsion forming section before the dispersed phase liquid enters the emulsion forming section, so the risk of the dispersed phase liquid blocking the emulsion forming section can be more effectively reduced.

In this embodiment, the dispersed phase liquid is moved to the inlet to the emulsion forming section, but a converse embodiment in which the continuous phase liquid is moved to the inlet to the emulsion forming section can also be envisioned. However, since the continuous phase liquid generally has an oil component as its main component and has a relatively high fluidity, it is preferable to move the dispersed phase liquid to the inlet from the viewpoint of ease of controlling the movement within the flow path.

In order to move the dispersed phase liquid to the emulsion forming section (or the inlet section to the emulsion forming section) through the dispersed phase liquid flow path filled with gas before contact, and to move the continuous phase liquid to the emulsion forming section through the continuous phase liquid flow path filled with gas, as described above, for example, the timing of supplying each phase liquid (dispersed phase liquid and continuous phase liquid, respectively) to each holding section (dispersed phase liquid holding section and continuous phase liquid holding section, respectively) and the movement speed of each phase liquid in the flow path can be adjusted.

When the dispersed phase liquid is an aqueous solution, it is preferable for the flow channel wall surface near the emulsion forming section to be hydrophobic, since this increases the time that the dispersed phase liquid remains at the inlet of the dispersed phase liquid flow channel to the emulsion forming section, and increases the time until the emulsion forming section is filled (blocked) with the dispersed phase liquid.

Selectively making the emulsion-forming section hydrophobic is preferable because it can prevent the dispersed phase liquid from entering the emulsion-forming section prematurely, but it may lead to increased manufacturing costs. On the other hand, if the entire flow path, including the dispersed phase liquid flow path, is made hydrophobic, the speed at which an aqueous solution with high surface tension can be introduced to the emulsion-forming section by capillary force tends to decrease. In this case, pressure can be applied to the dispersed phase liquid by the liquid delivery means and/or the dispensing means to promote its movement.

<Contact in the Dispersed Phase Liquid Retaining Section>
In another embodiment of the present disclosure, prior to contacting, the continuous phase liquid is moved through a gas-filled continuous phase liquid flow path, an emulsion forming section, and a dispersed phase liquid flow path to a dispersed phase liquid holding section.

Dispersed phase liquid generally has lower fluidity than continuous phase liquid, so if bubbles occur in a flow path filled with dispersed phase liquid, it is not easy to remove the bubbles. In particular, when negative pressure is used as the external liquid delivery driving force, it is not easy to apply liquid delivery pressure directly to the dispersed phase liquid, making it even more difficult to remove such bubbles.

In contrast, in the above embodiment in which the continuous phase liquid is moved to the dispersed phase liquid holding section through the continuous phase liquid flow path filled with gas, the emulsion forming section, and the dispersed phase liquid flow path before contact, there is no need to move the dispersed phase liquid in a flow path filled with gas before contact, which is thought to further reduce the risk of air bubbles being generated in the dispersed phase liquid in the dispersed phase liquid flow path. In this case, the generation of air bubbles can be further reduced by supplying the dispersed phase liquid to the dispersed phase liquid holding section by dripping, etc. As described below, the dispersed phase liquid can be supplied to the dispersed phase liquid holding section by a dispensing means.

In this embodiment, it is preferable to shorten the time from when the continuous phase liquid reaches the dispersed phase liquid holding section until the dispersed phase liquid is supplied to the dispersed phase liquid holding section, and/or the time from when the dispersed phase liquid is supplied to the dispersed phase liquid holding section until the liquid is started to be sent by the external liquid sending driving force, thereby reducing the amount of continuous phase liquid that flows back into the dispersed phase liquid holding section and reducing non-uniform droplets that are generated when the dispersed phase liquid and the continuous phase liquid come into contact with each other in the dispersed phase liquid holding section. In one advantageous embodiment of the present disclosure, the time from when the continuous phase liquid reaches the dispersed phase liquid holding section until the dispersed phase liquid is supplied to the dispersed phase liquid holding section is preferably 30 seconds or less, more preferably 20 seconds or less, and particularly preferably 10 seconds or less.

In addition, in a microchannel chip having an emulsion-holding channel, when the continuous phase liquid is moved to the dispersed phase liquid holding section as described above, it is preferable to prevent the continuous phase liquid from excessively filling the emulsion-holding channel before the application of the external liquid-transport driving force.

＜Move＞

As described above, in the present invention, the dispersed phase liquid and the continuous phase liquid are brought into contact with each other in the emulsion forming section or upstream thereof without using a bubble trap. Preferably, the flow resistance in the emulsion forming section and upstream thereof is sufficiently small so that the dispersed phase liquid and the continuous phase liquid can come into contact with each other without an external liquid delivery driving force.

In one embodiment of the present disclosure, prior to contact, the dispersed phase liquid and/or the continuous phase liquid are moved by capillary forces and/or liquid level differential pressure.

In this manner, the movement of the dispersed phase liquid and the continuous phase liquid can be easily controlled without the need for additional equipment.

As described above, for example, the desired capillary force can be obtained by appropriately setting the channel structure of the microchannel chip. Also, for example, the desired liquid level differential pressure can be obtained by adjusting the amount of liquid (particularly the liquid level height) supplied to each phase liquid holding section.

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

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

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

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

On the other hand, for example, when negative pressure is applied to the outlet when the dispersed phase liquid moves through the dispersed phase liquid flow path before the continuous phase liquid is supplied, the continuous phase liquid flow path and the continuous phase liquid holder are not blocked by liquid, so the flow path from the outlet to the continuous phase liquid holder is open to the outside air, and a negative pressure smaller than the negative pressure of the negative pressure supply source is applied to the dispersed phase liquid. This makes it easier to control the applied pressure to be small in order to accurately control the movement of minute amounts of dispersed phase liquid through the flow path, which is preferable as it makes it easier to ensure reproducibility as an apparatus.

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

Below, we will explain in more detail each of the components that make up the invention disclosed herein.

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

(Dispersed Phase Liquid)
The dispersed phase liquid is the liquid that makes up the droplets contained in the emulsion.

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

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

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

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

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

Commercially available fluorocarbons include FC-3283 (Fluorinert® manufactured by 3M Corporation), FC-40 (Fluorinert® manufactured by 3M Corporation), and HFE-7500 (3M ^™ Novec ^™ High Performance Fluids, manufactured by 3M Corporation).

When a fluorine-based dispersion medium, especially the above-mentioned fluorocarbons, is used as the continuous phase liquid, droplets can be generated particularly stably and quickly. In addition, because it has the characteristic of being extremely poorly compatible with polar and non-polar solvents, it is possible to suppress the problem of the components of the droplets in the emulsion moving to other droplets through the continuous phase liquid (crosstalk, contamination). In addition, when selecting a liquid with low surface tension and viscosity such as a hydrocarbon-based dispersion medium or silicone oil, there is generally an increased risk of it being a hazardous material such as flammable, but fluorine-based dispersion mediums are characterized by their high safety, so that they are used as fire extinguishing agents and cooling media.

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

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

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

The volume of the droplet is preferably large enough to hold approximately one target substance (e.g., one molecule). Specifically, the average volume is preferably 0.00001 nL or more, 0.0001 nL or more, 0.001 nL or more, 0.01 nL or more, 0.1 nL or more, or 1 nL or more, and/or 100 nL or less, 50 nL or less, or 10 nL or less. From the viewpoint of uniformly reacting the target substance in the droplet, it is preferable that the volume of the droplet formed is highly monodisperse. Specifically, monodisperse means that the coefficient of variation (CV) of the droplet volume is 20% or less, 10% or less, 5% or less, 2% or less, or 1% or less. In the following description, the droplets are treated as spherical to make the explanation easier to understand, but the same applies even if the droplets are non-spherical due to the flow channel structure or the surrounding flow.

It is preferable that the droplets have sufficient thermal stability to maintain their shape at least under the reaction temperature conditions of the target substance. As a specific example, in the detection process, when nucleic acid amplification is performed by the TRC method, it is preferable that the droplets have sufficient thermal stability to maintain their shape under temperature conditions of 40°C to 48°C, and when nucleic acid amplification is performed by the PCR method, it is preferable that the droplets have sufficient thermal stability to maintain their shape under temperature conditions of 50°C to 100°C.

<Microfluidic Chip>
The micro-channel chip of the present disclosure has a dispersed phase liquid holding section, a dispersed phase liquid flow path, a continuous phase liquid holding section, a continuous phase liquid flow path, an emulsion forming section, an emulsion flow path, and an outlet, and optionally an emulsion holding flow path. The dispersed phase liquid holding section is connected to the emulsion forming section via the dispersed phase liquid flow path, the continuous phase liquid holding section is connected to the emulsion forming section via the continuous phase liquid flow path, and the emulsion forming section is connected to the outlet via the emulsion flow path. The aspects in which the micro-channel chip has an emulsion holding flow path are as described above.

The dispersed phase liquid holding section, dispersed phase liquid flow path, continuous phase liquid holding section, continuous phase liquid flow path, emulsion forming section, emulsion flow path, and outlet, as well as the optional emulsion holding flow path, are fluidly connected to each other and together form a single flow path structure.

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

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

The size (width, depth, etc.) of the flow channel constituting the microchannel chip can be appropriately determined taking into consideration the volume of the desired droplets, and in particular the reaction form of the target substance. For example, when the target substance is a nucleic acid such as DNA or RNA, and the reaction of the target substance is a digital amplification reaction of the nucleic acid (amplification reaction at the single molecule level), it is necessary to produce droplets on the order of pL or nL, so the width and depth of the flow channel around the emulsion formation section are preferably in the range of 0.1 μm to 1000 μm, and in particular 1 μm to 300 μm.

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

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

The flow channels constituting the microchannel chip can have a flow channel wall surface with low affinity for at least the dispersed phase liquid. The microchannel chip may be fabricated using a material with low affinity for the dispersed phase liquid, and the portion corresponding to the flow channel wall surface may be surface treated with a material with low affinity for the dispersed phase liquid. For example, the microchannel chip may be fabricated using a polymer material such as PDMS (polydimethylsiloxane), acrylic, cycloolefin polymer, or PTFE (polytetrafluoroethylene), and the flow channel wall surface may be surface treated with a hydrocarbon-based silanizing agent, a fluorocarbon-based silanizing agent, or the like.

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

3 is a cross-sectional schematic diagram showing one embodiment of a dispersed phase liquid retaining portion according to the present disclosure. FIG. 3 is a schematic diagram for ease of understanding and is not drawn to scale. In FIG. 3, H is the height direction, and L is the length direction. The dispersed phase liquid retaining portion 300 shown in FIG. 3 has a well 31 and a hole portion 33. The well 31 is connected to the dispersed phase liquid flow path 115 via the hole portion 33. The well 31 and the hole portion 33 extend in the height direction (here, the vertical direction). The reference numeral 35 in FIG. 3 indicates an extension portion that is a joint between the bottom surface of the well and the hole portion. In the embodiment of FIG. 3, the bottom surface of the well 31 extends perpendicular to the extension direction of the hole portion 33 (particularly the side wall of the hole portion 33 near the extension portion 35).

With regard to the dispersed phase liquid holding section, it is preferable to further optimize it as follows:

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

From the viewpoint of reducing the change in the capillary force as described above, it is preferable that the hole and/or well have substantially no discontinuous shape on the wall surface and extend in the vertical direction. In addition, when the remaining amount of the dispersed phase liquid in the dispersed phase liquid holding portion becomes small, the gas-liquid interface substantially comes into contact with the bottom surface of the well of the holding portion, and the interface shape is likely to change. In order to reduce this effect, it is preferable that the diameter of the hole and/or well that is directly fluidly connected to the flow path is small, for example, 5 mm or less, more preferably 2 mm or less, and particularly preferably 1 mm or less. On the other hand, it is also possible to increase the amount of dispersed phase liquid held by expanding the diameter from the hole or well that is directly fluidly connected to the dispersed phase liquid flow path, but in this case, if the gas-liquid interface is present near the expansion portion, the gas-liquid interface shape is likely to change. Therefore, as one preferred embodiment, the shape of the expansion portion can be adjusted and/or the height of the hole or well that is directly fluidly connected to the flow path to the expansion portion can be reduced, for example, to 3 mm or less, more preferably 1 mm or less, and particularly preferably 0.5 mm or less. Furthermore, when the amount of dispersed phase liquid supplied is small and/or the external surface of the chip around the dispersed phase liquid holding portion has low affinity for the dispersed phase liquid, and the external liquid delivery driving force is the application of negative pressure to the outlet, it is preferable to configure only the hole and supply the dispersed phase liquid to the hole so that it forms a hemispherical shape, since this minimizes the contact area with the chip wall surface and suppresses changes in the shape of the gas-liquid interface even when the remaining amount of dispersed phase liquid becomes small.

(Supply of Dispersed Phase Liquid)
The method according to the present disclosure includes providing a dispersed phase liquid to a dispersed phase liquid holding portion.

To supply the dispersed phase liquid to the dispersed phase liquid holding section, a separate container (phase liquid holding container) can be used to hold the dispersed phase liquid. From the viewpoint of preventing the liquid from leaking during storage and operation, it is preferable that such a container is completely or variably sealed while holding the dispersed phase liquid.

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 can reduce the amount of residual dispersed phase liquid and can reduce the measurement time and/or contamination between reagents (dispersed phase liquids).

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

It is preferable that the dispensing means does not cause pressure fluctuations in the liquid holding portion. The dispensing means may be, for example, a pipette. It is preferable that the dispensing means (particularly the liquid outlet constituting the dispensing means) is not fluidically connected (sealed connected) to each holding portion, but is spatially separated.

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

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

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

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 flow channels corresponding to the two or more dispersed phase liquid holding sections.

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 flow path includes a first dispersed phase liquid flow path connected to the first dispersed phase liquid holding section, a second dispersed phase liquid flow path connected to the second dispersed phase liquid holding section, and a dispersed phase liquid junction section. The first dispersed phase liquid flow path and the second dispersed phase liquid flow path are each connected to an emulsion forming section via the dispersed phase liquid junction section.

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

In the microchannel chip having the above-mentioned dispersed phase liquid confluence section, when the dispersed phase liquid is moved to the emulsion forming section (or the inlet section to the emulsion forming section) through the dispersed phase liquid flow path filled with gas before contact between the dispersed phase liquid and the continuous phase liquid, and the continuous phase liquid is moved to the emulsion forming section through the continuous phase liquid flow path filled with gas, it is preferable that the first dispersed phase liquid and the second dispersed phase liquid supplied to the first dispersed phase liquid holding section and the second dispersed phase liquid holding section, respectively, are joined at the dispersed phase liquid confluence section.

If air bubbles cause blockage of the flow path at the dispersed phase liquid confluence, it may not be possible to produce an emulsion. In particular, when liquid is delivered under negative pressure, it is not easy to eliminate blockage of the flow path caused by air bubbles due to the nature of the dispersed phase liquid, which is generally water-soluble. By having the first dispersed phase liquid and the second dispersed phase liquid supplied to the first dispersed phase liquid holding section and the second dispersed phase liquid holding section, respectively, merge at the dispersed phase liquid confluence, it is possible to avoid such problems.

In addition, in the microchannel chip having the above-mentioned dispersed phase liquid confluence section, when the continuous phase liquid is moved to the dispersed phase liquid holding section through the continuous phase liquid flow path filled with gas, the emulsion forming section, and the dispersed phase liquid flow path before contact, it is preferable to move the continuous phase liquid to both the first dispersed phase liquid holding section and the second dispersed phase liquid holding section.

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

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

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

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

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

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

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

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

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

The emulsion forming section will be described with reference to the exemplary embodiment of FIG. 1. In the emulsion forming section 120 of FIG. 1, the continuous phase liquid flow path 111 consisting of two flow paths 112, 113 and the dispersed phase liquid flow path (more precisely, the dispersed phase liquid junction 116) are substantially perpendicular to each other. During the application of an external liquid delivery driving force (negative pressure), the continuous phase liquid flows into the emulsion forming section 120 from two substantially opposite directions, and the dispersed phase liquid flows into the emulsion forming section 120 in a direction substantially perpendicular to the inflow direction of the continuous phase liquid. As a result, in the emulsion forming section 120, droplets dispersed in the continuous phase (i.e., emulsion) are generated. In the emulsion filling method, the emulsion thus generated passes through the emulsion flow path 130 and enters the emulsion holding flow path 140, which is filled with gas (see FIG. 2).

The emulsion forming section may appropriately use a flow path that utilizes a common droplet generation method such as T-junction, flow-focus, co-flow, or step-emulsification. To generate emulsion quickly, multiple emulsion forming sections may be arranged in parallel. In addition, the emulsion forming section may be equipped with a serpentine flow path for stirring the droplets in the emulsion.

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

The emulsion flow path may have an expanded width and/or height downstream (toward the outlet) of the portion adjacent to the emulsion formation section, and/or may be serpentine. This is preferred because it can promote agitation within the droplets.

(Emulsion holding channel)
As described above, in one embodiment of the present disclosure, the microchannel chip has an emulsion holding channel. The emulsion holding channel is connected to the emulsion forming section via an emulsion channel and has a function of holding the emulsion generated in the emulsion forming section. The emulsion holding channel is also optionally connected to an outlet via an outlet-connected channel.

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

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

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

In one embodiment, it is intended to perform a detection process on the held emulsion. That is, the detection process described below can be performed on the emulsion held in the emulsion holding flow channel at will.

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

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

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

Preferably, the emulsion holding flow path is configured to be capable of performing a detection process on emulsion that is not open to the outside atmosphere. More specifically, for example, between the held emulsion and the detection means, there is a structure that isolates the emulsion from the outside atmosphere. This structure is made, for example, of a material that transmits light. In this case, among the emulsions held in the emulsion holding flow path, emulsions that are not subject to the detection process, for example, emulsions located at the end of the outlet side of the emulsion holding flow path, may be open to the outside atmosphere.

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

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

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

The volumes of a spherical droplet and a disk-shaped droplet (V _drop and V _disk [nL], respectively) are represented by the following formulas (1) and (2). Note that D _drop and D _disk in the following formulas (1) and (2) respectively represent the diameters of a spherical droplet and a disk-shaped droplet when the droplets held in the emulsion holding channel are observed from above in a normal use state of the microchannel chip. In addition, in formula (2), h represents the channel height of the emulsion holding channel.

Preferably, the droplets in the emulsion to be detected by the detection means (such as a camera) used in the detection process do not overlap each other, and in particular form a single layer in a plane perpendicular to the detection direction. In this case, the detection accuracy can be further improved.

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

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

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 to the dispersed phase liquid in the emulsion forming section. For example, if the flow rate ratio of the continuous phase liquid to the dispersed phase liquid is increased to stabilize the emulsion production, it is preferable to design the channel height to be larger than usual in order to pack the droplets tightly in the horizontal direction. For example, if the flow rate ratio of the continuous phase liquid to the dispersed phase liquid is 8 to 12, the height of the emulsion holding channel can be 2 to 4 times the diameter of the droplets.

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

When the application of negative pressure is stopped, the exhaust port can be gradually opened to the outside atmosphere, which may suppress backflow at the gas-liquid interface (gas-emulsion interface) when liquid transfer is stopped. For example, liquid transfer can be stopped gradually (e.g., 10 kPa → 5 kPa → 0 kPa) by providing a separate tank containing air between two valves (particularly solenoid valves). Liquid transfer can also be stopped by adjusting the opening speed of the solenoid valve so that the degree of opening increases gradually.

In addition, when stopping the liquid supply, it is preferable to increase the flow rate ratio of the continuous phase liquid or reduce the flow volume of the emulsion holding flow channel to increase the minimum distance between the gas-emulsion interface and all the droplets, since this makes it difficult for the droplets in the emulsion to flow out to the outlet and suppresses the adverse effects of the gas-emulsion interface on nearby droplets (aggregation and coalescence of droplets, inhibition of intra-droplet reactions and signal detection).

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

(Exhaust outlet)
The microchannel chip has an outlet that can be connected to an emulsion-holding channel and can also function as a negative pressure source connection for applying negative pressure to the microchannel chip.

The outlet is preferably adapted to be connected to a negative pressure source. In this case, it is preferable that the outlet is resistant to the applied pressure.

When the microchannel chip has an emulsion-holding channel, the outlet is usually located downstream of the emulsion-holding channel. By applying negative pressure to this outlet, the entire length or most of the emulsion-holding channel can be filled with the generated emulsion.

(Liquid Delivery)
The external liquid delivery driving force provides a driving force for generating an emulsion from the dispersed phase liquid and the continuous phase liquid in the emulsion forming section, and also provides a driving force for transporting the generated emulsion to the emulsion flow path (and optionally to the emulsion holding flow path).

In the method disclosed herein, negative pressure applied to the outlet is used as such an external liquid transfer driving force. From the viewpoint of device simplicity, it is preferable to keep each holding section at normal pressure while negative pressure is being applied to the outlet, but the pressure may be controlled to an emergency pressure state in order to stabilize the liquid transfer by negative pressure and/or to also serve as a sealing operation for holding the emulsion.

Negative pressure liquid delivery is preferred because it requires simpler equipment than positive pressure liquid delivery.

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

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

In addition, when using a microchannel chip with a small flow channel pressure drop resistance, applying positive pressure to each holding section may result in the dispersed phase liquid or continuous phase liquid unintentionally entering the flow channel due to unnecessary pressure when connecting to the liquid delivery means.

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

When applying negative pressure, for example, a pressure tank or a syringe pump can be used to aspirate fluid (e.g., gas or continuous phase liquid) in the channel of the microchannel chip through an outlet.

When a pressure tank is used as the negative pressure source, the volume of the pressure tank is preferably larger than the total volume of the flow path from the outlet to the pressure tank and the volume of the flow path of the microchannel chip. The pressure tank can also be designed so that it can be opened to the outside atmosphere (particularly the outside atmosphere). It is also preferable that the pressure inside the pressure tank is controlled by, for example, a pump. A pressure sensor may also be provided so that the pressure value inside the pressure tank can be monitored.

In addition, a monitoring means for monitoring the pressure value of the applied negative pressure can be used to check the liquid delivery status, for example, whether the emulsion is being generated without any problems.

In an embodiment in which droplet generation in the emulsion forming section and filling of the emulsion into the emulsion holding channel are performed simultaneously, it is preferable to control the movement of the interface (emulsion interface) between the gas (particularly air) and the emulsion (or continuous phase) in the emulsion holding channel. In particular, the pressure drop resistance during liquid transfer increases by the volume or length of the channel filled with emulsion. In this case, if the applied external liquid transfer driving force is kept constant (constant pressure difference), the movement speed or flow rate of the emulsion interface is likely to decrease when liquid transfer starts and stops. Therefore, it is preferable that the liquid transfer means is designed so that the pressure difference increases continuously or stepwise during liquid transfer. This can be controlled, for example, by the movement speed of the syringe pump, or pressure control can be performed by monitoring the pressure in the syringe or tank with a sensor.

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

For this reason, in the emulsion filling method, when the liquid supply is stopped, the gas-liquid interface is often present within the flow path or near the flow path opening of the outlet, and the sudden pressure fluctuation when the liquid supply is stopped (air flowing into the outlet in the case of negative pressure liquid supply) can cause the gas-liquid interface to flow back through the flow path, which can have a negative effect on emulsion retention (droplets flow out of the emulsion-retaining flow path, or droplets aggregate and coalesce near the gas-liquid interface).

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

To suppress such backflow when liquid delivery is stopped, it is preferable to reduce the liquid delivery pressure. For example, the pressure applied to the microchannel chip by the external liquid delivery driving force can be 30 kPa or less, 10 kPa or less, and particularly preferably 5 kPa or less. The pressure applied to the microchannel chip by the external liquid delivery driving force is, in particular, the negative pressure applied to the outlet.

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

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

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

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

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

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

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

For example, when the target substance is a nucleic acid (DNA, RNA) containing a specific sequence, and the reaction of the target substance is a reaction that amplifies this specific sequence, the components contained in the reaction solution include a primer containing a sequence homologous to a part of the specific sequence, a primer containing a sequence complementary to a part of the specific sequence, a detection probe containing a sequence homologous or complementary to a part of the specific sequence, polymerase, nucleotides, salts, and buffer components. It is preferable that the composition of the reaction solution is designed so that the target molecule, reaction substrate, enzyme, etc. are not decomposed, altered, or subjected to non-specific reactions, and glycerol, surfactants, etc. may be further added taking into account the behavior within the device.

＜Other methods＞

(Detection Means)
For detection of the reaction, for example, a detection means capable of detecting the product of the reaction can be further provided.

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

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

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

At least the part of the temperature control means that is in close proximity (preferably in close contact) with the microchannel chip is preferably made of a metal material with high thermal conductivity. In addition, when the microchannel chip is manufactured by bonding the substrate and the superstructure, it is preferable to make the thickness of the substrate and/or the superstructure in contact with the temperature control means thinner in order to more efficiently conduct heat to the channels provided in the microchannel chip. The temperature control means is required to at least control the temperature of the emulsion-holding channel, which is the reaction field of the target substance, but it is preferable to control the temperature of the phase liquid holding section and the channel as well in order to suppress nonspecific reactions of the target molecules. As a specific example, when the reaction of the target substance is a nucleic acid amplification reaction, the temperature control means controls the temperature in each holding section and channel so that it is higher than the reaction temperature of the target substance in the emulsion-holding channel, thereby reducing nonspecific annealing between primers/probes. In addition, when the bottom surface of the microchannel chip is heated to the reaction temperature by a temperature control means, and the top surface substrate of the microchannel chip is made of a light-transmitting material and transmitted light is detected from the top surface, it is preferable because the following can be easily performed on the device: evaluation of the position of the empty microchannel chip before supplying each phase liquid, evaluation of the flow channel structure and/or dirt inside and outside the chip; evaluation of the behavior inside the flow channel when supplying each phase liquid and/or the behavior of emulsion generation during liquid transfer; and improvement of the upper limit of quantification of digital detection using the signal detection results of the emulsion during the reaction.

The present invention will be described in more detail below using examples. The present invention is not limited to these descriptions.

<Fabrication of microfluidic chip>
The manufacturing method of the microchannel chips (No. 1 and No. 2) using photolithography and soft lithography techniques will be described below.

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

(2) Using a mask aligner (Ushio Inc.) and a chrome mask on which the channel pattern of the microchannel chip was formed, the channel pattern was formed on a photoresist film, and then the channel pattern was developed using an SU-8 Developer (Microchem Inc.) to create a template for the channels that make up the microchannel chip.

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

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

(5) The obtained polymer substrate was carefully peeled off from the mold, shaped with a cutter, and then a puncher was used to form the dispersed phase liquid holding portion, the continuous phase liquid holding portion, and the discharge port.

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

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

(Flow Channel Structure)
The microchannel chips (No. 1 and No. 2) had two dispersed phase liquid holders, a dispersed phase liquid flow path (having a first dispersed phase liquid flow path, a second dispersed phase liquid flow path, and a dispersed phase liquid junction), a continuous phase liquid holder, two continuous phase liquid flows, an emulsion forming section, an emulsion flow path, an emulsion holding flow path, and an outlet. The two dispersed phase liquid holders were connected to the emulsion forming section via the first or second dispersed phase liquid flow path, the continuous phase liquid holder was connected to the emulsion forming section via the two continuous phase liquid flows, the emulsion forming section was connected to the emulsion holding flow path via the emulsion flow path, and the emulsion holding flow path was connected to the outlet. The microchannel chips did not have a bubble trap.

Regarding the microchannel chip (No. 1), the first dispersed phase liquid flow channel and the second dispersed phase liquid flow channel are flow channels with a height of 100 μm, a width of 100 μm, and a length of 4200 μm, and are joined at the dispersed phase liquid joining section, narrowed to a flow channel width of 100 μm, and joined at the emulsion forming section. The two continuous phase liquid flow channels are each 380 μm wide with two bends, and are narrowed to a height of 100 μm and a width of 160 μm at the inlet to the emulsion forming section, with a total length of 30 mm.

In the emulsion formation section, the dispersed phase liquid junction and the two continuous phase liquid flow paths cross at an angle of 90 degrees. In the emulsion formation section, the reaction liquid and the immiscible liquid (oil) join together to form droplets.

The emulsion flow path is a straight flow path with a width of 80 μm and a length of 100 μm near the emulsion formation section, a width of 200 μm and a length of 700 μm downstream, and a stirring flow path with a width of 400 μm and a length of 11.5 mm including a meandering path composed of an arc curve with a radius of 300 μm downstream, which is connected to the emulsion holding flow path. The flow path height upstream of the emulsion holding flow path is 100 μm.

The emulsion-holding flow channel is a serpentine flow channel with a flow channel height of 160 μm, a width of 2 mm, and a length of 350 mm, and is directly connected to the outlet via an outlet-connected flow channel with a width of 2 mm and a length of 10 mm. The volume of the emulsion-holding flow channel was 91 μL.

(Microchannel chip: No. 2)
Microchannel chip No. 2 had the following structure.

The first dispersed phase liquid flow path and the second dispersed phase liquid flow path are flow paths with a width of 100 μm, a height of 50 μm, and a length of 2600 μm. They join at a dispersed phase liquid junction, narrow to a flow path width of 100 μm, and join at an emulsion forming section. The two continuous phase liquid flow paths are flow paths with a width of 300 μm, a height of 120 μm, and a length of 26 mm, each having two bends.

The emulsion forming section has a flow path height of 120 μm, and the dispersed phase liquid junction of the dispersed phase liquid flow path and the two continuous phase liquid flow paths cross at an angle of 90 degrees, bringing the joined reaction liquid into contact with the immiscible liquid (oil), forming droplets of the joined reaction liquid in the emulsion forming section or emulsion flow path.

The emulsion flow path is a flow path that directly intersects with the emulsion forming section, and is a straight flow path with a width of 80 μm and a length of 100 μm, and downstream of that, it is a straight flow path with a width of 200 μm and a length of 700 μm, and further downstream, it is a stirring flow path with a width of 400 μm and a length of 11.5 mm, including a meandering formed by an arc curve with a radius of 300 μm, and is connected to the emulsion holding flow path. The emulsion holding flow path is a meandering flow path with a flow path height of 160 μm, a width of 2 mm, and a length of 350 mm, and is directly connected to the outlet via an outlet connecting flow path with a width of 2 mm and a length of 10 mm. The volume of the emulsion holding flow path was 91 μL.

<Reference Example 1>
(Emulsion Creation and Maintenance)
Using the above-mentioned microchannel chip (No. 1), emulsions were generated and maintained according to the present invention, as detailed in the following (1) to (6).

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

(Starting solution)
36.8 mM Magnesium chloride 180.0 mM Potassium chloride 0.2% (w/v) Tween 20
18.0% (v/v) DMSO
5.0% (v/v) glycerol

(Reaction solution)
0.2% (w/v) Tween 20
300 nM Trehalose 5.0% (v/v) Glycerol

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

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

(4) Using a pipette, 20 μL of each of the reaction liquid and the starting liquid were dropped into the dispersed phase liquid holding section. After 40 seconds, 200 μL of oil was dropped into the continuous phase liquid holding section. The pipette used above was calibrated to a maximum allowable error of ±0.06 μL.

(5) 300 seconds after the contact between the dispersed phase liquid and the continuous phase liquid, the pressure in the tank connected to the outlet was adjusted to -5 kPa in advance, and negative pressure was applied to start droplet generation and retention. Note that each phase liquid retention section was open to atmospheric pressure during liquid transfer.

(6) After the oil-air interface (emulsion-air interface) reached the downstream end of the emulsion-holding flow path (approximately 150 seconds after the application of negative pressure), the negative pressure applied to the outlet was released to normal pressure, and the liquid flow was stopped.

(Non-uniform droplets)
A digital CMOS camera (ORCA-FLASH (trade name), Hamamatsu Photonics) was used to capture bright-field images of the emulsion formation area, and the number of droplets generated before application of negative pressure and their average volume (nL) were measured as a function of the elapsed time from contact between the dispersed phase liquid and the continuous phase liquid.

Similarly, the average volume (nL) of droplets generated after application of negative pressure was measured. The results are shown in Table 1.

As can be seen from Table 1, the longer the time from when the dispersed phase liquid comes into contact with the continuous phase liquid until the application of negative pressure (the liquid transfer preparation time), the more non-uniform droplets (droplets smaller in size than the droplets after the application of negative pressure) are formed.

<Reference Example 2>
Except for using the above microchannel chip (No. 2), the production, retention, and evaluation of emulsions were performed in the same manner as in Reference Example 1. The results are shown in Table 2.

As can be seen from Table 2, the longer the time between contact between the dispersed phase liquid and the continuous phase liquid and the application of negative pressure (the liquid transfer preparation time), the more non-uniform droplets (droplets larger in size than the droplets after the application of negative pressure) are formed.

10, 20 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 flow path 115 Second dispersed phase liquid flow path 116 Dispersed phase liquid junction section 117 Dispersed phase liquid flow path 120 Emulsion forming section 130 Emulsion flow path 140 Emulsion holding flow path 150 Outlet 300 Dispersed phase liquid holding section 31 Well 33 Hole 35 Expansion section W Width direction L Length direction H Height direction

Claims (13)

A method for producing an emulsion by supplying a dispersed phase liquid and a continuous phase liquid to a microchannel chip, comprising the steps of:
the microchannel chip has a dispersed phase liquid holding section, a dispersed phase liquid flow path, a continuous phase liquid holding section, a continuous phase liquid flow path, an emulsion forming section, an emulsion flow path, and an outlet;
the dispersed phase liquid holding unit is connected to the emulsion forming unit via the dispersed phase liquid flow path,
the continuous phase liquid holding unit is connected to the emulsion forming unit via the continuous phase liquid flow path,
the emulsion forming section is connected to the outlet through the emulsion flow path,
Supplying a dispersed phase liquid to the dispersed phase liquid holding unit;
Supplying a continuous phase liquid to the continuous phase liquid holding section; and
generating an emulsion including droplets composed of the dispersed phase liquid and a continuous phase composed of the continuous phase liquid in the emulsion forming section by applying a negative pressure to the outlet, and allowing the emulsion thus generated to enter the emulsion flow path;
Including,
contacting the dispersed phase liquid with the continuous phase liquid at or upstream of the emulsion formation section without using a bubble trap prior to the application of the negative pressure; and
administering said application of negative pressure within 30 seconds after said contact;
A method comprising: A negative pressure control means is fluidly connected to the outlet;
The negative pressure control means comprises a negative pressure source, a connection and a valve;
The negative pressure source is controlled to a constant negative pressure,
the connection is for fluidly connecting the negative pressure control means to the outlet, and the valve is disposed between the negative pressure source and the connection.
The method of claim 1. The method of claim 2, characterized in that the negative pressure control means is fluidly connected to the outlet prior to the contacting of the dispersed phase liquid and the continuous phase liquid. The method according to any one of claims 1 to 3, characterized in that the application of the negative pressure is stopped by connecting the exhaust port to the outside atmosphere. The method according to any one of claims 1 to 4, wherein the resistance of the emulsion forming section and the flow path upstream thereof is sufficiently small so that the dispersed phase liquid and the continuous phase liquid can come into contact with each other without an external liquid delivery driving force. Prior to said contacting,
The dispersed phase liquid is moved to the emulsion forming section through the dispersed phase liquid flow path filled with gas, and
The method according to any one of claims 1 to 5, characterized in that the continuous phase liquid is moved to the emulsion forming section through the continuous phase liquid flow path in a state filled with gas. The method according to claim 6, wherein the dispersed phase liquid and/or the continuous phase liquid are moved by capillary force and/or liquid level differential pressure. The method according to claim 6 or 7, characterized in that a negative pressure is applied to the outlet, thereby moving the dispersed phase liquid through the dispersed phase liquid flow path filled with gas to the emulsion forming section before the contact. The method according to any one of claims 1 to 5, characterized in that, prior to the contact, the continuous phase liquid is moved to the dispersed phase liquid holding section through the continuous phase liquid flow path, the emulsion forming section, and the dispersed phase liquid flow path, all of which are filled with gas. The method of claim 9, wherein the continuous phase liquid is moved by capillary force and/or liquid level differential pressure. the microchannel chip further has an emulsion-holding channel,
the emulsion forming section is connected to the emulsion holding flow path via the emulsion flow path, and the emulsion holding flow path is connected to the discharge port,
applying a negative pressure to the outlet, in the emulsion forming section, to generate an emulsion containing droplets composed of the dispersed phase liquid and a continuous phase composed of the continuous phase liquid, and transporting the emulsion thus generated through the emulsion flow path to the emulsion holding flow path filled with gas;
The method according to any one of claims 1 to 10, comprising: The method of claim 11, characterized in that the magnitude of the negative pressure applied to the outlet is 30 kPa or less. The method according to claim 11 or 12, characterized in that the emulsion production speed in the emulsion forming section is 5 droplets/second or more.

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