JP7298828B2 - Microdroplet manufacturing device and manufacturing method - Google Patents

Description

The present invention relates to a microfluidic control device and a manufacturing method thereof. More specifically, it is a manufacturing mechanism for microdroplets used in life science research, and is concerned with cost reduction, speedup, and simplification.

In recent years, in the field of life science research, research and development are underway aiming at medical applications by devising microfluidic devices (microfluidic chips) based on microfluidics. In particular, there is an increasing need for producing microdroplets (emulsions) in biotechnology research fields, diagnostic research fields, and pharmaceutical production technology research, and various flow path patterns are being designed and widely applied.

Among them, the development of next-generation sequencers has increased the ability to decode multiple types of gene sequences at once, and after gene amplification using microdroplets as one reaction container, each microdroplet is destroyed and the components inside are mixed again. It became possible to decode each sequence even if it was analyzed by applying it to the apparatus.

Alternatively, after gene amplification is performed using a microdroplet as one reaction container, only those that have been successfully amplified inside the droplet are illuminated with a fluorescent dye. It is now possible to detect how many molecules originally existed in units of one molecule.

Furthermore, by combining it with cell sorting technology, we have applications such as the high-precision, one-by-one sorting of microorganisms, immune cells, etc. Attempts have also been made to pick a sample using a microdropper under a microscope.

A microfluidic chip having branched channels is generally used to produce such microdroplets.

A microfluidic chip with branched channels has an injection port for an oil-phase liquid, which is a continuous phase, and an injection port for an aqueous-phase liquid, which contains cells, etc. and is a dispersed phase. It is common to connect a thin and flexible tube such as a (trademark) tube and feed the liquid using a pressure pump (air pressure, water pressure, hydraulic pressure, or mechanical type).

Alternatively, each injection port of the microfluidic chip is formed into a well, and after the liquid is stored in the well, a lid with an air inlet is placed, and air is sent from the inlet and pressure is applied to send the liquid. There is a way to do it.

However, such conventional methods have the following problems.

(1) It requires a liquid delivery system that is relatively expensive and difficult to operate and adjust.

(2) Use a microfluidic chip with branched channels that are difficult to control.

(3) While using a low-volume microchannel, a considerable amount of dead volume is generated due to bubble removal and initial condition determination.

Due to these problems, users who can use this technology are limited to researchers with knowledge of science and engineering or researchers who can purchase expensive automated equipment.

In such a background, the technique of the following literature exists as a means to solve the problem of (1).

Patent No. 4410040 Japanese Patent Application Laid-Open No. 2019-005700

The microfluidic chip described in Patent Document 1 is made of an elastic polymeric material such as rubber, and has ports that open at a plurality of ends of the microchannel, communicating with the microchannel. The decompression chamber is shielded from the outside, and after degassing, liquid is introduced from the opening in the atmosphere and flows into the flow path, and the decompression chamber is sealed. It dissolves in the material and creates a pressure difference between it and the opening. As long as this pressure difference exists, the liquid flows into the decompression chamber.

Patent document 2 is a mechanism that enhances the decompression effect in order to further develop the effect of patent document 1, and by forming a mesh-like microstructure space in a decompression part communicating with a microchannel, a closed The area of the space is increased, the speed of absorption into the chip body is increased, and a larger pressure difference is produced than in Patent Document 1. Along with this, the speed at which the liquid flows increases, and the speed at which microdroplets are produced also increases. In addition, it is expected that the high absorption rate will continue in the fine structure space.

In this way, the technology of Patent Documents 1 and 2 can solve the problem (1), but the problem of (2) using the branched flow path and generating the dead volume (3) is solved. I can't do it.

The present invention has been made in view of the circumstances of the above-mentioned prior art, and the problem and the solution thereof are not to use an expensive liquid transfer device, so that connection to a microfluidic chip is not required, and the dead volume is minimized. With the vacuum pack, the cost of reagents can be reduced by minimizing the cost of reagents, making it inexpensive, and microdroplets can be produced at high speed without using branch channels, and experiments can be performed easily and immediately without knowledge of microfluidics. It is an object of the present invention to provide a packaged microdroplet manufacturing device.

Common methods for introducing liquids into microfluidic chips include using a syringe pump and a system in which compressed air is gradually released with a valve.

When using a syringe pump, it is necessary to connect it to a tube. Therefore, it is devised to attach a silicon tube to the microfluidic chip or connect a fluorine-based tube via a connector. After that, prepare as many syringe pumps as the number of types of liquid samples to be introduced, suck the liquid sample from the tip of each syringe, connect the tip of the syringe to the tube coming from the microfluidic chip, set the syringe on the motor device, The liquid is sent by driving the motor.

When using a compressed air and valve system, there is a high-pressure chamber first, and compressed air flows into the chamber from a tube connected to the lid. The lid is opened to set a container containing a liquid sample therein, the tip of the tube extending from the lid is placed in the space in the container, and the high-pressure chamber is closed. In this case, after preparing and setting the apparatus for each type of liquid sample, the liquid is sent by controlling the opening and closing of the valve.

In any device, the volume and weight of each device are several hundred to several thousand times greater than that of a microfluidic chip, and the pump system occupies most of the experimental components. In addition, even if researchers prepare these devices individually, it costs several hundred thousand yen, and already systemized products have a market price of 2 million to 3 million yen.

In addition, when using the device as described above, if liquids are flowed simultaneously from a plurality of tubes into the microfluidic chip, air bubbles may be trapped in the channels, changing the flow of the liquid and adversely affecting the experimental results. occurs frequently. For this reason, it is common to first perform an operation to remove air bubbles, but the amount of reagent used for this purpose can become a dead volume.

In addition, the reagents used for the production of microdroplets are expensive, ranging from 2,000 to 3,000 yen per gram per gram.

In addition, numerical values such as the Reynolds number and the cross-sectional area of the channel are used for channel design when manufacturing microdroplets in a branched channel. , the conditions change moment by moment depending on the temperature, humidity, material, type of liquid, etc. at the time of the experiment. This becomes more conspicuous as the shape of the branch flow path becomes more complicated, and is optimized by adjusting the flow rate of the liquid transfer device.

However, since advanced know-how is required for such adjustments, it is necessary to move away from the original biological experiment and make full use of fluid dynamics and liquid manipulation technology, and it may be far from the purpose. be.

As described above, there are many problems in producing microdroplets using conventional microfluidic chips, and despite the high demand, many researchers in life science research have never used them.

The inventor of the present invention has conducted extensive research in the above-described conventional technology, and found that microdroplets can be produced at high speed at the boundary between the microchannel and the droplet reservoir instead of the conventional branched channel. (Figs. 1-3).

This phenomenon is caused by the fact that the oil phase flows into the depressurized droplet reservoir (FIG. 5) of the microdroplet production device (FIG. 4) through the microchannel due to the suction force, and the material of the microfluidic chip and the oil phase Due to the familiarity, the oil phase fills the bottom wall of the droplet reservoir to create a structure that blocks the exit of the microchannel, and the water phase tries to flow into the droplet reservoir by the same suction force as described above. When entering the oil phase, the water phase forms a lump at the contact portion as shown in FIG.

As shown in FIG. 6, the water phase suddenly changed to a narrowed shape near the outlet of the microchannel just before it turned into droplets, and then partly appeared in the reservoir where the pressure was released. Due to the suction force, the narrowed part that passed through the lump shape is cut into the oil phase using the step at the upper part of the flow path, and becomes spherical.

The minimum materials required to create this phenomenon are a plate-like microfluidic chip having a single microchannel, and a liquid that communicates with both ends of the microchannel and opens on the opposite side of the channel. An introduction part and a droplet storage part, a channel lid that closes the microchannel, a negative pressure lid that closes the droplet storage part, an oil phase that serves as a continuous phase for producing droplets, and an aqueous phase , is.

Microfluidic chips used here require transparency and low fluorescence because microdroplet fabrication involves microscopy. Although the material is not particularly limited as long as it has such properties, the most commonly used material is PDMS, which is applied to a semiconductor manufacturing technology to make a mold, and the above liquid substance at room temperature is placed in the mold. is flowed, hardened by high temperature treatment (70°C or higher), and peeled off to create a microstructure with high transferability. In addition, it can be said to be an optimal material because it has the property of absorbing gas.

In addition, the microfluidic chip has one or more non-branched channel structures that are straight, curved, or bent lines on one side of the wide surface of the plate-like structure, and liquid is introduced to one end. It has a liquid inlet opening to the bottom and a droplet reservoir at the other end.

The size of the channel is not particularly limited, but considering that the size of droplets to be produced is around 10 μm to 1000 μm, the cross-sectional area is 1 mm ² or less, more preferably 0.5 mm ² or less, and more preferably 0.5 mm 2 or less. is 0.2 mm ² or less.

In addition, on the surface of the microfluidic chip opposite to the channel structure, a micropattern is formed by laser processing or template transfer, although the shape and method are not particularly limited. Especially in this case, a grid shape is preferable.

The size and shape of the liquid introduction part are not particularly limited, but a cylindrical shape with a diameter of 1 mm or more is desirable for ease of liquid introduction with a pipette.

The size and shape of the liquid reservoir are not particularly limited, but in order to efficiently produce uniform droplets, the cross-sectional area of the liquid reservoir is at least twice the cross-sectional area of the flow path, more preferably It is 4 times or more, more preferably 10 times or more.

The channel cover that closes the microchannel is preferably made of a material that is not compatible with the aqueous phase that will be the dispersed phase. . The material is not particularly limited as long as it has such properties, but transparent plastics such as PDMS, COP, PMMA, polystyrene, polycarbonate, polypropylene, polyolefin, and polyethylene, or glass with a hydrophobic coating in the case of glass. is more preferable.

When decompressing the micropattern section and the liquid droplet storage opening formed on the surface opposite to the channel structure of the microfluidic chip, one sheet or each section needs a negative pressure lid. However, it is sufficient if it has a smooth plate shape, and it is more preferable if it has transparency and low fluorescence for microscopic observation. Although the material is not particularly limited as long as it has such properties, it may be a transparent plastic such as PDMS, COP, PMMA, polystyrene, polycarbonate, polypropylene, polyolefin, or polyethylene, or glass.

The negative pressure lid may have a micropattern formed thereon by laser processing or mold transfer, although the shape and technique are not particularly limited. It is preferable to have such a shape, and a square shape is particularly preferable from the viewpoint of ease of preparation.

A through hole may be formed in the negative pressure lid in order to collect the droplets produced from the droplet reservoir. It is preferable that it is closed with a seal or the like.

The oil phase is not particularly limited as long as it is not compatible with the aqueous phase that will be the dispersed phase, but fluorine-based oils are preferred, and perfluoroethers and perfluorocarbons are particularly preferred.

Further, the oil phase should preferably contain a surfactant, and its kind and amount are not particularly limited as long as it dissolves in the oil phase.

The aqueous phase is not particularly limited as long as it is not compatible with the oil phase, but is preferably pure water, aqueous solution, emulsion, cell suspension, biological extract, or blood.

Further, the aqueous phase may contain a surfactant, and the type and amount thereof are not particularly limited as long as they do not interfere with droplet production.

In order to produce droplets, the droplets are produced by decompressing the microdroplet production apparatus, introducing an oil phase from a liquid introduction part, and then introducing an aqueous phase. The decompression condition is not particularly limited, but it is preferably carried out at an absolute pressure of 500 Pa or less for 2 hours or more, more preferably 350 Pa or less for 6 hours or more.

Usability is improved by providing the reduced pressure to the user in a vacuum-packaged state using a vacuum packaging machine. The packaging material used in the vacuum packaging is not particularly limited, but in order to maintain the vacuum state for a long time, it is preferably made of nylon, polyethylene, or aluminum, or more preferably a composite material of these having a multilayer structure. .

For reference, a diagram of the prior art is shown below. A T-shaped channel diagram (Fig. 13) used in the production of microdroplets, a photographic image of microdroplet production using the T-shaped channel (Fig. 14), and a cross-channel diagram (Fig. 15) ) and a photographic image (FIG. 16) of producing microdroplets using a cross-shaped channel.

The present invention has the above configuration, does not use an expensive device, and does not require connection to a microfluidic chip, which is the same as Document 2, but also minimizes the dead volume and saves the cost of reagents. To provide a vacuum-packed micro-droplet manufacturing apparatus which is inexpensive, can manufacture micro-droplets at high speed without using a branch channel, and can be easily and immediately experimented without knowledge of microfluid dynamics. It becomes possible to

FIG. 10 is a photographic image of a connecting portion between a droplet reservoir and a channel immediately after droplet production is started using the microdroplet production apparatus; FIG. 10 is a photograph image showing about 30 droplets produced at the connecting portion; FIG. 5 is a photographic image showing about 50 droplets produced at the connecting portion; 1 is a top perspective view of a microdroplet manufacturing device; FIG. It is the droplet storage part of the microdroplet manufacturing device circled in FIG. 6 is an enlarged view of the droplet reservoir of FIG. 5; FIG. It is a top view of a microdroplet manufacturing apparatus. It is a side view of a microdroplet manufacturing apparatus. 1 is a plan view of a microfluidic chip; FIG. FIG. 4 is a plan view of the negative pressure lid; It is the figure which vacuum-packed the microdroplet production apparatus. It is a photograph image view of a microdroplet manufacturing apparatus. FIG. 3 is a diagram of a T-shaped channel used in general droplet production. FIG. 14 is a photographic image of droplets being produced in the same channel as in FIG. 13; FIG. 2 is a diagram of a criss-cross channel used in typical droplet production; FIG. 16 is a photographic image of droplets being produced in the same channel as in FIG. 15; FIG. 2 is a flow channel structure diagram of the microfluidic chip used in Example 1. FIG. FIG. 4 is a photographic image of a grid-like micropattern formed on the surface opposite to the flow path using a laser processing machine. FIG. 3 is a diagram of the microdroplet production apparatus before installation in a desiccator, in which the channel cover and the negative pressure cover are brought into close contact with each other. FIG. 10 is a photographic image of the droplet reservoir during production of microdroplets using the microdroplet production apparatus; FIG. 10 is a flow channel structure diagram of a microfluidic chip used in Example 2. FIG. FIG. 4 is a photographic image of a grid-like micropattern formed on the surface opposite to the flow path using a laser processing machine. FIG. 10 is a photographic image of the microdroplet manufacturing apparatus used in Example 2, which is vacuum-packed. FIG. 10 is a photographic image of the droplet reservoir during production of microdroplets using the microdroplet production apparatus; FIG. 10 is a flow channel structure diagram of a microfluidic chip used in Example 3. FIG. It is a photographic image view of the microfluidic chip. FIG. 4 is a photographic image of a grid-like micropattern formed using a laser processing machine. FIG. 10 is a photographic image of the droplet reservoir during production of microdroplets using the microdroplet production apparatus; FIG. 10 is a photographic image of a droplet reservoir, a flow path, and a wide flow path between them of the microdroplet production apparatus used in Example 4. FIG.

EXAMPLES The present invention will now be described more specifically based on examples. However, the present invention is not limited to the following examples.

Example 1 Droplet production using a microdroplet production apparatus depressurized by a desiccator 1-1) Fabrication of microfluidic chip (FIG. 17) A microfluidic chip having a flow channel structure shown in FIG. It was produced by the process. The material of the microfluidic chip is PDMS. The channel has a width of 250 μm and a height of 31 μm. Next, a grid-like micropattern for generating a large pressure difference was formed on the surface opposite to the channel using a laser processing machine (FIG. 18).
The obtained chip body was brought into close contact with a polycarbonate substrate that serves as a channel lid, and a through-hole was made with a φ5 mm punch for the droplet introduction part and a φ6 mm punch for the droplet storage part so as to communicate with both ends of the channel. formed.

1-2) Production of Microdroplets A slide glass serving as a negative pressure cover was brought into close contact with the droplet reservoir and the laser-processed micropattern (FIG. 19), and then placed in a desiccator to reduce the pressure. After reaching an absolute pressure of 350Pa, close the valve between the pump and the desiccator to create a state where there is no pressure leakage, let stand for 6 hours, remove the chip from the desiccator, place it on the desk with the opening of the liquid introduction part facing up. I got on. Immediately after that, 10 μL of the oil phase (RTFLS-2%10 emulseo.com) was introduced from the liquid introduction part using a micropipette, and after about 10 seconds from the same liquid introduction part as the above, the blue-colored aqueous phase (Indigo Carmin CI-73015 Tokyo Chemical Industry) 40 μL was introduced with a pipette and placed on the oil phase.
In the droplet reservoir, the suction force is applied in the direction of the negative pressure lid (upward in this case), but the oil phase has a strong interaction with the microfluidic chip material, and the specific gravity of the oil phase used is high (1.1 to 1.2). Therefore, it accumulates at the end near the bottom wall of the liquid reservoir and blocks the outlet of the channel. When the water phase flows into the droplet reservoir while maintaining this state, it passes through a lump shape and is torn off by the step at the outlet of the flow path and the oil phase to form droplets.
FIG. 20 shows the observation of the communicating portion between the droplet reservoir and the flow path in this state. Droplets were produced continuously at high speed for 25 seconds. The droplet diameter was about 170 μm and the production rate was 640 droplets/sec.

Example 2 Droplet production using a microdroplet production apparatus depressurized by a vacuum packaging machine 2-1) Fabrication of microfluidic chip A microfluidic chip having the flow channel structure shown in FIG. It was produced by the process. The material of the microfluidic chip is PDMS. The channel has a width of 250 μm and a height of 31 μm. Next, a grid-like micropattern for generating a large pressure difference was formed on the surface opposite to the channel using a laser processing machine (FIG. 21).
The obtained chip body was brought into close contact with a polycarbonate substrate that serves as a channel lid, and a through-hole was made with a φ5 mm punch for the droplet introduction part and a φ6 mm punch for the droplet storage part so as to communicate with both ends of the channel. was formed (FIG. 22).

2-2) Manufacture of microdroplets After attaching a slide glass as a negative pressure lid so as to close the droplet reservoir and the laser-processed micropattern, poly-pack for vacuum (HEIKO nylon-poly barrier type SPN-1217). It was placed inside a vacuum packaging machine and decompressed. After reaching an absolute pressure of 350 Pa, the reduced pressure state was maintained for about 20 seconds, and a vacuum pack was obtained after automatic sealing (Fig. 23). After 15 hours, the vacuum pack was opened, 10 μL of the oil phase (RTFLS-2%10) was introduced from the liquid introduction part using a micropipette, and after about 20 seconds, a blue-colored aqueous phase was introduced from the same liquid introduction part as above (Indigo Carmin) 40 μL was introduced by pipette and placed on top of the oil phase.
FIG. 24 shows the observation of the connecting portion between the droplet reservoir and the flow path in this state. Droplets were produced continuously over 600 seconds. The droplet diameter was about 170 μm and the production rate was 30 droplets/sec.

Example 3 Micropattern arrangement change from Example 1 (microfluidic chip → negative pressure lid)
3-1) Production of Microfluidic Chip A microfluidic chip having the channel structure shown in FIG. 25 was produced by a standard soft lithography process. The material of the microfluidic chip is PDMS. The channel has a width of 40 μm and a height of 50 μm.
After the obtained chip main body and the channel cover were brought into close contact with each other, a through hole was formed by a punch of φ4 mm in the droplet introduction part and φ5 mm in the droplet storage part so as to communicate with both ends of the channel. (Fig. 26). Next, a micropattern for generating a large pressure difference was applied to the negative pressure lid side made of PDMS using laser processing (Fig. 27).

3-2) Production of fine droplets After the above-described laser-processed negative pressure lid was brought into close contact with the droplet reservoir so as to cover it, it was placed in a desiccator and depressurized. After reaching an absolute pressure of 350 Pa, it was placed in a desiccator and decompressed. After reaching an absolute pressure of 350 Pa, the valve between the pump and the desiccator was closed to create a pressure-leakage-free state. Immediately after that, 10 μL of the oil phase (RTFLS-2%10 emulseo.com) was introduced from the liquid introduction part using a micropipette, and after about 20 seconds from the same liquid introduction part as the above, the blue-colored aqueous phase (Indigo Carmin) was introduced by pipette and placed on top of the oil phase.
FIG. 28 shows the observation of the communicating portion between the droplet reservoir and the channel in this state. Droplets were produced continuously over 300 seconds. The droplet diameter was about 120 μm and the production rate was 100 droplets/sec.

Example 4 Confirmation of Oil Phase Membrane Structure and Stepped Structure Required for Producing Droplets As in Example 1, a microfluidic chip with a channel structure on one side and a laser-processed surface on the opposite side was fabricated. A through-hole of φ4 mm was made in the liquid introduction part and a through-hole of φ6 mm in the droplet storage part by a punch. A polycarbonate substrate was used as the channel lid, and a slide glass was used as the negative pressure lid. A semicircular structure wider than the channel was constructed between the droplet reservoir and the channel. In the droplet reservoir, the suction force is applied in the direction of the negative pressure lid, but the oil phase has a strong interaction with the microfluidic chip material, and the specific gravity of the oil phase used is high (1.1 to 1.2), so the liquid reservoir It accumulates at the edge near the bottom wall and blocks the outlet of the channel. Due to such a structure, the oil phase does not remain at the connecting portion between the channel and the semicircular structure, and droplets are not produced at this connecting portion. A droplet is produced at the junction (FIG. 29).

[Mode for carrying out the invention]
1. Microdroplet manufacturing device

2. Microfluidic chip channel structure Liquid introduction section Droplet storage section
Micropattern structure

3. Hydrophobic/ water-repellent plate-like structure for channel cover

4. Negative pressure lid micropattern structure Retained droplet recovery hole Recovery hole seal

5. vacuum pack bag

6. Oil phase oil Surfactant

7. aqueous phase

1. Microdroplet manufacturing device 10. Liquid driving means (means for driving the liquid by the decompressed space)
11. Liquid driver

2. microfluidic chip 21 . Microfluidic chip side micropattern 22 . Flow path structure 23. liquid introduction part 24 . droplet reservoir 25 . liquid inlet seal

3. flow path lid

4. Negative pressure lid 41 . Negative pressure lid side micropattern 42 . droplet recovery hole 43 . Droplet collection hole seal

50. Hitodama shape 51 . Upper step 52 . Oil phase (film)
53. manufactured droplets

6. vacuum pack

Claims (4)

a plate-like chip body made of synthetic resin that absorbs gas;
one channel without branches formed on one surface of the chip body ;
a plate-like channel lid that closes the channel;
a liquid introduction part that communicates with one end of the channel and opens on a surface opposite to the surface on which the channel is formed;
a droplet reservoir that communicates with the other end of the channel and opens on a surface opposite to the surface on which the channel is formed;
a negative pressure lid for sealing the droplet reservoir and a step at a connecting portion between the droplet reservoir and the flow channel,
By introducing the oil phase from the liquid introduction part after depressurizing the chip body,
Having a structure in which an oil phase is accumulated at the edge of the droplet storage part and the flow path outlet part is completely covered with the oil phase,
By introducing the aqueous phase from the liquid introduction part,
micro droplets are produced at a connection portion having a step between the flow path and the droplet reservoir ;
A microfluidic chip, wherein the decompression is performed by a vacuum packaging machine, and the microfluidic chip is packaged in a highly airtight packaging material . 2. The microfluidic chip according to claim 1, wherein the depressurization is performed under conditions of an absolute pressure of 500 Pa or less and 2 hours or more. a plate-like chip body made of synthetic resin that absorbs gas;
one channel without branches formed on one surface of the chip body ;
a plate-like channel lid that closes the channel;
a liquid introduction part that communicates with one end of the channel and opens on a surface opposite to the surface on which the channel is formed;
a droplet reservoir that communicates with the other end of the channel and opens on a surface opposite to the surface on which the channel is formed;
a negative pressure lid for sealing the droplet reservoir and a step at a connecting portion between the droplet reservoir and the flow channel,
By introducing the oil phase from the liquid introduction part after depressurizing the chip body,
Having a structure in which an oil phase is accumulated at the edge of the droplet storage part and the flow path outlet part is completely covered with the oil phase,
By introducing the aqueous phase from the liquid introduction part,
micro droplets are produced at a connection portion having a step between the flow path and the droplet reservoir ;
The method for producing microdroplets , wherein the depressurization is performed by a vacuum packaging machine, and the droplets are packaged with a highly airtight packaging material . 4. The method of manufacturing microdroplets according to claim 3, wherein the decompression is performed under conditions of an absolute pressure of 500 Pa or less and 2 hours or more.

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