Liquid drop container and method of using the same

Abstract

The present invention discloses a droplet container, which is used to hold droplets that can float on a continuous phase; the container includes a droplet accommodating space and a continuous phase accommodating space; the droplet accommodating space has a solution inlet and outlet, and the solution inlet and outlet allow the droplets to enter and exit; the droplet accommodating space is used to hold the droplets and is connected to the continuous phase accommodating space through a continuous phase outlet; the continuous phase outlet allows the continuous phase to pass through but does not allow the droplets to pass through; the container disclosed by the present invention is suitable for collecting, storing and removing droplets, can effectively reduce the contact between the droplets and the container, and reduce adhesion loss and container residue.

Description

Technical Field

[0001] The present invention relates to the field of droplet microfluidics, and in particular to a droplet container and a method for using the same. Background Art

[0002] A microfluidic chip, also known as a lab-on-a-chip, integrates multiple functions used in biological and chemical experiments, including sample preparation, reaction, detection, and separation, onto a single chip measuring a few square centimeters, effectively acting as a miniature laboratory. With its advantages of miniaturization, integration, and automation, microfluidic chips have enormous potential for applications in biological sample processing and rapid disease diagnosis, and have achieved significant development in recent years.

[0003] Droplet microfluidics is an important branch of the microfluidic platform and a new technology for manipulating tiny volumes of liquid, namely droplets. Droplets are formed by one fluid within another immiscible carrier fluid, and the essence of their occurrence is emulsification. Depending on the different roles of the two immiscible fluids in the droplet formation process, they are respectively called the continuous phase and the dispersed phase (discontinuous phase); the dispersed phase is the fluid dispersed into droplets, and the continuous phase is the fluid that acts as a carrier for the droplets. Depending on whether the monolayer emulsified dispersed phase belongs to the aqueous phase or the oily phase, droplets can be divided into O / W type (oil-in-water type) droplets and W / O type (water-in-oil type) droplets, among which: O / W type droplets refer to oil droplets formed with the oil phase as the dispersed phase and the aqueous phase as the continuous phase, and W / O type droplets refer to water droplets produced with the aqueous phase as the dispersed phase and the oil phase as the continuous phase.

[0004] Droplets have the advantages of small size, low diffusion, no cross contamination, and fast reaction speed, and can be used for high-throughput analysis. At the same time, due to the small size of a single droplet, the volume is usually in the range of nanoliters to picoliters (10 -9 ~10 -12 L), a large number of droplets can be lost during the experiment due to adsorption in small-volume containers or system residues. In particular, the number of target droplets obtained after droplet sorting is often extremely small (tens to hundreds) and precious. Directly collecting these target droplets in a centrifuge tube (or syringe) will result in significant droplet loss due to surface adsorption and other effects. Summary of the Invention

[0005] In order to reduce the amount of liquid droplets remaining in the container after the container is adsorbed or sampled, the present invention provides a liquid droplet container.

[0006] The purpose of the present invention is to be solved by the following technical solutions:

[0007] One aspect of the present invention provides a droplet container for containing droplets capable of floating on a continuous phase. The container comprises a droplet-receiving space and a continuous phase-receiving space; the droplet-receiving space has a solution inlet and outlet that allow the droplets to enter and exit; the droplet-receiving space is used to contain the droplets and is connected to the continuous phase-receiving space via a continuous phase outlet; the continuous phase outlet allows the continuous phase to pass but not the droplets; a conical space is defined above the droplet-receiving space, and the solution inlet and outlet are connected to the conical space.

[0008] Droplets that can float on the continuous phase are generally W / O type droplets, such as fluorinated oil droplets that encapsulate single cells, but they may also be O / W / O droplets or other types that can float on the continuous phase.

[0009] The droplet accommodating space is generally smaller than the volume of the entire droplet container, and has the function of gathering droplets, which can gather the droplets in the entire container into the droplet accommodating space and reduce the contact between the droplets and the container wall.

[0010] The phrase "the continuous phase outlet allows the continuous phase to pass through but does not allow the droplets to pass through" here does not only mean that the size of the continuous phase outlet only allows the continuous phase to pass through but does not allow the droplets to pass through; it also means that the position of the continuous phase outlet allows the continuous phase to pass through but the droplets will not pass through this position (such as Example 2); a filter membrane can also be set to prevent the droplets from passing through.

[0011] The top refers to the upper part in the vertical direction, and here it refers to the vertical uppermost end of the droplet holding space, which is a conical space. The conical space refers to a spatial structure whose inner diameter gradually decreases from large to small. It is not limited to a cone, and can be a semi-conical or one-third cone, or a pyramid, trumpet, or other structures. It should be understood that the conical space of the present invention provides a slope or bevel structure, and the slope or bevel structure can change the direction of buoyancy (such as Example 1), so as to generate an oblique upward force acting on the droplets, so that the droplets gather and reach the outlet. If there is no slope or bevel structure, for example Figure 3 If the space in the middle and upper part is cylindrical, the droplets will be squeezed to the circular surface under the action of the upward buoyancy and will find it difficult to reach the outlet position.

[0012] Currently, during the experimental process, after droplet preparation or sorting, they will be mixed with a large amount of continuous phase (mixed solution). Generally, technicians will use 1.5ml or other volume centrifuge tubes to hold these mixed solutions. Due to the large volume of the continuous phase, the liquid surface of the mixed solution is large. Therefore, these droplets are in a "wide" liquid surface, and the force of their mutual collision is strong, which can easily cause droplet breakage; the area of the container wall is large, and the number of droplets that contact and adhere to the container wall is large; it is difficult to operate and difficult to completely remove these droplets later. Through the container of the present invention, after the droplet mixed solution enters the droplet holding space through the solution inlet and outlet, the droplets gather in the smaller droplet holding space, while the continuous phase can reach the continuous phase holding space through the continuous phase outlet. In this way, the droplets in the mixed solution containing more continuous phase can be gathered in one place, reducing the mutual collision effect, while reducing the area of the container wall that can contact the droplets and reducing adhesion. In addition, the conical space located above the droplet holding space can further cause the droplets to gather. When taking droplets, one only needs to raise the liquid level of the continuous phase, and the droplets will gather in the conical space and flow out from the solution inlet and outlet connected to the conical space. The operation is convenient and greatly reduces droplet residue.

[0013] Preferably, the solution inlet and outlet are located at the top of the conical space, where the top refers to the highest point in the vertical direction.

[0014] Preferably, the continuous phase outlet is located at the bottom end of the droplet receiving space, where the bottom end refers to the lowest point in the vertical direction.

[0015] Preferably, a filter membrane is provided at the continuous phase outlet, and the filter membrane allows the continuous phase to pass through but does not allow the droplets to pass through.

[0016] Furthermore, the container may be an integral structure or may be composed of different parts. For example, the container may include a container body and a droplet container, wherein the container body and the droplet container are engaged to form the continuous phase receiving space and the droplet receiving space.

[0017] Furthermore, the volume of the continuous phase in the mixed solution is generally larger than the volume of the droplets, so the droplet accommodation space is smaller than the continuous phase accommodation space.

[0018] In some embodiments, the continuous phase containing space is provided with an inlet communicating with the outside, and the inlet is used to inject the continuous phase into the continuous phase space.

[0019] In some embodiments, the container further includes a lid, and the lid is engaged with the drop container to form the conical space.

[0020] Preferably, the container is sealed except for the solution inlet and outlet and the inlet, which are open to the outside world. This reduces contact between the liquid in the container and the outside world, thereby reducing the risk of contamination. A piston or other sealing structure capable of blocking the solution inlet and outlet and the inlet may also be provided to seal the entire device for sealed storage of liquid droplets.

[0021] Furthermore, when the droplets are W / O type droplets, the continuous phase is an oil phase.

[0022] Another aspect of the present invention provides a method for using the above-mentioned container. The method is applicable to the above-mentioned container in an open state or a sealed state. The specific steps of use include:

[0023] (1) allowing the mixed solution containing the droplets to enter the container through the droplet inlet and outlet;

[0024] (2) A continuous phase is added to the container to raise the liquid level of the mixed solution, and the droplets reach the conical space and further flow out through the solution inlet and outlet.

[0025] If the above container is completely sealed except for the solution inlet and outlet and the inlet, it can also be used as follows:

[0026] (1) allowing the mixed solution containing the droplets to enter the container through the droplet inlet and outlet;

[0027] (2) Gas is injected through the inlet to increase the pressure in the continuous phase holding space, thereby forming a pressure difference between the continuous phase holding space and the droplet holding space. Under the action of this pressure difference, the liquid level in the droplet holding space rises, and the droplets reach the conical space and further flow out through the solution inlet and outlet.

[0028] The outflow rate of the droplets can be controlled by controlling the speed of adding the continuous phase or the speed of filling the gas. In some ways, the aperture of the solution inlet and outlet can also be controlled. For example, the aperture is made the same as the size of a single droplet, so that the droplets can only flow out one by one in sequence. This is particularly suitable for sampling when using a droplet sorting chip to sort droplets. The above-mentioned droplet inlet and outlet are both through the solution inlet port. In some ways, they can also be separated and the inlet and outlet are respectively set on the droplet holding space. For example, a larger inlet is set to facilitate a large amount of mixed solution to enter the container faster, and a smaller droplet outlet is set to allow the droplets to flow out in sequence or at a certain speed.

[0029] The advantages of the present invention are: the present invention provides a droplet container that can be used for collecting, storing and removing droplets; can gather droplets and reduce droplet adhesion or collision losses; facilitates droplet removal and reduces the amount of droplets remaining in the container; and is particularly suitable for containing trace or precious droplets. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 This is a schematic diagram of a liquid drop container provided in Example 1 of the present invention, a is a front view, and b is a cross-sectional view.

[0031] Figure 2 for Figure 1 Exploded view of the droplet container, a is the front view, b is the cross-sectional view.

[0032] Figure 3 for Figure 1 Schematic diagram of the droplet container working, a is a cross-sectional view of the mixed solution, and b is a cross-sectional view of the droplets flowing out after the continuous phase is injected.

[0033] Figure 4 This is a schematic diagram of another liquid drop container provided in Example 1 of the present invention, a is a front view, and b is a cross-sectional view.

[0034] Figure 5 This is a schematic diagram of a liquid drop container provided in Example 2 of the present invention, a is a front view, and b is a top view.

[0035] Figure 6 for Figure 5 Exploded view of the droplet container, a is the front view, b is the cross-sectional view.

[0036] Figure 7 for Figure 5 Schematic diagram of the droplet container working.

[0037] Figure 8 This is a schematic diagram of a liquid drop container provided in Example 3 of the present invention, a is a front view, and b is a cross-sectional view.

[0038] Figure 9 for Figure 8 Exploded view of the droplet container, a is the front view, b is the cross-sectional view.

[0039] Figure 10 for Figure 8 Schematic diagram of the droplet container working.

[0040] In the figure: 1-lid, 2-container body, 3-space, 31-conical space, 32-lower space, 6-conduit, 11-outlet, 111-inner opening, 112-outer opening, 113-outlet channel, 5-inlet, 51-inner surface of the inlet, 52-outer surface of the inlet, 53-inlet channel, 4-mixed solution, 41-droplet, 42-continuous phase, 8-droplet container, 81-container lid, 82-droplet accommodating space, 83-solution inlet and outlet, 84-continuous phase outlet, 21-opening, 22-continuous phase accommodating space, 85-tube channel. DETAILED DESCRIPTION

[0041] The present invention will be further described below with reference to the accompanying drawings and embodiments. It should be noted that the embodiments are merely detailed descriptions of the present invention and are not intended to limit the scope of protection of the present invention. All features disclosed in the embodiments of the present invention, or all steps in the methods or processes disclosed, except for mutually exclusive features and / or steps, may be combined in any manner and fall within the scope of protection of the present invention. Technologies not covered by the present invention may be implemented using existing technologies.

[0042] Example 1

[0043] (1) Figure 1 and Figure 2 The present invention provides a droplet container, which includes a lid 1 and a container body 2, and the container body 2 is a centrifuge tube structure. The lid 1 and the container body 2 form a space 3. The space 3 is composed of a conical space 31 in the upper part and a space 32 in the lower part. The conical space 31 is conical and has an outlet 11 at the top. The inner opening 111 of the outlet 11 opens into the conical space 31, and the outer opening 112 at the other end is connected to the outside world. There is also an inlet 5 on the space 3, the inner surface 51 of the inlet opens into the space 3, and the outer surface 52 of the inlet is connected to the outside world. The outlet 11 and the inlet 5 are formed by a conduit 6 passing through the outlet channel 113 and the inlet channel 53 on the lid 1, respectively.

[0044] like Figure 3 Mixed solution 4, comprising droplets 41 and a continuous phase 42, is located in space 3. The density of droplets 41 is lower than that of continuous phase 42, so they float above it. The continuous phase is injected through inlet 5, raising the liquid level of mixed solution 3. Droplets 41 reach conical space 31 and then flow out through outlet 11. This reduces contact between droplets and the container walls, lowering droplet adhesion losses. Furthermore, the presence of the continuous phase further reduces adhesion. Furthermore, this droplet container minimizes contact between droplets and the outside world, reducing the risk of contamination.

[0045] (2) Figure 4Another drop container provided by the present invention differs from (1) in that: the lid 1 has an outlet 11 but no inlet 5; the inlet 5 is located on the container body 2. The container body 2 is cylindrical and forms a space 3 with the lid 1. The space 3 consists of an upper conical space 31 and a lower space 32. The conical space 31 is semi-conical, and the lower space 32 is cylindrical.

[0046] Of course, the lid 1 and container 2 can also be designed as an integrated whole, and the mixed solution 3 can enter the container through the outlet 11 or the inlet 5. The lid 1 can be made of elastic silicone, and a sealing structure such as a rubber plug can be added to the outlet 11 and inlet 12 or the outlet channel 113 and the inlet channel 53. When not in use, the rubber plug can be added to store the mixed solution 3. The droplet container of the present invention can also be used for droplet collection, such as collecting target droplets after sorting. After collection, it can be directly used for storage and facilitates subsequent retrieval, greatly avoiding loss and contamination.

[0047] Example 2

[0048] like Figure 5 and Figure 6 The present invention provides a droplet container comprising a container body 2 and a droplet container 8. The container body 2 is a centrifuge tube structure with an opening 21 at its upper end. Droplet container 8 defines a droplet receiving space 82. A container cap 81 is located at its upper end. This cap 81 engages with the opening 21 to form a continuous phase receiving space 22. A continuous phase outlet 84 is located at the bottom of droplet receiving space 82, communicating with the continuous phase receiving space 22.

[0049] like Figure 7 , inject a small amount of continuous phase through the solution inlet and outlet 83, so that the continuous phase outlet 84 is immersed in the continuous phase ( Figure 7 a), and then use the container to collect the mixed solution 4 composed of the continuous phase and the target droplets during droplet sorting. The mixed solution 4 enters the droplet holding space 82 through the solution inlet and outlet 83. Since the droplet 41 has a low density, the droplet 41 floats above the continuous phase 42. The continuous phase 42 is located below the droplet holding space 82 and can enter the continuous phase holding space 72 through the continuous phase outlet 84 ( Figure 7 b) A filter membrane structure, such as a semipermeable membrane, can be added at the continuous phase outlet 84. This filter membrane only allows the continuous phase to pass through, but does not allow the droplets to pass through. Therefore, there is no need to inject a small amount of droplets before using the container. The container can be used directly to prevent the droplets from reaching the continuous phase holding space 22 through the continuous phase outlet 84.

[0050] To facilitate the outflow of gas in the continuous phase containing space 22, the container cover 81 is fitted with the opening 21 to allow gas to pass through. Preferably, a sealing structure can be provided at the opening 21 to keep the mixed solution 4 in a sealed environment after the droplets are collected, thereby reducing the risk of contamination and being suitable for storage.

[0051] Example 3

[0052] like Figure 8 and Figure 9 , is a droplet container provided by the present invention, which adds a lid 1 similar to that of Example 1 on the basis of the container of Example 2. The lid 1 and the droplet container 8 are fitted together to form a conical space 31. The conical space 31 is conical and has an outlet 11 at its top. The inner opening 111 of the outlet 11 opens to the top of the conical space 31, and the outer opening 112 at the other end is connected to the outside. There is also an inlet 5 on the continuous phase accommodating space 22, the inner surface 51 of the inlet opens to the continuous phase accommodating space 22, and the outer surface 52 of the inlet is connected to the outside. The outlet 11 is formed by a conduit passing through the outlet channel 113 on the lid 1, and the inlet 5 is formed by a conduit passing through the inlet channel 53 on the lid 1 and the tube channel 85 provided on the container cover 81 of the droplet container 8.

[0053] like Figure 10 , when the droplet container described in Example 2 collects the sorted target droplets ( Figure 10 a), when the droplet needs to be removed later, it is only necessary to inject the continuous phase through the inlet 5 to raise the solution level, and the droplet 41 reaches the conical space 31 and further flows out through the outlet 11 ( Figure 10 b). Alternatively, the joints between the container cover 81, the opening 21, and the lid 1 are all sealed. In this case, gas, such as air or an inert gas such as nitrogen, is injected through the inlet 5 to make the liquid surface pressure in the continuous phase receiving space 22 higher than the liquid surface pressure in the droplet receiving space 82. Under the action of the pressure difference, the droplets reach the conical space and further flow out through the outlet 11 ( Figure 10 c). Preferably, sealing plugs are added to the outlet 11 and the inlet 5 to completely seal the mixed solution 4 in the container, thereby reducing the risk of contamination. Furthermore, after the mixed solution 4 is loaded, an inert gas such as nitrogen, helium, argon, etc. is injected through the inlet 5 and then sealed to protect the mixed solution 4 for storage of droplets. It should be noted that the container cannot be inverted during storage to prevent droplets from entering the continuous phase accommodation space from the continuous phase outlet 84. To solve this problem, a filter membrane structure that allows the continuous phase to pass but does not allow droplets to pass can be added to the continuous phase outlet 84 as in Example 2.

[0054] It should be noted that the container body 2 in the above-mentioned embodiments 1-3 can be a centrifuge tube structure, or a combination structure of a cylinder, a cuboid or other two or more geometric shapes, and its internal volume can be 0.5ml, 1.5ml, 2ml, 5ml, 10ml, 15ml, 50ml or other specifications, which can be selected according to the amount of mixed solution actually collected. The container of the present invention is particularly suitable for collecting small-sized droplets, and therefore, 10ml and below specifications are preferred. The container body 2 and the droplet container 8 are preferably made of transparent materials, such as transparent plastic, for easy observation. In order to further reduce adhesion losses, the surfaces in the container that may come into contact with the droplets can be treated by inerting or the like to reduce the interaction force between the droplets and the inner wall.

Claims

1. A drop container, characterized in that: The container is used to hold droplets that can float on the continuous phase; The container includes a droplet accommodating space and a continuous phase accommodating space; the droplet accommodating space has a solution inlet and outlet, and the solution inlet and outlet allow the droplets to enter and exit; the droplet accommodating space is used to hold the droplets and is connected to the continuous phase accommodating space through a continuous phase outlet; the continuous phase outlet allows the continuous phase to pass through but does not allow the droplets to pass through; A conical space is located above the droplet accommodating space, and the solution inlet and outlet are connected to the conical space; and the continuous phase outlet is located at the bottom end of the droplet accommodating space.

2. The container according to claim 1, characterized in that The solution inlet and outlet are located at the top of the conical space.

3. The container according to claim 1, wherein A filter membrane is provided at the continuous phase outlet, and the filter membrane allows the continuous phase to pass through but does not allow the droplets to pass through.

4. The container according to claim 1, wherein The container includes a container body and a liquid drop container, and the container body and the liquid drop container are embedded to form the continuous phase accommodating space and the liquid drop accommodating space.

5. The container according to claim 1, wherein The continuous phase accommodating space is provided with an inlet communicating with the outside, and the inlet is used to inject the continuous phase into the continuous phase space.

6. The container according to claim 4, characterized in that The container further includes a cover, and the cover is engaged with the drop container to form the conical space.

7. The container according to claim 5, characterized in that The container is a sealed structure except that the solution inlet and outlet and the inlet are connected to the outside.

8. The container according to any one of claims 1 to 7, characterized in that The droplets are W / O type droplets, and the continuous phase is an oil phase.

9. A method for using the container according to claim 1, characterized in that: The specific steps include: (1) allowing the mixed solution containing the droplets to enter the container through the droplet inlet and outlet; (2) A continuous phase is added to the container to raise the liquid level of the mixed solution, and the droplets reach the conical space and further flow out through the solution inlet and outlet.

10. A method of using the container according to claim 7, characterized in that: The specific steps include: (1) allowing the mixed solution containing the droplets to enter the container through the droplet inlet and outlet; (2) Gas is injected through the inlet to increase the pressure in the continuous phase holding space, thereby forming a pressure difference between the continuous phase holding space and the droplet holding space. Under the action of this pressure difference, the liquid level in the droplet holding space rises, and the droplets reach the conical space and further flow out through the solution inlet and outlet.

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