Micro-droplet collecting device and collecting method

Abstract

The invention provides a micro-droplet collecting device and a method for collecting micro-droplets by using the device. The micro-droplet collecting device comprises a collecting part which is provided with a liquid distributing pipe, a plurality of collecting pipes and a plurality of liquid conveying pipes communicated with the liquid distributing pipe and the collecting pipes; a receiving opening for receiving micro-droplets is formed in the top of the liquid distribution pipe, and a plurality of liquid distribution openings corresponding to the collecting pipes are uniformly formed in the position close to the bottom in the circumferential direction; the top of the collecting pipe is provided with a collecting opening and a sealing cover matched with the collecting opening; one end of the liquid conveying pipe is limited on the inner side of the collecting opening and penetrates into the bottom of the collecting pipe through the collecting opening, and the other end of the liquid conveying pipe is communicated with the liquid separating opening corresponding to the liquid separating pipe; the fixing part is detachably mounted on the working platform; and the liquid distribution pipe is detachably placed and limited on the fixing part. When the method for collecting the micro-droplets is used for collecting the micro-droplets, tube replacement and oil replacement are not needed, the quality of the droplets is uniform and stable, and the experiment time is saved.

Description

Technical Field

[0001] The invention belongs to the field of microfluidics technology, and in particular relates to a collection device for collecting microdroplets generated by a microfluidics chip, and a method for collecting microdroplets using the device. Background Art

[0002] Microfluidics technology is one of the important branches of micro-electro-mechanical systems (MEMS), and is also one of the most rapidly developing multidisciplinary cross-cutting cutting-edge technologies. It has important applications in single-cell sequencing, life sciences, clinical medicine and other disciplines. Among them, single-cell sequencing is a new technology that has emerged in recent years. It can obtain the expression profile of the entire transcriptome from the level of a single cell, and perform high-throughput sequencing after amplification, so as to efficiently detect the gene expression level in a single cell. It has important application value in the diagnosis and treatment of tumors, the design of targeted drugs, the development and differentiation of stem cells and other fields.

[0003] Single-cell sequencing based on microfluidic technology can utilize micro-scale pipelines and control systems to efficiently and accurately operate and analyze single cells. For example, the 10× Genomics Chromium platform based on the principle of oil-in-water micro-reaction system is widely used. In terms of workflow, the 10× Genomics Chromium platform first uses microfluidic chips to capture single cells to ensure that each cell is separated and fixed in a specific position, then lyses each single cell, extracts RNA, synthesizes cDNA, amplifies the synthesized cDNA to build a library, and adds fragmentation tags such as barcode tags and unique molecular identifier (UMI) sequences, and then performs high-throughput sequencing to achieve analysis of the genome and transcriptome of single cells. In the above-mentioned four-step microfluidic experiment of lysis, multiple displacement amplification, fragmentation labeling, sequencing or dPCR, each droplet is a separate reaction unit. Therefore, the reaction ratio of the reaction system (such as lysis system, amplification system or fragmentation system) in the droplet to the nucleic acid concentration should always be consistent, which is crucial for the accuracy and reliability of the experimental results.

[0004] Taking the mainstream PCR instrument on the market as an example, please refer to Figure 1, its holding tank can only hold 200 μL or 500 μL centrifuge tubes, and there is currently no PCR instrument that can hold 1.5 mL centrifuge tubes. Therefore, in the actual operation process, the droplets generated by the microfluidic technology reaction in each step generally need to be re-collected in multiple 200 μL centrifuge tubes, and then the low-concentration generated oil at the bottom of the centrifuge tube is sucked out, and then the high-concentration generated oil is added to the centrifuge tube to stabilize the droplets, and finally the centrifuge tube that has completed the collection and oil change operation is placed in a 200 μL specification PCR instrument for the next reaction. However, there are many problems with this operation process.

[0005] (1) Quality issues: Please refer to Figure 2 In the above operation process, 50~70 μL of mineral oil needs to be pre-added to each 200 μL centrifuge tube to prevent dust from entering the droplet layer and the liquid in the droplet layer from volatilizing. Therefore, each 200 μL centrifuge tube can generally only hold less than 100 μL of the reaction solution containing droplets and low-concentration generated oil. The reaction solution of each step experiment is 200~600 μL, so usually no less than 3 200 μL centrifuge tubes are needed for collection. However, during the collection process, the operation of replacing a new 200 μL centrifuge tube often inevitably causes a sudden change in the pressure of the chip flow channel, so that the generated droplets are large and small, and droplets of different sizes are collected in different 200 μL centrifuge tubes, resulting in a decrease in the overall quality of the droplets and interference with the experimental results. At the same time, the change in flow rate or chip blockage during the tube replacement process will also cause the size of the droplets in each tube to be uneven. Furthermore, it generally takes a longer stabilization time to collect droplets with a diameter that is too large in the 200 μL centrifuge tube, which will cause the ratio of the reaction system to the nucleic acid concentration in the droplets to change. For example, during the lysis process, insufficient lysis or excessive lysis may occur, resulting in a decrease in the credibility of subsequent experimental results.

[0006] (2) Time issue: In actual operation, the collection time of a single 200 μL centrifuge tube is generally 20 minutes. After the collection is completed, a new tube is replaced, and the low-concentration generated oil collected at the bottom of the 200 μL centrifuge tube is removed using a pipette, and then the high-concentration generated oil used to stabilize the droplets is added again to stabilize the droplets for a period of time. Therefore, a series of operations such as changing tubes, changing oil, and stabilization will consume extra time.

[0007] (3) Space issues: Please refer to Figure 3The collection operation is performed on the microscope stage, and a 96-well plate is placed on the stage to fix the 200 μL centrifuge tube. However, the oversized 96-well plate not only occupies the limited experimental space, making the operable space smaller, but also only uses 3 to 8 wells at a time, which greatly wastes the experimental space. What's more serious is that in the limited experimental space, the 96-well plate is very easy to be touched by mistake. If the tube or oil is changed carelessly, the 96-well plate will vibrate or even overturn, causing the droplets in the 200 μL centrifuge tube to splash, or induce static electricity, resulting in insufficient droplet collection, droplet fusion, or droplet quality degradation in the experiment. Summary of the invention

[0008] In order to solve the problem of uneven droplet quality in the microfluidic droplet collection process in the prior art, the present invention provides a micro-droplet collection device and a method for collecting micro-droplets using the device. The collection process is stable, convenient and efficient, and the quality of the collected droplets is uniform.

[0009] The technical solution adopted by the present invention is: a micro-droplet collection device, comprising: The collecting part comprises a liquid dispensing tube, a plurality of collecting tubes and a plurality of liquid infusion tubes connecting the liquid dispensing tube and the collecting tube; the top of the liquid dispensing tube comprises a receiving port for receiving micro-droplets, and a plurality of liquid dispensing ports corresponding to the collecting tube are evenly arranged near the bottom in a circumferential direction; the top of the collecting tube comprises a collecting port and a sealing cover matching the collecting port; one end of the liquid infusion tube is limited to the inner side of the collecting port and penetrates into the bottom of the collecting tube through the collecting port, and the other end is connected to the liquid dispensing port corresponding to the liquid dispensing tube; The fixing part is detachably mounted on the working platform; the liquid dispensing tube is detachably placed and limited on the fixing part.

[0010] Specifically, the fixing part includes: A fixer, the fixing part is detachably mounted on the working platform through the fixer; The cantilever rod has an interface end close to the working platform and a storage end far from the working platform; the liquid dispensing tube is detachably placed and limited in the storage end; A joint, through which the interface end of the cantilever rod is detachably mounted on the fixture.

[0011] The micro-droplet collecting device provided by the present invention comprises a collecting part and a fixing part used for fixing the collecting part securely on an operating platform.

[0012] First, in order to solve the problem of unstable droplet quality caused by changing the tube during the collection process, the present invention provides a collection part that integrates receiving, liquid separation and collection, and the collection part is a one-piece disposable consumable. The collection part uses a larger capacity centrifuge tube (such as a 2 mL centrifuge tube or a 1.5 mL centrifuge tube) as a liquid separation tube, which is used to first receive about 200~600 μL of reaction liquid generated by the previous step reaction, and the reaction liquid contains micro-droplets to be collected and low-concentration generated oil for separating micro-droplets, and then the received reaction liquid is separated by gravity through a series of liquid separation ports evenly opened at the lower end of the collection tube through an infusion tube to several small-capacity collection tubes (such as 200 μL or 500 μL centrifuge tubes) that match the PCR instrument, so that the reaction liquid is evenly and stably distributed in several collection tubes, and each collection tube can collect about 50~100 μL of reaction liquid, and the number, size and quality of the droplets collected in each collection tube can be kept consistent. In this way, the device does not need to replace the 200 μL or 500 μL centrifuge tube used to collect microdroplets multiple times during the droplet collection process, thus avoiding the situation where the droplets fluctuate due to uneven pressure when changing tubes, and improving the overall quality of the droplets. In addition, droplets of uniform and stable size can further shorten the droplet stabilization time, avoiding changes in the ratio of the reaction system and nucleic acid concentration in the droplets, which may reduce the credibility of subsequent experimental results.

[0013] Secondly, in order to avoid the problem of droplet fusion caused by droplet splashing, unnecessary contact vibration, static electricity transmission, etc. before and after replacing the collection tube, the present invention also provides a fixing part for fixing the collection part securely on the working platform (such as the stage of a microscope). The fixing part is detachably fixed around the operating platform by a fixing device at the bottom in a clamping, clamping, bolting, etc. manner, and the cantilever rod for placing the collection part is connected to the fixing device through a joint, thereby realizing the secure placement of the collection part relative to the working platform to prevent the collection part from splashing or vibrating. Furthermore, in order to expand the experimental operation space, the present invention adopts the design of a cantilever rod, and the dispensing tube is detachably placed and limited in the storage end of the cantilever rod away from the operating platform in the height direction, and a plurality of collecting tubes naturally and evenly hang down under the dispensing tube under the action of gravity through the infusion tube, so that the collecting part as a whole is away from the working platform in the height direction, which not only saves the placement space of the 96-well plate for fixing the collecting tube, but also avoids the splashing of micro-droplets in the collecting tube due to the 96-well plate being accidentally touched and tipped over, and can also make the collecting part away from the hot microscope light source and the hand temperature of the experimenter, thereby avoiding the influence of temperature on the experimental results.

[0014] Furthermore, the cantilever rod has a deformable support rod and a protective cover sleeved outside the deformable support rod. In order to facilitate the experimenter to adjust the angle and direction of the cantilever rod according to the experimental requirements, so that the collection part placed at the storage end of the cantilever rod is in a suitable working position, a deformable material, such as iron wire, can be used as the support rod inside the cantilever rod, and a soft deformable material, such as rubber, can be used as the outer protective cover of the deformable support rod.

[0015] Furthermore, the joint is a rotary joint, and the storage end of the cantilever rod is circumferentially displaced with the rotary joint as the center. In order to facilitate the experimenter to adjust the angle and direction of the cantilever rod according to the experimental requirements, so that the collection part placed at the storage end of the cantilever rod is in a suitable working position, a rotary joint can be used as a connecting piece between the cantilever rod and the fixture, so that the experimenter can adjust the position of the collection part by rotating the cantilever rod.

[0016] Specifically, the collecting tube is any one of a 200 μL or 500 μL centrifuge tube, preferably a 200 μL centrifuge tube; the dispensing tube is any one of a 10 mL centrifuge tube, a 5 mL centrifuge tube, a 2 mL centrifuge tube, and a 1.5 mL centrifuge tube, preferably a 2 mL centrifuge tube or a 1.5 mL centrifuge tube, and more preferably a 1.5 mL centrifuge tube. It is understood that any container that can be used to safely store the reaction solution and avoid reaction with the microdroplets contained in the reaction solution and the generated oil can be used as the dispensing tube or collecting tube. Preferably, the dispensing tube or collecting tube is formed of a material that is easy to process and chemically stable, such as a thermoplastic material or a thermosetting material.

[0017] Specifically, one end of the infusion tube is limited inside the collection port by gluing or clamping. The collection tube is connected to the liquid distribution tube through the infusion tube, and the infusion tube and the collection port of the collection tube are glued or clamped. This limiting method not only fixes the collection tube, but also facilitates the subsequent separation of the collection tube from the infusion tube.

[0018] The present invention also provides a method for collecting micro-droplets using the micro-droplet collecting device, the method comprising the following steps: S1. The collecting unit is mounted on the working platform through the fixing unit, mineral oil is added to the liquid dispensing tube and the mineral oil flows into the corresponding collecting tubes through the infusion tubes, and the second generated oil is added to the collecting tubes; S2. The reaction solution containing the micro-droplets and the first generated oil is added to the separation tube, and then flows into the corresponding collection tubes through the infusion tubes; S3. After completing the collection of microdroplets, separate the collection tube from the infusion tube, remove the mixture of the first generated oil and the second generated oil at the bottom of the collection tube, and use the collection tube containing microdroplets and mineral oil for subsequent experiments.

[0019] Specifically, the concentration of the second generated oil is higher than that of the first generated oil.

[0020] Step S1 is a pretreatment process of the method, wherein adding mineral oil into the liquid separation tube can prevent dust and other impurities in the environment from entering the collected reaction liquid.

[0021] The collecting tube obtained in step S1 contains two layers of stratified liquid, wherein the lower layer is the second generated oil and the upper layer is mineral oil.

[0022] The collection tube obtained in step S2 contains three layers of stratified liquid, wherein the lower layer is a mixture of the first generated oil and the second generated oil, the middle layer is micro droplets, and the upper layer is mineral oil.

[0023] In general microfluidic experiments, for the sake of cost saving, the diluted low-concentration generated oil (i.e., the first generated oil in step S2) is used to separate the droplets in the previous reaction process. After the experiment, the reaction liquid containing microdroplets and low-concentration generated oil is collected together in a collection tube. After standing, the low-concentration generated oil that is layered to the lower layer of the collection tube needs to be absorbed and discarded, and then a higher-concentration generated oil is added to the collection tube to soak the just-collected droplets to stabilize them. This operation is called oil change. In order to solve the extra time consumption caused by a series of operations such as oil change and stabilization, the present invention pre-adds a second generated oil with a concentration higher than the first generated oil to the collection tube in step S1, so that after the reaction liquid flows into the collection tube, the low-concentration first generated oil is mixed with the high-concentration second generated oil, thereby forming a generated oil with a suitable concentration for stabilizing the droplets, achieving collection and stabilization at the same time, saving the operation time of oil change and stabilization.

[0024] Beneficial effects of the present invention: (1) Improving the quality of droplets: The present invention adopts a collection part that integrates receiving, liquid separation and collection. There is no need to replace the collection tube during the experiment. This not only avoids the situation where the droplets are large or small due to uneven pressure when replacing the collection tube, but also makes the droplets in each collection tube have the same quality. It also avoids the droplet splashing, unnecessary contact vibration, static electricity transmission, etc. that may be generated before and after replacing the collection tube, effectively preventing the droplet fusion, reduction in quantity and reduction in quality.

[0025] (2) Improvement of experimental space: The present invention adopts a fixing part with a cantilever rod design to securely fix the collecting part on the working platform, which not only expands the experimental operation space, but also keeps the droplets in the collecting part away from the heat source of the microscope, and avoids the use of large-volume and easily overturned 96-well plates, thereby preventing experimental failures caused by external factors.

[0026] (2) Saving experimental time: In the present invention, a high concentration of generated oil is pre-added into the collection tube to balance the concentration of generated oil in the reaction liquid. After mixing, generated oil with a suitable concentration that can be used to stabilize droplets is formed. The operations of oil change and droplet stabilization are completed while collecting, thus saving the operation time of oil change and stabilization. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 These are the mainstream 200 μL capacity PCR instruments, 1.5 mL centrifuge tubes, and 200 μL centrifuge tubes currently on the market.

[0028] Figure 2 There are three layers of liquid in the collection tube, from top to bottom: mineral oil - droplets - generated oil.

[0029] Figure 3 The experimental space on the microscope stage in the prior art has a messy experimental table and a small experimental space.

[0030] Figure 4 Schematic diagram of a micro-droplet collection device in an embodiment of the present invention.

[0031] Figure 5 It is a side view of the micro-droplet collection device in an embodiment of the present invention.

[0032] Figure 6 Schematic diagram of the rotation of the micro-droplet collecting device in an embodiment of the present invention.

[0033] Figure 7 Schematic diagram of the collecting part of the micro-droplet collecting device in an embodiment of the present invention.

[0034] Figure 8 Comparison of the mass of droplets collected by the original method (Comparative Example) and the droplets collected by the present invention (Example).

[0035] Among them, Figures 4 to 7 middle, 1-collecting part, 11-liquid dispensing tube, 111-receiving port, 112-liquid dispensing port, 12-collecting tube, 121-collecting port, 122-sealing cover, 13-infusion tube; 2-fixing part, 21-fixer, 211-clamp, 212-bolt, 22-cantilever rod, 221-interface end, 222-storage end, 23-connector. DETAILED DESCRIPTION

[0036] The following is an explanation of the embodiments of the present invention by specific specific examples, and those skilled in the art can easily understand other advantages and effects of the present invention from the contents disclosed in this specification. The present invention can also be implemented or applied by other different specific embodiments, and the details in this specification can also be modified or changed in various ways based on different viewpoints and applications without departing from the spirit of the present invention. It should be noted that, in the absence of conflict, the features in the following embodiments and embodiments can be combined with each other. In the embodiments of the present invention, the methods used are all conventional methods unless otherwise specified, and the reagents used can be obtained from commercial sources.

[0037] In a specific embodiment, the micro-droplet collection device comprises a collection portion 1 and a fixing portion for fixing the collection portion 1 securely on the working platform. First, in order to solve the problem of unstable droplet quality caused by changing the tube during the collection process, the present invention provides a collection part 1 that integrates receiving, liquid separation and collection. The collection part 1 is an integrally formed disposable consumable. Figure 7, the collecting part 1 has a liquid dispensing tube 11, four collecting tubes 12 and four infusion tubes 13 for connecting the liquid dispensing tube 11 with the collecting tube 12. In this embodiment, a 1.5 mL centrifuge tube is used as the liquid dispensing tube 11, and a receiving port 111 for receiving microdroplets is provided on the top thereof, and four liquid dispensing ports 112 corresponding to the collecting tube 12 are evenly arranged circumferentially at the same height near the bottom. The four collecting tubes 12 are all 200 μL centrifuge tubes matching the PCR instrument, and a collecting port 121 and a sealing cover 122 matching the collecting port 121 are provided on the top thereof. The four infusion tubes 13 are all made of plastic hoses for microfluidic experiments, and the hose diameter is adapted to the microdroplets. One end of the infusion tube 13 can be limited to the inner side of the collecting port 121 by gluing or clamping and penetrates into the bottom of the collecting tube 12 through the collecting port 121, and the other end is connected to the liquid dispensing port 112 corresponding to the liquid dispensing tube 11. In this embodiment, one end of the infusion tube 13 near the collection tube 12 is limited to the inner side of the collection port 12 by gluing. This limiting method plays a role in fixing the collection tube 12 on the one hand, and facilitates the subsequent separation of the collection tube 12 and the infusion tube 13 by shearing. Thus, under the action of gravity, the four collection tubes 12 can naturally and evenly fall below the liquid separation tube 11 through the infusion tube 13. In the process of collecting micro-droplets, about 200~600μL of the reaction liquid generated by the previous step reaction (the reaction liquid contains the micro-droplets to be collected and the low-concentration generated oil for separating the micro-droplets) is first received into the liquid separation tube 11, and then through a series of liquid separation ports 112 uniformly opened at the same height at the lower end of the collection tube 12, the liquid is separated into the four collection tubes 12 through the infusion tube 13 under the action of gravity, so that the reaction liquid is evenly and stably distributed at the bottom of each collection tube 12 to avoid the impact of the droplets on the droplets when they contact the liquid surface. Each collection tube 12 can collect approximately 50 to 100 μL of reaction solution, and the number, size, and quality of the droplets collected in each collection tube 12 can be kept consistent. In this way, the device does not need to replace the 200 μL or 500 μL centrifuge tube used to collect microdroplets multiple times during the droplet collection process, thereby avoiding the situation where the droplets fluctuate due to uneven pressure caused by changing tubes, thereby improving the overall quality of the droplets. In addition, droplets of uniform and stable size can further shorten the droplet stabilization time, avoiding changes in the ratio of the reaction system and nucleic acid concentration in the droplets, which may lead to a decrease in the credibility of subsequent experimental results.

[0038] It is understandable that the present invention does not limit the number of the collecting tubes 12 and the infusion tubes 13 , and a plurality of corresponding collecting tubes 12 and infusion tubes 13 may be provided according to experimental requirements.

[0039] In one or more embodiments, the collecting tube 12 can be any one of a 200 μL or 500 μL centrifuge tube, and a preferred embodiment is a 200 μL centrifuge tube; the dispensing tube 11 can also be any one of a 10 mL centrifuge tube, a 5 mL centrifuge tube, a 2 mL centrifuge tube, and a 1.5 mL centrifuge tube, and a preferred embodiment is a 2 mL centrifuge tube or a 1.5 mL centrifuge tube, and a more preferred embodiment is a 1.5 mL centrifuge tube. It is understood that any container that can be used to safely store the reaction solution and avoid reaction with the microdroplets contained in the reaction solution and the generated oil can be used as the dispensing tube 11 or the collecting tube 12. Preferably, the dispensing tube 11 or the collecting tube 12 is formed of a material that is easy to process and has stable chemical reactions, such as a thermoplastic material or a thermosetting material.

[0040] In a specific embodiment, please refer to Figure 4 and Figure 5 , Figure 7 The dispensing tube 11 of the collecting part 1 shown is detachably placed and limited on the fixing part 2, and the fixing part 2 includes a fixture 21, a cantilever rod 22 for placing the dispensing tube 11, and a joint 23 for connecting the fixture 21 and the cantilever rod 22. The fixing part 2 can be detachably mounted on the microscope stage through the fixture 21. This stable placement can avoid the problem of droplet fusion caused by droplet splashing, unnecessary contact vibration, static electricity transmission, etc. before and after replacing the collecting tube 12. In one or more specific embodiments, the fixture 21 can be detachably fixed around the operating platform by clamping, snapping, bolting, etc. Figure 4 In the illustrated embodiment, the fixture 21 includes a clamp 211 and a bolt 212 , so that the fixture 21 can be fixed on the stage by clamping.

[0041] In a specific embodiment, the cantilever rod 22 has an interface end 221 close to the working platform and a storage end 222 far from the working platform, and the liquid dispensing tube 11 is detachably placed and confined in the storage end 222. Figure 4 and Figure 5In order to expand the experimental operation space, the present invention adopts the design of a cantilever rod 22, and the dispensing tube 11 is detachably placed and limited in the storage end 222 of the cantilever rod 22 away from the operating platform in the height direction, and a plurality of collecting tubes 12 naturally and evenly fall below the dispensing tube 11 through the infusion tube 13 under the action of gravity, so that the collecting part 1 as a whole is away from the working platform in the height direction, which not only saves the placement space of the 96-well plate for fixing the collecting tube 12, but also avoids the splashing of micro-droplets in the collecting tube 12 due to the 96-well plate being accidentally touched and turned over, and can also keep the collecting part 1 away from the heating microscope light source and the body temperature of the experimenter's hands, avoiding the influence of temperature on the experimental results. In another specific embodiment, the cantilever rod 22 has a deformable support rod and a protective cover sleeved outside the deformable support rod. In order to facilitate the experimenters to adjust the angle and direction of the cantilever rod 22 according to the experimental requirements, so that the collecting part 1 placed at the storage end 222 of the cantilever rod 22 is in a suitable working position, deformable materials, such as iron wire, copper wire, etc., can be used as the deformable support rod inside the cantilever rod 22, and soft deformable materials, such as rubber, plastic, etc., can be used as the external protective cover of the deformable support rod.

[0042] In a specific embodiment, the interface end 221 of the cantilever rod 22 for placing the collecting part 1 is connected to the fixing device 21 through a joint 23. Figure 6 In this embodiment, the joint 23 is a rotary joint 23, and the storage end 222 of the cantilever rod 22 is circumferentially displaced with the rotary joint 23 as the center. In order to facilitate the experimenter to adjust the angle and direction of the cantilever rod 22 according to the experimental requirements, so that the collecting part 1 placed at the storage end 222 of the cantilever rod 22 is in a suitable working position, the rotary joint 23 can be used as a connecting piece between the cantilever rod 22 and the fixture 21, so that the experimenter can adjust the position of the collecting part 1 by rotating the cantilever rod 22.

[0043] In a specific embodiment, a method for collecting micro-droplets using the micro-droplet collecting device provided by the present invention is applied, and the method comprises the following steps: S1. The collecting part is mounted on the working platform through the fixing part, and mineral oil is added to the liquid separation tube to prevent dust and other impurities in the environment from entering the collected reaction liquid, and the mineral oil flows into the corresponding collecting tubes through the liquid infusion tubes, and the second generated oil is added to the collecting tubes; the resulting collecting tube contains two layers of stratified liquid, wherein the lower layer is the second generated oil and the upper layer is the mineral oil; S2. adding the reaction liquid containing the micro-droplets and the first generated oil into a liquid separation tube, and then flowing into the corresponding collection tubes through the liquid infusion tubes, wherein the concentration of the second generated oil is higher than that of the first generated oil; the resulting collection tube contains three layers of stratified liquid, wherein the lower layer is a mixture of the first generated oil and the second generated oil, the middle layer is the micro-droplets, and the upper layer is mineral oil; S3. After completing the collection of microdroplets, cut the glue point between the collection port and the infusion tube to separate the collection tube from the infusion tube, remove the mixture of the first generated oil and the second generated oil at the bottom of the collection tube, and use the collection tube containing microdroplets and mineral oil for subsequent experiments, such as placing the collection tube in a PCR instrument for PCR reaction after closing the sealing cover.

[0044] In general microfluidic experiments, for the sake of cost saving, diluted low-concentration generated oil (i.e., the first generated oil in step S2, the concentration can be 1×, 2×, or 3×) is used to separate the droplets in the previous reaction process. After the experiment, the reaction liquid containing microdroplets and low-concentration generated oil is collected together in a collection tube. After standing, the low-concentration generated oil that is stratified to the lower layer of the collection tube needs to be absorbed and discarded, and then the higher concentration generated oil (the concentration can be 4×, 5×, or 6×) in the collection tube is used to soak the newly collected droplets to stabilize them. This operation is called oil change. In order to solve the extra time consumption caused by a series of operations such as oil changing and stabilization, the present embodiment pre-adds a second generated oil (the concentration may be 8×, 9× or 10×) with a higher concentration than the first generated oil (the concentration may be 1×, 2× or 3×) into the collecting tube in step S1, so that after the reaction liquid flows into the collecting tube, the low-concentration first generated oil and the high-concentration second generated oil are mixed, thereby forming a generated oil with a suitable concentration (the concentration may be 4×, 5× or 6×) for stabilizing the droplets, thereby realizing collection and stabilization at the same time, saving the operation time of oil changing and stabilization.

[0045] Table 1. Mass distribution of collected droplets Comparative Example Example Proportion of unqualified small droplets with a diameter of ≤25 μm (%) 23.40% 2.5% Proportion of qualified droplets with diameters of 25 μm ≤ ≤ 30 μm (%) 74.60% 97.50% Proportion of unqualified large droplets with a diameter ≥30 μm (%) 2% 0 After collecting micro droplets according to the above method (Example), the quality and quantity of the droplets collected in each collection tube are uniform, and the quality comparison of the droplets collected according to the original method (Comparative Example) is shown in FIG. Figure 8, the proportion of qualified droplets can be increased by about 20% in each experiment. Referring to Table 1, taking the lysis experiment as an example, under a 20× objective lens, according to the standard that the diameter is less than 25 μm for unqualified small droplets, 25-30 μm for qualified droplets, and more than 30 μm for unqualified large droplets; the traditional method has about 26 unqualified small droplets, accounting for 23.4%, 82 qualified droplets, accounting for 74.6%, 2 unqualified large droplets, accounting for about 2%, and the total qualified rate is about 74.6%; in the method of the present invention, the proportion of unqualified small droplets is about 2.5%, there is no unqualified large droplets above 30 μm, and the total qualified rate is 97.5%.

[0046] The embodiments described above are merely descriptions of preferred implementations of the present invention and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope of the present invention.

Claims

1. A micro-droplet collecting device, characterized in that: include: The collecting part (1) comprises a liquid dispensing tube (11), a plurality of collecting tubes (12), and a plurality of liquid infusion tubes (13) connecting the liquid dispensing tube (11) and the collecting tube (12); the top of the liquid dispensing tube (11) comprises a receiving port (111) for receiving micro-droplets, and near the bottom thereof, a plurality of liquid dispensing ports (112) corresponding to the collecting tube (12) are evenly arranged in a circumferential direction; the top of the collecting tube (12) comprises a collecting port (121) and a sealing cover (122) matching the collecting port (121); one end of the liquid infusion tube (13) is located inside the collecting port (121) and penetrates into the bottom of the collecting tube (12) through the collecting port (121), and the other end thereof is connected to the liquid dispensing port (112) corresponding to the liquid dispensing tube (11); The fixing part (2) is detachably mounted on the working platform; the liquid dispensing tube (11) is detachably placed and limited on the fixing part (2).

2. The micro-droplet collecting device according to claim 1, characterized in that: The fixing part (2) comprises: A fixer (21), wherein the fixing portion (2) is detachably mounted on the working platform via the fixer (21); The cantilever rod (22) has an interface end (221) close to the working platform and a storage end (222) far from the working platform; the liquid dispensing tube (11) is detachably placed and confined in the storage end (222); A joint (23), wherein the interface end (221) of the cantilever rod (22) is detachably mounted on the fixer (21) via the joint (23).

3. The micro-droplet collecting device according to claim 4, characterized in that: The cantilever rod (22) comprises a deformable support rod and a protective sleeve sleeved outside the deformable support rod.

4. The micro-droplet collecting device according to claim 4, characterized in that: The joint (23) is a rotary joint (23), and the storage end (222) of the cantilever rod (22) performs circumferential displacement with the rotary joint (23) as the center of the circle.

5. The micro-droplet collecting device according to claim 1, characterized in that: The collection tube (12) is any one of a 200 μL or 500 μL centrifuge tube.

6. The micro-droplet collecting device according to claim 1, characterized in that: The dispensing tube (11) is any one of a 10 mL centrifuge tube, a 5 mL centrifuge tube, a 2 mL centrifuge tube, and a 1.5 mL centrifuge tube.

7. The micro-droplet collecting device according to claim 1, characterized in that: One end of the infusion tube (13) is positioned inside the collection port (121) by means of gluing or clamping.

8. A method for collecting micro-droplets using a collecting device, characterized in that: The micro-droplet collecting device as described in any one of claims 1 to 7 is used to collect the micro-droplets.

9. The method according to claim 8, characterized in that The method comprises the following steps: S1. The collecting unit is mounted on the working platform through the fixing unit, mineral oil is added to the liquid dispensing tube and the mineral oil flows into the corresponding collecting tubes through the infusion tubes, and the second generated oil is added to the collecting tubes; S2. The reaction solution containing the micro-droplets and the first generated oil is added to the separation tube, and then flows into the corresponding collection tubes through the infusion tubes; S3. After completing the collection of microdroplets, separate the collection tube from the infusion tube, remove the mixture of the first generated oil and the second generated oil at the bottom of the collection tube, and use the collection tube containing microdroplets and mineral oil for subsequent experiments.

10. The method according to claim 9, characterized in that The concentration of the second generated oil is higher than that of the first generated oil.

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