Microdroplet collection device, collection method

Abstract

The application 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, a distribution pipe, a plurality of collecting pipes and a plurality of liquid conveying pipes connecting the distribution pipe and the collecting pipes. The top of the distribution pipe is provided with a receiving port for receiving micro-droplets, and a plurality of distribution ports corresponding to the collecting pipes are uniformly arranged at the near bottom of the distribution pipe. The top of the collecting pipe is provided with a collecting port and a sealing cover matched with the collecting port. One end of the liquid conveying pipe is limited to the inside of the collecting port and extends into the bottom of the collecting pipe through the collecting port, and the other end is connected with the distribution port corresponding to the distribution pipe. The fixing part is detachably installed on a working platform, and the distribution pipe is detachably placed and limited on the fixing part. The method for collecting micro-droplets provided by the application can collect micro-droplets without changing the pipe and the oil, the quality of the droplets is uniform and stable, and the experimental time is saved.

Description

Technical Field

[0001] The present invention belongs to the field of microfluidics technology, and particularly 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 an important branch of micro-electro-mechanical systems (MEMS) and one of the most rapidly developing cutting-edge technologies in multidisciplinary cross-disciplinary science and technology. 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 at the level of a single cell, amplify it and perform high-throughput sequencing, thereby efficiently detecting 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, and the development and differentiation of stem cells.

[0003] Single-cell sequencing based on microfluidics technology utilizes microscale tubing and control systems to efficiently and precisely manipulate and analyze individual cells. For example, the 10× Genomics Chromium platform, based on an oil-in-water microreactor system, is widely used. The 10× Genomics Chromium platform first uses a microfluidic chip to capture individual cells, ensuring that each cell is isolated and fixed in a specific location. Each cell is then lysed, RNA is extracted, and cDNA is synthesized. The synthesized cDNA is amplified and a library is constructed, and fragmentation tags such as barcodes and unique molecular identifier (UMI) sequences are added. High-throughput sequencing is then performed, enabling analysis of the genome and transcriptome of a single cell. In the four-step microfluidic experiment described above—lysis, multiple displacement amplification, fragmentation labeling, sequencing, or dPCR—each droplet serves as a separate reaction unit. Therefore, maintaining consistent reaction ratios of the reaction systems (e.g., lysis, amplification, or fragmentation) and nucleic acid concentration within the droplet is crucial for accurate and reliable experimental results.

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

[0005] (1) Quality issues: Please refer to Figure 2 During the above-mentioned operation, 50–70 μL of mineral oil must be pre-added to each 200 μL centrifuge tube to prevent dust from entering the droplet layer and preventing evaporation of the liquid within the droplet layer. Therefore, each 200 μL centrifuge tube typically holds less than 100 μL of the reaction solution containing droplets and low-concentration generated oil. Since the reaction solution discharged from each experimental step ranges from 200 to 600 μL, at least three 200 μL centrifuge tubes are typically required for collection. However, during the collection process, swapping out a new 200 μL centrifuge tube inevitably causes sudden changes in chip flow pressure, resulting in erratic droplet sizes. Droplets of varying sizes are collected in different 200 μL centrifuge tubes, reducing overall droplet quality and interfering with experimental results. Furthermore, changes in flow rate during tube switching or chip clogging can also lead to uneven droplet size within each tube. Furthermore, droplets with excessively large diameters collected in a 200 μL centrifuge tube typically require longer stabilization times, which can alter the ratio of reaction system to nucleic acid concentration within the droplet. 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. Then, high-concentration generated oil is added to stabilize the droplets for a period of time. Therefore, a series of operations such as changing tubes, changing oil, and stabilizing the droplets will consume additional time.

[0007] (3) Space issues: Please refer to Figure 3The collection operation is performed on the microscope stage, with a 96-well plate placed on the stage to hold the 200 μL centrifuge tube. However, the overly large 96-well plate not only occupies the limited experimental space, reducing the operable space, but also greatly wastes experimental space since only 3 to 8 wells are used at a time. More seriously, in the limited experimental space, the 96-well plate is easily touched by mistake. If the tube or oil is not changed carefully, the 96-well plate will vibrate or even tip over, causing the droplets in the 200 μL centrifuge tube to spill or induce static electricity, resulting in insufficient droplet collection, droplet fusion, or reduced droplet quality. 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 collected droplets are of uniform quality.

[0009] The technical solution adopted by the present invention is: a micro-droplet collection device, comprising:

[0010] The collecting portion comprises a liquid dispensing tube, a plurality of collecting tubes, and a plurality of liquid infusion tubes connecting the liquid dispensing tubes and the collecting tubes; the top of the liquid dispensing tube has a receiving port for receiving micro-droplets, and near the bottom thereof, a plurality of liquid dispensing ports corresponding to the collecting tubes are evenly arranged circumferentially; the top of the collecting tube has 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 extends 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;

[0011] The fixing part is detachably mounted on the working platform; the liquid dispensing tube is detachably placed and restricted on the fixing part.

[0012] Specifically, the fixing portion includes:

[0013] A fixer, wherein the fixing portion is detachably mounted on the work platform through the fixer;

[0014] 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 confined in the storage end;

[0015] A joint, through which the interface end of the cantilever rod is detachably mounted on the fixer.

[0016] The micro-droplet collecting device provided by the present invention comprises a collecting part and a fixing part for securely fixing the collecting part on an operating platform.

[0017] First, to solve the problem of unstable droplet quality caused by changing tubes during the collection process, the present invention provides a collection unit that integrates receiving, liquid separation, and collection. The collection unit is a one-piece disposable consumable. The collection unit 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 to first receive approximately 200 to 600 μL of reaction liquid generated by the previous step of the reaction. The reaction liquid contains microdroplets to be collected and a low-concentration generated oil for separating the microdroplets. The received reaction liquid is then separated by gravity through a series of liquid separation ports evenly opened at the lower end of the collection tube through infusion tubes into several small-capacity collection tubes (such as 200 μL or 500 μL centrifuge tubes) compatible with the PCR instrument. The reaction liquid is evenly and stably distributed among the several collection tubes, and each collection tube can collect approximately 50 to 100 μL of reaction liquid. The number, size, and quality of the droplets collected in each collection tube can be kept consistent. This eliminates the need to frequently replace the 200 μL or 500 μL centrifuge tubes used to collect microdroplets during the droplet collection process. This prevents uneven pressure during tube changes, which can cause droplets to fluctuate in size, thereby improving the overall droplet quality. Furthermore, the uniform and stable size of the droplets further shortens the droplet stabilization time, preventing changes in the ratio of the reaction system to the nucleic acid concentration within the droplet, which could compromise the reliability of subsequent experimental results.

[0018] Secondly, to prevent droplet fusion caused by spillage, unnecessary contact vibration, and static electricity transmission before and after replacing the collection tube, the present invention also provides a fixing unit for securely securing the collection unit to an operating platform (such as a microscope stage). The fixing unit is removably secured to the operating platform via a fixture at the bottom, such as a clamp, snap, or bolt connection. The cantilevered rod, which houses the collection unit, is connected to the fixture via a joint, thereby ensuring the secure placement of the collection unit relative to the operating platform and preventing spillage or vibration. 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 confined in the storage end of the cantilever rod away from the operating platform in the height direction, and several collecting tubes naturally and evenly hang down under the dispensing tube through the infusion tube under the action of gravity, so that the entire collecting part is away from the working platform in the height direction. It 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 turned over, and can also keep 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.

[0019] Furthermore, the cantilever rod comprises a deformable support rod and a protective cover fitted over the deformable support rod. To facilitate the experimenter in adjusting the angle and direction of the cantilever rod according to experimental requirements, so that the collection unit placed at the storage end of the cantilever rod is in a suitable operating position, a deformable material, such as 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 protective cover outside the deformable support rod.

[0020] Furthermore, the joint is a rotary joint, and the storage end of the cantilever rod is circumferentially displaced about the rotary joint. To facilitate the experimenter to adjust the angle and direction of the cantilever rod according to experimental requirements, so that the collection unit placed at the storage end of the cantilever rod is in a suitable operating 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 unit by rotating the cantilever rod.

[0021] Specifically, the collection tube is either 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, or 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 and generated oil contained in the reaction solution can be used as the dispensing tube or collection tube. Preferably, the dispensing tube or collection tube is formed of a material that is easy to process and chemically stable, such as a thermoplastic material or a thermosetting material.

[0022] Specifically, one end of the infusion tube is secured inside the collection port by gluing or clamping. The collection tube is connected to the liquid distribution tube via the infusion tube, and the infusion tube and the collection port of the collection tube are glued or clamped together. This securing mechanism not only secures the collection tube, but also facilitates subsequent separation of the collection tube from the infusion tube.

[0023] The present invention also provides a method for collecting micro-droplets using the micro-droplet collecting device, the method comprising the following steps:

[0024] S1. The collecting portion is mounted on the work platform through the fixed portion, mineral oil is added to the dispensing tube and the mineral oil flows through the infusion tube into the corresponding collecting tube, and the second generated oil is added to the collecting tube;

[0025] S2. The reaction solution containing 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;

[0026] S3. After collecting the microdroplets, separate the collection tube from the infusion tube and remove the mixture of the first and second generated oils at the bottom of the collection tube. The collection tube containing the microdroplets and mineral oil is used for subsequent experiments.

[0027] Specifically, the concentration of the second generated oil is higher than that of the first generated oil.

[0028] Step S1 is a pretreatment process of the method, wherein mineral oil is added to the separation tube to prevent impurities such as dust from entering the collected reaction liquid.

[0029] The collection 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.

[0030] 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.

[0031] In general microfluidic experiments, for the sake of cost saving, a 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 the microdroplets and the 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. Then, a higher-concentration generated oil is added to the collection tube 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 change and stabilization, the present invention pre-adds a second generated oil with a higher concentration 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 and the high-concentration second generated oil are mixed, 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.

[0032] Beneficial effects of the present invention:

[0033] (1) Improving the quality of droplets: The present invention adopts a collection part that integrates receiving, separating and collecting. The collection tube does not need to be replaced during the experiment. It can not only avoid the situation where the droplets are large or small due to uneven pressure when replacing the collection tube, so that the droplets in each collection tube have the same quality, but also avoid the droplet splashing, unnecessary contact vibration, static electricity transmission, etc. that may be generated before and after replacing the collection tube, and effectively prevent the droplets from merging, reducing in number, and reducing in quality.

[0034] (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 a large and easily overturned 96-well plate, thereby preventing the failure of the experiment due to external factors.

[0035] (2) Saving experimental time: The present invention pre-adds high-concentration generated oil 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 the droplets is formed. The oil change and droplet stabilization operations are completed while collecting, saving the operation time of oil change and stabilization. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] 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.

[0037] Figure 2 There are three layers of liquid in the collection tube, from top to bottom: mineral oil - droplets - generated oil.

[0038] Figure 3 The experimental space on the microscope stage in the prior art has a messy experimental table and a small experimental space.

[0039] Figure 4 Schematic diagram of a micro-droplet collection device in an embodiment of the present invention.

[0040] Figure 5 It is a side view of the micro-droplet collection device in an embodiment of the present invention.

[0041] Figure 6 Schematic diagram of the rotation of the micro-droplet collection device in an embodiment of the present invention.

[0042] Figure 7 Schematic diagram of the collecting part of the micro-droplet collecting device in an embodiment of the present invention.

[0043] Figure 8 Comparison of the mass of droplets collected by the original method (comparative example) and the droplets collected by the present invention (example).

[0044] Among them, Figures 4 to 7 middle,

[0045] 1-collecting part, 11-dispensing tube, 111-receiving port, 112-dispensing port, 12-collecting tube, 121-collecting port, 122-sealing cover, 13-infusion tube;

[0046] 2-fixing part, 21-fixer, 211-clamp, 212-bolt, 22-cantilever rod, 221-interface end, 222-storage end, 23-connector. DETAILED DESCRIPTION

[0047] The following describes the embodiments of the present invention by specific embodiments. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments. The various details in this specification can also be modified or changed based on different viewpoints and applications without departing from the spirit of the present invention. It should be noted that, in the case of no conflict, the features in the following examples and embodiments can be combined with each other. In the embodiments of the present invention, unless otherwise specified, the methods used are all conventional methods, and the reagents used can be obtained from commercial sources.

[0048] In a specific embodiment, the micro-droplet collecting device comprises a collecting portion 1 and a fixing portion for fixing the collecting portion 1 securely on the working platform.

[0049] 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 a one-piece disposable consumable. Figure 7The collecting part 1 has a dispensing tube 11, four collecting tubes 12, and four infusion tubes 13 for connecting the dispensing tube 11 and the collecting tube 12. In this embodiment, a 1.5 mL centrifuge tube is used as the dispensing tube 11, which has a receiving port 111 for receiving microdroplets on the top, and four 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 that match the PCR instrument, and have a collecting port 121 and a sealing cover 122 that matches the collecting port 121 on the top. The four infusion tubes 13 are all made of plastic hoses used for microfluidic experiments. The diameter of the hose 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. The other end is connected to the dispensing port 112 corresponding to the dispensing tube 11. In this embodiment, the end of the infusion tube 13 near the collection tube 12 is secured inside the collection port 12 by gluing. This securing not only secures the collection tube 12 but also facilitates subsequent separation of the collection tube 12 from the infusion tube 13 by shearing. Thus, under the action of gravity, the four collection tubes 12 can naturally and evenly fall below the dispensing tube 11 via the infusion tube 13. During the microdroplet collection process, approximately 200 to 600 μL of reaction liquid generated from the previous reaction (containing the microdroplets to be collected and the low-concentration generated oil used to separate the microdroplets) is first received into the dispensing tube 11. Then, through a series of dispensing ports 112 uniformly arranged at the same height at the lower end of the collection tube 12, gravity forces the liquid through the infusion tube 13 into each of the four collection tubes 12. This ensures that the reaction liquid is evenly and stably distributed at the bottom of each collection tube 12, preventing impacts on the droplets when they come into contact with the liquid surface. Each collection tube 12 can collect approximately 50-100 μL of reaction solution, and the number, size, and quality of droplets collected in each collection tube 12 are consistent. This eliminates the need to repeatedly replace the 200 μL or 500 μL centrifuge tubes used to collect microdroplets during the droplet collection process. This avoids the uneven pressure caused by tube changes, which can cause droplets to fluctuate in size, and improves the overall droplet quality. Furthermore, droplets of uniform and stable size can further shorten the droplet stabilization time, preventing changes in the ratio of the reaction system to the nucleic acid concentration within the droplet, which could reduce the credibility of subsequent experimental results.

[0050] 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.

[0051] In one or more embodiments, the collection tube 12 can be any one of a 200 μL or 500 μL centrifuge tube, preferably a 200 μL centrifuge tube; the separation 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, 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 separation tube 11 or collection tube 12. Preferably, the separation tube 11 or collection tube 12 is formed of a material that is easy to process and chemically stable, such as a thermoplastic material or a thermosetting material.

[0052] 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 connector 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 method can avoid the problem of droplet fusion caused by droplet splashing, unnecessary contact vibration, static electricity transmission, etc. before and after the replacement of 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 to the stage by clamping.

[0053] 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 confined in the storage end 222 of the cantilever rod 22 away from the operating platform in the height direction. The multiple collection tubes 12 naturally and evenly hang down below the dispensing tube 11 under the action of gravity through the infusion tube 13, so that the entire collection part 1 is away from the operating platform in the height direction. This not only saves the placement space for the 96-well plate used to fix the collection tube 12, but also avoids the spillage of micro-droplets in the collection tube 12 due to the 96-well plate being accidentally touched and overturned, and can also keep the collection part 1 away from the hot microscope light source and the body temperature of the experimenter's hands, thereby 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.

[0054] In a specific embodiment, the interface end 221 of the cantilever rod 22 for placing the collecting part 1 is connected to the holder 21 via a joint 23. Figure 6 In this embodiment, the joint 23 is a rotary joint 23, and the receiving end 222 of the cantilever rod 22 is circumferentially displaced about the rotary joint 23. To facilitate the experimenter in adjusting the angle and direction of the cantilever rod 22 according to experimental requirements, so that the collecting unit 1 placed at the receiving end 222 of the cantilever rod 22 is in a suitable operating position, the rotary joint 23 is used as a connecting member between the cantilever rod 22 and the fixture 21, so that the experimenter can adjust the position of the collecting unit 1 by rotating the cantilever rod 22.

[0055] 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:

[0056] S1. The collection unit is mounted on a work platform via a fixed portion. Mineral oil is added to the liquid separation tubes to prevent dust and other impurities from entering the collected reaction liquid. The mineral oil is then flowed through the liquid transfer tubes into the corresponding collection tubes. The second generated oil is then added to the collection tubes. The resulting collection tubes contain two layers of liquid: the lower layer is the second generated oil, and the upper layer is mineral oil.

[0057] S2. The reaction mixture containing the microdroplets and the first generated oil is added to a separatory tube and then flows through a liquid transfer tube into a corresponding collection tube, 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 liquid: a lower layer of a mixture of the first and second generated oils, a middle layer of microdroplets, and an upper layer of mineral oil.

[0058] S3. After collecting the microdroplets, separate the collection tube from the infusion tube by cutting the glued joint between the collection port and the infusion tube. Remove the mixture of the first and second generated oils at the bottom of the collection tube. The collection tube containing the microdroplets and mineral oil can be used for subsequent experiments, such as closing the sealing cap and placing the collection tube in a PCR instrument for PCR reaction.

[0059] In general microfluidic experiments, for cost-saving purposes, diluted low-concentration generated oil (i.e., the first generated oil in step S2, which can have a concentration of 1×, 2×, or 3×) is used to separate the droplets in the previous reaction step. After the experiment, the reaction liquid containing the microdroplets and the low-concentration generated oil is collected together in a collection tube. After standing, the low-concentration generated oil that has stratified to the lower layer of the collection tube needs to be aspirated and discarded. Then, the newly collected droplets are immersed in the higher-concentration generated oil in the collection tube (the concentration can be 4×, 5×, or 6×) 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, this embodiment pre-adds a second generated oil (the concentration can be 8×, 9× or 10×) with a higher concentration than the first generated oil (the concentration can be 1×, 2× or 3×) into the collection tube in step S1. After the reaction liquid flows into the collection 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 can be 4×, 5× or 6×) for stabilizing the droplets, realizing collection and stabilization at the same time, saving the operation time of oil changing and stabilization.

[0060] Table 1. Mass distribution of collected microdroplets

[0061] Comparative Example Example Percentage 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% Percentage of unqualified large droplets with a diameter ≥30 μm (%) 2% 0

[0062] 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 of the droplets collected according to the original method (Comparative Example) is compared. Figure 8, the proportion of qualified droplets can be increased by approximately 20% per experiment. Referring to Table 1, taking the lysis experiment as an example, under a 20× objective lens, droplets with a diameter of less than 25 μm are considered unqualified small droplets, those with a diameter of 25-30 μm are qualified droplets, and those with a diameter of 30 μm or larger are considered unqualified large droplets. The traditional method resulted in approximately 26 unqualified small droplets, accounting for 23.4%, 82 qualified droplets, accounting for 74.6%, and 2 unqualified large droplets, accounting for approximately 2%, for a total qualified rate of approximately 74.6%. With the method of the present invention, unqualified small droplets accounted for approximately 2.5%, and there were no unqualified large droplets larger than 30 μm, resulting in a total qualified rate of 97.5%.

[0063] 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 fall within the scope of protection of the present invention.

Claims

1. A method for collecting microdroplets using a collection device, characterized in that: The collecting device comprises: 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 uniformly arranged circumferentially; 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 extends 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); 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); The method comprises the following steps: S1. The collecting portion is mounted on the work platform through the fixing portion, mineral oil is added to the liquid separation tube and the mineral oil flows into the corresponding collection tubes through the infusion tubes, and the second generated oil is added to the collection 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; the concentration of the second generated oil is higher than the first generated oil; S3. After completing the microdroplet collection, separate the collection tube from the infusion tube and remove the mixture of the first and second generated oils at the bottom of the collection tube. The collection tube containing the microdroplets and mineral oil is used for subsequent experiments.

2. The method according to claim 1, wherein The fixing portion (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 method according to claim 2, wherein The cantilever rod (22) comprises a deformable support rod and a protective sleeve sleeved outside the deformable support rod.

4. The method according to claim 2, wherein The joint (23) is a rotary joint (23), and the receiving end (222) of the cantilever rod (22) performs circumferential displacement with the rotary joint (23) as the center of the circle.

5. The method according to claim 1, wherein The collection tube (12) is any one of a 200 μL or a 500 μL centrifuge tube.

6. The method according to claim 1, wherein The separation 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 method according to claim 1, wherein One end of the infusion tube (13) is positioned inside the collecting port (121) by gluing or clamping.

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