A droplet sorting system and methods of using the same

Abstract

This invention relates to the field of droplet microfluidics, specifically to a droplet sorting system. The droplet sorting system disclosed in this invention includes a droplet sorting chip, a droplet sorter, and a computer system, capable of accurately and rapidly sorting droplets. The droplet sorting system shown is equipped with a monitoring module to monitor the sorting results, further ensuring the accuracy of the sorting results.

Description

Technical Field

[0001] This invention relates to the field of droplet microfluidics, specifically to a droplet sorting system and its usage method. Background Technology

[0002] Microfluidic chips, also known as lab-on-a-chip, are chips that integrate multiple functions for sample preparation, reaction, detection, and separation in biological and chemical experiments onto a single chip of a few square centimeters, essentially creating a miniature laboratory. With their advantages of miniaturization, integration, and automation, microfluidic chips have enormous application potential in fields such as biological sample processing and rapid disease diagnosis, and have achieved significant development in recent years.

[0003] Droplet microfluidics, an important branch of microfluidic platforms, is a novel technology for manipulating tiny volumes of liquid, namely droplets. Droplets are formed by one fluid within another immiscible carrier fluid; their formation is essentially an emulsification phenomenon. Based on the roles of the two immiscible fluids during droplet formation, they are referred to as 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 the droplet carrier. Depending on whether the monolayer emulsion dispersed phase is aqueous or oil-based, droplets can be classified as O / W (oil-in-water) droplets and W / O (water-in-oil) droplets. Specifically, O / W droplets are oil droplets formed with an oil phase as the dispersed phase and an aqueous phase as the continuous phase, while W / O droplets are water droplets formed with an aqueous phase as the dispersed phase and an oil phase as the continuous phase.

[0004] Droplets possess advantages such as small size, low diffusion, no cross-contamination, and fast reaction speed, making them suitable for high-throughput analysis. In practical applications, high-throughput droplet sorting is required, but individual droplets are small, typically ranging from nanoliters to picoliters (10⁻⁶). -9 ~10 -12 Within the range of L), the corresponding chip size used for droplet sorting is also relatively small, especially the inlet and channel in the sorting chip, whose size is generally at the same level as the droplet. If some external environment occurs or the liquid entering the microchannel contains impurities, it can significantly affect the sorting efficiency and effect of the liquid. Summary of the Invention

[0005] This invention reveals that even tiny particles of dust or fibers entering the microchannel can easily cause blockage, breakage, or fusion of the droplet sorting chip. Furthermore, during the droplet sorting process, minute changes in flow rate or other minor external forces can significantly impact the sorting results. How to accurately and effectively perform rapid droplet sorting has always been a technical challenge in this field. To sort mixed droplets accurately, quickly, and automatically acquire target droplets, this invention provides a droplet sorting system, including a droplet sorting chip and / or a droplet sorter for controlling or containing the chip.

[0006] A first aspect of the present invention provides a droplet sorting chip; the chip includes a droplet inlet, a spacer oil phase inlet, a high-voltage sorting electrode, a sorted droplet outlet, a waste liquid outlet, and channels connecting the inlets and outlets. The droplet inlet is used for the entry of a mixed solution containing target droplets, non-target droplets, and a continuous phase. The spacer oil phase inlet is used to inject the continuous phase, causing it to carry the mixed solution further forward, and can adjust the droplet spacing and control the droplet flow rate. The high-voltage sorting electrode is used to generate a non-uniform electric field, causing the target droplets to be subjected to dielectric force in this non-uniform electric field, thereby changing their flow direction and reaching the sorted droplet outlet. The sorted droplet outlet is used to collect the target droplets. The waste liquid outlet is used to collect the non-target droplets.

[0007] In some embodiments, the chip further includes a biased oil phase inlet for injecting a continuous phase to prevent non-target droplets from flowing into the sorting droplet outlet.

[0008] In some embodiments, the chip further includes a shielding electrode for shielding the non-uniform electric field generated by the high-voltage electrode, preventing the non-uniform electric field from interfering with other areas outside the target area.

[0009] In some embodiments, the chip includes a glass substrate and a PDMS chip fixed thereon, wherein the droplet inlet, spacer oil phase inlet, high-voltage sorting electrode, sorting droplet outlet, waste liquid outlet, channel, bias oil phase inlet, and shielding electrode are all disposed on the PDMS chip, such as by etching these microchannels or pore structures using soft photolithography.

[0010] In a second aspect, the droplet inlet of the droplet sorting chip has been improved.

[0011] During the design and use of the droplet sorting chip, the inventors discovered that the droplet inlet of the existing droplet sorting chip is easily blocked by impurities such as dust and fibers. At the same time, these impurities can also easily cause droplets to break or merge, ultimately leading to the droplet sorting process not being able to proceed normally or a reduction in droplet sorting efficiency and recovery rate.

[0012] In some embodiments, the chip of the present invention includes a cavity region, which is a space for containing impurities such as dust and fibers, thereby preventing impurity clogging. The size or number of cavity regions can be set according to the amount or size of the impurities that may be present. The size of the cavity region should be sufficient to accommodate the impurities, but it does not need to be too large, so as to reduce droplet retention in the cavity region and avoid losses.

[0013] In some embodiments, the chip includes an injection port for injecting a mixed solution containing target droplets, non-target droplets, and a continuous phase. Preferably, the cavity region is connected to or downstream of the injection port, allowing impurities to be directly trapped within the cavity region and preventing them from entering other parts of the chip. Alternatively, the chip may include a droplet generation unit, generating droplets that directly reach the sorting area without requiring an injection port. In this case, the cavity region can also be provided at the droplet generation unit to accommodate impurities that may enter the droplet generation unit.

[0014] In some embodiments, the chip includes a sieve-like structure located downstream of the injection port; after the solution enters the chip through the injection port, the sieve-like structure deflects the flow of impurities in the solution, directing them towards the cavity region, thus sorting the liquid as it passes through the sieve-like structure. The reason for this deflection will be explained in detail later.

[0015] At this point, if the mixed solution contains impurities such as fibers or dust, without a cavity area, these impurities will enter the area of ​​the sieve structure. This will block the gaps, micropores, or tiny slits of the sieve structure, potentially preventing droplets from passing through the blocked area. Without changing the pressure, this can cause droplets to break up, merge, or fail to sort efficiently, thus affecting subsequent sorting efficiency. This is especially true when the mixed solution contains rare or valuable target droplets; such breakage or merging reduces the number of droplets, consequently reducing the number of rare or valuable droplets obtained. The purpose of the sieve structure is to ensure that droplets are arranged continuously with relatively fixed intervals between them, facilitating subsequent screening efficiency. However, if the sieve structure is partially blocked, droplets may become discontinuous, for example, a section may only have oil phase flow without droplets, thus affecting subsequent screening efficiency.

[0016] Preferably, the sieve-like structure is angled at the end near the injection port, and the periphery of the angle is the cavity region.

[0017] Preferably, the injection port is directly connected to the cavity region, and the connection is rounded, which makes it easier for impurities to reach the cavity region.

[0018] In some embodiments, the sieve-like structure comprises cylinders and pores. The cylinders are arranged at regular intervals, forming a pore structure with uniformly spaced pores. The size of the cylinders and pores needs to be determined according to the size of the droplet; the droplet can pass through the pores but not the cylinders. The cylinders and pores alter the droplet's flow path, thereby changing its flow velocity, ultimately causing the droplets to flow out one after another at regular intervals. Impurities such as fibers are generally larger than the droplet, especially if their length exceeds that of the pores, making it difficult for them to enter the pores.

[0019] In some embodiments, the cavity region may be located at the top or bottom of the chip's depth direction, where the top and bottom of the depth direction refer to the vertical direction of the chip (e.g., ...). Figure 1 At both ends (perpendicular to the XY plane), compared to channels or sieve structures on the chip, the cavity region is located lower and / or higher in the depth direction of the chip. In other words, the height of the cavity region is greater than the height of the sieve structure. Figure 6 a). When the cavity region is located at the top of the chip's depth direction, it can be used to accommodate impurities with low density that float on the liquid surface. Figure 6 b); When the cavity region is located at the bottom end of the chip's depth direction, it can be used to accommodate impurities with high density that are located at the bottom of the liquid surface. Figure 6 c). At this point, the amount of impurities entering the sieve-like structure is reduced, thus preventing the clogging of the gaps caused by impurities. Of course, the cavity region can also be located simultaneously at the top and bottom ends of the chip's depth direction and at the same horizontal height. Figure 6 d) For example, downstream of the injection port, an upstream cavity region is provided in the sieve structure. The depth of the cavity region is greater than that of the sieve structure, the bottom end is deeper than that of the sieve structure, and the top end is higher than that of the sieve structure.

[0020] In some methods, the impurities may include fibers that may remain in the chip during chip manufacturing or fibers and dust introduced from the mixed solution, as well as other impurities such as glass fragments.

[0021] In a third aspect, the structure of the sorting channel of the droplet sorting chip has been improved.

[0022] The inventors discovered that existing droplet sorting chips often have a forked "Y"-shaped sorting channel, lacking a mechanism to prevent non-target droplets from entering the sorting droplet outlet. For example, patent 202110644418.6 (CN113477282A) uses a Y-shaped sorting channel: when a target droplet is detected, negative pressure is applied to change its direction of movement, causing it to flow towards the collection port; however, when non-target droplets reach the sorting area, there is no force preventing them from flowing towards the collection port or towards the waste outlet, allowing them to flow towards the collection port as well. This results in sorting failure or a mixture of non-target droplets and target droplets, leading to low sorting accuracy.

[0023] In some embodiments, the sorting channel of the present invention is a portion of a channel (e.g., Figure 1 and Figure 7 The sorting channel comprises a main channel, a target channel, a bias channel, a waste liquid channel, and a sorting section, and its shape is similar to an "X". The main channel and the bias channel are located upstream of the sorting channel, and the bias channel and the waste liquid channel are located downstream of the sorting channel. The main channel is used for the passage of a mixed solution, which includes droplets and a continuous phase. The bias channel is used for the passage of fluid and generates lateral resistance, which prevents non-target droplets from entering the target channel after passing through the sorting section from the main channel, thereby improving the sorting accuracy. The fluid in the bias channel can be a gas or a liquid. For example, nitrogen gas is introduced into the bias channel and blown towards the non-target droplets, causing them to flow towards the waste liquid channel. Preferably, the fluid is a liquid and is the same as the continuous phase in the mixed solution.

[0024] It should be noted that the lateral resistance at this point should not be too large, as excessive resistance may cause the droplets to collide with the channel wall and break up. If negative pressure is used for sorting as in patent 202110644418.6, this resistance should be less than the pressure acting on the target droplet. The magnitude of this resistance should be determined based on the droplets to be sorted, the chip structure, and the sorting force (such as magnetic force, hydrodynamic force, etc.). The magnitude of the resistance can be changed by adjusting the direction and size of the bias channel and the fluid velocity.

[0025] In some configurations, the main channel and the offset channel are parallel to each other and have the same dimensions. It should be noted that the channel cross-section refers to the cross-section of the channel in the vertical direction (e.g., with...). Figure 1 The cross-section in the Z direction perpendicular to the XY plane can be circular, semi-circular, rectangular, or other irregular polygons. "Parallel" here means that the main channel and the offset channel are parallel to each other in the flow path direction, and "same size" means that the size and shape of their channel cross-sections are the same.

[0026] In some embodiments, the fluid is a continuous phase, and the flow rate of the continuous phase in the bias channel is less than or equal to the flow rate of the mixed solution in the main channel.

[0027] In some embodiments, the bias channel forms a certain angle with the main channel, which is 0 to 90°.

[0028] In some embodiments, the chip further includes a high-voltage sorting electrode that conducts electricity to generate a non-uniform electric field, causing the target droplet to deflect to the target channel under the action of dielectric force.

[0029] In some embodiments, the chip includes a spaced oil phase inlet and a biased oil phase inlet, the biased oil phase inlet being used to pump a continuous phase into the main channel, and the biased oil phase inlet being used to pump fluid into the biased channel, preferably, the fluid being the same as the continuous phase.

[0030] In some embodiments, the fluid is the same as the continuous phase, and the chip includes an oil phase inlet that pumps the continuous phase into the main channel and the bias channel, the flow rate in the two channels being adjustable by changing the size of the main channel and the bias channel.

[0031] Preferably, the dimensions of the target channel and the waste liquid channel are both larger than the dimensions of the main channel and the offset channel (the dimensions refer to the size and shape of the vertical cross-section of the channel).

[0032] Preferably, the target channel and the waste liquid channel are interconnected through branch channels. These branch channels allow the continuous phase or the fluid to pass through, but not droplets. The branch channels enable liquid exchange between the target channel and the waste liquid channel, thereby balancing the pressure between the two channels and preventing a large pressure difference that could interfere with the droplet sorting process. The number of branch channels is greater than one, and the number and size of the branch channels should be determined based on the size of the droplet sorting chip, the size of the droplets to be sorted, and the channel dimensions.

[0033] In a fourth aspect, the droplet sorting chip provided by the present invention has a shielding electrode that shields against electric field interference.

[0034] In some methods, the chip utilizes dielectrophoresis to sort droplets. In this case, a ring of shielding electrodes can be placed around the periphery of the chip. Here, "ring" refers to a ring distributed around the periphery of the channels, microstructures, etc., within the chip (e.g., ...). Figure 1 During chip use, it is connected to the instrument's protective ground. This setup can shield the interfering electric field in each area, and the shielding electrodes are integrated into a single structure. Preparation is simple and convenient; just inject a liquid metal or electrolyte solution into the pre-etched channel.

[0035] For example, patent 201910470013.8 describes the use of two sorting electric fields. These fields not only generate a non-uniform electric field at the sorting location, but also reach other parts of the chip, such as the inlet and channels. In these areas, the dielectric force acting on the droplets can cause interference such as droplet collision and fusion, and changes in flow velocity. Furthermore, the particle sorting device utilizing dielectric force in patent 201310102904.0 also suffers from electric field interference. The shielding electrode of this invention shields the electric field interference outside the sorting location, allowing the droplet sorting process to proceed more stably and smoothly.

[0036] Preferably, the shielding electrode is a metal electrode.

[0037] In some embodiments, the metal electrode is formed by solidifying liquid metal.

[0038] In some embodiments, the metal electrode is composed of one or more of the following metals: indium, tin, and zinc.

[0039] In some embodiments, the metal electrode is an alloy with a melting point below 200°C, preferably between 40°C and 80°C, to facilitate liquid preparation of the alloy during electrode fabrication. For example, when the metal electrode is an indium-tin-zinc alloy, its melting point is between 40°C and 80°C. To prepare the metal electrode, the alloy only needs to be heated to 80°C to liquidate it, then poured into a pre-etched electrode channel. After cooling, the metal electrode is formed. This process avoids the use of high temperatures, reduces heating costs and the risk of burns, and shortens heating and cooling time. In some embodiments, such as the first aspect of the invention, the chip includes a droplet inlet, an oil phase inlet, a high-voltage sorting electrode, a sorting droplet outlet, a waste liquid outlet, and channels connecting the inlets and outlets. The shielding electrode is a ring of metal wire surrounding these structures with a depth greater than or equal to the channel.

[0040] In a fifth aspect, the present invention provides a droplet sorting system, the system comprising a droplet sorting chip, a droplet sorter, and a computer system.

[0041] The droplet sorting instrument includes a monitoring module, which includes an identification unit. The identification unit is used to collect information of the sorted droplets and transmit it to a computer system. The computer system determines whether the sorted droplets are target droplets.

[0042] In some embodiments, the identification unit is an optical camera, which is a CCD camera and / or a CMOS camera.

[0043] In some embodiments, the computer system includes a grayscale comparison program that compares grayscale information in images captured by an optical camera and determines whether the droplets have been successfully sorted.

[0044] In some embodiments, the computer system includes a deep learning model for recognizing droplet information images acquired by the CCD or CMOS camera to determine whether the droplets have been correctly sorted.

[0045] In some embodiments, the deep learning model includes VggNet, ResNet, and YOLOv, with YOLOv5 being preferred.

[0046] In some embodiments, the identification unit has fluorescence excitation and acquisition functions. By providing excitation light to the sorted droplets, if the droplet is the target droplet, it will generate fluorescence of a certain intensity. The fluorescence can then be acquired to determine whether the droplet has been correctly sorted.

[0047] In some methods, the identification unit collects information on each sorted droplet and transmits it to a computer system. The computer system determines whether each sorted droplet is a correctly sorted droplet and counts the number of correctly sorted and incorrectly sorted droplets. Here, a correctly sorted droplet is one that enters the target channel and is the same as the expected droplet, while an incorrectly sorted droplet is one that enters the target channel and is different from the expected droplet.

[0048] In some methods, when the computer system determines that the number of incorrectly sorted droplets or incorrectly sorted droplets has accumulated to a certain number or a certain proportion, it issues an early warning and / or sends a command to the droplet sorter to stop the droplet sorting process. In practical use, the threshold for issuing an early warning and / or stopping the droplet sorting process can be set according to specific needs. If the requirement for the proportion of correctly sorted droplets collected at the final sorted droplet outlet is high, such as 100%, the droplet sorting process can be stopped as soon as incorrect sorting is detected; if the requirement is lower, the droplet sorting process can be stopped after several sorting errors, such as 100 times. Alternatively, the cumulative proportion of incorrectly sorted droplets can be calculated, i.e., the ratio of the number of incorrectly sorted droplets to the total number of droplets passing through the target channel; when this proportion reaches a certain value, an early warning and / or the droplet sorting process can be stopped. For example, when the percentage of incorrectly sorted droplets is 1%, 2%, 3%, or 10%, the computer system issues a command to stop the droplet sorting process.

[0049] In some embodiments, the droplet sorter further includes a chip module, a fluorescence module, and an electrode driving module; the chip module is used to place the droplet sorting chip, the fluorescence module is used to excite and detect fluorescence, and the electrode driving module is used to provide high voltage to the droplet sorting chip.

[0050] In some embodiments, the droplet sorting chip is the droplet sorting chip provided in the first aspect of the present invention.

[0051] In some embodiments, the monitoring module further includes a pressure monitoring unit and a flow rate monitoring unit, wherein the pressure monitoring unit is used to monitor the pressure of the system and the flow rate monitoring unit is used to monitor the flow rate of the system.

[0052] In a sixth aspect, the present invention provides a method for sorting droplets.

[0053] In some embodiments, the method is performed using a droplet sorting system according to the fifth aspect of the present invention, which specifically includes the following steps:

[0054] S1. Place the droplet sorting chip in the chip module and set the sorting parameters through the computer system;

[0055] S2. The mixed solution, driven by the sample pressure pump, enters the droplet sorting chip through the droplet inlet and forms a sequentially arranged droplet stream. The continuous phase, driven by the spacer oil pressure pump, is injected through the spacer oil phase inlet. This continuous phase merges with the droplet stream, further arranging the droplets in the stream into individual, sequentially spaced droplets and driving the droplet stream forward. Simultaneously, the continuous phase is injected through the bias oil phase inlet and enters the bias channel.

[0056] S3. When the droplet to be sorted reaches the sorting area, the fluorescence module provides excitation light to irradiate the droplet, detects the fluorescence signal, amplifies the fluorescence signal, converts it into an electrical signal, and transmits it to the computer system. The computer system determines whether the droplet to be sorted is a target droplet based on the received electrical signal. If it is a target droplet, it outputs a command to the electrode drive module. The electrode drive module transmits high voltage to the high voltage sorting electrode, generating a non-uniform electric field. Under the action of the non-uniform electric field, a dielectric force is generated on the droplet to be sorted, causing the droplet to be sorted to deflect and flow towards the target channel. If it is a non-target droplet, the computer system determines that it is a non-target droplet and does not send a command to the electrode drive module. The droplet to be sorted flows towards the waste liquid channel. At this time, at the sorting area, the continuous phase entering from the bias channel can generate lateral resistance that hinders the droplet to be sorted from entering the target channel, preventing non-target droplets from accidentally flowing into the target channel.

[0057] S4. The identification unit collects information about the sorted droplets in the target channel and transmits it to the computer system. The computer system then determines whether the sorted droplets have been correctly sorted.

[0058] In some methods, during droplet sorting, the computer system statistically analyzes the number and proportion of correctly sorted droplets and can display and provide feedback on the system's operating status.

[0059] The advantages of this invention are:

[0060] I. The present invention provides a droplet sorting chip (1) The chip is provided with a cavity area for accommodating impurities, which can effectively reduce or avoid chip blockage problems; (2) The chip is provided with an anti-misflow structure, which improves the sorting accuracy; (3) The chip is provided with a ring of electrostatic shielding electrodes, which can prevent electric field interference and make the chip more stable when working.

[0061] II. The present invention provides a droplet sorting system (1) The system can perform fully automatic droplet sorting, reducing human operation; (2) The system is equipped with a monitoring mechanism to monitor the sorted droplets, increasing the stability and controllability of the system; (3) The system can count the number of sorted droplets or the proportion of target droplets without further statistics or confirmation; (4) The system can perform high-throughput droplet sorting with high accuracy. Attached Figure Description

[0062] Figure 1 A schematic diagram of a planar structure of a droplet sorting chip.

[0063] Figure 2 Schematic diagrams of two droplet inlet structures in the prior art.

[0064] Figure 3 Example 2 provides a schematic diagram of a droplet inlet structure.

[0065] Figure 4 A comparison of impurities entering the droplet inlet with a cavity structure (a) and without a cavity structure (b).

[0066] Figure 5 Figure 3 Micro-electron microscopy image of the droplet inlet structure during operation.

[0067] Figure 6 A schematic diagram showing the depth relationship between the cavity region and the sieve structure in Example 2.

[0068] Figure 7 Example 3 provides a schematic diagram of a sorting channel structure.

[0069] Figure 8 Example 3 provides another schematic diagram of a sorting channel structure.

[0070] Figure 9 A Y-shaped sorting channel.

[0071] Figure 10 A schematic diagram of a droplet sorting instrument provided by the present invention.

[0072] Figure 11 for Figure 10 A schematic diagram of the internal structure of a droplet separator.

[0073] Figure 12 for Figure 10 A schematic diagram of the internal structure of the droplet separator from another direction.

[0074] Figure 13 Grayscale captured images and the grayscale curve of the captured area versus time.

[0075] In the diagram: 1-Droplet inlet, 2-Biased oil phase inlet, 3-Interval oil phase inlet, 4-High-voltage sorting electrode, 5-Shielding electrode, 6-Sorted droplet outlet, 7-Waste liquid outlet, 8-Channel, 9-Impurity, 10-Droplet, 11-Injection port, 12-Neck channel, 111-Smooth setting, 13-Adjustment structure, 131-Sieve structure, 1311-Cylinder, 1312-Pore, 1313-Angle, 132-Cavity region, 81 - Sorting channel, 811- Main channel, 812- Target channel, 813- Bias channel, 814- Waste liquid channel, 815- Sorting section, 815a- Left side, 815b- Right side, 816- Branch channel, 100- Glass substrate, 101- PDMS chip, 200- Chip module, 300- Fluorescence module, 400- Pump drive module, 500- Electrode drive module, 600- Circuit module, 700- Monitoring module.

[0076] Detailed description

[0077] 1. Mixed solutions, target droplets, and non-target droplets

[0078] In this application, "mixed solution" refers to a solution containing droplets and a continuous phase. Droplets include target droplets and non-target droplets. Droplets can be one of W / O, O / W, W / O / W, O / W / O, or a combination of W / O and O / W / O, or O / W and W / O / W. The continuous phase can be a single oil phase or an aqueous phase, or it may be multiple miscible liquid reagents. Target droplets refer to the desired droplets, which in this application include droplets that can generate fluorescence of a certain intensity when passing through a sorting site, or droplets encapsulating target objects. Non-target droplets refer to other droplets in the mixed solution besides the target droplets. For example, a solution containing W / O type droplets (i.e., a mixed solution) can be prepared using a droplet generation chip. During droplet generation, droplets encapsulating target cells, proteins, etc., may be generated (i.e., target droplets), but other droplets without encapsulation or with incorrect encapsulation may also be generated (i.e., non-target droplets).

[0079] 2. Droplets to be sorted and droplets after sorting

[0080] In this application, "droplets to be sorted" refers to droplets that will be sorted by the droplet sorting chip, including droplets in a mixed solution, droplets located in the front channel of the sorting section, or droplets located at the droplet sorting section during the sorting process. "Post-sorted droplets" refers to droplets that have been sorted by the droplet sorting chip, including droplets that have reached the target channel after sorting, droplets collected from the sorted droplet outlet, or droplets that have reached the waste liquid channel. Detailed Implementation

[0081] 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 should not be construed as limiting the scope of protection of the present invention. All features disclosed in the embodiments of the present invention, or all steps in all disclosed methods or processes, except for mutually exclusive features and / or steps, can be combined in any way and are all within the scope of protection of the present invention. Any modifications made by those skilled in the art based on the technical concept of the present invention without creative effort should be within the scope of protection of the present invention. Technologies not covered in this invention can be implemented using existing technologies.

[0082] Example 1: Droplet Sorting Chip

[0083] like Figure 1 The present invention provides a droplet sorting chip, which consists of a glass substrate 100 and a PDMS chip 101 fixed thereon. The PDMS chip 101 is provided with a droplet inlet 1, a spacer oil phase inlet 3, a bias oil phase inlet 2, a high-voltage sorting electrode 4, a sorted droplet outlet 6, a waste liquid outlet 7, a channel 8 connecting each inlet and outlet, and a shielding electrode 5.

[0084] Droplet inlet 1 is used for the mixed solution containing droplets to enter. Interval oil phase inlet 3 is used to inject the continuous phase, allowing it to carry the mixed solution further forward and adjust the droplet spacing and control the droplet flow rate. Bias oil phase inlet 2 is used to inject the continuous phase, preventing non-target droplets from mistakenly flowing to the sorting droplet outlet. High-voltage sorting electrode 4 generates a non-uniform electric field, causing the target droplets to be subjected to dielectric force in this field, thus changing their flow direction and reaching the sorting droplet outlet 6. Sorting droplet outlet 6 is used to collect target droplets. Waste liquid outlet 7 is used to collect non-target droplets. Shielding electrode 5 shields the non-uniform electric field generated by the high-voltage electrode, preventing this non-uniform electric field from interfering with other areas outside the target region.

[0085] Example 2 Droplet Inlet

[0086] At the droplet inlet 1, the inventors discovered that the injection port 11 is connected to the adjustment structure 13 via a neck channel 12, and the adjustment structure 13 contains a sieve structure 131 (such as...). Figure 2a) During droplet sorting, partial or complete blockage of the chip can easily occur, preventing the sorting process from continuing. Electron microscopy revealed that impurities such as fibers blocked the neck channel 12, and that these impurities also entered the pores 1312 of the sieve structure 131, causing further blockage. Additionally, the droplet chamber structure shown in patent 202021628514.0 ( Figure 2 b) As droplet inlet 1, there is still a clogging problem.

[0087] These impurities may originate from the chip manufacturing process, such as PDMS fibers produced during PDMS cutting, or they may be impurities introduced from the mixed solution, such as dust, tiny particles, etc. To address these issues, this invention provides a novel design that effectively reduces impurity blockage of the chip, including blockage at the inlet and in the sieve structure 131 region, allowing droplets to enter the sieve structure 131 region more smoothly and arrange themselves in an orderly manner.

[0088] In order to provide a space for impurities such as fibers to prevent them from further entering the sorting chip, a cavity region 132 is provided in the sieve structure 131 region of the adjustment structure 13 to accommodate impurities such as fibers.

[0089] In some embodiments, the cavity region 132 is located within the inner perimeter of the triangular edge of the regulating structure 13, or on the outer periphery of the sieve structure 131 region. The cavity region 132 does not contain, or substantially does not contain, the sieve structure 131, but can be distributed around the periphery of the sieve structure 131 region and connected to it. When the mixed solution enters the sieve structure 131, droplets pass through the sieve structure 131 region, thus arranging themselves in an orderly manner. When the cavity region 132 is present, it is a blank space, while the sieve structure 131 region is sieve-shaped. The mixed solution encounters the sieve mesh, and the resistance it experiences entering and passing through the sieve structure 131 region is greater than the resistance to the mixed solution flowing into the cavity region 132. Impurities in the solution, especially fibrous impurities, preferentially enter the cavity region, reducing their chance of entering the sieve structure 132 region. This may be one reason why the cavity region 132 can accommodate impurities such as fibers and prevent them from entering the sieve structure 131. Generally, the length of fibrous impurities is greater than the diameter of the droplets. When the fibers enter the sieve structure 131 region, they will wrap around the cylinder 1311 in the sieve structure 131 region and be located in or block the pores 1312, which will hinder the flow of liquid. In addition, if the droplets directly contact these fibrous impurities that have entered the sieve structure 131, the droplets will break or merge, thereby reducing the number of target droplets obtained in subsequent sorting.

[0090] In some embodiments, the cavity region 132 for containing impurities is distributed on both sides of the inlet, and is located on the periphery of the sieve structure 131 region; alternatively, the cavity region 132 for containing impurities is distributed at the inlet, generally with or without branching at the inlet, and the mixed solution flowing in from the inlet enters the sieve structure 131 region, with a portion entering the cavity region 132 for containing impurities. For example... Figure 3 As shown, the cavity regions 132 that contain impurities are located on the periphery of the sieve structure 131 region, and are distributed in the shape of "human" or "eight". Of course, it is also feasible to have them only on one side.

[0091] In some ways, such as Figure 3 The upper end of the sieve structure 131 is also angled 1313 in the horizontal direction (XY direction). The area between the side of this angled 1313 and the angled side of the adjustment structure 13 is the cavity region 132. When the mixed solution enters the chip from the injection port 11, if there are fibrous impurities in the solution, these impurities are generally longer than the droplets and easily collide with the cylinder 1311, thus flowing to the cavity regions 132 on both sides and being retained. This effectively prevents fibrous impurities from entering the sieve structure 131. It should be noted that the cavity region 132 is directly connected to the injection port 11 of the mixed solution, and a structure that can change the flow direction of the mixed solution is set at the injection port 11. When the mixed solution or impurities in it come into contact with this structure, the flow direction changes and it first flows to the cavity region 132, and then enters the sieve structure 131 from the cavity region 132. Figure 4 As shown, when impurities 9 in the mixed solution enter the droplet sorting chip, if there is no cavity region 132, they will be blocked at the inlet; if there is an "eight"-shaped cavity region 132 between the corner 1313 and the corner side of the adjustment structure 13, the impurities 9 in the mixed solution will encounter the sharp corner of the corner 1313, and the flow direction will change from forward to sideways. After reaching the cavity region 132, they will be laterally stuck in the cavity region 132, thus preventing them from entering the sieve structure 131. The flow of droplets 10 and the retention of impurities 9 in actual use of this structure are as follows: Figure 5 As shown. Besides the angular shape of the triangle 1313, the angular corner of the triangle 1313 can also be rounded, which is more conducive to changing the flow direction of impurities. Of course, a small rectangular or frustum-shaped structure or other structures that can change the flow direction can also be provided at the front end of the injection port 11. The cavity region 132 is preferably the above-mentioned figure-eight shaped space, but it can also be other shapes such as a cuboid or cylinder.

[0092] like Figure 3The inlet 11 is directly connected to the regulating structure 13, avoiding blockage of the neck channel 12. In other words, the mixed liquid entering through the inlet can directly enter the regulating structure 13 without passing through a buffer area, allowing the liquid to enter the sieve structure 131 region with a larger surface area or a larger cross-sectional area. In fact, some droplets can also enter the sieve structure 131 region and be sorted through the interface between the cavity region 132 and the sieve structure 131 region.

[0093] In some configurations, the area where the adjustment structure 13 is located is smoothly connected to the injection port 11 (111). Figure 3 Instead of sharp corners, the rounded shape reduces forward resistance and further prevents blockage of the injection port 11. In particular, when the injection port 11 is flanked by cavity regions 132, the mixed solution entering from the injection port 11 enters the regulating structure 13 through the rounded setting 111, avoiding collisions between the mixed solution and sharp corners. This reduces the resistance experienced by the mixed solution and makes it easier to flow to the downstream regulating structure 13. The friction experienced by droplets or impurities in contact with the inner wall of the chip is also reduced when they come into contact with the rounded setting 111, making it easier to flow to the cavity regions 132 on both sides. In addition, the rounded setting 111 also makes the entrance of the cavity region 132 larger, facilitating the entry of the mixed solution.

[0094] The cavity region 132 is located at the top and / or bottom of the droplet sorting chip in the depth direction, such as the top and / or bottom of the sieve structure 131 in the vertical direction, or its depth and / or height is greater than the depth and / or height of the sieve structure 131. Figure 6 ).

[0095] In some embodiments, a cavity region 132 is provided in the depth direction or vertical direction (the direction perpendicular to the XY plane, denoted as the Z direction) of the regulating structure 13 at one end of the injection port 11. The cavity region 132 is located above the corresponding area on the periphery of the corner 1313 of the sieve structure 131. If there are fine impurities in the mixed solution, whose density is lower than that of the liquid, such as dust, these impurities will float on the surface of the solution. When they reach the cavity region 132, they can be retained by the cavity region 132, thereby preventing them from entering the sieve structure 131 or the downstream channel.

[0096] In some embodiments, the cavity region 132 is located below the corresponding region on the periphery of the corner 1313 of the sieve structure 131. If there are impurities with a density higher than that of the liquid in the mixed solution, such as small glass fragments, these impurities will be located at the bottom of the solution and can be retained by the cavity region 132 when they reach it, thereby preventing the impurities from entering the sieve structure 131 or the downstream channel.

[0097] In some embodiments, the cavity region 132 may be located directly at the top or bottom of the depth direction of the sieve structure 131, either upstream, midstream, or downstream. In this case, the mixed solution first reaches the sieve structure 131, but because impurities with higher or lower densities float at the top or bottom of the solution, the higher-density impurities will sink to the bottom and remain in the cavity region 132. When the sieve structure 131 is filled with solution, the lower-density impurities will float at the top, reach the cavity region 132, and be retained. This arrangement is particularly suitable for cases where impurities have higher or lower densities and relatively small volumes, such as dust, fine glass, or metal particles. Preferably, the cavity region 132 is located at the top or bottom of the upstream region of the sieve structure 131. In this case, the cavity region 132 is located upstream of the mixed solution flow path, allowing impurities to reach the cavity region 132 earlier and be retained, preventing impurities from flowing through more places or staying longer in the sieve structure 131, thus avoiding interference with more droplets. In some embodiments, the depth or height of the cavity region 132 is greater than the depth or height of the sieve structure 131. The cavity region 132 is located on the periphery of the sieve structure 131, and its depth and / or height may be greater than that of the sieve structure 131. In this case, after the mixed solution enters the chip through the injection port, it first easily reaches the cavity region 132. Here, impurities in the solution, including fibers, dust floating at the top of the solution, or small pieces of glass settled at the bottom of the solution, can be retained by the cavity region 132.

[0098] Example 3 Sorting Channel

[0099] 1. Sorting channel and its improvement

[0100] like Figure 7 The sorting channel 81 is shown. The sorting channel 81 consists of a main channel 811, a target channel 812, an offset channel 813, a waste liquid channel 814, and a sorting section 815 at the intersection of the channels.

[0101] The sequentially arranged droplets to be sorted flow forward in the main channel 811 under the influence of the spacer oil phase. When passing through the detector, if the detector detects that the droplet to be sorted is a target droplet, a voltage is applied through the high-voltage sorting electrode 4 to generate a dielectric force, causing the droplet to be sorted to be deflected at the sorting section 815 and enter the target channel 812. If the droplet to be sorted is a non-target droplet, it flows towards the waste liquid channel 814 under the influence of the spacer oil phase. At the same time, the bias oil phase enters the bias channel 813 through the bias oil phase inlet 2 and further flows into the target channel 812. This arrangement can generate a certain lateral resistance at the sorting section 815 to prevent non-target droplets from accidentally flowing into the target channel 812. The direction of this lateral resistance is from the bias channel 813 towards the main channel 812, that is, from left to right here. It is important to note that the resistance should not be too great. If it is too great, the droplet may crash into the channel wall to the right, causing it to break or deform. If it is greater than the dielectric force, the target droplet may not be deflected when it enters.

[0102] (1) In some methods, the bias oil phase and the spacer oil phase are the same and the same as the continuous phase. For example, when sorting W / O droplets of fluorinated oil type, both the bias oil phase and the spacer oil phase are fluorinated oil. The main channel 811, target channel 812, bias channel 813 and waste liquid channel 814 are all elliptical channels. The main channel 811 and bias channel 813 have the same dimensions (i.e., the shape and area of ​​the vertical channel cross section, denoted as R1) and are parallel to each other; the target channel 812 and waste liquid channel 814 have the same dimensions (the area of ​​the vertical channel cross section is denoted as R2) and are parallel to each other, with R2 being greater than R1. The flow rate of the fluorinated oil in the main channel 811 is less than or equal to the flow rate in the bias channel 813, which can effectively prevent non-target droplets from accidentally flowing into the target channel 812.

[0103] ① When the flow rate of fluorinated oil in the bias channel 813 is less than that in the main channel 811, the flow rate on the left side 815a of the sorting section 815 is less than that on the right side 815b. According to the Bernoulli effect, the pressure is lower at the location with higher flow rate and higher at the location with lower flow rate. Therefore, the pressure on the left side 815a is greater than that on the right side 815b, which will generate a force from left to right (i.e., lateral resistance) to prevent non-target droplets passing through the right side 815b from reaching the left side 815a and thus entering the target channel 812.

[0104] ② When the flow velocity of the fluorinated oil in the bias channel 813 is equal to that in the main channel 811, the pressure on the left side 815a is equal to that on the right side 815b. Non-target droplets continue to flow forward from the main channel 811 and reach the waste liquid channel 814 after passing through the sorting section. At the same time, if a non-target droplet flows from the right side 815b to the left side 815a, the non-target droplet will be subject to resistance from the bias channel 813 side to the main channel 811 side (i.e., lateral resistance from left to right) due to the fluorinated oil passing through the 815a area, thus preventing the non-target droplet from flowing to the left side 815a.

[0105] R2 being greater than R1 can make the flow velocity of the fluid in the target channel 812 and the waste liquid channel 814 lower than that in the main channel 811 and the bias channel 813, thereby reducing the influence of the resistance at the front end of the sorting section 815 on the flow velocity of the droplets and avoiding its possible interference with the sorting process of the droplets.

[0106] In some embodiments, a connecting branch channel 816 is provided between the target channel 812 and the waste liquid channel 814. This branch channel 816 allows liquid to pass through but does not allow droplets to pass through. The branch channel 816 allows for the exchange of liquids in the target channel 812 and the waste liquid channel 814, thereby making the pressures in the target channel 812 and the waste liquid channel 814 similar. This prevents excessive pressure in the target channel 812 from hindering the entry of target droplets or excessive pressure in the waste liquid channel 814 from hindering the entry of non-target droplets.

[0107] (2) Figure 8 The bias channel 813 forms a certain angle with the main channel 811, ranging from 0° to 90°. The biased oil phase entering through the bias channel 813 generates a left-to-right force (i.e., lateral resistance), which can prevent non-target droplets in the main channel 811 from mistakenly entering the target channel on the left. Similarly, the force cannot be too large, meaning the flow rate of the biased oil phase in the bias channel 813 should be controlled within a reasonable range: ① When the angle is large, the flow rate of the biased oil phase should be small; when the angle is small, the flow rate of the biased oil phase should be large. ② Simultaneously, this flow rate is related to the flow rate of the mixed solution in the main channel 811; the greater the flow rate of the mixed solution, the greater the flow rate; the smaller the flow rate of the mixed solution, the smaller the flow rate. ③ In addition, the magnitude of the dielectrophoretic force and the Bernoulli effect also need to be considered.

[0108] In summary, ① when a non-target droplet passes through the sorting section 815, the forces acting on it in the XY plane are divided into forces acting in the flow direction and forces perpendicular to the flow direction. The force perpendicular to the flow direction is directed from the bias channel 813 side towards the main channel 811 side (i.e., lateral resistance). Figure 7 and Figure 8(The force acts from left to right in the figure), so that the non-target droplets will not shift from the right side 815b to the left side 815a within the distance of passing through the sorting part 815. ② When the target droplet passes through the sorting part 815, in addition to the forces received by the non-target droplets, the target droplet is also affected by dielectrophoresis force. The resultant force is in the direction opposite to the acting force perpendicular to the flow direction and the lateral resistance, and can make the target droplet shift from the right side 815b to the left side 815a within the distance of passing through the sorting part 815, so as to further enter the target channel 812.

[0109] (3) Figure 1 The chip in [description] has two oil-phase inlets, namely the spaced oil-phase inlet 3 and the offset oil-phase inlet 2, which respectively control the liquid flow rates in the offset channel 813 and the main channel 811. The two inlets need to be respectively driven by power with pumps or the like. There will be errors in the power drive, which will cause a deviation between the actual flow rate in the channel and the set flow rate. And the pipelines of the droplet sorting chip are very thin, and even a tiny deviation will affect the sorting result. In addition, the two drives will also increase the cost. Therefore, the present invention provides another droplet sorting chip. The offset oil phase and the spaced oil phase of this chip both enter from the same inlet (oil-phase inlet). By adjusting the sizes of the offset channel 813 (the cross-sectional area Ra in the vertical direction of the channel) and the main channel 811 (the cross-sectional area Rb in the vertical direction of the channel), the flow rates of the offset channel 813 and the main channel 811 can be changed. When Ra is equal to Rb, the flow rates of the two channels are equal; when Ra > Rb, the flow rate of the offset channel 813 < the flow rate of the main channel 811; when Ra < Rb, the flow rate of the offset channel 813 > the flow rate of the main channel 811. It should be noted that after the mixed solution enters from the injection port 11, it is mixed with the oil phase entering the main channel 811 from the oil-phase inlet 100. The mixed solution may affect the flow rate. Depending on the injection speed of the mixed solution, the droplet density, etc., this influence may be negligible, or it may need to be considered and the inner diameter of the main channel 811 needs to be adjusted accordingly.

[0110] 2. Droplet sorting experiment

[0111] The inventors respectively used Figure 7 the "X"-type sorting channel ([experimental group]) and Figure 9 the "Y"-type sorting channel ([control group]) to conduct droplet sorting experiments.

[0112] Experimental group: Figure 7The main channel 811 and the bias channel 813 have a width of 54 μm and a depth of 60 μm. The sorting section 815 has a horizontal cross-section of 148 μm × 60 μm. The target channel 812 and the waste liquid channel 814 have a width of 128 μm and a depth of 60 μm. When sorting droplets (droplets include target droplets and non-target droplets; target droplets are W / O droplets containing the target substance, with a droplet diameter of approximately 45 μm, and the target substance is a single cell; non-target droplets do not contain the target substance), the flow rate of the main channel 811 is set to 20 μL / min, and the flow rate of the bias channel 813 is set to 20 μL / min. A mixed solution containing approximately 1 million droplets (the proportion of target droplets was approximately 1%) was sorted. Droplet solutions from 6 sorting droplet outlets were collected. The number of correctly sorted droplets (i.e., target droplets) and incorrectly sorted droplets (i.e., non-target droplets) were observed and counted using electron microscopy, and the proportion of correctly sorted droplets was calculated. The proportion of correctly sorted droplets was 99.5%.

[0113] Control group: Figure 9 The "Y"-shaped sorting channel consists of a main channel 811, a target channel 812, a waste liquid channel 814, and a sorting section 815 at the intersection of these channels. The main channel 811 has a width of 54 μm and a depth of 60 μm. The target channel 812 and the waste liquid channel 814 have a width of 128 μm and a depth of 60 μm. The horizontal cross-section of the sorting section 815 is irregularly shaped. All other aspects were handled in the same way as the experimental group for droplet sorting and statistical analysis. The correct droplet sorting rate was 92.9%.

[0114] The test results show that the sorting accuracy of the "X" type sorting channel is significantly higher than that of the "Y" type sorting channel. Its bias channel 813 can effectively prevent non-target droplets from flowing into the target channel 812.

[0115] Example 4 Shielding Electrode

[0116] Figure 1 The droplet sorting chip shown utilizes dielectric electrophoresis to sort droplets. Dielectric electrophoresis (DEP) refers to the effect where, when a particle in a fluid is subjected to a non-uniform electric field, its internal charge is induced to polarize, causing it to move in the positive or negative direction of the electric field gradient. During droplet sorting, a non-uniform electric field is applied by the high-voltage sorting electrode 4. This electric field propagates outward from the high-voltage electrode 4, decreasing in intensity. If the electric field is not shielded, it may cover the entire chip, causing interference. For example, if the droplet is located in an interfering electric field before entering the sorting channel 81, dielectric electrophoresis may occur, preventing the droplets from merging individually or forming irregular arrangements.

[0117] To avoid the influence of the interfering electric field, such as Figure 1A shielding electrode 5 is arranged around the chip. The shielding electrode 5 is a metal wire structure along the periphery of the droplet inlet 1, the spacer oil phase inlet 3, the bias oil phase inlet 2, the high-voltage sorting electrode 4, the sorting droplet outlet 6, the waste liquid outlet 7, and the channel 8. The metal wire is not closed at both ends and is connected to the instrument's protective ground wire during chip use to achieve the function of electric field shielding.

[0118] Existing technologies for fabricating droplet sorting chips involve first fabricating electrodes on a glass substrate, then etching the aforementioned droplet inlets, channels, and other structures onto a PDMS chip, and finally aligning the glass substrate and the PDMS chip to crosslink the PDMS chip onto the glass substrate. This process requires precise control of the positions of the electrodes and the channels and other structures on the PDMS chip, ensuring that the distances between the electrodes and the various structures on the PDMS chip are within a predetermined range. This fabrication process is complex, and the distances between the electrodes and the channels and other structures are difficult to control accurately. The shielding electrode provided by this invention allows the channel at the location of the shielding electrode 5 to be directly etched onto the PDMS chip. Liquid metal is then injected into this channel, and after cooling and solidification, the shielding electrode 5 is formed. Therefore, the alignment operation between the electrode and other structures can be avoided, the fabrication process is simple, and the position is controllable.

[0119] Compared to multiple discontinuously distributed shielding electrodes on a droplet sorting chip, the shielding electrode 5 in this application has a single integrated structure, requiring only a single filling of liquid metal, eliminating the need for multiple fillings and simplifying chip fabrication. The channels etched on the PDMS are extremely fine; during liquid metal filling, the amount of metal in the droplets must be precisely controlled to ensure complete filling without overflowing and causing waste or contamination. Therefore, a single filling simplifies and controls chip fabrication. Furthermore, during chip use, the shielding electrode 5 needs to be connected to the instrument's protective ground. The integrated shielding electrode 5 in this application only requires a single connection to the instrument's protective ground, simplifying operation; whereas multiple discontinuously distributed shielding electrodes require multiple connections, making the operation more cumbersome.

[0120] In some methods, the cross-section of the channel can be circular, semi-circular, rectangular, trapezoidal or other shapes, the depth of the channel is greater than or equal to that of the channel 8, and the shape of the shielding electrode 5 formed after the solidification of the injected droplet metal is the same as that of the channel, and its cross-section and depth are consistent with those of the channel.

[0121] In some methods, liquid salt solutions (e.g., 2M NaCl) can be used directly in the river channel instead of metal. While salt solutions can shield the electric field just like metals, the inventors found that: (1) if the salt solution is sealed in the river channel, it easily dries out and is difficult to encapsulate, thus increasing operational difficulty and potentially affecting the shielding effect. (2) If the salt solution is circulated in the river channel using a pump during chip use, although the encapsulation and drying problems can be avoided, more pumps and containers are needed, taking up space and increasing costs. Furthermore, the stability of the salt solution is low, and issues such as solution preparation and storage need to be considered. When using metal, only liquid metal needs to be injected into the river channel and allowed to solidify; minimal operation is required, and the subsequent use is stable. Therefore, a metal electrode is preferred for the shielding electrode 5.

[0122] In some methods, the metal electrode is an indium, tin, and zinc alloy with a melting point of 40°C to 80°C. Compared to high-melting-point metals such as copper and iron, the alloy only needs to be heated to 80°C to be in a liquid state during manufacturing. After being poured into the river, the cooling time is short and there is no risk of burns.

[0123] Example 5: Droplet Sorting System

[0124] 1. For example Figure 10-12 The present invention provides a droplet sorting instrument, which includes a chip module, a fluorescence module, a pump drive module, an electrode drive module, a circuit module, and a monitoring module.

[0125] The chip module houses the droplet sorting chip. The fluorescence module provides excitation light and collects fluorescence signals. The pump drive module, including a sample pressure pump, a spacer oil pressure pump, a bias oil pressure pump, and their drives and connections, is used to pump in the mixed solution or a continuous, equal-phase fluid. The circuit module connects the various modules to transmit signals or commands.

[0126] The monitoring module is used to monitor whether the droplet sorter is operating normally. The monitoring module includes a pressure monitoring unit and an identification unit. The pressure monitoring unit monitors whether the system pressure is normal, including the pump pressure of the sample pressure pump, interval oil pressure pump, and bias oil pressure pump. The identification unit identifies whether the droplets in the target channel are correctly sorted. This unit uses a CCD camera, which can quickly capture and record image data of each droplet in the target channel and transmit the acquired data to the computer system.

[0127] 2. Figure 1 Droplet sorting chip in Figure 10 The droplet sorting instrument and computer system constitute a droplet sorting system of the present invention. When using the system for droplet sorting, the following steps are included:

[0128] S1, will Figure 1 The droplet sorting chip is placed in the chip module. The parameters of each module are set through a computer system, including the flow rate of the sample pressure pump, the flow rate of the spacer oil pressure pump, the flow rate of the bias oil pressure pump, and the excitation wavelength.

[0129] S2. The droplet sorting process begins. Driven by the sample pressure pump, the mixed solution enters the droplet sorting chip through the droplet inlet and forms a sequentially arranged droplet stream in the main channel 811. The continuous phase is injected through the spacer oil inlet 3, driven by the spacer oil pressure pump. This continuous phase merges with the droplet stream, further arranging the droplets to be sorted in a sequential, individually spaced pattern, and driving the droplet stream forward. Simultaneously, the continuous phase is injected through the bias oil inlet 2, driven by the bias oil pressure pump, and enters the bias channel 813.

[0130] S3. When the droplet to be sorted reaches the front end of the sorting section 815, it is excited by the excitation light provided by the fluorescence module. If it is a target droplet, a fluorescence signal of a certain intensity is generated. This signal is detected and amplified by the fluorescence module, converted into an electrical signal, and transmitted to the computer system. The computer system determines whether it is a target droplet based on the received electrical signal. If it is a target droplet, it outputs a command to the electrode drive module. The electrode drive module transmits high voltage to the high-voltage sorting electrode, generating a non-uniform electric field. Under the action of the non-uniform electric field, a dielectric force is generated acting on the droplet, causing the droplet to deflect and flow towards the target channel. If it is a non-target droplet, no fluorescence is generated, or the received fluorescence signal is below the threshold. The computer system determines that it is a non-target droplet and does not send a command to the electrode drive module. Non-target droplets flow towards the waste liquid channel, and the biased oil phase provides resistance from the target channel to the waste liquid channel, or rather, lateral resistance that prevents non-target droplets from flowing into the target channel, effectively preventing non-target droplets from accidentally flowing into the target channel.

[0131] S4. The CCD camera acquires image information of the sorted droplets in the target channel and transmits it to the computer system, which then determines whether the droplets have been correctly sorted.

[0132] A computer system can determine whether a droplet has been correctly sorted using a grayscale comparison program. This program compares the grayscale value of the sorted droplet in the target channel of an image captured by a CCD camera with the grayscale values ​​of either an image containing incorrectly sorted droplets or a correctly sorted droplet. This comparison determines whether the droplet in the target channel is correctly sorted. Generally, correctly sorted droplets often contain target contents such as cells, proteins, and microspheres, and their grayscale value will be higher than that of incorrectly sorted droplets. For example... Figure 13 , Figure 13 Image 'a' is a photograph taken during droplet sorting; a grayscale comparison program can identify it. Figure 13 The grayscale value within the dashed box (collection area) can also be used to plot a curve showing the change in grayscale value over time in the collection area. Figure 13 b) and determine whether the droplets are correctly sorted by comparing and changing the grayscale.

[0133] Computer systems can also include deep learning models to determine whether droplets have been correctly sorted. Deep learning models are widely used in image recognition, such as face recognition [A review of deep learning-based face attribute recognition methods, Lai Xinyu et al., Computer Research and Development, 2021, 58(12), 2760-2782] and vehicle recognition [A review of the application of deep learning image recognition technology in vehicle detection and model recognition, Wang Ye, Artificial Intelligence and Recognition Technology, 2021, No. 6], which can accurately identify single or multiple complex individuals in an image. Deep learning models can be used to identify droplets in an image to determine whether they are target droplets. Compared with grayscale comparison methods, deep learning models can identify target droplets with different characteristics, including not only grayscale features but also droplet size, color, and shape information, resulting in higher accuracy. Deep learning models include VggNet, ResNet, and YOLOv. This invention selects YOLOv5, and the training and application of this model includes the following process: ① Taking pictures of the target droplet in advance to obtain an image of the target droplet; ② Labeling the features of the target droplet in the image (including grayscale, size, and contents); ③ Inputting the labeled droplet image into YOLOv5 for training to obtain the optimal model parameters; ④ Inputting the target droplet image into the trained YOLOv5 model to verify accuracy; ⑤ When performing droplet sorting, the image acquired by the CCD camera is input into the trained YOLOv5 model, which can then identify whether the droplet in the image is the target droplet.

[0134] Furthermore, the computational system can issue warnings or stop the droplet sorter based on the judgment results. The computer system can also statistically analyze the judgment results of the grayscale comparison program or deep learning model, such as counting the number of correctly sorted droplets passing through the target channel, the number of incorrectly sorted droplets, and the total number of droplets. It can further calculate the proportion of incorrectly sorted droplets to the total number of droplets, i.e., the error rate. When the error rate reaches a certain value, an warning is issued and / or the droplet sorting process is stopped. For example, the error rate could be 1%, 2%, 3%, or 10%, and the threshold for the error rate can be set according to the purity requirements of the final collected droplets. Alternatively, it can also issue warnings and / or stop the droplet sorting process when the number of incorrectly or incorrectly sorted droplets reaches a certain value.

[0135] The pressure monitoring unit in the droplet separator can also detect the pressure at various points in the system and transmit the pressure information to the computer system, which records and displays the pressure values. A safe pressure range can be set in the computer system; if the pressure value exceeds the range, an early warning can be issued or the droplet separator can be shut down. When the droplet separation system malfunctions, the pressure values ​​recorded by the computer system can also serve as reference data for troubleshooting.

Claims

1. A droplet sorting system, characterized in that, The system includes a droplet sorting chip, a droplet sorter, and a computer system. The droplet sorter includes a monitoring module, which in turn includes an identification unit. The identification unit collects information about the sorted droplets and transmits it to the computer system, which determines whether the droplets have been successfully sorted. The droplet sorting chip includes a droplet inlet and a cavity region. The droplet inlet allows a mixed solution to enter, and the mixed solution contains droplets. The cavity region is connected to the droplet inlet or is located downstream of the droplet inlet and is used to contain impurities. The droplet sorting chip includes an injection port for injecting a mixed solution, the mixed solution comprising droplets, and the cavity region located downstream of the injection port and capable of fluid communication with the injection port; The droplet sorting chip further includes a sieve structure located downstream of the injection port. The sieve structure is angled at one end near the injection port, and the periphery of the angle forms the cavity region. The mixed solution can enter the sieve structure through the injection port. The sieve structure includes cylinders and pores, with the cylinders arranged at intervals to form the pores. The pores allow the droplets to pass through. The cylinders and pores allow the droplets to flow out one after another at regular intervals. The cavity region is deeper than the sieve structure, with its bottom deeper and its top higher. The cavity region is located at the top and / or bottom of the chip in the depth direction. The injection port is directly connected to the cavity region, and the connection is smoothly rounded. The chip further includes a droplet inlet, a spacer oil phase inlet, a bias oil phase inlet, a high-voltage sorting electrode, a sorted droplet outlet, a waste liquid outlet, and a channel. The cavity region is located at the droplet inlet. The channel includes a sorting channel, which comprises a main channel, a target channel, a bias channel, and a waste liquid channel. The channel also includes a sorting section. The main channel, target channel, bias channel, and waste liquid channel converge at the sorting section. The main channel and bias channel are located upstream of the sorting channel, and the bias channel and waste liquid channel are located downstream of the sorting channel. The main channel is used for the passage of a mixed solution, which includes non-target droplets and a continuous phase. The bias channel is used for fluid passage and generates lateral resistance. The direction of the lateral resistance is from one side of the bias channel to one side of the main channel, preventing non-target droplets from entering the target channel after passing through the sorting section from the main channel. The vertical cross-sectional area of ​​the main channel and bias channel is smaller than the vertical cross-sectional area of ​​the target channel and waste liquid channel. A connecting branch channel is provided between the target channel and the waste liquid channel.

2. The system according to claim 1, characterized in that, The identification unit is an optical camera, which includes a CCD camera and / or a CMOS camera.

3. The system according to claim 1, characterized in that, The computer system includes a grayscale comparison program that can compare grayscale information in images captured by an optical camera and determine whether the droplets have been successfully sorted.

4. The system according to claim 1, characterized in that, The computer system includes a deep learning model, which is used to identify images containing droplet information acquired by the CCD camera and / or CMOS camera, thereby determining whether the droplets have been correctly sorted.

5. The system according to claim 1, characterized in that, The identification unit has fluorescence excitation and acquisition functions.

6. The system according to claim 1, characterized in that, The identification unit collects information on each sorted droplet and transmits it to the computer system. The computer system determines whether each sorted droplet is a correctly sorted droplet and counts the number of correctly sorted droplets and incorrectly sorted droplets.

7. The system according to claim 1, characterized in that, When the computer system determines that the sorted droplets are not correctly sorted droplets, or that the number of incorrectly sorted droplets reaches a certain number, or that the number of incorrectly sorted droplets reaches a certain proportion, it issues an early warning and / or sends a command to the droplet sorter to stop the droplet sorting process.

8. The system according to claim 1, characterized in that, The droplet sorter further includes a chip module, a fluorescence module, and an electrode driving module; the chip module is used to place the droplet sorting chip, the fluorescence module is used to excite and detect fluorescence, and the electrode driving module is used to provide high voltage to the droplet sorting chip.

9. The system according to claim 1, characterized in that, The monitoring module further includes a pressure monitoring unit and a flow rate monitoring unit. The pressure monitoring unit is used to monitor the pressure of the system, and the flow rate monitoring unit is used to monitor the flow rate of the system.

10. A method for droplet sorting using the system as described in claim 1, characterized in that, The droplet sorting instrument further includes a chip module, a fluorescence module, and an electrode driving module; the chip module is used to house the droplet sorting chip, the fluorescence module is used to excite and detect fluorescence, and the electrode driving module is used to provide high voltage to the droplet sorting chip; the droplet sorting chip includes a droplet inlet, a spacer oil phase inlet, a bias oil phase inlet, a high-voltage sorting electrode, and a channel; the channel includes a sorting channel, which consists of a main channel, a target channel, a bias channel, a waste liquid channel, and a sorting section; the main channel is used for the passage of a mixed solution, which includes non-target droplets, target droplets, and a continuous phase; The method includes the following steps: S1. Place the droplet sorting chip in the chip module and set the sorting parameters through the computer system; S2. The mixed solution enters the droplet sorting chip from the droplet inlet under the push of the sample pressure pump and forms a droplet flow arranged in sequence in the main channel; the continuous phase is injected from the interval oil phase inlet under the push of the interval oil pressure pump. The continuous phase merges with the droplet flow, further causing the droplets in the droplet flow to be arranged in sequence at a certain interval, and driving the droplet flow to continue to move forward. At the same time, the continuous phase is injected from the bias oil phase inlet and enters the bias channel. S3. When the droplet to be sorted reaches the sorting area, the fluorescence module provides excitation light to irradiate the droplet, detects the fluorescence signal, amplifies the fluorescence signal, converts it into an electrical signal, and transmits it to the computer system. The computer system determines whether the droplet to be sorted is a target droplet based on the received electrical signal and the sorting threshold set by the user. If it is a target droplet, the computer system outputs a command to the electrode drive module, which transmits high voltage to the high voltage sorting electrode to generate a non-uniform electric field. Under the action of the non-uniform electric field, a dielectric force is generated on the droplet to be sorted, causing the droplet to be sorted to deflect and flow towards the target channel. If it is a non-target droplet, the computer system determines that it is a non-target droplet and does not send a command to the electrode drive module. The droplet to be sorted flows towards the waste liquid channel. At this time, at the sorting area, the continuous phase entering from the bias channel can generate a lateral resistance that prevents the droplet to be sorted from entering the target channel, thus preventing non-target droplets from accidentally flowing into the target channel. S4. The identification unit collects information about the sorted droplets in the target channel and transmits it to the computer system. The computer system then determines whether the droplets have been successfully sorted.

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