A microfluidic chip for generating multiple particle single droplets

Abstract

This invention provides a microfluidic chip for generating various particle single droplets. By cleverly combining continuous U-shaped, V-shaped, and cross-shaped channels, and controlling the included angle of the V-shaped channels to be less than 90 degrees, and adopting a narrow constriction channel structure at the outlet end of the cross-shaped channel, the efficient, high-throughput, and high-stability preparation of single-cell single-bead (microsphere) droplets is achieved. The prepared droplets have a higher proportion of effective droplets, reducing the waste of expensive sample liquids. Moreover, the structure is simpler, more convenient and efficient, lower in cost, and easier to promote.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of microfluidic control, in particular, to a system for generating single droplets of multiple particles, and more particularly to a microfluidic chip for generating single droplets of multiple particles. BACKGROUND

[0002] Microfluidic chip (Microfluidic Chip) can integrate the basic operation units involved in the fields of chemistry and biology, such as sample preparation, reaction, separation, detection, and cell culture, sorting, and lysis. Through the design of flow channels of different shapes, different functions can be realized on the microfluidic chip. Therefore, it is also called Lab-on-a-Chip. Compared with traditional laboratories, microfluidic chips have the advantages of less reagent consumption, shorter reaction or analysis time, etc. Reducing the consumption of expensive reagents can control costs. And the shortening of time is conducive to reducing the experimental period. Combined with the chip size of square centimeter or even square millimeter, the experimental cost is greatly reduced in time and space.

[0003] The first modern "microfluidic" device can be traced back to 1975, when Terry et al. of Stanford University made the first small gas chromatograph on a silicon wafer. The key component of this instrument is a microchannel etched on a silicon wafer by microfabrication means. Its characteristics are small size and short analysis time. However, at that time, this silicon chip did not attract much attention. It was not until the late 1990s that Manz et al. proposed the concept of "micro total analysis system (micro total analysis system, μTAS)", which entered a period of rapid development. In the following 20 years, driven by the potential application prospect, the development speed of microfluidic technology has greatly accelerated. At the same time, the technical conditions for making microfluidic chips have reached a relatively mature period, providing technical support for the processing and popularization of microfluidic chips, and greatly reducing the cost of chip manufacturing.

[0004] The application field of microfluidic is very wide, and it has important application in chemistry, biology, medicine and many other fields.

[0005] In the field of modern drug development, traditional two-dimensional cell culture has gradually failed to meet the needs of drug testing in simulating the cell microenvironment. Three-dimensional cell culture using microfluidic technology has the advantages of controllability, easy observation, and more realistic simulation of cell in vivo microenvironment, which just makes up for this deficiency. It provides a new means for the study of cell physiology and pathology.

[0006] The micro-particles controlled by microfluidic technology can not only control the size, shape, monodispersity, shell thickness, and internal structure, shape and composition of the micro-particles, but also can endow the micro-particles with more diversified functions through the ingenious combination of the structure of the micro-particles and the functional components of the micro-particles, thereby providing new ideas and guidance for the design and development of new micro-particle functional materials.

[0007] In the field of chemical synthesis, traditional chemical synthesis is usually carried out in a large-volume container such as a flask or a beaker. Compared with the traditional chemical synthesis, the microfluidic chip has faster heat conduction, material diffusion and reaction process, can improve the selectivity of the reaction, and can select in micrometer level.

[0008] Meanwhile, the microfluidic technology has strong integration capability. Through reasonable design of the chip, the original work mode of cooperation of multiple instruments can be changed to integration on a small chip, and even the change of the experimental data can be directly reflected on the electronic device in real time through the data interface. The space for experiment and the time for data processing are greatly saved.

[0009] In the field of biochemistry, the dispersed droplets can be independently processed and manipulated. Each droplet can be used as an independent micro-reactor. The droplets have a large specific surface area, which is beneficial to improve the reaction rate or promote heat and mass exchange.

[0010] In the field of pharmacy, the droplets with specific properties are suitable for use as drug carriers in drug design, drug customization and various medical imaging technologies. Wearable chips can be used to provide quantitative and trace amounts of drugs to the human body. In medical imaging, various cells sealed in droplets can be imaged, detected, separated and counted.

[0011] In the field of biology, the droplets can encapsulate cells and serve as biological reactors. Cells can be encapsulated and cultured into tissues or organoids. It can also be used for cell sorting, such as sorting sperm and zygote cells, for artificial reproduction, including artificial insemination, in vitro fertilization, cloning and embryo splitting or cleavage.

[0012] It is well known that droplets are generated by a droplet generation chip, which has a flow channel intersection, i.e. a droplet generation site. The continuous phase and the dispersed phase enter the droplet generation site from different directions, and under the action of shear force, the continuous phase cuts the dispersed phase into droplets with uniform size. The size and frequency of the droplets are mainly determined by the geometry of the flow channel, the flow rate and the physical properties of the fluid, such as viscosity and surface tension. The existing methods for generating droplets include the use of electric field, thermal energy, pneumatic, acoustic control and magnetic field. The diameter of the droplets can be adjusted by changing the flow rate or applying pressure to the channel.

[0013] Multiple particle single droplet refers to containing two or more particles in each droplet generated. In order to meet the needs of biochemical experiments, there are devices and schemes for realizing multiple particle droplets as micro-reactors in the prior art. For example, the double particle droplet microfluidic chip involved in Dropseq and 10x (Fabrication of robust PDMS micro-structure with hydrophobic and antifouling properties), but these devices and schemes generally have problems such as high time cost, low effective flux or low effective droplet proportion.

[0014] Dropseq uses a double-cross flow channel to prepare double-particle droplets, and the proportion of effective droplets generated by the chip is only 0.1% of the total number of droplets. It takes 1 hour or more to collect droplets that meet the needs of a biochemical experiment, and the number of collected droplets is about 10 6 The above droplets, in which the effective droplets are only about 10 3 , are a waste of time and reagent cost.

[0015] The chip designed and manufactured by 10x company is said to have an effective droplet proportion of more than 65%, which is obtained by excluding all empty droplets, and is a result obtained by bioinformatics analysis of human and mouse cell mixtures. The actual proportion of effective droplets to total droplets is much lower than this value. In order to achieve a high single cell rate, a high bead concentration and a low cell concentration method is used, which results in a large amount of beads and oil being wasted.

[0016] In the process of generating multiple particle single droplets, a large number of invalid droplets are usually generated, so a droplet sorting system needs to be set up, which can sort out effective droplets from a large number of invalid droplets. The sorting system currently used is usually an AI cell sorting method based on image signal recognition. The image at the sorting site is collected by a high-speed camera and sent to a computer in real time for processing. The image size is usually about 1280x400, 8-bit grayscale image, and the size of each picture is 1280x400x8=4.1M. According to the frame rate of 500fps, 2.05GB of data needs to be transmitted per second. Limited by computer performance, the speed of AI processing pictures cannot be so fast. And when performing image signal recognition, multiple frames of pictures of the same object need to be transmitted to the computer for processing, so the flow rate of the object in the microfluidic channel is greatly limited.

[0017] The cell sorting based on microflow usually needs to exert external force on the target droplet at a proper position to make it enter the collection flow channel, and the implementation of the method is premised on that the droplet will flow to the waste liquid flow channel under the action of pressure in the natural condition. In addition to the structural design of the flow channel, the pressure difference between the waste liquid outlet and the collection outlet conduit also affects the flow direction. In the case of high flow rate, i.e. high liquid pressure in the flow channel, the pressure difference between the two outlets can be basically ignored. However, in the case of low flow rate, i.e. low liquid pressure in the flow channel, the pressure difference between the two outlets will have a very obvious influence on the flow direction of the droplet. If there is slight disturbance between the two outlets, the droplet will change the flow direction, which will greatly destroy the stability of the experiment.

[0018] Therefore, it is urgent to find a multi-particle single droplet generating device with simple structure, higher effective droplet proportion and lower cost, and a stable droplet sorting system under the condition of low flow rate in the flow channel, so as to be used for the preparation and sorting of multi-particle single droplets. SUMMARY

[0019] To solve the above problems, the application provides a microfluidic chip for generating multi-particle single droplets. Through the creative structural design, the continuous U-shaped flow channel, the V-shaped flow channel and the cross-shaped flow channel are ingeniously combined to complete the high-efficiency, high-throughput and high-stability preparation of single-cell single-beads (microspheres) droplets. The effective droplet proportion in the prepared droplets is higher, the waste of expensive sample liquid is reduced, the structure is simpler, the cost is lower, the high-quality and high-efficiency production of multi-particle single droplets is realized, and the application is suitable for industrial scale production.

[0020] Meanwhile, the application also provides a high-stability droplet sorting system. The flow channel width of the identification area is widened, and the liquid pressure in the flow channel is increased, so that the liquid pressure in the flow channel is increased without affecting the flow rate and spacing of the target object, the influence of the pressure difference between the outlets on the sorting system is reduced, the anti-interference performance is improved, the structure of the microfluidic chip is optimized, and the stability problem of the sorting system under the condition of low flow rate of the target object is solved.

[0021] The multi-particle single droplet of the application refers to that each internal droplet prepared contains multiple particles. In biochemical experiments, this kind of multi-particle single droplet is often used as a biological reactor. For example, in single-cell sequencing, a double-particle single droplet containing one single cell and one bead (microsphere) with primers is used to complete the sequencing in the droplet reaction chamber.

[0022] Effective droplet refers to that each kind of particle is contained in one single droplet.

[0023] In one aspect, the application provides a system for generating multiple particle single droplets, which comprises a V-shaped flow channel and a cross-shaped flow channel, the V-shaped flow channel is used for mixing particles, and the cross-shaped flow channel is used for droplet generation.

[0024] In some ways, the mixing of particles refers to that different kinds of particles are mixed together after flowing into the V-shaped flow channel through different flow channels.

[0025] In some ways, the droplet generation refers to that multiple particles and other reagents are encapsulated into droplets.

[0026] The double cross-shaped flow channel of Dropseq is designed based on the traditional double-layer wrapped droplet flow channel, and the main function is to generate W / O / W or O / W / O double-layer wrapped droplets by modifying the hydrophilicity and hydrophobicity of the two cross-shaped parts. However, this function does not match the purpose of generating the same-phase double-particle single-layer wrapped droplets (containing cells and microspheres in the water phase) required by Dropseq. In the actual use process, it can be obviously seen that the flow rate and proportion of the two components of the water phase are difficult to control and not intuitive in the process of generating droplets by the double cross-shaped flow channel, the external pressure required for the supply of the two components is quite different, and the stability of the generated droplets is poor.

[0027] Compared with the double cross-shaped flow channel of Dropseq, the V-shaped flow channel and the cross-shaped flow channel are combined to prepare multiple particle single droplets in the application. The V-shaped flow channel and the cross-shaped flow channel are designed, so that the flow channel environment of the two particle components in the water phase is completely consistent, and the proportion and flow rate of the two components can be intuitively displayed at the V-shaped flow channel convergence point before the cross-shaped flow channel. In the experimental test, the external pressure supplied is basically consistent, the stability of the generated droplets is obviously better than that of the double cross-shaped flow channel, and the proportion of the generated effective droplets is also higher. It is a more suitable flow channel design for generating multiple particle droplets.

[0028] Further, the system further comprises a continuous U-shaped flow channel, and the continuous U-shaped flow channel is used for arranging particles.

[0029] The arrangement of particles here refers to arranging particles in a single flow line in a particle suspension, which is used for arranging particles in a liquid phase to improve the effective single droplet rate, also known as sample focusing. It is an important link in cell counting, detection and separation chips and other devices, and directly affects the accuracy and efficiency of subsequent detection and sorting. It has important significance in the field of medical detection. The existing fluid focusing technology is mainly divided into two kinds of sheath liquid pinch flow focusing and non-sheath flow focusing.

[0030] Sheath flow focusing technology refers to adjusting the flow rate of one or more sheath flows to squeeze the particle flow containing the sample into a thin stream, which is realized by pressure driving. However, sheath flow focusing technology requires complex equipment control and the introduction of sheath flow, which not only increases the operation difficulty but also reduces the sample density.

[0031] Non-sheath flow focusing refers to using external force to laterally manipulate the suspended particles to the equilibrium position, such as applying inertial force or fluid hydrodynamic force. Carlo et al. designed a microchannel with a symmetrical sinusoidal structure, which uses the action of inertial force to focus the particles to the equilibrium position to become a single flow line. The method of non-sheath flow focusing is simple to use and does not require any additional instruments, which does not affect the activity and density of biological particles, and can realize high-throughput focusing, and can be widely used in particle separation and detection. The non-sheath flow focusing (inertial focusing) adopted by the present application realizes the single flow line arrangement of particles through a continuous U-shaped flow channel.

[0032] The continuous U-shaped flow channel can include multiple U-shaped flow channels (each U-shaped flow channel can constitute a complete U-shaped flow channel) connected to each other, and in actual use, a proper number of U-shaped flow channels can be selected to be connected in series as needed. Generally, 3-4 U-shaped flow channels are combined into a continuous U-shaped flow channel, which is used for the arrangement of a kind of particles, but this does not limit the number of U-shaped flow channels in the present application. It can be understood that any number of U-shaped flow channels can be selected to be connected in series to form a continuous U-shaped flow channel, which is within the protection scope of the present application.

[0033] The continuous U-shaped flow channel, the V-shaped flow channel and the cross-shaped flow channel are combined together in the present application, which realizes the efficient, high-throughput and high-stability preparation of multi-particle droplets. The effective droplet proportion in the prepared droplets is higher, the waste of expensive sample liquid is reduced, and the structure is simpler and the cost is lower.

[0034] Further, the number of continuous U-shaped flow channels is at least two, and each continuous U-shaped flow channel is provided with an inlet of a particle suspension.

[0035] According to the number of particle types contained in the prepared droplets, a corresponding number of continuous U-shaped flow channels can be provided for the arrangement of each type of particle. In some modes, the number of continuous U-shaped flow channels can be 2, 3, 4, 5, 6, 7, etc. If three types of particle single droplets are prepared, three corresponding continuous U-shaped flow channels are required to arrange the three types of particles respectively.

[0036] Further, the V-shaped flow channel includes at least two straight flow channels, and each straight flow channel is in communication with a continuous U-shaped flow channel.

[0037] In some ways, the V-shaped flow channel can also be composed of multiple V-shaped structures, and the number of straight-line flow channels in the V-shaped flow channel needs to be consistent with the number of continuous U-shaped flow channels, which can be 2, 3, 4, 5, 6, 7, etc., so that each straight-line flow channel receives the particle suspension flowing out of the corresponding continuous U-shaped flow channel.

[0038] Further, the angle between the two adjacent straight-line flow channels in the V-shaped flow channel is less than 90 degrees; the V-shaped flow channel includes two straight-line flow channels.

[0039] A large number of studies have shown that the angle between any two adjacent straight-line flow channels in the V-shaped flow channel needs to be less than 90 degrees, because if it is greater than 90 degrees, droplet generation will become difficult, the distance between the generated droplets will be too large and the frequency will be low.

[0040] Further, the angle between the two adjacent straight-line flow channels in the V-shaped flow channel is 30 degrees.

[0041] Further, the V-shaped flow channel includes a first end, a second end and a third end, wherein the first end and the second end are the inlet ends of the V-shaped flow channel, and the third end is the outlet end of the V-shaped flow channel; the third end is connected with the cross-shaped flow channel.

[0042] Further, the number of continuous U-shaped flow channels is two, which are a first continuous U-shaped flow channel and a second continuous U-shaped flow channel; one end of the first continuous U-shaped flow channel is provided with a first particle inlet, and the other end is connected with the first end of the V-shaped flow channel; one end of the second continuous U-shaped flow channel is provided with a second particle inlet, and the other end is connected with the second end of the V-shaped flow channel.

[0043] Further, the cross-shaped flow channel includes a first end, a second end, a third end and a fourth end (narrowed), the first end, the second end and the third end are the inlet ends of the cross-shaped flow channel, and the fourth end (narrowed) is the outlet end of the cross-shaped flow channel; the third end is connected with the outlet end of the V-shaped flow channel.

[0044] Further, the size of the fourth end (narrowed) of the cross-shaped flow channel is smaller than the size of other positions of the flow channel.

[0045] A large number of experiments have shown that when the fourth end (narrowed) of the cross-shaped flow channel adopts the flow channel structure of narrow narrowing, the distance between the prepared droplets can be obviously shortened, and smaller droplets can be generated faster, and the reason may be that because the generation of droplets is caused by the extrusion of oil phase on both sides, which causes the water phase in the middle to be unable to pass, the pressure gradually increases, and after reaching a threshold, the water phase will be extruded and release pressure, and then continue to be extruded by the oil phase, cycle, generate droplets, the port is narrowed, the water phase pressure will increase faster, and the pressure released each time will be smaller, so smaller droplets will be generated faster, and the distance will be smaller.

[0046] In some embodiments, the cross section of all flow channels of the system for generating a plurality of single particle droplets is rectangular. It is found that narrowing the width of the flow channel can shorten the distance between the prepared droplets and generate smaller droplets faster. However, since the preparation of flow channels with different heights is complicated, the width of the fourth end (narrowing) of the cross-shaped flow channel can be narrowed, and the height can remain unchanged.

[0047] In some embodiments, the width of the flow channel of the system for generating a plurality of single particle droplets is 125 microns, and the height is 100 microns. However, the width of the fourth end (narrowing) of the cross-shaped flow channel is narrowed to 40 microns, and the height remains 100 microns.

[0048] Further, the system further comprises an oil phase inlet, when the oil phase flows from the oil phase inlet, it is divided into two paths, and flows along the first oil phase flow channel and the second oil phase flow channel respectively; the first oil phase flow channel is connected with the first end of the cross-shaped flow channel, and the second oil phase flow channel is connected with the second end of the cross-shaped flow channel.

[0049] The above-mentioned microfluidic flow channel design provided by the present application can also be used in combination with other microfluidic structures.

[0050] Further, the system for generating a plurality of single particle droplets provided by the present application can further comprise a droplet sorting system, wherein the main flow channel has a width greater than that of other regions when located in the sorting region.

[0051] In some embodiments, the droplet sorting system can be connected to other droplet generation systems independently.

[0052] The greater the fluid pressure in the flow channel of the microfluidic chip, the stronger the anti-interference of the entire system. However, if the fluid pressure is increased to improve the anti-interference, the droplet flow rate will be too fast, which will bring great difficulty to the sorting of effective droplets. Therefore, for a microfluidic chip containing a droplet sorting system, the anti-interference cannot be improved simply by increasing the pressure of the flow channel.

[0053] The present application increases the width of the flow channel in the sorting region, so that the flow rate of the droplets in the flow channel remains unchanged in the case of increasing the liquid pressure, and the sorting can be successfully completed. The liquid pressure can be maintained, the anti-interference can be improved, the influence of the pressure difference between the outlets of the two sorting branch flow channels can be avoided, the stability of the sorting system can be maintained, and the sorting result is more accurate.

[0054] Further, the width of the main flow channel in the sorting region is 1.5 to 2 times the width of other regions.

[0055] In some embodiments, the degree to which the width of the main flow channel increases in the sorting region can be adjusted according to the hydraulic pressure and the flow rate of the droplets in the flow channel. Generally, the higher the hydraulic pressure, the stronger the anti-interference ability, but the hydraulic pressure cannot be increased indefinitely, and too high a hydraulic pressure can cause the flow rate to be too fast or the flow channel to crack under pressure. Therefore, the hydraulic pressure needs to be maintained within an appropriate range.

[0056] At the same time, the width cannot be increased indefinitely, and the greater the width, the slower the flow rate, which can also cause the spacing between the droplets to become narrower.

[0057] In some embodiments, the hydraulic pressure in the flow channel cannot exceed 500 mbar.

[0058] The sorting system provided in the present application increases the hydraulic pressure and the width of the flow channel in the sorting region, so that the width of the main flow channel in the sorting region is 1.5-2 times the width of the other regions, thereby maintaining the flow rate in the sorting region within a range suitable for sorting.

[0059] In some embodiments, the image acquisition speed suitable for sorting is about 200 frames per second, and 10-50 droplets pass through the sorting region per second, and each droplet flows in the widened region for about 4-6 frames.

[0060] Further, the main flow channel comprises a first end connected to the outlet end of the system for generating droplets and a second end connected to the branch flow channel after sorting is completed.

[0061] Further, the main flow channel is further provided with an oil phase inlet for introducing an oil phase to increase the spacing between the droplets.

[0062] The introduction of the oil phase can widen the spacing between the droplets, thereby providing further protection for maintaining accurate sorting.

[0063] Further, the width of the main flow channel after the oil phase inlet is greater than the width before the oil phase inlet.

[0064] Since the introduction of the oil phase can accelerate the flow channel due to the sudden increase in fluid, increasing the width after the oil phase inlet can eliminate the acceleration of the fluid and stabilize the flow rate.

[0065] Further, the width after the oil phase inlet is 1.5-2 times the width before the oil phase inlet, and the width of the main flow channel in the sorting region is 1.5-2 times the width after the oil phase inlet.

[0066] With the increase in width, the flow rate slows down, but the width cannot be increased indefinitely, because the greater the width, the slower the flow rate, which can also cause the spacing between the droplets to become narrower, and at this time, the pressure required to adjust the introduced oil phase to the original spacing increases, so the width also needs to be controlled within 2 times the width after the oil phase inlet.

[0067] Further, the main flow channel has a width of 125 microns before the oil phase inlet, a width of 200 microns after the oil phase inlet, and a width of 300 microns in the sorting region.

[0068] Further, the sorting region comprises a dielectrophoresis sorting mechanism, and the main flow channel and the branch flow channel have a constant depth.

[0069] In some embodiments, the width of the flow channel is changed to achieve stable flow rate during hydraulic pressure increase, and the main flow channel and the branch flow channel can have a constant depth, thereby facilitating the production of the microfluidic chip.

[0070] Further, the system for generating droplets is a system for generating single-cell droplets.

[0071] Further, the branch flow channel after sorting comprises an effective droplet flow channel and an ineffective droplet flow channel.

[0072] In some embodiments, the system for generating single-cell droplets is a system for generating single-cell single-bead double-particle droplets, which means that each prepared droplet contains multiple particles. In biochemical experiments, such multiple-particle single-droplet is often used as a biological reactor. For example, in single-cell sequencing, a double-particle single-droplet containing one single cell and one bead (microsphere) with primers is used to perform the reaction in the droplet reaction chamber to complete the sequencing.

[0073] An effective droplet refers to a multiple-particle single-droplet that contains exactly one particle of each type. For example, for the double-particle single-droplet used in single-cell sequencing, an effective droplet refers to a double-particle single-droplet that contains exactly one single cell and one bead. An ineffective droplet refers to a droplet that does not contain the desired multiple particles, or contains an incorrect number of particles, or the particles are prematurely lysed, etc.

[0074] The droplet sorting system provided by the application can be adapted to various flow channel designs without the need to change other architectures.

[0075] In some embodiments, the microfluidic flow channel design of the droplet sorting system provided by the application can also be used in combination with other microfluidic structures.

[0076] In another aspect, the application provides a microfluidic chip prepared by using the system for generating multiple-particle single-droplets.

[0077] Common microfluidic chip materials include PDMS, PMMA, PS, and other polymers. For PDMS, the common processing method is to use a photolithographic template to bond a microfluidic chip. For PMMA and other hard polymers, common processing methods include CNC, laser processing, mold injection, etc.

[0078] In some ways, the microfluidic chip provided by the application is sutured and sealed by PDMS (polydimethylsiloxane) with a concave pattern for forming a flow channel and flat corning glass.

[0079] The microfluidic chip for generating single droplets of multiple particles provided by the application has the following beneficial effects:

[0080] 1. Creatively using the combination of V-shaped flow channels and cross-shaped flow channels for preparing single droplets of multiple particles, compared with the double cross-shaped flow channels of Dropseq, the flow channel environment of the two particle components in the aqueous phase is completely consistent, and the ratio and flow rate of the two components can be intuitively displayed at the V-shaped flow channel junction before the cross, the stability of the generated droplets is obviously better than that of the double cross-shaped flow channels, and the effective droplet ratio is also higher, which is more than 10 times higher than that of Dropseq;

[0081] 2. By controlling the included angle between any two adjacent straight flow channels in the V-shaped flow channel to be less than 90 degrees, preferably 30 degrees, the frequency and spacing of the droplets are ensured, and effective droplets are more efficiently generated;

[0082] 3. By using continuous U-shaped flow channels to realize the single flow line arrangement of particles, and ingeniously combining the continuous U-shaped flow channels with the V-shaped flow channels and the cross-shaped flow channels, high-efficiency, high-throughput and high-stability preparation of multiple particle droplets is realized, the effective droplet ratio in the prepared droplets is higher, the loss of expensive sample liquid is reduced, the flexibility is better, and the structure is simpler;

[0083] 4. The narrow necked flow channel structure at the outlet end of the cross-shaped flow channel obviously shortens the spacing between the prepared droplets, and can generate smaller droplets faster, improving efficiency;

[0084] 5. The high-stability sorting system provided can maintain the flow rate in the identification area within a suitable range by widening the flow channel width of the sorting area when increasing the liquid pressure in the flow channel to improve the anti-interference performance, thereby maintaining the stability of the sorting system and making the sorting result more accurate;

[0085] 6. The high-stability sorting system provided can improve the anti-interference performance by increasing the liquid pressure in the flow channel to reduce the influence of the pressure difference between the outlets on the sorting system;

[0086] 7. The high-stability sorting system provided solves the stability problem of the sorting system at a low flow rate of the target object;

[0087] 8. The high-stability sorting system provided can adjust the flow channel width of the sorting area to change the pressure according to the demand

[0088] 9. The high-stability sorting system provided can be adapted to various flow channel designs without changing other architectures;

[0089] 10. The structure is simple, convenient, efficient, low in cost and easy to popularize. BRIEF DESCRIPTION OF DRAWINGS

[0090] Figure 1 A schematic diagram of the system for generating single droplets of multiple particles of Example 1;

[0091] Figure 2 A comparison diagram of the width of the fourth end (narrowing) of the cross-shaped flow channel of Example 4, wherein the left drawing has a width of 40 microns and the right drawing has a width of 100 microns;

[0092] Figure 3 A schematic diagram of the microfluidic chip for generating double-particle single droplets of Example 5;

[0093] Figure 4 A schematic diagram of the microfluidic chip for generating double-particle single droplets of Example 5;

[0094] Figure 5 A schematic diagram of the high-stability droplet sorting system in Example 6;

[0095] Figure 6 A schematic diagram of the high-stability droplet sorting system connected to the single-droplet system for generating single-cell single-beads in Example 6;

[0096] Figure 7 A droplet sorting system with a constant sorting region width in Example 7. DETAILED DESCRIPTION

[0097] The preferred embodiments of the present application will be further described in detail below with reference to the drawings, and it should be noted that the following embodiments are intended to facilitate the understanding of the present application and do not limit the present application in any way. The raw materials and equipment used in the specific embodiments of the present application are known products, which can be obtained by purchasing commercially available products.

[0098] Example 1 The system for generating single droplets of multiple particles provided by the present application

[0099] A schematic diagram of the system for generating single droplets of multiple particles provided by the present application is shown in Figure 1As shown, it includes a V-shaped flow channel 1, a cross-shaped flow channel 2, and a continuous U-shaped flow channel 3. The V-shaped flow channel 1 is used for particle mixing; different types of particles flow into the V-shaped flow channel through different channels and then mix together. The cross-shaped flow channel 2 is used for droplet generation, where multiple particles and other reagents can be encapsulated into droplets. The continuous U-shaped flow channel 3 can arrange the particles in the particle suspension into a single streamline, thereby improving the effective single droplet rate. The particle arrangement mentioned here refers to the inertial focusing effect of the continuous U-shaped flow channel, which arranges the particles in the liquid phase into rows, reducing the phenomenon of multiple identical particles being encapsulated in a droplet, and effectively improving the single-particle droplet rate at high concentrations.

[0100] The continuous U-shaped flow channel 3 can contain multiple U-shaped flow channels 4. Each U-shaped flow channel 4 can form a complete U-shape and be interconnected. In specific applications, an appropriate number of U-shaped flow channels 4 can be selected and connected end to end as needed. In this embodiment, four U-shaped flow channels 4 are combined to form a continuous U-shaped flow channel 3 for particle arrangement. In this embodiment, there are two continuous U-shaped flow channels 3, namely a first continuous U-shaped flow channel 11 and a second continuous U-shaped flow channel 12. One end of the first continuous U-shaped flow channel 11 is provided with a first particle inlet 9, and the other end is connected to the first end 6 of the V-shaped flow channel 1. One end of the second continuous U-shaped flow channel 12 is provided with a second particle inlet 10, and the other end is connected to the second end 7 of the V-shaped flow channel 1. The flow design provided in this embodiment is suitable for the generation of dual-particle single droplets.

[0101] The V-shaped flow channel 1 includes at least two straight flow channels 5, each of which is connected to a continuous U-shaped flow channel 3. The V-shaped flow channel 1 can also be composed of multiple V-shapes. The number of straight flow channels 5 in the V-shaped flow channel 1 must be consistent with the number of continuous U-shaped flow channels 3, so that each straight flow channel 5 receives the particle suspension flowing out from the corresponding continuous U-shaped flow channel 3. In this embodiment, the V-shaped flow channel 1 includes two straight flow channels 5, and the included angle 21 between the two straight flow channels 5 is less than 90 degrees. This is because if it is greater than 90 degrees, droplet generation will be more difficult, thus affecting the accuracy of subsequent experiments. The V-shaped flow channel 1 includes a first end 6, a second end 7, and a third end 8, where the first end 6 and the second end 7 are the inlet ends of the V-shaped flow channel 1, and the third end 8 is the outlet end of the V-shaped flow channel 1; the third end 8 is connected to the cross-shaped flow channel 2.

[0102] The cross-shaped flow channel 2 includes end I 13, end II 14, end III 15, end IV (constriction) 16 and droplet outlet end 17. End I 13, end II 14 and end III 15 are the inlet ends of the cross-shaped flow channel, and end IV (constriction) 16 is the outlet end of the cross-shaped flow channel; end III is connected to the outlet end of the V-shaped flow channel.

[0103] Preferably, the dimension of the fourth end (narrowing) 16 of the cross-shaped flow channel 2 is smaller than the dimensions of other parts of the flow channel. When the fourth end (narrowing) of the cross-shaped flow channel adopts a narrow-narrowing flow channel structure, the spacing between the prepared droplets can be significantly shortened, and smaller droplets can be generated more quickly. The reason may be that droplet generation is due to the oil phase on both sides squeezing, which prevents the water phase in the middle from passing through. The pressure gradually increases, and after reaching a threshold, the water phase will be squeezed out and release pressure, and then continue to be squeezed by the oil phase, cycling and generating droplets. When this opening is narrowed, the water phase pressure increases more quickly, and the pressure released each time is also smaller, so smaller droplets are generated more quickly and the spacing is smaller. In this embodiment, the cross-section of all the flow channels in the system for generating single droplets of various particles is rectangular, with a channel width of 125 micrometers and a height of 100 micrometers. However, at the fourth end (narrowing) 16 of the cross-shaped flow channel, the width of the flow channel is narrowed to 40 micrometers, while the height remains at 100 micrometers.

[0104] Preferably, the system for generating dual-particle single droplets provided in this embodiment further includes an oil phase inlet 18. When the oil phase flows in from the oil phase inlet 18, it is divided into two paths and flows along the first oil phase channel 19 and the second oil phase channel 20, respectively. The first oil phase channel 19 is connected to the first end 13 of the cross-shaped channel 2, and the second oil phase channel 20 is connected to the second end 14 of the cross-shaped channel 2.

[0105] Example 2: Fabrication and Use of a Microfluidic Chip for the Generation of Two-Particle Single Droplets

[0106] This embodiment completes the fabrication of a microfluidic chip according to the flow channel design provided in Embodiment 1, and includes the following steps:

[0107] A. Silicon wafer motherboard fabrication

[0108] The fabrication of silicon wafer master plates can be divided into seven steps: cleaning, homogenization, pre-baking, UV lithography, post-baking, settling, and development.

[0109] (1) Cleaning

[0110] To prevent air bubbles and particles from forming on the adhesive layer after the spin coating process, the cleanliness of the silicon wafer surface is crucial for the adhesion between the adhesive layer and the wafer. A plasma adhesive remover is used to clean the silicon wafer surface. This process utilizes active components in the plasma, such as ions, electrons, active groups, and excited-state nuclides, to treat the silicon wafer surface, thereby achieving the cleaning purpose.

[0111] (2) Spin coating

[0112] Apply SU-8 adhesive evenly to the polished surface of the silicon wafer and complete the spin coating process using a spin coater. Select the appropriate spin coating program based on the desired adhesive layer thickness. During operation, the spin coater uses a vacuum to hold the silicon wafer in place via the grooves of the support frame. Therefore, the back of the silicon wafer must be kept flat during spin coating; otherwise, insufficient vacuum may cause the wafer to fly off the platform, damaging the instrument and materials. During normal operation, the support frame holds the silicon wafer and rotates at high speed, using centrifugal force to evenly coat the wafer with adhesive.

[0113] After the program stops normally, remove the silicon wafer and let it stand for more than 1 hour. The standing process allows the adhesive layer to become smoother.

[0114] During this period, clean the spin coater, replace the spin coater foil, and prepare the materials for the next experiment.

[0115] (3) Pre-baking

[0116] The purpose of pre-baking is to solidify the SU-8 adhesive, evaporate the solvent, increase the concentration of the photoinitiator, and ensure a more thorough optical-chemical reaction during exposure. Additionally, due to the self-leveling properties of SU-8, pre-baking results in a smoother adhesive layer, facilitating subsequent experiments. Insufficient pre-baking time leads to incomplete SU-8 adhesive solidification, causing adhesion to the photomask and contaminating both the silicon wafer and the photomask. Insufficient solvent evaporation results in a low photoinitiator concentration and insufficient acid catalyst generated after exposure, leading to incomplete cross-linking during post-baking and ultimately incomplete etched patterns. Excessive pre-baking time results in excessive solvent evaporation, an excessively high photoinitiator concentration, and an overabundance of acid catalyst after exposure, causing the adhesive layer to detach from the silicon wafer after development.

[0117] (4) Ultraviolet lithography

[0118] Ultraviolet (UV) lithography is the most crucial step in creating the silicon wafer master. Using a UV lithography machine, the pre-baked silicon wafer is exposed to near-ultraviolet light generated by a mercury lamp. This light is absorbed by the photoresist, producing a strong acid that acts as a photocatalyst for the cross-linking reaction of the SU-8 photoresist during post-baking. The exposure time depends on various factors, including the thickness of the photoresist layer, the energy output of the mercury lamp, and the type of photoresist used.

[0119] (5) Post-baking

[0120] After UV lithography, the silicon wafer is baked on a temperature-controlled stage for a period of time. The purpose of post-baking is to allow the photoresist in the exposed areas to undergo a full cross-linking reaction. The photoresist in the shaded areas will not react because there is no acid catalyst; therefore, the solubility of the photoresist in the developer differs between the exposed and shaded areas after post-baking. After post-baking, a clear flow channel pattern should be observed on the surface of the silicon wafer.

[0121] (6) Let stand

[0122] The silicon wafer is left to stand in the air to stabilize the flow channel pattern.

[0123] (7) Development

[0124] The developer must be selected based on the type of photoresist used, with the selection criterion being a significant difference in the solubility of the photoresist before and after exposure in the developer. The developer used for this chip is PGMEA (Propylene glycol monomethyl ether acetate). After the cross-linking reaction, the epoxy groups in the SU-8 photoresist form a network structure, increasing its molecular weight and resulting in very low solubility in PGMEA. In contrast, SU-8 photoresist that has not undergone the cross-linking reaction can dissolve significantly in PGMEA. Therefore, rinsing the silicon wafer with the developer leaves a fine flow channel pattern on the wafer. The development time must be strictly controlled. If the time is too short, the unexposed areas will not dissolve completely, leaving unwanted residue; if the time is too long, the exposed areas will dissolve, damaging the integrity of the flow channel structure.

[0125] Because the flow channels fabricated by the SU-8 have a high aspect ratio, material transfer is restricted, which can easily lead to overdevelopment of the surface layer, resulting in pattern erosion, while the bottom layer is not fully developed, leaving residual adhesive. To solve these problems, the culture dish needs to be continuously agitated during the development process to ensure PGMEA flow, accelerate the transfer of fresh PGMEA to the bottom of the layer, and speed up the removal of residual adhesive.

[0126] After development with the developer, a fixer is needed to fix the solution. The purpose of fixing is to remove residual developer to avoid damaging the developed flow channels. Isopropanol was used as the fixer in this experiment. For the same reason, the petri dish also needs to be continuously shaken during fixing to ensure complete removal of the developer.

[0127] After fixing, the image can be heated at a suitable temperature for a period of time to stabilize it.

[0128] Once all the above steps are completed, you will obtain the finished silicon wafer master, which should be properly stored for subsequent production.

[0129] B. Microfluidic chip fabrication

[0130] (1) Mixing

[0131] Weigh out an appropriate amount of PDMS main agent and mix it evenly with the curing agent (the common ratio is 10:1).

[0132] (2) Defoaming

[0133] Place the mixed PDMS into a vacuum oven, evacuate and let it stand to remove air bubbles (if there are still air bubbles on the surface after standing, they can be blown away with a blower).

[0134] (3) Molding

[0135] Use tape to surround the edge of the silicon wafer template, pour in an appropriate amount of defoamed PDMS, and bake at 80℃ for more than 1.5 hours.

[0136] (4) Drilling holes

[0137] Remove the cured PDMS, drill holes at the designed locations, and cut the chip according to the designed structure.

[0138] (5) Bonding

[0139] Place the PDMS and glass substrate to be bonded into the plasma surface treatment machine with the bonding side facing up, start the preset program, and after the program is completed, quickly align and bond the chip, gently extruding large air bubbles.

[0140] (6) Baking

[0141] Bake at approximately 80℃ to restore the hydrophobicity of PDMS. After baking, the desired chip is obtained. When not in use, store in a sealed container to prevent dust accumulation.

[0142] The fabricated microfluidic chip was used to generate a dual-particle single droplet:

[0143] 1. Prepare reagent 1 (the specific components depend on the experimental requirements; in this example, it is a cell suspension, and the components are: T2 cells), reagent 2 (the specific components depend on the experimental requirements; in this example, it is a bead suspension, and the components are: PMMA microspheres), and oil phase (fluorinated oil containing surfactants);

[0144] 2. Place each reagent into a 15ml centrifuge tube (use a syringe if the volume is small), and connect the air pump. After expelling air from the tubing, connect it to the chip inlet;

[0145] 3. Oil phase inlet 17 connects to the oil phase;

[0146] 4. The first particle inlet 9 connects to the cell suspension;

[0147] 5. The second particle inlet 10 is connected to the bead suspension;

[0148] 6. The droplet outlet end 17 is connected to the collection tube;

[0149] 7. Turn on the air pump switch and adjust it until it generates droplets of appropriate size, and the flow rate ratio of reagent 1 and reagent 2 meets the requirements.

[0150] Example 3: Effect of the angle of the V-shaped flow channel on the preparation of various particle single droplets

[0151] In this embodiment, a microfluidic chip with dual particles and single droplets was designed and generated according to Embodiment 2. The included angles between the two straight channels in the V-shaped channel are 120 degrees, 90 degrees, 60 degrees and 30 degrees respectively. Under high-speed photography, it was found that the channels with 120 degrees, 90 degrees and 60 degrees were more difficult to adjust the droplet generation than the channel with 30 degrees. Moreover, the generated droplets had a lower frequency and larger spacing. Therefore, the preferred included angle is 30 degrees. The results are shown in Table 1.

[0152] Table 1. Effect of the angle of the V-shaped flow channel on the preparation of various particle single droplets

[0153] Angle (degrees) Drop frequency (Hz) Drop spacing (pm) Ease of generation Proportion of effective drops (%) 30 70 600 Easy 10.92 60 52 3024 Generally 9.84 90 48 4219 Generally 9.02 120 30 5282 More difficult 8.53

[0154] As shown in Table 1, the angle significantly affects the frequency, spacing, ease of droplet generation, and effective droplet ratio. At 120 degrees, droplet generation is noticeably more difficult, with a lower droplet frequency, larger spacing, and a lower effective droplet ratio. This is likely because a larger angle increases the likelihood of head-on collisions between cells and beads, thus affecting effective droplet generation. As the angle decreases, the droplet generation frequency gradually increases, the spacing gradually decreases, and droplet generation becomes easier, while also increasing the effective droplet ratio. However, considering that excessively small angles can negatively impact processing accuracy and chip stability, and are difficult to fabricate, 30 degrees is optimally chosen as the V-channel angle due to its ease of processing, high droplet frequency, and small spacing.

[0155] Example 4: Effect of narrowing at the outlet end of the cross-shaped flow channel

[0156] This embodiment designs and generates a microfluidic chip with two particles and a single droplet according to Embodiment 2. The width of the fourth end (narrowing) of the cross-shaped channel is 125 μm, 100 μm, 60 μm, and 40 μm, respectively. The effect of the narrowing at the outlet end of the cross-shaped channel on the generated droplet is investigated. The results are shown in Table 2, with a comparison graph showing the widths of 100 μm and 40 μm. Figure 2 As shown, the width of the left image is 40 micrometers, and the width of the right image is 100 micrometers.

[0157] Table 2. Effect of narrowing at the outlet end of the cross-shaped flow channel on the preparation of various particle single droplets

[0158] Outlet width (pm) Drop diameter (pm) Generation drop frequency (Hz) Proportion of effective drops (%) 125 108 10 10.93 100 94 18 10.77 60 49 33 10.82 40 28 52 10.81

[0159] As shown in Table 2, the width of the fourth end (narrowing) of the cross-shaped flow channel has a significant impact on the diameter, velocity, and effective droplet ratio of the generated double-particle single droplets. When the width remains unchanged at 125 micrometers, the generated droplet diameter is large, and the generated droplet velocity is slow, resulting in low efficiency. As the width gradually narrows, the generated droplet diameter gradually decreases, and the generated droplet velocity gradually increases. This is because droplet generation is caused by the compression of the oil phase on both sides, which prevents the water phase in the middle from passing through. The pressure gradually increases, and after reaching a threshold, the water phase will be squeezed out and release pressure, and then continue to be squeezed by the oil phase, cycling and generating droplets. When the opening narrows, the water phase pressure increases more rapidly, and the pressure released each time also decreases, so smaller droplets are generated more quickly with smaller spacing.

[0160] from Figure 2 It can be seen more clearly and intuitively that when the width of the fourth end (narrowing) of the cross-shaped flow channel is 40 micrometers ( Figure 2 (Left), compared to a width of 100 micrometers ( Figure 2 (Right) The resulting droplet spacing is smaller, and the droplets are directly smaller. It can be seen that when the width is narrowed to 40 micrometers, more smaller droplets can be generated more quickly.

[0161] Furthermore, as the width of the fourth end (narrowing) continues to shrink, the size becomes too small, causing great inconvenience to the manufacturing process. Therefore, the optimal width is 40 micrometers.

[0162] In summary, narrowing the width of the fourth end (constriction) of the cross-shaped flow channel can significantly shorten the spacing between the prepared droplets and generate smaller droplets more quickly, with a preferred width of 40 micrometers.

[0163] Example 5: Performance Comparison of Microfluidic Chips with Different Flow Channel Designs

[0164] This embodiment uses a microfluidic chip designed and generated according to Example 2, which consists of two particles and a single droplet. Figure 3 As shown, a corresponding microfluidic chip was designed according to the Dropseq double cross-shaped flow channel. Figure 4The Dropseq chip also features a continuous U-shaped channel for particle alignment, with the outlet of the second cross-shaped channel narrowed to bring the two chips closer together. The key difference lies in the microfluidic chip's use of a V-shaped channel + cross-shaped channel, while the Dropseq chip uses a cross-shaped channel + cross-shaped channel. Both chips use the same cell suspension (T2 cells), beads (PMMA microspheres), and oil phase reagent (fluorinated oil containing surfactants). Pressure was adjusted to approximate the speeds of both chips, and droplet generation was performed on both chips. The preparation of dual-particle single-droplets was examined, and the number and proportion of single-cell single-bead droplets generated by the Dropseq chip's double cross-shaped channel and the chip of this invention within the same time period were statistically analyzed. The results are shown in Table 3.

[0165] Table 3. Fabrication of dual-particle single droplets using different chips

[0166] Chip used Single-cell single-beads drop number Total drop number Proportion Double-cross flow channel (Experiment 1) 67 920 7.28％ Double-cross flow channel (Experiment 2) 70 1027 6.82％ The present invention (Experiment 1) 367 3424 10.72％ The present invention (Experiment 2) 403 3419 11.79％

[0167] As shown in Table 3, the experimental results indicate that within the same time frame, the number of droplets generated by this invention is more than three times that of the double-cross channel chip, the total number of single-cell single-bead droplets is approximately six times that of the Dropseq chip, and the proportion of single-cell single-bead droplets is 1.2 to 1.5 times that of the Dropseq chip. It is evident that the chip prepared using this invention significantly increases the effective droplet ratio and accelerates the droplet generation speed.

[0168] Furthermore, the cell and bead concentrations in Dropseq are low, resulting in an effective droplet percentage of only 1‰. This embodiment, after concentration adjustment, achieves approximately 6-7%. Compared to Dropseq, this invention increases the effective droplet percentage to over 10%. Simultaneously, compared to a 10x chip, this invention, using a high-speed camera for recording and statistics during operation, achieves an effective droplet percentage of over 65% compared to non-empty droplets, with even consumption of beads and cells, reducing reagent waste. The 10x chip can only perform experiments with a fixed reagent volume, and the total amount of effective droplets collected cannot be flexibly adjusted. This invention, however, allows for control of the number of effective droplets collected through collection time, offering greater flexibility.

[0169] Simultaneously, the stability of the two chips in the preparation of dual-particle droplets was investigated. The specific steps were as follows: during the preparation of dual-particle droplets, sampling was performed every 10 minutes to obtain dual-particle droplets. The particle size was measured using a dynamic light scattering particle size analyzer, and this was repeated three times. The test results showed that the chip structure provided by this invention exhibited better sustained stability. After 40 minutes of continuous operation, the particle size obtained was comparable to the initial value, with a polydispersity index (PDI) of less than 0.05. In contrast, the particle size prepared by the dual-cross-shaped flow channel chip was unstable, with a PDI greater than 0.5. It is evident that using the chip provided by this invention to generate dual-particle droplets not only results in a higher effective droplet ratio and faster speed but also greater stability.

[0170] Example 6: High-stability droplet sorting system and microfluidic chip provided by the present invention

[0171] A schematic diagram of the high-stability droplet sorting system 101 provided in this embodiment is shown below. Figure 5 As shown, the system includes a main channel 102 and branch channels 103. The width of the main channel 102 in the sorting region 104 is greater than the width of other regions. To eliminate the impact of the pressure difference between the two outlets 105 and 106 of the two branch channels 103 on droplet sorting, we need to increase the hydraulic pressure within the channels to improve the overall system's anti-interference capability. However, increasing the fluid pressure will cause the droplet velocity to be too high, making it extremely difficult to sort effective droplets. Therefore, even with increased fluid pressure, the droplet velocity within the sorting region 104 must remain constant. By increasing the width of the channels in the sorting region 104, the droplet velocity within the channels remains constant even with increased hydraulic pressure, thus successfully completing the sorting process, maintaining hydraulic pressure, improving anti-interference capability, avoiding the impact of the pressure difference at the outlets of the two sorting branch channels 103, and maintaining the stability of the sorting system, resulting in more accurate sorting results.

[0172] Preferably, the width of the main channel 102 in the sorting area 104 is 1.5 to 2 times the width of other areas.

[0173] like Figure 2 As shown, the main flow channel 102 includes a first end 107 and a second end 108. The first end 107 is connected to the outlet end 110 of the system 109 used to generate droplets, and the second end 108 is connected to the branch flow channel 103 after sorting. The main flow channel 102 is also provided with an oil phase inlet 111 for introducing oil phase to increase the spacing between droplets, thereby facilitating sorting. The sorting area 104 is provided with a dielectric electrophoresis sorting mechanism 114, and the branch flow channel 103 after sorting includes an effective droplet flow channel 115 and an ineffective droplet flow channel 116.

[0174] In this embodiment, the width of the main channel 102 after the oil phase inlet 112 is greater than the width before the oil phase inlet 113. "Before the oil phase inlet" in this embodiment refers to the period before the oil phase enters the fluid, while "after the oil phase inlet" refers to the process where the oil phase is introduced into the fluid and mixes with the existing fluid. Since the introduction of the oil phase causes the flow channel to accelerate due to the sudden increase in fluid volume, increasing the width after the oil phase inlet can eliminate this acceleration and stabilize the flow rate. In this embodiment, the width of the main channel 102 after the oil phase inlet 112 is 1.5 to 2 times the width of the oil phase inlet 113, and the width of the main channel 102 in the sorting area 104 is 1.5 to 2 times the width of the main channel 102 after the oil phase inlet 112.

[0175] Preferably, in this embodiment, the width of the main channel 102 is 125 micrometers before the oil phase inlet 113 and 200 micrometers after the oil phase inlet 112. The width of the sorting area 104 is 300 micrometers. The depth of each channel remains unchanged at 100 micrometers, which facilitates the fabrication of the microfluidic chip.

[0176] The extent to which the width of the main channel 102 increases in the sorting area 104 can be adjusted according to the hydraulic pressure and droplet flow rate within the channel. Generally, higher hydraulic pressure results in stronger anti-interference performance, but the hydraulic pressure cannot be increased indefinitely. Excessive hydraulic pressure can lead to excessively high flow rates or the channel cracking under pressure. Therefore, the hydraulic pressure must be maintained within a suitable range. Typically, the hydraulic pressure within the channel should not exceed 500 mbar; in this embodiment, the hydraulic pressure within the channel can be controlled to not exceed 150 mbar.

[0177] The droplet analysis system 101 provided in this embodiment maintains the flow velocity within the sorting region 104 within a range suitable for sorting by appropriately increasing the hydraulic pressure and widening the flow channel of the sorting region 4. In this embodiment, the suitable image acquisition speed for sorting is approximately 200 frames per second, while 10 to 50 droplets pass through the sorting region per second, with each droplet flowing within the widened region for approximately 4 to 6 frames.

[0178] The droplet sorting system 101 provided by this invention can be adapted to various flow channel designs without requiring changes to other architectures. In this embodiment, the droplet sorting system 101 is connected to the droplet generating system 109 provided in Embodiment 1. The droplet generating system 109 is a single-droplet system that generates single-cell single-beads (e.g., Figure 6 It is used to separate effective droplets from ineffective droplets in a multi-particle single droplet.

[0179] Example 7: Comparison of different droplet sorting systems

[0180] This embodiment employs a droplet sorting system (first group and second group) where the sorting area width remains constant. Figure 7The main channel 102 has a width of 125 micrometers before the oil phase inlet 113 and a width of 200 micrometers after the oil phase inlet 112; the sorting region 104 has a width of 200 micrometers. Simultaneously, it is used in conjunction with the system for generating multi-particle single droplets using the highly stable droplet sorting system provided in Example 6 (third group). Figure 6 The width of the main channel 102 is 125 micrometers before the oil phase inlet 113 and 200 micrometers after the oil phase inlet 112, and the width of the sorting area 104 is 300 micrometers. This is compared with the case where the width of the sorting area 104 is 460 micrometers (the fourth group, where the width of the main channel 102 is 125 micrometers before the oil phase inlet 113 and 200 micrometers after the oil phase inlet 112). The changes in fluid hydraulic pressure and flow rate in the two systems were detected, and the results are shown in Table 4.

[0181] Table 4. Effects of different droplet sorting systems on hydraulic pressure and flow velocity in the sorting zone

[0182]

[0183] As can be seen from Table 4, adopting the following... Figure 7 In the droplet sorting system shown, with a sorting area width of 200 micrometers, the flow velocity in the sorting area is 56 μm / ms when the hydraulic pressure is low (first group). As the hydraulic pressure increases (second group), the flow velocity in the sorting area rapidly rises to 85 μm / ms. Using a system such as... Figure 6 When the droplet sorting system shown increases the sorting area width to 300 micrometers (Group 3), although the hydraulic pressure rises to a level comparable to Group 2, the flow rate remains stable at 56 μm / ms, thus not affecting the sorting efficiency and accuracy. As the sorting area width continues to increase (460 micrometers), the flow rate continues to decrease to 38 μm / ms at the same hydraulic pressure level, and the rate of decrease in flow rate slows down. However, the width cannot be increased indefinitely, because the larger the width, the slower the flow rate, which also leads to a narrower droplet spacing. This would increase the pressure required to adjust the oil phase to the original spacing. Therefore, the preferred width of the sorting area is currently 300 micrometers.

[0184] While the present invention has been disclosed above, it is not limited thereto. Its application scope in the field of microfluidics can be expanded accordingly. Any person skilled in the art can make various modifications and alterations without departing from the spirit and scope of the invention; therefore, the scope of protection of the present invention should be determined by the scope defined in the claims.

Claims

1. A system for generating multiple particle single droplets and increasing the proportion of effective droplets, characterized in that, It includes V-shaped flow channels and cross-shaped flow channels. The V-shaped flow channels are used for particle mixing, and the cross-shaped flow channels are used for droplet generation. The included angle between two adjacent straight flow channels in the V-shaped flow channel is 30 degrees. The multi-particle single droplet is a double-particle single droplet. An effective droplet means that each double-particle single droplet must contain exactly one single cell and one bead.

2. The system as described in claim 1, characterized in that, It also includes a continuous U-shaped flow channel for arranging particles.

3. The system as described in claim 2, characterized in that, The number of continuous U-shaped channels is at least two, and each continuous U-shaped channel is provided with an inlet for a particle suspension.

4. The system as described in claim 3, characterized in that, The V-shaped flow channel includes at least two straight flow channels, each of which is connected to a U-shaped flow channel.

5. The system as described in claim 4, characterized in that, The V-shaped flow channel includes a first end, a second end, and a third end, wherein the first end and the second end are the inlet ends of the V-shaped flow channel, and the third end is the outlet end of the V-shaped flow channel; the third end is connected to the cross-shaped flow channel.

6. The system as described in claim 5, characterized in that, There are two continuous U-shaped flow channels, namely a first continuous U-shaped flow channel and a second continuous U-shaped flow channel; one end of the first continuous U-shaped flow channel is provided with a first particle inlet, and the other end is connected to the first end of the V-shaped flow channel; one end of the second continuous U-shaped flow channel is provided with a second particle inlet, and the other end is connected to the second end of the V-shaped flow channel.

7. The system as described in claim 6, characterized in that, The cross-shaped flow channel includes end I, end II, end III, and end IV, with end IV being a constricted opening. Ends I, II, and III are the inlet ends of the cross-shaped flow channel, and end IV is the outlet end of the cross-shaped flow channel. End III is connected to the outlet end of the V-shaped flow channel.

8. The system as described in claim 7, characterized in that, The dimension of the fourth end of the cross-shaped flow channel is smaller than the dimensions of other parts of the flow channel.

9. The system as described in claim 8, characterized in that, The system also includes an oil phase inlet. When the oil phase flows in from the oil phase inlet, it is divided into two paths, which flow along the first oil phase flow channel and the second oil phase flow channel, respectively. The first oil phase flow channel is connected to the first end of the cross-shaped flow channel, and the second oil phase flow channel is connected to the second end of the cross-shaped flow channel.

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