A droplet generator

Abstract

The application provides a droplet generator, which is characterized in that: a large corner continuous U-shaped flow channel is arranged on an encoding microsphere flow channel, the radius of the corner of the continuous U-shaped flow channel is controlled, and the accumulation of microfibers in the encoding microsphere suspension at the corner of the continuous U-shaped flow channel is prevented to avoid blockage; a buffer groove body is arranged on the encoding microsphere flow channel, which is used for pre-arrangement of the encoding microspheres and increases the controllability of the flow rate of the encoding microspheres; a buffer groove body is arranged on the oil phase flow channel, which is used for preventing the oil phase from penetrating into the water phase due to too high flow rate and increasing the controllability of the flow rate of the oil phase; and finally, the high-efficiency, high-throughput and high-stability preparation of single-cell single-encoding microsphere droplets is realized, the proportion of effective droplets is increased, the waste of expensive cell samples is reduced, the structure is simpler, the cost is lower, and the application is suitable for industrial application.

Description

Technical Field

[0001] This invention relates to the field of microfluidics, and more specifically, to a droplet generator for generating multi-particle droplets, and particularly to a multi-particle droplet generator with a coded microsphere channel having a large-angle continuous U-shaped channel, and both the coded microsphere channel and the oil phase channel having buffer grooves. Background Technology

[0002] Microfluidic chips, also known as lab-on-a-chip devices, integrate basic operational units used in chemistry and biology, including sample preparation, reaction, separation, detection, cell culture, sorting, and lysis. By designing various flow channels, different functions can be achieved on a microfluidic chip. Compared to traditional labs, microfluidic chips offer advantages such as lower reagent consumption and shorter reaction or analysis times. Reducing the consumption of expensive reagents controls costs, while shortening the time required shortens experimental cycles. Combined with chip sizes on the order of square centimeters or even square millimeters, this significantly reduces experimental costs in both time and space. Microfluidics has numerous applications, playing a crucial role in chemistry, biology, medicine, and many other fields.

[0003] Microfluidic technology can control microparticles, allowing for precise control over their size, shape, monodispersity, shell thickness, and internal structure, shape, and composition. Furthermore, the ingenious combination of microparticle structure and the various functional components that make up the microparticles can endow them with more diverse functions, thus providing new ideas and guidance for the design and development of microparticle functional materials.

[0004] In the biological field, droplets can encapsulate cells and act as bioreactors. Cells can be encapsulated and cultured into tissues or organoids. They can also be used for cell sorting, such as sorting sperm and fertilized eggs, for artificial reproduction, including artificial insemination, in vitro fertilization, cloning, and embryo division or cleavage. In the field of biochemistry, dispersed droplets can be handled and manipulated independently. Each droplet can function as an independent microreactor.

[0005] Multi-particle droplets refer to droplets containing two or more types of particles, including cells and coded microspheres (microspheres). To meet the needs of biochemical experiments, existing technologies have developed devices and solutions for realizing multi-particle droplets as microreactors. Examples include the dual-particle droplet microfluidic chips involved in Dropseq and 10x (US10745742B2). However, these existing devices and solutions generally suffer from problems such as easy clogging of the coded microsphere channels, difficulty in controlling the flow rate of the coded microspheres within the channels, and easy infiltration of the oil phase into the aqueous phase, resulting in adverse outcomes such as low effective throughput or a small proportion of effective droplets.

[0006] Therefore, there is an urgent need to find a multi-particle droplet generator with a simple structure that can effectively control the flow rate and flow resistance of the coded microspheres and the oil phase, is not prone to clogging the coded microsphere channels, prevents the oil phase from penetrating into the water phase, and has a higher effective droplet ratio. Summary of the Invention

[0007] To address the aforementioned issues, this invention provides a droplet generator. It utilizes a continuous U-shaped flow channel with large bends in the coded microsphere flow channel, controlling the radius of the bends to ensure sharp turns and prevent microfibers in the coded microsphere suspension from accumulating and clogging at these bends. The length of the continuous U-shaped flow channel and the inclination angle of the straight flow channel connecting the two bends are also controlled. Simultaneously, a buffer tank is incorporated into the coded microsphere flow channel for pre-arranging the coded microspheres, increasing the controllability of the flow rate. A buffer tank is also included in the oil phase flow channel, ensuring that the oil phase must first fill the buffer tank before entering the droplet generation channel. This prevents the oil phase from seeping into the aqueous phase due to excessive flow rate, further increasing the controllability of the oil phase flow rate. Ultimately, this achieves efficient, high-throughput, and high-stability preparation of single-cell, single-coded microsphere droplets, increasing the effective droplet ratio, reducing waste of expensive cell samples, and offering a simpler, lower-cost structure suitable for industrial applications.

[0008] The multi-particle droplets described in this invention refer to droplets in which each droplet contains multiple particles. In biochemical experiments, such multi-particle droplets are often used as bioreactors. For example, in single-cell sequencing, a dual-particle droplet is needed, containing both a single cell and a primer-encoding microsphere (microsphere, such as PMMA microspheres), allowing the reaction to occur within the droplet reaction chamber and sequencing to be completed.

[0009] An effective droplet is a multi-particle droplet that contains exactly one of each type of particle. For example, in the dual-particle droplets used for single-cell sequencing, an effective droplet means that each dual-particle droplet must contain exactly one single cell and one coding microsphere.

[0010] The single-cell, single-coded microsphere droplet generator requires two aqueous phases and one oil phase. The two aqueous phases are the coded microsphere suspension and the cell suspension, respectively. The coded microsphere suspension and the cell suspension are first mixed, and then introduced into the droplet generation site along with the oil phase. Under the action of shear force, the oil phase cuts the mixture of coded microspheres and cells into droplets of uniform size.

[0011] Existing single-cell single-coded microsphere droplet generators have the following three problems: First, the use of ordinary continuous U-shaped channels in the coded microsphere flow channels makes them prone to clogging; second, for coded microsphere suspensions, using only continuous U-shaped channels to control the flow rate of coded microspheres in the suspension results in poor control; and third, for the oil phase, even adjusting the oil phase inlet pressure to control the oil phase flow rate results in poor control.

[0012] Regarding the first issue, the conventional continuous U-shaped flow channel for coded microspheres is prone to clogging. This is mainly because: when preparing single-cell, single-coded microsphere droplets, the cell suspension, coded microsphere suspension, and oil phase need to be introduced into the microfluidic system to generate droplets. Both the cell suspension and oil phase can be filtered through a membrane to ensure they are free of impurities such as dust and microfibers, preventing clogging when passing through the continuous U-shaped flow channel and effectively controlling flow resistance and velocity. However, the coded microspheres in the coded microsphere suspension have a relatively large diameter, typically around 50 μm, making filtration by a membrane impossible. This results in the retention of some dust, microfibers, and other impurities in the coded microsphere suspension.

[0013] Continuous U-shaped channels can be used to control the flow rate of liquid within the channel and to arrange particles. This refers to arranging particles in a particle suspension into a single streamline, improving the effective single droplet rate, also known as sample focusing. It is a crucial step in devices such as cell counting, detection, and separation chips, directly affecting the accuracy and efficiency of subsequent detection and sorting, and is of great significance in the field of medical testing. However, existing continuous U-shaped channels are composed of a series of regular U-shaped channels connected end-to-end, with relatively small turning angles at the bends. This makes them unsuitable for coded microsphere suspensions. During continuous use, impurities such as dust and microfibers in the coded microsphere suspension tend to accumulate at the bends of the continuous U-shaped channel, causing blockages and severely affecting the droplet preparation efficiency and quality. This invention solves the problem of clogging at bends in coded microsphere suspensions by setting up a continuous U-shaped flow channel with large bends, controlling the radius of the bends to a suitable range, and preventing dust, microfibers and other impurities from accumulating at the bends by controlling the flow rate.

[0014] Regarding question two, numerous studies have demonstrated that while continuous U-shaped channels can largely control the flow rate of the coded microsphere suspension, they cannot effectively control the uniform spacing between each coded microsphere. Maintaining uniform spacing between each coded microsphere ensures that each microsphere and each cell is precisely encapsulated under the shear force of the oil phase, thus producing single-cell, single-coded microsphere droplets. This is the decisive condition for truly increasing the effective droplet ratio.

[0015] Because cell samples are extremely precious and scarce, and because cells are small in size, cell suspensions are almost entirely aqueous. Therefore, the only way to maximize the effective droplet ratio is by controlling the flow rate of the cell suspension, making precise control over the flow of each individual cell difficult. Encoded microspheres, on the other hand, are cheaper, more abundant, and larger in size, making them easier to control. To ensure that each cell suspension encounters a specific coded microsphere and is successfully encapsulated during outflow, we need to not only control the flow rate but, more importantly, control the spacing between each coded microsphere. This ensures that each cell, upon passing through, is combined with a corresponding coded microsphere and encapsulated into a droplet. This approach fully utilizes the cell sample, avoids wasting expensive cells, and maximizes the effective droplet ratio.

[0016] The coded microspheres (hydrogel microspheres) in the coded microsphere suspension have a relatively large diameter, and each coded microsphere occupies a large proportion of the suspension. The flow behavior of each coded microsphere in the flow channel affects the flow resistance of the suspension. Therefore, the coded microsphere suspension cannot be treated as a simple aqueous phase, and the flow rate cannot be controlled solely by a continuous U-shaped flow channel. Since the flow resistance of the coded microsphere suspension is closely related to the flow behavior of the coded microspheres in the flow channel, the flow resistance can be altered by changing the flow channel structure, thereby enhancing the control over the flow behavior of the coded microspheres. This invention adds a buffer tank to the coded microsphere flow channel. When the coded microsphere suspension enters the buffer tank, the flow resistance gradually decreases as the tank width gradually increases, reaching its minimum at the bottom. Therefore, for the coded microspheres to flow out from the bottom of the tank, they need to overcome the sudden increase in flow resistance at the bottom to enter the coded microsphere flow channel. Therefore, initially, the coded microspheres do not easily flow out from the bottom of the tank, allowing the coded microspheres in the suspension to pre-align within the buffer tank, achieving a re-aggregation effect. Once the tank is full, the pressure on the coded microspheres is sufficient to overcome the sudden increase in flow resistance at the bottom of the tank, at which point each coded microsphere will flow out from the bottom of the tank at an equal distance with the fluid. Since the change in flow resistance is the same for each coded microsphere, their outflow velocity through the tank is also the same, effectively allowing each coded microsphere to flow out at the same interval. This increases the controllability of the flow rate of each coded microsphere in the suspension, thereby improving the efficiency and quality of effective droplet preparation.

[0017] Regarding question three, numerous studies have demonstrated that oil-phase fluids, due to their inherent characteristics, are highly susceptible to backflow into the aqueous phase. Therefore, if the oil phase flow rate is adjusted solely by regulating the inlet pressure, a slight decrease in the aqueous phase pressure can easily lead to backflow. This invention incorporates a buffer tank and a continuous U-shaped flow channel for the oil phase. The continuous U-shaped flow channel increases flow resistance, and the buffer tank further enhances control over the oil phase flow rate. When the oil phase enters the buffer tank, the flow resistance decreases due to the gradual increase in tank width, causing the tank to fill initially. Exiting the tank requires overcoming this sudden increase in flow resistance. Therefore, the oil phase only exits the tank after it has filled, once the pressure is sufficient to overcome this sudden increase. This adds an extra layer of control to the oil phase, increasing flow resistance and significantly enhancing the controllability of the oil phase flow rate. This improves the resistance to backflow into the aqueous phase and significantly reduces the likelihood of backflow.

[0018] In addition, for cell suspensions, since the cells in the cell suspension are very small in volume, each cell occupies a very small proportion of the cell suspension, and the flow resistance effect of each cell on the cell suspension is almost negligible, it is more similar to the aqueous phase. Therefore, there is no need to set up a buffer tank for pre-arrangement of the cell suspension.

[0019] On one hand, the present invention provides a droplet generator, comprising an encapsulated biomaterial channel, a coded microsphere channel, and an oil phase channel; characterized in that the coded microsphere channel includes an arc-shaped channel, which comprises one or more arc-shaped channels with different radii, wherein at least one arc-shaped channel has a radius that is more than 6 times the width of the coded microsphere channel; wherein the coded microsphere channel is further provided with a coded microsphere buffer groove, and the flow resistance of the coded microsphere fluid in the coded microsphere buffer groove is less than the flow resistance in the coded microsphere channel; the biomaterial may be selected from one or more of cells, nucleic acids, proteins, antibodies, or antibody fragments.

[0020] In some embodiments, the biomaterial may be selected as cells, and the droplet generator includes cell channels, coded microsphere channels, and oil phase channels.

[0021] In some embodiments, the coded microsphere flow channel provided by the present invention, which includes an arc-shaped flow channel, is equivalent to setting a continuous U-shaped flow channel with a large bend on the coded microsphere flow channel. At each bend of the continuous U-shaped flow channel with a large bend, the radius of the bend is more than 6 times the width of the flow channel.

[0022] The large bend mentioned in this invention refers to a bend in the flow channel connecting two flow channels with a large turning radius. The flow channel at the bend can be a regular arc-shaped flow channel, or it can be an arc-shaped flow channel composed of multiple arc-shaped flow channels with different radii, but at least one of the arc-shaped flow channels must have a radius that is more than 6 times the width of the flow channel.

[0023] The shape and structure of the continuous U-shaped flow channel (arc flow channel) of the coded microsphere suspension described in this invention are different from the shape and structure of the traditional continuous U-shaped flow channel. It adopts a large-amplitude corner at the turning point, and the upper and lower flow channels connecting the turning point can be parallel to each other or not.

[0024] In some configurations, the upper and lower channels connecting the arc-shaped flow channel are parallel to each other, but the distance between the upper and lower channels increases due to the presence of a large bend.

[0025] In some other configurations, the upper and lower channels connecting the arc-shaped flow channel are not parallel to each other, and the extensions of the two channels extending towards the bend intersect at an acute angle.

[0026] This invention solves the problem of clogging at bends in coded microsphere suspensions by using a continuous U-shaped flow channel (arc-shaped flow channel) with large bends. This allows the radius of the bends to be controlled within a suitable range, preventing the accumulation of dust, microfibers, and other impurities at the bends, especially when the coded microsphere suspension passes through a bend with a larger radius. In other words, at a certain flow rate (0.01-0.1 μL / s), when the coded microspheres pass through a bend with a larger radius, dust, microfibers, and other impurities are completely washed away, preventing accumulation at the bend. However, at the same flow rate, if the bend radius is smaller, dust, microfibers, and other impurities in the coded microsphere suspension may not be carried away in time and gradually deposit. As more and more dust and microfibers accumulate, the flow resistance increases, the flow rate decreases, and the deposition phenomenon worsens, leading to clogging over time.

[0027] Numerous studies have demonstrated that when the flow rate of the coded microsphere suspension is (0.01-0.1 μL / s), as long as the radius of the bend (arc channel) of the continuous U-shaped channel is controlled to be more than 6 times the width of the coded microsphere channel, impurities such as dust and microfibers in the coded microsphere suspension cannot accumulate at the bend, thus ensuring the long-term smooth operation of the coded microsphere channel.

[0028] All coded microsphere channels have the same width. The continuous U-shaped channel is part of the coded microsphere channel, and the width of the continuous U-shaped channel is the same as the width of the other parts of the coded microsphere channel.

[0029] Because the flow resistance of the coded microsphere buffer tank is less than that of the coded microsphere flow channel, and the flow resistance needs to be overcome to overcome the sudden increase in flow resistance at the bottom of the tank before it can flow out, the coded microspheres are pre-arranged in the buffer tank. By controlling the flow resistance at the inlet of the coded microsphere suspension, when the flow rate of the coded microsphere suspension is (0.01-0.1μL / s), it can be achieved that after the tank is filled, the coded microspheres overcome the sudden increase in flow resistance at the bottom of the tank and flow out with the fluid at equal distances.

[0030] Furthermore, the radius of the arc-shaped flow channel is 6 to 20 times the width of the flow channel.

[0031] It is understandable that the radius of the turning point of the continuous U-shaped flow channel (arc flow channel) cannot be increased indefinitely. The layout of the entire microfluidic system must also be taken into account, as well as the appropriate control of the flow resistance, so as to control the flow rate of the coded microspheres and ensure the generation of effective droplets.

[0032] Furthermore, the radius of the arc-shaped flow channel is 10 times the width of the flow channel.

[0033] Numerous experimental results have demonstrated that when the radius of the bend in a continuous U-shaped flow channel (arc-shaped flow channel) is 10 times the width of the flow channel, it can ensure that impurities such as dust and microfibers in the coded microsphere suspension cannot accumulate at the bend, and can also better control the flow resistance, thereby helping to stabilize the flow velocity of the coded microspheres and further improve the generation efficiency of effective droplets.

[0034] Furthermore, the extensions of the two straight flow channels connected to the bend intersect at an acute angle.

[0035] It is understandable that when the upper and lower flow channels at the bend are not parallel to each other, that is, when the extension lines of the two flow channels extending towards the bend intersect at an acute angle, impurities such as dust and microfibers in the coded microsphere suspension are less likely to clog at the bend.

[0036] Furthermore, there is one or more arc-shaped flow channels.

[0037] It's understandable that the more curved flow channels there are, the more effectively the flow resistance can be controlled.

[0038] Furthermore, there are two arc-shaped flow channels.

[0039] To ensure the large bends and turns of the continuous U-shaped flow channel, too many bends (arc-shaped flow channels) cannot be set in the continuous U-shaped flow channel. At the same time, since the large-angle bends and other straight flow channels can already control the flow resistance smoothly, there is no need to set too many bends (arc-shaped flow channels). Generally, two bends are set, one on the left and one on the right.

[0040] Furthermore, the straight flow channels connecting the arc-shaped flow channels are arranged horizontally, upwardly, or downwardly.

[0041] The straight flow channel connecting the two bends can be horizontal, or slightly upward or downward inclined, thereby increasing flow resistance and making it easier to control the flow velocity. In this invention, "horizontal" means that when the droplet generator containing the continuous U-shaped flow channel is placed vertically, the straight flow channel connecting the two bends is parallel to the horizontal plane of the ground, that is, perpendicular to the vertically downward direction of the center of gravity. "Upward" refers to the upstream direction of the fluid; "downward" refers to the downstream direction of the fluid.

[0042] Furthermore, the straight flow channel connecting the two arc-shaped flow channels is arranged at an upward angle, and the arc-shaped flow channel and the straight flow channel connected to the arc-shaped flow channel form a large-angle continuous U-shaped flow channel of the coded microsphere flow channel.

[0043] Furthermore, the large-angle continuous U-shaped flow channel includes an inlet section straight flow channel and an outlet section straight flow channel, wherein the angle between the inlet section straight flow channel and the inlet section of the coded microsphere flow channel is greater than or equal to 90 degrees.

[0044] While ensuring the continuous U-shaped flow channel has large bends and turns, the connection between the continuous U-shaped flow channel and other straight flow channels also needs to have large bends and turns. This can not only allow the coded microsphere suspension to smoothly transition from other straight flow channels to the continuous U-shaped flow channel, and from the continuous U-shaped flow channel to other straight flow channels, but also prevent the blockage of impurities such as dust and microfibers in the coded microsphere suspension.

[0045] Furthermore, the angle between the straight section of the outlet section of the large-angle continuous U-shaped flow channel and the outlet section of the coded microsphere flow channel is greater than or equal to 90 degrees.

[0046] Understandably, when the upper and lower flow channels connecting the bend are parallel to each other, the angle between the straight inlet flow channel and the inlet section of the coded microsphere flow channel, or the angle between the straight outlet flow channel and the outlet section of the coded microsphere flow channel, can be set to 90 degrees or greater than 90 degrees, thus ensuring a sharp bend. However, when the upper and lower flow channels at the bend are not parallel to each other, that is, when the extension lines of the two flow channels extending towards the bend intersect at an acute angle, the angle between the straight inlet flow channel and the inlet section of the coded microsphere flow channel, or the angle between the straight outlet flow channel and the outlet section of the coded microsphere flow channel, is preferably greater than 90 degrees.

[0047] Furthermore, the width of the coded microsphere flow channel is 50-100μm, and the radius of the turning point of the large-angle continuous U-shaped flow channel is 500-1000μm.

[0048] Furthermore, the width of the coded microsphere channel is 50 μm, and the radius of the turning point of the large-angle continuous U-shaped channel is 500 μm.

[0049] Furthermore, the total length of the large-angle continuous U-shaped flow channel is 15,000-25,000 μm; wherein the straight flow channel connecting the two bends is inclined upwards, with an angle of 10-15 degrees with the horizontal line, and a length of 4,000-6,000 μm; the length of the straight flow channel in the inlet section is 4,000-6,000 μm, and the angle with the inlet section of the coded microsphere flow channel is 90-180 degrees; the length of the straight flow channel in the outlet section is 4,000-6,000 μm, and the angle with the outlet section of the coded microsphere flow channel is 90-180 degrees.

[0050] Furthermore, the total length of the large-angle continuous U-shaped flow channel is 20,000 μm; the straight flow channel connecting the two bends is inclined upwards, with an angle of 10 to 15 degrees with the horizontal line, and a length of 6,240 μm; the length of the straight flow channel in the inlet section is 5,000 μm, and the angle between it and the inlet section of the coded microsphere flow channel is 120 degrees; the length of the straight flow channel in the outlet section is 5,000 μm, and the angle between it and the outlet section of the coded microsphere flow channel is 120 degrees.

[0051] By controlling the total length of the continuous U-shaped flow channel and the length of each straight flow channel within the continuous U-shaped flow channel, it is possible to further control the flow resistance more precisely and adjust the flow rate more precisely, thereby improving the effective droplet generation rate.

[0052] Furthermore, the volume of the coded microsphere buffer groove is larger than the volume of the coded microsphere channel of the same length.

[0053] Furthermore, the width of the coded microsphere buffer groove is greater than the width of the coded microsphere channel, and / or the depth of the coded microsphere buffer groove is greater than the depth of the coded microsphere channel.

[0054] Furthermore, the width of the coded microsphere buffer groove is 5 to 15 times the width of the coded microsphere channel, and the depth is 2 to 5 times the depth of the coded microsphere channel.

[0055] Furthermore, the shape of the coded microsphere buffer groove gradually increases from top to bottom, and then rapidly shrinks back downwards.

[0056] The shape of the coded microsphere buffer tank has a significant impact on controlling the flow rate of the coded microspheres. It gradually increases in size from top to bottom and then rapidly shrinks downwards, so that the flow resistance of the coded microsphere suspension entering the buffer tank gradually decreases first. After passing through the buffer tank, the flow resistance increases rapidly, which allows the coded microspheres to pre-arrange in the buffer tank and achieve the effect of coded microsphere re-aggregation. After the tank is full, the coded microspheres will overcome the sudden increase in flow resistance from the bottom of the tank and flow out with the fluid at equal distances.

[0057] The coded microsphere buffer groove is shaped like a water droplet with smooth edges, which can avoid dead volume and ensure that all the coded microspheres stored in it flow out in sequence without remaining inside.

[0058] The size of the coded microsphere buffer groove directly affects its effectiveness in pre-arranging coded microspheres and controlling the spacing between them. Controlling the width of the buffer groove to 5–15 times the width of the coded microsphere channel and the depth to 2–5 times the depth of the channel ensures better performance and guarantees high-efficiency and high-quality droplet generation.

[0059] Furthermore, the coded microsphere flow channel includes a continuous U-shaped flow channel, and the coded microsphere buffer groove is located below the continuous U-shaped flow channel.

[0060] Furthermore, the width of the coded microsphere channel is 40-100μm and the depth is 40-100μm; the width of the widest part of the coded microsphere buffer groove is 200-1500μm, the depth of the deepest part is 200-1500μm, and the length is 200-2000μm.

[0061] Furthermore, the width of the coded microsphere channel is 50 μm and the depth is 50 μm; the width of the widest part of the coded microsphere buffer groove is 500 μm, the depth of the deepest part is 150 μm, and the length is 1280 μm.

[0062] Furthermore, an oil phase buffer tank is provided on the oil phase flow channel, and the flow resistance of the oil phase in the oil phase buffer tank is less than the flow resistance in the oil phase flow channel.

[0063] Furthermore, the oil phase flow channel is divided into a first oil phase flow channel and a second oil phase flow channel, and each of the first oil phase flow channel and the second oil phase flow channel is provided with an oil phase buffer tank.

[0064] Furthermore, the first oil phase flow channel and the second oil phase flow channel include a continuous U-shaped flow channel, and the oil phase buffer tank is located on the continuous U-shaped flow channel.

[0065] Setting a continuous U-shaped flow channel on the oil phase flow channel can help control the flow rate and enhance the control effect on the oil phase flow rate.

[0066] Furthermore, the volume of the oil phase buffer tank is greater than the volume of an oil phase flow channel of the same length.

[0067] Furthermore, the width of the oil phase buffer tank is greater than the width of the oil phase flow channel, and / or the depth of the oil phase buffer tank is greater than the depth of the oil phase flow channel.

[0068] Furthermore, the width of the oil phase buffer tank is 5 to 15 times the width of the oil phase flow channel, and the depth is 2 to 5 times the depth of the oil phase flow channel.

[0069] Furthermore, the shape of the oil phase buffer tank gradually increases from top to bottom, and then rapidly shrinks back downwards.

[0070] The oil phase buffer tank acts like a reservoir, ensuring that the oil phase fills the buffer tank before flowing into the subsequent oil phase channels. The size of the oil phase buffer tank directly affects its effectiveness in increasing the controllability of the oil phase flow rate and reducing resistance to the oil phase's penetration into the water phase. Maintaining the width of the oil phase buffer tank to 5–15 times the width of the oil phase channel and the depth to 2–5 times the depth of the oil phase channel better ensures the buffer tank's effectiveness, further guaranteeing the high efficiency and high quality of droplet formation.

[0071] Furthermore, the width of the oil phase flow channel is 40-100μm and the depth is 40-100μm; the width of the widest part of the oil phase buffer tank is 200-1500μm, the depth of the deepest part is 200-1500μm, and the length is 200-2000μm.

[0072] Furthermore, the width of the oil phase flow channel is 40 μm and the depth is 40 μm; the width of the oil phase buffer tank at its widest point is 400 μm, the depth at its deepest point is 150 μm, and the length is 1280 μm.

[0073] Furthermore, the cell flow channel and the encoded microsphere flow channel constitute the first cross-shaped flow channel, and the output channel of the first cross-shaped flow channel and the oil phase flow channel constitute the second cross-shaped flow channel.

[0074] Furthermore, the distance between the first cross-shaped flow channel and the second cross-shaped flow channel should be maintained at 20-500 μm.

[0075] Studies have shown that the length of the output channel between the first and second cross-shaped flow channels must be kept within a certain range in order to better leverage the role of oil phase shearing and encapsulation in forming effective droplets. This is mainly because a certain time interval is required between each shearing and encapsulation of the oil phase to form droplets. The distance between the first and second cross-shaped flow channels needs to be matched as closely as possible with the time interval between each shearing and encapsulation of the oil phase to form droplets, so as to further improve the efficiency of generating effective droplets.

[0076] Furthermore, the width and / or depth of the outlet section of the second cross-shaped flow channel is greater than the width and / or depth of the outlet end.

[0077] Studies have shown that when the width and / or depth of the outlet section of the second cross-shaped flow channel is greater than the width and / or depth of the outlet end, the generated droplets can flow out more quickly. This is mainly due to the increased width and / or depth, which reduces flow resistance and increases flow velocity.

[0078] In some configurations, the width of the outlet section of the second cross-shaped flow channel is 1.5 to 3 times the width of the outlet end.

[0079] Increasing the width and / or depth reduces flow resistance and increases flow velocity, but the flow velocity cannot be too high, otherwise it will cause difficulties in subsequent droplet sorting. When the width of the outlet section of the second cross-shaped flow channel is 1.5 to 3 times the width of the outlet end, more effective droplets can be obtained more efficiently, and the flow resistance and flow velocity are within a controllable range.

[0080] The droplet generator with a buffer tank provided by the present invention has the following beneficial effects:

[0081] 1. By setting up a large-angle continuous U-shaped flow channel and controlling the radius of the turning point within an appropriate range, the coded microsphere suspension can pass through a turning point with a larger turning radius. By controlling the flow rate, impurities such as dust and microfibers cannot accumulate at the turning point, thus solving the problem of coded microsphere suspension clogging at the turning point after long-term operation.

[0082] 2. A buffer groove for coded microspheres is set on the flow channel of the coded microspheres, so that the coded microspheres are pre-arranged in the buffer groove. After the groove is filled, the coded microspheres will overcome the sudden increase in flow resistance from the bottom of the groove and flow out with the fluid at equal distances. This helps to accurately control the spacing of the coded microspheres, increases the controllability of the flow rate of each coded microsphere in the coded microsphere suspension, and improves the preparation efficiency and quality of effective droplets.

[0083] 3. By combining a large-angle continuous U-shaped flow channel and a buffer groove for the coded microspheres, the spacing between the coded microspheres can be precisely controlled over a long period of time. The flow resistance and flow rate of the coded microsphere suspension can also be effectively controlled over a long period of time, which greatly increases the proportion of effective droplets and has a significant synergistic effect.

[0084] 4. An oil phase buffer tank is set up on the oil phase flow channel, so that the oil phase must fill the oil phase buffer tank first before it can continue to flow to the subsequent oil phase flow channel. This adds an extra layer of control to the oil phase, increases the flow resistance, significantly increases the controllability of the oil phase flow rate, increases the resistance to the oil phase penetrating into the water phase, significantly reduces the possibility of the oil phase penetrating into the water phase, and further increases the effective droplet ratio.

[0085] 5. By maintaining an appropriate distance between the first and second cross-shaped flow channels, and making the width of the outlet section of the second cross-shaped flow channel 1.5 to 3 times the width of the outlet end, effective droplets can be generated more efficiently.

[0086] 6. Increase flow resistance, precisely control flow rate, and increase the proportion of effective droplets;

[0087] 7. Simple structure, convenient and efficient, low cost, and easy to promote. Attached Figure Description

[0088] Figure 1This is a schematic diagram of the coded microsphere flow channel structure in the droplet generator of Example 1;

[0089] Figure 2 This is a schematic diagram of the overall structure of the droplet generator in Example 1;

[0090] Figure 3 This is a schematic diagram of the overall structure of the droplet generator;

[0091] Figure 4 This is a schematic diagram of the structure of the first and second cross-shaped flow channels;

[0092] Figure 5 This is a schematic diagram showing the flow of cells and beads in the first and second cross-shaped flow channels. Detailed Implementation

[0093] The preferred embodiments of the present invention will be described in further detail below with reference to the accompanying drawings. It should be noted that the following embodiments are intended to facilitate understanding of the present invention and are not intended to limit it in any way. The raw materials and equipment used in the specific embodiments of the present invention are all known products and were obtained by purchasing commercially available products.

[0094] Example 1: Droplet generator provided by the present invention

[0095] A schematic diagram of the droplet generator provided in this embodiment is shown below. Figures 1-3 As shown, where Figure 1 A schematic diagram of a large-angle continuous U-shaped flow channel structure for coding microsphere flow channels; Figure 2 A schematic diagram of the coded microsphere flow channel structure in a droplet generator; Figure 3 This is a schematic diagram of the overall structure of the droplet generator.

[0096] like Figures 1-3 As shown, the single-cell single-coded microsphere droplet generator includes a coded microsphere channel 1, a cell channel 2, and an oil phase channel 3. The coded microsphere channel 1 is provided with a large-angle continuous U-shaped channel 110 and a coded microsphere buffer groove 4.

[0097] like Figure 1 As shown, the large-angle continuous U-shaped flow channel 110 of the coded microsphere flow channel 1 includes a first bend 101, a second bend 102, a straight flow channel 103 connecting the two bends, an inlet section straight flow channel 104, and an outlet section straight flow channel 105. The bend radius 106 of the first bend 101 is greater than 6 times the width of the coded microsphere flow channel 1, and the bend radius 7 of the second bend 102 is also greater than 6 times the width of the coded microsphere flow channel 1. Because the coded microspheres have a relatively large diameter, generally around 50 μm, it is impossible to use a filter membrane with a pore size smaller than 50 μm for filtration, resulting in the retention of some dust, microfibers, and other impurities in the coded microsphere suspension. When using a conventional continuous U-shaped flow channel (such as...) Figure 3 In the continuous U-shaped flow channel 108 of the cell flow channel 2, during continuous use, impurities such as dust and microfibers in the coded microsphere suspension tend to accumulate gradually at the bends of the continuous U-shaped flow channel, causing blockage and seriously affecting the preparation efficiency and quality of droplets. This embodiment addresses this by setting a large-angle continuous U-shaped flow channel 110 for the coded microsphere suspension, controlling the radius of the bends (first bend 101 or second bend 102) to a suitable range. When the coded microsphere suspension passes through a bend with a larger radius, at a certain flow rate, impurities such as dust and microfibers cannot accumulate at the bend, thus solving the problem of blockage at the bends of the coded microsphere suspension after long-term operation.

[0098] For example, in this embodiment, when the flow rate of the coded microsphere suspension is 0.01-0.1 μL / s, when the coded microsphere suspension passes through a bend with a larger turning radius at this flow rate, all impurities such as dust and microfibers will be washed away, preventing them from accumulating at the bend. However, at the same flow rate, if the turning radius is smaller, the dust and microfibers in the coded microsphere suspension may not have enough time to be carried away and will gradually deposit. As more and more dust and microfibers accumulate, the flow resistance increases, the flow rate decreases, and the deposition phenomenon is further aggravated, leading to blockage after prolonged operation. Numerous studies have shown that when the flow rate of the coded microsphere suspension is 0.01-0.1 μL / s, as long as the turning radius of the continuous U-shaped flow channel 110 is controlled to be more than 6 times the width of the coded microsphere flow channel 1, the dust and microfibers in the coded microsphere suspension cannot accumulate at the bend, thus ensuring the long-term smooth operation of the coded microsphere flow channel 1.

[0099] Both the first bend 101 and the second bend 102 are arc-shaped flow channels. They can be regular arcs or arc-shaped flow channels composed of multiple arc-shaped flow channels with different radii. However, at least one arc-shaped flow channel must have a radius that is more than 6 times the width of the flow channel.

[0100] The straight flow channel 103 and the inlet section straight flow channel 104 may be parallel or non-parallel, and the straight flow channel 103 and the outlet section straight flow channel 105 may be parallel or non-parallel. In this embodiment, the straight flow channel 103 and the inlet section straight flow channel 104 are not parallel, and the straight flow channel 103 and the inlet section straight flow channel 104 intersect each other at an acute angle along the extension line of the first bend 101; the straight flow channel 103 and the outlet section straight flow channel 105 are not parallel, and the straight flow channel 103 and the outlet section straight flow channel 105 intersect each other at an acute angle along the extension line of the second bend 102.

[0101] Preferably, the turning radius of the large-angle continuous U-shaped channel 110 is 6 to 20 times the width of the coded microsphere channel 1. This is mainly because the turning radius of the continuous U-shaped channel 110 cannot be increased indefinitely. The layout of the entire microfluidic system, the space occupied by the channels, and cost control must also be considered. Appropriate control of flow resistance is also necessary to control the flow velocity of the coded microspheres and ensure the generation of effective droplets. In this embodiment, the turning radius of the large-angle continuous U-shaped channel 110 is 10 times the width of the coded microsphere channel 1. Experiments have shown that when the turning radius of the continuous U-shaped channel 110 is 10 times the width of the coded microsphere channel 1, it ensures that impurities such as dust and microfibers in the coded microsphere suspension cannot accumulate at the turning point, and it also better controls the flow resistance, thereby helping to stabilize the flow velocity of the coded microspheres and further improving the generation efficiency of effective droplets.

[0102] The straight flow channel 103 connecting the two bends can be arranged horizontally or slightly inclined upwards. In this embodiment, as... Figure 1 As shown, the straight flow channel 103 connecting the two bends is slightly inclined upward (the upward direction refers to the upstream direction of the fluid), which helps to increase the flow resistance and is more conducive to stable control of the flow rate.

[0103] Preferred, such as Figure 1 and 2 As shown, the angle 111 between the inlet section of the large-angle continuous U-shaped channel 110 (straight channel 104) and the inlet section of the coded microsphere channel 109 is greater than 90 degrees (the inlet section of the coded microsphere channel 109 is directly connected to the inlet of the coded microsphere channel); the angle 113 between the outlet section of the large-angle continuous U-shaped channel 110 (straight channel 105) and the outlet section of the coded microsphere channel 112 is greater than 90 degrees. This arrangement ensures that while the continuous U-shaped channel 110 has large bends, the connection points between the continuous U-shaped channel 110 and other straight channels also have large bends, avoiding bends less than 90 degrees. This design allows the coded microsphere suspension to smoothly transition from other straight channels to the continuous U-shaped channel 110, and from the continuous U-shaped channel 110 to other straight channels, while also preventing clogging by dust, microfibers, and other impurities in the coded microsphere suspension.

[0104] Preferably, in this embodiment, the width of the coded microsphere channel 1 is 50 μm, and the radius of the turning point of the large-angle continuous U-shaped channel 110 is 500 μm. The width of all coded microsphere channels 1 is the same, and the continuous U-shaped channel 110 is a part of the coded microsphere channel 1. The width of the continuous U-shaped channel 110 is also the same as the width of other parts of the coded microsphere channel 1.

[0105] Preferably, the total length of the large-angle continuous U-shaped flow channel 110 is 20,000 μm; the straight flow channel 103 connecting the two bends is inclined upwards, forming an angle 114 of 15 degrees with the horizontal line, and has a length of 6,240 μm; the inlet section straight flow channel 104 has a length of 5,000 μm, forming an angle 111 of 120 degrees with the inlet section 109 of the coded microsphere flow channel; the outlet section straight flow channel 105 has a length of 5,000 μm, forming an angle 113 of 120 degrees with the outlet section 112 of the coded microsphere flow channel. By controlling the total length of the continuous U-shaped flow channel 110 and the length of each straight flow channel in the continuous U-shaped flow channel 110, it is possible to further help to more accurately control the flow resistance and more accurately adjust the flow rate, thereby improving the effective droplet generation rate.

[0106] like Figure 2 and 3 The widest part of the coded microsphere buffer tank 4 is 5 to 15 times the width of the coded microsphere flow channel 1, and the deepest part is 2 to 5 times the depth of the coded microsphere flow channel 1. The shape of the coded microsphere buffer tank 4 gradually increases from top to bottom and then rapidly shrinks downwards, resembling a teardrop shape with smooth edges. The shape of the coded microsphere buffer tank 4 has a very important influence on controlling the flow rate of the coded microspheres. The gradual increase in size from top to bottom and then the rapid shrinking downwards causes the flow resistance of the coded microsphere suspension entering the coded microsphere buffer tank 4 to gradually decrease first. After passing through the coded microsphere buffer tank 4, the flow resistance increases rapidly, causing the coded microspheres to pre-arrange themselves in the coded microsphere buffer tank 4. Once the coded microsphere buffer tank 4 is full, the coded microspheres overcome the suddenly increased flow resistance from the bottom of the coded microsphere buffer tank 4 and flow out with the fluid at equal distances. The smooth edges of the coded microsphere buffer tank 4 avoid dead volume, ensuring that all the coded microspheres stored in it flow out in sequence and do not remain inside. The size of the coded microsphere buffer groove 4 directly affects its effectiveness in pre-arranging coded microspheres and controlling the spacing between them. Controlling the width of the coded microsphere buffer groove 4 to be 5 to 15 times the width of the coded microsphere channel 1, and the depth of its deepest point to be 2 to 5 times the depth of the coded microsphere channel 1, better ensures the effectiveness of the coded microsphere buffer groove 4 and guarantees high-efficiency and high-quality generation of effective droplets.

[0107] Preferably, the coded microsphere flow channel 1 further includes a large-angle continuous U-shaped flow channel 110, and the coded microsphere buffer tank 4 is located below the large-angle continuous U-shaped flow channel 110. The coded microsphere suspension first passes through the large-angle continuous U-shaped flow channel 110 from the coded microsphere inlet 20, and then enters the coded microsphere buffer tank 4. It can be understood that the proper functioning of the coded microsphere buffer tank 4 is closely related to its properties. It must be in a vertically downward straight flow channel to achieve the pre-arrangement of coded microspheres and precise control of the spacing of the discharged coded microspheres through its gradually increasing and then rapidly contracting shape. However, the large-angle continuous U-shaped flow channel 110 is entirely a curved flow channel and has no vertically downward straight flow channel. Therefore, the coded microsphere buffer tank 4 cannot be set on the large-angle continuous U-shaped flow channel 110, but can only be set on the vertical straight flow channel 6 behind it. In this embodiment, the width of the coded microsphere channel 1 is 50 μm and the depth is 50 μm; the width of the coded microsphere buffer groove 4 at its widest point is 500 μm, the depth at its deepest point is 150 μm, and the length is 1280 μm.

[0108] like Figure 2 An oil phase buffer tank 7 is provided on the oil phase flow channel 3. The oil phase flow channel 3 is divided into a first oil phase flow channel 8 and a second oil phase flow channel 9, and each of the first oil phase flow channel 8 and the second oil phase flow channel 9 is provided with an oil phase buffer tank 7. Both the first oil phase flow channel 8 and the second oil phase flow channel 9 are provided with continuous U-shaped flow channels for the oil phase, and the oil phase buffer tank 7 is located on the continuous U-shaped flow channel for the oil phase.

[0109] Preferably, the width of the oil phase buffer tank 7 is 5 to 15 times the width of the oil phase flow channel 3, and the depth is 2 to 5 times the depth of the oil phase flow channel 3. The shape of the oil phase buffer tank 7 gradually increases from top to bottom and then rapidly shrinks downwards, with smooth boundaries. The oil phase buffer tank 7 acts like a reservoir, ensuring that the oil phase must first fill the oil phase buffer tank 7 before continuing to flow into the subsequent oil phase flow channel 3. The size of the oil phase buffer tank 7 directly affects its effect in increasing the controllability of the oil phase flow rate and improving the resistance to the oil phase penetrating into the water phase. Controlling the width of the oil phase buffer tank 7 to 5 to 15 times the width of the oil phase flow channel and the depth to 2 to 5 times the depth of the oil phase flow channel better ensures the effectiveness of the oil phase buffer tank 7, further guaranteeing the high efficiency and high quality of effective droplet generation. In this embodiment, the width of the oil phase flow channel 3 is 40 μm and the depth is 40 μm; the width of the oil phase buffer tank 7 at its widest point is 400 μm, the depth at its deepest point is 150 μm, and the length is 1280 μm.

[0110] The droplet generator in this embodiment adopts a double cross droplet generator. The cell channel 2 and the coded microsphere channel 1 form the first cross-shaped channel 11. The output channel 12 of the first cross-shaped channel 11 and the oil phase channel 3 form the second cross-shaped channel 13.

[0111] like Figure 3As shown, the coded microsphere channel 1 and the cell channel 2 constitute the first cross-shaped channel 11. Because cell samples are very precious and scarce, and because cells are small in size, the cell suspension is almost equivalent to an aqueous phase. Therefore, it is difficult to precisely control the flow of each individual cell by controlling the flow rate of the cell suspension to maximize the proportion of effective droplets. In contrast, coded microspheres are inexpensive, abundant, and larger in size, making them easier to control. To ensure that the cell suspension encounters a coded microsphere at the first cross-shaped channel 11 each time it flows out and is successfully encapsulated, we need to control not only the flow rate of the cell suspension but, more importantly, the spacing between each coded microsphere. This ensures that each cell, upon passing through, is combined with a corresponding coded microsphere and encapsulated into a droplet. This approach fully utilizes the cell sample, avoids wasting expensive cell samples, and maximizes the proportion of effective droplets. The output channel 12 of the first cross-shaped flow channel 11 and the oil phase flow channel 3 form the second cross-shaped flow channel 13. After passing through the second cross-shaped flow channel 13, the encoded microspheres and cells are encapsulated and sheared by the oil phase to generate droplets.

[0112] like Figure 4 The length of the output channel 12 between the first cross-shaped flow channel 11 and the second cross-shaped flow channel 13 must be maintained between 20 and 500 μm to better utilize the oil phase shearing and encapsulation to form effective droplets. This is mainly because a certain time interval is required between each shearing and encapsulation of the oil phase to form droplets. The distance between the first cross-shaped flow channel 11 and the second cross-shaped flow channel 13 needs to be matched as closely as possible to the time interval between each shearing and encapsulation of the oil phase to form droplets, so as to further improve the efficiency of generating effective droplets. In this embodiment, the length of the output channel 12 between the first cross-shaped flow channel and the second cross-shaped flow channel is 80 μm.

[0113] Preferably, the width and / or depth of the outlet section 14 of the second cross-shaped flow channel 13 is greater than the width and / or depth of the outlet end 15. When the width and / or depth of the outlet section 14 of the second cross-shaped flow channel 13 is greater than the width and / or depth of the outlet end 15, the generated droplets can flow out more quickly. This is mainly because the increased width and / or depth reduces flow resistance and increases flow velocity. While the increased width and / or depth reduces flow resistance and increases flow velocity, the flow velocity cannot be too high, otherwise it will cause difficulties in subsequent droplet sorting. When the width of the outlet section 14 of the second cross-shaped flow channel 13 is 1.5 to 3 times the width of the outlet end 15, more effective droplets can be obtained more efficiently, and the flow resistance and flow velocity are within a controllable range. In this embodiment, the width of the outlet section of the second cross-shaped flow channel is twice the width of the outlet end, i.e., 100 μm.

[0114] Example 2: Effect of the radius at the bend of the continuous U-shaped flow channel with large corners on the preparation of single-cell single-code microsphere droplets

[0115] This embodiment uses the single-cell single-coded microsphere droplet generator provided in Example 1 to prepare single-cell single-coded microsphere droplets. The radius of the turning point of the continuous U-shaped flow channel with large bends in the coded microsphere flow channel is 3 times (150 μm), 6 times (300 μm), 10 times (500 μm), 15 times (750 μm), 20 times (1000 μm), and 25 times (1250 μm) of the flow channel width, respectively. The pressure at the inlet of the coded microsphere flow channel is controlled at 3.0 psi, the pressure at the inlet of the cell flow channel is 4.5 psi, and the pressure at the inlet of the oil phase flow channel is 8.5 psi. The coded microspheres in the suspension are 6% polyethylene glycol hydrogel microspheres (50–55 μm), and the cell phase consists of 10000 HEK293T cells (ATCC) suspended in 100 μl of cell buffer (50 mM Tris, 75 mM KCl, 3 mM... The mixture consisted of MgCl2, 13% Optiprep (Sigma; D1556), pH 8.3, and an oil phase of fluorinated oil containing 2% surfactant FS10 (the following examples all used the same coded microspheres, cells, and oil phase). The flow resistance and velocity changes in the flow channel were detected, and whether there was any blockage after 1 hour of continuous operation were examined. Samples were taken to compare the total number of generated droplets and the number of effective droplets, and the proportion of effective droplets was calculated. The effect of the radius of the turning point of the large-angle continuous U-shaped flow channel on the preparation of single-cell single-coded microsphere droplets was investigated. The results are shown in Table 1.

[0116] Table 1. Effect of turning radius on the preparation of single-cell, single-code microsphere droplets

[0117]

[0118] As shown in Table 1, when the radius of the bend is three times the width of the flow channel, significant blockage occurs after one hour of continuous operation, leading to a gradual increase in flow resistance, a gradual decrease in flow velocity, a decrease in droplet generation rate, and a significant decrease in the proportion of effective droplets. When the radius of the bend increases to six times the width of the flow channel, under the flow velocity generated by the inlet pressure of the coded microspheres, impurities such as dust and microfibers in the coded microsphere suspension are difficult to accumulate at the bend, thus avoiding blockage and keeping the flow resistance and flow velocity stable, generating more droplets, and increasing the proportion of effective droplets. As the radius of the bend continues to increase, the flow resistance decreases, the flow velocity increases, and the number of generated droplets increases, but due to the excessively high flow velocity, the control difficulty increases, and the proportion of generated effective droplets decreases. In addition, considering the processing and overall flow channel layout, the radius of the bend should not be too large. In this embodiment, the preferred radius of the bend is 10 times the width of the flow channel, at which point the flow velocity is stably maintained at 0.060 μL / s, and the proportion of effective droplets reaches 71.06%.

[0119] Example 3: Effect of the upward tilt angle of the straight flow channel connecting the two bends on the preparation of single-cell single-code microsphere droplets

[0120] This embodiment uses the single-cell single-code microsphere droplet generator provided in Example 1 to prepare single-cell single-code microsphere droplets. The radius of the bend in the large-angle continuous U-shaped flow channel is 10 times the width of the flow channel. The straight flow channel connecting the two bends is inclined upward at angles of 0 degrees, 5 degrees, 10 degrees, 15 degrees, 20 degrees, 25 degrees, and 30 degrees, respectively. The pressure at the inlet of the coded microsphere flow channel is controlled at 3.0 psi, the pressure at the inlet of the cell flow channel is 4.5 psi, and the pressure at the inlet of the oil phase flow channel is 8.5 psi. The changes in flow resistance and flow velocity of the coded microsphere flow channel are detected, and whether there is any blockage after 1 hour of continuous operation is checked. The total number of generated droplets and the number of effective droplets are compared, and the proportion of effective droplets is calculated. The influence of the upward inclination angle of the straight flow channel connecting the two bends in the large-angle continuous U-shaped flow channel on the preparation of single-cell single-code microsphere droplets is investigated. The results are shown in Table 2.

[0121] Table 2. Effect of the upward tilt angle of the straight flow channel connecting the two bends on the preparation of single-cell, single-coding microsphere droplets.

[0122]

[0123] As shown in Table 2, the upward inclination angle of the straight flow channel connecting the two bends has a significant impact on the proportion of effective droplets generated. This is likely due to the different flow resistances generated by different inclination angles, which affect the flow velocity and thus the generation of effective droplets. When the upward inclination angle of the straight flow channel at the two bends is 5 degrees, the flow is relatively stable after 1 hour of continuous operation, with no obvious blockage, a relatively fast flow velocity, and a relatively fast droplet generation rate, with an effective droplet ratio of 11.12%. As the inclination angle increases, the flow resistance increases, the flow velocity decreases, the flow velocity slows down, making it easier to control, and the effective droplet ratio also increases significantly. However, when the inclination angle reaches 30 degrees, the flow resistance is large, the flow velocity decreases significantly, leading to blockage again, and the effective droplet ratio drops rapidly. Therefore, in a continuous U-shaped flow channel with large bends, the upward inclination angle of the straight flow channel connecting the two bends is preferably 10-15 degrees, with 15 degrees being the most preferred, resulting in the highest effective droplet ratio of 72.72%.

[0124] Example 4: Effect of the total length of the continuous U-shaped flow channel with large bends on the preparation of single-cell single-code microsphere droplets

[0125] In this embodiment, the single-cell single-code microsphere droplet generator provided in Example 1 is used to prepare single-cell single-code microsphere droplets. The radius of the turning point of the large-angle continuous U-shaped flow channel is 10 times the width of the flow channel. The straight flow channel connecting the two turns is inclined upward at an angle of 15 degrees. The total lengths of the large-angle continuous U-shaped flow channel are 10000μm, 15000μm, 20000μm, 25000μm, and 30000μm, respectively. The ratio of the length of the straight flow channel at the inlet section to the length of the straight flow channel connecting the two turns to the length of the straight flow channel at the outlet section is 1:1.25:1. The pressure at the inlet of the coded microsphere channel was controlled at 3.0 psi, the pressure at the inlet of the cell channel at 4.5 psi, and the pressure at the inlet of the oil phase channel at 8.5 psi. The changes in flow resistance and flow velocity of the coded microsphere channel were detected, and whether there was any blockage after 1 hour of continuous operation was checked. The total number of generated droplets and the number of effective droplets were compared, and the proportion of effective droplets was calculated. The effect of the total length of the continuous U-shaped channel with large bends on the preparation of single-cell single-coded microsphere droplets was investigated. The results are shown in Table 3.

[0126] Table 3. Effect of total length of continuous U-shaped flow channel with large bends on the preparation of single-cell single-code microsphere droplets

[0127]

[0128] As shown in Table 3, the total length of the large-angle continuous U-shaped flow channel also has a certain impact on the proportion of effective droplets generated. This may be because different total lengths result in different flow resistances, which in turn affect the flow velocity and thus the generation of effective droplets. When the total length of the large-angle continuous U-shaped flow channel is 1000 μm, it is relatively stable after 1 hour of continuous operation, with no obvious blockage, but the flow velocity is relatively fast, the droplet generation rate is fast, and the proportion of effective droplets is slightly low. As the total length of the large-angle continuous U-shaped flow channel increases, the flow resistance increases, the flow velocity decreases, the flow velocity slows down, and it is easier to control, and the proportion of effective droplets also increases. However, when the total length reaches 3000 μm, due to the large flow resistance, the flow velocity decreases significantly, and the proportion of effective droplets also decreases significantly. Therefore, the preferred total length of the large-angle continuous U-shaped flow channel is 1500–2500 μm, with 2000 μm being the most preferred, generating the highest proportion of effective droplets.

[0129] Example 5: Effect of the size of the coded microsphere buffer groove on the preparation of single-cell single-coded microsphere droplets

[0130] This embodiment uses the droplet generator provided in Embodiment 1. The straight flow channels connecting the two bends are inclined upward at an angle of 15 degrees. The total length of the continuous U-shaped flow channel at the large bend is 2000 μm, and it is equipped with coded microsphere buffer grooves. The dimensions of the coded microsphere buffer grooves are set as follows: the width at the widest point is 3 times (150 μm), 5 times (250 μm), 10 times (500 μm), 15 times (750 μm), and 20 times (1000 μm) of the width of the coded microsphere flow channel 1, respectively; the depth at the deepest point is 1 time (50 μm) of the depth of the coded microsphere flow channel 1. The dimensions of the coded microsphere buffer tanks were set as follows: 2 times (100 μm), 3 times (150 μm), 5 times (250 μm), and 7 times (350 μm), all with a length of 1280 μm. The specific dimensions of the coded microsphere buffer tanks are shown in Table 1. The pressure at the inlet of the coded microsphere channel was controlled at 3.0 psi, the pressure at the inlet of the cell channel was 4.5 psi, and the pressure at the inlet of the oil phase channel was 8.5 psi. The tanks were run continuously for 1 hour to investigate the effect of the size of the coded microsphere buffer tanks on the preparation of single-cell single-coded microsphere droplets. The flow rate of the coded microspheres was the flow rate of the coded microspheres at the outlet of the coded microsphere buffer tank. The results are shown in Table 4.

[0131] Table 4. Effect of the size of the coded microsphere buffer groove on the preparation of single-cell single-coded microsphere droplets.

[0132]

[0133] As shown in Table 4, the size of the coded microsphere buffer tank has a significant impact on the preparation of single-cell, single-coded microsphere droplets. When the size is small, the re-aggregation of the coded microspheres is poor, and the coded microspheres may flow out from the bottom of the buffer tank before they are fully pre-arranged. This also leads to deviations in the spacing control after the coded microspheres flow out, resulting in a faster flow rate and affecting the proportion of finite droplets. When the size is too large, the time it takes for the coded microspheres to flow out of the buffer tank and reach the cross-shaped position is much slower than that of the cellular phase, causing sample waste. Therefore, the optimal size of the coded microsphere buffer tank is 10 times the width of the coded microsphere channel 1 (500 μm) at its widest point and 3 times the depth of the coded microsphere channel 1 (150 μm) at its deepest point, at which point the effective droplet proportion reaches 72.69%.

[0134] Example 6: Effect of the size of the oil phase buffer tank on the preparation of single-cell, single-code microsphere droplets

[0135] This embodiment uses the droplet generator provided in Embodiment 1. The straight flow channels connecting the two bends are inclined upward at an angle of 15 degrees. The total length of the continuous U-shaped flow channel at the large bend is 2000 μm, and a coded microsphere buffer tank is provided. The dimensions of the coded microsphere buffer tank are 500 μm at its widest point and 150 μm at its deepest point. The dimensions of the oil phase buffer tank are set as follows: the widths are 3 times (120 μm), 5 times (200 μm), 10 times (400 μm), 15 times (600 μm), and 20 times (800 μm) of the width of the oil phase flow channel 3, respectively. The depths of the oil phase flow channels were 1 (50 μm), 2 (100 μm), 3 (150 μm), 5 (250 μm), and 7 (350 μm) times the depth of the oil phase flow channel 3, respectively, and the length of each channel was 1280 μm. The specific dimensions of the oil phase buffer tank are shown in Table 2. The pressure at the inlet of the coded microsphere flow channel was controlled at 3.0 psi, the pressure at the inlet of the cell flow channel was 4.5 psi, and the pressure at the inlet of the oil phase flow channel was 8.5 psi. The tank was run continuously for 1 hour to investigate the effect of the size of the oil phase buffer tank on the preparation of single-cell single-coded microsphere droplets. The results are shown in Table 5.

[0136] Table 5. Effect of the size of the oil phase buffer tank on the preparation of single-cell, single-code microsphere droplets.

[0137]

[0138] As shown in Table 5, the size of the oil phase buffer tank also has a significant impact on the preparation of single-cell, single-code microsphere droplets. When the size is small, the effect on controlling the oil phase flow rate is weak; when the size is too large, the oil phase flow rate is too slow. Therefore, the preferred size of the oil phase buffer tank is 10 times the width of the oil phase flow channel 3 (400 μm) at its widest point and 3 times the depth of the oil phase flow channel 3 (150 μm), at which point the effective droplet ratio reaches 72.85%.

[0139] Example 7: The Influence of the Combined Design of a Large-Corner Continuous U-Shaped Flow Channel and a Coded Microsphere Buffer Groove on the Preparation of Single-Cell Single-Coded Microsphere Droplets

[0140] In this embodiment, three sets of droplet generators were used. The first set used the droplet generator with the combination design of large-angle continuous U-shaped flow channel and coded microsphere buffer groove provided in Example 1. The second set used a droplet generator with only large-angle continuous U-shaped flow channel. The third set used a droplet generator with only coded microsphere buffer groove. The fourth set used an existing ordinary continuous U-shaped flow channel without coded microsphere buffer groove. The generators were run continuously for 1 hour to investigate the effect of the combination design of large-angle continuous U-shaped flow channel and coded microsphere buffer groove on the preparation of single-cell single-coded microsphere droplets. The results are shown in Table 6.

[0141] Table 6. Effect of the combined design of the large-angle continuous U-shaped flow channel and the coded microsphere buffer groove on the preparation of single-cell single-coded microsphere droplets.

[0142]

[0143] As shown in Table 6, when the large-angle continuous U-shaped channel (Group 2) or the coded microsphere buffer tank (Group 3) is used alone in the coded microsphere channel, compared with the combined design of the large-angle continuous U-shaped channel and the coded microsphere buffer tank (Group 1), although the total number of droplets prepared is greater, the number of effective droplets is very small, resulting in a significant waste of expensive samples. This demonstrates that the combined design of the large-angle continuous U-shaped channel and the coded microsphere buffer tank has a significant synergistic effect in increasing the proportion of effective droplets, saving samples, reducing costs, and exhibiting very obvious beneficial effects.

[0144] Example 8: Effect of the distance between the second and first cross-shaped flow channels on the preparation of single-cell, single-code microsphere droplets.

[0145] This embodiment uses the droplet generator provided in Example 1. The straight flow channels connecting the two bends are inclined upwards at an angle of 15 degrees. The total length of the continuous U-shaped flow channel at the large bend is 20,000 μm. A coded microsphere buffer tank is provided, with a maximum width of 500 μm and a maximum depth of 150 μm. The oil phase buffer tank has a maximum width of 400 μm and a maximum depth of 150 μm. The distances between the second and first cross-shaped flow channels are 20 μm, 40 μm, 60 μm, 80 μm, 150 μm, 350 μm, and 500 μm, respectively. The pressure at the inlet of the coded microsphere flow channel is controlled at 3.0 psi, the pressure at the inlet of the cell flow channel is 4.5 psi, and the pressure at the inlet of the oil phase flow channel is 8.5 psi. After continuous operation for 1 hour, the effect of the distance between the second and first cross-shaped flow channels on the preparation of single-cell, single-coded microsphere droplets is investigated. The results are shown in Table 7.

[0146] Table 7. Effect of the distance between the second and first cross-shaped flow channels on the preparation of single-cell single-code microsphere droplets.

[0147]

[0148] As shown in Table 7, the distance between the second and first cross-shaped channels also has a significant impact on the preparation of single-cell single-code microsphere droplets. When the distance is less than 20 μm or greater than 500 μm, the proportion of finite droplets decreases significantly. Therefore, the distance between the second and first cross-shaped channels must be strictly controlled within 20-500 μm. The main reason is that a certain time interval is required between each shearing and encapsulation of the oil phase to form droplets. The distance between the first and second cross-shaped channels needs to be matched as closely as possible with the time interval between each shearing and encapsulation of the oil phase to form droplets, so as to further improve the efficiency of generating effective droplets.

[0149] Example 9: Effect of the width of the second cross-shaped flow channel droplet nozzle on the preparation of single-cell, single-code microsphere droplets

[0150] This embodiment uses the droplet generator provided in Embodiment 1. The straight flow channels connecting the two bends are inclined upward at an angle of 15 degrees, and the total length of the continuous U-shaped flow channel at the large bend is 2000 μm. It also includes a coded microsphere buffer tank with a maximum width of 500 μm and a maximum depth of 150 μm. The oil phase buffer tank has a maximum width of 400 μm and a maximum depth of 150 μm. The distance between the second cross-shaped flow channel and the first cross-shaped flow channel is 80 μm, wherein the droplet nozzle width of the second cross-shaped flow channel (outlet end 15) is... Figure 4 The microsphere diameters were 40 μm, 50 μm, 60 μm, 70 μm, and 80 μm, respectively. The pressure at the inlet of the coded microsphere channel was controlled at 3.0 psi, the pressure at the inlet of the cell channel at 4.5 psi, and the pressure at the inlet of the oil phase channel at 8.5 psi. After continuous operation for 1 hour, the width of the droplet nozzle in the second cross-shaped channel was examined. Figure 4 The effect of ) on the preparation of single-cell single-code microsphere droplets is shown in Table 8.

[0151] Table 8. Effect of the width of the second cross-shaped flow channel droplet nozzle section on the preparation of single-cell, single-code microsphere droplets.

[0152]

[0153] As shown in Table 8, the width of the second cross-shaped flow channel nozzle has a significant impact on the efficiency of preparing single-cell, single-coded microsphere droplets. When the width is less than 60 μm, the droplet velocity is slow, resulting in low droplet preparation efficiency. Conversely, when the width is greater than 60 μm, the droplet velocity increases, improving droplet preparation efficiency, but the proportion of effective droplets decreases. This is mainly due to the increased width and / or depth, which reduces flow resistance and increases flow velocity. However, excessively high flow velocity leads to a significant increase in the velocity of the coded microspheres, resulting in multiple coded microspheres within the droplet and a decrease in the proportion of effective droplets. When the width of the outlet section of the second cross-shaped flow channel is 60 μm, more effective droplets can be obtained more efficiently, and both flow resistance and flow velocity remain within a controllable range.

[0154] 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 droplet generator, comprising an encapsulated biomaterial channel, an coded microsphere channel, and an oil phase channel; characterized in that, The coded microsphere flow channel includes an arc-shaped flow channel, which comprises one or more arc-shaped flow channels with different radii. The radius of the arc-shaped flow channel is more than 6 times the width of the coded microsphere flow channel. The extensions of two straight flow channels connected to the arc-shaped flow channel intersect at an acute angle. The straight flow channel connecting the two arc-shaped flow channels is arranged at an upward inclination. The arc-shaped flow channel and the straight flow channel connected to the arc-shaped flow channel form a large-angle continuous U-shaped flow channel of the coded microsphere flow channel. The coded microsphere flow channel is also provided with a coded microsphere buffer groove, and the flow resistance of the coded microsphere fluid in the coded microsphere buffer groove is less than the flow resistance in the coded microsphere flow channel. The shape of the coded microsphere buffer groove gradually increases from top to bottom and then rapidly shrinks downward. The biomaterial can be selected from one or more of cells, nucleic acids, proteins, antibodies, or antibody fragments.

2. The droplet generator as described in claim 1, characterized in that, The radius of the arc-shaped flow channel is 6 to 20 times the width of the flow channel.

3. The droplet generator as described in claim 2, characterized in that, The radius of the arc-shaped flow channel is 10 times the width of the flow channel.

4. The droplet generator as described in claim 3, characterized in that, The arc-shaped flow channel includes one or more.

5. The droplet generator as described in claim 4, characterized in that, The arc-shaped flow channels are set in two or more intervals.

6. The droplet generator as described in claim 5, characterized in that, The large-angle continuous U-shaped flow channel includes an inlet section straight flow channel and an outlet section straight flow channel, wherein the angle between the inlet section straight flow channel and the inlet section of the coded microsphere flow channel is greater than or equal to 90 degrees.

7. The droplet generator as described in claim 6, characterized in that, The angle between the straight section of the arc-shaped flow channel and the outlet section of the coded microsphere flow channel is greater than or equal to 90 degrees.

8. The droplet generator according to any one of claims 1 to 7, characterized in that, The volume of the coded microsphere buffer groove is larger than the volume of the coded microsphere channel of the same length.

9. The droplet generator as described in claim 8, characterized in that, The width of the coded microsphere buffer groove is greater than the width of the coded microsphere channel, and / or the depth of the coded microsphere buffer groove is greater than the depth of the coded microsphere channel.

10. The droplet generator as claimed in claim 9, characterized in that, The width of the coded microsphere buffer groove is 5 to 15 times the width of the coded microsphere channel, and the depth is 2 to 5 times the depth of the coded microsphere channel.

11. The droplet generator as claimed in claim 10, characterized in that, The coded microsphere buffer groove is located downstream of the arc-shaped flow channel.

12. The droplet generator as claimed in claim 11, characterized in that, The oil phase flow channel is provided with an oil phase buffer tank, and the flow resistance of the oil phase in the oil phase buffer tank is less than that in the oil phase flow channel.

13. The droplet generator as described in claim 12, characterized in that, The oil phase flow channel is divided into a first oil phase flow channel and a second oil phase flow channel, and each of the first oil phase flow channel and the second oil phase flow channel is provided with an oil phase buffer tank.

14. The droplet generator as described in claim 13, characterized in that, The first oil phase flow channel and the second oil phase flow channel include arc-shaped flow channels, and the oil phase buffer tank is located upstream of the arc-shaped flow channel.

15. The droplet generator as described in claim 14, characterized in that, The volume of the oil phase buffer tank is larger than the volume of an oil phase flow channel of the same length.

16. The droplet generator as claimed in claim 15, characterized in that, The width of the oil phase buffer tank is greater than the width of the oil phase flow channel, and / or the depth of the oil phase buffer tank is greater than the depth of the oil phase flow channel.

17. The droplet generator as claimed in claim 16, characterized in that, The width of the oil phase buffer tank is 5 to 15 times the width of the oil phase flow channel, and the depth is 2 to 5 times the depth of the oil phase flow channel.

18. The droplet generator as claimed in claim 17, characterized in that, The shape of the oil phase buffer tank gradually increases from top to bottom, and then quickly shrinks back downwards.

19. The droplet generator as claimed in claim 18, characterized in that, The encapsulated biomaterial channels and coded microsphere channels form the first cross-shaped channel, and the output channel of the first cross-shaped channel and the oil phase channel form the second cross-shaped channel.

20. The droplet generator as claimed in claim 19, characterized in that, The distance between the first cross-shaped flow channel and the second cross-shaped flow channel is 20-500μm.

21. The droplet generator as described in claim 20, characterized in that, The width and / or depth of the outlet section of the second cross-shaped flow channel is greater than the width and / or depth of the outlet end.

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