DROPLET GENERATOR

Abstract

The present invention provides a droplet generator. The droplet generator includes a large-corner continuous U-shaped flow channel on the encoded microsphere flow channel. Controlling the turning radius of the continuous U-shaped flow channel prevents the blockage resulted from accumulation of microfibers in the encoded microsphere suspension at the turn of the continuous U-shaped flow channel. A buffer tank is arranged at the encoded microsphere flow channel, which is used for the pre-arrangement of the encoded microspheres, to increase the controllability of the flow rate of the encoded microspheres. The buffer tank is provided in the oil-phase flow channel to prevent the oil phase from infiltrating into the aqueous phase due to excessive flow rate, and increase the controllability of the oil phase flow rate. The droplet generator has simple structure, low cost, high-throughput, and high-stability, and may be used to prepare single-cell single-encoded microsphere droplets.

Claims

1. A droplet generator, comprising a wrapped biological material flow channel, an encoded microsphere flow channel and an oil-phase flow channel; wherein the encoded microsphere flow channel comprises an arc-shaped flow channel, the arc-shaped flow channel comprises one or more segments of circular arc-shaped flow channels with different radii, at least one of the circular arc-shaped flow channels has a radius that is more than 6 times the width of the encoded microsphere flow channel; wherein, the encoded microsphere flow channel is also provided with an encoded microsphere flow channel tank, the flow resistance of the encoded microsphere fluid in the encoded microsphere buffer tank is less than the flow resistance of the encoded microsphere flow channel; the biological material can be selected from one or more of cells, nucleic acids, proteins, antibodies or antibody fragments.

2. The droplet generator according to claim 1, wherein the radius of the circular arc-shaped flow channel is 6 to 20 times the width of the flow channel.

3. The droplet generator according to claim 2, wherein the radius of the circular arc-shaped flow channel is 10 times the width of the flow channel.

4. The droplet generator according to claim 1, wherein the extension lines of two straight flow channels that are connected with the arc-shaped flow channel intersect at an acute angle.

5. The droplet generator according to claim 4, wherein there are one or more arc-shaped flow channels.

6. The droplet generator according to claim 5, wherein two or more than two of the arc-shaped flow channels are spaced.

7. The droplet generator according to claim 6, wherein the straight flow channel connecting the two arc-shaped flow channels is arranged horizontally or arranged to be inclined upwardly or inclined downwardly.

8. The droplet generator according to claim 7, wherein the straight flow channel connecting the two arc-shaped flow channels is arranged to be inclined upwardly, the arc-shaped flow channel and the straight flow channel that is connected with the arc-shaped flow channel form a large-corner continuous U-shaped flow channel of the encoded microsphere flow channel.

9. The droplet generator according to claim 8, wherein the large-corner continuous U-shaped flow channel comprises an inlet segment straight flow channel and an outlet segment straight flow channel, and the angle between the inlet segment straight flow channel and the inlet segment of the encoded microsphere flow channel is greater than or equal to 90 degrees.

10. The droplet generator according to claim 9, wherein the angle between the outlet segment straight flow channel of the arc-shaped flow channel and the outlet segment of the encoded microsphere flow channel is greater than or equal to 90 degrees.

11. The droplet generator according to claim 1, wherein the volume of the encoded microsphere buffer tank is greater than the volume of the encoded microsphere flow channel of the same length.

12. The droplet generator according to claim 11, wherein the width of the encoded microsphere buffer tank is greater than the width of encoded microsphere flow channel, and/or the depth of the encoded microsphere buffer tank is greater than the depth of the encoded microsphere flow channel.

13. The droplet generator according to claim 12, wherein the width of the encoded microsphere buffer tank is 5 to 15 times the width of the encoded microsphere flow channel, and the depth of the encoded microsphere buffer tank is 2 to 5 times the depth of the encoded microsphere flow channel.

14. The droplet generator according to claim 13, wherein the shape of the encoded microsphere buffer tank gradually enlarges from top to bottom, and then rapidly retracts downwardly.

15. The droplet generator according to claim 14, wherein the encoded microsphere buffer tank is located downstream of the arc-shaped flow channel.

16. The droplet generator according to claim 15, wherein 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.

17. The droplet generator according to claim 16, wherein 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.

18. The droplet generator according to claim 17, wherein the first oil-phase flow channel and the second oil-phase flow channel comprise an arc-shaped flow channel, and the oil phase buffer tank is located upstream of the arc-shaped flow channel.

19. The droplet generator according to claim 18, wherein the volume of the oil phase buffer tank is greater than the volume of the oil-phase flow channel of the same length.

20. The droplet generator according to claim 19, wherein the width of the oil phase buffer tank is 5 to 15 times the width of the oil-phase flow channel, and the depth of the oil phase buffer tank is 2 to 5 times the depth of the oil-phase flow channel.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0082] FIG. 1 is a schematic diagram of a structure of an encoded microsphere flow channel in a droplet generator of Example 1;

[0083] FIG. 2 is a schematic diagram of an overall structure of a droplet generator of Example 1.

[0084] FIG. 3 is a schematic diagram of an overall structure of a droplet generator.

[0085] FIG. 4 is an enlarged view of a specific structure of a droplet generator.

[0086] FIG. 5 is a schematic diagram of a process flow of droplet wrapping.

DETAILED DESCRIPTION

[0087] The preferred embodiments of the present invention will be further described in detail below with reference to the accompanying drawings. It should be noted that the following embodiments are intended to facilitate the understanding of the present invention, but do not constitute any limitation on the present invention. The raw materials and equipment used in the specific embodiments of the present invention are all known products, and are commercially available.

Example 1 Droplet Generator of the Present Invention

[0088] The schematic diagram of a droplet generator provided by the example is shown as FIGS. 1 to 3, wherein FIG. 1 is a schematic diagram of a large-corner continuous U-shaped flow channel of an encoded microsphere flow channel; FIG. 2 is a schematic diagram of an encoded microsphere flow channel in a droplet generator; and FIG. 3 is a schematic diagram of an overall structure of a droplet generator.

[0089] As shown as FIGS. 1 to 3, a single-cell single-encoded microsphere droplet generator comprises an encoded microsphere flow channel 1, a cell flow channel 2 and an oil-phase flow channel 3, wherein the encoded microsphere flow channel 1 is provided with a large-corner continuous U-shaped flow channel 110 and an encoded microsphere buffer tank 4.

[0090] As shown in FIG. 1, the large-corner continuous U-shaped flow channel 110 of the encoded microsphere flow channel 1 comprises a first turn 101, a second turn 102, a straight flow channel 103 connecting two turns, an inlet segment straight flow channel 104 and an outlet segment straight flow channel 105, wherein the size of a turning radius 106 of the first turn 101 is 6 times greater than the width of the encoded microsphere flow channel 1, and the size of a turning radius 7 of the second turn 102 is also 6 times greater than the width of the encoded microsphere flow channel 1. Because the diameter of encoded microsphere is larger and is about 50 μm generally, a filter membrane with the pore diameter of less than 50 μm cannot be adopted for filtration, and impurities such as dust and microfibers can be remained in encoded microsphere suspension. When a common continuous U-shaped flow channel (such as a continuous U-shaped flow channel 108 of the cell flow channel 2 in FIG. 3) is adopted, the impurities such as the dust and the microfibers in the encoded microsphere suspension are easy to gradually accumulate at the turns of the continuous U-shaped flow channel in the continuous using process to cause blockage so as to seriously influence the preparation efficiency and the quality of droplets. In this example, the radius of the turns (the first turn 101 or the second turn 102) is controlled in a proper large range by arranging the continuous U-shaped flow channel 110 with large turns for the encoded microsphere suspension, and when the microsphere suspension passes through the turning radius, the impurities such as the dust and the microfibers cannot be accumulated at the turns at a certain flow rate, so that the problem that the encoded microsphere suspension is easy to block at the turns after long-time operation is solved.

[0091] For example, in this example, when the flow rate of encoded microsphere suspension is 0.01-0.1 μL/s, impurities such as dust and microfibers in the encoded microsphere suspension are completely washed away when the encoded microsphere suspension passes through a turning with a larger turning radius at the flow rate, and the impurities cannot be accumulated at the turning; however, at the same flow rate, if the turning radius is smaller, the impurities such as the dust and the microfibers in the encoded microsphere suspension are possibly not taken away in time and are gradually deposited, the flow resistance is larger and the flow rate is smaller and smaller along with more and more deposition of the impurities such as the dust and the microfibers, the deposition phenomenon is further aggravated, and blockage can be caused after long-time operation. A large number of studies have demonstrated that, when the flow rate of the encoded microsphere suspension is 0.01-0.1 μL/s, the impurities such as the dust and the microfibers in the encoded microsphere suspension cannot be accumulated at the turning only by controlling the turning radius of the continuous U-shaped flow channel 110 to be more than 6 times the width of an encoded microsphere flow channel 1, so that long-time smooth operation of the encoded microsphere flow channel 1 is guaranteed. Particularly, under the condition that some fibers are longer, the fibers can be curled up to block a micro-channel, and the length of the fibers is generally larger than the width or the depth of the micro-channel.

[0092] The first turn 101 and the second turn 102 are both arc-shaped flow channels, can be in regular arc shapes, and can also be arc-shaped flow channels composed of a plurality of sections of circular arc-shaped flow channels with different radii, but the radius of at least one section of arc shape must be more than 6 times the width of the flow channels.

[0093] The straight flow channel 103 and the inlet segment straight flow channel 104 can be parallel or not parallel, and the straight flow channel 103 and the outlet segment strain flow channel 105 can be parallel or not parallel. In this example, the straight flow channel 103 and the inlet segment straight flow channel 104 are not parallel, and the straight flow channel 103 and the inlet segment straight flow channel 104 intersect with each other along the extension line of the first turn 101 to form an acute angle; the straight flow channel 103 and the outlet segment strain flow channel 105 are not parallel, and the straight flow channel 103 and the outlet segment strain flow channel 105 intersect with each other along the extension line of the second turn 102 to form an acute angle.

[0094] Preferably, the turning radius of the large-corner continuous U-shaped flow channel 110 is 6 to 20 times the width of the encoded microsphere flow channel 1. The turning radius of the continuous U-shaped flow channel 110 cannot be increased infinitely, the layout arrangement of a whole microfluidic system, the space area occupied by the flow channel and cost control need to be considered, meanwhile, proper control over flow resistance needs to be guaranteed, and therefore the flow speed of encoded microsphere flow is controlled, and generation of effective droplets is guaranteed. In this example, the turning radius of the large-corner continuous U-shaped flow channel 110 is 10 times the width of the encoded microsphere flow channel 1. As is proved by experiments, when the turning radius of the continuous U-shaped flow channel 110 is 10 times the width of the encoded microsphere flow channel 1, it can be guaranteed that dust, microfibers and other impurities in encoded microsphere suspension cannot be accumulated at the turning, the flow resistance can be better controlled, and therefore the flow speed of the encoded microsphere flow is stabilized, and the generation efficiency of the effective droplets is further improved.

[0095] The straight flow channel 103 connecting two turns can be arranged horizontally or arranged in a slightly upward inclined manner. In this example, as shown in FIG. 1, the straight flow channel 103 connecting the two turns is slightly inclined upwardly (the upward direction is the upstream direction of a fluid), so that the flow resistance can be improved, and the flow rate can be controlled more stably.

[0096] Preferably, as shown in FIG. 1 and FIG. 2, the angle 111 between the inlet segment straight flow channel 104 and an encoded microsphere flow channel inlet segment 109 of the large-corner continuous U-shaped flow channel 110 is larger than 90 degrees (the encoded microsphere flow channel inlet segment 109 is directly connected with an encoded microsphere flow channel inlet 125), and the angle 13 between the outlet segment straight flow channel 105 and the encoded microsphere flow channel outlet segment 112 of the large-corner continuous U-shaped flow channel 110 is larger than 90 degrees. According to the above arrangement, it is guaranteed that the continuous U-shaped flow channel 110 is bent greatly, and meanwhile the connecting positions of the continuous U-shaped flow channel 110 and other straight light flow channels need to be bent greatly, so that the phenomenon that the continuous U-shaped flow channel 110 is bent by less than 90 degrees is avoided. By means of the design, the encoded microsphere suspension can be smoothly transited from other straight flow channels to the continuous U-shaped flow channel 110 and from the continuous U-shaped flow channel 110 to other straight flow channels, and blocking caused by impurities such as dust and microfibers in the encoded microsphere suspension can be prevented.

[0097] Preferably, In this example, the width of the encoded microsphere flow channel 1 is 50 μm, and the turning radius of the large-corner continuous U-shaped flow channel 110 is 500 μm. The widths of all encoded microsphere flow channels 1 are the same, the continuous U-shaped flow channel 110 is a part of the encoded microsphere flow channels 1, and the width of the continuous U-shaped flow channel 110 is the same as the width of the other part of the encoded microsphere flow channels 1.

[0098] Preferably, the total length of the large-corner continuous U-shaped flow channel 110 is 20,000 μm; the straight flow channel 103 connecting two turns inclines upwards, an angle 114 between the straight flow channel and the horizontal line is 15 degrees, and the length of the straight flow channel is 6,240 μm; the length of the inlet segment straight flow channel 104 is 5,000 μm, and an angle 111 between the inlet segment straight flow channel and the encoded microsphere flow channel inlet segment 109 is 120 degrees; the length of the outlet segment straight flow channel 105 is 5,000 μm, and an angle 113 between the outlet segment strain flow channel and the encoded microsphere flow channel outlet segment 112 is 120 degrees. 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, the flow resistance can be further helped to be controlled more accurately, the flow rate is helped to be adjusted more accurately, and therefore the generation rate of effective droplets is increased.

[0099] As shown in FIG. 2 and an FIG. 3, the width of the widest part of the encoded microsphere buffer tank 4 is 5 to 15 times the width of the encoded microsphere flow channel 1, and the depth of the deepest part of the encoded microsphere buffer tank is 2 to 5 times the depth of the encoded microsphere flow channel 1; the shape of the encoded microsphere buffer tank 4 gradually enlarges from top to bottom and then rapidly retracts downwardly, the whole is similar to a water drop shape, and the boundary is smooth. The shape of the encoded microsphere buffer tank 4 has a very important influence on control over the flow rate of encoded microsphere, the shape of the encoded microsphere buffer tank gradually enlarges from top to bottom and then rapidly retracts downwardly, the flow resistance of encoded microsphere suspension entering the encoded microsphere buffer tank 4 is gradually reduced, the flow resistance is rapidly increased after the encoded microsphere buffer tank passes through the encoded microsphere buffer tank 4, and therefore the encoded microspheres are pre-arranged in the encoded microsphere buffer tank 4; after the encoded microsphere buffer tank 4 is filled, each encoded microsphere overcomes the suddenly-increased flow resistance from the lower end of the encoded microsphere buffer tank 4 one by one and flows out along with fluid at equal intervals. The boundary of the encoded microsphere buffer tank 4 is smooth, the dead volume can be avoided, and the encoded microsphere stored in the encoded microsphere buffer tank 4 can flow out in sequence and cannot stay in the encoded microsphere buffer tank. The size of the encoded microsphere buffer tank 4 can directly influence the effects of pre-arrangement of the encoded microspheres and spacing control of the encoded microspheres of the encoded microsphere buffer tank 4. The width of the widest part of the encoded microsphere buffer tank 4 is 5 to 15 times the width of the encoded microsphere flow channel 1, the depth of the deepest part of the encoded microsphere buffer tank is 2 to 5 times the depth of the encoded microsphere flow channel 1, the effect of the encoded microsphere buffer tank 4 can be better guaranteed, and efficient and high-quality generation of droplets is guaranteed.

[0100] Preferably, the encoded microsphere flow channel 1 further comprises a large-corner continuous U-shaped flow channel 110, an encoded microsphere buffer tank 4 is located below the large-corner continuous U-shaped flow channel 110, and encoded microsphere suspension passes through the large-corner continuous U-shaped flow channel 110 from the encoded microsphere inlet 20 and then enters the encoded microsphere buffer tank 4. It can be understood that the encoded microsphere buffer tank 4 plays a role normally, has a close relation with the property, and can achieve pre-arrangement of encoded microspheres and accurate control over the spacing of discharging the encoded microspheres only through the shape that the encoded microsphere buffer tank gradually enlarges downwards and then rapidly retracts downwardly in a vertically downward straight flow channel, the large-corner continuous U-shaped flow channel 110 is completely a bent flow channel, no vertically downward straight flow channel exists, and therefore the encoded microsphere buffer tank 4 cannot be arranged on the large-corner continuous U-shaped flow channel 110 and can only be arranged on a vertical straight flow channel 6 behind the large-corner continuous U-shaped flow channel. In this example, the width of the encoded microsphere flow channel 1 is 50 μm, and the depth of the encoded microsphere flow channel is 50 μm; the width of the widest portion of the encoded microsphere buffer tank 4 is 500 μm, the depth of the deepest portion of the encoded microsphere buffer tank is 150 μm, and the length of the deepest portion of the encoded microsphere buffer tank 4 is 1,280 μm.

[0101] As shown in FIG. 2, an oil-phase flow channel 3 is provided with an oil phase buffer tank 7. 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 the first oil-phase flow channel 8 and the second oil-phase flow channel 9 are respectively provided with an oil phase buffer tank 7. The first oil-phase flow channel 8 and the second oil-phase flow channel 9 are respectively provided with an oil-phase continuous U-shaped flow channel 10, and the oil phase buffer tank 7 is positioned on the oil-phase continuous U-shaped flow channel 10.

[0102] 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 of the oil phase buffer tank is 2 to 5 times the depth of the oil-phase flow channel 3; the shape of the oil phase buffer tank 7 gradually enlarges from top to bottom and then is rapidly retracts downwardly, and the boundary is smooth. The oil phase buffer tank 7 is equivalent to a reservoir, so that the oil phase must fill the oil phase buffer tank 7 firstly and then flows to the oil-phase flow channel 3 behind. The size of the oil phase buffer tank 7 directly affects the effects of increasing the controllability of the oil phase buffer tank 7 to the flow rate of the oil phase and improving the resistance of the oil phase to reversely permeate into a water phase. The width of the oil phase buffer tank 7 is controlled to be 5 to 15 times the width of the oil-phase flow channel, and the depth of the oil phase buffer tank is controlled to be 2 to 5 times the depth of the oil-phase flow channel, so that the effects achieved by the oil phase buffer tank 7 can be better ensured, and high-efficiency and high-quality generation of effective droplets is further ensured. In this example, the width of the oil-phase flow channel 3 is 40 μm, and the depth of the oil-phase flow channel is 40 μm; the width of the widest part of the oil phase buffer tank 7 is 400 μm, the depth of the deepest part of the oil phase buffer tank is 150 μm, and the length of the oil phase buffer tank is 1,280 μm.

[0103] The droplet generator of this example adopts a double cross-shaped droplet generator, a first cross-shaped flow channel 11 is formed by a cell flow channel 2 and an encoded microsphere flow channel 1, and a second cross-shaped flow channel 13 is formed by an output channel 12 of the first cross-shaped flow channel 11 and the oil-phase flow channel 3.

[0104] As shown in FIG. 3, the encoded microsphere flow channel 1 and the cell flow channel 2 form a first cross-shaped flow channel 11. Because a cell sample is very precious and rare, and the cell volume is small, the cell suspension is almost like an aqueous phase, and the ratio of effective drops can be increased as much as possible only by controlling the flow rate of the cell suspension, so that the flow condition of each cell is difficult to accurately control; and the encoded microspheres are low in price, large in amount, large in volume and easy to control. In order to ensure that the first cross-shaped flow channel 11 can just meet one encoded microsphere and is smoothly wrapped each time when the cell suspension flows out, the flow rate of the cell suspension needs to be controlled as much as possible, and more importantly, the spacing between each encoded microsphere and the encoded microsphere needs to be controlled as much as possible, so that each cell can be combined with the corresponding encoded microsphere and is wrapped into a droplet when passing through the corresponding encoded microsphere, and thus the cell sample can be fully utilized, the waste of expensive cell samples cannot be caused, and the ratio of effective drops can be increased. The output channel 12 of the first cross-shaped flow channel 11 and the oil-phase flow channel 3 form a second cross-shaped flow channel 13, and after passing through the second cross-shaped flow channel 13, the encoded microspheres and the cells are wrapped by an oil phase and are sheared to generate droplets.

[0105] As shown in FIG. 4, the length of an output channel 12 between the first cross-shaped flow channel 11 and the second cross-shaped flow channel 13 must be kept at 20-500 μm, so that the effect of forming effective droplets by shearing and wrapping an oil phase can be better exerted, and the main reason is that a certain time interval is needed between the droplets formed by shearing and wrapping the oil phase every time, and the spacing between the first cross-shaped flow channel 11 and the second cross-shaped flow channel 13 needs to be matched with the time interval between the droplets formed by shearing and wrapping the oil phase every time as much as possible, so that the efficiency of generating the effective droplets can be further improved. In the, the length of the output channel 12 between the first cross-shaped flow channel 11 and the second cross-shaped flow channel 13 is 80 μm.

[0106] Preferably, the width and/or depth of the outlet segment flow channel 14 of the second cross-shaped flow channel 13 is larger than that of the outlet end 15. When the width and/or depth of the outlet segment flow channel 14 of the second cross-shaped flow channel 13 is larger than that of the outlet end 15, generated droplets can flow out more quickly, and due to the fact that the width and/or depth is increased, the flow resistance is reduced, and the flow speed is increased. Due to the fact that the width and/or depth is increased, the flow resistance is reduced, the flow speed is increased, but the flow speed cannot be too high, otherwise, subsequent droplet sorting is difficult, when the width of the outlet segment flow channel 14 of the second cross-shaped flow channel 13 is 1.5 to 3 times that of the outlet end 15, more effective droplets can be obtained more efficiently, and the flow resistance and the flow speed are both within a controllable range. In this example, the width of the outlet segment flow channel of the second cross-shaped flow channel is twice that of the outlet end, namely 100 μm.

Example 2 Effect of the Turning Radius of a Large-Corner Continuous U-Shaped Flow Channel on Preparation of Single-Cell Single-Encoded Microsphere Droplets

[0107] In this example, the single-cell single-encoded microsphere droplet generator provided by Example 1 is used to prepare single-cell single-encoded microsphere droplets, wherein the turning radius of the large-corner continuous U-shaped flow channel of the encoded microsphere flow channel is respectively 3 times (150 μm), 6 times (300 μm), 10 times (500 μm), 15 times (750 μm), 20 times (1,000 μm) and 25 times (1,250 μm) of the width of the flow channel; the pressure at the inlet of the encoded microsphere flow channel is controlled at 3.0 psi; the pressure at the inlet of a cell flow channel is controlled at 4.5 psi; the pressure at the inlet of an oil-phase flow channel is controlled at 8.5 psi; the encoded microsphere in the encoded microsphere suspension adopts 6% polyethylene glycol hydrogel microspheres (50-55 μm); the cell phase is that 10000 HEK293T cells (ATCC) are suspended in 100 μm of cell buffer (50 mM Tris, 75 mM KCl, 3 mM MgCl2, 13% Optiprep (Sigma; D1556), and the pH is 8.3); the oil phase is fluorinated oil containing 2% of surfactant FS10 (the following examples adopt consistent encoded microsphere, cell and oil phases); the flow resistance and flow rate changes of the flow channel and whether the blocking condition exists after continuous operation for 1 hour are detected, samples are taken to compare the total number of generated droplets and the number of generated effective droplets, the ratio of effective droplets is calculated, the effect of the turning radius of the large-corner continuous U-shaped flow channel on the preparation of the single-cell single-encoded microsphere droplets is investigated, and the results are shown in Table 1.

TABLE-US-00001 TABLE 1 Effect of turning radius on preparation of single- cell single-encoded microsphere droplets Flow of Ratio of Turning Changes encoded Total number Number of effective radius of flow microsphere of sampled effective droplets (μm) resistance (μL/s) Blockage droplets droplets (%) 150 Increased 0.015 Blocked 159814 27680 17.32 flow resistance 300 Stable flow 0.030 None 206013 106076 51.49 resistance 500 Stable flow 0.050 None 219814 156200 71.06 resistance 750 Stable flow 0.063 None 220141 132833 60.34 resistance 1000 Stable flow 0.069 None 225587 117350 52.02 resistance 1250 Stable flow 0.075 None 234435 90656 38.67 resistance

[0108] As shown in Table 1, when the turning radius is 3 times the width of the flow channel, an obvious blocking phenomenon occurs after continuous operation for one hour, so that the flow resistance gradually rises, the flow speed gradually decreases, the droplet generation speed decreases, and the proportion of effective droplets is also obviously decreased; when the turning radius rises to 6 times the width of the flow channel, impurities such as dust and microfibers in encoded microsphere dispersion are difficult to accumulate at the turning at the flow speed generated by the pressure of an encoded microsphere inlet, so that the blocking phenomenon is avoided, the flow resistance and the flow speed are always kept stable, more droplets are generated, and the ratio of effective droplets is also increased; and with the continuous increase of the turning radius, the flow resistance becomes smaller, the flow speed is increased, the number of generated droplets is increased, but the control difficulty is increased due to the too high flow speed, and the Ratio of the generated effective droplets is decreased. In addition, considering from the aspects of processing and the overall layout of the flow channel, the turning radius is not suitable for being too large, the turning radius is preferably 10 times the width of the flow channel, at the moment, the flow speed is stably kept at 0.060 μL/s, and the ratio of effective droplets reaches 71.06%.

Example 3 Effect of the Angle of Upward Inclination of the Straight Flow Channel Connecting Two Turnings on Preparation of Single-Cell Single-Encoded Microsphere Droplets

[0109] In this example, the single-cell single-encoded microsphere droplet generator provided by Example 1 is adopted to prepare single-cell single-encoded microsphere droplets, wherein the turning radius of the large-corner continuous U-shaped flow channel is 10 times the width of the flow channel, the angle of upward inclination of the straight flow channel connecting two turnings is 0, 5, 10, 15, 20, 25 and 30 degrees respectively. The pressure at the inlet of the encoded 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 flow resistance and flow rate change condition of the encoded microsphere flow channel and whether the blocking condition exists after continuous operation for 1 hour are detected, samples are taken to compare the total number of generated droplets and the number of generated effective droplets, the ratio of effective droplets is calculated, the effect of the angle of upward inclination of the straight flow channel at the two turnings of the large-corner continuous U-shaped flow channel on the preparation of the single-cell single-encoded microsphere droplets is investigated, and the results are shown in Table 2.

TABLE-US-00002 TABLE 2 Effect of the angle of upward inclination of the straight flow channel connecting the two turnings on the preparation of single-cell single-encoded microsphere droplets Flow of Ratio of Angle of Changes encoded Total number Number of effective inclination of flow microsphere of sampled effective droplets (degrees) resistance (μL/s) Blockage droplets droplets (%) 0 Stable flow 0.050 None 229508 117324 51.12 resistance 5 Stable flow 0.049 None 227461 140753 61.88 resistance 10 Stable flow 0.046 None 219282 157993 72.05 resistance 15 Stable flow 0.042 None 217327 158040 72.72 resistance 20 Stable flow 0.038 None 198492 122549 61.74 resistance 25 Stable flow 0.031 None 174623 90193 51.65 resistance 30 Gradually 0.013 Blocked 129332 35683 27.59 increased flow resistance

[0110] As shown in Table 2, the angle of upward inclination of the straight flow channel connecting the two turnings has a great influence on the generation ratio of the effective droplets. The reason is that, different flow resistance is produced at different angles of upward inclination, which influences the flow rate and thus influences the generation of effective droplets. When the angle of upward inclination of the straight flow channel at the two turnings is 5 degrees, it is stable after 1 hour of continuous operation, and there is no obvious blockage, the flow rate is faster, the droplet generation speed is faster, and the proportion of effective droplets is 11.12%; with the increase of the angle of inclination, the flow resistance increases, the flow rate decreases, the flow rate becomes slower, and it is easier to control, and the proportion of effective droplets also increases significantly; but when the angle of inclination reaches 30 degrees, the flow resistance increases, the flow rate drops significantly, causing reappearance of the blockage phenomenon, and the ratio of effective droplets drops rapidly. Therefore, the angle of upward inclination of the straight flow channel connecting the two turnings in the large-corner continuous U-shaped flow channel is preferably 10-15 degrees, most preferably 15 degrees, and the ratio of generated effective droplets is the highest, up to 72.72%.

Example 4 Effect of the Total Length of the Large-Corner Continuous U-Shaped Flow Channel on the Preparation of Single-Cell Single-Encoded Microsphere Droplets

[0111] In this example, the single-cell single-encoded microsphere droplet generator provided by Example 1 is used to prepare single-cell single-encoded microsphere droplets. The turning radius of the large-corner continuous U-shaped flow channel is 10 times the width of the flow channel. The angle of upward inclination of the straight flow channel at the two turnings is 15 degrees, and the total length of the large-corner continuous U-shaped flow channel is 10,000 μm, 15,000 μm, 20,000 μm, 25,000 μm, 30,000 μm, respectively, wherein the ratio of the length of the inlet segment straight flow channel to the length of the straight flow channel connecting two turnings to the linear length of the outlet segment is 1:1.25:1. The pressure at the inlet of the encoded microsphere flow channel is controlled at 3.0 psi, the pressure at the inlet of the cell flow channel is controlled at 4.5 psi, and the pressure at the inlet of the oil-phase flow channel is controlled at 8.5 psi. The flow resistance and flow rate changes of the encoded microsphere flow channel and whether the blocking condition exists after continuous operation for 1 hour are detected, samples are taken to compare the total number of generated droplets and the number of generated effective droplets, the ratio of effective droplets is calculated, the effect of the total length of the large-corner continuous U-shaped flow channel on the preparation of the single-cell single-encoded microsphere droplets is investigated, and the results are shown in Table 3.

TABLE-US-00003 TABLE 3 Effect of the total length of the large-corner continuous U-shaped flow channel on the preparation ofsingle-cell single-encoded microsphere droplets Flow of Ratio of Total Changes encoded Total number Number of effective length of flow microsphere of sampled effective droplets (μm) resistance (μL/s) Blockage droplets droplets (%) 10000 Stable flow 0.0068 None 227032 136923 60.31 resistance 15000 Stable flow 0.0061 None 219817 156883 71.37 resistance 20000 Stable flow 0.0052 None 216735 158108 72.95 resistance 25000 Stable flow 0.0047 None 206211 145420 70.52 resistance 30000 Stable flow 0.0031 None 195754 120741 61.68 resistance

[0112] As shown in Table 3, the total length of the large-corner continuous U-shaped flow channel also exerts certain effect on the ratio of generating effective droplets. The reason may be that, the total length is different, the flow resistance produced is different, thereby influencing the flow rate and the generation of effective droplets. When the total length of the large-corner continuous U-shaped flow channel is 1000 μm, it is stable after 1 hour of continuous operation, and there is no obvious blocking phenomenon, but the flow rate is faster, the droplet generation speed is faster, and the proportion of effective droplets is slightly lower; as the total length of the large-corner continuous U-shaped flow channel increases, the flow resistance increases, the flow rate decreases, and it is easier to control, and the proportion of effective droplets also increases; but when the total length reaches 3,000 μm, because of a large flow resistance, the flow rate decreases obviously and the ratio of effective droplets also decreases obviously. Thus, the total length of the large-corner continuous U-shaped flow channel is preferably 1,500-2,500 μm, and most preferably 2,000 μm, and the ratio of generated effective droplets is the highest.

Example 5 Effect of the Size of Encoded Microsphere Buffer Tank on the Preparation of Single-Cell Single-Encoded Microsphere Droplets

[0113] In this example, the droplet generator provided by Example 1 is used. The angle of upward inclination of the straight flow channel at two turnings is 15 degrees. The total length of the large-corner continuous U-shaped flow channel is 2000 μm, and an encoded microsphere buffer tank is provided. Wherein the size of the encoded microsphere buffer tank is set as follows: the width at the widest part is 3 times (150 μm), 5 times (250 μm), 10 times (500 μm), 15 times (750 μm), and 20 times (1,000 μm) of the width of the encoded microsphere flow channel 1 respectively, the depth at the deepest part is 1 time (50 μm), 2 times (100 μm), 3 times (150 μm), 5 times (250 μm) and 7 times (350 μm) of the depth of encoded microsphere flow channel 1 respectively, the length is 1,280 μm. The size of the encoded microsphere buffer tank is set as shown in Table 1. The pressure at the inlet of the encoded microsphere flow channel is controlled at 3.0 psi; the pressure at the inlet of the cell flow channel is controlled at 4.5 psi; the pressure at the inlet of an oil-phase flow channel is controlled at 8.5 psi; after continuous operation for 1 hour, the effect of the size of the encoded microsphere buffer tank on the preparation of single-cell single-encoded microsphere droplets is investigated, wherein the flow rate of the encoded microspheres is the flow rate of the encoded microspheres at the outlet of the encoded microsphere buffer tank, and the results are shown in Table 4.

TABLE-US-00004 TABLE 4 Effect of the size of the encoded microsphere buffer tank on the preparation of single-cell single-encoded microsphere droplets Width at Depth at Flow rate Ratio of the widest the deepest of encoded Total number Number of effective Serial part part microspheres of sampled effective droplets No. (μm) (μm) (μL/s) droplets droplets (%) 1 150 50 0.060 58038 27789 47.88 2 300 100 0.056 62423 38127 61.08 3 500 150 0.050 67157 48816 72.69 4 750 250 0.046 64072 39308 61.35 5 1000 350 0.040 57781 34663 59.99

[0114] As shown in Table 4, the size of the encoded microsphere buffer tank has a great influence on the preparation of single-cell single-encoded microsphere droplets. When the size is small, the re-aggregation effect of the encoded microspheres is poor, and it is possible that, the encoded microspheres have not been well pre-arranged in the buffer tank before flowing out of the lower end of the buffer tank. There is also a deviation in the spacing control effect of the encoded microspheres after flowing out, and the encoded microspheres have a faster flow rate, which affects the proportion of effective droplets. When the size is too large, the time of the encoded microsphere flowing out of the buffer tank and reaching the cross position will be much slower than that of the cell phase, resulting in waste of samples. Therefore, the size of the encoded microsphere buffer tank is preferably 10 times the width of the encoded microsphere flow channel 1 (500 μm) at the widest part, and the depth of the deepest part is 3 times the depth of the encoded microsphere flow channel 1 (150 μm). At this time, the ratio of effective droplets reaches 72.69%.

Example 6 Effect of Size of Oil Phase Buffer Tank on the Preparation of Single-Cell Single-Encoded Microsphere Droplets

[0115] In this example, the droplet generator provided by Example 1 is used. The angle of upward inclination of the straight flow channel at the two turnings is 15 degrees. The total length of the large-corner continuous U-shaped flow channel is 2,000 μm, and an encoded microsphere buffer tank is provided, in which the size of the encoded microsphere buffer tank is 500 μm at the widest part, 150 μm at the deepest part. The size of the oil phase buffer tank is set as follows: the width is (120 μm), 5 times (200 μm), 10 times (400 μm), 15 times (600 μm), 20 times (800 μm) of the width of the oil-phase flow channel 3, and the depth is 1 time (50 μm), 2 times (100 μm), and 3 times (150 μm), 5 times (250 μm), 7 times (350 μm) of the depth of the oil-phase flow channel 3, and the length is 1280 μm. The size of the oil phase buffer tank is set as shown in Table 2. The pressure at the inlet of the encoded microsphere flow channel is controlled at 3.0 psi; the pressure at the inlet of the cell flow channel is controlled at 4.5 psi; the pressure at the inlet of an oil-phase flow channel is controlled at 8.5 psi; after continuous operation for 1 hour, the effect of the size of the oil phase buffer tank on the preparation of single-cell single-encoded microsphere droplets is investigated. The results are shown in Table 5.

TABLE-US-00005 TABLE 5 Effect of the size of the oil phase buffer tank on the preparation of single-cell single-encoded microsphere droplets Width at Depth at Flow rate Ratio of the widest the deepest of oil Number of Number of effective Serial part part phase effective effective droplets No. (μm) (μm) (μL/s) droplets droplets (%) 1 120 50 0.040 62452 30014 48.06 2 200 100 0.034 62673 40825 65.14 3 400 150 0.030 67966 49513 72.85 4 600 250 0.026 64114 35666 55.37 5 800 350 0.020 67868 27799 40.96

[0116] As shown in Table 5, the size of the oil phase buffer tank also has a great influence on the preparation of single-cell single-encoded microsphere droplets. When the size is small, the control effect on the oil phase flow rate is small; when the size is too large, the oil phase flow rate is too slow. Therefore, the size of the oil phase buffer tank is preferably 10 times the width of the oil-phase flow channel 3 (400 μm) at the widest part, and the depth of the deepest part is 3 times the depth of the oil-phase flow channel 3 (150 μm). At this time, the ratio of effective droplets reaches 72.85%.

Example 7 Effect of Combined Design of a Large-Corner Continuous U-Shaped Flow Channel and an Encoded Microsphere Buffer Tank on the Preparation of Single-Cell Single-Encoded Microsphere Droplets

[0117] In this embodiment, three groups of droplet generators are used respectively. The first group uses a droplet generator with the combination of the large-corner continuous U-shaped flow channel and the encoded microsphere buffer tank provided by Example 1, the second group uses a droplet generator containing the large-corner continuous U-shaped flow channel only, and the third group uses a droplet generator containing the encoded microsphere buffer tank only, and the fourth group uses an existing common continuous U-shaped flow channel and a droplet generator without an encoded microsphere buffer tank. After operating for 1 hour continuously, the effect of the combined design of the large-corner continuous U-shaped flow channel and the encoded microsphere buffer tank on the preparation of single-cell single-encoded microsphere droplets is investigated. The results are shown in Table 6.

TABLE-US-00006 TABLE 6 Effect of combined design of a large-corner continuous U-shaped flow channel and an encoded microsphere buffer tank on the preparation of single-cell single-encoded microsphere droplets Flow rate of encoded Ratio of microsphere Total number Number of effective suspension of sampled effective droplets Group (μL/s) droplets droplets (%) First group 0.051 67452 49280 73.06 Second group 0.065 72673 18270 25.14 Third group 0.088 107966 24670 22.85 Fourth group 0.105 114114 17539 15.37 (blank control)

[0118] As shown in Table 6, when a large-corner continuous U-shaped flow channel (the second group) or an encoded microsphere buffer tank (the third group) is used alone in the encoded microsphere flow channel, compared with the combined design of a large-corner continuous U-shaped flow channel and an encoded microsphere buffer tank (the first group) in the encoded microsphere flow channel, although the total number of prepared droplets is large, the combined design of the large-corner continuous U-shaped flow channel and the encoded microsphere buffer tank has the synergistic effect of significantly improving the ratio of effective droplets, save the samples and reduce the costs, with very obvious beneficial effects.

Example 8 Effect of the Spacing Between the Second Cross-Shaped Flow Channel and the First Cross-Shaped Flow Channel on the Preparation of Single-Cell Single-Encoded Microsphere Droplets

[0119] In this example, the droplet generator provided by Example 1 is used. The angle of upward inclination of the straight flow channel connecting the two turnings is 15 degrees. The total length of the large-corner continuous U-shaped flow channel is 20000 μm, and an encoded microsphere buffer tank is provided. The encoded microsphere buffer tank has a width of 500 μm at the widest part and a depth of 150 μm at the deepest part. The size of the oil phase buffer tank is 400 μm wide at the widest part and 150 μm deep at the deepest part. Wherein, the spacing between the second cross-shaped flow channel and the first cross-shaped flow channel is 20 μm, 40 μm, 60 μm, 80 μm, 150 μm, 350 μm and 500 μm respectively. The pressure at the inlet of the encoded microsphere flow channel is controlled at 3.0 psi; the pressure at the inlet of a cell flow channel is controlled at 4.5 psi; the pressure at the inlet of an oil-phase flow channel is controlled at 8.5 psi. After operating for 1 hour continuously, the effect of the spacing between the second cross-shaped flow channel and the first cross-shaped flow channel on the preparation of single-cell single-encoded microsphere droplets is investigated. The results are shown in Table 7.

TABLE-US-00007 TABLE 7 Effect of the spacing between the second cross-shaped flow channel and the first cross-shaped flow channel on the preparation of single-cell single-encoded microsphere droplets Ratio of Total number Number of effective Serial Spacing of sampled effective droplets No. (μm) droplets droplets (%) 1 10 65845 18476 28.06 2 20 67321 29345 43.59 3 60 66349 39969 60.24 4 80 66678 48555 72.82 5 200 67962 42714 62.85 6 500 67411 27888 41.37 7 600 65878 19737 29.96

[0120] As shown in Table 7, the spacing between the second cross-shaped flow channel and the first cross-shaped flow channel also has a great influence on the preparation of single-cell single-encoded microsphere droplets. When the distance is less than 20 μm or greater than 500 μm, the proportion of limited droplets is reduced obviously, therefore, the spacing between the second cross-shaped flow channel and the first cross-shaped flow channel must be strictly controlled at 20-500 μm. The main reason is that a certain time interval is required between each shearing and wrapping of the oil phase to form droplets. The spacing between the first cross-shaped flow channel and the second cross-shaped flow channel needs to match the time interval between each shearing and wrapping of the oil phase to form droplets, so as to further improve the efficiency of generating effective droplets.

Example 9 Effect of the Droplet Nozzle Width of the Second Cross-Shaped Flow Channel on the Preparation of Single-Cell Single-Encoded Microsphere Droplets

[0121] In this example, the droplet generator provided by Example 1 is used. The angle of upward inclination of the straight flow channel at two turnings is 15 degrees. The total length of the large-corner continuous U-shaped flow channel is 2,000 μm, and an encoded microsphere buffer tank is provided. The size of the encoded microsphere buffer tank is 500 μm wide at the widest part and 150 μm deep at the deepest part. The size of the oil phase buffer tank is 400 μm wide at the widest part and 150 μm deep at the deepest part. The spacing between the shaped flow channel and the first cross-shaped flow channel is 80 μm, respectively, and the droplet nozzle width (outlet end 15) (FIG. 4) of the second cross-shaped flow channel is 40 μm, 50 μm, 60 μm, 70 μm and 80 μm, respectively. The pressure at the inlet of the encoded microsphere flow channel is controlled at 3.0 psi; the pressure at the inlet of the cell flow channel is controlled at 4.5 psi; the pressure at the inlet of an oil-phase flow channel is controlled at 8.5 psi; after continuous operation for 1 hour, the effect of the droplet nozzle width (FIG. 4) of the second cross-shaped flow channel on the preparation of single-cell single-encoded microsphere droplets is investigated. Results are shown in Table 8.

TABLE-US-00008 TABLE 8 Effect of the droplet nozzle width of the second cross-shaped flow channel on the preparation of single-cell single-encoded microsphere droplets Droplet Droplet Ratio of suspension preparation effective Serial Width flow rate efficiency droplets No. (μm) (μL/s) (droplet/s) (%) 1 40 0.032 520 38.24% 2 50 0.040 641 58.34% 3 60 0.054 934 72.82% 4 70 0.062 1152 51.64% 5 80 0.075 1349 46.27%

[0122] As shown in Table 8, the droplet nozzle width of the second cross-shaped flow channel has a great influence on the preparation efficiency of single-cell single-encoded microsphere droplets. When the width is less than 60 μm, the flow rate of the droplet is slower, and the efficiency of droplet preparation is lower; when the width is more than 60 μm, the flow rate of droplets accelerates, and the efficiency of droplet preparation increases, but the ratio of effective droplets decreases. The main reason is that, with the increase in the width and/or depth, the flow resistance decreases and the flow rate accelerates. However, when the flow rate is too fast, the flow rate of the encoded microspheres is increased significantly, and phenomenon of multiple encoded microspheres appears in the droplets, and the ratio of effective droplets decreases. When the width of the outlet segment flow channel of the second cross-shaped flow channel is 60 μm, more effective droplets can be obtained more efficiently, and the flow resistance and flow rate are all within the controllable range.

[0123] Although the present invention is disclosed above, the present invention is not limited thereto. The present invention can be expanded according to the application ranges in the field of microfluidics. Any person skilled in the art can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be based on the scope as claimed by the appended claims.