Chip and device for preparing emulsion droplets

Abstract

The present invention relates to the field of microfluidic chips, and particularly to a chip and a device for preparing emulsion droplets. The chip comprises a substrate, and the substrate includes a droplet generation component 1, a main flow channel 2 and an outlet 3 which are connected in sequence. The droplet generation component 1 includes a continuous phase injection end 4, a continuous phase flow channel 5, a dispersed phase injection end 6, a dispersed phase flow channel 7 and a droplet generation end 8; the continuous phase injection end 4 is communicated with the continuous phase flow channel 5; the dispersed phase injection end 6 is communicated with the dispersed phase flow channel 7; the continuous phase flow channel 5, the dispersed phase flow channel 7 and the main flow channel 2 intersect and communicate at the droplet generation end 8; a reagent injection end 9 is arranged on the side wall of the main flow channel 2; the reagent injection end 9 is arranged between the droplet generation end 8 and the outlet 3; an alternating current electrode 10 is further arranged on the other side wall of the main flow channel 2. The detection amount that can be achieved by the chip provided by the present invention is more than 20 times higher than that of the prior art. Thus, the processing amount of the chip for samples can be greatly increased, and the time for pretreatment and detection analysis can be shortened.

Description

Technical Field

[0001] The present invention relates to the field of microfluidic chips, and particularly to a chip and a device for preparing emulsion droplets. Background Art

[0002] Emulsification is a stable system formed by water, oil, and surfactant in appropriate proportions. Microemulsification technology uses microelectromechanical technology to fabricate microstructures and generate microemulsion droplets. The principle is to use hydrodynamic focusing to converge the continuous phase and dispersed phase liquids. When the shear force of the continuous phase is greater than the surface tension of the shear force of the dispersed phase, the dispersed phase liquid breaks to form w / o or o / w emulsion droplets (as Figure 1 shown).

[0003] Emulsion droplets can be widely used in microscale reagent synthesis, microscale material synthesis, microscale molecular reaction and analysis (such as DNA, protein), single cell encapsulation, single cell separation, single cell capture, single cell sequencing, and single cell proteomics analysis. When performing these microscale droplet analysis processes, various reaction reagents, synthetic substances, etc. with different requirements often need to be incorporated. For example, appropriate proportions of synthetic substances need to be incorporated for microscale reagent synthesis, dNTP and amplification primers need to be incorporated for microscale molecular amplification (DNA amplification), and cell lysis solution and molecular diagnostic reagents for DNA amplification are required for single cell analysis. However, whether the microdroplets are processed by the merge mode or injection mode for the mixing of sample droplets and reagents, the throughput is usually very low, generally about 5 - 50 μL / hr. If these external synthetic substances are injected at a high flow rate (>50 μL / min), the incorporation amount will be insufficient or too low due to the too short contact time, or even the merge or injection cannot be performed due to the too short contact time. Therefore, low throughput is often the bottleneck and Achilles' heel of the droplet microfluidic system. Summary of the Invention

[0004] In view of this, the present invention provides a chip and a device for preparing emulsion droplets. The detection throughput that can be achieved by this chip is more than 20 times higher than that of the prior art. Thus, the processing amount of the chip for samples can be greatly increased, and the time for pretreatment and detection analysis can be shortened.

[0005] To achieve the above-mentioned invention objectives, the present invention provides the following technical solutions:

[0006] The present invention provides a chip for preparing emulsion droplets, which includes a substrate. The substrate is provided with a droplet generation component (1), a main flow channel (2), and an outlet (3) connected in sequence. The droplet generation component 1 includes a continuous phase injection end 4, a continuous phase flow channel 5, a dispersed phase injection end 6, a dispersed phase flow channel 7, and a droplet generation end 8;

[0007] The continuous phase injection end 4 communicates with the continuous phase flow channel 5;

[0008] The dispersed phase injection end 6 communicates with the dispersed phase flow channel 7;

[0009] The continuous phase flow channel 5, the dispersed phase flow channel 7, and the main flow channel 2 intersect and communicate at the droplet generation end 8;

[0010] A reagent injection end 9 is provided on the side wall of the main flow channel 2; the reagent injection end 9 is provided between the droplet generation end 8 and the outlet 3;

[0011] Another side wall of the main flow channel 2 is also provided with an alternating current electrode 10.

[0012] In some specific embodiments of the present invention, the connection line between the reagent injection end 9 and the alternating current electrode 10 is perpendicular to the main flow channel 2.

[0013] In some specific embodiments of the present invention, the flow channel width of the reagent injection area 11 is smaller than the width of other areas of the main flow channel 2.

[0014] In some specific embodiments of the present invention, 1 / 5 droplet diameter ≤ the flow channel width of the reagent injection area 11 ≤ 4 / 5 droplet diameter; 1 / 10 the width of the main flow channel 2 ≤ the flow channel width of the reagent injection area 11 ≤ 3 / 4 the width of the main flow channel 2;

[0015] The droplets are formed by the fusion of the mobile phase and the dispersed phase.

[0016] In some specific embodiments of the present invention, the main flow channel 2 is further provided with a shunt pipeline 12; one end of the shunt pipeline 12 is provided between the droplet generation end 8 and the reagent injection area 11, and the other end of the shunt pipeline 12 is provided between the reagent injection area 11 and the outlet 3.

[0017] In some specific embodiments of the present invention, 1 / 10 droplet diameter ≤ the width of the shunt pipeline 12 ≤ 1 / 2 droplet diameter;

[0018] The droplets are formed by the fusion of the mobile phase and the dispersed phase.

[0019] In some specific embodiments of the present invention, a cylindrical structure 13 is further provided in the flow channel of the shunt pipeline 12;

[0020] When the number of the columnar structures 13 is 1, a microchannel 14 is formed between the columnar structure 13 and the side wall of the shunt pipe 12;

[0021] When the number of the columnar structures 13 > 1, microchannels 14 are formed between and / or between the columnar structures 13 and the side wall of the shunt pipe 12;

[0022] 1 / 10 droplet diameter ≤ the width of the microchannel 14 ≤ 1 / 2 droplet diameter;

[0023] The droplet is formed by the fusion of the continuous phase and the dispersed phase.

[0024] The present invention also provides an application of the chip in the preparation of emulsion droplets.

[0025] On this basis, the present invention also provides applications of the chip in microscale reagent synthesis, microscale material synthesis, microscale molecular reaction and analysis, single-cell sequencing, and single-cell proteomics analysis.

[0026] The present invention also provides a kit, including the chip and acceptable reagents.

[0027] The present invention also provides a device, including the chip and auxiliary components.

[0028] In some specific embodiments of the present invention, the auxiliary component includes a pump.

[0029] The present invention also provides a method for preparing emulsion droplets. Based on the chip of the present invention, the continuous phase is injected into the continuous phase flow channel 5 through the continuous phase injection end 4, and the dispersed phase is injected into the dispersed phase flow channel 7 through the dispersed phase injection end 6;

[0030] The continuous phase flows through the continuous phase flow channel 5, the dispersed phase flows through the dispersed phase flow channel 7, and the continuous phase and the dispersed phase meet and fuse at the droplet generation end 8 to form droplets;

[0031] The droplets flow through the main flow channel 2, reagents are injected at the reagent injection end 9, an alternating current electric field is applied by the alternating current electrode 10, the reagents and the droplets fuse to form the emulsion droplets, and the emulsion droplets are collected at the outlet 3.

[0032] In some specific embodiments of the present invention, the flow rate of the continuous phase is 100 μL / hr to 10 mL / hr, and the flow rate of the dispersed phase is 10 μL / hr to 1 mL / hr.

[0033] In some specific embodiments of the present invention, 1 / 10 droplet flow rate ≤ the flow rate of the reagent ≤ droplet flow rate.

[0034] In some specific embodiments of the present invention, 1 / 5 mobile phase flow rate ≥ disperse phase flow rate ≥ 1 / 50 mobile phase flow rate.

[0035] The present invention provides a controllable microfluidic emulsification chip. A continuous phase and a disperse phase are respectively injected to generate w / o (water in oil) microemulsion droplets, such that each w / o droplet contains only a single cell to be tested or reagent to be reacted. After the droplets are formed, one or more reagent injection ends are arranged on the rear-end pipeline, and with the structural design and the application of an alternating current electric field, the reagent to be added can be injected into the droplets. The microemulsion droplets prepared by the chip provided by the present invention can encapsulate single cells, and provide detections such as DNA hybridization polymerase chain reaction amplification, sequencing, single-cell immunoassay, etc. for single cells in a microenvironment, RNA sequencing, and RNA expression quantitative analysis. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for the description of the embodiments or the prior art.

[0037] Figure 1 FIG. 1 shows a schematic diagram of the generation of microemulsion droplets. When the shear force of the continuous phase is greater than the surface tension of the shear force of the disperse phase, the disperse phase liquid breaks to form microemulsion droplets.

[0038] FIG. 2(a) shows a schematic diagram of a microemulsion droplet reagent injection chip. After injecting the continuous phase - oil (Oil) and the disperse phase - sample (Sample) at the front end to generate droplets, a structure is designed at the rear end - such that the reagent (Reagent) can be injected after the droplets enter the structure; FIG. 2(b) is a schematic diagram of droplet injection. When the droplet passes through the reagent (Reagent) injection end 9, an alternating current electric field is applied by the alternating current electrode 10 arranged below to change the surface tension of the droplet, and then the reagent (Reagent) enters the droplet.

[0039] FIG. 3(a) shows a schematic diagram of an experimental operation flow; FIG. 3(b) is a simple explanatory diagram of the experimental steps.

[0040] FIG. 4(a) is a schematic diagram of a method for injecting reaction reagents into microdroplets using a non-contact electric field; FIG. 4(b1) shows a larger shunt pipe 12, with a cylindrical structure 13 provided in front of the shunt pipe to prevent droplets from entering the shunt pipe 12 under the influence of pressure; FIG. 4(b2) shows multiple narrower shunt pipes 12, which can shunt the pressure in segments when the velocity of high-flow droplets is relatively fast, slow down the velocity of the droplets in the reagent injection area 11, and thus increase the electric field interaction time between the droplets and the reagent and the reagent injection time;

[0041] Figure 5 It shows that a structure is used to cause deformation of the droplets when they enter the reagent injection end 9, increasing the contact area between the droplets and the reagent injection end 9;

[0042] FIG. 6 shows a schematic diagram of reagent injection; FIG. 6(a) shows that when a relatively small pressure is applied to the reagent injection end 9, the injection amount of the reagent containing the dye is relatively small; FIG. 6(b) shows that when a relatively large pressure is applied to the reagent injection end 9, the injection amount of the reagent containing the dye is relatively large;

[0043] FIG. 7 shows a diagram of the droplet state at the outlet end 3 after the reagent is injected into the droplets, showing that the reagent has been fully incorporated into the droplets and quickly achieved a mixed and uniform state at the outlet; among them, FIG. 7(a) shows that no reagent is injected when an electric field is applied; FIG. 7(b) shows that the reagent injection flow rate is 1 / 10 of the droplet flow rate, FIG. 7(c) shows that the reagent injection flow rate is 1 / 5 of the droplet flow rate; FIG. 7(d) shows that the reagent injection flow rate is 1 / 2.5 of the droplet flow rate; the results show that the amount of reagent injected into the droplets can be regulated according to different injection flow rates to produce concentrations with different requirements;

[0044] Figure 8 It shows a schematic diagram of a microemulsified droplet reagent injection chip;

[0045] Among them, 1 - droplet generation component; 2 - main flow channel; 3 - outlet; 4 - continuous phase injection end; 5 - continuous phase flow channel; 6 - dispersed phase injection end; 7 - dispersed phase flow channel; 8 - droplet generation end; 9 - reagent injection end; 10 - alternating current electrode; 11 - reagent injection area; 12 - shunt pipe (FIG. 4(b1, b2)); 13 - cylindrical structure (FIG. 4(b1)); 14 - microchannel (FIG. 4(b2)). Specific embodiments

[0046] The present invention discloses a chip and a device for preparing emulsion droplets. Those skilled in the art can draw on the content of this article and appropriately modify process parameters to achieve it. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art, and they are all considered to be included in the present invention. The methods and applications of the present invention have been described through preferred embodiments. Relevant personnel can obviously make changes or appropriate modifications and combinations to the methods and applications described herein without departing from the content, spirit and scope of the present invention to implement and apply the technology of the present invention.

[0047] Taking single-cell whole-genome sequencing as an example: The methods for obtaining single cells include gradient dilution method, laser capture microdissection, or flow cytometry to screen cells. After obtaining the cells by these methods, artificial operations are used to add liquid such as cell lysis buffer and reaction reagents and then enter the subsequent analysis. During the operation process, human operation errors cannot be avoided, and mostly tissue samples or cell populations are used for analysis. By calculating the average value of the cells in the sample population, the heterogeneity between cells and the characteristics of individual cells are often ignored, and the uniqueness of each cell cannot be truly reflected. The present invention provides a microfluidic emulsification chip that separately injects a continuous phase and a disperse phase to generate w / o (water in oil) microemulsion droplets, so that each w / o droplet contains only a single cell to be detected or a reagent to be reacted. After the droplet is formed, one or more reagent injection ports are arranged on the rear pipeline, and with the structural design and the application of an alternating current electric field, the reagent to be added can be injected into the droplet. The microemulsion droplets can encapsulate single cells, isolate single cells, and capture single cells, providing detections such as DNA hybridization polymerase chain reaction amplification, sequencing, single-cell immunological analysis, RNA sequencing, and RNA expression quantitative analysis for single cells in a microenvironment.

[0048] The chip provided by the present invention is a droplet generation structure, and a reagent injection port 9 is arranged at the rear end (as shown in Fig. 2(a)). Among them, the continuous phase is Oil, the disperse phase is the sample, and the injected liquid can be a reagent or other liquid to be added to the droplet. When the droplet passes through the opening of the reagent injection port 9, an AC electric field can be applied by the electrode to change the surface tension between the droplet and the reagent injection port 9, so that the reagent is injected into the droplet (as shown in Fig. 2(b)).

[0049] The experimental operation flow chart and simple description of this case are shown in Figure 3. A pump is used to inject the sample and oil-phase droplets into the chip. Hydrodynamic focusing is used to converge the sample (dispersed-phase liquid) and oil-phase liquid (continuous-phase liquid) to generate micro-droplets. Then, a pump is used to inject the reagent into the chip at the injection end. When the micro-droplets pass through the injection end, an AC electric field is applied to change the surface tension between the micro-droplets and the reagent injection end, enabling the reagent to be injected into the micro-droplets.

[0050] In order to be able to regulate the amount of reagent injected according to requirements, a pressure balance structure - a shunt pipe 12 is set in the pipeline to adjust the rate at which the droplets pass through the reagent injection end 9, and the pipeline is narrowed to deform the droplets to increase the contact area between the droplets and the injection end. The amount of reagent injected is adjusted by these two methods. The chip provided by the present invention can simultaneously complete the formation of microemulsion droplets, the injection of reagents, the mixing of reagents, and the collection of products. If multiple reagent additions are required, the chip provided by the present invention can also connect multiple injection ends in series, provide multiple reagent addition pipelines, reduce human operation errors and reagent consumption, and greatly increase the sample processing speed and accuracy.

[0051] In addition, the amount of reagent injected into the sampled droplets can be easily regulated by controlling the pressure at the reagent end. As shown in Figures 6(a,b) and Figure 7(a) blank injection group (without injection), in the state where no voltage is applied, the reagent cannot use the electric field to change the surface tension of the droplets to allow the reagent to enter. When an AC electric field with a frequency of 10 - 1000 kHz and above 100 Vpp is applied, the reagent can be injected into the sampled droplets. In Figures 7(b), (c), and (d), 10 pL, 20 pL, and 40 pL of reagent are injected at the injection end flow rates of 50 μL / hr, 100 μL / hr, and 200 μL / hr respectively, and the sample flow rate is 300 μL / hr at this time.

[0052] The present invention compresses the sampled droplet through structural design so that it can present a flat shape when passing through the injection region 11, thereby increasing the contact area between the sample droplet and the injection port, and greatly increasing the injection efficiency of the reagent (injection rate / area of the injection region ~ square function). In addition, a bypass fluidic channel 12 is designed before and after the injection region, so that a part of the oil phase flow is diverted to the bypass channel 12, greatly reducing the speed of the sample droplet in the injection region 11 (as shown in Figure 8 ), thereby increasing the contact time of the reagent injected into the sample droplet (injection rate / time ~ linear function). Thus, through the cooperation of these two structural designs (contact area increased x contact time increased), the injection efficiency of the reagent into the sample droplet can be increased approximately exponentially (square increase). Therefore, in cooperation with the front-end droplet formation part 1, high-throughput on-chip droplet formation and reagent injection can be achieved. The experimental results of the examples show that it is possible to successfully inject reagent volumes of 100 pL and 50 pL into microdroplets with a volume of 0.2 nL (diameter ~ 70 μm) at high flow rates of 300 μL / hr and 700 μL / hr. The throughput that can be achieved by a single chip is more than 20 times higher than that of the prior art. Thus, the processing capacity of the chip for samples can be greatly increased, and the time for pretreatment and detection analysis can be shortened.

[0053] The chip provided by the present invention is provided with a reagent injection end 9 at the main channel 2 after the droplet is formed. When the droplet passes through the reagent injection end 9, an electric field is applied to change the surface tension between the droplet and the reagent injection end 9, so that the reagent is injected into the droplet, and thus a reaction occurs.

[0054] In some embodiments, the diameter of the pipeline is reduced before the main flow channel 2 of the chip provided by the present invention enters the reagent injection end 9. Preferably, 1 / 5 droplet diameter ≤ the flow channel width of the reagent injection area 11 ≤ 4 / 5 droplet diameter; preferably, 1 / 10 main flow channel 2 width ≤ the flow channel width of the reagent injection area 11 ≤ 3 / 4 main flow channel 2 width, so that the droplet is deformed into a relatively long and narrow droplet, and this deformation can increase the contact area between the droplet and the reagent injection end 9, thereby increasing the injection volume of the droplet. The oil phase injection rate is 100 μL / hr to 10 mL / hr, and the sample injection rate is 10 μL / hr to 1 mL / hr.

[0055] In other embodiments, the setting of the bypass channel 12 can divert the volume of the oil phase liquid, slow down the speed of the droplet passing through the reagent injection end 9, and thereby increase the injection amount of the reagent. The injection amount of the reagent is regulated by the bypass channel 12. Preferably, 1 / 10 droplet diameter ≤ the width of the bypass channel 12 ≤ 1 / 2 droplet diameter.

[0056] In some embodiments, the bypass channel 12 can also be shunted by a bypass channel 12 with the same width as the main flow channel 2 plus a cylindrical structure 13. The setting of the cylindrical structure 13 can prevent the droplet from detaching from the main flow channel 2. 1 / 10 droplet diameter ≤ the gap of the cylindrical structure 13 ≤ 1 / 2 droplet diameter.

[0057] In other embodiments, when the number of the cylindrical structures 13 is 1, the cylindrical structure 13 and the side wall of the bypass channel 12 form a microchannel 14;

[0058] When the number of the cylindrical structures 13 > 1, microchannels 14 are formed between the cylindrical structures 13 and / or between the cylindrical structure 13 and the side wall of the bypass channel 12;

[0059] 1 / 10 droplet diameter ≤ the width of the microchannel 14 ≤ 1 / 2 droplet diameter.

[0060] When the droplet flow rate is fixed, the injection amount of the reagent can also be regulated by adjusting the pressure and flow rate of the reagent injection end, thereby increasing the throughput of the overall droplet formation and reagent injection. 1 / 10 droplet flow rate ≤ the flow rate of the injected reagent ≤ the droplet flow rate.

[0061] By switching an electric field (alternating current, above 100 Vpp, 10 - 1000 KHz), a reagent can be selectively injected into a specific sampled droplet.

[0062] Integrating droplet formation, narrowing the channel width of the reagent injection region (channel width < droplet diameter) to squeeze the sampled droplet into a flattened shape, and combining with the setting of the bypass channel 12, droplets can be generated simultaneously on a single chip at an extremely high sample processing speed and appropriate doses of reagents can be injected online.

[0063] The chip, components, reagents, and raw materials used in the device for preparing emulsion droplets provided by the present invention can all be purchased on the market.

[0064] The present invention will be further illustrated below in conjunction with embodiments:

[0065] Embodiment 1

[0066] The present invention provides a chip for preparing emulsion droplets. The chip includes a substrate, and the substrate is provided with a droplet generation component 1, a main channel 2, and an outlet 3 connected in sequence. The droplet generation component 1 includes a continuous phase injection end 4, a continuous phase channel 5, a dispersed phase injection end 6, a dispersed phase channel 7, and a droplet generation end 8; the continuous phase injection end 4 is communicated with the continuous phase channel 5; the dispersed phase injection end 6 is communicated with the dispersed phase channel 7; the continuous phase channel 5, the dispersed phase channel 7, and the main channel 2 meet and communicate at the droplet generation end 8; a reagent injection end 9 is provided on the side wall of the main channel 2; the reagent injection end 9 is provided between the droplet generation end 8 and the outlet 3; an alternating current electrode 10 is further provided on the other side wall of the main channel 2. The connection line between the reagent injection end 9 and the alternating current electrode 10 is perpendicular to the main channel 2. As shown in Figures 2(a) and 2(b).

[0067] Embodiment 2

[0068] The present invention provides a chip for preparing emulsified droplets. The chip includes a substrate, and the substrate is provided with a droplet generation component 1, a main flow channel 2, and an outlet 3 that are connected in sequence. The droplet generation component 1 includes a continuous phase injection end 4, a continuous phase flow channel 5, a dispersed phase injection end 6, a dispersed phase flow channel 7, and a droplet generation end 8; the continuous phase injection end 4 is communicated with the continuous phase flow channel 5; the dispersed phase injection end 6 is communicated with the dispersed phase flow channel 7; the continuous phase flow channel 5, the dispersed phase flow channel 7, and the main flow channel 2 intersect and communicate at the droplet generation end 8; a reagent injection end 9 is provided on the side wall of the main flow channel 2; the reagent injection end 9 is provided between the droplet generation end 8 and the outlet 3; an alternating current electrode 10 is further provided on the other side wall of the main flow channel 2. The connecting line between the reagent injection end 9 and the alternating current electrode 10 is perpendicular to the main flow channel 2.

[0069] The flow channel width of the reagent injection area 11 is smaller than the width of other areas of the main flow channel 2.

[0070] 1 / 5 droplet diameter ≤ the flow channel width of the reagent injection area 11 ≤ 4 / 5 droplet diameter; 1 / 10 the width of the main flow channel 2 ≤ the flow channel width of the reagent injection area 11 ≤ 3 / 4 the width of the main flow channel 2; the droplets are formed after the mobile phase and the dispersed phase are fused. As shown in Figure 4(a).

[0071] Example 3

[0072] The present invention provides a chip for preparing emulsified droplets. The chip includes a substrate, and the substrate is provided with a droplet generation component 1, a main flow channel 2, and an outlet 3 that are connected in sequence. The droplet generation component 1 includes a continuous phase injection end 4, a continuous phase flow channel 5, a dispersed phase injection end 6, a dispersed phase flow channel 7, and a droplet generation end 8; the continuous phase injection end 4 is communicated with the continuous phase flow channel 5; the dispersed phase injection end 6 is communicated with the dispersed phase flow channel 7; the continuous phase flow channel 5, the dispersed phase flow channel 7, and the main flow channel 2 intersect and communicate at the droplet generation end 8; a reagent injection end 9 is provided on the side wall of the main flow channel 2; the reagent injection end 9 is provided between the droplet generation end 8 and the outlet 3; an alternating current electrode 10 is further provided on the other side wall of the main flow channel 2. The connecting line between the reagent injection end 9 and the alternating current electrode 10 is perpendicular to the main flow channel 2.

[0073] The flow channel width of the reagent injection area 11 is smaller than the width of other areas of the main flow channel 2.

[0074] 1 / 5 droplet diameter ≤ the flow channel width of the reagent injection area 11 ≤ 4 / 5 droplet diameter; 1 / 10 the width of the main flow channel 2 ≤ the flow channel width of the reagent injection area 11 ≤ 3 / 4 the width of the main flow channel 2; the droplets are formed after the mobile phase and the dispersed phase are fused.

[0075] The main flow channel 2 is also provided with a flow splitting pipeline 12; one end of the flow splitting pipeline 12 is arranged between the droplet generation end 8 and the reagent injection area 11, and the other end of the flow splitting pipeline 12 is arranged between the reagent injection area 11 and the outlet 3. The number of the flow splitting pipelines is at least 1. And 1 / 10 droplet diameter ≤ the width of the flow splitting pipeline 12 ≤ 1 / 2 droplet diameter; the droplets are formed by the fusion of the mobile phase and the dispersed phase. As shown in FIGS. 4(b1) and 4(b2).

[0076] Embodiment 4

[0077] The present invention provides a chip for preparing emulsion droplets, the chip includes a substrate, the substrate is provided with a droplet generation component 1, a main flow channel 2 and an outlet 3 which are connected in sequence, the droplet generation component 1 includes a continuous phase injection end 4, a continuous phase flow channel 5, a dispersed phase injection end 6, a dispersed phase flow channel 7 and a droplet generation end 8; the continuous phase injection end 4 is communicated with the continuous phase flow channel 5; the dispersed phase injection end 6 is communicated with the dispersed phase flow channel 7; the continuous phase flow channel 5, the dispersed phase flow channel 7 and the main flow channel 2 meet and communicate at the droplet generation end 8; the side wall of the main flow channel 2 is provided with a reagent injection end 9; the reagent injection end 9 is arranged between the droplet generation end 8 and the outlet 3; the other side wall of the main flow channel 2 is also provided with an alternating current electrode 10. The connecting line between the reagent injection end 9 and the alternating current electrode 10 is perpendicular to the main flow channel 2.

[0078] The flow channel width of the reagent injection area 11 is smaller than the width of other areas of the main flow channel 2.

[0079] 1 / 5 droplet diameter ≤ the flow channel width of the reagent injection area 11 ≤ 4 / 5 droplet diameter; 1 / 10 the width of the main flow channel 2 ≤ the flow channel width of the reagent injection area 11 ≤ 3 / 4 the width of the main flow channel 2; the droplets are formed by the fusion of the mobile phase and the dispersed phase.

[0080] The main flow channel 2 is also provided with a flow splitting pipeline 12; one end of the flow splitting pipeline 12 is arranged between the droplet generation end 8 and the reagent injection area 11, and the other end of the flow splitting pipeline 12 is arranged between the reagent injection area 11 and the outlet 3. And 1 / 10 droplet diameter ≤ the width of the flow splitting pipeline 12 ≤ 1 / 2 droplet diameter; the droplets are formed by the fusion of the mobile phase and the dispersed phase.

[0081] A columnar structure 13 is also arranged in the flow channel of the flow splitting pipeline 12;

[0082] When the number of the columnar structures 13 is 1, the columnar structure 13 and the side wall of the flow splitting pipeline 12 form a micro-channel 14;

[0083] When the number of the columnar structures 13 > 1, the spaces between the columnar structures 13 and / or the columnar structures 13 and the side wall of the flow splitting pipeline 12 form a micro-channel 14;

[0084] 1 / 10 droplet diameter ≤ the width of the micro-channel 14 ≤ 1 / 2 droplet diameter;

[0085] The droplets are formed after the continuous phase and the dispersed phase are fused.

[0086] As shown in FIGS. 4(b1) and 4(b2).

[0087] Preparation method of emulsion droplets in Example 5

[0088] Based on the chip as shown in Example 4 (the injection area is designed as FIG. 4(b)), the continuous phase is injected into the continuous phase flow channel 5 through the continuous phase injection end 4, and the dispersed phase is injected into the dispersed phase flow channel 7 through the dispersed phase injection end 6; the continuous phase flows through the continuous phase flow channel 5, the dispersed phase flows through the dispersed phase flow channel 7, and the continuous phase and the dispersed phase meet and fuse at the droplet generation end 8 to form droplets.

[0089] The droplets flow through the main flow channel 2, a reagent is injected at the reagent injection end 9, an alternating current electrode 10 applies an alternating current electric field (>100 Vpp, >10 kHz), the reagent and the droplets fuse to form emulsion droplets, and they are collected at the outlet 3.

[0090] The flow rate of the continuous phase is 1 mL / hr, and the flow rate of the dispersed phase is 100 μL / hr.

[0091] 1 / 10 droplet flow rate ≤ flow rate of the reagent ≤ droplet flow rate.

[0092] Preparation method of emulsion droplets in Example 6

[0093] Based on the chip as shown in Example 4 (the injection area is designed as FIG. 4(b)), the continuous phase is injected into the continuous phase flow channel 5 through the continuous phase injection end 4, and the dispersed phase is injected into the dispersed phase flow channel 7 through the dispersed phase injection end 6; the continuous phase flows through the continuous phase flow channel 5, the dispersed phase flows through the dispersed phase flow channel 7, and the continuous phase and the dispersed phase meet and fuse at the droplet generation end 8 to form droplets.

[0094] The droplets flow through the main flow channel 2, a reagent is injected at the reagent injection end 9, an alternating current electrode 10 applies an alternating current electric field (>300 Vpp, >10 kHz), the reagent and the droplets fuse to form emulsion droplets, and they are collected at the outlet 3.

[0095] The flow rate of the continuous phase is 5 mL / hr, and the flow rate of the dispersed phase is 500 μL / hr.

[0096] 1 / 10 droplet flow rate ≤ flow rate of the reagent ≤ droplet flow rate.

[0097] As Figure 5 shown.

[0098] Preparation method of emulsion droplets in Example 7

[0099] Based on the chip shown in Embodiment 4 (the injection area is designed as shown in Fig. 4(b)), the continuous phase is injected into the continuous phase flow channel 5 through the continuous phase injection end 4, and the dispersed phase is injected into the dispersed phase flow channel 7 through the dispersed phase injection end 6; the continuous phase flows through the continuous phase flow channel 5, the dispersed phase flows through the dispersed phase flow channel 7, and the continuous phase and the dispersed phase meet and merge at the droplet generation end 8 to form droplets.

[0100] The droplets flow through the main flow channel 2, a reagent is injected at the reagent injection end 9, and an alternating current electric field (>500 Vpp, >10 kHz) is applied by the alternating current electrode 10. The reagent and the droplets merge to form emulsified droplets, which are collected at the outlet 3.

[0101] The flow rate of the mobile phase is 10 mL / hr, and the flow rate of the dispersed phase is 1 mL / hr.

[0102] 1 / 10 droplet flow rate ≤ reagent flow rate ≤ droplet flow rate.

[0103] Control group

[0104] The structural design of the original microfluidic chip and the method for preparing emulsified droplets and injecting reagents are shown in Fig. 2(b) (without the design of flattening the structure of the droplets and without the design of oil splitting). Based on the chip shown in Embodiment 1 (the injection area is designed as shown in Fig. 2(b)), the continuous phase is injected into the continuous phase flow channel 5 through the continuous phase injection end 4, and the dispersed phase is injected into the dispersed phase flow channel 7 through the dispersed phase injection end 6; the continuous phase flows through the continuous phase flow channel 5, the dispersed phase flows through the dispersed phase flow channel 7, and the continuous phase and the dispersed phase meet and merge at the droplet generation end 8 to form droplets.

[0105] The droplets flow through the main flow channel 2, a reagent is injected at the reagent injection end 9, and an alternating current electric field (300 Vpp, 100 KHz) is applied by the alternating current electrode 10. The reagent and the droplets merge to form emulsified droplets, which are collected at the outlet 3.

[0106] The flow rate of the mobile phase is 100 μL / hr, and the flow rate of the dispersed phase is 10 μL / hr.

[0107] 1 / 10 droplet flow rate ≤ reagent flow rate ≤ droplet flow rate.

[0108] Comparison of the preparation of emulsified droplets in Example 8

[0109] Experimental groups 1-4: Prepare emulsified droplets according to Examples 5-8 respectively;

[0110] Control group: Prepare emulsified droplets according to the structure and method of the microfluidic chip described in the control group of the present invention, and the comparison results are shown in Table 1.

[0111] Table 1

[0112]

[0113]

[0114] Example 9

[0115] The chips provided in Embodiments 1 to 4 of the present invention can easily control the amount of reagent injected into the sampled droplet by controlling the pressure at the reagent end. As shown in FIGS. 6(a, b) and FIG. 7(a) (without injection), in the state where no voltage is applied, the reagent cannot enter by changing the surface tension of the droplet using an electric field. When an alternating electric field with a frequency of 300 kHz and 300 Vpp is applied, the reagent can be injected into the sampled droplet. When injecting 10 pL, 20 pL, and 40 pL of reagent at the reagent injection end with flow rates of 50 μL / hr, 100 μL / hr, and 200 μL / hr respectively as shown in FIGS. 7(b), (c), and (d), the sample flow rate is 500 μL / hr at this time.

[0116] The above are only the preferred embodiments of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and modifications can be made, and these improvements and modifications should also be regarded as the protection scope of the present invention.

Claims

1. Chip for preparing emulsion droplets, characterized in that, the chip comprises a substrate, and the substrate is provided with a droplet generation component (1), a main flow channel (2) and an outlet (3) which are connected in sequence. The droplet generation component (1) comprises a continuous phase injection end (4), a continuous phase flow channel (5), a dispersed phase injection end (6), a dispersed phase flow channel (7) and a droplet generation end (8); the continuous phase injection end (4) is communicated with the continuous phase flow channel (5); the dispersed phase injection end (6) is communicated with the dispersed phase flow channel (7); the continuous phase flow channel (5), the dispersed phase flow channel (7) and the main flow channel (2) meet and communicate at the droplet generation end (8); a reagent injection end (9) is arranged on the side wall of the main flow channel (2); the reagent injection end (9) is arranged between the droplet generation end (8) and the outlet (3); an alternating current electrode (10) is further arranged on the other side wall of the main flow channel (2); the flow channel width of the reagent injection area (11) is smaller than the width of other areas of the main flow channel (2); the main flow channel (2) is further provided with a shunt pipeline (12); one end of the shunt pipeline (12) is arranged between the droplet generation end (8) and the reagent injection area (11), and the other end of the shunt pipeline (12) is arranged between the reagent injection area (11) and the outlet (3); 1 / 10 droplet diameter ≤ the width of the shunt pipeline (12) ≤ 1 / 2 droplet diameter; the droplet is formed after the mobile phase and the dispersed phase are fused; when the droplet passes through the opening of the reagent injection end (9), an AC electric field can be applied by the alternating current electrode (10) to change the surface tension between the droplet and the reagent injection end (9), so that the reagent is injected into the droplet; by providing an alternating current, turning on and off an electric field of more than 100Vpp and 10 - 1000KHz, the reagent can be selectively injected into the droplet.

2. The chip according to claim 1, characterized in that, the connecting line between the reagent injection end (9) and the alternating current electrode (10) is perpendicular to the main flow channel (2).

3. The chip according to claim 1 or 2, characterized in that, the flow channel width of the reagent injection area (11) is smaller than the width of other areas of the main flow channel (2); 1 / 5 droplet diameter ≤ the flow channel width of the reagent injection area (11) ≤ 4 / 5 droplet diameter; 1 / 10 the width of the main flow channel (2) ≤ the flow channel width of the reagent injection area (11) ≤ 3 / 4 the width of the main flow channel (2); the droplet is formed after the mobile phase and the dispersed phase are fused; a columnar structure (13) is further arranged in the flow channel of the shunt pipeline (12); 1 / 10 droplet diameter ≦ the gap between the columnar structure (13) and the side wall of the shunt pipeline (12) ≦ 1 / 2 droplet diameter.

4. The chip according to claim 3, characterized in that, the number of the columnar structures (13) is 1, and the columnar structure (13) and the side wall of the shunt pipeline (12) form a microchannel (14); 1 / 10 droplet diameter ≤ the width of the microchannel (14) ≤ 1 / 2 droplet diameter; The droplet is formed by the fusion of the continuous phase and the dispersed phase.

5. The chip according to claim 3, wherein, the number of the columnar structures (13) > 1, and microchannels (14) are formed between the columnar structures (13) and / or between the columnar structures (13) and the side wall of the shunt pipe (12); 1 / 10 of the droplet diameter ≤ the width of the microchannel (14) ≤ 1 / 2 of the droplet diameter; The droplet is formed by the fusion of the continuous phase and the dispersed phase.

6. The application of the chip according to any one of claims 1 to 5 in the preparation of emulsified droplets.

7. The application of the chip according to any one of claims 1 to 5 in the synthesis of trace reagents, the synthesis of micromaterials, the reaction and analysis of trace molecules, the encapsulation of single cells, the separation of single cells, the capture of single cells, the sequencing of single cells and the analysis of single cell proteomics.

8. A kit, wherein, it includes the chip according to any one of claims 1 to 5 and acceptable reagents.

9. A device, wherein, it includes the chip according to any one of claims 1 to 5 and auxiliary components.

10. The device according to claim 9, wherein, the auxiliary component includes a pump.

11. A method for preparing emulsified droplets, wherein, based on the chip according to any one of claims 1 to 5, the continuous phase is injected into the continuous phase flow channel (5) through the continuous phase injection end (4), and the dispersed phase is injected into the dispersed phase flow channel (7) through the dispersed phase injection end (6); the continuous phase flows through the continuous phase flow channel (5), the dispersed phase flows through the dispersed phase flow channel (7), and the continuous phase and the dispersed phase meet and fuse at the droplet generation end (8) to form a droplet; the droplet flows through the main flow channel (2), a reagent is injected at the reagent injection end (9), an alternating electric field is applied by the alternating current electrode (10), the reagent and the droplet fuse to form the emulsified droplet, and the emulsified droplet is collected at the outlet (3).

12. The preparation method according to claim 11, wherein, the flow rate of the continuous phase is 100 μL / hr to 10 mL / hr, and the flow rate of the dispersed phase is 10 μL / hr to 1 mL / hr.

13. The preparation method according to claim 11 or 12, wherein, 1 / 10 of the droplet flow rate ≤ the flow rate of the injected reagent ≤ the droplet flow rate.

Images