CN112439467A - Chip and device for preparing emulsion droplets - Google Patents
Chip and device for preparing emulsion droplets Download PDFInfo
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- CN112439467A CN112439467A CN201910807870.2A CN201910807870A CN112439467A CN 112439467 A CN112439467 A CN 112439467A CN 201910807870 A CN201910807870 A CN 201910807870A CN 112439467 A CN112439467 A CN 112439467A
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- 239000007788 liquid Substances 0.000 claims abstract description 104
- 239000000758 substrate Substances 0.000 claims abstract description 14
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- 238000004445 quantitative analysis Methods 0.000 description 2
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
- B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
- B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
- B01L3/5027—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip
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Abstract
The invention relates to the field of microfluidic chips, in particular to a chip and a device for preparing emulsion droplets. The chip comprises a substrate, wherein the substrate comprises a liquid drop generating component 1, a main runner 2 and an outlet 3 which are connected in sequence, and the liquid drop generating component 1 comprises a continuous phase injection end 4, a continuous phase runner 5, a dispersed phase injection end 6, a dispersed phase runner 7 and a liquid drop generating end 8; the continuous phase injection end 4 is communicated with the continuous phase flow passage 5; the dispersed phase injection end 6 is communicated with the dispersed phase flow channel 7; the continuous phase flow channel 5, the disperse phase flow channel 7 and the main flow channel 2 are communicated at a liquid drop generating end 8 in a crossing way; 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 channel 2 is also provided with an alternating current electrode 10. The detection amount of the chip provided by the invention is more than 20 times that of the chip in the prior art, so that the sample processing amount of the chip can be greatly increased, and the time for pretreatment and detection analysis is shortened.
Description
Technical Field
The invention relates to the field of microfluidic chips, in particular to a chip and a device for preparing emulsion droplets.
Background
Emulsification is a stable system formed by water, oil and surfactant in a proper proportion, the micro-emulsification technology is to make a micro-structure by micro-electro-mechanical technology and generate micro-emulsification droplets, the principle is that fluid dynamic focusing (hydrodynamic focusing) is utilized to collect continuous phase and dispersed phase liquid, and when the shearing force of the continuous phase is larger than the surface tension of the shearing force of the dispersed phase, the dispersed phase liquid is broken to form w/o or o/w emulsification droplets (as shown in figure 1).
The emulsion droplets can be widely applied to synthesis of trace reagents, synthesis of micron materials, reaction and analysis of trace molecules (such as DNA and protein), single cell coating, single cell separation, single cell capture, single cell sequencing, single cell protein plastid analysis and the like, and the analysis process of the trace droplets usually needs to be mixed with various reaction reagents, compounds and the like with different requirements, for example, the synthesis of the trace reagents needs to be mixed with a compound with a proper proportion, the amplification of the trace molecules (DNA amplification) needs to be mixed with dNTP and an amplification primer (amplification primer) and the like, and the analysis of the single cells needs to be carried out by DNA amplification cell lysate and molecular diagnostic reagents and the like. However, the throughput (throughput) of mixing the sample droplet (sample droplet) with the reagent (reagent) in the micro-droplet whether in the fusion mode or injection mode is usually very low, typically about 5-50 μ L/hr, and if the additive composition is injected at a high flow rate (>50 μ L/min), the low throughput (low-throughput) is often the bottleneck and gate of the droplet microfluidic system because the contact time is too short and the incorporation is insufficient or too low, even the contact time is too short and the combination (fusion) or injection (injection) cannot be performed.
Disclosure of Invention
In view of the above, the present invention provides a chip and an apparatus for preparing emulsion droplets. The detection amount (through put) of the chip is more than 20 times higher than that of the prior art, so that the processing amount of the chip on a sample can be greatly increased, and the time for pretreatment and detection analysis is shortened.
In order to achieve the above object, the present invention provides the following technical solutions:
the invention provides a chip for preparing emulsified liquid drops, which comprises a substrate, wherein the substrate is provided with a liquid drop generating component (1), a main flow channel (2) and an outlet (3) which are sequentially connected, and the liquid drop generating component 1 comprises a continuous phase injection end 4, a continuous phase flow channel 5, a disperse phase injection end 6, a disperse phase flow channel 7 and a liquid drop generating 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 are communicated with each other at the droplet generation end 8 in a crossing manner;
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.
In some embodiments of the invention, the connection line of the reagent injection end 9 and the ac electrode 10 is perpendicular to the main channel 2.
In some embodiments of the present invention, the reagent injection region 11 has a flow channel width smaller than that of other regions of the main flow channel 2.
In some embodiments of the invention, the 1/5 droplet diameter is ≦ the channel width of the reagent injection region 11 ≦ 4/5 droplet diameter; 1/10 the width of the main channel 2 is not more than the width of the main channel 2 of the reagent injection area 11 is not more than 3/4;
the liquid drop is formed after the mobile phase and the disperse phase are fused.
In some particular embodiments of the invention, the main flow channel 2 is further provided with a flow distribution channel 12; one end of the flow distribution pipe 12 is provided between the droplet generation end 8 and the reagent injection region 11, and the other end of the flow distribution pipe 12 is provided between the reagent injection region 11 and the outlet 3.
In some embodiments of the invention, the 1/10 droplet diameter is ≦ the width of the diversion conduit 12 ≦ 1/2 droplet diameter;
the liquid drop is formed after the mobile phase and the disperse phase are fused.
In some embodiments of the present invention, a columnar structure 13 is further disposed in the flow channel of the flow dividing pipe 12;
when the number of the columnar structures 13 is 1, the columnar structures 13 and the side wall of the shunt pipeline 12 form a micro-channel 14;
when the number of the columnar structures 13 is more than 1, micro channels 14 are formed between the columnar structures 13 and/or between the columnar structures 13 and the side walls of the shunt pipelines 12;
1/10 the diameter of the liquid drop is not more than 1/2 of the width of the micro-channel 14;
the liquid drop is formed after the mobile phase and the disperse phase are fused.
The invention also provides the application of the chip in preparing emulsion droplets.
On the basis, the invention also provides the application of the chip in trace reagent synthesis, micron material synthesis, trace molecule reaction and analysis, single cell sequencing and single cell protein plastid analysis.
The invention also provides a kit comprising the chip and an acceptable reagent.
The invention also provides a device comprising the chip and the auxiliary component.
In some embodiments of the invention, the auxiliary component comprises a pump.
The invention also provides a preparation method of the emulsified liquid drop, based on the chip, the continuous phase is injected into the continuous flow channel 5 through the continuous phase injection end 4, and the disperse phase is injected into the disperse phase flow channel 7 through the disperse 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 are converged and fused at the droplet generation end 8 to form a droplet;
the droplets flow through the main flow channel 2, a reagent is injected into the reagent injection end 9, an alternating current electric field is applied by the alternating current electrode 10, the reagent and the droplets are fused to form the emulsified droplets, and the emulsified droplets are collected at the outlet 3.
In some embodiments of the invention, the rate of the mobile phase is from 100 μ L/hr to 10mL/hr and the rate of the dispersed phase is from 10 μ L/hr to 1 mL/hr.
In some embodiments of the invention, the 1/10 droplet flow rate ≦ the flow rate of the reagent ≦ the droplet flow rate.
In some embodiments of the invention, the 1/5 mobile phase flow rate ≧ the dispersed phase flow rate ≧ 1/50 mobile phase flow rate.
The invention provides a controllable microfluid emulsification chip, which is used for respectively injecting a continuous phase (continuous phase) and a disperse phase (dispersed phase) to generate w/o (water in oil) micro-emulsified liquid drops, so that each w/o liquid drop only has a single cell to be detected or a reagent to be reacted, and after the liquid drop (drop) is formed, one or more reagent injection ends (injection) are arranged on a rear end pipeline, and the matching structure design and alternating current electric field application are carried out to ensure that the reagent to be added can be injected into the liquid drop (drop). The micro-emulsified liquid drops prepared by the chip provided by the invention can be coated with single cells, and the single cells can be used for carrying out DNA hybridization polymerase chain reaction amplification, sequencing, single cell immunoassay and other detection, RNA sequencing and RNA expression quantitative analysis in a microenvironment.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below.
FIG. 1 is a schematic diagram showing the generation of micro-emulsified droplets, wherein when the shear force of the continuous phase is greater than the surface tension of the shear force of the dispersed phase, the liquid in the dispersed phase breaks to form micro-emulsified droplets;
fig. 2(a) shows a schematic diagram of a micro-emulsion droplet Reagent injection chip, after a continuous phase-Oil (Oil) and a dispersed phase-Sample (Sample) are injected at the front end to generate a droplet (droplet), a structure is designed at the rear end, so that the droplet (droplet) can be injected with a Reagent (Reagent) after entering the structure; fig. 2(b) is a schematic diagram of droplet (droplet) injection, when the droplet (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 (droplet), so that the Reagent (Reagent) enters the droplet (droplet);
FIG. 3(a) shows a flow chart of the experimental operation; FIG. 3(b) is a simplified explanatory diagram of the experimental procedure;
FIG. 4(a) is a schematic diagram of a method for injecting reagents into microdroplets with a non-contact electric field; fig. 4(b1) shows a larger distribution pipe 12, in which a column structure 13 is arranged in front of the distribution pipe to prevent liquid drops from entering the distribution pipe 12 under the influence of pressure; fig. 4(b2) shows a plurality of narrow diversion pipes 12, which can segment diversion pressure when the speed of the high-flow-rate droplet (droplet) is fast, and slow down the speed of the droplet (droplet) in the Reagent injection region 11, thereby increasing the electric field action time and the Reagent injection time of the droplet (droplet) and the Reagent (Reagent);
FIG. 5 shows that the structure is used to deform the liquid droplet when entering the reagent injection end 9, increasing the contact area between the liquid droplet and the reagent injection end 9;
FIG. 6 shows a schematic of reagent injection; FIG. 6(a) shows that the amount of the reagent containing the stain injected is small when the reagent injection end 9 applies a small pressure; FIG. 6(b) shows that the amount of the reagent containing the stain is injected more when the reagent injection end 9 applies a larger pressure;
FIG. 7 is a diagram showing the state of the droplets at the outlet 3 after the injection of the reagents into the droplets, showing that the reagents have been sufficiently melted into the droplets at the outlet and rapidly mixed uniformly; wherein FIG. 7(a) shows that no reagent has been injected by applying an electric field; FIG. 7(b) reagent injection flow rate is 1/10 droplet flow rate, and FIG. 7(c) reagent injection flow rate is 1/5 droplet flow rate; FIG. 7(d) reagent injection flow rate of 1/2.5 droplet flow rate; the result shows that the amount of the reagent for injecting the liquid drops can be regulated and controlled according to different injection flow rates, and different required concentrations are generated;
FIG. 8 shows a schematic diagram of a micro-emulsion droplet reagent injection chip;
wherein, 1-a droplet generating member; 2-a main runner; 3-an outlet; 4-continuous phase injection end; 5-continuous phase flow channel; 6-dispersed phase injection end; 7-dispersed phase flow channel; 8-a droplet generation end; 9-reagent injection end; 10-alternating current electrode; 11-reagent injection zone; 12-a shunt conduit (fig. 4(b1, b 2)); 13-columnar structure (fig. 4(b 1)); 14-micro flow channel (FIG. 4(b 2)).
Detailed Description
The invention discloses a chip and a device for preparing emulsion droplets, and the technical personnel in the field can appropriately improve the technological parameters by referring to the content. It is expressly intended that all such similar substitutes and modifications which would be obvious to one skilled in the art are deemed to be included in the invention. While the methods and applications of this invention have been described in terms of preferred embodiments, it will be apparent to those of ordinary skill in the art that variations and modifications in the methods and applications described herein, as well as other suitable variations and combinations, may be made to implement and use the techniques of this invention without departing from the spirit and scope of the invention.
Taking single cell whole genome sequencing as an example: the single cell obtaining method includes a gradient dilution method, a Laser capture microdissection technology (Laser capture microdissection) or a flow cytometer to screen cells, and after obtaining the cells, the method adds liquids such as cell lysate, reaction reagents and the like by manual operation and then enters subsequent analysis, so that manual operation errors cannot be avoided in the operation process, most of the methods utilize a tissue sample or a cell group to carry out analysis, and the average value is obtained by operating the cells of the sample group, so that the heterogeneity among the cells and the characteristics of single cell individuals are often ignored, and the uniqueness of each cell cannot be truly reflected. The invention provides a controllable microfluid emulsification chip, which is used for respectively injecting a continuous phase (continuous phase) and a disperse phase (dispersed phase) to generate w/o (water in oil) micro-emulsified liquid drops, so that each w/o liquid drop only has a single cell to be detected or a reagent to be reacted, and after the liquid drop (drop) is formed, one or more reagent injection ends (injection) are arranged on a rear end pipeline, and the matching structure design and alternating current electric field application are carried out to ensure that the reagent to be added can be injected into the liquid drop (drop). The micro-emulsified liquid drops can coat single cells, separate the single cells and capture the single cells, and the single cells are provided for detection such as DNA hybridization polymerase chain reaction amplification, sequencing, single cell immunoassay and the like, RNA sequencing and RNA expression quantitative analysis in a microenvironment.
The chip provided by the invention is a droplet (droplet) generating structure, and a Reagent injection end 9 is arranged at the rear end, as shown in fig. 2(a), wherein the Continuous phase (Continuous phase) is Oil, the Disperse phase (Disperse phase) is a sample (sample), and the injection liquid can be a Reagent (Reagent) or other liquid to be added to the droplet (droplet). When the droplet (droplet) passes through the opening of the Reagent injection end 9, the AC electric field can be applied by the electrodes to change the surface tension of the droplet (droplet) and the Reagent injection end 9, so that the Reagent (Reagent) is injected into the droplet (droplet) (as shown in fig. 2 (b)).
As shown in fig. 3, a sample and oil phase droplets are injected into a chip by a pump, the sample (dispersed phase liquid) and the oil phase liquid (continuous phase liquid) are collected by hydrodynamic focusing to generate micro droplets, a reagent is injected into the chip at an injection end by the pump, and an AC electric field is applied when the micro droplets pass through the injection end to change the surface tension between the micro droplets and the reagent injection end, so that the reagent can be injected into the micro droplets.
In order to regulate the amount of injected Reagent according to the requirement, a pressure balance structure, namely a shunt pipeline 12 is arranged in the pipeline, the speed of the liquid drop (droplet) passing through the Reagent injection end 9 is regulated, the pipeline is reduced to deform the liquid drop (droplet) so as to increase the contact area between the liquid drop (droplet) and the injection end, and the injection amount of the Reagent (Reagent) is regulated by the liquid drop (droplet) and the injection end. The chip provided by the invention can simultaneously complete the formation of micro-emulsion droplets, the injection of reagents, the mixing of the reagents and the collection of products. If the reagent needs to be added for multiple times, the chip provided by the invention can be connected with a plurality of injection ends in series, so that a plurality of reagent adding pipelines are provided, the manual operation error and the reagent consumption are reduced, and the sample processing speed and the sample processing accuracy are greatly increased.
In addition, the amount of the Reagent (Reagent) injected into the sample droplet (sample droplet) can be easily controlled by the pressure control of the Reagent (Reagent) end, as shown in fig. 6(a, b) and fig. 7(a) blank injection group (without injection), the Reagent (Reagent) can not change the surface tension of the droplet (droplet) by the electric field when no voltage is applied, the Reagent (Reagent) can enter, when an alternating current electric field with frequency of 10-1000 kHz and 100Vpp or more is applied, the Reagent can be injected into the sample droplet, and the flow rates of the injection ends of fig. 7(b), (c) and (d) are respectively 50 μ L/hr, 100 μ L/hr and 200 μ L/hr, 10pL, 20pL and 40pL reagents (Reagent) are injected, and the sample flow rate is 300 μ L/hr.
The present invention utilizes the structural design to compress the sample droplet (sample droplet) to be flat when passing through the reagent injection region 11(injection region), and further increases the contact area between the sample droplet (sample droplet) and the injection port (injection port), so that the injection efficiency of the reagent (reagent) is greatly increased (injection rate/injection region injection/area-square function). In addition, a bypass channel 12 is designed before and after the injection region (injection region), so that part of the oil phase (oil) flows to the bypass channel 12, the speed of the sample droplet (sample droplet) in the reagent injection region 11(injection region) is greatly reduced (as shown in fig. 8), and the contact time (injection rate/time-linear function) of the reagent (reagent) injected into the sample droplet (sample droplet) is further increased, so that the efficiency of the reagent (reagent) injected into the sample droplet (sample droplet) can be approximated to the geometric growth (sample droplet) by the cooperation of the two structural designs (connected x sample increment). Therefore, high throughput chip droplet formation and reagent injection (high throughput on-chip droplet formation and reagent injection) can be achieved in cooperation with the front end droplet generation section 1(droplet formation part). The experimental results of the examples show that 100pL and 50pL of reagent (reagent volume) can be successfully injected into a droplet (microdroplet) with a volume of 0.2nL (diameter-70 μm) at high flow rates of 300 μ L/hr and 700 μ L/hr, and the detection amount (throughput) of a single chip is more than 20 times higher than that of the prior art, so that the throughput of the chip on a sample can be greatly increased, and the time for pretreatment and detection analysis can be shortened.
The chip provided by the invention is provided with a reagent injection end 9 at the main flow channel 2 part formed by the liquid drop (drop), so that an electric field is applied when the liquid drop (drop) passes through the reagent injection end 9, the surface tension of the liquid drop (drop) and the reagent injection end 9 is changed, and the reagent is injected into the liquid drop (drop) to generate reaction.
In some embodiments, the diameter of the main channel 2 of the chip provided by the present invention is reduced before entering the reagent injection end 9, and preferably, the diameter of the 1/5 droplet is less than or equal to the diameter of the 4/5 droplet in the reagent injection area 11; preferably, the width of main flow channel 2 of 1/10 ≦ the width of main flow channel 2 of reagent injection region 11 ≦ 3/4, so that the droplet is deformed into a relatively long and narrow droplet, and this deformation increases the contact area between the droplet and reagent injection end 9, and thus increases the droplet injection volume (drop). The oil phase injection rate is 100 mu L/hr-10 mL/hr, and the sample injection rate is 10 mu L/hr-1 mL/hr.
In other embodiments, the diversion pipe 12(Bypass channel) is arranged to divert the volume of the oil phase liquid, so as to slow down the speed of the liquid drop (droplet) passing through the reagent injection end 9, thereby increasing the injection amount of the reagent (reagent), and the reagent injection amount is controlled by the diversion pipe 12. Preferably, the 1/10 droplet diameter is less than or equal to the width of the distribution pipe 12 is less than or equal to 1/2 droplet diameter.
In some embodiments, the diversion channel 12(Bypass channel) can also divert liquid through the diversion channel 12 having the same width as the main channel 2 and the pillar structure 13, and the pillar structure 13 can prevent liquid drops from separating from the main channel 2. 1/10 droplet diameter < gap of the columnar structure 13 < 1/2 droplet diameter.
In other embodiments, when the number of the columnar structures 13 is 1, the columnar structures 13 and the side wall of the shunt pipe 12 form a micro flow channel 14;
when the number of the columnar structures 13 is more than 1, micro channels 14 are formed between the columnar structures 13 and/or between the columnar structures 13 and the side walls of the shunt pipelines 12;
1/10 the diameter of the liquid drop is less than or equal to the width of the micro-channel 14 is less than or equal to 1/2 liquid drop diameter.
Under the condition that the flow rate of the liquid drops is fixed, the injection amount of the reagent (reagent) can be regulated and controlled by adjusting the pressure and the flow rate of the reagent injection end, so that the throughput of the whole field formation and the reagent injection (repeat formation + reagent injection) is improved. 1/10 droplet flow rate is less than or equal to the flow rate of injected reagent.
By switching an electric field (AC, above 100Vpp, 10-1000 KHz), a reagent (reagent) can be selectively injected into a specific sample droplet (sample droplet).
Integrating droplet formation (droplet formation) and reducing the channel width (droplet diameter) of the reagent injection region (reagent injection region) allows the sample droplet (sample droplet) to be squeezed into a flat shape in accordance with the arrangement of the bypass channel 12(bypass channel), allowing simultaneous droplet generation at very high sample processing speeds on a single chip and in-line injection of a suitable dose of reagent (reagent).
The chip and the device for preparing the emulsified liquid drop provided by the invention are available on the market, and all the components, the reagents and the raw materials are available on the market.
The invention is further illustrated by the following examples:
example 1
The invention provides a chip for preparing emulsified liquid drops, which comprises a substrate, wherein the substrate is provided with a liquid drop generating component 1, a main flow channel 2 and an outlet 3 which are sequentially connected, and the liquid drop generating 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 liquid drop generating end 8; the continuous phase injection end 4 is communicated with the continuous phase flow passage 5; the dispersed phase injection end 6 is communicated with the dispersed phase flow channel 7; the continuous phase flow channel 5, the disperse phase flow channel 7 and the main flow channel 2 are communicated at a liquid drop generating end 8 in a crossing way; 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 channel 2 is also provided with an alternating current electrode 10. The connection line of the reagent injection end 9 and the alternating current electrode 10 is perpendicular to the main flow channel 2. As shown in fig. 2(a) and 2 (b).
Example 2
The invention provides a chip for preparing emulsified liquid drops, which comprises a substrate, wherein the substrate is provided with a liquid drop generating component 1, a main flow channel 2 and an outlet 3 which are sequentially connected, and the liquid drop generating 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 liquid drop generating end 8; the continuous phase injection end 4 is communicated with the continuous phase flow passage 5; the dispersed phase injection end 6 is communicated with the dispersed phase flow channel 7; the continuous phase flow channel 5, the disperse phase flow channel 7 and the main flow channel 2 are communicated at a liquid drop generating end 8 in a crossing way; 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 channel 2 is also provided with an alternating current electrode 10. The connection line of the reagent injection end 9 and the alternating current electrode 10 is perpendicular to the main flow channel 2.
The flow channel width of the reagent injection region 11 is smaller than the width of the other regions of the main flow channel 2.
1/5 the diameter of the liquid drop is not more than 4/5 liquid drop diameter; 1/10 the width of the main flow channel 2 is not more than the width of the main flow channel 2 of the reagent injection area 11 is not more than 3/4; the liquid drop is formed by the fusion of a mobile phase and a disperse phase. As shown in fig. 4 (a).
Example 3
The invention provides a chip for preparing emulsified liquid drops, which comprises a substrate, wherein the substrate is provided with a liquid drop generating component 1, a main flow channel 2 and an outlet 3 which are sequentially connected, and the liquid drop generating 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 liquid drop generating end 8; the continuous phase injection end 4 is communicated with the continuous phase flow passage 5; the dispersed phase injection end 6 is communicated with the dispersed phase flow channel 7; the continuous phase flow channel 5, the disperse phase flow channel 7 and the main flow channel 2 are communicated at a liquid drop generating end 8 in a crossing way; 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 channel 2 is also provided with an alternating current electrode 10. The connection line of the reagent injection end 9 and the alternating current electrode 10 is perpendicular to the main flow channel 2.
The flow channel width of the reagent injection region 11 is smaller than the width of the other regions of the main flow channel 2.
1/5 the diameter of the liquid drop is not more than 4/5 liquid drop diameter; 1/10 the width of the main flow channel 2 is not more than the width of the main flow channel 2 of the reagent injection area 11 is not more than 3/4; the liquid drop is formed by the fusion of a mobile phase and a disperse phase.
The main runner 2 is also provided with a shunt pipeline 12; one end of the flow distribution pipe 12 is provided between the droplet generation end 8 and the reagent injection region 11, and the other end of the flow distribution pipe 12 is provided between the reagent injection region 11 and the outlet 3. The number of the shunt tubes is at least 1. The 1/10 liquid drop diameter is not more than the width of the diversion pipeline 12 and not more than 1/2 liquid drop diameter; the liquid drop is formed by the fusion of a mobile phase and a disperse phase. As shown in fig. 4(b1) and 4(b 2).
Example 4
The invention provides a chip for preparing emulsified liquid drops, which comprises a substrate, wherein the substrate is provided with a liquid drop generating component 1, a main flow channel 2 and an outlet 3 which are sequentially connected, and the liquid drop generating 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 liquid drop generating end 8; the continuous phase injection end 4 is communicated with the continuous phase flow passage 5; the dispersed phase injection end 6 is communicated with the dispersed phase flow channel 7; the continuous phase flow channel 5, the disperse phase flow channel 7 and the main flow channel 2 are communicated at a liquid drop generating end 8 in a crossing way; 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 channel 2 is also provided with an alternating current electrode 10. The connection line of the reagent injection end 9 and the alternating current electrode 10 is perpendicular to the main flow channel 2.
The flow channel width of the reagent injection region 11 is smaller than the width of the other regions of the main flow channel 2.
1/5 the diameter of the liquid drop is not more than 4/5 liquid drop diameter; 1/10 the width of the main flow channel 2 is not more than the width of the main flow channel 2 of the reagent injection area 11 is not more than 3/4; the liquid drop is formed by the fusion of a mobile phase and a disperse phase.
The main runner 2 is also provided with a shunt pipeline 12; one end of the flow distribution pipe 12 is provided between the droplet generation end 8 and the reagent injection region 11, and the other end of the flow distribution pipe 12 is provided between the reagent injection region 11 and the outlet 3. The 1/10 liquid drop diameter is not more than the width of the diversion pipeline 12 and not more than 1/2 liquid drop diameter; the liquid drop is formed by the fusion of a mobile phase and a disperse phase.
A columnar structure 13 is also arranged in the flow passage of the flow distribution pipeline 12;
when the number of the columnar structures 13 is 1, the columnar structures 13 and the side wall of the shunt pipeline 12 form a micro-channel 14;
when the number of the columnar structures 13 is more than 1, micro-channels 14 are formed among the columnar structures 13 and/or between the columnar structures 13 and the side walls of the shunt pipelines 12;
1/10 the diameter of the liquid drop is not more than 1/2 of the width of the micro-channel 14;
the liquid drop is formed by the fusion of a mobile phase and a disperse phase.
As shown in fig. 4(b1) and 4(b 2).
EXAMPLE 5 method for preparing emulsion droplets
Based on the chip as shown in example 4 (the injection region is designed as fig. 4(b)), the continuous phase is injected into the continuous 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 are converged and fused at the droplet generation end 8 to form droplets.
The droplets flow through the main channel 2, a reagent is injected into the reagent injection end 9, an alternating current electric field (>100Vpp, >10kHz) is applied by the alternating current electrode 10, the reagent and the droplets are fused to form emulsified droplets, and the emulsified droplets are collected at the outlet 3.
The rate of the mobile phase was 1mL/hr and the rate of the dispersed phase was 100. mu.L/hr.
1/10 droplet flow Rate the flow rate of the reagent is less than or equal to the droplet flow rate.
Example 6 preparation of emulsified droplets
Based on the chip as shown in example 4 (the injection region is designed as fig. 4(b)), the continuous phase is injected into the continuous 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 are converged and fused at the droplet generation end 8 to form droplets.
The droplets flow through the main channel 2, reagent is injected into the reagent injection end 9, an alternating current electric field (>300Vpp, >10kHz) is applied by the alternating current electrode 10, the reagent and the droplets are fused to form emulsified droplets, and the emulsified droplets are collected at the outlet 3.
The rate of the mobile phase was 5mL/hr and the rate of the dispersed phase was 500. mu.L/hr.
1/10 droplet flow Rate the flow rate of the reagent is less than or equal to the droplet flow rate.
As shown in fig. 5.
Example 7 method for preparing emulsified liquid droplets
Based on the chip as shown in example 4 (the injection region is designed as fig. 4(b)), the continuous phase is injected into the continuous 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 are converged and fused at the droplet generation end 8 to form droplets.
The droplets flow through the main channel 2, reagent is injected into the reagent injection end 9, an alternating current electric field (>500Vpp, >10kHz) is applied by the alternating current electrode 10, the reagent and the droplets are fused to form emulsified droplets, and the emulsified droplets are collected at the outlet 3.
The rate of the mobile phase was 10mL/hr and the rate of the dispersed phase was 1 mL/hr.
1/10 droplet flow Rate the flow rate of the reagent is less than or equal to the droplet flow rate.
Control group
The structure design of the original microfluidic chip and the preparation of emulsified droplets and the injection method of reagents are shown in fig. 2(b) (the design of non-structure squashed droplets, the design of non-oil shunt). Based on the chip as shown in example 1 (the injection region is designed as fig. 2(b)), the continuous phase is injected into the continuous 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 are converged and fused at the droplet generation end 8 to form droplets.
The droplets flow through the main channel 2, reagent is injected at the reagent injection end 9, an alternating current electric field (300Vpp, 100KHz) is applied by the alternating current electrode 10, and the reagent and the droplets are fused to form emulsified droplets which are collected at the outlet 3.
The rate of the mobile phase was 100. mu.L/hr and the rate of the dispersed phase was 10. mu.L/hr.
1/10 droplet flow Rate the flow rate of the reagent is less than or equal to the droplet flow rate.
Example 8 comparison of emulsion droplet preparation
Experiment groups 1-4: emulsion droplets were prepared according to examples 5 to 8, respectively;
control group: emulsified droplets were prepared 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.
TABLE 1
Example 9
The chips provided in embodiments 1 to 4 of the present invention can easily control the amount of the Reagent (Reagent) injected into the sample droplet (sample droplet) by controlling the pressure at the Reagent (Reagent) end, as shown in fig. 6(a, b) and fig. 7(a) the blank injection group (without injection), the Reagent (Reagent) cannot change the surface tension of the droplet (Reagent) by the electric field in the state of no voltage application, the Reagent (Reagent) enters the sample droplet (sample), when an alternating electric field with a frequency of 300kHz and 300Vpp is applied, the Reagent can be injected into the sample droplet, and the flow rates at the Reagent injection ends of fig. 7(b), (c) and (d) are 50 μ L/hr, 100 μ L/hr and 200 μ L/hr, respectively, 10pL, 20pL and 40pL reagents (Reagent) are injected, and the sample flow rate is 500 μ L/hr.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.
Claims (15)
1. The chip for preparing the emulsified liquid drops is characterized by comprising a substrate, wherein the substrate is provided with a liquid drop generating component (1), a main flow channel (2) and an outlet (3) which are sequentially connected, and the liquid drop generating component (1) comprises a continuous phase injection end (4), a continuous phase flow channel (5), a disperse phase injection end (6), a disperse phase flow channel (7) and a liquid drop generating 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) are communicated with each other at the liquid drop generating end (8) in a crossing manner;
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);
and an alternating current electrode (10) is also arranged on the other side wall of the main flow channel (2).
2. The chip according to claim 1, wherein the connection line of the reagent injection end (9) and the alternating current electrode (10) is perpendicular to the main channel (2).
3. The chip according to claim 1 or 2, wherein the reagent injection region (11) has a flow channel width smaller than the width of the other regions of the main flow channel (2).
4. The chip of claim 3, wherein the 1/5 droplet diameter is equal to or less than 4/5 droplet diameter of the flow channel width of the reagent injection region (11); 1/10 the width of the main channel (2) is not more than the channel width of the reagent injection area (11) is not more than 3/4 the width of the main channel (2);
the liquid drop is formed after the mobile phase and the disperse phase are fused.
5. A chip according to claim 3 or 4, characterized in that the main channel (2) is further provided with a flow-dividing duct (12); one end of the shunt pipe (12) is arranged between the droplet generation end (8) and the reagent injection area (11), and the other end of the shunt pipe (12) is arranged between the reagent injection area (11) and the outlet (3).
6. The chip of claim 5, wherein 1/10 droplet diameter is ≦ the width of the diversion conduit (12) is ≦ 1/2 droplet diameter;
the liquid drop is formed after the mobile phase and the disperse phase are fused.
7. The chip according to claim 5, wherein a columnar structure (13) is further arranged in the flow channel of the shunt pipe (12);
if the number of the columnar structures (13) is 1, the columnar structures (13) and the side wall of the shunt pipeline (12) form a micro-channel (14);
if the number of the columnar structures (13) is more than 1, micro-channels (14) are formed among the columnar structures (13) and/or between the columnar structures (13) and the side wall of the shunt pipeline (12);
1/10 the diameter of the liquid drop is less than or equal to the width of the micro-channel (14) is less than or equal to 1/2 liquid drop diameter;
the liquid drop is formed after the mobile phase and the disperse phase are fused.
8. Use of a chip according to any one of claims 1 to 7 for the preparation of emulsified droplets.
9. Use of the chip of any one of claims 1 to 7 for synthesis of micro-reagents, synthesis of micro-materials, micro-molecular reactions and analysis, single cell coating, single cell separation, single cell capture, single cell sequencing and single cell protein plastomic analysis.
10. Kit comprising a chip according to any one of claims 1 to 7 and acceptable reagents.
11. Device, characterized in that it comprises a chip according to any one of claims 1 to 7 and an auxiliary component.
12. The apparatus of claim 11, wherein the auxiliary component comprises a pump.
13. A method for producing emulsified droplets, characterized in that, based on the chip according to any one of claims 1 to 7, a continuous phase is injected into the continuous flow channel (5) through the continuous phase injection port (4), and a dispersed phase is injected into the dispersed phase flow channel (7) through the dispersed phase injection port (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 are converged and fused at the droplet generation end (8) to form a droplet;
the liquid drops flow through the main flow channel (2), reagents are injected into the reagent injection end (9), an alternating current electric field is applied by the alternating current electrode (10), the reagents and the liquid drops are fused to form the emulsified liquid drops, and the emulsified liquid drops are collected at the outlet (3).
14. The method of claim 13, wherein the rate of the mobile phase is 100 μ L/hr to 10mL/hr and the rate of the dispersed phase is 10 μ L/hr to 1 mL/hr.
15. The method of claim 13 or 14, wherein the 1/10 droplet flow rate is equal to or less than the flow rate of the injected reagent is equal to or less than the droplet flow rate.
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