A centrifugal microfluidic chip for simultaneously generating droplets of different sizes

Abstract

The present application relates to the technical field of microfluidic chip, and disclose a centrifugal microfluidic chip for generating different size droplets simultaneously, including a liquid distribution channel for uniformly distributing sample to multiple droplet generation units, the design of a quantitative pool ensures the fixed volume of liquid and the consistency of the number of droplet generation, the chip contains a sample inlet and two continuous phase reagent inlets, the sample is precisely distributed to the quantitative pool by centrifugal force and enters the droplet generation units respectively, the first droplet generation unit is used for generating large size droplets with a diameter of 100-200 mu m, and the second droplet generation unit is used for generating small size droplets with a diameter of 10-100 mu m, the microfluidic chip can realize the preparation of ultrahigh throughput droplets, the generated droplets are guided to the collection area by centrifugal force to form a regular single-layer arrangement, the chip has wide application in the fields of biomedical research, material science, chemical synthesis and environmental monitoring, especially in the preparation of fluorescent microspheres, which can improve the experimental efficiency and result consistency.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of microfluidic chips, in particular to a centrifugal microfluidic chip for generating droplets of different particle sizes simultaneously. BACKGROUND

[0002] Microfluidic technology has a wide application prospect in biomedical, chemical synthesis, environmental monitoring and other fields by precisely controlling and manipulating fluid at microscale. The microfluidic chip for generating droplets usually adopts flow focusing method, cross injection method and drop flow method to form droplets by shear force. The flow focusing method forms droplets at the focusing point through multi-channel structure, and utilizes the pressure applied by the continuous phase to the dispersed phase to produce uniform and controllable droplets. The cross injection method realizes droplet generation through T-shaped cross structure, which is suitable for simple droplet generation application and easy to manufacture and operate. The drop flow method relies on the shear force of the continuous phase fluid to form droplets at the nozzle, which is particularly suitable for droplet generation under low flow rate and high viscosity fluid conditions. These methods make the droplet generation process more precise and controllable, suitable for various experimental needs and meet the high requirements of modern scientific research on microscale sample processing.

[0003] It is of great significance and value to construct a microfluidic chip capable of generating droplets of different particle sizes simultaneously. First, it provides diversity and flexibility, which can handle samples with different needs in the same experiment, improve the high-throughput screening ability of the experiment and the consistency of the results. For example, in the process of drug screening, large droplets can be generated for drug delivery and small droplets for single cell analysis, thereby greatly improving the experimental efficiency. Secondly, such chip has wide application in biomedical research, material science, chemical synthesis and environmental monitoring. For example, droplets of different particle sizes can simulate different biological environments, such as the microstructure of extracellular matrix, helping to study cell behavior and drug response. In material science, it can be used to synthesize micrometer or nanometer materials with specific size and structure, such as nanoparticles and microspheres. In chemical synthesis, droplets of different sizes can provide different reaction time and conditions, thereby optimizing the synthesis path and improving the yield and purity. In environmental monitoring, it can be used to capture and analyze pollutants of different sizes.

[0004] Therefore, we propose a centrifugal microfluidic chip for generating droplets of different particle sizes simultaneously. By realizing the simultaneous generation of droplets of different particle sizes, this microfluidic chip will promote further innovation and progress in scientific research and industrial application, opening up new research and application prospects. SUMMARY

[0005] In order to make up for the shortcomings of the prior art, the present application provides the following technical solutions: a chip including a bottom sheet and a cover sheet in sealing cooperation with the bottom sheet, a through hole is provided at the center of the microfluidic chip, and a positioning notch is provided at the inner edge of the through hole for fixing the chip on a centrifugal device.

[0006] Preferably, the chip comprises one sample inlet and two continuous phase reagent inlets, the sample inlet is used to accommodate up to 150 μL of sample, which is directed to the sample reservoir through the distribution channel, the continuous phase reagent inlets are used to add continuous phase reagent.

[0007] Preferably, the sample reservoir is connected to a first vent, and the sample is precisely dispensed from the first reservoir into two different volumes of the first and second metering reservoirs through a centrifugation step.

[0008] Preferably, the first metering reservoir is provided with a second vent, and the sample in the first metering reservoir is introduced into the first and second droplet generation units through centrifugal force, which are used to generate large and small droplets respectively, and the first and second droplet generation units are connected to the overflow reservoir through corresponding microchannels.

[0009] Preferably, the first droplet generation unit comprises 1-10 first micro-nozzle structures or first micro-nozzle structure arrays, which are used to generate large droplets with a target diameter of 100-200 μm; the second droplet generation unit comprises 5-30 second micro-nozzle structure arrays, which are used to generate small droplets with a target diameter of 10-100 μm.

[0010] Preferably, the bottom sheet and the cover sheet of the microfluidic chip are firmly attached using double-sided tape with the required adhesive strength, which can withstand conventional heating temperatures and have no significant adverse effects on the reactions performed.

[0011] Preferably, the droplets generated by the first and second droplet generation units are guided to the droplet collection area by centrifugal force, forming a regular single-layer arrangement.

[0012] Preferably, the uniform distribution of droplets in the collection area is controlled by adjusting the rotation frequency and fluid flow rate.

[0013] Preferably, the method comprises the following steps:

[0014] Step one: add the dispersed phase reagent through the sample inlet, and the continuous phase reagent through the first and second oil inlets into the first and second oil reservoirs;

[0015] Step two: then start the centrifugation program for microsphere preparation at 30 g for 15 s, which transfers the fluorescent solution and monomer solution to the first and second reservoirs and the first and second oil reservoirs;

[0016] Step three: rotate at 80 g for 20 s, which transfers the fluorescent solution to the second and first metering reservoirs, and the monomer solution to the first and second droplet generation units;

[0017] Step four: the rotation speed is 181g, and after 0.5s, the rotation speed is adjusted to 80g, and the preparation of the droplets is completed for 300s.

[0018] Advantages

[0019] Compared with the prior art, the present application provides a centrifugal microfluidic chip for simultaneously generating droplets of different particle sizes, which has the following advantages:

[0020] 1. The present application designs a microfluidic chip for generating droplets by double-volume centrifugal stepwise emulsification, which uniformly distributes the sample to multiple droplet generation units through the liquid distribution channel, and ensures the consistency of the number of droplet generation by fixing the volume of the liquid through the design of the quantitative pool.

[0021] 2. The microfluidic chip of the present application can realize the preparation of ultra-high throughput droplets, and the generated droplets are guided to the collection area by centrifugal force to form a regular single-layer arrangement.

[0022] 3. The chip of the present application has wide application in the fields of biomedical research, material science, chemical synthesis and environmental monitoring, especially in the preparation of fluorescent microspheres, which can improve the experimental efficiency and consistency of results. BRIEF DESCRIPTION OF DRAWINGS

[0023] Figure 1 The present application is a centrifugal microfluidic chip for simultaneously generating droplets of different particle sizes.

[0024] Figure 2 The present application is a centrifugal microfluidic chip for simultaneously generating droplets of different particle sizes.

[0025] Figure 3 The present application is a centrifugal microfluidic chip for simultaneously generating droplets of different particle sizes.

[0026] Figure 4 The present application is a centrifugal microfluidic chip for simultaneously generating droplets of different particle sizes.

[0027] Figure 5 The present application is a centrifugal microfluidic chip for simultaneously generating droplets of different particle sizes.

[0028] Figure 6This is a fluorescence spectrum of fluorescent microspheres prepared by a centrifugal microfluidic chip capable of simultaneously generating droplets of different sizes, according to the present invention.

[0029] In the diagram: 1. Sample inlet; 2. Liquid distribution channel; 3. First vent; 4. Sample reservoir; 5. First oil inlet; 6. First reservoir; 7. Second vent; 8. First metering tank; 9. Second oil inlet; 10. First oil reservoir; 11. Second metering tank; 12. Waste liquid tank; 13. Second oil reservoir; 14. First droplet generation unit; 15. Second reservoir; 16. First micro-nozzle structure; 17. Second droplet generation unit; 18. Second micro-nozzle structure; 19. Overflow tank. Detailed Implementation

[0030] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0031] like Figures 1-2 As shown, a centrifugal microfluidic chip for simultaneously generating droplets of different sizes includes a microfluidic chip substrate and a cover plate that seals with the substrate. Both are firmly bonded using double-sided adhesive with the required bonding strength, capable of withstanding conventional heating temperatures, and without significantly adversely affecting the reactivity. A through hole is provided in the center of the microfluidic chip, and a positioning notch is provided at the inner edge of the through hole. The through hole and the positioning notch work together to fix and position the chip, and only one centrifuge is needed to complete the centrifugal distribution of the sample.

[0032] like Figure 1 As shown, multiple independent droplet generating components are provided on the film. The five independent droplet generating components are connected by a liquid distribution channel 2. One end of the liquid distribution channel 2 is connected to the sample inlet 1 provided on the film, and the other end is connected to the droplet generating component provided on the film. For example, when the required sample volume is between 20 and 200 microliters, the diameter of the sample inlet 1 can be between 0.5 mm and 200 mm.

[0033] like Figure 2As shown, the sample can enter the sample reservoir 4 through the liquid separation channel 2 under the action of centrifugal force, the sample reservoir 4 is connected with the first exhaust hole 3, the sample in the first reservoir 6 can enter the second quantitative pool 11 and the first quantitative pool 8 corresponding to the first droplet generation unit 14 and the second droplet generation unit 17 through centrifugation, and the excess sample enters the waste pool 12, the quantitative pool is provided with the second exhaust hole 7, with the change of the centrifugal speed, the sample in the second quantitative pool 11 enters the first droplet generation unit 14 through the first micro-nozzle structure 16, the first droplet generation unit 14 is filled with the continuous phase reagent through the first oil inlet hole 5 and the first oil reservoir 10, the sample in the first quantitative pool 8 enters the second droplet generation unit 17 through the second reservoir 15 and the second micro-nozzle structure 18, and the second droplet generation unit 17 is filled with the continuous phase reagent through the second oil inlet hole 9 and the second oil reservoir 13, and the first droplet generation unit 14 and the second droplet generation unit 17 and the first oil reservoir 10 and the second oil reservoir 13 are connected with the overflow pool 19 through the corresponding micro-channel.

[0034] As shown in the figure, Figure 3 The first droplet generation unit 14 comprises a single or multiple first micro-nozzle structure 16 or a first micro-nozzle structure 16 array, the first micro-nozzle structure 16 has an outlet size of 75 μm, and the nozzle depth is 70 μm.

[0035] As shown in the figure, Figure 4 The second droplet generation unit 17 comprises an array of 5-30 second micro-nozzle structures 18, the second micro-nozzle structure 18 has an outlet size of 11 μm, and the second micro-nozzle structure 18 has a depth of 11 μm.

[0036] The microfluidic chip design comprises a sample inlet and two continuous phase inlets, which ensures the smooth flow and input of the sample and the continuous phase, the sample inlet can accommodate up to 150 μL of sample, which is guided to the sample reservoir 4 through the liquid separation channel 2, and the sample is accurately distributed into the first quantitative pool 8 and the second quantitative pool 11 through the overflow metering structure, the two continuous phase inlets are used to add fluorinated oil to support droplet generation, the first quantitative pool 8 and the second quantitative pool 11 distribute the sample to two regions with volumes of 10.5 μL and 2.5 μL respectively through a centrifugation step, and then the sample is transferred to the first droplet generation unit 14 and the second droplet generation unit 17 (DGU) to generate droplets with large particle size (100-200 μm) and small particle size (10-100 μm), and the droplets are guided to the collection area by centrifugal force after droplet generation to form a regular single-layer arrangement.

[0037] The microfluidic chip provided by the application can develop various applications for various reactions, such as digital detection, microsphere / liposome preparation, single cell separation, etc., and the following will take the preparation of fluorescent microspheres as an example to illustrate the use process:

[0038] Suitable fluorescent dyes such as FITC (fluorescein isothiocyanate) are selected, polymer monomers such as acrylate or methacrylate are usually used, and cross-linking agents such as divinylbenzene (DVB) are usually used, water, ethanol or dimethyl sulfoxide (DMSO) are usually used as solvents, suitable surfactants such as Span 80 are selected for stabilizing droplet generation, and suitable thermal initiators or photoinitiators such as azobisisobutyronitrile (AIBN) or potassium persulfate (KPS) are selected for initiating polymerization;

[0039] The microfluidic chip is cleaned with isopropyl alcohol and deionized water to ensure that the inside of the channel is clean and dust-free, and the chip surface is treated with oxygen plasma to increase hydrophilicity and facilitate liquid flow. The chip and pressure sensitive film are placed in a hot pressing device and hot pressed at a suitable temperature and pressure. Typical hot pressing conditions are 70-100℃, pressure 0.1-0.5MPa, duration 5-10 minutes.

[0040] The monomer material, initiator and stabilizer are dissolved in a suitable solvent to form a monomer solution, which is used as the continuous phase for microsphere preparation. The fluorescent dye is dissolved in a solvent to form a fluorescent solution, which is used as the dispersed phase for microsphere preparation.

[0041] The fluorescent solution is added through the sample inlet 1, and the monomer solution is added through the first oil inlet 5 and the second oil inlet 9 into the first oil reservoir 10 and the second oil reservoir 13 respectively. Then the centrifugation program for microsphere preparation is started, with a speed of 30g for 15s. The fluorescent solution and the monomer solution are all transferred to the first liquid reservoir 6 and the second liquid reservoir 15, and the first oil reservoir 10 and the second oil reservoir 13. The speed is adjusted to 80g for 20s, and the fluorescent solution is transferred to the second dosing tank 11 and the first dosing tank 8, and the monomer solution is transferred to the first droplet generation unit 14 and the second droplet generation unit 17. After a speed of 181g for 0.5s, the speed is adjusted to 80g, and the preparation of microspheres is continued for 300s.

[0042] The generated droplets are collected in a heating device (such as a heating plate or an oven) for polymerization, and the temperature is usually between 60-80℃, and the time is determined by the specific monomer and initiator. If a photoinitiator is used, the droplets are cured by UV irradiation after collection, and the irradiation time and intensity are determined by the specific initiator and fluorescent dye. The cured fluorescent microspheres are separated by centrifuge and washed several times with deionized water or a suitable solvent to remove unreacted monomers and surfactants.

[0043] As shown in Figure 5 , the morphology and uniformity of the microspheres are observed using a fluorescence microscope. The fluorescent microspheres prepared by the first droplet generation unit 14 have a particle size of about 200μm Figure 5 (A), and the fluorescent microspheres prepared by the second droplet generation unit 17 have a particle size of about 30μm Figure 5 (B).

[0044] As shown in Figure 6 The fluorescence intensity of the microspheres was determined by a fluorescence spectrometer, and the fluorescence emission peak of the prepared microspheres was single, which was the characteristic peak of FITC (fluorescein isothiocyanate), and exhibited excellent fluorescence intensity.

[0045] It should be noted that, in this document, the terms such as first and second are used merely to distinguish one entity or operation from another, and do not necessarily require or imply any such actual relationship or order between such entities or operations. Moreover, the terms "comprising", "containing" or any other variant thereof are intended to cover non-exclusive inclusion, so that a process, method, article or apparatus including a series of elements not only includes those elements, but also includes other elements not explicitly listed, or further includes elements inherent in such a process, method, article or apparatus.

[0046] Although the embodiments of the present application have been shown and described, it should be understood by those of ordinary skill in the art that various changes, modifications, substitutions and variations can be made thereto without departing from the principles and spirit of the present application, and the scope of the present application is defined by the appended claims and their equivalents.

[0047] Finally, it should be noted that: the above embodiments are only used to illustrate the technical solutions of the present application, and not to limit it; although the present application has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that they can still modify the technical solutions recorded in the foregoing embodiments, or make equivalent substitutions for part or all of the technical features; and these modifications or substitutions do not make the essence of the corresponding technical solutions deviate from the scope of the technical solutions of the embodiments of the present application.

Claims

1. A centrifugal microfluidic chip for simultaneously generating droplets of different sizes, characterized in that: The chip comprises a base sheet and a cover sheet sealed to the base sheet, a through hole is arranged in the center of the microfluidic chip, and a positioning notch is arranged at the inner edge of the through hole for fixing the chip on a centrifugal device; The chip comprises a sample inlet and two continuous phase reagent inlets, the sample inlet is used to accommodate up to 150 μL of sample, and the sample is guided to a sample reservoir (4) through a distribution channel (2), and the continuous phase reagent inlet is used to add a continuous phase reagent; The sample reservoir (4) is connected to the first vent hole (3), and the sample reservoir (4) is connected to the first reservoir (6), and the sample is accurately dispensed from the first reservoir (6) into two volume different first and second quantitative pools (8) and (11) through a centrifugal step; The sample in the second quantitative pool (11) enters the first droplet generation unit (14) through the first micro-nozzle structure (16), the first droplet generation unit (14) is filled with a continuous phase reagent through the first oil inlet hole (5) and the first oil reservoir (10), the sample in the first quantitative pool (8) enters the second droplet generation unit (17) through the second oil reservoir (15) and the second micro-nozzle structure (18), and the second droplet generation unit (17) is filled with a continuous phase reagent through the second oil inlet hole (9) and the second oil reservoir (13), the first quantitative pool (8) is provided with a second vent hole (7) for generating droplets of large and small particle sizes, respectively, and the first and second droplet generation units (14) and (17) are connected to the first and second oil reservoirs (10) and (13) through corresponding micro-channels and overflow pools (19); The first droplet generation unit (14) comprises 1-10 first micro-nozzle structures (16) or an array of first micro-nozzle structures (16); and the second droplet generation unit (17) comprises an array of 5-30 second micro-nozzle structures (18).

2. The centrifugal microfluidic chip for simultaneously generating droplets of different sizes according to claim 1, wherein: The base sheet and the cover sheet of the microfluidic chip are firmly attached by using double-sided adhesive tape with the required bonding strength, which can withstand conventional heating temperatures and has no significant adverse effects on the reactions performed.

3. The centrifugal microfluidic chip for simultaneously generating droplets of different sizes according to claim 1, wherein: The droplets generated by the first and second droplet generation units (14) and (17) are guided to the droplet collection area by centrifugal force, forming a regular single-layer arrangement.

4. The centrifugal microfluidic chip for simultaneously generating droplets of different sizes according to claim 3, wherein: By adjusting the rotation frequency and fluid flow rate, the uniform distribution of droplets in the collection area is controlled.

5. The centrifugal microfluidic chip for simultaneously generating droplets of different diameters according to any one of claims 1 to 4, characterized in that: The method comprises the following steps: Step one: add the fluorescent solution through the sample inlet (1), and add the monomer solution through the first and second oil inlet holes (5) and (9) into the first and second oil reservoirs (10) and (13), respectively; Step two: then start the centrifugal program for microsphere preparation, at a speed of 30g for 15s, to transfer the fluorescent solution and the monomer solution to the first and second reservoirs (6) and (15) and the first and second oil reservoirs (10) and (13); Step three: rotate at a speed of 80g for 20s to transfer the fluorescent solution to the second quantitative pool (11) and the first quantitative pool (8), and transfer the monomer solution to the first and second droplet generation units (14) and (17); Step four: speed 181 g for 0.5 s, then adjust the speed to 80 g for 300 s, complete the preparation of the droplet.

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