Centrifugal micro-fluidic chip capable of simultaneously generating liquid drops with different particle sizes

Abstract

The invention relates to the technical field of micro-fluidic chips, and discloses a centrifugal micro-fluidic chip capable of simultaneously generating liquid drops with different particle sizes, the centrifugal micro-fluidic chip comprises a liquid separation flow channel which uniformly distributes a sample to a plurality of liquid drop generation units, through the design of a quantitative pool, the liquid volume is ensured to be fixed, and the consistency of the number of generated liquid drops is ensured; the chip comprises a sample inlet and two continuous phase reagent inlets, a sample is accurately distributed to the quantitative pools under the action of centrifugal force and respectively enters the liquid drop generation units, the first liquid drop generation unit is used for generating large-particle-size liquid drops with the diameter of 100-200 microns, the second liquid drop generation unit is used for generating small-particle-size liquid drops with the diameter of 10-100 microns, and the sample inlet is communicated with the continuous phase reagent inlets. The micro-fluidic chip can be used for preparing ultrahigh-flux liquid drops, the generated liquid drops are guided to a collecting area through centrifugal force to form regular single-layer arrangement, and the chip is widely applied to the fields of biomedical research, material science, chemical synthesis, environmental monitoring and the like, and can be used for improving the experiment efficiency and the result consistency especially in the aspect of preparing fluorescent microspheres.

Description

Technical Field

[0001] The invention relates to the technical field of microfluidic chips, in particular to a centrifugal microfluidic chip capable of simultaneously generating droplets of different particle sizes. Background Art

[0002] Microfluidic technology has broad application prospects in biomedicine, chemical synthesis, environmental monitoring and other fields by accurately controlling and manipulating fluids at a microscale. Microfluidic chips that generate droplets usually use methods such as flow focusing, cross injection and dripping. Droplets are formed through shear force. The flow focusing method forms droplets at the focusing point through a multi-channel structure, and uses the pressure applied by the continuous phase to the dispersed phase to produce uniform and controllable droplets. The cross injection method achieves droplet generation through a T-shaped cross structure, which is suitable for simple droplet generation applications and is easy to manufacture and operate. The dripping method relies on the shear force of the continuous phase fluid to form droplets at the nozzle, and 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 a variety of experimental needs, and meet the high requirements of modern scientific research for micro-scale sample processing.

[0003] Constructing a microfluidic chip that can simultaneously generate droplets of different particle sizes is of great significance and value. First, it provides diversity and flexibility, and can process samples with different needs in the same experiment, improving the high-throughput screening capabilities of the experiment and the consistency of the results. For example, in the drug screening process, large droplets can be generated simultaneously for drug delivery and small droplets can be generated for single-cell analysis, thereby greatly improving the experimental efficiency. Secondly, this chip has a wide range of applications in biomedical research, materials science, chemical synthesis, and environmental monitoring. For example, droplets of different particle sizes can simulate different biological environments, such as the microstructure of the extracellular matrix, to help study cell behavior and drug response. In materials science, it can be used to synthesize micron or nanomaterials with specific sizes and structures, such as nanoparticles and microspheres. In chemical synthesis, droplets of different sizes can provide different reaction times and conditions, thereby optimizing the synthesis path and improving yield and purity. In environmental monitoring, it can be used to capture and analyze pollutants of different sizes.

[0004] To this end, we propose a centrifugal microfluidic chip that can simultaneously generate droplets of different particle sizes. By achieving the simultaneous generation of droplets of different particle sizes, this microfluidic chip will promote further innovation and progress in scientific research and industrial applications, and open up new research and application prospects. Summary of the invention

[0005] In order to make up for the shortcomings of the prior art, the present invention provides the following technical solution: it includes a chip base and a cover sealably matched with the base, 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 for fixing the chip on the centrifugal device.

[0006] Preferably, the chip comprises a sample inlet and two continuous phase reagent inlets, the sample inlet is used to accommodate samples up to 150 μL and guide the samples to the sample reservoir through the liquid separation channel, and the continuous phase reagent inlet is used to add continuous phase reagents.

[0007] Preferably, the sample reservoir is connected to the first exhaust port, and the sample is accurately distributed from the first reservoir into a first quantitative reservoir and a second quantitative reservoir of two different volumes through a centrifugation step.

[0008] Preferably, the first quantitative pool is provided with a second exhaust hole, and the sample in the first quantitative pool enters the first droplet generating unit and the second droplet generating unit through centrifugal force, which are used to generate droplets of large particle size and small particle size, respectively. The first droplet generating unit and the second droplet generating unit are connected to the first oil storage tank and the second oil storage tank through corresponding microchannels and the overflow tank.

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

[0010] Preferably, the bottom sheet and cover sheet of the microfluidic chip are firmly attached using double-sided adhesive tape that has the required bonding strength, can withstand conventional heating temperatures, and has no significant adverse effect on the reactivity being performed.

[0011] Preferably, the droplets generated by the first droplet generating unit and the second droplet generating unit are guided to the droplet collecting area by centrifugal force to form a regular single-layer arrangement.

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

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

[0014] Step 1: Add the dispersed phase reagent through the injection hole, and the continuous phase reagent enters the first oil reservoir and the second oil reservoir through the first oil inlet hole and the second oil inlet hole respectively;

[0015] Step 2: Then start the centrifugation procedure for microsphere preparation at a speed of 30 g for 15 seconds to transfer all the fluorescent solution and the monomer solution to the first liquid reservoir and the second liquid reservoir and the first oil reservoir and the second oil reservoir;

[0016] Step 3: rotating at 80 g for 20 s, transferring the fluorescent solution to the second quantitative pool and the first quantitative pool, and transferring the monomer solution to the first droplet generation unit and the second droplet generation unit;

[0017] Step 4: Rotate at 181 g for 0.5 s, then adjust the rotation speed to 80 g for 300 s to complete the preparation of droplets. The bottom film and cover film of the microfluidic chip are firmly attached with double-sided tape that has the required bonding strength, can withstand conventional heating temperatures, and has no significant adverse effect on the reactivity being carried out.

[0018] Beneficial Effects

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

[0020] 1. The present invention designs a microfluidic chip that generates droplets using a dual-volume centrifugal step-by-step emulsification method. The sample is evenly distributed to multiple droplet generation units through a liquid separation channel, and the liquid volume is fixed through the design of a quantitative pool, thereby ensuring the consistency of the number of droplet generation.

[0021] 2. The microfluidic chip of the present invention 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 monolayer arrangement.

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

[0023] Figure 1 This is a schematic diagram of the overall structure of a centrifugal microfluidic chip for simultaneously generating droplets of different particle sizes according to the present invention;

[0024] Figure 2 A schematic diagram of an independent droplet generation component of a centrifugal microfluidic chip capable of simultaneously generating droplets of different particle sizes according to the present invention;

[0025] Figure 3 It is an enlarged schematic diagram of a droplet generation unit 1 in a centrifugal microfluidic chip for simultaneously generating droplets of different particle sizes according to the present invention;

[0026] Figure 4 It is an enlarged schematic diagram of a droplet generation unit 2 in a centrifugal microfluidic chip for simultaneously generating droplets of different particle sizes according to the present invention.

[0027] Figure 5 This is a photograph under a microscope of a centrifugal microfluidic chip for simultaneously generating droplets of different particle sizes after generating droplets of different particle sizes.

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

[0029] In the figure: 1. injection hole; 2. liquid separation channel; 3. first exhaust hole; 4. sample liquid storage tank; 5. first oil inlet hole; 6. first liquid storage tank; 7. second exhaust hole; 8. first quantitative tank; 9. second oil inlet hole; 10. first oil storage tank; 11. second quantitative tank; 12. waste liquid tank; 13. second oil storage tank; 14. first droplet generating unit; 15. second liquid storage tank; 16. first micro nozzle structure; 17. second droplet generating unit; 18. second micro nozzle structure; 19. overflow tank. DETAILED DESCRIPTION

[0030] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0031] like Figure 1-2 As shown, a centrifugal microfluidic chip for simultaneously generating droplets of different particle sizes comprises a microfluidic chip bottom sheet and a cover sheet sealed with the bottom sheet, both of which are firmly attached with double-sided adhesive tape having the required bonding strength, being able to withstand conventional heating temperatures, and having no significant adverse effect on the reactivity being performed. 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. The through hole and the positioning notch together play a fixing and positioning role, and only one centrifuge is required to complete the centrifugal distribution of the sample.

[0032] like Figure 1 As shown, a plurality of independent droplet generating components are provided on the bottom film, and five independent droplet generating components are connected by a liquid separation channel 2, one end of the liquid separation channel 2 is connected to the injection hole 1 provided on the bottom film, and the other end is connected to the droplet generating component provided on the bottom film. For example, when the required sample volume is between 20 and 200 microliters, the diameter of the injection hole 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 to 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 generating unit 14 and the second droplet generating unit 17 through centrifugation, and the excess sample enters the waste liquid pool 12. The quantitative pool is provided with a second exhaust hole 7. As the centrifugal speed changes, the sample in the second quantitative pool 11 enters the first droplet generating unit 14 through the first micro-nozzle structure 16. The first droplet generating 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 generating unit 17 through the second liquid reservoir 15 and the second micro-nozzle structure 18. The second droplet generating unit 17 is filled with a continuous phase reagent through the second oil inlet hole 9 and the second oil reservoir 13. The first droplet generating unit 14 and the second droplet generating unit 17 and the first oil reservoir 10 and the second oil reservoir 13 are connected to the overflow pool 19 through corresponding micro-channels.

[0034] like Figure 3 As shown, the first droplet generating unit 14 includes a single or multiple first micro-nozzle structures 16 or an array of first micro-nozzle structures 16 , and the outlet size of the first micro-nozzle structure 16 is 75 μm, and the nozzle depth is 70 μm.

[0035] like Figure 4 As shown, the second droplet generating unit 17 comprises an array of 5 to 30 second micro-nozzle structures 18, the outlet size of the second micro-nozzle structure 18 is 11 μm, and the depth of the second micro-nozzle structure 18 is 11 μm;

[0036] The microfluidic chip design includes a sample inlet and two continuous phase inlets to ensure 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 to 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 areas with volumes of 10.5 μL and 2.5 μL respectively through a centrifugation step, and then 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). After the droplets are generated, they are guided to the collection area by centrifugal force to form a regular single-layer arrangement;

[0037] The microfluidic chip provided by the present invention can be developed for a variety of applications to perform a variety of reactions, such as digital detection, microsphere / liposome preparation, single cell separation, etc. The following takes the preparation of fluorescent microspheres as an example to illustrate its use process:

[0038] Select a suitable fluorescent dye, such as FITC (fluorescein isothiocyanate), usually use a polymer monomer, such as acrylate or methacrylate, and a cross-linking agent such as divinylbenzene (DVB), usually use water, ethanol or dimethyl sulfoxide (DMSO) as a solvent, select a suitable surfactant: such as Span80, for stable droplet formation, select a suitable thermal initiator or photoinitiator, such as azobisisobutyronitrile (AIBN) or potassium persulfate (KPS) to initiate the polymerization reaction;

[0039] Clean the microfluidic chip with isopropanol and deionized water to ensure that the inside of the channel is clean and dust-free. Treat the chip surface with oxygen plasma to increase hydrophilicity to facilitate liquid flow. Place the chip and pressure-sensitive film in a hot pressing device and perform hot pressing bonding at appropriate temperature and pressure. Typical hot pressing conditions are 70-100°C, pressure 0.1-0.5MPa, and duration 5-10 minutes.

[0040] The monomer material, the initiator and the stabilizer are dissolved in a suitable solvent to prepare a monomer solution, which is used as a continuous phase for preparing the microspheres; the fluorescent dye is dissolved in a solvent to prepare a fluorescent solution, which is used as a dispersed phase for preparing the microspheres;

[0041] The fluorescent solution is added through the injection hole 1, and the monomer solution enters the first oil reservoir 10 and the second oil reservoir 13 through the first oil inlet hole 5 and the second oil inlet hole 9, respectively. Then, the centrifugation procedure for microsphere preparation is started, the speed is 30g for 15s, and 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 80g for 20s, and the fluorescent solution is transferred to the second quantitative pool 11 and the first quantitative pool 8, and the monomer solution is transferred to the first droplet generation unit 14 and the second droplet generation unit 17. After the speed is 181g for 0.5s, the speed is adjusted to 80g, and the speed is continued for 300s to continue the preparation of microspheres;

[0042] The generated droplets are collected in a heating device (such as a heating plate or an oven) for polymerization reaction. The common temperature is between 60-80°C, and the time depends on the specific monomer and initiator. If a photoinitiator is used to collect the droplets and then cure them by ultraviolet light, the illumination time and intensity depend on the specific initiator and fluorescent dye. The cured fluorescent microspheres are separated by a centrifuge and washed multiple times with deionized water or an appropriate solvent to remove unreacted monomers and surfactants.

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

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

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

[0046] Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention 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 invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A centrifugal microfluidic chip for simultaneously generating droplets of different particle sizes, characterized in that: The microfluidic chip comprises a bottom plate of the chip and a cover plate which is sealed with the bottom plate. A through hole is arranged at 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 the centrifugal device.

2. A centrifugal microfluidic chip for simultaneously generating droplets of different particle sizes according to claim 1, characterized in that: The chip comprises a sample inlet and two continuous phase reagent inlets. The sample inlet is used to accommodate a sample of up to 150 μL and guide the sample to a sample storage tank (4) through a liquid separation channel (2). The continuous phase reagent inlet is used to add a continuous phase reagent.

3. A centrifugal microfluidic chip for simultaneously generating droplets of different particle sizes according to claim 2, characterized in that: The sample liquid reservoir (4) is connected to the first exhaust hole (3), and the sample is accurately distributed from the first liquid reservoir (6) into a first quantitative reservoir (8) and a second quantitative reservoir (11) of two different volumes through a centrifugal step.

4. A centrifugal microfluidic chip for simultaneously generating droplets of different particle sizes according to claim 3, characterized in that: The first quantitative pool (8) is provided with a second exhaust hole (7). The sample in the first quantitative pool (8) enters the first droplet generation unit (14) and the second droplet generation unit (17) through centrifugal force, and is used to generate droplets of large particle size and small particle size, respectively. The first droplet generation unit (14) and the second droplet generation unit (17) are connected to the first oil storage pool (10) and the second oil storage pool (13) through corresponding microchannels and an overflow pool (19).

5. A centrifugal microfluidic chip for simultaneously generating droplets of different particle sizes according to claim 4, characterized in that: The first droplet generating unit (14) comprises 1 to 10 first micro-nozzle structures (16) or an array of first micro-nozzle structures (16) for generating large-particle droplets with a target diameter of 100 to 200 μm; the second droplet generating unit (17) comprises an array of 5 to 30 second micro-nozzle structures (18) for generating small droplets with a target diameter of 10 to 100 μm.

6. A centrifugal microfluidic chip for simultaneously generating droplets of different particle sizes according to claim 1, characterized in that: The bottom sheet and cover sheet of the microfluidic chip are firmly attached by double-sided adhesive having the required bonding strength, being able to withstand conventional heating temperatures, and having no significant adverse effect on the reactivity being performed.

7. A centrifugal microfluidic chip for simultaneously generating droplets of different particle sizes according to claim 1, characterized in that: The droplets generated by the first droplet generating unit (14) and the second droplet generating unit (17) are guided to the droplet collecting area by centrifugal force to form a regular single-layer arrangement.

8. A centrifugal microfluidic chip for simultaneously generating droplets of different particle sizes according to claim 7, characterized in that: By adjusting the rotation frequency and fluid flow rate, the uniform distribution of droplets in the collection area can be controlled.

9. A centrifugal microfluidic chip for simultaneously generating droplets of different particle sizes according to any one of claims 1 to 8, characterized in that: The following steps are involved: Step 1: Add the dispersed phase reagent through the injection hole (1), and the continuous phase reagent enters the first oil reservoir (10) and the second oil reservoir (13) through the first oil inlet hole (5) and the second oil inlet hole (9) respectively; Step 2: Then start the centrifugation procedure for microsphere preparation at a speed of 30 g for 15 seconds, and transfer all the fluorescent solution and monomer solution to the first liquid reservoir (6) and the second liquid reservoir (15) and the first oil reservoir 10 and the second oil reservoir 13; Step 3: rotating at 80 g for 20 seconds, transferring the fluorescent solution to the second quantitative pool (11) and the first quantitative pool (8), and transferring the monomer solution to the first droplet generation unit (14) and the second droplet generation unit (17); Step 4: Rotate at 181 g for 0.5 s, then adjust the rotation speed to 80 g for 300 s to complete the preparation of droplets. The bottom film and cover film of the microfluidic chip are firmly attached with double-sided tape that has the required bonding strength, can withstand conventional heating temperatures, and has no significant adverse effect on the reactivity being carried out.

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