Marine microorganism particle multi-stage sorting device based on double telescopic micro-droplet obstacles

Abstract

The invention provides a marine microorganism particle multi-stage sorting device based on double telescopic micro-droplet obstacles. The marine microorganism particle multi-stage sorting device comprises a micro-channel layer, a sample marine microorganism particle inlet, a sheath flow inlet, an oil inlet, a first sorting area, a second sorting area and an outlet, wherein the sample marine microorganism particle inlet, the sheath flow inlet, the oil inlet, the first sorting area, the second sorting area and the outlet are formed in the micro-channel layer. A mixed particle sample is injected into the sample inlet; oil is pumped into the oil inlet, arc-shaped micro-droplets are formed in the micro-channel through pressure, and the distance between the micro-droplets and the micro-channel can be adjusted according to different sizes of sorted particles. DC dielectrophoresis is applied to the first sorting area and the second sorting area, two external electrodes act in a micro-channel, a non-uniform electric field is generated at micro-droplets, and marine microorganism particles sorted by the sorting area enter different sample outlets based on the size according to the difference of dielectrophoresis force. Therefore, the multi-stage sorting of the marine microorganism particles is realized.

Description

Technical Field

[0001] The present invention relates to the technical field of marine ship ballast water microbial analysis, and in particular to a multi-stage sorting device for marine microbial particles based on double retractable micro-droplet barriers. Background Art

[0002] Dielectrophoresis is the movement of particles caused by the interaction between the inhomogeneous electric field and the induced effective dipole moment of the particles. The magnitude of the dielectrophoretic force is proportional to the particle size, and the direction of the force is determined by the dielectric properties of the particles. When subjected to a positive dielectrophoretic force, the particles move to areas with high electric field strength; when subjected to a negative dielectrophoretic force, the particles move to areas with low electric field strength. In a DC electric field, a spatially inhomogeneous electric field is generated through a specially designed structure, such as using obstacles (microdroplets) of electrically insulating materials to make polarizable particles move in an inhomogeneous electric field. This is the insulating dielectrophoresis technology.

[0003] Shipping is an important way to spread non-native species, harmful organisms and pathogens. Because ships add a large amount of ballast water to control their heel, trim, draft and stability during navigation. Ships' ballast water contains a large number of microorganisms, including marine bacteria, viruses, fungi, microscopic algae, etc. After these microorganisms are sucked into the ballast tank, they are equivalent to being in a closed ecosystem. Most of the microorganisms die because they cannot adapt to changes in factors such as temperature and salinity and the treatment of the ballast water treatment system. However, some microorganisms can still survive. These surviving microorganisms often have extremely strong vitality and competitiveness. Once discharged into a suitable environment, uncontrollable "avalanche-like" reproduction may occur, causing biological invasion. Therefore, the detection of ballast water before discharge is an important link to prevent the spread of microorganisms in the water and cause serious damage to the marine environment.

[0004] Traditional ballast water microbial detection methods include staining, fluorescence characteristic detection, flow cytometry, optical imaging detection, etc. The above traditional ballast water microbial detection methods have the disadvantages of being time-consuming, labor-intensive, expensive, susceptible to external influences, and low accuracy. Therefore, developing efficient ballast water microbial detection methods and quickly and efficiently screening and separating microorganisms in ballast water have become the focus of research.

[0005] In recent years, with the continuous development of microfluidic chip technology, it has become possible to detect microorganisms in ballast water on the chip. Using insulating barriers instead of metal electrodes can make the manufacturing process simpler and cheaper; its solid chemically inert structure will not have electrochemical effects on organisms or fluids; very high electric fields can be applied during the electrolysis process without generating bubbles. Two retractable microdroplet barriers are used to separate microorganisms of different sizes. The biggest advantage of this design is that the size of each microdroplet barrier can be controlled, providing an electric field gradient that dynamically adapts to various separation requirements, and the same chip can be used multiple times to sort microbial particles of different sizes. It has the advantages of miniaturization, high throughput, small sample requirements, and low reagent consumption. Summary of the invention

[0006] According to the technical problems raised above, a multi-stage sorting device for marine microorganism particles based on double retractable micro-droplet barriers is provided. The device of the present invention is composed of a sample marine microorganism particle inlet, a sheath flow inlet, an oil inlet, a first sorting area, a second sorting area, and an outlet. The sample inlet injects a mixed particle sample. The oil inlet pumps oil into the oil inlet, and arc-shaped micro-droplets are formed in the microchannel by pressure, and the spacing between the micro-droplets and the microchannel can be adjusted according to the size of the sorted particles. Both the first sorting area and the second sorting area use direct current dielectrophoresis, and two external electrodes act in the microchannel to generate an uneven electric field at the micro-droplet. The marine microorganism particles sorted in the sorting area enter different sample outlets based on their size and the size of the dielectrophoretic force, so as to achieve multi-stage sorting of marine microorganism particles.

[0007] The technical means adopted by the present invention are as follows:

[0008] A multi-stage sorting device for marine microbial particles based on double retractable micro-droplet barriers comprises: a microchannel layer; wherein:

[0009] A microfluidic channel is provided on the microchannel layer; the two ends of the microfluidic channel are connected to the sheath flow inlet and the third sample outlet; one side wall of the microfluidic channel is connected to the sample inlet channel, the first sample outlet channel and the second sample outlet channel; the other end of the sample inlet channel is connected to the sample inlet; the other end of the first sample outlet channel is connected to the first sample outlet; the other end of the second sample outlet channel is connected to the second sample outlet; the other side wall of the microfluidic channel is connected to the first oil inlet channel and the second oil inlet channel; the other end of the first oil inlet channel is connected to the first oil inlet; the first oil inlet is connected to the first oil inlet; the other end of the second oil inlet channel is connected to the second oil inlet; the second oil inlet is connected to the second oil inlet; a first micro droplet is formed at the connection between the microfluidic channel and the first oil inlet channel; a second micro droplet is formed at the connection between the microfluidic channel and the second oil inlet channel; a positive power supply insertion port is provided at the sample inlet; a negative power supply insertion port is provided at the third sample outlet;

[0010] During sorting, the sheath liquid is passed into the sheath flow inlet, and the sample liquid is passed into the sample inlet. The sheath liquid gathers the sample liquid near the first micro-droplet, so that the marine microbial particles are subjected to a greater dielectrophoretic force. A first angle is set between the microfluidic channel and the first sample outlet to complete the sorting of marine microbial particles with large size differences, separate the largest size of marine microbial particles, and complete the first-level sorting. After the smaller size of marine microbial particles after the initial screening pass through the second micro-droplet, a second angle is set between the microfluidic channel and the second sample outlet, so that marine microbial particles of different sizes can enter different outlets after passing through the second sorting area, separate the middle size and the smallest size of marine microbial particles, and complete multi-level sorting.

[0011] Furthermore, the positive power supply insertion port is connected to the positive pole of the DC power supply, and a high gradient non-uniform electric field is generated at the intersection of the microfluidic channel and the first micro-droplet; the negative power supply insertion port is connected to the negative pole of the DC power supply, and a high gradient non-uniform electric field is generated at the intersection of the microfluidic channel and the second micro-droplet.

[0012] Furthermore, the arc-shaped microdroplets formed in the first oil inlet channel, the second oil inlet channel and the microfluidic channel are different in size and are used to dynamically adjust the electric field gradient; by controlling the spacing between the first microdroplets, the second microdroplets and the microfluidic channel, multi-stage sorting of marine microbial particles is performed, and the spacing between the first microdroplets, the second microdroplets and the microfluidic channel can be adjusted according to the different sizes of the sorted marine microbial particles.

[0013] Furthermore, the channel structure connecting the microfluidic channel and the first oil inlet and the channel structure connecting the microfluidic channel and the second oil inlet are T-shaped structures.

[0014] Furthermore, by applying pressure through an injection pump, the oil is pumped into the oil reservoir through the first oil inlet and the second oil inlet, and the oil reservoir is sealed by the first cover plate and the second cover plate, so that the oil can form a first micro-droplet at the T-shaped structure where the microfluidic channel and the first oil inlet are connected; and form a second micro-droplet at the T-shaped structure where the microfluidic channel and the second oil inlet are connected.

[0015] Furthermore, the power positive electrode insertion port and the power negative electrode insertion port are connected to a DC power supply for applying a DC signal for screening and separation of marine microbial particles.

[0016] Furthermore, the microchannel layer, the first cover plate and the second cover plate are all made of PDMS material and are manufactured by a photolithography casting process.

[0017] Compared with the prior art, the present invention has the following advantages:

[0018] 1. The present invention provides a multi-stage sorting device for marine microbial particles based on double retractable micro-droplet barriers, which utilizes two retractable micro-droplet barriers to separate microorganisms of different sizes. The size of each micro-droplet barrier can be controlled, providing an electric field gradient that dynamically adapts to various separation requirements. The same chip can be used multiple times to sort microbial particles of different sizes.

[0019] 2. The present invention provides a multi-stage sorting device for marine microbial particles based on double retractable micro-droplet barriers, which does not require too much from the experimenter, and only requires mastering the correct experimental method.

[0020] 3. The present invention provides a multi-stage sorting device for marine microbial particles based on a double retractable micro-droplet barrier, which does not require sample pretreatment, has low cost, flexible design, short reaction time and can be integrated with other functions.

[0021] Based on the above reasons, the present invention can be widely promoted in the fields of marine ship ballast water microbial analysis and the like. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative labor.

[0023] Figure 1 Schematic diagram of the microchannel layer of the present invention.

[0024] Figure 2 This is a 3D schematic diagram of the microchannel layer of the device of the present invention.

[0025] In the figure: 1, sheath flow inlet; 2, sample inlet; 3, microchannel layer; 4, first microdroplet; 5, first oil inlet; 6, first oil inlet; 7, first cover; 8, microfluidic channel; 9, first angle; 10, first sample outlet; 11, second microdroplet; 12, second oil inlet; 13, second oil inlet; 14, second cover; 15, second angle; 16, second sample outlet; 17, third sample outlet; 18, negative power supply plug-in port; 19, positive power supply plug-in port. DETAILED DESCRIPTION

[0026] In order to enable those skilled in the art to better understand the scheme of the present invention, the technical scheme in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings 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 should fall within the scope of protection of the present invention.

[0027] It should be noted that the terms "first", "second", etc. in the specification and claims of the present invention and the above-mentioned drawings are used to distinguish similar objects, and are not necessarily used to describe a specific order or sequence. It should be understood that the data used in this way can be interchanged where appropriate, so that the embodiments of the present invention described herein can be implemented in an order other than those illustrated or described herein. In addition, the terms "including" and "having" and any variations thereof are intended to cover non-exclusive inclusions, for example, a process, method, system, product or device that includes a series of steps or units is not necessarily limited to those steps or units that are clearly listed, but may include other steps or units that are not clearly listed or inherent to these processes, methods, products or devices.

[0028] like Figure 1 , 2 As shown, the present invention provides a multi-stage sorting device for marine microbial particles based on double retractable micro-droplet barriers, comprising: a microchannel layer 3, wherein:

[0029] A microfluidic channel 8 is provided on the microchannel layer 3; the two ends of the microfluidic channel 8 are connected to the sheath flow inlet 1 and the third sample outlet 17; one side wall of the microfluidic channel 8 is connected to the sample inlet channel, the first sample outlet channel and the second sample outlet channel; the other end of the sample inlet channel is connected to the sample inlet 2; the other end of the first sample outlet channel is connected to the first sample outlet 10; the other end of the second sample outlet channel is connected to the second sample outlet 16; the other side wall of the microfluidic channel 8 is connected to the first oil inlet channel and the second oil inlet channel; the other end of the first oil inlet channel is connected to the first oil inlet 5; the first oil inlet 5 is connected to the first oil inlet 6; the other end of the second oil inlet channel is connected to the second oil inlet 12; the second oil inlet 12 is connected to the second oil inlet 13; a first micro droplet 4 is formed at the connection between the microfluidic channel 8 and the first oil inlet channel; a second micro droplet 11 is formed at the connection between the microfluidic channel 8 and the second oil inlet channel; a positive power supply insertion port 19 is provided at the sample inlet 2; a negative power supply insertion port 18 is provided at the third sample outlet 17;

[0030] During sorting, the sheath liquid is passed into the sheath inlet 1, and the sample liquid is passed into the sample inlet 2. The sheath liquid gathers the sample liquid near the first micro-droplet 11, so that the marine microbial particles are subjected to a greater dielectrophoretic force; a first angle 9 is set between the microfluidic channel 8 and the first sample outlet to complete the sorting of marine microbial particles with large size differences, separate the marine microbial particles of the largest size, and complete the first-level sorting; after the smaller-sized marine microbial particles after the initial screening pass through the second micro-droplet 11, a second angle 15 is set between the microfluidic channel 8 and the second sample outlet 16, so that marine microbial particles of different sizes can enter different outlets after passing through the second sorting area, separate the marine microbial particles of the intermediate size and the smallest size, and complete the multi-level sorting. In this embodiment, the first angle 9 and the second angle 15 are both set to 45°.

[0031] In this embodiment, the magnitude of the dielectrophoretic force is proportional to the particle size, and the difference in dielectrophoretic force can be used to separate marine microbial particles of different sizes. First, a high-gradient non-uniform electric field is generated at the intersection of the first micro-droplet and the microfluidic channel to perform separation, thereby achieving the separation of marine microbial particles with large size differences. After the first-level sorting, the largest-sized marine microbial particles flow out of the first sample outlet 10, and the remaining marine microbial particles flow to the second sorting area; in the second sorting area, a high-gradient non-uniform electric field is generated at the intersection of the second micro-droplet and the microfluidic channel to perform separation, thereby achieving the separation of marine microbial particles with small size differences. Marine microbial particles of intermediate size and minimum size flow out of the second sample outlet 16 and the third sample outlet 17, respectively, to achieve multi-stage sorting.

[0032] During specific implementation, as a preferred embodiment of the present invention, the positive electrode insertion port 19 of the power supply is connected to the positive electrode of the DC power supply, and a high-gradient inhomogeneous electric field is generated at the intersection of the microfluidic channel 8 and the first micro-droplet 4; the negative electrode insertion port 18 of the power supply is connected to the negative electrode of the DC power supply, and a high-gradient inhomogeneous electric field is generated at the intersection of the microfluidic channel 8 and the second micro-droplet 11.

[0033] In specific implementation, as a preferred embodiment of the present invention, the arc-shaped microdroplets formed in the first oil inlet channel, the second oil inlet channel and the microfluidic channel 8 are of different sizes, which are used to dynamically adjust the electric field gradient; by controlling the spacing between the first microdroplets 4, the second microdroplets 11 and the microfluidic channel 8, multi-stage sorting of marine microbial particles is performed, and the spacing between the first microdroplets 4, the second microdroplets 11 and the microfluidic channel 8 can be adjusted according to the different sizes of the sorted marine microbial particles.

[0034] In specific implementation, as a preferred embodiment of the present invention, the channel structure connecting the microfluidic channel 8 and the first oil inlet 5 and the channel structure connecting the microfluidic channel 8 and the second oil inlet 12 are T-shaped structures.

[0035] During specific implementation, as a preferred embodiment of the present invention, pressure is applied by an injection pump, and the oil is pumped into the oil reservoir through the first oil inlet 6 and the second oil inlet 12. The oil reservoir is sealed by the first cover plate 7 and the second cover plate 14, so that the oil can form a first micro-droplet 4 at the T-shaped structure where the microfluidic channel 8 and the first oil inlet 5 are connected; and a second micro-droplet 11 is formed at the T-shaped structure where the microfluidic channel 8 and the second oil inlet 12 are connected.

[0036] In specific implementation, as a preferred embodiment of the present invention, the power positive electrode insertion port 19 and the power negative electrode insertion port 18 are connected to a DC power supply to apply a DC signal for screening and separation of marine microbial particles.

[0037] In specific implementation, as a preferred embodiment of the present invention, the substrate of the multi-stage sorting device for marine microbial particles based on double retractable micro-droplet barriers is a silicon wafer, and its manufacturing process includes the following steps:

[0038] Cleaning: Rinse the glass slides with acetone and deionized water and blow dry with nitrogen to remove surface impurities.

[0039] Apply photoresist: Place the glass slide in the center of the spin coater, then pour an appropriate amount of photoresist into the center of the glass slide, and finally set the rotation speed and time according to the required height of the chip so that the photoresist evenly covers the surface of the glass slide.

[0040] Pre-baking: Place the glass slide with the photoresist spin-coated on a heater to heat it, and then cool it down after heating to allow the photoresist to take shape.

[0041] Exposure: After the cooling process is completed, place the glass slide under the exposure machine, place the mask negative on the glass slide, and set the exposure time.

[0042] Post-baking: Place the exposed glass slide on a heater to heat it, and then cool it down after heating to increase the colloid toughness of the photoresist and make it less likely to break.

[0043] Development: After post-baking, the glass slide is immersed in the developer. At this time, the photoresist in the unexposed area will be washed away by the developer, and the pattern in the exposed area will be retained.

[0044] In specific implementation, as a preferred embodiment of the present invention, the microchannel layer 8, the first cover sheet (7) and the second cover sheet 14 are all made of PDMS material and are manufactured by photolithography casting process. The manufacturing process includes the following steps:

[0045] Vacuuming: Pour liquid PDMS and curing agent into a clean glass in a ratio of 10:1, stir evenly with a stirring rod, and then place it in a vacuum box and let it stand to remove the air in the glass to prevent bubbles from forming after PDMS is cured.

[0046] Pouring and curing: After vacuuming, pour PDMS on the substrate of the device separation chip, then put it into the oven and let it stand, waiting for the PDMS to cure.

[0047] Cleaning: Trim the cured PDMS chip according to the graphic size, align the puncher with the inlet and outlet of the chip to punch holes, and then place the chip and the glass slide in a plasma cleaner for cleaning.

[0048] Bonding: The glass slide and PDMS chip are taken out of the plasma cleaner one after another, and the two are quickly bonded.

[0049] This embodiment also provides a sorting method based on the above-mentioned marine microbial particle multi-stage sorting device based on double retractable micro-droplet barriers, comprising the following steps:

[0050] S1. Place the bonded marine microbial particle multi-stage sorting device into a plasma cleaning machine for plasma cleaning to improve the hydrophilicity of the channel and facilitate liquid flow;

[0051] S2, insert the platinum electrode into the positive electrode insertion port 19 of the power supply and the negative electrode insertion port 18 of the power supply, and connect the DC power supply with a wire;

[0052] S3, injecting a solution containing mixed sample marine microorganism particles into the sample inlet 2 with a syringe pump, injecting sheath liquid into the sheath flow inlet (1), pumping oil into the first oil inlet 5 and the second oil inlet 12 by pressure, and controlling the size of the microdroplets;

[0053] S4. Use a microscope to observe the separation effect of marine microbial particles. In the dielectrophoresis multi-stage sorting area, adjust the size of the microdroplets and the voltage to improve the multi-stage sorting efficiency.

[0054] Example

[0055] In this embodiment, fluorescent polystyrene (PS) particles with diameters of 5, 10, and 15 μm were used to replace marine microbial particles, and multi-stage sorting of marine microbial particles based on double retractable micro-droplet barriers was performed. Specifically:

[0056] First, two customized injection pumps are used to push the oil through the first oil inlet 6 and the second oil inlet 13, and then slowly pressurized until the oil reaches the T-shaped structure to form two microdroplets. A solution mixed with 5, 10, and 15 μm PS particle samples is injected into the sample inlet 2. After the sample solution enters the channel, a DC voltage is applied to the platinum electrodes inserted into the positive power supply insertion port 19 and the negative power supply insertion port 18. The voltage can be adjusted according to the size of the target marine microbial particles.

[0057] When the three fluorescent PS particles pass through the first sorting area, they are all subjected to negative-dielectrophoretic force, and a high-gradient non-uniform electric field is generated at the intersection of the first micro-droplet and the microfluidic channel. The larger the particle volume, the greater the negative-dielectrophoretic force it is subjected to. Therefore, the 15μm PS particles are separated and flow into the first sample outlet 10, and the remaining PS particles continue to flow forward and enter the second sorting area. When passing through the second sorting area, the 5 and 10μm PS particles are still subjected to negative-dielectrophoretic force. When passing through the second sorting area, a high-gradient non-uniform electric field is generated at the intersection of the second micro-droplet and the microfluidic channel. When the PS particles pass through the second micro-droplet, the 10μm PS particles are subjected to a larger negative-dielectrophoretic force and flow into the second sample outlet 16, while the 5μm PS particles are subjected to a smaller negative-dielectrophoretic force and flow into the third sample outlet 17.

[0058] 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 multi-stage sorting device for marine microbial particles based on double retractable micro-droplet barriers, characterized in that: include: A microchannel layer (3), wherein: A microfluidic channel (8) is provided on the microchannel layer (3); two ends of the microfluidic channel (8) are connected to a sheath flow inlet (1) and a third sample outlet (17); one side wall of the microfluidic channel (8) is connected to a sample liquid inlet channel, a first sample outlet channel, and a second sample outlet channel; the other end of the sample liquid inlet channel is connected to a sample liquid inlet (2); the other end of the first sample outlet channel is connected to a first sample outlet (10); the other end of the second sample outlet channel is connected to a second sample outlet (16); the other side wall of the microfluidic channel (8) is connected to a first oil inlet channel and a second oil inlet channel; the other end of the first oil inlet channel is connected to a first sample outlet (10); the other end of the second sample outlet channel is connected to a second sample outlet (16); the other side wall of the microfluidic channel (8) is connected to a first oil inlet channel and a second oil inlet channel; the other end of the first oil inlet channel is connected to a first sample outlet (11); the other end of the second sample outlet channel is connected to a second sample outlet (16); the other side wall of the microfluidic channel (8) is connected to a first oil inlet channel and a second oil inlet channel; the other end of the first oil inlet channel is connected to a first sample outlet (11); the other end of the first sample outlet ... first sample outlet channel is connected to a first sample outlet (11); the other end of the first sample outlet channel is connected to a first sample outlet (11); the other end of the first sample outlet channel is connected to a first One end is connected to a first oil inlet (5); the first oil inlet (5) is connected to a first oil inlet (6); the other end of the second oil inlet channel is connected to a second oil inlet (12); the second oil inlet (12) is connected to a second oil inlet (13); a first micro-droplet (4) is formed at the connection between the microfluidic channel (8) and the first oil inlet channel; a second micro-droplet (11) is formed at the connection between the microfluidic channel (8) and the second oil inlet channel; a positive power supply insertion port (19) is provided at the sample inlet (2); and a negative power supply insertion port (18) is provided at the third sample outlet (17); During sorting, sheath liquid is introduced into the sheath flow inlet (1), and sample liquid is introduced into the sample liquid inlet (2). The sheath liquid gathers the sample liquid near the first micro-droplet (11), so that the marine microbial particles are subjected to a greater dielectrophoretic force. A first angle (9) is provided between the microfluidic channel (8) and the first sample outlet, so that the marine microbial particles with large size differences are sorted, and the marine microbial particles with the largest size are separated, so as to complete the primary sorting. After the marine microbial particles with smaller size after the initial screening pass through the second micro-droplet (11), a second angle (15) is provided between the microfluidic channel (8) and the second sample outlet (16), so that the marine microbial particles with different sizes can enter different outlets after passing through the second sorting area, so as to separate the marine microbial particles with intermediate size and the smallest size, so as to complete the multi-stage sorting.

2. The multi-stage sorting device for marine microorganism particles based on double retractable micro-droplet barriers according to claim 1 is characterized in that: The power positive electrode insertion port (19) is connected to the positive electrode of the DC power supply, and a high-gradient non-uniform electric field is generated at the intersection of the microfluidic channel (8) and the first micro-droplet (4); the power negative electrode insertion port (18) is connected to the negative electrode of the DC power supply, and a high-gradient non-uniform electric field is generated at the intersection of the microfluidic channel (8) and the second micro-droplet (11).

3. The multi-stage sorting device for marine microorganism particles based on double retractable micro-droplet barriers according to claim 1 is characterized in that: The arc-shaped micro-droplets formed in the first oil inlet channel, the second oil inlet channel and the microfluidic channel (8) are of different sizes and are used to dynamically adjust the electric field gradient; by controlling the spacing between the first micro-droplets (4), the second micro-droplets (11) and the microfluidic channel (8), multi-stage sorting of marine microbial particles is performed, and the spacing between the first micro-droplets (4), the second micro-droplets (11) and the microfluidic channel (8) can be adjusted according to the different sizes of the sorted marine microbial particles.

4. The multi-stage sorting device for marine microorganism particles based on double retractable micro-droplet barriers according to claim 1 is characterized in that: The channel structure connecting the microfluid channel (8) and the first oil inlet (5) and the channel structure connecting the microfluid channel (8) and the second oil inlet (12) are T-shaped structures.

5. The multi-stage sorting device for marine microorganism particles based on double retractable micro-droplet barriers according to claim 1 is characterized in that: By applying pressure through an injection pump, oil is pumped into the oil reservoir through a first oil inlet (6) and a second oil inlet (12), and the oil reservoir is sealed by a first cover sheet (7) and a second cover sheet (14), so that the oil can form a first micro-droplet (4) at a T-shaped structure where a microfluidic channel (8) and a first oil inlet (5) are connected; and a second micro-droplet (11) is formed at a T-shaped structure where a microfluidic channel (8) and a second oil inlet (12) are connected.

6. The multi-stage sorting device for marine microorganism particles based on double retractable micro-droplet barriers according to claim 1 is characterized in that: The power positive electrode insertion port (19) and the power negative electrode insertion port (18) are connected to a direct current power source and are used to apply a direct current signal for screening and separating marine microbial particles.

7. The multi-stage sorting device for marine microorganism particles based on double retractable micro-droplet barriers according to claim 1 is characterized in that: The microchannel layer (8), the first cover plate (7) and the second cover plate (14) are all made of PDMS material and are manufactured using a photolithography casting process.

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