A multi-stage sorting device and method for marine microbial particles based on a U-shaped microfluidic chip

Abstract

The present invention provides a multi-stage sorting device and method for marine microbial particles based on a U-shaped microfluidic chip. The device includes a first sorting zone, a U-shaped microchannel, and a second sorting zone. The first sorting zone includes a sample inlet, a ratchet structure, an oil inlet, a first microfluidic straight channel, a sample outlet, and a positive power supply insertion port. The second sorting zone includes two sample outlets, a negative power supply insertion port, and a second microfluidic straight channel. The oil and sample particles form microdroplets at the connection between the oil channel and the first microfluidic straight channel. Due to the different dielectrophoretic forces required for particle separation, the microdroplets have different sizes. Both the first and second sorting zones use direct current dielectrophoresis. Two external electrodes act in the microchannel to generate a non-uniform electric field at the microdroplets. Marine microbial particles sorted in the sorting zones enter different sample outlets based on their size and the magnitude of the dielectrophoretic force, thereby achieving multi-stage sorting of marine microbial particles.

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 and method for marine microbial particles based on a U-shaped microfluidic chip. Background Art

[0002] Dielectrophoresis (DEP) is the motion of particles caused by the interaction between a nonuniform electric field and the induced effective dipole moment of the particles. The magnitude of the DEP force is proportional to the particle size, and the direction of the force is determined by the particle's dielectric properties. When subjected to a positive DEP force, particles move toward areas of high electric field strength; when subjected to a negative DEP force, particles move toward areas of low electric field strength. Insulating DEP technology involves creating a spatially nonuniform electric field in a DC electric field through specially designed structures, such as obstacles (microdroplets) made of electrically insulating materials, to enable polarizable particles to move within the nonuniform electric field.

[0003] Shipping is a significant vector for the spread of non-native species, harmful organisms, and pathogens. This is because ships carry large quantities of ballast water to control their heel, trim, draft, and stability during navigation. Ships' ballast water contains numerous microorganisms, including marine bacteria, viruses, fungi, and microscopic algae. Once these microorganisms are drawn into the ballast tanks, they are effectively placed in a closed ecosystem. Most die due to their inability to adapt to changes in temperature, salinity, and other factors, as well as the treatment required by the ballast water treatment system. However, some microorganisms are able to survive, and these surviving microorganisms often possess extremely strong vitality and competitiveness. Once released into a suitable environment, they may reproduce uncontrollably, causing an "avalanche-like" bioinvasion. Therefore, testing ballast water before discharge is crucial to preventing the spread of microorganisms in the water and the resulting serious damage to the marine environment.

[0004] Traditional methods for ballast water microbial detection include staining, fluorescence detection, flow cytometry, and optical imaging. These methods suffer from drawbacks such as being time-consuming and labor-intensive, requiring expensive instruments, susceptible to external influences, and exhibiting low accuracy. Therefore, developing efficient ballast water microbial detection methods to rapidly and effectively screen and isolate microorganisms in ballast water has become a research priority.

[0005] With the continuous development of microfluidic chip technology in recent years, it has become possible to detect microorganisms in ballast water on-chip. Replacing metal electrodes with insulating barriers can make the manufacturing process simpler and cheaper. Its robust, chemically inert structure does not electrochemically affect organisms or fluids. During the electrolysis process, very high electric fields can be applied without generating bubbles. The design utilizes U-shaped channels and deformable microdroplets to separate microorganisms of different sizes. The greatest advantage of this design is that it eliminates the stagnation areas caused by sharp turns in conventional channels and allows for control of droplet size, providing an electric field gradient that dynamically adapts to various separation requirements. Furthermore, the same chip can be used multiple times to sort microbial particles of different sizes. It has the advantages of miniaturization, high throughput, low sample requirements, and low reagent consumption. Summary of the Invention

[0006] To address the aforementioned technical issues, a multi-stage sorting device and method for marine microbial particles based on a U-shaped microfluidic chip is provided. This invention utilizes a U-shaped microchannel to eliminate the stagnant areas caused by sharp turns in conventional channels, enabling localized electric field amplification and preventing particle adhesion to the sidewalls. Both the first and second sorting zones utilize direct current dielectrophoresis. Two external electrodes act within the microfluidic straight channel to generate a non-uniform electric field at the microdroplets. Marine microbial particles sorted in the sorting zones then enter different sample outlets based on size and the magnitude of the dielectrophoretic force exerted on them, achieving multi-stage sorting of marine microbial particles.

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

[0008] A multi-stage sorting device for marine microbial particles based on a U-shaped microfluidic chip comprises: a microchannel layer, a first sorting zone arranged on the microchannel layer, a U-shaped microchannel, and a second sorting zone, wherein:

[0009] The first sorting area includes a sample inlet, a ratchet structure, an oil inlet, a first microfluidic straight channel, a first sample outlet, and a power supply positive electrode insertion port; wherein:

[0010] The sample inlet is connected to one end of the ratchet structure, the other end of the ratchet structure is connected to one end of the first microfluidic straight channel, and the other end of the first microfluidic straight channel is connected to one end of the U-shaped microchannel; the oil inlet is connected to a side wall of the first microfluidic straight channel near the second sorting area through the oil channel, and microdroplets are formed at the connection between the oil channel and the first microfluidic straight channel; the first sample outlet is connected to a side wall near the second sorting area at a certain angle through the sample channel and is arranged behind the oil inlet; the positive power supply insertion port is arranged at the sample inlet, and the positive power supply insertion port is connected to the positive electrode of the DC power supply, generating a high-gradient non-uniform electric field through the microfluidic channel and the U-shaped microchannel;

[0011] The second sorting area includes a second sample outlet, a third sample outlet, a power supply cathode insertion port, and a second microfluidic straight channel, wherein:

[0012] One end of the second microfluidic straight channel is connected to the other end of the U-shaped microchannel, and the other end of the second microfluidic straight channel is connected to the third sample outlet; the second sample outlet is connected to a side wall away from the first sorting area at a certain angle through the sample channel; the negative power supply insertion port is set at the third sample outlet, and the negative power supply insertion port is connected to the negative pole of the DC power supply.

[0013] Furthermore, the sample inlet injects a mixed particle sample, and the oil inlet pumps in oil, forming arc-shaped microdroplets in the first microfluidic straight channel through pressure. Due to the different dielectrophoretic forces required for particle separation, the sizes of the arc-shaped microdroplets formed in the first microfluidic straight channel are different, which is used to dynamically adjust the electric field gradient.

[0014] Furthermore, the distance between the arc-shaped micro-droplets and the first microfluidic straight channel is adjusted according to the size of the sorted marine microbial particles. By controlling the distance between the arc-shaped micro-droplets and the first microfluidic straight channel, multi-stage sorting of marine microbial particles is performed.

[0015] Furthermore, the ratchet structure is used to focus the flow of marine microbial particles at the middle position of the channel.

[0016] Furthermore, a first angle is set between the first microfluidic straight channel and the first sample outlet to complete the sorting of marine microbial particles with large size differences, separate the marine microbial particles with the smallest size, and complete the first-level sorting; the solution of larger-sized marine microbial particles after the initial screening passes through the U-shaped microchannel and then flows through the second microfluidic straight channel; a second angle is set between the second microfluidic straight channel and the second sample outlet to facilitate marine microbial particles of different sizes to enter the second sample outlet and the third sample outlet after passing through the second sorting area, separate the marine microbial particles of intermediate size and largest size, and complete multi-level sorting.

[0017] Furthermore, a cover plate is provided on the oil inlet, and the oil is pumped into the oil reservoir through the oil inlet, and the cover plate is used to seal the oil reservoir.

[0018] 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.

[0019] Furthermore, the microchannel layer and the cover plate are both made of PDMS material and are manufactured using a photolithography casting process.

[0020] The present invention also provides a method for multi-stage sorting of marine microbial particles based on the multi-stage sorting device of marine microbial particles based on the U-shaped microfluidic chip, comprising the following steps:

[0021] 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;

[0022] S2. Insert the platinum electrode into the positive and negative power supply ports and connect them to a DC power supply with a wire.

[0023] S3, injecting a solution containing mixed sample marine microbial particles into the sample inlet using a syringe pump, pumping oil into the oil inlet by pressure, and controlling the size of the microdroplets;

[0024] 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 voltage to improve the multi-stage sorting efficiency.

[0025] Compared with the prior art, the present invention has the following advantages:

[0026] 1. The present invention provides a multi-stage sorting device for marine microbial particles based on a U-shaped microfluidic chip. Its U-shaped microchannel eliminates the stagnation area caused by sharp turns in ordinary channels, can achieve local amplification of the electric field, and prevent particles from adhering to the side walls. Its adjustable microdroplets can dynamically adjust the electric field gradient, and can simultaneously sort marine microbial particles of various sizes.

[0027] 2. The multi-stage sorting device for marine microbial particles based on a U-shaped microfluidic chip provided by the present invention is simple to operate, lightweight and portable, and can achieve rapid and accurate sorting without the need to label marine microbial particles. It is low-cost, flexible in design, has a short reaction time, and can be integrated with other functions.

[0028] 3. The multi-stage sorting device for marine microbial particles based on a U-shaped microfluidic chip provided by the present invention does not require too many experimental personnel, and only requires mastering the correct experimental methods.

[0029] 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

[0030] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. 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 any creative labor.

[0031] Figure 1 It is a schematic diagram of the structure of the device of the present invention.

[0032] Figure 2 Schematic diagram of the microchannel layer structure of the device of the present invention.

[0033] Figure 3 This is a structural diagram of the oil inlet portion of the device of the present invention.

[0034] In the figure: 1. Sample inlet; 2. Ratchet structure; 3. Microdroplet; 4. Oil channel; 5. Oil inlet; 6. First microfluidic straight channel; 7. First angle; 8. First sample outlet; 9. U-shaped microchannel; 10. Second sample outlet; 11. Third sample outlet; 12. Negative power supply port; 13. Positive power supply port; 14. Microchannel layer; 15. Cover plate; 16. Second angle; 17. Second microfluidic straight channel. DETAILED DESCRIPTION

[0035] It should be noted that, in the absence of conflict, the embodiments and features of the embodiments of the present invention can be combined with each other. The present invention will be described in detail below with reference to the accompanying drawings and in combination with the embodiments.

[0036] In order to make the purpose, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions 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. The following description of at least one exemplary embodiment is actually only illustrative and is in no way intended to limit the present invention and its application or use. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

[0037] It should be noted that the terms used herein are only for describing specific embodiments and are not intended to limit the exemplary embodiments according to the present invention. As used herein, unless the context clearly indicates otherwise, the singular form is intended to include the plural form. In addition, it should be understood that when the terms "comprise" and / or "include" are used in this specification, they indicate the presence of features, steps, operations, devices, components and / or combinations thereof.

[0038] Unless otherwise specifically stated, the relative arrangement of the parts and steps, numerical expressions and numerical values ​​described in these embodiments do not limit the scope of the present invention. At the same time, it should be clear that, for ease of description, the sizes of the various parts shown in the drawings are not drawn according to the actual proportional relationship. The techniques, methods and equipment known to ordinary technicians in the relevant fields may not be discussed in detail, but where appropriate, the techniques, methods and equipment should be considered as part of the authorization specification. In all examples shown and discussed here, any specific value should be interpreted as merely exemplary and not as a limitation. Therefore, other examples of the exemplary embodiments may have different values. It should be noted that similar numbers and letters represent similar items in the following figures, so once an item is defined in one figure, it does not need to be further discussed in subsequent figures.

[0039] In the description of the present invention, it should be understood that the directions or positional relationships indicated by directional words such as "front, back, up, down, left, right", "horizontal, vertical, vertical, horizontal" and "top, bottom" are usually based on the directions or positional relationships shown in the accompanying drawings. They are only for the convenience of describing the present invention and simplifying the description. Unless otherwise specified, these directional words do not indicate or imply that the device or element referred to must have a specific direction or be constructed and operated in a specific direction. Therefore, they cannot be understood as limiting the scope of protection of the present invention: the directional words "inside and outside" refer to the inside and outside relative to the outline of each component itself.

[0040] For ease of description, spatially relative terms such as "above", "above", "on the upper surface of", "above", etc. may be used herein to describe the spatial positional relationship of a device or feature to other devices or features as shown in the figures. It should be understood that spatially relative terms are intended to include different orientations of the device in use or operation in addition to the orientation described in the figures. For example, if the device in the drawings is inverted, the device described as "above other devices or structures" or "above other devices or structures" will be positioned as "below other devices or structures" or "below their position devices or structures". Thus, the exemplary term "above" can include both "above" and "below". The device can also be positioned in other different ways (rotated 90 degrees or in other orientations), and the spatially relative descriptions used here are interpreted accordingly.

[0041] In addition, it should be noted that the use of terms such as "first" and "second" to limit components is only for the convenience of distinguishing the corresponding components. Unless otherwise stated, the above terms have no special meaning and therefore cannot be understood as limiting the scope of protection of the present invention.

[0042] like Figure 1 、2 As shown, the present invention provides a multi-stage sorting device for marine microbial particles based on a U-shaped microfluidic chip, comprising: a microchannel layer 14, a first sorting area arranged on the microchannel layer 14, a U-shaped microchannel 9, and a second sorting area, wherein:

[0043] The first sorting area includes a sample inlet 1, a ratchet structure 2, an oil inlet 5, a first microfluidic straight channel 6, a first sample outlet 8 and a power positive electrode insertion port 13; wherein:

[0044] The sample inlet 1 is connected to one end of the ratchet structure 2, the other end of the ratchet structure 2 is connected to one end of the first microfluidic straight channel 6, and the other end of the first microfluidic straight channel 6 is connected to one end of the U-shaped microchannel 9; the oil inlet 5 is connected to a side wall of the first microfluidic straight channel 6 near the second sorting zone through the oil channel 4, and microdroplets 3 are formed at the connection between the oil channel 4 and the first microfluidic straight channel 6; the first sample outlet 8 is connected to a side wall near the second sorting zone at a certain angle through the sample channel and is arranged behind the oil inlet 5; the positive power supply insertion port 13 is provided at the sample inlet 1, and the positive power supply insertion port 13 is connected to the positive pole of the DC power supply, generating a high-gradient non-uniform electric field through the microfluidic channel 6 and the U-shaped microchannel 9;

[0045] The second sorting area includes a second sample outlet 10, a third sample outlet 11, a power supply negative electrode insertion port 12, and a second microfluidic straight channel 17, wherein:

[0046] One end of the second microfluidic straight channel 17 is connected to the other end of the U-shaped microchannel 9, and the other end of the second microfluidic straight channel 17 is connected to the third sample outlet 11; the second sample outlet 10 is connected at a certain angle to the side wall away from the first sorting area through the sample channel; the negative power supply insertion port 12 is set at the third sample outlet 11, and the negative power supply insertion port 12 is connected to the negative pole of the DC power supply.

[0047] In this embodiment, the magnitude of the dielectrophoretic force is proportional to particle size. Dielectric electrophoretic force differences can be exploited to separate marine microbial particles of varying sizes. First, a high-gradient, non-uniform electric field is generated at the intersection of the microdroplets 3 and the first microfluidic straight channel 6 to achieve separation, enabling the sorting of marine microbial particles with significantly varying sizes. After the primary sorting stage, the smallest marine microbial particles flow out of the first sample outlet 8, while the remaining marine microbial particles flow into the second sorting zone. Within the second sorting zone, the intermediate-sized and largest marine microbial particles flow out of the second sample outlet 10 and third sample outlet 11, respectively, achieving multi-stage sorting.

[0048] In specific implementation, as a preferred embodiment of the present invention, the sample inlet 1 injects a mixed particle sample, and the oil inlet 5 pumps in oil, and arc-shaped microdroplets 3 are formed in the first microfluidic straight channel 6 by pressure. Due to the different dielectrophoretic forces required for particle separation, the sizes of the arc-shaped microdroplets 3 formed in the first microfluidic straight channel 6 are different, which is used to dynamically adjust the electric field gradient.

[0049] In specific implementation, as a preferred embodiment of the present invention, the spacing between the arc-shaped micro-droplets 3 and the first microfluidic straight channel 6 is adjusted according to the different sizes of the sorted marine microbial particles. By controlling the spacing between the arc-shaped micro-droplets 3 and the first microfluidic straight channel 6, multi-stage sorting of marine microbial particles is performed.

[0050] In specific implementation, as a preferred embodiment of the present invention, the ratchet structure 2 is used to focus the flow of marine microbial particles at the middle position of the channel.

[0051] In specific implementation, as a preferred embodiment of the present invention, a first angle 7 is set between the first microfluidic straight channel 6 and the first sample outlet 8 to complete the sorting of marine microbial particles with large size differences, separate the marine microbial particles of the smallest size, and complete the first-level sorting; after the initial screening, the larger-sized marine microbial particle solution passes through the U-shaped microchannel 9 and flows through the second microfluidic straight channel 17; a second angle 16 is set between the second microfluidic straight channel 17 and the second sample outlet 10 to facilitate the entry of marine microbial particles of different sizes into the second sample outlet 10 and the third sample outlet 11 after passing through the second sorting area, separating the marine microbial particles of intermediate size and the largest size, and completing multi-stage sorting. In this embodiment, the first angle 7 is set to -135°, and the second angle 16 is set to -45°.

[0052] In specific implementation, as a preferred embodiment of the present invention, a cover plate 15 is provided on the oil inlet 5, and the oil is pumped into the oil reservoir through the oil inlet 5. The cover plate 15 is used to seal the oil reservoir.

[0053] In specific implementation, as a preferred embodiment of the present invention, the power positive electrode insertion port 13 and the power negative electrode insertion port 12 are connected to a DC power supply to apply a DC signal for screening and separation of marine microbial particles.

[0054] The substrate of the microchannel layer 14 is a silicon wafer, and its production includes the following steps:

[0055] Cleaning: Rinse the glass slides with acetone and deionized water and blow dry with nitrogen to remove surface impurities.

[0056] Apply photoresist: Place the glass slide in the center of the spin coater, then pour an appropriate amount of photoresist on the center of the glass slide. Finally, set the rotation speed and time according to the required height of the chip to make the photoresist evenly cover the surface of the glass slide.

[0057] 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 be heated and formed.

[0058] 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.

[0059] Post-baking: Place the exposed glass slide on a heater to heat it, and then cool it down after heating to improve the colloidal toughness of the photoresist and make it less likely to break.

[0060] Development: After post-baking, the glass slide is immersed in a developer. The photoresist in the unexposed area will be washed away by the developer, leaving the pattern in the exposed area.

[0061] In a specific implementation, as a preferred embodiment of the present invention, the microchannel layer 14 and the cover sheet 15 are both made of PDMS material and are manufactured using a photolithography casting process. The manufacturing process includes the following steps:

[0062] Vacuuming: Pour liquid PDMS and curing agent into a clean glass at 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.

[0063] 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 for a while until the PDMS solidifies.

[0064] Cleaning: Trim the cured PDMS chip according to the pattern size, align the puncher with the inlet and outlet of the chip to punch holes, and then place the chip and glass slide in a plasma cleaner for cleaning.

[0065] Bonding: The glass slide and PDMS chip were taken out of the plasma cleaner one after another and the two were quickly bonded.

[0066] The embodiment of the present invention further provides a method for multi-stage sorting of marine microbial particles based on the multi-stage sorting device of marine microbial particles based on the U-shaped microfluidic chip, comprising the following steps:

[0067] 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;

[0068] S2. Insert the platinum electrode into the positive electrode insertion port 13 and the negative electrode insertion port 12 of the power supply and connect them to the DC power supply with a wire;

[0069] S3, injecting a solution containing mixed sample marine microbial particles into the sample inlet 1 using a syringe pump, and pumping oil into the oil inlet 5 by pressure, while controlling the size of the microdroplets;

[0070] 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 voltage to improve the multi-stage sorting efficiency.

[0071] Example

[0072] In this example, fluorescent polystyrene (PS) particles with diameters of 3, 12, and 15 μm were used instead of marine microbial particles to perform multi-stage sorting of marine microbial particles based on a U-shaped microfluidic chip. In this example, the width of the first microfluidic straight channel 6 and the second microfluidic straight channel 17 is 500 μm, the width of the sample inlet 1 is 500 μm, the width of the first sample outlet 8 is 500 μm, the width of the second sample outlet 10 is 400 μm, the width of the third sample outlet 11 is 500 μm, the width of the oil channel 4 is 200 μm, and the width of the U-shaped microchannel 9 is 500 μm.

[0073] Specifically, a custom syringe pump is used to push oil through the oil inlet 5. Then, pressure is slowly applied until the oil reaches the junction of the oil channel 4 and the first microfluidic straight channel 6, forming microdroplets. A solution containing a mixture of 3, 12, and 15 μm PS particle samples is injected into the sample inlet 1. After the sample solution enters the channel, a DC voltage is applied to the platinum electrodes inserted into the positive and negative power supply ports 13 and 12. The voltage can be adjusted according to the size of the target marine microbial particles.

[0074] When passing through the first sorting region, all three types of fluorescent PS particles are subject to a negative dielectrophoretic force, generating a high-gradient, non-uniform electric field at the intersection of the microdroplet and the first microfluidic straight channel 6. The smaller the particle, the smaller the negative dielectrophoretic force. Therefore, the 3μm PS particles are separated and flow into the first sample outlet 8, while the remaining PS particles continue to flow forward and enter the second sorting region. When passing through the second sorting region, the 12μm and 15μm PS particles are still subject to a negative dielectrophoretic force. When passing through the U-shaped microchannel 9, a non-uniform electric field is generated to prevent the particles from adhering to the sidewalls. When the PS particles pass through the angle, the 12μm PS particles experience a smaller negative dielectrophoretic force and flow into the second sample outlet 10, while the 15μm PS particles experience a larger negative dielectrophoretic force and flow into the third sample outlet 11.

[0075] 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 above embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the above 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 a U-shaped microfluidic chip, characterized in that: include: A microchannel layer (14), a first sorting area arranged on the microchannel layer (14), a U-shaped microchannel (9), and a second sorting area, wherein: The first sorting area includes a sample inlet (1), a ratchet structure (2), an oil inlet (5), a first microfluidic straight channel (6), a first sample outlet (8) and a power supply positive electrode insertion port (13); wherein: The sample inlet (1) is connected to one end of the ratchet structure (2), the other end of the ratchet structure (2) is connected to one end of the first microfluidic straight channel (6), and the other end of the first microfluidic straight channel (6) is connected to one end of the U-shaped microchannel (9); the oil inlet (5) is connected to a side wall of the first microfluidic straight channel (6) near the second sorting zone through the oil channel (4), and a micro droplet (3) is formed at the connection between the oil channel (4) and the first microfluidic straight channel (6); the first sample outlet (8) is connected to a side wall near the second sorting zone at a certain angle through the sample channel and is arranged behind the oil inlet (5); the positive electrode insertion port (13) of the power supply is arranged at the sample inlet (1), and the positive electrode insertion port (13) of the power supply is connected to the positive electrode of the DC power supply, and a high-gradient non-uniform electric field is generated through the microfluidic channel (6) and the U-shaped microchannel (9); the ratchet structure (2) is used to focus the marine microbial particle flow at the middle position of the channel; The second sorting area includes a second sample outlet (10), a third sample outlet (11), a power supply negative electrode insertion port (12) and a second microfluidic straight channel (17), wherein: One end of the second microfluidic straight channel (17) is connected to the other end of the U-shaped microchannel (9), and the other end of the second microfluidic straight channel (17) is connected to the third sample outlet (11); the second sample outlet (10) is connected to a side wall away from the first sorting area at a certain angle through the sample channel; the negative power supply insertion port (12) is set at the third sample outlet (11), and the negative power supply insertion port (12) is connected to the negative pole of the DC power supply.

2. The multi-stage sorting device for marine microbial particles based on a U-shaped microfluidic chip according to claim 1, characterized in that: The sample inlet (1) injects a mixed particle sample, and the oil inlet (5) pumps oil into the oil, and arc-shaped micro-droplets (3) are formed in the first microfluidic straight channel (6) by pressure. Due to the different dielectrophoretic forces required for particle separation, the sizes of the arc-shaped micro-droplets (3) formed in the first microfluidic straight channel (6) are different, which is used to dynamically adjust the electric field gradient.

3. The multi-stage sorting device for marine microbial particles based on a U-shaped microfluidic chip according to claim 1, characterized in that: The distance between the arc-shaped micro-droplets (3) and the first microfluidic straight channel (6) is adjusted according to the size of the marine microbial particles to be sorted. By controlling the distance between the arc-shaped micro-droplets (3) and the first microfluidic straight channel (6), multi-stage sorting of marine microbial particles is performed.

4. The multi-stage sorting device for marine microbial particles based on a U-shaped microfluidic chip according to claim 1, characterized in that: A first angle (7) is provided between the first microfluidic straight channel (6) and the first sample outlet (8), so as to complete the sorting of marine microbial particles with large size differences, separate the marine microbial particles with the smallest size, and complete the first-stage sorting; the marine microbial particle solution with larger size after the initial screening passes through the U-shaped microchannel (9) and then flows through the second microfluidic straight channel (17); a second angle (16) is provided between the second microfluidic straight channel (17) and the second sample outlet (10), so as to facilitate the marine microbial particles of different sizes to enter the second sample outlet (10) and the third sample outlet (11) after passing through the second sorting area, separate the marine microbial particles with intermediate size and the largest size, and complete the multi-stage sorting.

5. The multi-stage sorting device for marine microbial particles based on a U-shaped microfluidic chip according to claim 1, characterized in that: A cover plate (15) is provided on the oil inlet (5), and oil is pumped into the oil reservoir through the oil inlet (5). The cover plate (15) is used to seal the oil reservoir.

6. The multi-stage sorting device for marine microbial particles based on a U-shaped microfluidic chip according to claim 1, characterized in that: The power positive electrode insertion port (13) and the power negative electrode insertion port (12) are connected to a DC power supply and are used to apply a DC signal for screening and separation of marine microbial particles.

7. The multi-stage sorting device for marine microbial particles based on a U-shaped microfluidic chip according to claim 1, characterized in that: The microchannel layer (14) and the cover plate (15) are both made of PDMS material and are manufactured using a photolithography casting process.

8. A method for multi-stage sorting of marine microbial particles using the multi-stage sorting device for marine microbial particles based on a U-shaped microfluidic chip according to any one of claims 1 to 7, characterized in that: The steps include: 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; S2, insert the platinum electrode into the positive electrode insertion port (13) and the negative electrode insertion port (12) of the power supply and connect them to the DC power supply with a wire; S3, injecting a solution containing mixed sample marine microbial particles into the sample inlet (1) using a syringe pump, pumping oil into the oil inlet (5) by pressure, and controlling the size of the microdroplets; 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 voltage to improve the multi-stage sorting efficiency.

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