Micro-fluidic chip for sorting circulating tumor cells based on double-helix structure and sorting method of micro-fluidic chip

Abstract

The invention discloses a micro-fluidic chip for sorting circulating tumor cells based on a double-helix structure and a sorting method thereof, the micro-fluidic chip has the double-helix structure comprising an inner outlet, and the micro-fluidic chip comprises a primary screening channel for primarily focusing and separating circulating tumor cells; the secondary screening channel is directly connected with the primary screening channel and is used for carrying out secondary focusing separation on the circulating tumor cells subjected to primary screening; wherein the primary screening channel is separated into two branches after rotating into the chip, one branch is connected with the middle outlet and used for discharging unfocused particles and redundant liquid, and the other branch is connected with the secondary screening channel so as to continue focusing and separation of the particles; the micro-fluidic chip is provided with an inlet for injecting a liquid sample and three outlets, namely a middle outlet, a particle collection outlet and a waste outlet, so that the injection of the liquid sample and the collection of separated particles are realized. According to the invention, high-throughput screening of the circulating tumor cells is realized, and the separation efficiency is improved.

Description

Technical Field

[0001] The present invention belongs to the technical field of rare cell enrichment and screening, and in particular relates to a microfluidic chip based on a double helix structure for sorting circulating tumor cells and a sorting method thereof. Background Art

[0002] Current cell sorting technologies are mainly based on cell diameter, including microcolumn / micropore screening, deterministic lateral displacement (DLD), and microfluidic chips using fluid mechanics or external electric, magnetic, and acoustic fields. Microcolumn and micropore chips achieve separation by physically intercepting circulating tumor cells with larger diameters. DLD chips arrange microcolumns to cause cells to move laterally, thereby collecting target cells at specific outlets. Microfluidic chips with external magnetic and electric fields use the difference in force on cells of different diameters to separate tumor cells.

[0003] Inertial sorting technology separates tumor cells through the inertial lift force exerted on cells in a direct current channel. However, in order to improve the separation efficiency, researchers introduced the Dean vortex sorting chip (DFF), which generates Dean drag force through spiral and serpentine structures, allowing cells of different sizes to move between the inner walls of the channel to achieve more precise separation. The size of the Dean drag force is closely related to the particle size, causing cells with larger diameters to tend to focus on the inner wall of the channel, while smaller cells focus on the outer wall.

[0004] However, the shortcomings of the existing technology include: generally involving external fields, such as magnetic fields, electric fields, etc., and the integration of additional components will make chip manufacturing more complicated, and cannot meet the requirements of efficient separation of multi-ml blood samples in clinical applications. Microcolumn, micropore and DLD chips involve a large number of microcolumns or micropore structures, which will lead to easy blockage during cell sorting and cause greater flow resistance. When facing the above problems, filter chips and DLD chips with microcolumn structures may cause blockage, resulting in lower cell separation efficiency and purity over time, lower accuracy of CTC counting due to interception failure, and inability to perform downstream detection and analysis of CTC rupture and enriched cells.

[0005] For devices that perform separation based on inertial sorting technology, when different inertial sorting technologies are coupled (e.g. spiral + serpentine), a mismatch in the flow rates required by different structures may occur, resulting in the addition of additional sheath liquid inlet channels, increasing the overall flow resistance of the entire sorting channel and increasing the integration complexity of the sorting device. Summary of the invention

[0006] In order to solve the technical problem that circulating tumor cells in the blood are mixed with nearly a million times the background cells and are difficult to be efficiently separated, the present invention proposes a microfluidic chip based on a double helix structure for sorting circulating tumor cells and a sorting method thereof, so as to solve the problems existing in the above-mentioned prior art.

[0007] To achieve the above objectives, in a first aspect, the present invention provides a microfluidic chip for sorting circulating tumor cells based on a double helix structure, wherein the microfluidic chip has a double helix structure and comprises:

[0008] The primary screening channel is used for preliminary focused separation of circulating tumor cells;

[0009] The secondary screening channel is directly connected to the primary screening channel and is used for secondary focusing separation of circulating tumor cells after primary screening;

[0010] The primary screening channel is separated into two branches after rotating into the inside of the chip, one branch is connected to the middle outlet for discharging unfocused particles and excess liquid, and the other branch is connected to the secondary screening channel to continue focusing and separating the particles;

[0011] The microfluidic chip is designed with an inlet for the injection of liquid samples and three outlets, namely a middle outlet, a particle collection outlet and a waste outlet, to achieve the injection of liquid samples and the collection of separated particles.

[0012] Preferably, the width of the primary screening channel is greater than the width of the secondary screening channel to achieve preliminary separation of particles of different diameters.

[0013] Preferably, the design of the intermediate outlet allows adjustment of the flow rate to optimize retention of the particles in the secondary screening channel.

[0014] Preferably, the flow rate adjustment is achieved through a flow resistance tube connected to the middle outlet, and the length and diameter of the flow resistance tube are adjusted as needed.

[0015] Preferably, the channel spacing of the microfluidic chip is 400 μm, the initial spiral radius is 3.3 mm, and the middle S-bend is adjusted to a symmetrical structure.

[0016] Preferably, the microfluidic chip is made of PDMS material, and the chip is formed and functionalized through a preparation process.

[0017] Preferably, the inlet, the particle collection outlet and the waste outlet are all arranged outside the microfluidic chip to achieve direct integration with other devices.

[0018] In a second aspect, the present invention further provides a microfluidic chip sorting method, based on the microfluidic chip for sorting circulating tumor cells based on a double helix structure described in the first aspect, for separating circulating tumor cells, comprising the following steps:

[0019] Injecting the sample to be separated into the inlet of the microfluidic chip;

[0020] Using the primary screening channel of the microfluidic chip to perform preliminary focusing separation on the cells in the sample to be separated;

[0021] guiding the primary screened cells to the secondary screening channel of the microfluidic chip for secondary focusing separation;

[0022] discharging unfocused particles and excess liquid through a middle outlet of the microfluidic chip;

[0023] The focused particles discharged from the particle collection outlet of the microfluidic chip are collected.

[0024] Preferably, the cross-section of the primary screening channel is designed to be rectangular, with a low aspect ratio between width and height, allowing particles of different diameters to be focused at different positions in the flow channel, thereby meeting the separation requirements of particles of different sizes.

[0025] Preferably, the cross-section of the secondary screening channel is designed to be rectangular, and its width is narrower than that of the primary screening channel, which is conducive to focusing of small-diameter particles to achieve more precise particle or cell separation.

[0026] Preferably, the intermediate outlet is connected to a flow resistance tube to adjust the flow rate to optimize the retention rate of the particles in the secondary screening channel.

[0027] Preferably, the length and diameter of the flow resistance tube are adjusted as required to accommodate different flow rates and particle separation efficiencies.

[0028] Preferably, the double helix structure is designed so that cells with larger diameters are subjected to higher inertial lift, thereby being more easily focused near the inner wall of the channel.

[0029] Preferably, during the sorting process, smaller cells are susceptible to the Dean force and tend to be focused near the outer wall, thereby achieving separation from cells with larger diameters.

[0030] Preferably, the sorting method further comprises connecting the chip based on biochemical principles with a microfluidic chip to perform secondary screening to improve the removal rate of background cells.

[0031] Compared with the prior art, the present invention has the following advantages and technical effects:

[0032] The present invention provides a microfluidic chip for sorting circulating tumor cells based on a double helix structure, the microfluidic chip having a double helix structure with an inner outlet, comprising: a primary screening channel for preliminary focusing and separation of circulating tumor cells; a secondary screening channel directly connected to the primary screening channel for secondary focusing and separation of circulating tumor cells after the primary screening; wherein the primary screening channel is separated into two branches after rotating to the inside of the chip, one branch is connected to the middle outlet for discharging unfocused particles and excess liquid, and the other branch is connected to the secondary screening channel to continue focusing and separation of particles; the microfluidic chip is designed with an inlet for injection of liquid samples, and three outlets, namely the middle outlet, the particle collection outlet and the waste outlet, to realize the injection of liquid samples and the collection of separated particles.

[0033] The present invention realizes high-throughput screening of circulating tumor cells and improves separation efficiency through the design of double helix structure; the design of primary screening channel and secondary screening channel allows circulating tumor cells to undergo two focusing separations, thereby improving separation accuracy. The present invention designs an inlet for injection of liquid samples and three outlets for collection of separated particles, which simplifies the operation process and improves the convenience and efficiency of the experiment. Compared with the multiple treatments of cell modification in active technology separation, the simplicity of the passive separation technology of the microfluidic chip of the present invention can reduce the errors of different experiments to a certain extent, and obtain a sorting chip with relatively stable performance.

[0034] Compared with the prior art, the microfluidic chip provided by the present invention does not involve additional processing of cells, and there is no collision between cells and microstructures, which ensures the activity of cells and lays the foundation for downstream gene and protein analysis of sorted cells. Compared with the existing double-helix technology, the present invention adds research on the middle S-bend, and the forces of the particles are still balanced when they flow through the spiral junction, preventing the particles from defocusing, thereby improving the focusing effect of different particles and reducing the overlap of particle trajectories. In addition, the microfluidic chip provided by the present invention does not add a sheath liquid channel, and can balance the flow required by the two-stage channels while ensuring the sorting efficiency. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] The drawings constituting a part of the present application are used to provide a further understanding of the present application. The illustrative embodiments and descriptions of the present application are used to explain the present application and do not constitute an improper limitation on the present application. In the drawings:

[0036] Figure 1 A plan view of a double helix chip according to an embodiment of the present invention;

[0037] Figure 2 A physical picture of a double helix chip according to an embodiment of the present invention;

[0038] Figure 3Schematic diagram of the fluorescence trajectory of 15 μm microspheres (left) and 10 μm microspheres (right) at a flow rate of 1.7 mL / min according to an embodiment of the present invention;

[0039] Figure 4 Schematic diagram of the relationship between the retention rate of 15 μm microspheres and the length of the flow resistance tube according to an embodiment of the present invention;

[0040] Figure 5 Schematic diagram of the distribution of a single microsphere solution (left) and a microsphere mixed solution (right) at each outlet at a flow rate of 1.7 mL / min in an embodiment of the present invention. DETAILED DESCRIPTION

[0041] It should be noted that, in the absence of conflict, the embodiments and features in the embodiments of the present application can be combined with each other. The present application will be described in detail below with reference to the accompanying drawings and in combination with the embodiments.

[0042] It should be noted that the steps shown in the flowcharts of the accompanying drawings can be executed in a computer system such as a set of computer executable instructions, and that, although a logical order is shown in the flowcharts, in some cases, the steps shown or described can be executed in an order different from that shown here.

[0043] First, the technical terms involved in the following embodiments are explained.

[0044] A microfluidic chip with a double helix structure including an inner outlet is a microfluidic chip with a double helix channel, and generally includes components such as a chip inlet, a double helix chip, a red blood cell outlet, a white blood cell outlet, a fluid outlet, and a cell filter membrane.

[0045] The design features of this microfluidic chip include:

[0046] Double helix channel: The double helix channel is opened along two parallel chip tangent directions to form two parallel and opposite openings, one opening is connected to the chip inlet, and the other opening is connected to the red blood cell outlet, the white blood cell outlet and the fluid outlet respectively.

[0047] High throughput: Microfluidics technology can concentrate multiple steps of sample detection on a small chip, integrating these operation steps through the combination of flow channel size and curvature, microvalves, and cavity design, ultimately making the entire detection integrated into a miniaturized and automated process.

[0048] Automation: Microfluidic chip technology has the characteristics of small size, low composition, high efficiency, automation and integration, and can complete complex fluid operation systems and functional unit modules on a chip of a few square centimeters3.

[0049] Applications of this microfluidic chip include:

[0050] Blood analysis: It can be used to separate and detect blood components, such as sorting and counting red blood cells and white blood cells.

[0051] Biomedical research: In the biomedical field, microfluidic chips can be used for cell culture, drug screening, and disease modeling.

[0052] In conclusion, the double-helix microfluidic chip with an inner outlet has broad application prospects in fields such as biomedical research and blood analysis.

[0053] Embodiment 1

[0054] In order to solve the problem that circulating tumor cells in the blood are mixed with nearly a million times of background cells and are difficult to be separated efficiently, this embodiment chooses to develop a spiral structure to achieve high-throughput cell screening. In order to improve the sorting accuracy of ordinary monopolar spirals, the spiral is designed as a double spiral structure to allow the particles or cells to be sorted to be focused twice.

[0055] This embodiment provides a microfluidic chip for sorting circulating tumor cells based on a double helix structure, wherein the microfluidic chip has a double helix structure with an inner outlet, and comprises:

[0056] The primary screening channel is used for preliminary focused separation of circulating tumor cells;

[0057] The secondary screening channel is directly connected to the primary screening channel and is used for secondary focusing separation of circulating tumor cells after primary screening;

[0058] Specifically, the spiral chip as a double helix structure includes two spiral structures. The first spiral structure is a rectangular cross-section as the primary screening channel, with a channel width of 350μm and a height of 90μm. After the spiral rotates from the outside to the inside, it separates the channel 200μm away from the inside of the chip. The outer 150μm is connected to the middle outlet to discharge excess liquid and unfocused particles, and the inner 200μm is directly connected to the lower spiral channel for the second focusing separation of particles. The cross-section of the second spiral structure is rectangular, with a width of 200μm and a height of 90μm.

[0059] The primary screening channel is separated into two branches after rotating into the inside of the chip, one branch is connected to the middle outlet for discharging unfocused particles and excess liquid, and the other branch is connected to the secondary screening channel to continue focusing and separating the particles;

[0060] The microfluidic chip is designed with an inlet for the injection of liquid samples and three outlets, namely a middle outlet, a particle collection outlet and a waste outlet, to achieve the injection of liquid samples and the collection of separated particles.

[0061] Compared with the common double helix structure, the microfluidic chip structure of this embodiment has fewer spiral turns. In addition, the double helix structure includes a solution inlet and three outlets, which ensures that the solution injection inlet and the final outlet are outside the chip, thereby ensuring that the structure can be directly integrated with other devices.

[0062] In this embodiment, Figure 1 , Figure 2 As shown, a plan view of a double helix structure is designed using Solidworks software, where circles are drawn at the middle outlet, outer outlet, and inner outlet to determine the punching area. The middle outlet is connected to a 0.18×1.6mm flow resistance tube, and the other outlets are connected to 0.5×1.6mm tubes.

[0063] The double helix structure plan was delivered to SMIC Qiheng for chip mold production, and the PDMS mold was used to produce the corresponding PDMS chip. After cutting, it was bonded to the plasma-treated glass slide, and then the cannula was inserted to complete the production of the entire injection chip.

[0064] There are certain requirements for the insertion parameters of the middle outlet so that it can match the secondary spiral flow resistance. Flow resistance balance study: The first method is to directly insert a 0.15×0.2mm tube into the middle outlet, but after using a 0.3mm puncher to punch a hole, the PDMS itself shrinks, which makes it impossible to insert 0.15×0.2mm smoothly, and the tube will be folded and damaged during the insertion process. Replacing a puncher with a larger aperture can allow the tube to enter smoothly, but PDMS needs to be used for sealing, and blockage will occur due to the thin diameter of the tube. The second method is to use a cannula method. A 0.25×1.2mm tube can find a suitable puncher to insert into the chip, and then insert the thin tube 0.15×0.2mm into 0.25×1.2mm. The part where the two tubes meet needs to be immersed in PDMS and wait for it to solidify, so as to achieve the purpose of no leakage in the entire chip system and balance the flow resistance. The third method is to directly connect the third spiral part with a tube on the outside, but this operation will cause a sharp increase in the overall flow resistance, resulting in the inability to inject liquid into the chip.

[0065] Through actual experiments of these three methods, a simple and optimal method for adjusting the flow resistance was tested, namely the third method. Therefore, this embodiment directly uses a flow resistance injection tube (0.18×1.6mm) with a sufficiently large outer diameter and a sufficiently small inner diameter to match the secondary spiral flow resistance. This method meets the flow resistance requirements of the secondary spiral.

[0066] Furthermore, the width of the primary screening channel is greater than the width of the secondary screening channel to achieve preliminary separation of particles of different diameters.

[0067] Furthermore, the design of the intermediate outlet allows the flow rate to be adjusted to optimize the retention of particles in the secondary screening channel.

[0068] Specifically, this embodiment explores a low-cost means of resistance balance to ensure that the flow resistance at the middle outlet of the double helix structure matches the flow resistance of the secondary helix. Under the premise of ensuring the sorting efficiency, the double helix structure does not need to adjust the chip flow resistance by the chip flow channel structure, and can directly use the PTFE tube to match the flow resistance of the secondary helix from the outside. This operation reduces the flow resistance burden of the chip and can adjust the flow velocity distribution of the secondary helix according to the length of the flow resistance tube, optimizing the final retention rate of 15μm microspheres. Due to its existence, large-volume samples can be concentrated in advance and some particles that are not effectively focused can be removed.

[0069] The chip also has a good liquid concentration effect. Assuming the total inlet liquid is 1mL, since the middle outlet and the outer outlet discharge similar amounts of liquid, about 800μL of liquid can be discarded. Finally, nearly 100-200μL of liquid is obtained from the microsphere collection port, and the original sample is concentrated by 5-10 times.

[0070] Furthermore, the flow rate adjustment is achieved through a flow resistance tube connected to the middle outlet, and the length and diameter of the flow resistance tube are adjusted as needed.

[0071] Furthermore, the channel spacing of the microfluidic chip is 400 μm, the initial spiral radius is 3.3 mm, and the middle S-bend is adjusted to a symmetrical structure.

[0072] Furthermore, the microfluidic chip is made of PDMS material, and the chip is formed and functionalized through a preparation process.

[0073] Specifically, in the chip manufacturing process, polydimethylsiloxane (PDMS) prepolymer and curing agent are first mixed in a mass ratio of 10:1, stirred with a glass rod for 10 minutes, and then placed in a vacuum pump to remove bubbles in the mixture. Pour it into the mold that has been made, and continue to vacuumize to remove tiny bubbles. The mold is placed horizontally in an 80°C oven for 1 hour, and taken out after the PDMS is cured and naturally cooled to room temperature. After cooling, the PDMS is peeled off, cut into appropriate sizes with a cutter, and holes are punched at the channel outlet and entrance with a puncher, and the PDMS microstructure chip is cleaned with a traceless cleaning tape. At the same time, the glass slide is rinsed with anhydrous ethanol and deionized water, and wiped dry until there is no moisture. The glass slide and the PDMS microstructure chip are placed in a plasma cleaner for modification. After completion, the chip and the glass slide are bonded, and gently pressed to ensure that there are no bubbles at the bonding point. After completion, it is placed in an oven for 30 minutes to dry to enhance the bonding effect. Insert a tube at the punching point to complete the microfluidic chip production. If an experiment is required, connect it to the syringe for sample injection.

[0074] Furthermore, the inlet, the particle collection outlet and the waste outlet are all arranged outside the microfluidic chip to achieve direct integration with other devices.

[0075] Specifically, since separation based solely on diameter differences will result in incomplete removal of background cells, the chip will continue to be connected to a chip based on biochemical principles for secondary screening. Therefore, the final outlet flow rate of 100-200 μL / min can better match the chip for sorting based on biochemical principles.

[0076] This embodiment provides a method for adjusting the flow resistance directly through the length of the flow resistance tube, which has a suitable outer diameter and has a capillary function. Balancing the flow resistance reduces the clogging loss problem during capillary insertion and reduces the extra steps required for the cannulation process. At the same time, the chip can concentrate the original liquid by 5-10 times.

[0077] This embodiment designs a double helix structure chip with a middle outlet for cell sorting. The spiral chip exhibits excellent sorting effects. The removal rates of single 5μm and 10μm microsphere solutions reached 85.57% and 90.58% respectively, and the retention rate of single 15μm microspheres reached 85.82%. At the same time, in the mixed solution of the three microsphere solutions, the removal rates of 5μm and 10μm were 83.99% and 82.94%, and the retention rate of 15μm microspheres was 83.32%.

[0078] Qualitative performance analysis of double helix chips, such as Figure 3 As shown. Microspheres of different diameters are used to simulate the distribution of different cells in the microfluidic chip, where 5μm microspheres simulate red blood cells, 10μm microspheres simulate white blood cells, and 15μm microspheres simulate tumor cells. Take a 5mL centrifuge tube and add an appropriate amount of deionized water, then use a pipette to take an appropriate amount of microsphere suspension and add it to the centrifuge tube, use a vortex oscillator to mix evenly, and configure it into a polystyrene microsphere suspension of a specific concentration. To prevent the mutual aggregation and nonspecific adsorption of microspheres, a surfactant is added to the suspension. Fix the chip on top of an inverted microscope and adjust the magnification to 20 times. The channel structure and microsphere particles can be clearly seen under the bright field. After assembling the injection pump and the collection device, switch to the fluorescence module corresponding to the microsphere, and select the appropriate exposure time to record the trajectory of the microsphere. Aim at the outlet to take a photo or video to record the distribution of microspheres during the sorting process.

[0079] Intermediate outlet flow resistance tube optimization experiment, such as Figure 4As shown in the figure. The change in the length of the flow resistance tube (inner diameter × outer diameter: 0.18 × 1.6 mm) connected to the middle outlet affects the flow of the secondary spiral of the chip, which causes the flow rate of the secondary spiral to change, and the sorting efficiency of the microspheres by the secondary spiral to change accordingly. The use of length optimization experiments can find the most suitable parameters and obtain a higher retention efficiency of 15μm microspheres. When the length is 50cm, the retention rate of 15um microspheres is the highest.

[0080] Quantitative performance analysis of double helix chips, such as Figure 5 As shown. 5μm, 10μm and 15μm fluorescent microsphere solutions were injected respectively, the target microsphere concentrations and the corresponding solution volumes at different outlets after sorting were measured, and the distribution of different microspheres at each outlet was calculated. After the three microsphere mixed solutions were configured and injected into the chip, the volumes of different outlets and the three microsphere concentrations were also measured, and the retention rate of 15μm microspheres and the removal rates of 10μm and 5μm microspheres were calculated. The fourth version of the chip has a removal rate of more than 80% for single 5μm and 10μm microsphere solutions, and a retention rate of more than 80% for single 15μm microspheres. The chip was used to sort a mixed solution of the three microsphere solutions, and the retention rate and removal rate of the three microspheres also reached 80%, basically achieving the sorting requirements for microspheres of different diameters.

[0081] Embodiment 2

[0082] This embodiment provides a sorting method of a microfluidic chip, which is based on the microfluidic chip for sorting circulating tumor cells based on a double helix structure described in Embodiment 1, and is used to separate circulating tumor cells, comprising the following steps:

[0083] Injecting the sample to be separated into the inlet of the microfluidic chip;

[0084] Using the primary screening channel of the microfluidic chip to perform preliminary focusing separation on the cells in the sample to be separated;

[0085] guiding the primary screened cells to the secondary screening channel of the microfluidic chip for secondary focusing separation;

[0086] discharging unfocused particles and excess liquid through a middle outlet of the microfluidic chip;

[0087] The focused particles discharged from the particle collection outlet of the microfluidic chip are collected.

[0088] The microfluidic chip sorting method provided in this embodiment has all the advantages of the microfluidic chip based on the double helix structure for sorting circulating tumor cells provided in the first embodiment.

[0089] Furthermore, the cross-section of the primary screening channel is designed to be rectangular, and the width and height have a low aspect ratio, allowing particles of different diameters to be focused at different positions in the flow channel to meet the separation requirements of particles of different sizes.

[0090] Furthermore, the cross-section of the secondary screening channel is designed to be rectangular, and its width is narrower than that of the primary screening channel, which is conducive to focusing of small-diameter particles to achieve more precise particle or cell separation.

[0091] Furthermore, the intermediate outlet is used to adjust the flow rate through a connected flow resistance tube to optimize the retention rate of the particles in the secondary screening channel.

[0092] Furthermore, the length and diameter of the flow resistance tube can be adjusted as required to adapt to different flow rates and particle separation efficiencies.

[0093] Furthermore, the double helix structure design causes cells with larger diameters to be subjected to higher inertial lift, making it easier for them to focus near the inner wall of the channel.

[0094] Furthermore, during the sorting process, smaller cells are susceptible to the Dean force and tend to focus near the outer wall, thereby achieving separation from cells with larger diameters.

[0095] Furthermore, the sorting method also includes connecting the chip based on biochemical principles with a microfluidic chip to perform secondary screening to improve the removal rate of background cells.

[0096] The above are only preferred specific implementations of the present application, but the protection scope of the present application is not limited thereto. Any changes or substitutions that can be easily thought of by a person skilled in the art within the technical scope disclosed in the present application should be included in the protection scope of the present application. Therefore, the protection scope of the present application should be based on the protection scope of the claims.

Claims

1. A microfluidic chip for sorting circulating tumor cells based on a double helix structure, characterized in that: The microfluidic chip has a double helix structure with an inner outlet, comprising: The primary screening channel is used for preliminary focused separation of circulating tumor cells; A secondary screening channel, directly connected to the primary screening channel, for performing secondary focusing separation on circulating tumor cells after primary screening; The primary screening channel is separated into two branches after rotating into the inside of the chip, one branch is connected to the middle outlet for discharging unfocused particles and excess liquid, and the other branch is connected to the secondary screening channel to continue focusing and separating the particles; The microfluidic chip is designed with an inlet for the injection of liquid samples and three outlets, namely a middle outlet, a particle collection outlet and a waste outlet, to achieve the injection of liquid samples and the collection of separated particles.

2. The microfluidic chip for sorting circulating tumor cells based on a double helix structure according to claim 1, characterized in that: The width of the primary screening channel is greater than that of the secondary screening channel to achieve preliminary separation of particles with different diameters.

3. The microfluidic chip for sorting circulating tumor cells based on a double helix structure according to claim 1, characterized in that: The design of the intermediate outlet allows the flow rate to be adjusted to optimize the retention of particles in the secondary screening channel.

4. The microfluidic chip for sorting circulating tumor cells based on a double helix structure according to claim 3, characterized in that: The flow rate adjustment is achieved by a flow resistance tube connected to the middle outlet, and the length and diameter of the flow resistance tube are adjusted as required.

5. The microfluidic chip for sorting circulating tumor cells based on a double helix structure according to claim 1, characterized in that: The channel spacing of the microfluidic chip is 400 μm, the initial spiral radius is 3.3 mm, and the middle S-bend is adjusted to a symmetrical structure.

6. The microfluidic chip for sorting circulating tumor cells based on a double helix structure according to claim 1, characterized in that: The microfluidic chip is made of PDMS material, and the chip is formed and functionalized through a preparation process.

7. The microfluidic chip for sorting circulating tumor cells based on a double helix structure according to claim 1, characterized in that: The inlet, the particle collection outlet and the waste outlet are all arranged outside the microfluidic chip to achieve direct integration of other devices.

8. A microfluidic chip sorting method, characterized in that: The microfluidic chip for sorting circulating tumor cells based on a double helix structure according to any one of claims 1 to 7 is used to separate circulating tumor cells, comprising the following steps: Injecting the sample to be separated into the inlet of the microfluidic chip; Using the primary screening channel of the microfluidic chip to perform preliminary focusing separation on the cells in the sample to be separated; guiding the primary screened cells to the secondary screening channel of the microfluidic chip for secondary focusing separation; discharging unfocused particles and excess liquid through a middle outlet of the microfluidic chip; The focused particles discharged from the particle collection outlet of the microfluidic chip are collected.

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