Cascade viscoelastic micro-fluidic chip and method for preparing, sorting and enriching circulating tumor cells

Abstract

The invention provides a cascade viscoelastic micro-fluidic chip and a method for preparing, sorting and enriching circulating tumor cells. The cascade viscoelastic micro-fluidic chip comprises a cell sorting module, a transition section and a cell concentration module. The cell sorting module is composed of an inlet I, an inlet II, an annular bifurcated branch, a straight channel section I, an amplification section, a serpentine channel I and an outlet I; the cell concentration module is composed of a second straight channel section, a first bifurcation structure, a second serpentine channel, a second outlet, a third straight channel section, a second bifurcation structure, a third serpentine channel, a third outlet, a fourth straight channel section, a third bifurcation structure, a fourth outlet and a fifth outlet. The cell sorting module and the cell concentration module are cascaded through a transition section. The invention breaks through the bottleneck that the traditional sorting technology can be compatible with downstream biological analysis only by intervening manual centrifugation and other operations at the rear end, and can provide powerful technical support for developing a tumor liquid biopsy integrated system.

Description

Technical Field

[0001] This invention relates to microfluidic cell manipulation technology, specifically to a cascaded viscoelastic microfluidic chip and a method for preparing, sorting, and enriching circulating tumor cells. Background Technology

[0002] The vast majority of cancer-related deaths are caused by tumor metastasis. If patients can receive timely treatment in the early stages of metastasis, the number of cancer deaths can be significantly reduced. Therefore, in-depth research into metastasis mechanisms and the development of early precision diagnosis and treatment technologies are crucial means to combat cancer in the future. However, early metastatic lesions are often "hidden" due to their extremely low tumor burden, making it difficult for traditional imaging techniques to accurately locate the lesions and for tumor tissue to be extracted and analyzed through biopsy. This greatly limits research on early molecular diagnosis of tumor metastasis, precision treatment, and the mechanisms of cancer development and progression.

[0003] Circulating tumor cells (CTCs) are seed cells that detach from primary or secondary tumor sites and enter the peripheral blood circulation, existing in the peripheral blood even in the early stages of tumor metastasis. Therefore, CTC liquid biopsy technology overcomes the limitation of traditional tissue biopsy techniques, which rely on images to detect the tumor. Furthermore, thanks to its non-invasive nature, convenient sampling, and dynamic monitoring capabilities, this technology enables real-time and precise capture of tumor biological information, thus providing strong technical support for early cancer diagnosis and clinical intervention.

[0004] However, the direct and precise separation of target CTCs from human whole blood remains challenging due to the complex flow dynamics within microchannels and interference from high concentrations of red blood cells. Furthermore, the ultimate goal of sorting CTCs from blood is downstream bioanalysis, which often requires high concentrations of CTC solutions. Traditional cell sorting techniques typically require the use of sheath fluid for cell pre-focusing, inevitably leading to significant dilution of the CTC sample. Therefore, to meet the CTC concentration requirements of downstream bioanalysis, further enrichment and concentration of CTCs is usually achieved after using existing sorting techniques through centrifugation or labeling. Developing advanced instruments capable of accurately sorting CTCs from blood and subsequently performing molecular marker analysis is a crucial direction for the development of liquid biopsy technology. However, reliance on centrifugation devices and manual intervention remains a key technological bottleneck in the development of integrated systems. Therefore, developing a novel microfluidic chip capable of integrating CTC sorting and concentration can provide strong technical support for the development of integrated CTC liquid biopsy systems, thereby contributing to early precision diagnosis and treatment of tumors and research on metastasis mechanisms.

[0005] Patent application CN120394110A discloses a multi-channel viscoelastic microfluidic chip, its manufacturing method, and a method for isolating MCF-7 cells. The multi-channel viscoelastic microfluidic chip includes a first multi-stage shunt module, a pipette, a second multi-stage shunt module, a single-stage viscoelastic microfluidic submodule, and a third multi-stage shunt module. The first multi-stage shunt module is connected to one inlet of the single-stage viscoelastic microfluidic submodule via the pipette; the second multi-stage shunt module is connected to the other inlet of the single-stage viscoelastic microfluidic submodule; and the outlet of the single-stage viscoelastic microfluidic submodule is connected to the third multi-stage shunt module. However, this patent cannot completely solve the existing technical problems, nor can it meet the needs of this invention. Summary of the Invention

[0006] To address the shortcomings of existing technologies, the purpose of this invention is to provide a cascaded viscoelastic microfluidic chip and a method for preparing, sorting, and enriching circulating tumor cells.

[0007] The cascaded viscoelastic microfluidic chip provided by the present invention includes a sequentially cascaded cell sorting module, a transition section, and a cell concentration module;

[0008] The cell sorting module includes an inlet 1, an inlet 2, a circular bifurcation branch, a straight channel section 1, an amplification section, a serpentine channel 1, and an outlet 1. Inlet 1 and inlet 2 are respectively connected to the inlet ends of the circular bifurcation branch. The outlet end of the circular bifurcation branch is connected to the inlet end of the straight channel section 1. The outlet end of the straight channel section 1 is connected to the inlet end of the amplification section. The amplification section has a central outlet channel and two side outlet channels. The central outlet channel of the amplification section is connected to the inlet end of the transition section through the serpentine channel 1. The two side outlet channels of the amplification section merge and connect to outlet 1. The cell concentration module includes a second straight channel section, a first-stage concentration unit, a second-stage concentration unit, and a third-stage concentration unit; the outlet end of the transition section is connected to the inlet end of the second straight channel section; the outlet end of the second straight channel section is connected to the inlet end of the first-stage concentration unit. The first-stage concentration unit includes a bifurcation structure one, a serpentine channel two, and an outlet two; the bifurcation structure one includes an intermediate branch and two lateral branches, the inlet end of the bifurcation structure one is connected to the outlet end of the straight channel section two, the intermediate branch of the bifurcation structure one is connected to the inlet end of the second-stage concentration unit through the serpentine channel two, and the two lateral branches of the bifurcation structure one are connected to the outlet two after merging; The second-stage concentration unit includes a straight channel section three, a bifurcation structure two, a serpentine channel three, and an outlet three; the inlet end of the straight channel section three is connected to the outlet end of the serpentine channel two; the outlet end of the straight channel section three is connected to the inlet end of the bifurcation structure two; the bifurcation structure two includes an intermediate branch and two lateral branches, the intermediate branch of the bifurcation structure two is connected to the inlet end of the third-stage concentration unit through the serpentine channel three, and the two lateral branches of the bifurcation structure two merge and connect to the outlet three; The third-stage concentration unit includes a straight channel section four, a bifurcation structure three, an outlet four, and an outlet five; the inlet end of the straight channel section four is connected to the outlet end of the serpentine channel three; the outlet end of the straight channel section four is connected to the inlet end of the bifurcation structure three; the bifurcation structure three includes one intermediate branch and two lateral branches, the intermediate branch of the bifurcation structure three is connected to the outlet five, and the two lateral branches of the bifurcation structure three merge and connect to the outlet four.

[0009] Preferably, the channel height of the chip is 50 μm; the length of the first straight channel segment is 30 mm and the width is 120 μm; the length of the second straight channel segment is 10 mm and the width is 50 μm; the length of the third straight channel segment is 15 mm and the width is 50 μm; and the length of the fourth straight channel segment is 25 mm and the width is 50 μm.

[0010] Preferably, the total width of the amplification section is 1080 μm; the width of the middle outlet channel of the amplification section is 280 μm; and the width of each of the two side outlet channels of the amplification section is 356 μm.

[0011] Preferably, the first bifurcation structure, the second bifurcation structure, and the third bifurcation structure have the same geometric structure; the width of the two lateral branches of the first bifurcation structure is 50 μm, and the angle between the two lateral branches and the middle branch is 40°.

[0012] Preferably, the first serpentine channel has a width of 50 μm and a length of 12,500 μm to 13,500 μm; the second serpentine channel has a width of 50 μm and a length of 21,200 μm to 22,200 μm; and the third serpentine channel has a width of 50 μm and a length of 22,100 μm to 23,100 μm.

[0013] Preferably, the second inlet is used to inject a polyethylene oxide solution with a concentration of 0.12% to 0.14%, wherein the polyethylene oxide has a molecular weight of 600 kDa.

[0014] Preferably, the injection flow rate of inlet one is 1.5. Up to 2 The injection flow rate at inlet two is 15. Up to 20 .

[0015] The method for fabricating a cascaded viscoelastic microfluidic chip according to the present invention includes the following steps: Step S1: Mix polydimethylsiloxane and its curing agent at a mass ratio of 10:1, and stir for 8 to 10 minutes until the mixture is homogeneous to obtain a mixture; Step S2: Place the mixture in a vacuum environment and evacuate for 50 to 70 minutes to remove air bubbles; Step S3: Provide a mold with the chip channel pattern engraved on its surface, pour the mixture processed in step S2 onto the surface of the mold to cover the pattern, and then vacuum again for 20 to 30 minutes; Step S4: Place the mold and the mixture on its surface in an environment of 80°C to 90°C and heat to cure for 60 to 80 minutes; Step S5: Peel the cured polydimethylsiloxane from the mold, cut it into shape, and punch holes at the positions corresponding to the inlet 1, inlet 2, outlet 1, outlet 2, outlet 3, outlet 4, and outlet 5; Step S6: Plasma treatment is performed on the channel surface of the perforated polydimethylsiloxane sheet and the surface of a substrate. The treatment conditions are: after the cavity pressure is reduced to 200 Pa, glow discharge treatment is performed for 50 seconds. Step S7: The channel surface of the plasma-treated polydimethylsiloxane sheet is bonded to the surface of the substrate and heated to bond at 80°C to 85°C for 30 to 40 minutes. Step S8: Insert the conduit into the hole made in step S5, apply the mixture of polydimethylsiloxane and curing agent to the interface, and reheat at 80°C to 90°C for 60 to 80 minutes to seal the interface.

[0016] Preferably, in step S1, the amount of polydimethylsiloxane used is 32 g, and the amount of curing agent used is 3.2 g.

[0017] The method for sorting and enriching circulating tumor cells using a cascaded viscoelastic microfluidic chip according to the present invention includes the following steps: Step P1: Prepare a polyoxyethylene solution with a concentration of 0.12% to 0.14% as a sheath fluid, wherein the molecular weight of the polyoxyethylene is 600 kDa; Step P2: The blood sample containing circulating tumor cells and the polyethylene oxide solution are simultaneously injected into the chip through inlet one and inlet two using a syringe pump; the injection flow rate of the blood sample is 1.5. Up to 2 The injection flow rate of the polyethylene oxide solution is 15. Up to 20 ; Step P3: Collect the enriched circulating tumor cell suspension from outlet five.

[0018] Compared with the prior art, the present invention has the following beneficial effects: (1) The cascaded viscoelastic microfluidic chip of the present invention can generate additional elastic force on cells by relying on the viscoelastic effect of PEO solution, thereby realizing the sorting of MCF-7 tumor cells from blood in a microfluidic chip, and then the MCF-7 cell solution is concentrated stepwise in subsequent structures to increase its concentration, and finally a high-purity, high-concentration MCF-7 cell solution can be directly obtained to meet the requirements of downstream single-cell immunoblotting and other analytical techniques. (2) Since the process of sorting and concentrating MCF-7 from blood samples only relies on the action of viscoelastic fluid in the channel, it does not require centrifugation devices and manual intervention as traditional sorting techniques do. Therefore, it can provide technical support for the development of integrated systems for tumor liquid biopsy. Attached Figure Description

[0019] Other features, objects, and advantages of the present invention will become more apparent from the following detailed description of non-limiting embodiments with reference to the accompanying drawings: Figure 1 This is a schematic diagram illustrating the working principle of the cascaded viscoelastic microfluidic chip for sorting and enriching circulating tumor cells in an embodiment of the present invention.

[0020] Figure 2 This is a schematic diagram of cell movement in the cascaded viscoelastic microfluidic chip channel in an embodiment of the present invention.

[0021] Figure 3 This is an overall design diagram of the cascaded viscoelastic microfluidic chip in an embodiment of the present invention.

[0022] Figure 4 This is a partial design diagram of the cascaded viscoelastic microfluidic chip in an embodiment of the present invention at the intersection of the straight channel segment and the circular bifurcation branch.

[0023] Figure 5 This is a partial design diagram of the cascaded viscoelastic microfluidic chip in the amplification section of an embodiment of the present invention.

[0024] Figure 6 This is a partial design diagram of the cascaded viscoelastic microfluidic chip in the embodiments of the present invention at bifurcation structure one, bifurcation structure two, and bifurcation structure three.

[0025] Figure 7 This is a partial design diagram of the cascaded viscoelastic microfluidic chip in a serpentine channel in an embodiment of the present invention.

[0026] Figure 8 This is a partial design diagram of the cascaded viscoelastic microfluidic chip in the second serpentine channel of this invention.

[0027] Figure 9 This is a partial design diagram of the cascaded viscoelastic microfluidic chip at three points in the serpentine channel in an embodiment of the present invention.

[0028] The annotations in the attached figures are explained as follows: 1-Cell sorting module; 11-Inlet 1; 12-Inlet 2; 13-Circular bifurcation branch; 14-Straight channel section 1; 15-Amplification section; 16-Serpentine channel 1; 17-Outlet 1; 2-Transition section; 3-Cell concentration module; 31-Straight channel section 2; 32-Bifurcation structure 1; 33-Straight channel section 3; 34-Serpentine channel 2; 35-Outlet 2; 36-Bifurcation structure 2; 37-Serpentine channel 3; 38-Straight channel section 4; 39-Outlet 3; 310-Bifurcation structure 3; 311-Outlet 5; 312-Outlet 4. Detailed Implementation

[0029] The present invention will now be described in detail with reference to specific embodiments. These embodiments will help those skilled in the art to further understand the present invention, but do not limit the invention in any way. It should be noted that those skilled in the art can make several changes and improvements without departing from the concept of the present invention. These all fall within the protection scope of the present invention.

[0030] Example like Figure 1 As shown, this invention proposes a cascaded viscoelastic microfluidic chip for sorting and enriching circulating tumor cells, mainly comprising three parts: a cell sorting module 1, a transition section 2, and a cell concentration module 3. The cell sorting module 1 consists of an inlet 11, an inlet 2 12, a circular bifurcated branch 13, a straight channel section 14, an amplification section 15, a serpentine channel 16, and an outlet 17. The cell concentration module 3 consists of a straight channel section 2 31, a bifurcated structure 1 32, a serpentine channel 2 34, an outlet 2 35, a straight channel section 3 33, a bifurcated structure 2 36, a serpentine channel 3 37, an outlet 39, a straight channel section 4 38, a bifurcated structure 310, an outlet 4 312, and an outlet 5 311. The cell sorting module 1 and the cell concentration module 3 are cascaded through the transition section 2.

[0031] The first inlet 11 and the second inlet 12 are respectively connected to the inlet end of the circular bifurcation branch 13; the outlet end of the circular bifurcation branch 13 is connected to the inlet end of the straight channel section 14; the outlet end of the straight channel section 14 is connected to the inlet end of the enlarged section 15; the enlarged section 15 is provided with a middle outlet channel and two side outlet channels, the middle outlet channel of the enlarged section 15 is connected to the inlet end of the transition section 2 through the serpentine channel 16, and the two side outlet channels of the enlarged section 15 merge and are connected to the outlet 17; The cell concentration module 3 includes a second straight channel section 31, a first-stage concentration unit, a second-stage concentration unit, and a third-stage concentration unit; the outlet end of the transition section 2 is connected to the inlet end of the second straight channel section 31; the outlet end of the second straight channel section 31 is connected to the inlet end of the first-stage concentration unit. The first-stage concentration unit includes a bifurcation structure 32, a serpentine channel 34, and an outlet 35. The bifurcation structure 32 includes a central branch and two lateral branches. The inlet end of the bifurcation structure 32 is connected to the outlet end of the straight channel section 31. The central branch of the bifurcation structure 32 is connected to the inlet end of the second-stage concentration unit through the serpentine channel 34. The two lateral branches of the bifurcation structure 32 merge and connect to the outlet 35. The second-stage concentration unit includes a straight channel section 33, a bifurcation structure 36, a serpentine channel 37, and an outlet 39. The inlet end of the straight channel section 33 is connected to the outlet end of the serpentine channel 34. The outlet end of the straight channel section 33 is connected to the inlet end of the bifurcation structure 36. The bifurcation structure 36 includes an intermediate branch and two lateral branches. The intermediate branch of the bifurcation structure 36 is connected to the inlet end of the third-stage concentration unit through the serpentine channel 37. The two lateral branches of the bifurcation structure 36 merge and connect to the outlet 39. The third-stage concentration unit includes a straight channel section four 38, a bifurcation structure three 310, an outlet four 312, and an outlet five 311; the inlet end of the straight channel section four 38 is connected to the outlet end of the serpentine channel three 37; the outlet end of the straight channel section four 38 is connected to the inlet end of the bifurcation structure three 310; the bifurcation structure three 310 includes one intermediate branch and two lateral branches, the intermediate branch of the bifurcation structure three 310 is connected to the outlet five 311, and the two lateral branches of the bifurcation structure three 310 merge and connect to the outlet four 312.

[0032] like Figure 2 As shown, when cells in a blood sample reach the beginning of the straight channel segment 14 via the circular bifurcation branch 13, they are arranged on both sides of the straight channel segment 14 under the influence of the sheath fluid. Subsequently, the cells are lifted by inertial lift. Elastic force and viscous drag Under the combined action of these factors, blood cells gradually migrate towards the center line of the channel in the straight channel segment 14. Due to the small size of blood cells, Unable to overcome The repulsive effect prevents blood cells from fully penetrating the sample-sheath fluid interface and maintaining equilibrium near the interface. MCF-7 cells, due to their larger size, ... Under the action of being able to offset The repulsive effect allows the cells to completely penetrate the sample-sheath fluid interface and enter the intermediate sheath fluid, eventually balancing near the channel midline. Therefore, when blood cells and MCF-7 cells reach the end of the straight channel segment 14, a significant lateral positional difference occurs, which is further amplified after the cells enter the amplification segment 15. This causes blood cells to flow out of the microfluidic chip from the outlet 17, while MCF-7 cells enter the cell concentration module 3 from the transition segment 2. In the straight channel segment 31, because the MCF-7 cells are completely immersed in a 0.13% PEO solution, Enough to offset By focusing MCF-7 cells at the centerline of the channel, the cells flow into subsequent structures along the central channel as they pass through the first bifurcation structure 32, while some cell suspension flows out of the microfluidic system from the side channels, thus concentrating the MCF-7 cells. Based on the same principle, MCF-7 cells are sequentially concentrated as they flow through the second bifurcation structure 36 and the third bifurcation structure 310, achieving a high-proportion concentration of the sorted MCF-7 cells. Therefore, the cascaded viscoelastic microfluidic chip proposed in this invention can directly recover a high-purity, high-concentration MCF-7 cell solution at the outlet 311.

[0033] like Figures 3-9As shown, the channel height in the cascaded viscoelastic microfluidic system is 50 μm. The lengths of the first straight channel segment 14, the second straight channel segment 31, the third straight channel segment 33, and the fourth straight channel segment 38 are 30 mm, 10 mm, 15 mm, and 25 mm, respectively, and the channel widths are 120 μm, 50 μm, 50 μm, and 50 μm, respectively. The total width of the amplification segment 15 is 1080 μm, and the widths of the middle outlet channel and the two side outlet channels are 280 μm and 356 μm, respectively. The width of the two side channels of the bifurcation structure is 50 μm, and the angle between the two side channels and the middle channel is 40°. The bifurcation structure one, the bifurcation structure two, and the bifurcation structure three have the same geometric design parameters. The width of the serpentine channel one, the serpentine channel two, and the serpentine channel three is 50 μm, and the lengths are 12500 μm~13500 μm, 21200 μm~22200 μm, and 22100 μm~23100 μm, respectively.

[0034] Working principle of the invention: A cascaded viscoelastic microfluidic chip for directly sorting and enriching MCF-7 cells from blood comprises three parts: a cell sorting module 1, a transition section 2, and a cell concentration module 3. Cell sorting module 1 consists of inlet 11, inlet 2 12, a circular bifurcation branch 13, a straight channel section 14, an amplification section 15, a serpentine channel 16, and an outlet 17. Cell concentration module consists of straight channel section 2 31, bifurcation structure 1 32, serpentine channel 2 34, outlet 2 35, straight channel section 3 33, bifurcation structure 2 36, serpentine channel 3 37, outlet 3 39, straight channel section 4 38, bifurcation structure 3 310, outlet 4 312, and outlet 5 311. Cell sorting module 1 and cell concentration module 3 are cascaded through transition section 2. Blood samples and PEO solutions are injected into the cascaded viscoelastic microfluidic chip through inlet 11 and inlet 2 12, respectively. Blood cells and MCF-7 cells, under the influence of sheath fluid, flow out through outlet 17 and into cell concentration module 3 through the straight channel section 14 and amplification section 15, respectively. In the straight channel section 2 31, MCF-7 cells are focused on the channel centerline due to the elastic force from the PEO solution. This causes the MCF-7 cells to flow into subsequent structures along the middle branch when flowing through bifurcation structure 1 32, bifurcation structure 2 36, and bifurcation structure 3 310, while some of the cell carrier fluid flows out from the side branches, achieving stepwise concentration of the sorted MCF-7 cell solution.

[0035] Those skilled in the art will understand that, in addition to implementing the system, apparatus, and their modules provided by this invention in purely computer-readable program code, the same program can be implemented in the form of logic gates, switches, application-specific integrated circuits, programmable logic controllers, and embedded microcontrollers by logically programming the method steps. Therefore, the system, apparatus, and their modules provided by this invention can be considered a hardware component, and the modules included therein for implementing various programs can also be considered structures within the hardware component; alternatively, modules for implementing various functions can be considered both software programs implementing the method and structures within the hardware component.

[0036] Specific embodiments of the present invention have been described above. It should be understood that the present invention is not limited to the specific embodiments described above, and those skilled in the art can make various changes or modifications within the scope of the claims, which do not affect the essence of the present invention. Unless otherwise specified, the embodiments and features described in this application can be arbitrarily combined with each other.

Claims

1. A cascaded viscoelastic microfluidic chip, characterized in that, It includes a sequentially cascaded cell sorting module, a transition section, and a cell concentration module; The cell sorting module includes an inlet 1, an inlet 2, a circular bifurcation branch, a straight channel section 1, an amplification section, a serpentine channel 1, and an outlet 1. Inlet 1 and inlet 2 are respectively connected to the inlet ends of the circular bifurcation branch. The outlet end of the circular bifurcation branch is connected to the inlet end of the straight channel section 1. The outlet end of the straight channel section 1 is connected to the inlet end of the amplification section. The amplification section has a central outlet channel and two side outlet channels. The central outlet channel of the amplification section is connected to the inlet end of the transition section through the serpentine channel 1. The two side outlet channels of the amplification section merge and connect to outlet 1. The cell concentration module includes a second straight channel section, a first-stage concentration unit, a second-stage concentration unit, and a third-stage concentration unit; the outlet end of the transition section is connected to the inlet end of the second straight channel section; the outlet end of the second straight channel section is connected to the inlet end of the first-stage concentration unit. The first-stage concentration unit includes a bifurcation structure one, a serpentine channel two, and an outlet two; the bifurcation structure one includes an intermediate branch and two lateral branches, the inlet end of the bifurcation structure one is connected to the outlet end of the straight channel section two, the intermediate branch of the bifurcation structure one is connected to the inlet end of the second-stage concentration unit through the serpentine channel two, and the two lateral branches of the bifurcation structure one are connected to the outlet two after merging; The second-stage concentration unit includes a straight channel section three, a bifurcation structure two, a serpentine channel three, and an outlet three; the inlet end of the straight channel section three is connected to the outlet end of the serpentine channel two; the outlet end of the straight channel section three is connected to the inlet end of the bifurcation structure two; the bifurcation structure two includes an intermediate branch and two lateral branches, the intermediate branch of the bifurcation structure two is connected to the inlet end of the third-stage concentration unit through the serpentine channel three, and the two lateral branches of the bifurcation structure two merge and connect to the outlet three; The third-stage concentration unit includes a straight channel section four, a bifurcation structure three, an outlet four, and an outlet five; the inlet end of the straight channel section four is connected to the outlet end of the serpentine channel three; the outlet end of the straight channel section four is connected to the inlet end of the bifurcation structure three; the bifurcation structure three includes one intermediate branch and two lateral branches, the intermediate branch of the bifurcation structure three is connected to the outlet five, and the two lateral branches of the bifurcation structure three merge and connect to the outlet four.

2. The cascaded viscoelastic microfluidic chip according to claim 1, characterized in that, The chip has a channel height of 50 μm; the first straight channel segment has a length of 30 mm and a width of 120 μm; the second straight channel segment has a length of 10 mm and a width of 50 μm; the third straight channel segment has a length of 15 mm and a width of 50 μm; and the fourth straight channel segment has a length of 25 mm and a width of 50 μm.

3. The cascaded viscoelastic microfluidic chip according to claim 1, characterized in that, The total width of the amplification section is 1080 μm; the width of the middle outlet channel of the amplification section is 280 μm; and the width of each of the two side outlet channels of the amplification section is 356 μm.

4. The cascaded viscoelastic microfluidic chip according to claim 1, characterized in that, The first bifurcation structure, the second bifurcation structure, and the third bifurcation structure have the same geometric structure; the width of the two lateral branches of the first bifurcation structure is 50 μm, and the angle between the two lateral branches and the middle branch is 40°.

5. The cascaded viscoelastic microfluidic chip according to claim 1, characterized in that, The first serpentine channel has a width of 50 μm and a length of 12,500 μm to 13,500 μm; the second serpentine channel has a width of 50 μm and a length of 21,200 μm to 22,200 μm; the third serpentine channel has a width of 50 μm and a length of 22,100 μm to 23,100 μm.

6. The cascaded viscoelastic microfluidic chip according to claim 1, characterized in that, The second inlet is used to inject a polyethylene oxide solution with a concentration of 0.12% to 0.14%, wherein the polyethylene oxide has a molecular weight of 600 kDa.

7. The cascaded viscoelastic microfluidic chip according to claim 1, characterized in that, The injection flow rate at inlet one is 1.

5. Up to 2 The injection flow rate at inlet two is 15. Up to 20 .

8. A method for fabricating a cascaded viscoelastic microfluidic chip as described in any one of claims 1 to 7, characterized in that, Includes the following steps: Step S1: Mix polydimethylsiloxane and its curing agent at a mass ratio of 10:1, and stir for 8 to 10 minutes until the mixture is homogeneous to obtain a mixture; Step S2: Place the mixture in a vacuum environment and evacuate for 50 to 70 minutes to remove air bubbles; Step S3: Provide a mold with the chip channel pattern engraved on its surface, pour the mixture processed in step S2 onto the surface of the mold to cover the pattern, and then vacuum again for 20 to 30 minutes; Step S4: Place the mold and the mixture on its surface in an environment of 80°C to 90°C and heat to cure for 60 to 80 minutes; Step S5: Peel the cured polydimethylsiloxane from the mold, cut it into shape, and punch holes at the positions corresponding to the inlet 1, inlet 2, outlet 1, outlet 2, outlet 3, outlet 4, and outlet 5; Step S6: Plasma treatment is performed on the channel surface of the perforated polydimethylsiloxane sheet and the surface of a substrate. The treatment conditions are: after the cavity pressure is reduced to 200 Pa, glow discharge treatment is performed for 50 seconds. Step S7: The channel surface of the plasma-treated polydimethylsiloxane sheet is bonded to the surface of the substrate and heated to bond at 80°C to 85°C for 30 to 40 minutes. Step S8: Insert the conduit into the hole made in step S5, apply the mixture of polydimethylsiloxane and curing agent to the interface, and reheat at 80°C to 90°C for 60 to 80 minutes to seal the interface.

9. The chip fabrication method according to claim 8, characterized in that, In step S1, the amount of polydimethylsiloxane used is 32 g, and the amount of curing agent used is 3.2 g.

10. A method for sorting and enriching circulating tumor cells using a cascaded viscoelastic microfluidic chip as described in any one of claims 1 to 7, characterized in that, Includes the following steps: Step P1: Prepare a polyoxyethylene solution with a concentration of 0.12% to 0.14% as a sheath fluid, wherein the molecular weight of the polyoxyethylene is 600 kDa; Step P2: The blood sample containing circulating tumor cells and the polyethylene oxide solution are simultaneously injected into the chip through inlet one and inlet two using a syringe pump; the injection flow rate of the blood sample is 1.

5. Up to 2 The injection flow rate of the polyethylene oxide solution is 15. Up to 20 ; Step P3: Collect the enriched circulating tumor cell suspension from outlet five.

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