Multi-channel viscoelastic micro-fluidic chip, manufacturing method and method for separating MCF-7 cells

Abstract

The invention provides a multi-channel viscoelastic micro-fluidic chip, a manufacturing method and a method for separating MCF-7 cells. The multi-channel viscoelastic micro-fluidic chip comprises a first multi-stage shunting module, a pipette, a second multi-stage shunting module, a single-stage viscoelastic micro-fluidic sub-module and a third multi-stage shunting module, the first multi-stage shunting module is connected with one inlet of the single-stage viscoelastic micro-fluidic sub-module through a pipette; the second multi-stage shunting module is connected with the other inlet of the single-stage viscoelastic micro-fluidic sub-module; and the outlet of the single-stage viscoelastic micro-fluidic sub-module is connected with the third multi-stage shunting module. According to the method, the PEO solution is used as sheath fluid, and the action of elastic force is introduced to cells, so that CTC can be directly separated from undiluted human blood in an ultrahigh-flux manner, and the limitation that erythrocytes need to be split and a blood sample needs to be diluted by dozens of times before cell separation in traditional inertial microfluidics is broken through; and the complexity of operation and the risk of sample pollution are reduced.

Description

Technical Field

[0001] The present invention relates to the high-throughput sorting technology of CTCs. Specifically, it relates to a multi-channel viscoelastic microfluidic chip, a manufacturing method and a method for separating MCF-7 cells. In particular, it relates to a multi-channel viscoelastic microfluidic chip for ultra-high-throughput cell sorting. Background Art

[0002] Tumor metastasis is one of the main causes of cancer-related deaths. If cancer patients can receive timely treatment in the early stage of metastasis, the number of cancer-related deaths can be reduced by about one-third. Therefore, developing early and precise tumor diagnosis and treatment technologies and studying the mechanism of tumor metastasis are important means to combat cancer. However, the tumor burden in patients at the early stage of metastasis is extremely low, making it difficult to accurately locate the metastatic foci through traditional imaging techniques. As a result, it is impossible to extract tumor cells at this location using the puncture biopsy technique, which greatly hinders the research on the occurrence, development, and metastasis mechanisms of cancer.

[0003] Circulating tumor cells (CTCs) refer to tumor cells that detach from the tumor lesion site and enter the peripheral blood through epithelial-mesenchymal transition. Currently, a large number of studies have shown that CTCs exist in the peripheral blood of patients at the early stage of tumor metastasis. Therefore, the CTC liquid biopsy technology can break through the limitation of traditional tissue biopsy technology, which is only applicable to solid tumors detectable by imaging. With the advantages of non-invasiveness, easy sampling, and convenient dynamic observation, it can comprehensively and real-time analyze the biological information of tumors and has unique advantages in early tumor diagnosis and treatment guidance.

[0004] Isolating CTCs from patient blood is a critical component of CTC liquid biopsy technology. However, due to interference from the extremely high concentration of red blood cells (the concentration of red blood cells is approximately 108 times that of CTCs), isolating CTCs at ultra-high throughput from undiluted human blood remains a significant challenge. Isolation techniques can be primarily categorized into three types: active, passive, and hybrid. Active techniques utilize driving forces generated by external multi-physical fields to isolate CTCs from blood samples. While they offer precise and personalized manipulation, they rely on highly complex and expensive systems, significantly increasing the difficulty of further back-end technology integration and research costs. Furthermore, the high-precision manipulation of active methods often comes at the expense of sample processing throughput, making them difficult to implement in clinical research. Passive techniques, owing to their advantages of high throughput, small size, and low cost, have become a research hotspot. These techniques utilize forces generated by interactions between cells, fluids, and microstructures to drive cells, eliminating the need for external active control modules. However, passive techniques typically require red blood cell lysis and dozens of dilutions of the blood sample prior to sorting, significantly increasing operational complexity and the risk of sample contamination. Furthermore, their throughput remains insufficient to meet clinical requirements. In addition, passive technologies may cause problems such as clogging and excessive cell stress, which can seriously affect the integrity and activity of cells. Hybrid systems usually refer to the integration of multiple sorting submodules or the embedding of active control elements in the original system. Due to the coupling constraints between the submodules, the separation performance of the hybrid system is usually not significantly improved, and the embedding of active control elements often requires more complex preparation processes and higher development costs. Therefore, the proposal of a technology that can quickly sort CTCs directly from undiluted human blood is of great significance for the study of tumor metastasis mechanisms and drug development.

[0005] In summary, how to quickly isolate CTCs directly from undiluted human blood is a technical challenge that needs to be solved urgently. Summary of the Invention

[0006] In view of the defects in the prior art, the present invention aims to provide a multi-channel viscoelastic microfluidic chip, a manufacturing method and a method for isolating MCF-7 cells.

[0007] According to the present invention, a multi-channel viscoelastic microfluidic chip is provided, comprising 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;

[0008] The first multi-stage flow diversion module is connected to an inlet of the single-stage viscoelastic microfluidic submodule via a pipette;

[0009] The second multi-stage flow diversion module is connected to another inlet of the single-stage viscoelastic microfluidic submodule;

[0010] The outlet of the single-stage viscoelastic microfluidic sub-module is connected to the third multi-stage shunt module.

[0011] Preferably, the first multi-stage shunt module includes a first inlet, a first multi-stage shunt channel, and a first outlet;

[0012] The number of the first inlets is 1, and the first inlet is a sheath fluid inlet;

[0013] The first inlet is connected to a plurality of first outlets through the first multi-stage shunt channel;

[0014] The pipette, the first outlet, and the single-stage viscoelastic microfluidic sub-module correspond one by one; one end of the pipette is connected to the first outlet; the other end is connected to the single-stage viscoelastic microfluidic sub-module.

[0015] Preferably, the second multi-stage shunt module includes a second inlet, a second multi-stage shunt channel, and a first outlet channel;

[0016] The number of the second inlets is 1, and the second inlet is a blood sample inlet;

[0017] The second inlet is connected to a plurality of first outlet channels through the second multi-stage shunt channel;

[0018] The first outlet channels correspond to the single-stage viscoelastic microfluidic sub-module one by one, and the first outlet channels are connected to the single-stage viscoelastic microfluidic sub-module.

[0019] Preferably, the single-stage viscoelastic microfluidic sub-module includes a third inlet, an annular bifurcation branch, a straight channel section, an amplification section, a second outlet channel, a third outlet channel, a serpentine channel, and a second outlet;

[0020] One end of the third inlet is connected to the pipette, and the other end is connected to the annular bifurcation branch. The annular bifurcation branch is connected to the amplification section through the straight channel section. The third outlet channel and the serpentine channel are both connected to the amplification section. The second outlet is located at the outlet end of the serpentine channel.

[0021] Preferably, the third multi-stage shunt module includes a third multi-stage shunt channel and a third outlet;

[0022] The third outlet channel is connected to the third outlet through the third multi-stage shunt channel, and the number of the third outlets is one.

[0023] Preferably, the multi-channel viscoelastic microfluidic chip is a multi-channel viscoelastic microfluidic chip for ultra-high-throughput cell sorting, and the pipette is a pipette.

[0024] Preferably, the serpentine channel is used to balance the additional flow resistance caused by the third multi-stage shunt module. The width of the serpentine channel is 60 μm, and the length is 4000 μm.

[0025] Preferably, the sheath fluid flowing into the first inlet is a PEO solution with a concentration of 0.08% w / v to 0.13% w / v;

[0026] The flow rates at the first inlet and the second inlet are 120 mL·h -1 to 150 mL·h -1 and 12 mL·h -1 to 15 mL·h -1 .

[0027] A preparation method of a multi-channel viscoelastic microfluidic chip according to the present invention is used to prepare the multi-channel viscoelastic microfluidic chip, and includes the following steps:

[0028] Step 1: Mix polydimethylsiloxane and polydimethylsiloxane curing agent in a mass ratio of 10:1, and stir with a glass rod for 8 minutes to 12 minutes to make them fully mixed;

[0029] Step 2: Place the mixed solution in Step 1 in a vacuum drying dish, evacuate to remove the bubbles in the mixed solution, and the time is 40 minutes to 60 minutes;

[0030] Step 3: Place the silicon wafer with the photoresist pattern of the multi-channel viscoelastic microfluidic chip channels in a culture dish, pour the mixed solution after removing bubbles in Step 2, and after the mixed solution evenly and completely covers the silicon wafer, place the culture dish in a vacuum drying dish and evacuate to further remove the bubbles in the mixed solution, and the time is 20 minutes to 30 minutes;

[0031] Step 4: Take out the culture dish in Step 3, place it in an oven for curing, set the oven temperature to 85 °C, and the time is 60 minutes to 80 minutes;

[0032] Step 5: Take out the culture dish in Step 4, slowly peel off the cured polydimethylsiloxane from the silicon wafer, cut it into regular shapes, and use a punch to punch holes at the positions of the first inlet, the second inlet, the third inlet, the first outlet, the second outlet, and the third outlet;

[0033] Step 6: Put the polydimethylsiloxane and the glass slide in Step 5 into a plasma cleaner, turn on the vacuum pump connected to the plasma cleaner, when the pressure in the cavity of the plasma cleaner drops to 200 Pa, stop evacuating and turn on the glow discharge, start timing when purple-red glow appears in the vacuum chamber, terminate the glow discharge after 50 seconds and take the two out;

[0034] Step 7: Closely attach the glass slide in Step 6 to the patterned surface of polydimethylsiloxane, gently press to remove the air bubbles between the surfaces, and place it on a heating table to enhance the bonding effect. The temperature of the heating table is set at 85°C and the time is 20 minutes to 30 minutes;

[0035] Step 8: Insert a pipette into the punched position of the multi-channel viscoelastic microfluidic chip, pour a small amount of polydimethylsiloxane mixture at this position, and place it in an oven for curing to enhance the sealing performance at the punched position. The temperature of the oven is set at 85°C and the time is 60 minutes to 80 minutes.

[0036] A method for separating MCF-7 cells provided by the present invention, using the multi-channel viscoelastic microfluidic chip described above, further includes the following steps:

[0037] Step 1: Pour 1 g of 600 kDa PEO powder into 50 mL of deionized water and swing for 35 hours to prepare a mother liquor with a PEO concentration of 2% w / v;

[0038] Step 2: Dilute the PEO mother liquor with a concentration of 2% w / v into a working solution with a PEO concentration of 0.13% w / v as the sheath fluid;

[0039] Step 3: Use two precision syringe pumps to inject the sheath fluid with a PEO concentration of 0.13% w / v and the human blood sample into the multi-channel viscoelastic microfluidic chip from the first inlet and the second inlet respectively. The flow rates at the first inlet and the second inlet are 145.6 mL·h -1 and 14.56 mL·h -1 .

[0040] Step 4: Collect MCF-7 cells from the second outlet.

[0041] Compared with the prior art, the present invention has the following beneficial effects:

[0042] 1. By using the PEO solution as the sheath fluid, the present invention introduces the action of elastic force on the cells, and can directly sort out CTCs from undiluted human blood, breaking through the limitation that traditional inertial microfluidics requires lysing red blood cells and diluting the blood sample by dozens of times before cell sorting, and reducing the complexity of operation and the risk of sample contamination.

[0043] 2. By connecting multiple viscoelastic microfluidic sub-modules in parallel, the present invention greatly improves the sorting throughput of CTCs and can process 14.56 mL·h -1 of undiluted blood samples within 1 hour.

[0044] 3. The present invention realizes the sorting of CTCs by utilizing internal fluid forces, greatly reducing the probability of direct collision between cells and microstructures, and improving the integrity and viability of the sorted cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0045] Other features, objects, and advantages of the present invention will become more apparent by reading the detailed description of the non-limiting embodiments with reference to the following drawings:

[0046] Figure 1 It is a working schematic diagram of the multi-channel viscoelastic microfluidic chip in the present invention.

[0047] Figure 2 It is a schematic structural diagram of the single-stage viscoelastic microfluidic sub-module in the present invention.

[0048] Figure 3 It is a schematic diagram of the movement of cells in the single-stage viscoelastic microfluidic sub-module in the present invention.

[0049] Figure 4 It is an overall design diagram and an enlarged layout diagram of the multi-channel viscoelastic microfluidic chip in the present invention.

[0050] Figure 5 It is a sorting experiment result diagram of polystyrene microspheres in the enlarged section in the present invention.

[0051] Figure 6 It is a sorting experiment result diagram of cells in the enlarged section in the present invention.

[0052] The figures show:

[0053] DETAILED DESCRIPTION OF THE EMBODIMENTS

[0054] The present invention will be described in detail below with reference to specific embodiments. The following embodiments will help those skilled in the art to further understand the present invention, but do not limit the present invention in any form. It should be noted that those of ordinary skill 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.

[0055] The present invention provides a multi-channel viscoelastic microfluidic chip, as Figure 1-6 shown, including a first multi-stage shunt module 1, a pipette 2, a second multi-stage shunt module 3, a single-stage viscoelastic microfluidic sub-module 4, and a third multi-stage shunt module 5; the first multi-stage shunt module 1 is connected to an inlet of the single-stage viscoelastic microfluidic sub-module 4 through the pipette 2; the second multi-stage shunt module 3 is connected to another inlet of the single-stage viscoelastic microfluidic sub-module 4; the outlet of the single-stage viscoelastic microfluidic sub-module 4 is connected to the third multi-stage shunt module 5.

[0056] The first multi-stage shunt module 1 includes a first inlet 11, a first multi-stage shunt channel 12, and a first outlet 13; the number of the first inlets 11 is 1, and the first inlet 11 is a sheath fluid inlet; the first inlet 11 is connected to a plurality of first outlets 13 through the first multi-stage shunt channel 12; the pipette 2, the first outlet 13, and the single-stage viscoelastic microfluidic sub-module 4 correspond to each other one by one; one end of the pipette 2 is connected to the first outlet 13; the other end is connected to the single-stage viscoelastic microfluidic sub-module 4.

[0057] The second multi-stage shunt module 3 includes a second inlet 31, a second multi-stage shunt channel 32, and a first outlet channel 33; the number of the second inlets 31 is 1, and the second inlet 31 is a blood sample inlet; the second inlet 31 is connected to a plurality of first outlet channels 33 through the second multi-stage shunt channel 32; the first outlet channels 33 and the single-stage viscoelastic microfluidic sub-module 4 correspond to each other one by one, and the first outlet channels 33 are connected to the single-stage viscoelastic microfluidic sub-module 4.

[0058] The single-stage viscoelastic microfluidic sub-module 4 includes a third inlet 41, an annular bifurcation branch 42, a straight channel section 43, an amplification section 44, a second outlet channel 45, a third outlet channel 46, a serpentine channel 47, and a second outlet 48; one end of the third inlet 41 is connected to the pipette 2, and the other end is connected to the annular bifurcation branch 42, the annular bifurcation branch 42 is connected to the amplification section 44 through the straight channel section 43, the third outlet channel 46 and the serpentine channel 47 are both connected to the amplification section 44, and the second outlet 48 is located at the outlet end of the serpentine channel 47.

[0059] The third multi-stage shunt module 5 includes a third multi-stage shunt channel 51 and a third outlet 52; the third outlet channel 46 is connected to the third outlet 52 through the third multi-stage shunt channel 51, and the number of the third outlets 52 is one.

[0060] Specifically, the first outlet 13 of the first multi-stage shunt module 1 and the third inlet 41 of the single-stage viscoelastic microfluidic sub-module 4 are connected through the pipette 2, the second multi-stage shunt channel 32 and the single-stage viscoelastic microfluidic sub-module 4 are connected through the first outlet channel 33, and the second outlet channel 45 and the second outlet 48 are connected through the serpentine channel 47.

[0061] More specifically, the sheath fluid flows into the first multi-stage shunt channel 12 through the first inlet 11, and then flows into eight single-stage viscoelastic microfluidic sub-modules 4 from the third inlet 41 through the pipette 2. The human blood sample flows into the second multi-stage shunt module 3 from the second inlet 31 and then into eight single-stage viscoelastic microfluidic sub-modules 4. Under the action of the sheath fluid, the CTCs and blood cells in the blood sample will flow into the second outlet channel 45 and the third outlet channel 46 respectively after passing through the straight channel section 43 and the amplification section 44, and finally flow out of the microfluidic chip from the second outlet 48 and the third outlet 52.

[0062] In a preferred example, the multi-channel viscoelastic microfluidic chip is a multi-channel viscoelastic microfluidic chip for ultra-high-throughput cell sorting, and the pipette 2 is a pipette. The serpentine channel 47 is used to balance the additional flow resistance caused by the third multi-stage shunt module 5. The width of the serpentine channel 47 is 60 μm and the length is 4000 μm. The sheath fluid flowing into the first inlet 11 is a PEO solution with a concentration of 0.08% w / v to 0.13% w / v. Specifically, the sheath fluid flowing into the first inlet 11 is prepared by dissolving the powder of the high molecular polymer polyethylene oxide (PEO) in PBS buffer, and its concentration is 0.08% w / v to 0.13% w / v. The flow rates at the first inlet 11 and the second inlet 31 are 120 mL·h -1 ~150 mL·h -1 and 12 mL·h -1 ~15 mL·h -1 .

[0063] In the present invention, an injection pump can be used to inject the sheath fluid and the human blood sample into the multi-channel viscoelastic microfluidic chip from the first inlet 11 and the second inlet 31 respectively. The flow rates at the first inlet 11 and the second inlet 31 are 145.6 mL·h -1 and 14.56 mL·h -1 .

[0064] As Figure 3 shown, the human blood sample will reach the beginning of the straight channel section 43 after passing through the circular bifurcation branch 42. The sheath fluid flows into the first inlet 11, and after passing through the first multi-stage shunt module 1 and the pipette 2, it will flow into the straight channel section 43 from the third inlet 41 together with the blood sample. Since the flow rate of the sheath fluid is 10 times that of the blood sample, the blood cells and CTCs will be focused on both sides of the channel under the action of the sheath fluid when they first enter the straight channel section 43. The cells will be subject to inertial lift elastic force and viscous drag force F d ~a p joint action, and Fi and F e plays a leading role in the movement of cells. When cells move from both sides of the channel to the sample-sheath fluid interface position, relatively small blood cells are balanced at the sample-sheath fluid interface position under the repulsive force of F e , while relatively large CTCs can counteract the repulsive force of F i under the action of F e , enabling it to completely penetrate the sample-sheath fluid interface and enter the middle sheath fluid. At the same time, the direction of F e also changes to point to the middle of the channel, pushing the CTCs further towards the center of the channel and reaching equilibrium. Therefore, blood cells and CTCs will be located at different lateral equilibrium positions when moving to the end of the straight channel section 43, and this position difference will be further enlarged after the cells enter the amplification section 44, so that the CTCs and blood cells flow into the second outlet channel 45 and the third outlet channel 46 respectively, and finally flow out of the multi-channel viscoelastic microfluidic chip from the second outlet 48 and the third outlet 52. Among them, the above-mentioned F i and F e are the inertial lift and the elastic force

[0065] As Figure 4 shown, in a preferred example, the channel height in the multi-channel viscoelastic microfluidic chip is 50μm, the length and width of the straight channel section 43 are 10mm and 120μm respectively, the width of the amplification section 44 is 1080μm, the widths of the second outlet channel 45 and the third outlet channel 46 are 280μm and 356μm respectively, and the width of the circular bifurcation branch 42 is 220μm.

[0066] As Figures 5-6 shown, the multi-channel viscoelastic microfluidic chip has obvious sorting effects on both the 10μm / 20μm polystyrene microsphere mixed solution and the undiluted human blood sample.

[0067] The working principle of the present invention:

[0068] After the human blood sample flows into the multi-channel viscoelastic microfluidic chip from the second inlet 31, it will flow into the straight channels 43 of 8 single-stage viscoelastic microfluidic sub-modules 4 through the multi-stage shunt module two 3 and the circular bifurcation branch 42 respectively. The sheath fluid flows in from the first inlet 11, and after passing through the first multi-stage shunt module 1, the pipette 2 and the third inlet 41, it will enter the straight channel section 43 together with the blood sample in the 8 single-stage viscoelastic microfluidic sub-modules 4. When the blood cells and CTCs move to the end of the straight channel section 43, they will be located at different lateral equilibrium positions, and this position difference will be further amplified after the cells enter the amplification section 44, so that the CTCs and blood cells flow into the second outlet channel 45 and the third outlet channel 46 respectively, and finally flow out of the multi-channel viscoelastic microfluidic chip from the second outlet 48 and the third outlet 52, achieving the goal of ultra-high-throughput sorting of CTCs directly from undiluted human blood samples.

[0069] By using the PEO solution as the sheath fluid in the multi-channel viscoelastic microfluidic chip of the present invention, an elastic force is introduced to the cells, and CTCs can be sorted directly from undiluted human blood with ultra-high throughput, breaking through the limitations of traditional inertial microfluidics that require lysing red blood cells and diluting the blood sample by dozens of times before cell sorting, and reducing the complexity of the operation and the risk of sample contamination.

[0070] By connecting multiple viscoelastic microfluidic sub-modules in parallel in the multi-channel viscoelastic microfluidic chip of the present invention, the sorting throughput of CTCs is greatly improved, and 14.56 mL·h can be processed within 1 hour -1 of undiluted blood samples.

[0071] The viscoelastic microfluidic sub-module of the present invention is mainly composed of a straight channel section. Compared with the traditional spiral channel inertial microfluidic chip, it has the advantages of simple structure and small size, greatly reducing the overall size of the system after parallel design.

[0072] The multi-channel viscoelastic microfluidic chip of the present invention uses the internal fluid force to sort CTCs, greatly reducing the probability of direct collision between cells and microstructures, and improving the integrity and activity of the sorted cells.

[0073] The present invention also provides a preparation method of a multi-channel viscoelastic microfluidic chip for preparing the multi-channel viscoelastic microfluidic chip, including the following steps:

[0074] Step 1: Mix polydimethylsiloxane and polydimethylsiloxane curing agent in a mass ratio of 10:1, and stir with a glass rod for 8 minutes to 12 minutes to make it fully mixed;

[0075] Step 2: Place the mixed solution in step 1 in a vacuum drying dish, evacuate to remove the bubbles in the mixed solution for 40 minutes to 60 minutes;

[0076] Step 3: Place the silicon wafer with the lithography resist pattern of the multi-channel viscoelastic microfluidic chip channels in a petri dish, pour the mixed solution after removing bubbles in Step 2 into it. After the mixed solution evenly and completely covers the silicon wafer, place the petri dish in a vacuum drying dish and evacuate it to further remove the bubbles in the mixed solution for 20 to 30 minutes.

[0077] Step 4: Take out the petri dish in Step 3 and place it in an oven for curing. Set the oven temperature to 85°C and the time to 60 to 80 minutes.

[0078] Step 5: Take out the petri dish in Step 4, slowly peel off the cured polydimethylsiloxane from the silicon wafer, cut it into regular shapes, and use a punch to punch holes at the positions of the first inlet 11, the second inlet 31, the third inlet 41, the first outlet 13, the second outlet 48, and the third outlet 52.

[0079] Step 6: Put the polydimethylsiloxane and the glass slide in Step 5 into a plasma cleaner, turn on the vacuum pump connected to the plasma cleaner. When the pressure in the cavity of the plasma cleaner drops to 200 Pa, stop evacuating and turn on the glow discharge. Start timing when purple-red glow appears in the vacuum chamber. Terminate the glow discharge after 50 seconds and take the two out.

[0080] Step 7: Closely attach the glass slide in Step 6 to the patterned surface of the polydimethylsiloxane, gently press to remove the bubbles between the surfaces, and place it on a heating table to strengthen the bonding effect. Set the heating table temperature to 85°C and the time to 20 to 30 minutes.

[0081] Step 8: Insert a pipette into the punched holes of the multi-channel viscoelastic microfluidic chip, pour a small amount of polydimethylsiloxane mixed solution at these positions, and place it in an oven for curing to strengthen the sealing performance at the punched hole positions. Set the oven temperature to 85°C and the time to 60 to 80 minutes.

[0082] The present invention also provides a method for separating MCF-7 cells. Using the multi-channel viscoelastic microfluidic chip described above, it further includes the following steps:

[0083] Step 1: Pour 1 g of 600 kDa (kiloDalton) PEO powder into 50 mL of deionized water, and slowly swing it for 35 hours to prepare a mother liquor with a PEO concentration of 2% w / v.

[0084] Step 2: Dilute the PEO mother liquor with a concentration of 2% w / v into a working solution with a PEO concentration of 0.13% w / v as the sheath fluid.

[0085] Step 3: Use two precision syringe pumps to inject the sheath fluid with a PEO concentration of 0.13% w / v and the human blood sample into the multi-channel viscoelastic microfluidic chip from the first inlet and the second inlet respectively. The flow rates at the first inlet and the second inlet are 145.6 mL·h -1 and 14.56 mL·h -1 .

[0086] Step 4: Collect MCF-7 cells from the second outlet.

[0087] In this solution, the first multi-stage shunt module 1 and the second multi-stage shunt module 3 are designed in parallel. In the traditional design solution, the microfluidic chip usually adopts the soft lithography process. The sheath fluid inlet in each sub-module needs to be connected to a multi-stage shunt device. However, the traditional design idea is to use a three-way valve or make a two-layer PDMS structure. The former usually cannot achieve uniform flow shunting, and the latter process is very complex. Therefore, in this solution, a shunt channel is introduced at the leftmost end of the structure, and then connected to the sheath fluid inlet through a Teflon tube of the same length to achieve uniform flow distribution, overcoming the technical prejudice and proposing a new parallel solution.

[0088] In summary, to overcome the existing technical bottlenecks, the purpose of the present invention is to provide a multi-channel viscoelastic microfluidic chip that can ultra-high-throughput sort CTCs from undiluted human blood. Specifically, the present invention discloses a multi-channel viscoelastic microfluidic chip that can directly ultra-high-throughput sort circulating tumor cells (CTCs) from undiluted human blood, which can quickly sort CTCs from an undiluted human blood sample, and has the advantages of high sorting purity, no need for cell modification, simple operation, and small structural size.

[0089] In the description of the present application, it should be understood that the orientation or positional relationship indicated by the terms "upper", "lower", "front", "rear", "left", "right", "vertical", "horizontal", "top", "bottom", "inner", "outer", etc. is based on the orientation or positional relationship shown in the drawings, and is only for the convenience of describing the present application and simplifying the description, rather than indicating or implying that the device or element referred to must have a specific orientation, be constructed and operated in a specific orientation, and therefore should not be construed as a limitation of the present application.

[0090] The specific embodiments of the present invention have been described above. It should be understood that the present invention is not limited to the above specific embodiments, and those skilled in the art can make various changes or modifications within the scope of the claims, which does not affect the essence of the present invention. Without conflict, the embodiments of the present application and the features in the embodiments can be combined with each other arbitrarily.

Claims

1. A multiplexed viscoelastic microfluidic chip, characterized in that It includes a first multi-stage shunt module (1), a pipette (2), a second multi-stage shunt module (3), a single-stage viscoelastic microfluidic sub-module (4), and a third multi-stage shunt module (5); The first multi-stage shunt module (1) is connected to an inlet of the single-stage viscoelastic microfluidic sub-module (4) through the pipette (2); The second multi-stage shunt module (3) is connected to another inlet of the single-stage viscoelastic microfluidic sub-module (4); The outlet of the single-stage viscoelastic microfluidic sub-module (4) is connected to the third multi-stage shunt module (5).

2. The multi-channel viscoelastic microfluidic chip according to claim 1, wherein The first multi-stage shunt module (1) includes a first inlet (11), a first multi-stage shunt channel (12), and a first outlet (13); The number of the first inlets (11) is 1, and the first inlet (11) is a sheath fluid inlet; The first inlet (11) is connected to a plurality of first outlets (13) through the first multi-stage shunt channel (12); The pipette (2), the first outlet (13), and the single-stage viscoelastic microfluidic sub-module (4) correspond one by one; one end of the pipette (2) is connected to the first outlet (13); the other end is connected to the single-stage viscoelastic microfluidic sub-module (4).

3. The multi-channel viscoelastic microfluidic chip according to claim 1, wherein The second multi-stage shunt module (3) includes a second inlet (31), a second multi-stage shunt channel (32), and a first outlet channel (33); The number of the second inlets (31) is 1, and the second inlet (31) is a blood sample inlet; The second inlet (31) is connected to a plurality of first outlet channels (33) through the second multi-stage shunt channel (32); The first outlet channels (33) correspond to the single-stage viscoelastic microfluidic sub-module (4) one by one, and the first outlet channels (33) are connected to the single-stage viscoelastic microfluidic sub-module (4).

4. The multi-channel viscoelastic microfluidic chip according to claim 1, characterized in that The single-stage viscoelastic microfluidic sub-module (4) includes a third inlet (41), an annular bifurcation branch (42), a straight channel section (43), an amplification section (44), a second outlet channel (45), a third outlet channel (46), a serpentine channel (47), and a second outlet (48); One end of the third inlet (41) is connected to the pipette (2), and the other end is connected to the annular bifurcation branch (42). The annular bifurcation branch (42) is connected to the amplification section (44) through the straight channel section (43). The third outlet channel (46) and the serpentine channel (47) are both connected to the amplification section (44). The second outlet (48) is located at the outlet end of the serpentine channel (47).

5. The multi-channel viscoelastic microfluidic chip according to claim 1, characterized in that, The third multi-stage shunt module (5) includes a third multi-stage shunt channel (51) and a third outlet (52); The third outlet channel (46) is connected to the third outlet (52) through the third multi-stage shunt channel (51), and the number of the third outlets (52) is one.

6. The multi-channel viscoelastic microfluidic chip according to claim 1, wherein The multi-channel viscoelastic microfluidic chip is a multi-channel viscoelastic microfluidic chip for ultra-high-throughput cell sorting, and the pipette (2) is a pipette.

7. The multi-channel viscoelastic microfluidic chip according to claim 4, wherein, The serpentine channel (47) is used to balance the additional flow resistance caused by the third multi-stage shunt module (5). The width of the serpentine channel (47) is 60 μm, and the length is 4000 μm.

8. The multi-channel viscoelastic microfluidic chip according to claim 3, characterized in that the sheath fluid flowing into the first inlet (11) is a PEO solution with a concentration of 0.08% w / v to 0.13% w / v; The flow rates at the first inlet (11) and the second inlet (31) are 120 mL·h -1 ~150 mL·h -1 and 12 mL·h -1 ~15 mL·h -1 .

9. A preparation method of a multi-channel viscoelastic microfluidic chip, characterized in that, The method for preparing the multi-channel viscoelastic microfluidic chip according to any one of claims 1-8 includes the following steps: Step 1: Mix polydimethylsiloxane and polydimethylsiloxane curing agent in a mass ratio of 10:1, and stir with a glass rod for 8 minutes to 12 minutes to make it fully mixed; Step 2: Place the mixed solution in step 1 in a vacuum drying dish, evacuate to remove the bubbles in the mixed solution for 40 minutes to 60 minutes; Step 3: Place the silicon wafer with the photoresist pattern of the multi-channel viscoelastic microfluidic chip channels in a culture dish, pour the mixed solution after removing bubbles in step 2, and after the mixed solution evenly and completely covers the silicon wafer, place the culture dish in a vacuum drying dish and evacuate to further remove the bubbles in the mixed solution for 20 minutes to 30 minutes; Step 4: Take out the culture dish in step 3, place it in an oven for curing, set the oven temperature to 85 °C, and the time is 60 minutes to 80 minutes; Step 5: Take out the culture dish in step 4, slowly peel off the cured polydimethylsiloxane from the silicon wafer, cut it into regular shapes, and use a punch to punch holes at the positions of the first inlet (11), the second inlet (31), the third inlet (41), the first outlet (13), the second outlet (48), and the third outlet (52); Step 6: Put the polydimethylsiloxane and the glass slide in step 5 into a plasma cleaner, turn on the vacuum pump connected to the plasma cleaner, stop evacuating when the pressure in the cavity of the plasma cleaner drops to 200 Pa, turn on the glow discharge, start timing when a purplish-red glow appears in the vacuum chamber, terminate the glow discharge after 50 seconds and take the two out; Step 7: Closely attach the glass slide in step 6 to the patterned surface of the polydimethylsiloxane, gently press to remove the bubbles between the surfaces, and place it on a heating table to strengthen the bonding effect, set the heating table temperature to 85 °C, and the time is 20 minutes to 30 minutes; Step 8: Insert a pipette into the punched position of the multi-channel viscoelastic microfluidic chip, pour a small amount of polydimethylsiloxane mixed solution at this position, place it in an oven for curing to strengthen the sealing performance at the punched position, set the oven temperature to 85 °C, and the time is 60 minutes to 80 minutes.

10. A method for separating MCF-7 cells, characterized in that, Using the multi-channel viscoelastic microfluidic chip according to any one of claims 1-8, further includes the following steps: Step 1: Pour 1 g of 600 kDa PEO powder into 50 mL of deionized water and swing for 35 hours to prepare a mother liquor with a PEO concentration of 2% w / v; Step 2: Dilute the PEO mother liquor with a concentration of 2% w / v into a working solution with a PEO concentration of 0.13% w / v as the sheath fluid; Step 3: Use two precision syringe pumps to inject sheath fluid with a PEO concentration of 0.13% w / v and a human blood sample into the multiplex viscoelastic microfluidic chip from the first inlet and the second inlet respectively. The flow rates at the first inlet and the second inlet are 145.6 mL·h -1 and 14.56 mL·h -1 . Step 4: Collect MCF-7 cells from the second outlet.

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