A cascade viscoelastic microfluidic chip and its preparation method and application

Abstract

The present invention provides a cascade viscoelastic microfluidic chip, a preparation method and an application, which belong to the field of CTC sorting technology, and include three parts: a blood filtration module, a transition section and a CTC sorting module. The blood filtration module consists of an inlet one, an inlet two, a circular bifurcated branch one, a straight channel section one, an amplifying section one and an outlet one, and the CTC sorting module consists of an inlet three, a circular bifurcated branch two, a straight channel section two, an amplifying section two, an outlet two and an outlet three. The blood filtration module and the CTC sorting module are cascaded in series using the transition section. The present invention adopts the above-mentioned cascade viscoelastic microfluidic chip, a preparation method and an application. The cascade viscoelastic microfluidic chip has the advantages of large sample processing throughput, high sorting purity, small structural size, no need for cell modification and simple operation, and can simultaneously sort out two types of CTCs, A549 and MCF-7, from undiluted whole blood samples.

Description

Technical Field

[0001] The present invention relates to the technical field of CTC high-throughput sorting, and in particular to a cascade viscoelastic microfluidic chip, a preparation method and an application thereof. Background Art

[0002] Cancer metastasis is the leading cause of cancer-related deaths. Promptly treating patients in the early stages of metastasis can reduce cancer deaths by approximately 30%. Therefore, studying the mechanisms of cancer metastasis and developing early, precision diagnosis and treatment technologies are key to humanity's future fight against cancer. However, in the early stages of metastasis, patients' tumor burden is extremely low, making it difficult to locate metastatic lesions using traditional imaging methods and extract tumor cells through puncture biopsies. This significantly limits molecular diagnosis and precision treatment of early-stage metastatic tumors, as well as research into the mechanisms of cancer occurrence, development, and metastasis.

[0003] Circulating tumor cells (CTCs) refer to tumor cells that have shed from primary or metastatic tumor lesions, undergone epithelial-mesenchymal transition, and entered the peripheral blood circulation. A large number of studies have shown that CTCs are already present in the peripheral blood at the low tumor load stage. Therefore, CTC liquid biopsy technology can break the limitation that tissue biopsy is only applicable to solid tumor tissues that can be detected by imaging. At the same time, with its non-invasive, convenient sampling, and dynamic observation characteristics, it can display the biological information of the tumor in real time, comprehensively, and accurately, showing unique advantages in early cancer diagnosis and treatment guidance.

[0004] However, due to the interference of the high abundance of blood cells in peripheral blood, high-throughput and accurate isolation of CTCs remains a huge challenge. Active manipulation cell sorting technology, because it relies on external forces, usually requires the use of complex instruments and equipment and the introduction of additional manual operations to complete CTC sorting. Therefore, active manipulation cell sorting methods generally face problems such as high technical cost, difficulty in small-scale modularization, and complex operation. Existing passive manipulation cell sorting technology can avoid the disadvantages of the above-mentioned active manipulation methods, but it usually requires red blood cell lysis and blood dilution dozens of times before sorting, which makes it impossible to directly sort CTCs from whole blood samples. Sample processing throughput is still limited, and passive sorting technology also has problems such as high cell shear stress and severe clogging. In addition, most current studies can only achieve the isolation of a specific type of CTC from blood, and have not yet achieved the direct sorting of multiple types of CTCs from whole blood at the same time. Therefore, the proposal of a new cell sorting technology that can quickly, accurately, and directly isolate multiple types of CTCs from whole blood is of great significance for the study of cancer metastasis mechanisms and the development of anticancer drugs. Summary of the Invention

[0005] The present invention aims to provide a cascade viscoelastic microfluidic chip, a preparation method, and an application thereof. The cascade viscoelastic microfluidic chip has the advantages of high sample processing throughput, high sorting purity, small structural size, no need for cell modification, and simple operation. It can also realize the simultaneous sorting of two types of CTCs, A549 and MCF-7, from undiluted whole blood samples.

[0006] To achieve the above objectives, the present invention provides a cascade viscoelastic microfluidic chip, comprising a blood filtration module, a transition section, and a CTC sorting module, wherein the transition section cascades the blood filtration module and the CTC sorting module in series;

[0007] The blood filtration module includes an inlet 1, an inlet 2, a circular bifurcated branch 1, a straight channel section 1, and an amplifying section 1, which are connected in sequence. The initial end of the circular bifurcated branch 1 is connected to the terminal end of the inlet 1, the terminal end of the circular bifurcated branch 1 is connected to the straight channel section 1, the terminal end of the amplifying section 1 is connected to the initial end of the transition section, and the amplifying section 1 is further connected to two symmetrically arranged outlets 1.

[0008] The CTC sorting module includes an inlet three, a circular bifurcated branch two, a straight channel section two, an amplifying section two, an outlet two and an outlet three. The initial end of the circular bifurcated branch two is connected to the end of the transition section, the end of the circular bifurcated branch two is connected to the straight channel section two, the outlet two and the outlet three are both connected to the end of the amplifying section two, the outlet three is connected to the middle position of the amplifying section two, and there are two outlets two, which are symmetrically arranged about the outlet three.

[0009] Preferably, the height of all channels in the cascade viscoelastic microfluidic chip is 50 μm, the length and width of the straight channel section 1 and the straight channel section 2 are 38 mm and 120 μm respectively, the width of the amplification section 1 and the amplification section 2 is 1080 μm, the width of the outlet 1 and the outlet 2 is 356 μm, and the width of the outlet 3 is 280 μm;

[0010] The whole blood sample in the inlet 1 is introduced into the straight channel section 1 together with the sheath fluid 1 in the inlet 2 through the circular bifurcation branch 1 with a ring width of 120 μm. The blood cell-free sample solution flowing out of the blood filtration module is introduced into the straight channel section 2 together with the sheath fluid 2 in the inlet 3 after passing through the transition section.

[0011] Preferably, both sheath liquid 1 and sheath liquid 2 are prepared by dissolving powder of a high molecular weight polymer polyethylene oxide in deionized water to obtain a PEO solution, wherein the concentration of sheath liquid 1 is 0.04% to 0.06% w / v, and the concentration of sheath liquid 2 is 0.13% to 0.17% w / v.

[0012] Preferably, the flow rates at the inlet one, the inlet two and the inlet three are 1.2-1.8 mL / h, 8-10 mL / h and 15-17 mL / h respectively.

[0013] The present invention also provides a method for preparing the above-mentioned cascade viscoelastic microfluidic chip, comprising the following steps:

[0014] S1. Mix polydimethylsiloxane and polydimethylsiloxane curing agent in a mass ratio of 10:1, and stir with a glass rod for 7 to 10 minutes to fully mix them to obtain a polydimethylsiloxane mixed solution;

[0015] S2, placing the polydimethylsiloxane mixture obtained in step S1 in a vacuum drying dish, and removing bubbles in the polydimethylsiloxane mixture by vacuuming to obtain a polydimethylsiloxane mixture after the bubbles are removed;

[0016] S3. Place the silicon wafer with the cascaded viscoelastic microfluidic chip channel pattern in a cell culture dish and introduce the polydimethylsiloxane mixture after the bubbles are removed in step S2. After the polydimethylsiloxane mixture completely and evenly covers the surface of the silicon wafer, place it in a vacuum drying dish again and evacuate the vacuum to remove bubbles between the silicon wafer and the bottom of the cell culture dish.

[0017] S4, placing the cell culture dish in step S3 in an oven for curing;

[0018] S5. Take out the cell culture dish from step S4, separate the solidified polydimethylsiloxane from the silicon wafer, cut it into a cuboid shape, and use a punch to punch holes at inlet 1, inlet 2, inlet 3, outlet 1, outlet 2, and outlet 3;

[0019] S6. Use transparent tape to remove the patterned surface of the polydimethylsiloxane and the surface of the glass slide in step S5, and place the polydimethylsiloxane and the glass slide together into a plasma cleaning machine;

[0020] S7. Turn on the vacuum pump connected to the plasma cleaning machine in step S6. When the pressure in the plasma cleaning chamber drops to 200 Pa, stop evacuating and turn on the glow light. Start timing when a purple-red glow appears in the vacuum chamber. Stop the glow light after 50 seconds and remove the two.

[0021] S8. Closely fit the patterned surface of the polydimethylsiloxane to the glass slide, gently press to remove air bubbles between the surfaces, and then place on a heating table to further strengthen the bonding effect;

[0022] S9. Insert the polytetrafluoroethylene capillary into the punched hole of the cascade viscoelastic microfluidic chip, and pour the polydimethylsiloxane mixture at this position, and place it in an oven to enhance the sealing performance of all outlet and inlet positions.

[0023] Preferably, in step S2, the vacuuming time is 50 to 70 minutes.

[0024] Preferably, in step S3, the vacuuming time is 20 to 30 minutes.

[0025] Preferably, in step S4 and step S9, the oven temperature is set to 80° C. and the drying time is set to 50 to 60 minutes.

[0026] Preferably, in step S8, the temperature of the heating stage is set to 85° C., and the time is 40 to 50 minutes.

[0027] The present invention also provides a use of the above-mentioned cascade viscoelastic microfluidic chip for simultaneously separating A549 and MCF-7 from whole blood, comprising the following steps:

[0028] a. Pour 600 kDa PEO powder into 50 mL of deionized water and shake for 28 to 30 hours to prepare a stock solution with a PEO concentration of 1% to 2% w / v;

[0029] b. Dilute the 1% to 2% w / v stock solution into a working solution with a PEO concentration of 0.04% to 0.06% w / v and a working solution with a PEO concentration of 0.13% to 0.17% w / v, which serve as sheath liquid 1 and sheath liquid 2, respectively;

[0030] c. Whole blood samples spiked with A549 and MCF-7, 0.04%-0.06% w / v working solution, and 0.13%-0.17% w / v working solution were injected into the cascade viscoelastic microfluidic chip from inlet 1, inlet 2, and inlet 3, respectively, using three syringe pumps. The flow rates at inlet 1, inlet 2, and inlet 3 were 1.2-1.8 mL / h, 8-10 mL / h, and 15-17 mL / h, respectively.

[0031] d. Collect A549 and MCF-7 from outlet 2 and outlet 3, respectively.

[0032] Therefore, the present invention adopts the above-mentioned cascade viscoelastic microfluidic chip and its preparation method and application, and the beneficial technical effects are as follows:

[0033] (1) The cascade viscoelastic microfluidic chip of the present invention generates an additional elastic force on cells in the blood by introducing a PEO solution, breaking the limitation of traditional inertial microfluidics that the fluid medium must be a Newtonian fluid. It can directly sort CTCs from the blood, avoiding the operations of lysing red blood cells and diluting samples before sorting, thereby improving the sample processing throughput and making the operation simpler.

[0034] (2) The cascade viscoelastic microfluidic chip of the present invention utilizes a transition section to connect the blood filtration module and the CTC sorting module in series, enabling simultaneous sorting of both A549 and MCF-7 CTCs, thus overcoming the technical bottleneck of existing research that can only sort a specific CTC.

[0035] (3) The cascade viscoelastic microfluidic chip of the present invention has a simple structure and a smaller area compared to the traditional spiral microfluidic chip structure, which is conducive to further improving the sorting throughput through parallel design;

[0036] (4) The cascade viscoelastic microfluidic chip of the present invention utilizes the force exerted by the fluid on cells to achieve sorting, which effectively reduces the probability of cells directly colliding with the microstructure, can reduce cell damage during the sorting process, and improve the integrity of cells after sorting. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 This is a working principle diagram of the cascade viscoelastic microfluidic chip;

[0038] Figure 2 Schematic diagram of the motion in the channel of the cascade viscoelastic microfluidic chip;

[0039] Figure 3 This is the overall design diagram of the cascade viscoelastic microfluidic chip;

[0040] Figure 4 This is a partial design diagram of the cascade viscoelastic microfluidic chip at the beginning of the straight channel section 1 and the straight channel section 2;

[0041] Figure 5 This is a partial design diagram of the cascade viscoelastic microfluidic chip at the amplification section one and amplification section two.

[0042] Reference numerals

[0043] 1. Blood filtration module; 11. Inlet 1; 12. Inlet 2; 13. Circular bifurcation branch 1; 14. Straight channel section 1; 15. Amplification section 1; 16. Exit 1; 2. Transition section; 3. CTC sorting module; 31. Inlet 3; 32. Circular bifurcation branch 2; 33. Straight channel section 2; 34. Amplification section 2; 35. Exit 2; 36. Exit 3. DETAILED DESCRIPTION

[0044] The technical solution of the present invention is further described below with reference to the accompanying drawings and embodiments.

[0045] Unless otherwise defined, technical or scientific terms used in the present invention shall have the same meaning as commonly understood by one of ordinary skill in the art to which the present invention belongs.

[0046] Example 1

[0047] like Figure 1 As shown, the present invention proposes a cascade viscoelastic microfluidic chip, comprising a blood filtration module 1, a transition section 2, and a CTC sorting module 3. The transition section 2 cascades the blood filtration module 1 and the CTC sorting module 3 in series.

[0048] The blood filtration module 1 includes an inlet 11, an inlet 2 12, a circular bifurcated branch 13, a straight channel section 14, and an amplifying section 15, which are connected in sequence. The initial end of the circular bifurcated branch 13 is connected to the terminal end of the inlet 11, the terminal end of the circular bifurcated branch 13 is connected to the straight channel section 14, and the terminal end of the amplifying section 15 is connected to the initial end of the transition section 2. The amplifying section 15 is also connected to two symmetrically arranged outlets 16.

[0049] The CTC sorting module 3 includes an inlet three 31, a circular bifurcated branch 2 32, a straight channel section 2 33, an amplifying section 2 34, an outlet two 35 and an outlet three 36. The initial end of the circular bifurcated branch 2 32 is connected to the end of the transition section 2, the end of the circular bifurcated branch 2 32 is connected to the straight channel section 2 33, both outlet two 35 and outlet three 36 are connected to the end of the amplifying section 2 34, outlet three 36 is connected to the middle position of the amplifying section 2 34, and there are two outlets 2 35, which are symmetrically arranged about outlet three 36.

[0050] Three syringe pumps were used to inject whole blood samples, sheath fluid 1 and sheath fluid 2 into the cascade viscoelastic microfluidic chip from inlet 1 11, inlet 2 12 and inlet 3 31, respectively. The flow rates at inlet 1 11, inlet 2 12 and inlet 3 31 were 1.5 mL / h, 9 mL / h and 16 mL / h, respectively.

[0051] Both sheath liquid 1 and sheath liquid 2 are prepared by dissolving powder of a high molecular weight polymer polyethylene oxide in deionized water to obtain PEO solutions, wherein the concentration of sheath liquid 1 is 0.04% to 0.06% w / v, and the concentration of sheath liquid 2 is 0.13% to 0.17% w / v.

[0052] like Figure 2 As shown, the whole blood sample will reach the beginning of the straight channel section 14 through the circular bifurcated branch 13. Since the sheath fluid 1 is introduced from the inlet 2 12, the cells will be subject to inertial lift when moving in the straight channel section 14. elastic force and viscous drag F d ~a p The combined effect of F i and F e Play a leading role; p is the cell diameter. Due to the small size of red blood cells and white blood cells, eDue to the repulsive effect of the sample-sheath interface, A549 and MCF-7 cannot completely penetrate the sample-sheath interface and eventually reach equilibrium near the interface. i Under the action of F e The repulsive effect causes the two CTCs to enter the intermediate sheath fluid, while F e The original direction of the channel wall will change to the center of the channel, pushing A549 and MCF-7 to migrate further toward the center of the channel, reaching an equilibrium position. As a result, blood cells and the two types of CTCs will have a lateral position difference in straight channel section 14. This difference is further amplified when the cells enter amplification section 15, causing red blood cells and white blood cells to flow into outlet 16, while A549 and MCF-7 enter CTC sorting module 3 through transition section 2, achieving separation of blood cells and the two types of CTCs.

[0053] like Figure 2 As shown, A549 and MCF-7 can be separated in the CTC sorting module 3 and flow out from the second outlet 35 and the third outlet 36 respectively. The sorting principle is the same as that of the above-mentioned blood cells and the two CTCs.

[0054] like Figures 3 to 5 As shown, the length and width of the straight channel section 14 and the straight channel section 2 33 are 38 mm and 120 μm respectively, the width of the enlarged section 15 and the enlarged section 2 34 is 1080 μm, the width of the outlet 16 and the outlet 2 35 is 356 μm, the width of the outlet 3 36 is 280 μm, the width of the circular bifurcation branch 1 13 and the circular bifurcation branch 2 32 is 120 μm, and the height of all channels in the cascade viscoelastic microfluidics is 50 μm.

[0055] The working principle of the present invention is as follows:

[0056] After a whole blood sample enters the cascade viscoelastic microfluidic chip through inlet 11, it follows the sheath fluid flowing in through inlet 2 12 into straight channel segment 14 in blood filtration module 1. Blood cells and two types of CTCs experience lateral positional differences in straight channel segment 14, which are further amplified after the cells enter amplification segment 15. This causes red blood cells and white blood cells to flow out of outlet 16, while A549 and MCF-7 enter CTC sorting module 3 through transition segment 2. Based on the same principle, A549 and MCF-7, after passing through straight channel segment 2 33 and amplification segment 2 34, are split and flow out of outlet 2 35 and outlet 3 36, respectively, achieving the goal of simultaneously sorting A549 and MCF-7 from the whole blood sample.

[0057] Therefore, the present invention adopts the above-mentioned cascade viscoelastic microfluidic chip, preparation method and application. The cascade viscoelastic microfluidic chip has the advantages of large sample processing throughput, high sorting purity, small structural size, no need for cell modification and simple operation, and can simultaneously sort out two types of CTCs, A549 and MCF-7, from undiluted whole blood samples; the sorted A549 and MCF-7 are used for downstream analysis, including immunoblotting, fluorescence flow cytometry, and mass spectrometry flow cytometry.

[0058] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention rather than to limit the same. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that they can still modify or replace the technical solutions of the present invention with equivalents, and these modifications or equivalent replacements cannot cause the modified technical solutions to deviate from the spirit and scope of the technical solutions of the present invention.

Claims

1. A cascade viscoelastic microfluidic chip, characterized in that: It includes a blood filtration module, a transition section and a CTC sorting module, wherein the transition section connects the blood filtration module and the CTC sorting module in series; The blood filtration module includes an inlet 1, an inlet 2, a circular bifurcated branch 1, a straight channel section 1, and an amplifying section 1, which are connected in sequence. The initial end of the circular bifurcated branch 1 is connected to the terminal end of the inlet 1, the terminal end of the circular bifurcated branch 1 is connected to the straight channel section 1, the terminal end of the amplifying section 1 is connected to the initial end of the transition section, and the amplifying section 1 is further connected to two symmetrically arranged outlets 1. The CTC sorting module includes an inlet three, a circular bifurcated branch two, a straight channel section two, an amplifying section two, an outlet two, and an outlet three. The initial end of the circular bifurcated branch two is connected to the end of the transition section, the end of the circular bifurcated branch two is connected to the straight channel section two, the outlet two and the outlet three are both connected to the end of the amplifying section two, and the outlet three is connected to the middle position of the amplifying section two. There are two outlets two, and they are symmetrically arranged with respect to the outlet three. The annular bifurcation branch 1 and the sheath fluid 1 in the inlet 2 are introduced into the straight channel section 1 together, and the annular bifurcation branch 2 and the sheath fluid 2 in the inlet 3 are introduced into the straight channel section 2 together.

2. The cascade viscoelastic microfluidic chip according to claim 1, characterized in that: The height of all channels in the cascade viscoelastic microfluidic chip is 50 μm, the length and width of the straight channel section 1 and the straight channel section 2 are 38 mm and 120 μm respectively, the width of the amplification section 1 and the amplification section 2 is 1080 μm, the width of the outlet 1 and the outlet 2 is 356 μm, and the width of the outlet 3 is 280 μm; The whole blood sample in the inlet 1 is introduced into the straight channel section 1 together with the sheath fluid 1 in the inlet 2 through the circular bifurcation branch 1 with a ring width of 120 μm. The blood cell-free sample solution flowing out of the blood filtration module is introduced into the straight channel section 2 together with the sheath fluid 2 in the inlet 3 after passing through the transition section.

3. The cascade viscoelastic microfluidic chip according to claim 2, characterized in that: Both sheath liquid 1 and sheath liquid 2 are prepared by dissolving polyethylene oxide powder in deionized water to obtain PEO solutions, wherein the concentration of sheath liquid 1 is 0.04% to 0.06% w / v, and the concentration of sheath liquid 2 is 0.13% to 0.17% w / v.

4. The cascade viscoelastic microfluidic chip according to claim 3, characterized in that: The flow rates at the first inlet, the second inlet and the third inlet are 1.2-1.8 mL / h, 8-10 mL / h and 15-17 mL / h respectively.

5. A method for preparing the cascade viscoelastic microfluidic chip according to any one of claims 1 to 4, characterized in that: The following steps are involved: S1. Mix polydimethylsiloxane and polydimethylsiloxane curing agent in a mass ratio of 10:1, and stir with a glass rod for 7 to 10 minutes to fully mix them to obtain a polydimethylsiloxane mixed solution; S2, placing the polydimethylsiloxane mixture obtained in step S1 in a vacuum drying dish, and removing bubbles in the polydimethylsiloxane mixture by vacuuming to obtain a polydimethylsiloxane mixture after the bubbles are removed; S3. Place the silicon wafer with the cascaded viscoelastic microfluidic chip channel pattern in a cell culture dish and introduce the polydimethylsiloxane mixture after the bubbles are removed in step S2. After the polydimethylsiloxane mixture completely and evenly covers the surface of the silicon wafer, place it in a vacuum drying dish again and evacuate the vacuum to remove bubbles between the silicon wafer and the bottom of the cell culture dish. S4, placing the cell culture dish in step S3 in an oven for curing; S5. Take out the cell culture dish from step S4, separate the solidified polydimethylsiloxane from the silicon wafer, cut it into a cuboid shape, and use a punch to punch holes at inlet 1, inlet 2, inlet 3, outlet 1, outlet 2, and outlet 3; S6. Use transparent tape to remove the patterned surface of the polydimethylsiloxane and the surface of the glass slide in step S5, and place the polydimethylsiloxane and the glass slide together into a plasma cleaning machine; S7. Turn on the vacuum pump connected to the plasma cleaning machine in step S6. When the pressure in the plasma cleaning chamber drops to 200 Pa, stop evacuating and turn on the glow light. Start timing when a purple-red glow appears in the vacuum chamber. Stop the glow light after 50 seconds and remove the two. S8. Closely fit the patterned surface of the polydimethylsiloxane to the glass slide, gently press to remove air bubbles between the surfaces, and then place on a heating table to further strengthen the bonding effect; S9. Insert the polytetrafluoroethylene capillary into the punched hole of the cascade viscoelastic microfluidic chip, and pour the polydimethylsiloxane mixture at this position, and place it in an oven to enhance the sealing performance of all outlet and inlet positions.

6. The method for preparing a cascade viscoelastic microfluidic chip according to claim 5, characterized in that: In step S2, the vacuuming time is 50 to 70 minutes.

7. The method for preparing a cascade viscoelastic microfluidic chip according to claim 5, characterized in that: In step S3, the vacuuming time is 20 to 30 minutes.

8. The method for preparing a cascade viscoelastic microfluidic chip according to claim 5, characterized in that: In step S4 and step S9, the oven temperature is set to 80° C. and the time is set to 50 to 60 minutes.

9. The method for preparing a cascade viscoelastic microfluidic chip according to claim 5, characterized in that: In step S8, the temperature of the heating stage is set to 85° C. for 40 to 50 minutes.

10. Use of the cascade viscoelastic microfluidic chip according to claim 1 for simultaneous separation of A549 and MCF-7 from whole blood, characterized in that: The following steps are involved: a. Pour 600 kDa PEO powder into deionized water and shake for 28 to 30 hours to prepare a stock solution with a PEO concentration of 1% to 2% w / v; b. Dilute the 1% to 2% w / v stock solution into a working solution with a PEO concentration of 0.04% to 0.06% w / v and a working solution with a PEO concentration of 0.13% to 0.17% w / v, which serve as sheath liquid 1 and sheath liquid 2, respectively; c. Whole blood samples spiked with A549 and MCF-7, 0.04%-0.06% w / v working solution, and 0.13%-0.17% w / v working solution were injected into the cascade viscoelastic microfluidic chip from inlet 1, inlet 2, and inlet 3, respectively, using three syringe pumps. The flow rates at inlet 1, inlet 2, and inlet 3 were 1.2-1.8 mL / h, 8-10 mL / h, and 15-17 mL / h, respectively. d. Collect A549 and MCF-7 from outlet 2 and outlet 3, respectively.

Images