A drug screening chip and drug screening method based on droplet microfluidics

Abstract

This invention discloses a drug screening chip and method based on droplet microfluidics. The chip includes a substrate and a microchannel layer. The substrate includes a glass layer and a bonding layer attached thereto. Both the bonding layer and the microchannel layer are made of PDMS material and are bonded together. Several microchannels are photolithographically etched on the side of the microchannel layer bonded to the bonding layer. Each microchannel has an inlet and an outlet port at both ends. Several capture units are uniformly arranged along the inlet direction on the microchannel. Each capture unit includes a connected droplet orifice 1 and droplet orifice 2, both of which are cylindrical. The diameter and depth of droplet orifice 1 are larger than those of droplet orifice 2, and droplet orifice 1 is positioned closer to the inlet port, while droplet orifice 2 is positioned closer to the outlet port. The method of this invention can generate a uniform 3D tumor cell spheroid array in the chip and can simultaneously study different concentrations and types of anticancer drugs, thereby achieving high-throughput screening of anticancer drugs.

Description

Technical Field

[0001] This invention relates to the field of drug screening, and in particular to a drug screening chip and method based on droplet microfluidics. Background Technology

[0002] Cancer is a leading cause of death worldwide, accounting for nearly 10 million deaths in 2020. The most common cancers include breast cancer, lung cancer, colon cancer, rectal cancer, and prostate cancer. Taking breast cancer as an example, current treatments mainly include surgical resection, radiation therapy, and drug therapy. However, the recurrence rate after breast cancer surgery is around 40%. Radiation therapy can improve local control rates but has no significant impact on survival rates and often causes adverse neurological effects. Therefore, improving the effectiveness of drug therapy is one of the important tasks in current breast cancer treatment. Since the development cycle for new drugs is generally over 10 years, improving drug screening efficiency is particularly crucial.

[0003] Currently, the main models used for drug screening include 2D tumor models, 3D tumor models, in vitro models based on microfluidic chips, and animal models. Among them, microfluidic chips have attracted widespread attention due to their advantages such as high throughput, low cost, and low reagent consumption. The integration advantage of microfluidic chips is very significant, enabling multiple steps such as cell culture and drug efficacy detection to be integrated into a single chip. Combining 3D tumor models with microfluidic technology to construct drug screening platforms is currently a research hotspot. How to manipulate microfluidics to achieve the formation of 3D tumor models and to detect drug effects and efficacy on tumor cells is the key and challenging aspect of this research. The emergence of droplet microfluidic technology has made it possible to realize the processing, reaction, observation, and detection of multiple independent units on the same chip.

[0004] Currently, a common method involves using hydrodynamics to generate water-in-oil droplets of preset sizes containing different types or concentrations of substances within a droplet generator. Each microdroplet can function as an independent microreactor. To achieve independent control of a single droplet, it is anchored using micropores of appropriate size, fixing it to a specific area. However, these micropores are mostly distributed in large, open chambers, requiring a large number of droplets to maintain the anchoring rate, resulting in waste. Furthermore, when generating microdroplets containing 3D tumor microspheres using a droplet generator, the number of cells encapsulated in each droplet is affected by a Poisson distribution, making it difficult to guarantee a uniform cell count in each droplet. This makes it challenging to form tumor microspheres of uniform size. The uniformity of the 3D tumor model size plays a crucial role in subsequent analysis; for example, in drug processing, the size of the tumor spheres determines the drug penetration and absorption efficiency. Therefore, providing a microfluidic chip and its detection method that can stably generate uniformly sized 3D tumor microdroplets and possess efficient capture and precise control capabilities for microdroplets is essential. Summary of the Invention

[0005] To address the aforementioned technical problems, this invention provides a drug screening chip and method based on droplet microfluidics, which can generate a highly uniform 3D tumor cell sphere array in the chip and simultaneously achieve the goal of studying different concentrations and types of anticancer drugs.

[0006] To achieve the above objectives, the technical solution of the present invention is as follows:

[0007] A drug screening chip based on droplet microfluidics includes a substrate and a microchannel layer. The substrate includes a glass layer and a bonding layer attached thereto. Both the bonding layer and the microchannel layer are made of PDMS material. The bonding layer and the microchannel layer are bonded together after being treated by a plasma cleaner. Several microchannels are photolithographically etched on the side of the microchannel layer bonded to the bonding layer. Sample inlet and sample outlet are provided at both ends of the microchannel. Several capture units are uniformly formed on the microchannel along the sample inlet direction. Each capture unit includes a connected droplet orifice 1 and droplet orifice 2. Both droplet orifice 1 and droplet orifice 2 are cylindrical. The diameter and depth of droplet orifice 1 are larger than those of droplet orifice 2. Droplet orifice 1 is located closer to the sample inlet, and droplet orifice 2 is located closer to the sample outlet.

[0008] In the above scheme, the diameter of the first droplet orifice is 300-500μm.

[0009] Preferably, the first droplet orifice has a diameter of 300 μm and a depth of 300 μm; the second droplet orifice has a diameter of 180 μm and a depth of 100 μm, and the first droplet orifice and the second droplet orifice partially intersect each other for a length of 20 μm.

[0010] Preferably, the microchannel has a width of 500 μm and a depth of 50 μm.

[0011] Preferably, 100 capture units are evenly distributed on the microchannel.

[0012] A drug screening method based on droplet microfluidics, employing a drug screening chip based on droplet microfluidics as described above, includes the following steps:

[0013] (1) Use fluorinated oil to flush the microchannel at high speed to completely remove the gas in the microchannel;

[0014] (2) After the microchannel is completely occupied by fluorinated oil, the cell concentrate is introduced into the microchannel through the injection port;

[0015] (3) When the cell concentrate completely occupies the microchannel, fluorinated oil with surfactant is introduced to cut off the cell concentrate at the capture unit. The oil phase wraps the water phase to form cell droplets with the same shape as the capture unit. The flow rate of fluorinated oil with surfactant is adjusted to flush the cell droplets to adjust the size of the cell droplets until a droplet with a diameter equivalent to the diameter of droplet orifice one is formed, which wraps the cell.

[0016] (4) Place the chip loaded with the first array of droplets in a constant temperature incubator and incubate for more than 5 hours. The cells in the first droplet will aggregate to form a second droplet containing a cell ball. During the incubation process, ensure that the microchannel is always filled with fluorinated oil containing surfactant.

[0017] (5) Then, droplet three containing the drug is generated by the droplet generator and the droplet three is introduced into the microchannel through the chip injection port. The droplet three will enter the empty droplet two and be anchored by the droplet two.

[0018] (6) Then, a demulsifier is introduced to disrupt the oil-water interface, causing the second droplet containing cell spheres to fuse with the third droplet containing drug to form the fourth droplet. The drug in the third droplet will then come into contact with the cell spheres and exert its effect. Cell activity is verified using a live / dead kit to achieve drug screening.

[0019] In the above scheme, the cell concentrate uses the human breast cancer MCF-7 cell line, and the preparation method is as follows: MCF-7 cells cultured in RPMI 1640 complete culture medium until the bottom surface coverage is more than 90% are centrifuged at 2000 rpm for 4 min, and all supernatant is removed. The precipitate at the bottom is the cell concentrate.

[0020] In the above scheme, the specific method for generating drug-encapsulated droplets by the droplet generator is as follows: Fluorinated oil containing surfactant is introduced into the inlet A of the droplet generator at a rate of 1.5 mL / h, and drug is introduced into the inlet B of the droplet generator at a rate of 1.2 mL / h. The oil phase encapsulates the aqueous phase to generate droplets with a diameter between 160-180 μm containing drug. Then, the drug-encapsulated droplets are collected from the outlet C of the droplet generator.

[0021] In a further technical solution, the drug includes anthracyclines, taxanes, and antimetabolites.

[0022] In a further technical solution, fluorescent labeling of drugs with different colored fluorescent dyes can achieve simultaneous screening of drugs of different concentrations and types.

[0023] Through the above technical solution, the drug screening chip and drug screening method based on droplet microfluidics provided by the present invention have the following beneficial effects:

[0024] 1. The drug screening chip disclosed in this invention can form large droplets containing tumor cells and capture small droplets containing drugs by setting a capture unit composed of two droplet orifices of different sizes and depths. The connection of the two droplet orifices can realize the fusion of droplets containing tumor cells and droplets containing drugs, thereby realizing the effect of drugs on cells. Finally, cell activity is verified by a live / dead kit, thereby achieving the purpose of drug screening.

[0025] 2. The cylindrical droplet orifice design of the present invention fits the original shape of the droplet and has a greater depth, which can achieve stable droplet anchoring. Furthermore, due to the design of the narrow straight microchannel, the movement trajectory of the droplet is restricted, making it less likely to escape, thus improving the utilization rate of the micropore and avoiding the waste of a large number of droplets.

[0026] 3. The method of this invention utilizes the microfluidic chip involved in this invention. After completely filling the microchannel with cells, the cells are then cut using fluorinated oil. This ensures that each large droplet contains a basically the same number of cells, thus overcoming the problems of instability and easy breakage of large droplets in droplet generators. The droplets generated autonomously in the microchannel by this invention through self-emulsification are very stable and their size is highly consistent with the droplet orifice size, which is of great significance for the uniformity of subsequent 3D tumor model establishment.

[0027] 4. The present invention discloses a drug screening chip and drug screening method based on droplet microfluidics, which can generate a 3D tumor cell sphere array in the chip and simultaneously study different concentrations and types of anticancer drugs, thereby achieving high-throughput screening of anticancer drugs.

[0028] 5. The advantages of the droplet microfluidic-based drug screening chip and drug screening method provided by this invention are that each droplet pair is independent and does not affect each other. Drugs are fluorescently labeled with commonly used fluorescent dyes, such as allophycocyanin (APC) and fluorescein isothiocyanate (FITC). By using different colors of fluorescence to distinguish between them, it is possible to study different concentrations and types of drugs simultaneously. Attached Figure Description

[0029] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the description of the embodiments or the prior art will be briefly introduced below.

[0030] Figure 1 This is a schematic diagram of a drug screening chip based on droplet microfluidics disclosed in an embodiment of the present invention;

[0031] Figure 2 The diagram shows the capture unit. (a) is a perspective view, (b) is a side view, and (c) is a top view.

[0032] Figure 3 This is a flowchart of a drug screening method based on droplet microfluidics.

[0033] Figure 4 This is a schematic diagram of a droplet generator.

[0034] In the figure, 1. Substrate; 2. Microchannel layer; 3. Microchannel; 4. Inlet port; 5. Outlet port; 6. Capture unit; 7. Droplet orifice one; 8. Droplet orifice two; 9. Fluorinated oil one; 10. Cell concentrate; 11. Fluorinated oil two; 12. Droplet one; 13. Cell sphere; 14. Droplet two; 15. Droplet three; 16. Demulsifier; 17. Droplet four; 18. Inlet A; 19. Inlet B; 20. Outlet C. Detailed Implementation

[0035] The technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present invention.

[0036] This invention provides a drug screening chip based on droplet microfluidics, such as... Figure 1 As shown, it includes a substrate 1 and a microchannel layer 2. The substrate 1 includes a glass layer and an extremely thin bonding layer attached thereto. Both the bonding layer and the microchannel layer 2 are made of PDMS (polydimethylsiloxane) material. The bonding layer and the microchannel layer 2 are bonded together after the PDMS surface is treated by a plasma cleaning machine.

[0037] Several microchannels 3 are photolithographically etched on the side of the microchannel layer 2 bonded to the bonding layer. Each microchannel 3 has an inlet port 4 and an outlet port 5 at both ends. One hundred capture units 6 are uniformly arranged along the inlet direction on the microchannel 3. Each capture unit 6 includes a connected droplet orifice 1 7 and droplet orifice 2 8. Both droplet orifice 1 7 and droplet orifice 2 8 are cylindrical. The diameter and depth of droplet orifice 1 7 are larger than those of droplet orifice 2 8. The centers of droplet orifice 1 7 and droplet orifice 2 8 are located on the centerline of the microchannel, with droplet orifice 1 7 positioned closer to the inlet port 4 and droplet orifice 2 8 positioned closer to the outlet port 5. That is, the inlet direction is from droplet orifice 1 7 to droplet orifice 2 8. The microchannel layer utilizes multilayer photolithography to realize the aforementioned composite stepped capture unit 6.

[0038] In this embodiment, as Figure 2 As shown in (a), (b), and (c), the diameter D of droplet orifice 7 is 300 μm, and the depth H is 300 μm. To ensure that only a single small droplet is captured in droplet orifice 8, its depth cannot be too deep; therefore, the diameter d of droplet orifice 8 is designed to be 180 μm, and the depth h is 100 μm. To ensure that droplets of different sizes can contact each other, droplet orifice 7 and droplet orifice 8 partially intersect, with an intersection length of 20 μm; that is, along the injection direction, the total length of droplet orifice 7 and droplet orifice 8 is 360 μm. In this embodiment, the microchannel width W is 500 μm, and the depth M is 50 μm.

[0039] A drug screening method based on droplet microfluidics, such as Figure 3 As shown, the above-described drug screening chip based on droplet microfluidics includes the following steps:

[0040] (1) Insert the inlet tube and outlet tube into the inlet hole 4 and outlet hole 5, and use fluorinated oil HEF 7500, denoted as fluorinated oil-9, to flush the microchannel 3 at high speed to completely remove the gas in the microchannel 3.

[0041] (2) When the microchannel 3 is completely occupied by fluorinated oil-9, the cell concentrate 10 is introduced into the microchannel 3 through the injection port 4;

[0042] In this embodiment, cell concentrate 10 uses the human breast cancer MCF-7 cell line, and the preparation method is as follows: MCF-7 cells cultured in RPMI 1640 complete culture medium to a bottom surface coverage of more than 90% are centrifuged at 2000 rpm for 4 min, and all supernatant is removed. The precipitate at the bottom is cell concentrate 10.

[0043] (3) After the cell concentrate 10 completely occupies the microchannel 3, fluorinated oil HEF 7500 containing 1% French EmulseoFluoSurf neat surfactant is introduced, which is referred to as fluorinated oil II 11. This causes the cell concentrate 10 to be cut off at the capture unit. The oil phase wraps the water phase to form a cell droplet with the same shape as the capture unit. The flow rate of the fluorinated oil is then adjusted to flush the cell droplet and adjust the size of the cell droplet until a droplet I 12 with a diameter equivalent to the diameter of droplet orifice I is formed, which wraps the cell (the sample flow direction is from left to right).

[0044] (4) Place the chip loaded with the droplet-12 array in a constant temperature incubator at 37°C for more than 5 hours. The breast cancer cell line MCF-7 in the droplet-12 will aggregate to form a droplet-214 containing cell spheres 13, i.e., MCF-7 cell spheres. During the culture process, ensure that the microchannel is always filled with fluorinated oil 21.

[0045] (5) Then, droplets 315 containing the drug are generated by the droplet generator and introduced into the microchannel 3 through the chip injection port 4. The droplets 315 will enter the empty droplet port 28 and be anchored by the droplet port 28.

[0046] The specific method for generating drug-encapsulated droplets using a droplet generator is as follows: Figure 4 As shown, fluorinated oil HEF7500 containing 2% French Emulseo FluoSurf neat surfactant is introduced into the droplet generator inlet A18 at a rate of 1.5 mL / h, and drug is introduced into the droplet generator inlet B19 at a rate of 1.2 mL / h. The oil phase encapsulates the aqueous phase to generate droplets with a diameter between 160-180 μm containing the drug. Then, the drug-containing droplets are collected from the droplet generator outlet C20.

[0047] Currently, the most commonly used chemotherapy drugs for breast cancer include anthracyclines (such as doxorubicin), taxanes (such as paclitaxel), and antimetabolites (such as gemcitabine).

[0048] (6) Then, demulsifier 16 is introduced to disrupt the oil-water interface, so that droplet 2 14 containing cell spheres 13 and droplet 3 15 containing drugs are fused together to form droplet 4 17. The drug in droplet 3 15 will then come into contact with cell spheres 13 and exert its effect. Cell activity can be verified by the live / dead kit, which can directly reflect the effect of the drug and thus achieve the purpose of drug screening.

[0049] The advantage of the droplet microfluidic-based drug screening chip and method provided by this invention is that each droplet pair is independent and does not affect each other. The drugs are fluorescently labeled with commonly used fluorescent dyes, such as allophycocyanin (APC) and fluorescein isothiocyanate (FITC). By using different colors of fluorescence to distinguish between them, it is possible to study different concentrations and types of drugs simultaneously.

[0050] The above description of the disclosed embodiments enables those skilled in the art to make or use the invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A drug screening method based on droplet microfluidics, using a drug screening chip based on droplet microfluidics, characterized by, The chip includes a substrate and a microfluidic layer. The substrate includes a glass layer and a bonding layer attached thereto. Both the bonding layer and the microfluidic layer are made of PDMS material. The bonding layer and the microfluidic layer are bonded together after being treated by a plasma cleaner. Several microfluidic channels are photolithographically etched on the side of the microfluidic layer that is bonded to the bonding layer. Sample inlet and sample outlet are provided at both ends of the microfluidic channels. Several capture units are uniformly formed on the microfluidic channels along the sample inlet direction. Each capture unit includes a connected droplet orifice 1 and a droplet orifice 2. Both droplet orifice 1 and droplet orifice 2 are cylindrical. The diameter and depth of droplet orifice 1 are larger than those of droplet orifice 2. Droplet orifice 1 is located closer to the sample inlet, and droplet orifice 2 is located closer to the sample outlet. The method includes the following steps: (1) Use fluorinated oil to flush the microchannel at high speed to completely remove the gas in the microchannel; (2) After the microchannel is completely occupied by fluorinated oil, the cell concentrate is introduced into the microchannel through the injection port; (3) When the cell concentrate completely occupies the microchannel, fluorinated oil with surfactant is introduced to cut off the cell concentrate at the capture unit. The oil phase wraps the water phase to form cell droplets with the same shape as the capture unit. The flow rate of fluorinated oil with surfactant is adjusted to flush the cell droplets to adjust the size of the cell droplets until a droplet with a diameter equivalent to the diameter of droplet orifice one is formed, which wraps the cell. (4) Place the chip loaded with the first array of droplets in a constant temperature incubator and incubate for more than 5 hours. The cells in the first droplet will aggregate to form a second droplet containing a cell ball. During the incubation process, ensure that the microchannel is always filled with fluorinated oil containing surfactant. (5) Then, droplet three containing the drug is generated by the droplet generator and the droplet three is introduced into the microchannel through the chip sample inlet. The droplet three will enter the empty droplet orifice two and be anchored by the droplet orifice two. (6) Then, a demulsifier is introduced to disrupt the oil-water interface, causing the second droplet containing cell spheres to fuse with the third droplet containing drug to form the fourth droplet. The drug in the third droplet will then come into contact with the cell spheres and exert its effect. Cell activity is verified using a live / dead kit to achieve drug screening.

2. The droplet microfluidics-based drug screening method according to claim 1, characterized in that, The diameter of the first droplet orifice is 300-500 μm.

3. The drug screening method based on droplet microfluidics according to claim 2, characterized in that, The first droplet orifice has a diameter of 300 μm and a depth of 300 μm; the second droplet orifice has a diameter of 180 μm and a depth of 100 μm. The first droplet orifice and the second droplet orifice partially intersect each other, with an intersection length of 20 μm.

4. The drug screening method based on droplet microfluidics according to claim 1, characterized in that, The microchannel has a width of 500 μm and a depth of 50 μm.

5. The drug screening method based on droplet microfluidics according to claim 1, characterized in that, One hundred capture units are evenly distributed on the microchannel.

6. The drug screening method based on droplet microfluidics according to claim 1, characterized in that, The cell concentrate was prepared using the human breast cancer MCF-7 cell line as follows: MCF-7 cells cultured in RPMI 1640 complete medium until the bottom surface coverage was more than 90% were centrifuged at 2000 rpm for 4 min, and all supernatant was removed. The precipitate at the bottom was the cell concentrate.

7. The drug screening method based on droplet microfluidics according to claim 1, characterized in that, The specific method for generating drug-encapsulated droplets using a droplet generator is as follows: Fluorinated oil containing a surfactant is introduced into the inlet A of the droplet generator at a rate of 1.5 mL / h, and the drug is introduced into the inlet B of the droplet generator at a rate of 1.2 mL / h. The oil phase encapsulates the aqueous phase to generate drug-encapsulated droplets with a diameter between 160-180 µm. Then, the drug-encapsulated droplets are collected from the outlet C of the droplet generator.

8. The drug screening method based on droplet microfluidics according to claim 1, characterized in that, The drugs include anthracyclines, taxanes, and antimetabolites.

9. The drug screening method based on droplet microfluidics according to claim 1, characterized in that, By labeling drugs with fluorescent dyes of different colors, it is possible to screen drugs of different concentrations and types simultaneously.

Images