Liquid drop micro-fluidic chip based on micro-filtration array

Abstract

The invention provides a liquid drop micro-fluidic chip based on a micro-filtration array. The micro-filtration array structure is highly integrated in the water phase sample introduction area of the chip, synchronous filtration of cell suspension in the inflow process is achieved, newly formed cell blocks or fragments can be effectively intercepted and prevented from entering the liquid drop generation area, and therefore the liquid drop wrapping quality is guaranteed from the source. In an actual test, the droplet micro-fluidic chip has an efficient filtering capability, supports the droplet generation frequency of about 1000 Hz, has been verified in a 293T cell suspension system, has good stability and repeatability, and can be widely applied to single cell sequencing, cell screening and other high-throughput single cell analysis scenes.

Description

Technical Field

[0001] This invention belongs to the field of biology, specifically relating to the field of microfluidic chip technology, and more specifically to a droplet microfluidic chip based on a microfiltration array. Background Technology

[0002] With the development of single-cell omics and personalized medicine, single-cell analysis technology has shown great application potential in life sciences, early disease screening, and drug screening. Among them, single-cell encapsulation platforms based on microfluidic droplet technology have become an important foundation for building single-cell sequencing and cell screening systems due to their advantages such as high throughput, low sample consumption, and strong sealing.

[0003] In typical droplet microfluidic platforms, cell suspensions and oil phases must be precisely injected into the chip to generate oil-in-water droplets, ensuring that each droplet encapsulates a complete living cell. However, when cell sample pretreatment is insufficient, the cell fluid often contains a large number of impurities, debris, dead cell clusters, and cell aggregates. Traditional droplet microfluidic chips often experience droplet deformation, fusion failure, and even chip clogging during single-cell encapsulation experiments due to these impurities, thus affecting the stable generation of droplets. Each clogging requires interrupting the experiment, cleaning the apparatus, or replacing the chip, increasing experimental costs and reducing overall throughput and data continuity. Especially under medium-to-high throughput experimental conditions (>1000 Hz droplet frequency), even with standard centrifugation or membrane pretreatment, there is still a significant risk of clogging, severely limiting the application of chips in automated and high-throughput scenarios. Currently, most mainstream cell filtration methods are performed externally to the chip, such as using membrane filters, cell sieves, or centrifugation. However, these methods are cumbersome to operate, difficult to integrate into automated processes, and can easily damage cell viability, lacking real-time and high-throughput processing capabilities.

[0004] Therefore, there is an urgent need to develop a technology that can achieve efficient, gentle, and real-time cellular impurity filtration within a microfluidic chip, ensuring that only a single healthy cell is encapsulated in the droplet, thereby improving the accuracy and stability of downstream single-cell sequencing and functional analysis experiments. Summary of the Invention

[0005] To address the problem of clogging in traditional microfluidic chips during droplet generation experiments, this invention provides a droplet microfluidic chip based on a microfilter array.

[0006] To achieve the above objectives, the technical solution adopted by the present invention is as follows: a droplet microfluidic chip based on a microfiltration array, comprising a PDMS layer sealed on a substrate layer, wherein the PDMS layer is provided with an oil phase injection area, an oil phase introduction channel communicating with the oil phase injection area, an aqueous phase injection area, an aqueous phase introduction channel communicating with the aqueous phase injection area, a droplet generation area, a confluence channel communicating with the droplet generation area, and a droplet collection port communicating with the confluence channel; the oil phase introduction channel and the aqueous phase introduction channel are connected and converge in the droplet generation area; the aqueous phase injection area includes an aqueous phase inlet. The system includes an aqueous phase outlet and an aqueous phase filtration structure disposed between the aqueous phase inlet and the aqueous phase outlet; the aqueous phase inlet, the aqueous phase outlet, and the aqueous phase inlet channel are sequentially connected; the arrangement range of the aqueous phase filtration structure covers at least a portion of the flow channel cross-section of the aqueous phase injection area; the aqueous phase filtration structure includes multiple regularly arranged aqueous phase micro-columns and aqueous phase screening channels located between the aqueous phase micro-columns; the diameter D of the aqueous phase screening channel is the vertical straight-line distance between the sidewalls of two adjacent aqueous phase micro-columns, which is not less than the diameter of the target single cell and not greater than the minimum feature size of the target interceptor.

[0007] To address the clogging issue inherent in traditional microfluidic chips during droplet generation experiments, this invention proposes a microfluidic design integrating a filtration structure within the chip. The core idea is to physically intercept large particulate impurities, dead cell clusters, and cell aggregates in the cellular fluid by integrating a microscale solid-state filtration structure between the aqueous inlet and outlet, thereby improving channel flow and droplet generation stability. Figure 1-3 As shown, the microfluidic chip of this invention incorporates multiple array-shaped filtration structures in the aqueous sample introduction zone. Each filtration structure consists of regularly arranged micropillars with a certain spacing between them, allowing individual suspended cells to pass through smoothly while intercepting large particles or cell clusters at the front end. The micropillar array is arranged at an oblique angle relative to the aqueous flow direction, which increases the filtration path length and reduces the risk of clogging.

[0008] Preferably, the aqueous micro-column has a square cross-section, and the diagonal of the cross-section is parallel or perpendicular to the shortest flow path direction of the aqueous sample introduction zone, wherein the shortest flow path is a straight line between the aqueous inlet and the aqueous outlet. The micro-column array is arranged at an oblique angle relative to the aqueous flow direction, which increases the filtration path length and reduces the risk of clogging.

[0009] Preferably, the diameter D of the aqueous phase screening channel is 10~40 μm, more preferably 20 μm.

[0010] Preferably, the cross-sectional side length of the aqueous micro-column is 50~70 μm, more preferably 60 μm.

[0011] Preferably, the oil phase injection zone includes an oil phase inlet, an oil phase outlet, and an oil phase filter structure disposed between the oil phase inlet and the oil phase outlet; the oil phase inlet, oil phase outlet, and oil phase inlet channel are sequentially connected; the arrangement range of the oil phase filter structure covers at least a portion of the flow channel cross-section of the oil phase injection zone; the oil phase filter structure includes multiple regularly arranged oil phase micro-columns and oil phase screening channels located between the oil phase micro-columns; the oil phase micro-columns have a square cross-section and the diagonal of the cross-section is parallel or perpendicular to the shortest flow path direction of the oil phase injection zone, the shortest flow path being a straight path between the oil phase inlet and the oil phase outlet.

[0012] Preferably, the oil-phase micro-column comprises an oil-phase type I column and an oil-phase type II column; the cross-sectional area of ​​the oil-phase type I column is larger than that of the oil-phase type II column. In the oil-phase sample introduction area, this invention employs a multi-stage gradient filtration mechanism. By designing columns with different cross-sectional dimensions, a filtration hierarchy from sparse to dense is formed. This hierarchical filtration strategy further reduces the risk of clogging and alleviates pressure concentration on a single filtration surface, making it particularly suitable for high-frequency (>1000Hz) droplet generation scenarios, significantly improving the stability and reliability of the chip during long-term operation.

[0013] Preferably, the oil phase type I columns are arranged at equal intervals on the side relatively close to the oil phase inlet; the oil phase type II columns are arranged at equal intervals on the side relatively close to the oil phase outlet.

[0014] Preferably, the edge spacing between any two adjacent oil phase type I columns is greater than the edge spacing between the oil phase type II columns.

[0015] Preferably, the side length of the cross-section of the oil phase type I column is 1.5 to 2 times that of the side length of the cross-section of the oil phase type II column.

[0016] The present invention also provides a method for fabricating the droplet microfluidic chip based on a microfiltration array, comprising: Step 01: Prepare a PDMS layer using soft lithography; wherein the preparation method of the oil phase filtration structure and the water phase filtration structure includes: fabricating an oil phase filtration structure mold and a water phase filtration structure mold on a silicon wafer using SU-8, and forming the oil phase filtration structure and the water phase filtration structure after development on the PDMS layer; Step 02: The PDMS layer and the glass sheet are bonded together by oxygen plasma treatment to form the droplet microfluidic chip.

[0017] Among them, the oil phase filtration structure mold and the water phase filtration structure are patterned by drawing software during the chip design stage, and then transferred to the SU-8 mold by standard photolithography. Finally, they are molded into the microfluidic chip using PDMS replication technology. No additional assembly steps are required, and it has high integration and good reproducibility.

[0018] The beneficial effects of this invention are as follows: This invention highly integrates a microfiltration array structure within the aqueous sample inlet area of ​​the chip, achieving simultaneous filtration of cell suspensions during inflow. This design eliminates the traditional cumbersome sample pretreatment steps, simplifying the operation process and enabling real-time dynamic removal of impurities. It effectively intercepts newly formed cell clumps or fragments, preventing them from entering the droplet generation area, thus ensuring droplet encapsulation quality from the source. Furthermore, this invention allows for flexible configuration of the filtration array structure to address the differences in impurity characteristics in different fluid phases (such as aqueous and oil phases). For example, a two-stage filtration system can be used in the oil phase inlet area to handle complex impurities, while a fine filtration unit is used in the aqueous phase area. This differentiated configuration improves the targeting and overall efficiency of filtration, further expanding the chip's applicable scenarios and performance potential. In actual testing, this droplet microfluidic chip has the ability to efficiently filter dead cell clusters and impurities, supports a droplet generation frequency of approximately 1000 Hz, and has been validated in a 293T cell suspension system. It has good stability and reproducibility and can be widely used in single-cell sequencing, cell screening and other high-throughput single-cell analysis scenarios. Attached Figure Description

[0019] Figure 1 This is a schematic diagram of the droplet microfluidic chip in the embodiments of this application.

[0020] Figure 2 This is a schematic diagram of the aqueous phase injection zone structure in an embodiment of this application.

[0021] Figure 3 This is a schematic diagram of the oil phase injection zone structure in an embodiment of this application.

[0022] Figure 4 This is a microscope image of the aqueous sample injection area in an embodiment of this application.

[0023] Figure 5 This is a microscope image of the aqueous sample injection area in an actual experiment, as shown in the embodiments of this application.

[0024] Figure 6 Experimental images of a traditional chip without integrated filtering structure.

[0025] In the diagram: 11-Oil phase injection area, 12-Oil phase introduction channel, 21-Aqueous phase injection area, 22-Aqueous phase introduction channel, 31-Droplet generation area, 32-Merging channel, 33-Droplet collection port, 111-Oil phase inlet, 112-Oil phase outlet, 113-Oil phase filtration structure, 1131-Oil phase micro-column, 1131A-Oil phase type I column, 1131B-Oil phase type II column, 1132-Oil phase screening channel, 211-Aqueous phase inlet, 212-Aqueous phase outlet, 213-Aqueous phase filtration structure, 2131-Aqueous phase micro-column, 2132-Aqueous phase screening channel. Detailed Implementation

[0026] The following specific embodiments illustrate the implementation of the present invention. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and various details in this specification can be modified or changed based on different viewpoints and applications without departing from the spirit of the present invention. It should be noted that, unless otherwise specified, the following embodiments and features can be combined with each other. Unless otherwise specified, the methods used in the embodiments of the present invention are conventional methods, and the reagents used are commercially available.

[0027] This embodiment aims to provide a droplet microfluidic chip, including a PDMS layer sealed to a substrate. For example... Figure 1 As shown, the PDMS layer includes an oil phase injection zone 11 for injecting spacer oil and two aqueous phase injection zones 21 for injecting cell suspension. The injected spacer oil and cell suspension are transported to the droplet generation zone 31 through the corresponding oil phase introduction channel 12 and aqueous phase introduction channel 22, where the target single cell is efficiently encapsulated and stably generated with good dispersion of water-in-oil microdroplets.

[0028] refer to Figure 1 The aqueous phase injection area 21 includes an aqueous phase inlet 211, an aqueous phase outlet 212, and an aqueous phase filtration structure 213 disposed between the aqueous phase inlet 211 and the aqueous phase outlet 212. After the cell suspension is injected into the chip through the aqueous phase inlet 211, it passes through the aqueous phase filtration structure 213 to remove impurities such as cell clumps, aggregates, or fragments, and then flows out from the aqueous phase outlet 212 and into the aqueous phase introduction channel 22. Figure 2As can be seen, the arrangement of the aqueous phase filtration structure 213 covers the entire flow channel cross-section of the aqueous phase injection area 21, ensuring that all injected cell suspensions must pass through this aqueous phase filtration structure 213 before flowing to the aqueous phase outlet 212. Specifically, the aqueous phase filtration structure 213 includes multiple regularly arranged aqueous phase micropillars 2131 and aqueous phase screening channels 2132 located between the aqueous phase micropillars 2131. Further, the cross-section of the aqueous phase micropillars 2131 is designed as a square with a side length of 50~70 μm, more preferably 60 μm. The regular geometric shape of the cross-section of the aqueous phase micropillars 2131 ensures a high degree of uniformity in the entire microfiltration array. Because the cross-section of the square has completely equal side lengths and symmetry in all directions, the numerous aqueous screening channels 2132 formed by their regular arrangement maintain a high degree of geometric uniformity. This uniformity ensures that the resistance of each flow path is evenly distributed when the fluid passes through the array, effectively avoiding flow deviation or eddy currents that may occur due to differences in path resistance. This allows the fluid to pass through the entire filtration interface more smoothly and evenly, significantly improving the uniformity and stability of filtration efficiency. At the same time, this symmetrical and stable flow field also helps to reduce the potential damage to cells caused by fluid shear force and further reduces the risk of local accumulation of impurities in specific areas leading to blockage. Specifically, the distance between two adjacent aqueous micro-columns 2131, i.e., the width of the aqueous screening channel 2132, is designed to be 10~40 μm, such as 10 μm, 15 μm, 20 μm, 25 μm, 30 μm, 40 μm, more preferably 20 μm. This size only allows a single cell to pass through smoothly, while intercepting large particles or cell clusters at the front end to prevent them from entering subsequent flow channels. Understandably, the width of the aqueous phase screening channel 2132 can be customized according to the target cell type. For example, for larger cells (such as certain stem cells), a larger channel spacing can be designed; for smaller suspended cells, a denser structure can be used to intercept impurities. This high degree of customization allows the chip to adapt to the needs of different experimental scenarios, exhibiting good versatility and scalability. Furthermore, the diagonal of the square cross-section of the aqueous phase micropillar 2131 is parallel or perpendicular to the shortest flow path direction of the aqueous phase injection area 21, where the shortest flow path is a straight line between the aqueous phase inlet 211 and the aqueous phase outlet 212. That is, the micropillar array is arranged at an oblique angle relative to the aqueous phase flow direction, thereby increasing the filtration path length and reducing the risk of clogging.

[0029] refer to Figure 3This embodiment also adds an oil phase filtration structure 113 based on a microfiltration array to the oil phase injection area 11. Similar to the aqueous phase injection area 21, the oil phase injection area 11 includes an oil phase inlet 111, an oil phase outlet 112, and an oil phase filtration structure 113 disposed between the oil phase inlet 111 and the oil phase outlet 112. After the spacer oil is injected into the chip through the oil phase inlet 111, it passes through the oil phase filtration structure 113 to remove impurities in the oil phase and is adjusted to a flow rate matching the cell suspension. Then it flows out from the oil phase outlet 112 and enters the oil phase inlet channel 12. Similarly, the arrangement range of the oil phase filtration structure 113 covers the entire flow channel cross-section of the oil phase injection area 11. The oil phase filtration structure 113 includes a plurality of regularly arranged oil phase micropillars 1131 with a square cross-section and oil phase screening channels 1132 located between the oil phase micropillars 1131. The oil phase micro-column 1131 has a square cross-section, and the diagonal of the cross-section is parallel or perpendicular to the shortest flow path direction of the oil phase injection zone 11. The shortest flow path is a straight line between the oil phase inlet 111 and the oil phase outlet 112. Furthermore, in the oil phase injection zone 11, this invention employs a multi-stage gradient filtration mechanism. Specifically, the oil phase micro-column 1131 includes an oil phase type I column 1131A arranged at equal intervals on the side relatively closer to the oil phase inlet 111 and an oil phase type II column 1131B arranged at equal intervals on the side relatively closer to the oil phase outlet 112. The cross-sectional area of ​​the oil phase type I column 1131A is larger than the cross-sectional area of ​​the oil phase type II column 1131B. For example, the side length of the cross-section of the oil phase type I column 1131A is 1.5 to 2 times the side length of the cross-section of the oil phase type II column 1131B. Correspondingly, the edge spacing between any two adjacent oil phase type I pillars 1131A is greater than the edge spacing between the oil phase type II pillars 1131B. That is, the oil phase screening channel 1132 near the oil phase output port 112 has a smaller width than the oil phase screening channel 1132 near the oil phase inlet 111. By designing pillars with different cross-sectional dimensions and oil phase screening channels 1132 with different widths, a filtration hierarchy from sparse to dense is formed. This hierarchical filtration strategy further reduces the risk of clogging, alleviates the pressure concentration on a single filtration surface, and can further adjust the flow rate of the spacer oil to match the cell suspension. It is particularly suitable for high-frequency (>1000 Hz) droplet generation scenarios, significantly improving the stability and reliability of the chip during long-term operation.

[0030] This embodiment also provides a method for fabricating the droplet microfluidic chip based on a microfiltration array, comprising: Step 01: Prepare a PDMS layer using soft lithography; wherein the preparation method of the oil phase filtration structure and the water phase filtration structure includes: fabricating an oil phase filtration structure mold and a water phase filtration structure mold on a silicon wafer using SU-8, and forming the oil phase filtration structure and the water phase filtration structure after development on the PDMS layer; Step 02: The PDMS layer and the glass sheet are bonded together by oxygen plasma treatment to form the droplet microfluidic chip.

[0031] Specifically, the oil phase filtration structure mold and the water phase filtration structure are patterned using drawing software during the chip design stage and transferred to the SU-8 mold using standard photolithography. Finally, they are molded into the microfluidic chip using PDMS replication technology, without the need for additional assembly steps, and have high integration and good reproducibility.

[0032] Microscopic images of the aqueous sample introduction area of ​​the droplet microfluidic chip prepared by the above method are shown below. Figure 4 As shown, the actual morphology of the micron-scale filter structure array can be clearly observed under a microscope. This structure achieves physical interception of large particulate impurities and cell clusters in the cell suspension through a regular geometric design, while single cells can smoothly pass through the filtration zone and enter the droplet generation zone. The chip was tested in a 293T cell system (cDMEM culture medium, HFE-7500 fluorinated oil oil), confirming that it did not experience clogging issues after continuous operation for more than 30 minutes (>1000Hz droplet frequency), and the filter structure showed no deformation or detachment, demonstrating good stability. Figure 5 Microscopic images of the chip's aqueous sample introduction area during a real-world experiment using 293T cell suspension are shown. The images reveal an orderly array of micron-sized aqueous micropillars, with the aqueous screening channels having a diameter of approximately 40 µm. After cell fluid injection, this structure successfully intercepted impurities in the liquid. Numerous micron-sized particles were observed to be blocked before entering the subsequent channels, while the cell suspension passed through smoothly without clogging, effectively protecting the unobstructed flow of the droplet generation area. This image confirms the functionality and effectiveness of the filtration structure under actual operating conditions. Figure 6 Images show experiments conducted using a conventional chip without an integrated filter structure and 293T cell suspension. It is evident that impurities in the liquid directly enter the main channel and adhere to or remain on the channel walls, posing a significant risk of blockage. Two blockages occurred within just 15 minutes of testing. In practical use, this structure is prone to causing abnormal droplet formation, affecting normal chip operation and cell encapsulation efficiency.

[0033] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope of the present invention.

Claims

1. A droplet microfluidic chip based on a microfiltration array, comprising a PDMS layer sealed to a substrate, characterized in that, The PDMS layer is provided with an oil phase injection area (11), an oil phase introduction channel (12) connected to the oil phase injection area (11), an aqueous phase injection area (21), an aqueous phase introduction channel (22) connected to the aqueous phase injection area (21), a droplet generation area (31), a confluence channel (32) connected to the droplet generation area (31), and a droplet collection port (33) connected to the confluence channel (32); the oil phase introduction channel (12) and the aqueous phase introduction channel (22) are connected and converge in the droplet generation area (31); the aqueous phase injection area (21) includes an aqueous phase inlet (211), an aqueous phase outlet (212), and a collection port (33) provided at the aqueous phase inlet (211) and the aqueous phase outlet (212). A water phase filtration structure (213) between 212); the water phase inlet (211), water phase outlet (212), and water phase inlet channel (22) are connected in sequence; the arrangement range of the water phase filtration structure (213) covers at least part of the flow channel cross section of the water phase injection area (21); the water phase filtration structure (213) includes multiple regularly arranged water phase micro columns (2131) and water phase screening channels (2132) located between the water phase micro columns (2131); the diameter D of the water phase screening channel (2132) is the vertical straight-line distance between the side walls of two adjacent water phase micro columns (2131), which is not less than the diameter of the target single cell and not greater than the minimum feature size of the target interceptor.

2. The droplet microfluidic chip based on a microfiltration array according to claim 1, characterized in that, The aqueous micro-column (2131) has a square cross-section and the diagonal of the cross-section is parallel or perpendicular to the shortest flow path of the aqueous sample injection area (21). The shortest flow path is a straight line between the aqueous inlet (211) and the aqueous outlet (212).

3. A droplet microfluidic chip based on a microfiltration array according to claim 1, characterized in that, The diameter D of the aqueous phase screening channel (2132) is 10~40 μm.

4. A droplet microfluidic chip based on a microfiltration array according to claim 2, characterized in that, The cross-sectional side length of the aqueous micro-column (2131) is 50~70 μm.

5. A droplet microfluidic chip based on a microfiltration array according to claim 1, characterized in that, The oil phase injection area (11) includes an oil phase inlet (111), an oil phase outlet (112), and an oil phase filter structure (113) disposed between the oil phase inlet (111) and the oil phase outlet (112); the oil phase inlet (111), the oil phase outlet (112), and the oil phase inlet channel (12) are connected in sequence; the arrangement range of the oil phase filter structure (113) covers at least part of the flow channel cross section of the oil phase injection area (11); the oil phase filter structure (113) includes a plurality of regularly arranged oil phase micro columns (1131) and an oil phase screening channel (1132) located between the oil phase micro columns (1131); the oil phase micro columns (1131) have a square cross section and the diagonal of the cross section is parallel or perpendicular to the shortest flow path direction of the oil phase injection area (11), and the shortest flow path is a straight path between the oil phase inlet (111) and the oil phase outlet (112).

6. A droplet microfluidic chip based on a microfiltration array according to claim 5, characterized in that, The oil phase micro-column (1131) includes an oil phase type I column (1131A) and an oil phase type II column (1131B); the cross-sectional area of ​​the oil phase type I column (1131A) is larger than the cross-sectional area of ​​the oil phase type II column (1131B).

7. A droplet microfluidic chip based on a microfiltration array according to claim 6, characterized in that, The oil phase type I columns (1131A) are arranged at equal intervals on one side relatively close to the oil phase inlet (111); the oil phase type II columns (1131B) are arranged at equal intervals on one side relatively close to the oil phase outlet (112).

8. A droplet microfluidic chip based on a microfiltration array according to claim 6, characterized in that, The edge spacing between any two adjacent oil phase type I columns (1131A) is greater than the edge spacing between the oil phase type II columns (1131B).

9. A droplet microfluidic chip based on a microfiltration array according to claim 6, characterized in that, The side length of the cross-section of the oil phase type I column (1131A) is 1.5 to 2 times that of the side length of the cross-section of the oil phase type II column (1131B).

10. A method for fabricating a droplet microfluidic chip based on a microfilter array as described in any one of claims 1 to 9, characterized in that, include: Step 01: Prepare the PDMS layer using soft lithography. The preparation methods of the oil phase filtration structure and the water phase filtration structure include: fabricating the oil phase filtration structure mold and the water phase filtration structure mold on the silicon wafer using SU-8, and forming the oil phase filtration structure and the water phase filtration structure after development on the PDMS layer. Step 02: The PDMS layer and the glass sheet are bonded together by oxygen plasma treatment to form the droplet microfluidic chip.

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