Cell capture and pairing microfluidic chip

Abstract

The application provides a cell array capture and pairing microfluidic chip, which comprises cell pair units arranged in an array, each cell pair unit being provided with a large micro-trap, three small micro-traps arranged in a sinking interval in the large micro-trap, three groups of electrode pairs and shielding electrodes between adjacent small micro-traps, and the small micro-traps being located between corresponding electrode pairs. The cell capture and pairing microfluidic chip can capture three kinds of cells on the chip in sequence, realize large-scale triple cell array, and arrange the three kinds of cells in the array in one of each kind of cell and three cells as a group for pairing, so that great convenience and possibility are provided for studying three-cell paracrine and other intercellular interactions or cell fusion.

Description

Technical Field

[0001] This invention relates to the technical field of micro-nano fabrication and life sciences, and in particular to a cell capture and pairing microfluidic chip. Background Technology

[0002] Through long-term research, it has been discovered that even cells of the same species exhibit individual differences in cell biology research; this property is known as cellular heterogeneity. In traditional biological cell experiments, population analysis can mask the differences between individual cells. Therefore, studying cellular heterogeneity at the single-cell level allows for a clearer understanding of cells and the study of the life processes of various organisms. For example, cancer is a highly heterogeneous disease; studying the heterogeneity of cancer cells can lead to a better understanding of the occurrence and development of cancer, enabling more precise treatment for patients.

[0003] Cellular heterogeneity is widespread in various cellular life processes such as differentiation, immunity, and metabolism. Single-cell analysis can avoid the problem of individual differences being masked in averaging experiments, thus enabling the study of cellular heterogeneity. Cells themselves are living units containing a wealth of information. Current research on cells includes fields such as genomics, transcriptomics, metabolomics, endocrinology, and cell-cell interactions. As cell research deepens, the wide range of research subjects within cells demands high-throughput analytical capabilities for single-cell analysis.

[0004] Microfluidic chips are experimental chips capable of manipulating and analyzing trace amounts of fluids on an area of ​​a few square centimeters or even smaller. Using microfluidic technology for biological or chemical analysis can reduce the research scale, decrease experimental errors, improve detection sensitivity, and reduce reagent or sample loss. For single-cell and even subcellular level analysis, microfluidic chips are an ideal experimental platform.

[0005] For single-cell analysis on microfluidic chips, cell capture is the first step. Currently, there are two main methods for single-cell capture on microfluidic chips: active and passive. Passive methods include microstructure filtration and fluid shear force methods, which work by capturing cells to specific regions based on the differences in microstructures or cells under different hydrodynamic conditions. Active methods include dielectrophoresis, optical tweezers, acoustic tweezers, magnetic bead sorting, and antigen-antibody labeling, which control cell movement by applying external forces or biomarkers. Passive cell capture methods have low sensitivity and require a large number of cells; the cells may also be deformed or damaged due to the special microstructures. Active cell capture methods often require the addition of specific markers, which can affect downstream single-cell analysis.

[0006] Dielectrophoresis (DEP) is a technique developed based on electrophoresis. It is a special type of electrophoresis, also known as two-dimensional electrophoresis. Dielectrophoresis refers to the phenomenon of neutral particles moving relative to a liquid in a non-uniform electric field. Using dielectrophoresis, we can design different structures and apply an external high-frequency electric field to make cells move directionally within a microfluidic chip. This is a high-throughput, label-free, and non-contact cell capture method.

[0007] Currently, an increasing number of studies are arranging cells into single-cell arrays on microfluidic chips for single-cell analysis. Forming multiple cell arrays on a chip allows for multiplex analysis of various cell types. Parallel experiments enhance the control and avoid experimental errors caused by differences between chips when samples are loaded onto multiple chips.

[0008] Currently, there are not many methods to achieve multiple cell arrays, so it is necessary to develop a microfluidic chip with multiple cell arrays. Summary of the Invention

[0009] The purpose of this invention is to provide a cell capture and pairing microfluidic chip that can capture three types of cells and provides great possibilities for studying the heterogeneity of the same type of cells.

[0010] The present invention provides a cell capture and pairing microfluidic chip, which includes an array of cell pair units. Each cell pair unit has a large micro-trap, three small micro-traps located in the large micro-trap and spaced downwards, three sets of electrode pairs, and a shielding electrode located between adjacent small micro-traps. The small micro-traps are located between corresponding electrode pairs.

[0011] Furthermore, the large micro-well is elliptical in shape.

[0012] Furthermore, the cell capture and pairing microfluidic chip also includes buffer regions located on both sides of the cell capture region.

[0013] Furthermore, the three micro-wells are arranged in sequence as a first micro-well, a second micro-well, and a third micro-well; the shielding electrode includes a first shielding electrode located between the first and second micro-wells, and a second shielding electrode located between the second and third micro-wells.

[0014] Furthermore, the three sets of electrode pairs are a first set of electrode pairs, a second set of electrode pairs, and a third set of electrode pairs. The first set of electrode pairs includes a first upper electrode and a first lower electrode located on both sides of the first micro-well, respectively. The second set of electrode pairs includes a second upper electrode and a second lower electrode located on both sides of the second micro-well, respectively. The third set of electrode pairs includes a third upper electrode and a third lower electrode located on both sides of the third micro-well, respectively. The first shielding electrode is located between the first set of electrode pairs and the second set of electrode pairs, and the second shielding electrode is located between the second set of electrode pairs and the third set of electrode pairs.

[0015] Furthermore, the three sets of electrode pairs and the two shielding electrodes constitute an electrode unit, and each electrode unit has multiple large micro-wells in the lateral direction.

[0016] Furthermore, the cell capture and pairing microfluidic chip has N electrode units in the longitudinal direction, and the N electrode units form an electrode pair array, where N is a positive integer; the cell capture and pairing microfluidic chip also includes four sets of metal structure pairs located on both sides of it, the four sets of metal structure pairs include a first set of metal structure pairs connected to the first set of electrode pairs, a second set of metal structure pairs connected to the two shielding electrodes, a third set of metal structure pairs connected to the second set of electrode pairs, and a fourth set of metal structure pairs connected to the third set of electrode pairs.

[0017] Furthermore, the cell capture and pairing microfluidic chip also includes a glass substrate, on which the electrode units are located.

[0018] Furthermore, the large and small micro-trap structures, nested in size, serve as a means for cell fixation.

[0019] Furthermore, the large micro-well is encapsulated with an encapsulation material to encapsulate the flow channel.

[0020] The cell capture and pairing microfluidic chip of this invention can capture three types of cells sequentially on the chip to achieve a large-scale triple cell array. In the array, one of each type of cell is selected and arranged in pairs of three, which can provide great convenience and possibilities for studying intercellular interactions such as paracrine function or cell fusion. Attached Figure Description

[0021] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0022] Figure 1 This is a front view of the microfluidic chip in an embodiment of the present invention;

[0023] Figure 2 This is a schematic diagram of the structure of a single cell pair unit within a microfluidic chip according to an embodiment of the present invention;

[0024] Figure 3 This is a partial schematic diagram of a microfluidic chip according to an embodiment of the present invention;

[0025] Figure 4 This is a schematic diagram of steps S1 to S5 of the manufacturing process of a microfluidic chip according to an embodiment of the present invention;

[0026] Figure 5 This is a schematic diagram of steps S6 to S8 of the manufacturing process of the microfluidic chip according to an embodiment of the present invention.

[0027] Figure 6 This is a top view of the manufacturing process of a microfluidic chip according to an embodiment of the present invention, with reserved through holes;

[0028] Figure 7 This is a top view showing the manufacturing process of a microfluidic chip according to an embodiment of the present invention, with copper exposed.

[0029] Figure 8 This is a schematic diagram of a cell experiment using a microfluidic chip according to an embodiment of the present invention;

[0030] Figure 9 This is a schematic diagram comparing the natural light and fluorescence synthesis of a triple cell array captured by a microfluidic chip according to an embodiment of the present invention. Detailed Implementation

[0031] Embodiments of the present invention are described in detail below, examples of which are illustrated in the accompanying drawings, wherein the same or similar reference numerals denote the same or similar elements or elements having the same or similar functions throughout. The embodiments described below with reference to the accompanying drawings are exemplary and intended to explain the present invention, and should not be construed as limiting the present invention.

[0032] In the description of this invention, it should be understood that the terms "length," "width," "upper," "lower," "front," "rear," "left," "right," "vertical," "horizontal," "top," "bottom," "inner," and "outer," etc., indicating orientation or positional relationships, are based on the orientation or positional relationships shown in the accompanying drawings and are only for the convenience of describing the invention and simplifying the description, and do not indicate or imply that the device or element referred to must have a specific orientation, or be constructed and operated in a specific orientation, and therefore should not be construed as a limitation of the invention. Furthermore, in the description of this invention, "a plurality of" means two or more, unless otherwise explicitly specified.

[0033] This invention discloses a cell capture and pairing microfluidic chip, such as... Figure 1 and Figure 2As shown, it includes a glass substrate 100, an electrode layer on the glass substrate 100, a small micro-well, a large micro-well 10 and an encapsulation layer, wherein the electrode layer includes three sets of electrode pairs and two shielding electrodes, wherein the three sets of electrode pairs are arranged sequentially, the small micro-well is located between each set of electrode pairs, and each shielding electrode is located between adjacent sets of electrode pairs.

[0034] A schematic diagram of the internal macroscopic structure of the cell capture and pairing microfluidic chip is shown below. Figure 3 As shown, it includes a buffer region 1 and a cell capture region 2, wherein the width of the buffer region 1 is 4 mm to 6 mm (preferably 5 mm), and the length and width of the cell capture region 2 are 0.3 cm to 0.7 cm (preferably 0.5 cm) and 0.8 cm to 1.2 cm (preferably 1 cm), respectively.

[0035] The buffer region serves as a pre- and post-positioned area for cell capture and pairing microfluidic chips. The buffer region is located on both sides of the cell capture region 2 to ensure a stable injection flow rate.

[0036] The cell capture area 2 contains an array of M*N cell pairs (M and N are both positive integers, M can be 72 and N can be 28). The vertical spacing of each cell pair is 0.3mm to 0.6mm (preferably 0.4mm), and the horizontal spacing of each cell pair is 0.1mm to 0.3mm (preferably 0.2mm).

[0037] Each cell pair unit contains an elliptical large micro-trap 10, three small micro-traps 11, 12, and 13 located within the large micro-trap 10 and spaced downwards, three sets of electrode pairs, and shielding electrodes located between adjacent small micro-traps. Cells are captured by the three small micro-traps.

[0038] The large micro-trap 10 has a length of 250 μm to 270 μm (preferably 260 μm), a width of 110 μm to 130 μm (preferably 120 μm), and a depth of 20 μm to 35 μm (preferably 27 μm). The small micro-trap has a diameter of 13 μm to 17 μm (preferably 15 μm) and a depth of 1 μm to 6 μm (preferably 1.2 μm). The size of the micro-trap is adjusted according to the average particle size of the experimental cell species. In this embodiment, the size is determined using human lung cancer epithelial cells A549 according to the present invention.

[0039] The three micro-wells are arranged in sequence as the first micro-well 11, the second micro-well 12, and the third micro-well 13.

[0040] The shielding electrode includes a first shielding electrode 51 located between the first micro-well 11 and the second micro-well 12, and a second shielding electrode 52 located between the second micro-well 12 and the third micro-well 13. The shielding electrode can avoid the mutual inductance effect between each pair of electrodes when the electrodes are energized to generate dielectric electrophoretic force to capture cells (i.e., the situation where cells are captured by the induced electric field generated in adjacent unenergized electrode pairs after the electrode pair is energized).

[0041] The three electrode pairs are designated as a first electrode pair 20, a second electrode pair 30, and a third electrode pair 40. The first electrode pair 20 includes a first upper electrode 21 and a first lower electrode 22 located on both sides of the first micro-well 11, respectively. The second electrode pair 30 includes a second upper electrode 31 and a second lower electrode 32 located on both sides of the second micro-well 12, respectively. The third electrode pair 40 includes a third upper electrode 41 and a third lower electrode 42 located on both sides of the third micro-well 13, respectively. The spacing between each electrode pair is 4 μm to 8 μm (preferably 6 μm).

[0042] An electrode unit consists of three sets of electrode pairs and two shielding electrodes, i.e., eight parallel electrodes. Each electrode unit has M large micro-wells 10 (specifically 72) in the lateral direction. The length of a single electrode in each electrode unit is 2.3 cm to 2.6 cm (preferably 2.5 cm), and the spacing between two adjacent electrode units is 4 μm to 8 μm (preferably 6 μm).

[0043] The embodiments of this invention use human lung cancer epithelial cells A549. In the embodiments, the width of a single electrode is 7 micrometers, and the distance between the shielding electrode and the corresponding electrode is 6 micrometers.

[0044] The human lung cancer cell line A549 was established in 1972 by Giard DJ through lung cancer tissue transplantation, originating from a 58-year-old Caucasian male. The A549 cell line can synthesize lecithin rich in unsaturated fatty acids via the cytidine diphosphatidylcholine pathway; it is keratin-positive. The cell capture and pairing microfluidic chip has N electrode units (up to 28) in the longitudinal direction, forming an electrode pair array.

[0045] The cell capture and pairing microfluidic chip also includes four sets of metal structure pairs located on both sides of the chip. Specifically, the four sets of metal structure pairs include a first set of metal structure pairs connected to the first set of electrode pairs 20, a second set of metal structure pairs connected to the shielding electrode, a third set of metal structure pairs connected to the second set of electrode pairs 30, and a fourth set of metal structure pairs connected to the third set of electrode pairs 40.

[0046] like Figure 7As shown, the first group of metal structure pairs includes a first left metal structure 611 located on the left side of the cell capture and pairing microfluidic chip and connected to the first upper electrode 21, and a first right metal structure 612 located on the right side of the cell capture and pairing microfluidic chip and connected to the first lower electrode 22; the second group of metal structure pairs includes a second left metal structure 621 located on the left side of the cell capture and pairing microfluidic chip and connected to the first shielding electrode 51, and a second right metal structure 622 located on the right side of the cell capture and pairing microfluidic chip and connected to the second shielding electrode 52; the third group of metal structure pairs includes a third left metal structure 631 located on the left side of the cell capture and pairing microfluidic chip and connected to the second upper electrode 31, and a third right metal structure 632 located on the right side of the cell capture and pairing microfluidic chip and connected to the second lower electrode 32; the fourth group of metal structure pairs includes a third left metal structure 641 located on the left side of the cell capture and pairing microfluidic chip and connected to the third upper electrode 41, and a fourth right metal structure 642 located on the right side of the cell capture and pairing microfluidic chip and connected to the third lower electrode 42.

[0047] N electrode units form an integrated control structure through four sets of metal structures on both sides, that is, the metal structures on both sides connect the electrode pairs array; one of the metal structure pairs connected to the three sets of electrode pairs is made of ITO. In this embodiment, the first set of metal structure pairs is made of ITO, and the two sets of metal structure pairs connected to the other two sets of electrode pairs are made of copper or other conductive metals (copper is selected in this example), which can save costs.

[0048] like Figure 3 As shown, the large micro-trap 10 and the small micro-traps 11, 12, and 13 form a nested micro-trap structure for cell fixation. During operation, cells enter the cell capture region 2 from the inlet. Electrode units in the same layer can be energized simultaneously to generate positive permittivity electrophoresis force to capture cells and pull them down into the small micro-traps 11, 12, and 13, forming a cell array. Cells that are not captured leave the cell capture region 2 from the outlet.

[0049] Because PDMS encapsulation material has good biocompatibility and is easy to observe, PDMS encapsulation material is used to encapsulate the flow channels in the large micro-trap 10, and the height of the flow channels is 45μm to 65μm (preferably 55μm).

[0050] This invention also discloses a method for manufacturing a cell capture and pairing microfluidic chip, comprising the following steps:

[0051] S1: As Figure 4 As shown in part (a), an ITO electrode layer 101 is deposited on a glass substrate 100, and then the glass substrate 100 with the ITO electrode layer 20 is cleaned to ensure that the surface of the ITO electrode layer 101 remains clean and has a certain degree of adhesion.

[0052] For step S1, the specific method for cleaning the glass substrate 100 with the ITO electrode layer 101 is as follows: A square ITO electrode layer 101 with a sheet resistance of 20Ω and a side length of 8cm and the glass substrate 100 are placed in a cleaning rack and immersed in a mixture of hydrogen peroxide, ammonia, and deionized water. The ratio of hydrogen peroxide, ammonia, and deionized water is 1:1:6, respectively. The mixture is then heated in a 70°C water bath for 40 minutes before being removed. After removal, the surface of the ITO electrode layer 101 is sprayed with a water gun, and the moisture is dried with nitrogen before being placed in a silicon wafer cassette for storage. The purpose of surface cleaning is to remove various particles and impurities from the surface of the ITO electrode layer 101 and to increase the surface energy to ensure a smooth and sufficiently adhesive photoresist film formation during subsequent spin-coating.

[0053] S2: After cleaning, the surface of the glass substrate 100 of the ITO electrode layer 101 is irradiated with ultraviolet (UV) for 30 minutes to remove impurities and dangling bonds on the surface of the ITO electrode layer 101 and at the same time further increase the surface energy of the ITO electrode layer 101 so that the photoresist has sufficient adhesion to adhere to the surface of the ITO electrode layer 101.

[0054] S3: As Figure 4 As shown in sections (b) to (d), positive photoresist 102 is spin-coated onto the ITO electrode layer 101 to create a photoresist pattern of a certain height in the shape of an electrode as a wet etching protective layer.

[0055] Before spin-coating the positive photoresist 102, the glass substrate 100 with the ITO electrode layer 101 is first baked on a hot plate at 180°C for 20 minutes to remove residual moisture on the surface of the ITO electrode layer 101. Then, the positive photoresist 102 is spin-coated in a spin coater with the spin parameters in Table 1.

[0056] Table 1 Spin coating parameters for positive photoresist

[0057]

[0058] S4: As Figure 4 As shown in parts (c) and (f), the pattern formed by S3 and the mask 200 are placed together in a photolithography machine (not shown) for exposure to form the electrode layer pattern 103;

[0059] After spin coating, the ITO electrode layer 102 is first pre-baked at 95°C for 2 minutes on a hot plate; then the ITO electrode layer 102 is fixed onto an alignment sheet (not shown), as follows. Figure 3 As shown in sections (c) and (d), the ITO electrode layer 102 and the mask 200 are placed together in the lithography machine and adjusted to a fixed position for exposure, with an exposure dose of 40 mJ / cm². 2 ;like Figure 4As shown in sections (e) and (f), after exposure, the image is immersed in a suitable amount of developer for 30 seconds to remove the positive photoresist 103. It is then rinsed with deionized water and dried with nitrogen. Finally, it is heated on a hot plate at 150°C for 2 minutes to harden the film, forming the electrode layer pattern 103. The electrode layer pattern 103 specifically includes three sets of electrode pairs and two shielding electrodes. The electrode layer pattern 103 is detailed as follows: Figure 1 and Figure 2 As shown.

[0060] Specifically, after forming the electrode layer pattern 103, wet etching is performed using an ITO etching solution. The purpose is to cleanly etch the ITO electrode layer 101 in areas without photoresist protection, leaving the desired electrode layer pattern 103. The photoresist is removed by washing with acetone to obtain the electrode layer pattern. The specific method is as follows: First, the ITO etching solution is heated to 35°C in a water bath. Then, the electrode layer pattern is immersed in the ITO etching solution for 150 seconds. Ideally, it should be quickly transferred to a beaker containing deionized water to dilute the etching solution. Finally, it is rinsed with water, dried, and stored.

[0061] After etching, the positive photoresist 102 needs to be removed to obtain the bare electrodes. This can be done by sonicating in acetone solution for 1 minute followed by direct air drying. After obtaining the electrode layer, the resistance between each integrated electrode pair needs to be tested with a multimeter. If there is continuity between each electrode pair, the electrode fabrication has failed; if there is no continuity, it can be used for the next step of manufacturing. Before the next photolithography step, the sample still needs to be irradiated with ultraviolet light for 30 minutes and baked at 180°C for 20 minutes to ensure sufficient adhesion between the photoresist and the sample.

[0062] S5: As Figure 4 As shown in section (g), a first negative photoresist 104 is spin-coated onto the electrode layer pattern 103 using a hard contact photolithography process, as follows: Figure 1 and 2 As shown, micro-trapholes 11, 12, and 13 were fabricated;

[0063] Specifically, firstly, the first negative photoresist 104 is spin-coated onto the electrode layer pattern 103 according to Table 2, and then pre-baked on a hot plate at 95°C for 5 minutes. After pre-baking, it needs to be left to stand for 20 minutes to allow the first negative photoresist 104 to dry completely. Because the exposure window of the micro-well is relatively small, to prevent unnecessary overexposure caused by ultraviolet light reflection within the glass substrate 100, a layer of black wallpaper (not shown) is adhered to the back of the glass substrate 100 for light absorption; Figure 4 As shown in section (g), the mask 105 with circular structures of different sizes and the electrode layer pattern with the first negative photoresist 104 are then placed into the photolithography machine sequentially. Alignment is performed using alignment marks on the mask 105 and the electrode layer pattern 103, followed by hard-contact exposure at an exposure dose of 150 mJ / cm.2 When ultraviolet light irradiates the unshielded first negative photoresist 104, the first negative photoresist 104 generates acid, causing polymer cross-linking, which can remain in the developer. After exposure, it is placed on a hot plate at 95°C for 2 minutes for post-baking, and then allowed to cool to room temperature. After cooling, development is performed by immersing the sample in the developer, during which the container needs to be shaken evenly to allow the developer to flow. After 60 seconds, it is removed, fixed with isopropanol, and then dried with nitrogen. Finally, it is heated on a hot plate at 150°C for 2 minutes to harden the film, so that the first negative photoresist 104 is completely set and stabilized.

[0064] Table 2 Spin-coating parameters of the first negative photoresist for the microwell layer

[0065]

[0066] The thickness of the microwell layer fabricated with the first negative photoresist according to the rotation speeds in Table 2 is approximately 1.2 micrometers.

[0067] After S5 was completed, the cell capture and paired microfluidic chip was placed in a step tester for testing. The test showed that the thickness of the micro-trap was about 1.2 μm. The pore size of the micro-trap was measured in an optical microscope to see if it met the expectations.

[0068] S6: As Figure 5 As shown in section (a), each electrode pair is integrated using magnetron sputtering copper plating technology.

[0069] Specifically, in the manufacturing process of micro-traplets, such as Figure 6 As shown, vias 201 are pre-drilled at the ends of these electrodes, meaning there is no first negative photoresist 104 at the electrode ends, allowing copper to grow directly on the ITO electrodes. To protect the micro-well region and the areas already integrated with ITO from being affected, high-temperature tape is applied to unnecessary areas of the sample to block copper growth, leaving only exposed areas. Figure 7 The copper (metal structure pairs) in the sputtering process are 621, 622, 631, 632, 641, and 642. The sputtering process is carried out in an Ar atmosphere with a gas flow rate of 50 sccm and a sputtering power of 100 W, which can sputter a layer of copper (metal structure pairs) 621, 622, 631, 632, 641, and 642 of about 370 nm. The thickness requirement of the copper is not particularly precise, only ensuring conductivity of the parts to be integrated is required.

[0070] After sputtering is completed, the three sets of electrode pairs are tested for conductivity again to ensure that each electrode pair is not conductive before proceeding to the next step. This is to ensure the smooth generation of dielectric electrophoresis force in subsequent cell experiments.

[0071] S7: As Figure 5As shown in parts (b) and (c), a large microwell 10 is fabricated by performing negative photoresist lithography again on the small microwell using hard contact lithography.

[0072] Specifically, according to Table 3, the second negative photoresist was spin-coated onto the micro-well, then pre-baked at 65°C for 1 minute on a hot plate, and then transferred to a 95°C hot plate for another 5 minutes of baking. After pre-baking, the sample needed to be left to stand for 20 minutes to allow the photoresist to dry completely. Similarly, a layer of black wallpaper was adhered to the back of the glass substrate 100 for light absorption. Next, the mask with the micro-well structure and the sample were placed sequentially into the lithography machine, aligned using alignment marks on the mask and electrodes, and then subjected to hard-contact exposure at an exposure dose of 180 mJ / cm². 2 After exposure, the sample was placed on a hot plate at 65°C for 1 minute for post-baking, and then transferred to a hot plate at 95°C for another 6 minutes for baking. After cooling to room temperature, development was performed by immersing the sample in SU-8 developer for 60 seconds. The sample was then removed, fixed with isopropanol, and dried with nitrogen. Finally, the sample was heated on a hot plate at 150°C for 2 minutes to harden the film, ensuring that the second negative photoresist was completely set and stabilized. After the above steps were completed, a large micro-well 10 with a thickness of approximately 25 μm was obtained. After microscopic inspection under an optical microscope to verify that the micro-well size met expectations, the fabrication of the chip's main microstructure was completed.

[0073] Table 3 Spin-coating parameters of the second negative photoresist for the large micro-well.

[0074]

[0075] S8: As Figure 5 As shown in section (d), PDMS (Polydimethylsiloxane) material forms an encapsulation layer 301 for encapsulation, thereby creating a sealed space for fluid to pass through.

[0076] First, the silicon wafer is ultrasonically treated with acetone, isopropanol, and water solutions sequentially for 2 minutes each. Then, it is irradiated with ultraviolet light for 30 minutes and baked at 180°C for 20 minutes to ensure sufficient adhesion between the photoresist and the electrode layer pattern. After the silicon wafer returns to room temperature, the third negative photoresist (not shown) is spin-coated onto the wafer according to Table 4. It is then baked on a 65°C hot plate for 3 minutes and 30 seconds, followed by baking on a 95°C hot plate for 9 minutes and 30 seconds. After the initial baking, the wafer is allowed to stand for 30 minutes to allow the third negative photoresist to dry completely. Finally, ultraviolet hard contact exposure is performed using a film mask at an exposure dose of 200 mJ / cm². 2After exposure, post-baking is performed, first baking on a hot plate at 65℃ for 1 min 30 s, then baking on a hot plate at 95℃ for 6 min 30 s. After post-baking, it is allowed to stand at room temperature, then developed in developer for 7 minutes, followed by rinsing with isopropanol and drying. Finally, after development, it is hardened on a hot plate at 150℃ for 10 min to complete the photolithography process. Then, it is treated with FDTS (1H,1H,2H,2H-perfluorodecyltrichlorosilane) to prevent the encapsulation layer 301 from sticking to the template during curing, thus obtaining a template for PDMS casting. The height of the flow channel inside the encapsulation layer 301 is approximately 55 μm, as determined by a step meter test.

[0077] Table 4 Spin coating parameters of the third negative photoresist

[0078]

[0079] PDMS and its curing agent are mixed at a mass ratio of 10:1 and stirred for 5 minutes to ensure thorough mixing. Then, the mixture is placed in a vacuum dish and vacuumed to remove air bubbles. After all air bubbles are removed, the prepared PDMS is cast into a soft photolithography mold with a channel structure. After baking in an oven at 65°C for 3 hours, the PDMS solidifies, is removed, demolded, and cut to the appropriate size. Then, holes are drilled at the liquid inlet and outlet and a conduit is connected.

[0080] The prepared PDMS encapsulation layer 301 is placed on the microfluidic chip, and oxygen plasma (O2 Plasma) bonding is performed on it for 1 minute in a plasma cleaner, thus completing the encapsulation of the microfluidic chip. The microfluidic chip is formed through the above steps.

[0081] After encapsulation, the electrodes are attached with conductive tape so that the signal generator can clamp them to apply the signal. Finally, the microfluidic chip is fixed by a fixture (not shown).

[0082] The general procedure for cell experiments involving cell capture and pairing with microfluidic chips is as follows: Figure 8As shown: In the cell experiment, shielding electrodes 51 and 52 are always grounded. A cell suspension containing a low-conductivity buffer is introduced at a pulsed flow rate. A high-frequency alternating current is applied to the first electrode pair array via a signal generator, while the remaining electrode pairs are grounded. This generates a positive permittivity electrophoretic force to capture the first type of cells into the micro-trap. Then, the low-conductivity buffer is introduced to clear any uncaptured cells, forming a single-cell array. Maintaining the high-frequency alternating current on the first electrode pair array fixes the first type of cells. Then, a second cell suspension containing a low-conductivity buffer is introduced at a pulsed flow rate. The second electrode pair array is energized, while the remaining electrodes are grounded, capturing cells into their corresponding micro-traps. The same buffer as before is introduced to clear any uncaptured cells, forming a double array of captured cells. Then, while maintaining the energization of the two electrode pairs that have already captured cells, the last electrode pair array is energized, with the shielding wire grounded. Ultimately, the three types of cells can form a triple-cell array on the chip.

[0083] Human lung cancer epithelial cells A549 were used in the experiment to verify the role of cell capture and pairing with microfluidic chips. The experiment required the construction of a microfluidic experimental platform, including a fluorescence macroscopic zoom microscope and display screen, two syringe pumps, and three signal generators. The selected experimental cells were then cultured autonomously and stained with fluorescence.

[0084] During the experiment, a micropump was equipped with a low conductivity buffer solution (hereinafter referred to as the first micropump), and a micropump was equipped with a cell suspension containing a low conductivity buffer solution (hereinafter referred to as the second micropump). The two liquids were connected to the chip through a micro three-way tube, and the displacement of the micropump was then controlled by the program and software to inject the sample.

[0085] First, a pure buffer solution is uniformly introduced using the first micropump to ensure that the liquid inside the chip is free of air bubbles. Then, the micropump is stopped, and the second micropump is started to pulse-feed the first cell suspension into the fabricated microfluidic chip. Once the cells have entered the working area, a high-frequency AC signal (V0) is applied to the first electrode array via a signal generator. PP =18V, the fluid flow rate is faster at the first point, so the highest voltage is applied to ensure stable grasping), the other electrodes are grounded, and the first type of cell is captured to form a single cell array.

[0086] Then, the pulse displacement of the second micropump is stopped, maintaining the electrical parameters of the first electrode pair array. The first micropump is then activated to purge excess uncaptured suspended cells with buffer solution. The cells mounted on the second micropump are replaced, and the second micropump is activated again. When the second type of cells enters the capture area, a high-frequency alternating current (V) is applied to the second electrode pair array. PP =12V, the flow rate at the second point is relatively stable, reducing the voltage reduces the influence of mutual inductance on the other electrodes), the other electrodes are grounded, and the second type of suspended cells are captured.

[0087] Next, the pulse displacement of the second micropump was stopped, maintaining the electrical parameters of the first and second electrode pairs arrays. The first micropump was then activated to purge excess uncaptured suspended cells with buffer solution. The cells mounted on the second micropump were replaced, and the second micropump was activated again. When the third type of cell entered the capture area, a high-frequency alternating current (V0.05) was applied to the third electrode pair array. PP =10V, the liquid flow rate is the slowest at the third point, so the minimum voltage is applied. If the voltage is too high, it will cause multiple cells to be trapped in a micro-trap, which is not conducive to the experiment. Capture suspended cells.

[0088] Finally, stop the pulse displacement of the second micropump, maintain the electrical parameters of the first, second, and third electrode arrays, start the first micropump to purge buffer to clear excess uncaptured suspended cells, and take a fluorescence field photograph for record-keeping and subsequent experimental analysis.

[0089] Through the above cell experiments, cells were stained with red, green, and blue colors to distinguish different cell arrays. Triple cell arrays were successfully captured on the chip. We randomly selected 7×8 cell units for observation. To facilitate observation, the images of the three fluorescence channels were synthesized. A comparison of the natural light field and the synthesized fluorescence image is shown below. Figure 9 As shown (scale bar is 200 micrometers).

[0090] It can be clearly seen that the cells are all located in the small micro-trap. Most cell pairs contain exactly three cells (one red, one green, and one blue) (considered as successful pairing). Based on a rough calculation of the successful pairing efficiency in the selected area, a total of 39 / 56 cell pairs were successfully paired, with a pairing success rate of approximately 70%. Therefore, this cell capture and pairing microfluidic chip has the capability to construct large-scale triple cell arrays, and the utility of this invention has achieved its intended purpose.

[0091] The cell capture and pairing microfluidic chip of this invention is a microfluidic chip that uses dielectrophoresis to capture triple cell arrays. By performing red, green and blue fluorescence staining on the cells, the array of red, green and blue cells arranged in groups of three can be clearly seen under a fluorescence microscope. This verifies that the cell capture and pairing microfluidic chip has achieved its design expectations. At the same time, the arrayed cell arrangement can provide a great possibility for studying the heterogeneity of the same type of cells.

[0092] This type of cell offers good controllability and manageability. The cell positions in the array are fixed and controllable, and batch operations and analyses of cells can be performed, thereby reducing the time and reagent costs associated with parallel operations and minimizing experimental errors.

[0093] The cell capture and pairing microfluidic chip of this invention can capture three types of cells sequentially on the chip to achieve a large-scale triple cell array. In the array, one of each type of cell is selected and arranged in pairs of three, which can provide great convenience and possibilities for studying intercellular interactions such as paracrine function or cell fusion.

[0094] The above description discloses only one preferred embodiment of the present invention, and should not be construed as limiting the scope of the present invention. Those skilled in the art will understand that all or part of the processes of the above embodiments can be implemented, and equivalent changes made in accordance with the claims of the present invention are still within the scope of the invention.

Claims

1. A cell capture and pairing microfluidic chip, characterized in that, It includes an array of cell pair units, each cell pair unit having a large micro-trap (10), three small micro-traps (11, 12, 13) located in the large micro-trap (10) and arranged at intervals, three sets of electrode pairs, and shielding electrodes located between adjacent small micro-traps, the small micro-traps being located between corresponding electrode pairs; The three micro-wells are arranged in sequence as a first micro-well (11), a second micro-well (12), and a third micro-well (13); the shielding electrode includes a first shielding electrode (51) located between the first micro-well (11) and the second micro-well (12), and a second shielding electrode (52) located between the second micro-well (12) and the third micro-well (13); The three sets of electrode pairs are the first set of electrode pairs (20), the second set of electrode pairs (30), and the third set of electrode pairs (40). The first set of electrode pairs (20) includes a first upper electrode (21) and a first lower electrode (22) located on both sides of the first micro-well (11); the second set of electrode pairs (30) includes a second upper electrode (31) and a second lower electrode (32) located on both sides of the second micro-well (12); the third set of electrode pairs (40) includes a third upper electrode (41) and a third lower electrode (42) located on both sides of the third micro-well (13); the first shielding electrode (51) is located between the first set of electrode pairs (20) and the second set of electrode pairs (30), and the second shielding electrode (52) is located between the second set of electrode pairs (30) and the third set of electrode pairs (40). The cell capture and pairing microfluidic chip forms a cell array according to the following process: The first shielding electrode 51 and the second shielding electrode 52 are always grounded. After a cell suspension containing a low conductivity buffer solution is introduced at a pulsed flow rate, a high-frequency alternating current is applied to the first set of electrode pairs by a signal generator. The remaining electrode pairs are grounded, generating a positive permittivity electrophoretic force to grab the first type of cell into the microtrap. Then, a low-conductivity buffer solution is introduced to clear the cells that were not captured, thus forming a single cell array. Maintaining the high-frequency alternating current on the first set of electrode pairs array keeps the first type of cell in place; Then, a second cell suspension containing a low conductivity buffer solution is introduced at a pulsed flow rate. The second set of electrode pairs is energized, and the other electrodes are grounded to capture cells into the corresponding microtrap. The same buffer solution as before was introduced to clear the cells that were not captured, and at this time the cells captured before and after formed a double array; Then, while keeping the two sets of electrodes that have captured cells powered on, power is supplied to the last set of electrode pairs array, and the shielding wire is grounded. The three types of cells eventually form a triple cell array on the chip. The cell capture and pairing microfluidic chip also includes buffer regions located on both sides of the cell capture region; Three sets of electrode pairs and two shielding electrodes constitute an electrode unit, and each electrode unit has multiple large micro-wells in the lateral direction. The cell capture and pairing microfluidic chip has N electrode units in the longitudinal direction, and the N electrode units form an electrode pair array, where N is a positive integer; the cell capture and pairing microfluidic chip also includes four sets of metal structure pairs located on both sides of it, the four sets of metal structure pairs include a first set of metal structure pairs connected to the first set of electrode pairs (20), a second set of metal structure pairs connected to the two shielding electrodes, a third set of metal structure pairs connected to the second set of electrode pairs (30), and a fourth set of metal structure pairs connected to the third set of electrode pairs (40); The cell capture and pairing microfluidic chip also includes a glass substrate, on which the electrode units are located; The large micro-trap (10) and small micro-trap (11, 12, 13) form a nested micro-trap structure for cell fixation; The large micro-well (10) is encapsulated with encapsulation material for the flow channel; The large micro-well (10) is elliptical in shape.

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