Micro-fluidic chip for screening and capturing cells and application of micro-fluidic chip

Abstract

The invention provides a micro-fluidic chip for cell screening and capturing and application, a closed structure is formed by bonding a flow channel part and a recognition molecule fixing part, a liquid inlet, a shunting area, a wall-attached capturing area and a liquid outlet are integrated in the closed space inside the micro-fluidic chip, the shunting area is composed of a plurality of parallel micro-channels, and the flow channel part and the recognition molecule fixing part are connected in parallel. The liquid inlet, the flow dividing area, the wall-attached capturing area and the liquid outlet are sequentially communicated according to the flow direction of fluid and are arranged on the flow channel part, and the recognition molecule fixing part serves as a substrate of the wall-attached capturing area; on the premise of not depending on a high-energy field and complex disturbance, the micro-fluidic chip gives consideration to efficient specific contact, cell low loss and system flux / maintainability, is used for efficiently screening and capturing target cells, and can be widely applied to key screening links in cell sorting, upstream preparation of immunotherapy and biological manufacturing.

Description

Technical Field

[0001] This invention relates to the field of microfluidics and bioseparation technology, and in particular to a microfluidic chip and its application for cell screening and capture. Background Technology

[0002] Cell screening and sorting are core technologies in modern biomedical research and biomanufacturing processes. Their core objective is to isolate target cell subpopulations or single-cell clones from complex biological samples using high-throughput, low-damage, and high-specificity methods, providing high-quality starting materials for subsequent cell culture, functional evaluation, and molecular mechanism analysis. Their technological value is concentrated in key areas such as cell biology research, immunotherapy and personalized medicine, and the industrialization of biopharmaceuticals.

[0003] Existing cell screening technologies mainly include four categories: fluorescence activated cell sorting (FACS), magnetic sorting (MACS), label-free physical sorting (based on properties such as size / deformation / density / dielectricity / acoustics), and microfluidic surface affinity trapping. Among these, FACS supports multi-parameter sorting, offering high purity and large throughput, but its equipment purchase and maintenance costs are high, and the process requires fluorescence staining and compensation adjustments. The high-speed shear force and pressure pulsation generated by the nozzle sheath flow can easily damage cell viability and affect cell function; the aseptic operation process is complex, and buffer consumption is high. Magnetic sorting operates under mild conditions, causing minimal cell damage, and is easily scaled up; however, the labeling dimension is limited, typically only able to sort based on 1-2 surface markers, often requiring multiple rounds of enrichment; magnetic particles easily remain on the cell surface, exhibiting significant non-specific enrichment, which may affect downstream experimental results or related product properties. Label-free physical sorting technologies do not require chemical labeling or biological modification, reducing exogenous perturbation to cells; however, the engineering implementation difficulty and maturity of different technologies vary greatly; when the physical characteristics of target cells and background cells overlap, the sorting resolution is significantly insufficient; some technologies require strong electric fields or rely on complex driving systems, which may impose stress on cells, and there are also limitations in system compatibility. Microfluidic surface affinity trapping technology has a high degree of device miniaturization, is easy to integrate, and offers flexible selection of recognition molecules (receptors / ligands, aptamers, peptides, glycosyl binding elements, etc. can be used to construct a target cell-specific functional layer on the channel surface); however, since laminar flow is dominant within microchannels, cells mostly slide along the middle of the velocity profile, resulting in short effective contact time and limited contact area with the functional layer on the channel surface; to enhance the impact / perturbation between cells and the functional layer, existing technologies often introduce microstructures such as serpentine channels and columnar arrays, but this leads to increased channel pressure drop and is prone to clogging and cleaning difficulties, making it difficult to achieve a balance between throughput, purity, and maintainability.

[0004] Based on the above situation, existing solutions generally face the following common challenges: a) The dilemma of capture efficiency versus flux: reducing the flow rate can prolong contact but lose flux; increasing the flow rate reduces contact and binding stability; b) Complex processes and high time costs: multiple rounds of labeling / elution / re-enrichment lead to the accumulation of time, reagents, and errors; c) Cell activity and phenotypic perturbation: high shear, strong electric / acoustic fields, or complex chemical treatments can easily cause functional decline or phenotypic drift; d) Unfriendly to low abundance surface features: when the copy number of the target cell surface feature is low, the limited contact area / time significantly reduces the "hit rate"; e) Structural clogging and regeneration difficulties: microstructures that enhance perturbation are prone to accumulating impurities and cell clumps, affecting continuous operation and reuse.

[0005] Therefore, achieving efficient and specific contact, scalable throughput, and good maintainability / recyclability without sacrificing cell viability and system stability is a pressing technical challenge that needs to be addressed at present. Summary of the Invention

[0006] The technical problem to be solved by the present invention is to provide a microfluidic chip for cell screening and capture.

[0007] Another technical problem to be solved by the present invention is to provide the application of the above-mentioned microfluidic chip for cell screening and capture.

[0008] The technical solution adopted in this invention is: A microfluidic chip for cell screening and capture comprises a channel portion 5 and a recognition molecule immobilization portion 6. The channel portion 5 and the recognition molecule immobilization portion 6 are bonded to form a closed structure. The enclosed internal space integrates four functional units: a liquid inlet 1, a diversion region 2, an adherent capture region 3, and a liquid outlet 4. The diversion region 2 is composed of several parallel microchannels. The liquid inlet 1, diversion region 2, adherent capture region 3, and liquid outlet 4 are sequentially connected according to the fluid flow direction and are disposed on the channel portion 5. The recognition molecule immobilization portion 6 serves as the substrate of the adherent capture region 3 and is the carrier for constructing the cell-specific recognition functional layer. The adherent capture region 3 adopts a geometrically confined, controlled, lightly pressed design and is the core region for achieving cell-specific capture based on the functional layer of the recognition molecule immobilization portion 6. Its key parameters are as follows: The vertical distance between the flow channel portion 5 and the recognition molecule fixation portion 6, corresponding to the adhesion capture region 3, is defined as the channel height H1. The channel height H1 is 80%–95% of the equivalent diameter of the target cell in suspension. For example, for lung cancer tumor cells (PC-9) with a diameter of 10 μm, the channel height is set to 8–9.5 μm; for breast cancer tumor cells (such as MCF-7) with a diameter of 15 μm, the channel height is set to 12–14 μm. The equivalent diameter of the target cell in suspension is taken as the equivalent diameter of the suspension state for spherical / elliptical cells or the long axis diameter for rod cells, ensuring that the cells produce mild and reversible microscale deformation (deformation rate <20%), avoiding cell rupture or functional damage. The width of a single parallel microchannel contained in the diversion region 2 (the lateral width of a single parallel channel in the wall-attached capture region 3) is taken as the channel width W. The channel width W is 50 to 200 μm (adjusted according to the number of parallel channels N, N=64 to 256, and the total width matches the inlet / outlet size) to ensure that the fluid flows in a laminar state (Reynolds number Re<100) and avoids non-specific impacts caused by turbulence. The extension dimension of the adherent capture region 3 in the fluid flow direction (the longitudinal length of the adherent capture region 3) is taken as the channel length L. The channel length L is 500-2000 μm. Combined with the set flow rate, the cell's adherence time in this region can be controlled within 0.5-5 seconds, ensuring the interaction window of the recognition molecules.

[0009] Preferably, in the microfluidic chip for cell screening and capture described above, the diversion region 2 consists of 128 parallel microchannels (i.e., the number of microchannels is 128), which is used to divert the main stream (referring to the complete fluid that enters from the liquid inlet 1 of the chip and has not yet been distributed by the diversion region 2), thereby ensuring the total throughput while reducing the flow rate and shear force of a single channel and reducing the risk of blockage.

[0010] Preferably, in the microfluidic chip for cell screening and capture described above, the adherent capture region 3 is a rectangular cavity structure located in the center of the parallel microchannels.

[0011] The microfluidic chip described above for cell screening and capture introduces a geometrically confined, controlled, mildly pressurized design. Within the wall-attached capture region 3 (as the key capture region), the channel's geometric height is designed to be slightly lower than the equivalent diameter of the suspended cell. Under set flow rate and pressure difference, this induces mild, reversible microscale deformation in the cells, guiding them to form a stable near-wall contact with the surface functional layer that has specific recognition capabilities, thereby overcoming the core bottleneck of existing technologies.

[0012] Preferably, in the microfluidic chip for cell screening and capture described above, the flow channel portion 5 is made of PDMS, thermoplastic polymers (such as COC, PMMA), silicon dioxide, or silicon wafers, taking into account both processing convenience and fluid compatibility.

[0013] Preferably, in the microfluidic chip for cell screening and capture described above, the material of the recognition molecule immobilization part 6 is silicon dioxide or a glass slide (rectangular glass slide), which facilitates stable surface chemical modification (APTES-glutaraldehyde crosslinking) and ensures the immobilization efficiency and specificity of the recognition molecules.

[0014] Preferably, in the microfluidic chip for cell screening and capture described above, the method for constructing a cell-specific recognition functional layer on the surface of the flow channel portion 5 and the recognition molecule immobilization portion 6 is as follows: (1) Base amination (APTES modification) Pretreatment: The immobilized portion 6 of the recognition molecule was rinsed sequentially with ultrapure water and isopropanol, and then dried with nitrogen. Oxygen plasma treatment: Oxygen plasma treatment is performed together with flow channel section 5, and the bond is formed immediately after treatment to form a closed channel; APTES modification: 1% to 10% (preferably 5%) of anhydrous ethanol solution of APTES (3-aminopropyltriethoxysilane) is introduced into the channel through liquid inlet 1 to ensure that the solution fills the wall-adhering trapping region 3 and forms a dense amino (-NH2) layer on the surface of the recognition molecule immobilization part 6. Post-treatment: Anhydrous ethanol and deionized water are sequentially introduced through liquid inlet 1 to rinse the channel. After being dried with nitrogen, baking is performed to promote the condensation of silicon-oxygen bonds (Si-O-Si) and enhance the stability of the amino layer. (2) Aldehyde grouping (glutaraldehyde crosslinking) Solution preparation: Prepare a 1% to 5% (preferably 2.5%) aqueous solution of glutaraldehyde using deionized water; Aldehyde modification: The above-mentioned glutaraldehyde aqueous solution is introduced into the channel through liquid inlet 1, filling the wall-adhering capture region 3, and incubated at room temperature in the dark; the aldehyde group at one end of the glutaraldehyde undergoes a Schiff base reaction (-C=N-) with the -NH2 on the surface of the recognition molecule fixation part 6, while the free aldehyde group (-CHO) is retained at the other end, forming a "substrate-APTES-glutaraldehyde-CHO" intermediate layer. To terminate the reaction: flush the channel with PBS buffer (pH 7.4) through liquid inlet 1 to remove unreacted glutaraldehyde; (3) EGFR antibody fixation Antibody dilution: Dilute the anti-human EGFR monoclonal antibody with PBS (pH 7.4) to a final concentration of 1–5 μg / mL; Antibody immobilization: Place the flow channel portion 5 and the recognition molecule immobilization portion 6 horizontally, and inject the diluted EGFR antibody solution through the liquid inlet 1, ensuring that the adhesion and capture area 3 is completely filled; seal the liquid inlet 1 and the liquid outlet 4, and incubate overnight in a humidified chamber to allow the free amino groups (lysine residues) of the antibody to form covalent Schiff base bonds with the basal aldehyde group (-CHO), thereby achieving directional antibody immobilization (exposing the antigen-binding domain within the channel). Rinse to remove free antibodies: Flow PBS (pH 7.4) into the channel through liquid inlet 1 to thoroughly rinse the channel and remove uncovalently bound free antibodies.

[0015] The above-mentioned microfluidic chip for cell screening and capture is used in the screening and capture of cells.

[0016] The beneficial effects of this invention are: The aforementioned microfluidic chip for cell screening and capture is designed around geometrically confined, controlled, and gentle pressure bonding. Without relying on high-energy fields or complex perturbations, it balances efficient and specific contact, low cell damage, and system throughput / maintainability. It is used for the efficient screening and capture of target cells and can be widely applied in key screening stages of cell sorting, upstream preparation for immunotherapy, and biomanufacturing. Specifically: (1) The microfluidic chip can significantly improve the effective contact area and contact time: near-wall contact and local flattening increase the probability of interaction at the recognition site, which is especially beneficial for the capture of target cells with low abundance surface features; (2) Achieving high binding stability under low shear conditions: By guiding cells to adhere to the near wall through geometric confinement, and with precise control of flow rate and pressure, it not only ensures that cells pass through the capture area in an orderly manner, but also avoids shear damage caused by simply relying on high flow rate to increase the frequency of impact, thus significantly reducing the risk of cell breakage. (3) The structure is modular and cascaded: it can be connected in series and parallel with fishbone / finger comb type multi-branch unit (shunting area (2) or parallel branch module based on shunting area (2)) to achieve re-contact-recapture and high throughput compatibility; at the same time, it is easy to set up bypass and conical transition to alleviate blockage and improve cleanability; (4) Simple engineering implementation and controllable cost: compatible with PDMS, thermoplastic polymers or silicon-based processing; regular structure, easy to replicate, and convenient for disposable consumables and large-scale production. Attached Figure Description

[0017] Figure 1 This is a top view of the microfluidic chip for cell screening and capture described in this invention, showing the overall shape of the chip and the layout of its internal channels. Figure 2This is a top view of the functional units of the microfluidic chip for cell screening and capture described in this invention, showing the planar layout and key dimensions (L, W) of each functional unit, where: 1 is the liquid inlet, 2 is the diversion area, 3 is the wall-adhering capture area, 4 is the liquid outlet, L is the length of the wall-adhering capture area, and W is the width of the parallel microchannels. Figure 3 This is a side view of the microfluidic chip for cell screening and capture described in this invention, used to show the physical components and height relationships of the chip, wherein: 5 is the flow channel, 6 is the molecule recognition and fixation part, H1 is the channel height of the wall-adhering capture region 3, and H2 is the channel height of the shunt region 2. Figure 4 This is a schematic diagram of cell deformation in the microfluidic chip for cell screening and capture described in this invention, showing the morphological changes of cells before and after entering the adherent capture region (channel height H1): H1 is the channel height of the adherent capture region 3 (the cell is in a compressed state after entering), and H2 is the channel height of the diversion region 2 (the cell is in a suspended state in the diversion region when it has not entered the capture region). H1 is slightly smaller than H2, reflecting the design of "geometric confinement - controlled mild compression". Figure 5 This is a schematic diagram of the cell screening process of the microfluidic chip for cell screening and capture described in this invention, wherein: (a) is the initial suspension state (without contact with the recognition molecule fixation part) of the cells after being distributed by the shunt region and moving towards the adherent capture region; (b) is the capture state of the cells after being deformed by geometric confinement and specifically binding to the EGFR antibody on the surface of the recognition molecule fixation part (in the figure, the "Y" shaped structure is the antibody, and the "antigen" is the EGFR molecule on the cell surface). Figure 6 This is a schematic diagram of the actual microfluidic chip for cell screening and capture described in this invention, showing the actual fabrication form of the chip and the external connection method of the fluid pathway. Detailed Implementation

[0018] To enable those skilled in the art to clearly understand the technical solution of the present invention, the technical solution will be described in detail below with reference to the accompanying drawings and specific embodiments.

[0019] Example 1 Core Design and Construction of Specific Functional Layers of Microfluidic Chips like Figure 1-4As shown, the microfluidic chip for cell screening and capture consists of a flow channel portion 5 and a recognition molecule immobilization portion 6. The flow channel portion 5 and the recognition molecule immobilization portion 6 are bonded together to form a closed structure. The enclosed internal space integrates four functional units: a liquid inlet 1, a diversion region 2, a wall-adhering capture region 3, and a liquid outlet 4. The diversion region 2 consists of 128 parallel microchannels, used to divert the main flow (referring to the complete fluid entering from the liquid inlet 1 of the chip that has not yet been distributed by the diversion region 2), ensuring the total throughput while reducing... Single-channel flow rate and shear force reduce the risk of clogging; the liquid inlet 1, diversion region 2, wall-attached trapping region 3, and liquid outlet 4 are sequentially connected according to the fluid flow direction and are arranged on the flow channel section 5. The recognition molecule fixation part 6 serves as the base of the wall-attached trapping region 3 and is the carrier for constructing the cell-specific recognition functional layer. The wall-attached trapping region 3 has a rectangular cavity structure and is located in the center of the parallel microchannels. The wall-attached trapping region 3 adopts a geometrically confined-controlled light pressure design and is the core area for achieving cell-specific capture based on the functional layer of the recognition molecule fixation part 6. 1.1 Chip Core Structure Design (Geometric Confinement - Controlled Light Pressing) In this embodiment, an adhesion capture region 3 was designed for human breast cancer cells MDA-MB-231 (suspension diameter 15-20 μm, high EGFR expression): Channel height H1: Corresponding Figure 3 Between the middle channel portion 5 and the recognition molecule fixation portion 6, Figure 2 Vertical spacing corresponding to the wall-mounted capture area 3 Figure 4 The vertical dimension of the channel when the cell is compressed; designed to be 80% to 95% of the equivalent diameter (15 to 20 μm) of the MDA-MB-231 cell suspension state, specifically 15 μm; this height can cause the cell to produce about 15% microscale deformation (deformation rate <20%), avoiding cell rupture or functional damage, and can completely recover to spherical shape within 10 seconds after the constraint is removed; Channel height H2: Corresponding Figure 3 Between the middle channel portion 5 and the recognition molecule fixation portion 6, Figure 2 Vertical spacing corresponding to the middle diversion area 2 Figure 4 The vertical dimension of the channel when the cells are in suspension; the value is 10-30 μm, and in this embodiment, the value is 20 μm to ensure the mechanical stability of the flow channel structure and avoid channel deformation after bonding.

[0020] Channel width W: corresponding Figure 2The width of a single parallel channel in the diversion area 2 is 100 μm, with N = 128 parallel channels (total width 12.8 mm, matching the 13 mm inner diameter of liquid inlet 1 / liquid outlet 4). This ensures that the fluid is in a laminar flow state (Reynolds number Re≈50<100), avoiding non-specific impacts of turbulence on cells. In addition, the spacing between adjacent parallel channels is 20 μm. Channel length L: corresponding Figure 2 The longitudinal extension size of the adhesion capture region 3 is specifically set at 1000 μm. When combined with a single-channel flow rate of 2 μL / min (total flow rate of 256 μL / min for 128 channels), the adhesion contact time of MDA-MB-231 cells in this region can be stably controlled at 2 seconds, which precisely meets the specific interaction window between the EGFR antibody and the EGFR molecules on the cell surface (neither too long a contact time will lead to flux loss, nor too short a contact time will affect the binding efficiency). 1.2 Construction of Surface-Specific Recognition Functional Layer (APTES-Glutaraldehyde Crosslinking Method) All modification steps are completed by passing liquid into the channel after the flow channel 5 and the recognition molecule immobilization portion 6 are bonded together to form a closed channel (not by modifying the substrate first and then bonding). The operation is as follows: 1.2.1 Substrate amination (APTES modification) Pretreatment: Rinse the glass slide / silica substrate (recognition molecule immobilization part 6) sequentially with ultrapure water and isopropanol, and then dry it with nitrogen; Oxygen plasma treatment: Oxygen plasma treatment is carried out together with flow channel 5 (power 60W, time 25 seconds, oxygen inlet flow rate 3mL / min), and the channel is immediately bonded after treatment to form a closed channel; APTES modification: 5% (1% to 10% is acceptable) of APTES (3-aminopropyltriethoxysilane) anhydrous ethanol solution is introduced into the channel through liquid inlet 1 to ensure that the solution fills the wall-attached capture region 3. The solution is incubated at 37°C for 2 hours to form a dense amino (-NH2) layer on the surface of the recognition molecule immobilization part 6. Post-processing: The channel is rinsed sequentially with anhydrous ethanol and deionized water through liquid inlet 1 (5 minutes each / 3-5 minutes each). After drying with nitrogen, the chip is placed on a hot plate at 120°C and baked for 30 minutes to promote silicon-oxygen bond (Si-O-Si) condensation and enhance the stability of the amino layer.

[0021] 1.2.2 Aldehydeization (glutaraldehyde crosslinking) Solution preparation: Prepare a 2.5% (1% to 5% is acceptable) glutaraldehyde aqueous solution with deionized water (prepare immediately before use to avoid oxidation of the aldehyde group); Aldehyde modification: The above glutaraldehyde solution is introduced into the channel through liquid inlet 1, filling the wall-attached trapping region 3, and incubated at room temperature in the dark for 1 hour; the aldehyde group at one end of the glutaraldehyde reacts with the -NH2 on the surface of the recognition molecule fixation part 6 in a Schiff base reaction (-C=N-), while the free aldehyde group (-CHO) is retained at the other end, forming a "substrate-APTES-glutaraldehyde-CHO" intermediate layer. To terminate the reaction: Gently flush the channel three times with PBS buffer (pH 7.4) through liquid inlet 1 to remove unreacted glutaraldehyde and prevent residual aldehyde groups from nonspecifically binding to cells.

[0022] 1.2.3 EGFR antibody fixation Antibody dilution: Take 20 μL of anti-human EGFR monoclonal antibody and dilute it to 500 μL with 0.1M PBS (pH 7.4). Mix gently to avoid bubbles. Antibody immobilization: Place the chip horizontally and inject 200 μL of diluted EGFR antibody solution through liquid inlet 1, ensuring that the adhesion and capture area 3 is completely filled; seal liquid inlet 1 and liquid outlet 4, and incubate overnight (approximately 14 hours) in a humidified chamber at 4°C, allowing the free amino groups (lysine residues) of the antibody to form covalent Schiff base bonds with the aldehyde group (-CHO), thus achieving directional antibody immobilization (exposing the antigen-binding domain within the channel). Rinsing to remove free antibodies: The next day, use a 1mL syringe to slowly introduce 4mL of PBS (pH 7.4) into the channel through the liquid inlet (1) at a flow rate of 0.5mL / min to thoroughly rinse the channel and remove uncovalently bound free antibodies.

[0023] 1.3 Verification of Functional Layer Modification Effect The detection method using fluorescently labeled secondary antibody specific binding was employed, and the validation standards were quantified and the operation was standardized. Validation steps: Blocking (inject 80 μL of 2% BSA / PBST (PBST is 0.1M PBS containing 0.05% Tween-20, pH 7.4) solution, incubate at room temperature for 1.5 hours) → Washing (wash 4 times with PBST, 1 mL each time) → Fluorescent secondary antibody binding (inject 80 μL of 1:800 diluted CoraLite488–conjugated Goat Anti-Mouse IgG (H+L), incubate at room temperature for 1.5 hours) → Washing (wash 4 times with PBST) → Fluorescence detection (fluorescence microscope, excitation 488 nm / emission 515 nm, ImageJ quantitative analysis).

[0024] The control group underwent the same procedure.

[0025] Blank control group: Unmodified glass substrate, excluding substrate autofluorescence or non-specific adsorption of secondary antibody; EGFR antibody-free control group: APTES-glutaraldehyde modified with PBS was used to replace the antibody to exclude non-specific binding of the glutaraldehyde layer; Non-specific antibody control group: Immobilized anti-GFP monoclonal antibody (same concentration from mouse) to verify the binding specificity of the secondary antibody; Control group without secondary antibody after blocking: No secondary antibody was added after blocking and washing to exclude blocking solution / antibody autofluorescence; Judgment criteria: The fluorescence intensity of the experimental group is ≥3 times that of the blank control group and ≥2 times that of the control group without EGFR antibody, and the fluorescence signal distribution in the adhesion capture area (3) is uniform (variance <20%), and the modification is judged to be successful.

[0026] The fabrication method of the microfluidic chip used for cell screening and capture described above is as follows: (1) Preparation of materials Flow channel section 5: PDMS, wherein the mass ratio of PDMS substrate (PDMS prepolymer, providing the main structure of the material) to PDMS curing agent is 10:1 (thermoplastic polymers (such as COC, PMMA), silica or silicon wafers can also be used). Molecular fixation part 6: Glass slide (silicon dioxide can also be used); Auxiliary materials: IP-S photosensitive resin, 3-(trimethoxysilyl)propyl methacrylate, APTES, release agent (1H,1H,2H,2H-perfluorodecyltrichlorosilane).

[0027] (2) Preparation steps Glass substrate pretreatment (silanization): Clean the substrate with isopropanol and blow dry → plasma treatment → immerse in a mixture containing 50 mL ethanol, 250 μL 3-(trimethoxysilyl)propyl methacrylate, and 1.5 mL acetic acid (glacial acetic acid: water = 1:10) (≥5 minutes) → isopropanol cleaning → evaporation of moisture by heating on a hot plate at 120°C; Negative mold preparation: Using IP-S photosensitive resin as the material, a negative mold is prepared on the pretreated substrate by two-photon lithography → propylene glycol monomethyl ether acetate development (>5 minutes, accompanied by shaking) → oxygen plasma treatment → chemical vapor deposition release agent (to form a hydrophobic layer). PDMS runner molding: Mix PDMS substrate and PDMS curing agent in a 10:1 ratio → vacuum for 3-5 minutes to remove air bubbles → pour into negative mold → cure for 24 hours → demold and cut; Bonding and sealing: The PDMS flow channel and the glass slide are treated with oxygen plasma (parameters: 60W, 25 seconds, 3mL / minO2) → immediately bonded, forming a closed channel (corresponding to...). Figure 3 layered structure Figure 6 (In physical form).

[0028] Example 2 Cell screening application process of microfluidic chips (Objective: To clarify the actual operation steps of the chip, corresponding to...) Figure 5-6 ) 2.1 Preparation of Experimental Materials Chip: The microfluidic chip prepared in Example 1 (128 parallel channels, 3 channels of the adhesion capture region highly adapted to the target cells MDA-MB-231, and 6 recognition molecule immobilization parts modified with EGFR antibody). Reagents: PBS buffer (pH 7.4), containing 1 mM Mg 2+ Serum-free DMEM medium, 1% BSA / PBS, 0.5 mM EDTA / PBS; Instruments: Precision syringe pump (flow rate accuracy ±0.1μL / min), inverted fluorescence microscope (with image analysis function), cell counting chamber.

[0029] 2.2 Filtering steps, such as Figure 5 As shown 2.2.1 Chip Preprocessing Degassing: Draw 1 mL of PBS buffer into a syringe and connect it to liquid inlet 1 via a silicone tubing. Set the syringe pump flow rate to 0.5 mL / h and flush the channel for 5 minutes until no more air bubbles flow continuously from liquid outlet 4 (ensure the channel is free of air bubbles; tubing connections are as follows). Figure 6 (as shown) Environmental balancing: Replace the syringe with one containing 1 mM Mg 2+ The serum-free DMEM medium was used to continue washing for 5 minutes at a flow rate of 0.5 mL / h to maintain EGFR conformational stability.

[0030] 2.2.2 Sample loading and capture, such as Figure 5 As shown Sample preparation: Adjust the target cell suspension MDA-MB-231 to a concentration of 5 × 10⁻⁶. 5 Cells / mL (viable cell rate >95%), filtered through a 40μm cell sieve to remove clumps; Loading parameters: The cell suspension is injected into the chip through liquid inlet 1 via an injection pump. The total flow rate = single channel flow rate × number of parallel channels (single channel 1~5μL / min, preferably 2μL / min; 128 channels correspond to a total flow rate of 256μL / min). The operating pressure difference is controlled at 10±2kPa (5~20kPa is acceptable). Capture process: Cells are distributed to each parallel channel (corresponding to) via shunt region 2. Figure 5 (a) In its initial suspended state, after entering the adherent capture region 3, it undergoes slight deformation due to geometric confinement, adheres to the near-wall, and specifically binds to the EGFR antibody on the surface of the recognition molecule immobilization part 6 (corresponding to...). Figure 5(b) of the combination state).

[0031] 2.2.3 Elution and Collection Uncaptured cells elution: Turn off the syringe pump, replace the syringe with 1% BSA / PBS, and rinse at a low speed of 0.2 mL / h for 2 minutes. Collect the eluent (labeled "uncaptured fraction"). Target cell collection: Connect the syringe to the liquid outlet (4) in reverse, draw PBS containing 0.5 mM EDTA, flush the channel in reverse at a flow rate of 1 mL / h, collect the eluent (labeled "capture component"), and the target cells are obtained.

[0032] Example 3 Validation of EGFR-positive cell capture efficiency based on channel height 3.1 Experimental Design Experimental group: The chip described in Example 1 (adherent capture region 3 channels, height 15μm, adapted to MDA-MB-231 cells with a diameter of 15-20μm, producing slight compression, such as...) Figure 4 (as shown) Non-pressure control group: The channel structure is the same as the experimental group, except that the height of the 3 channels in the adhering capture area is 25 μm (significantly larger than the maximum cell diameter of 20 μm, allowing cells to pass through in suspension without any deformation). Blank control group: channel height 15μm, recognized 6 unmodified EGFR antibody (treated with APTES-glutaraldehyde only), excluding non-specific physical capture; Repeat each group three times to ensure the reliability of the results.

[0033] 3.2 Experimental Procedure and Result Analysis 3.2.1 Experimental Procedure Referring to the “chip preprocessing → sample loading → elution and collection” steps in Example 2, the total flow rate was uniformly controlled at 256 μL / min and the operating pressure difference was 10 ± 2 kPa. The cell state was observed in real time using an inverted fluorescence microscope.

[0034] 3.2.2 Key Results Cell deformation and contact area: The cells in the experimental group exhibited a "flattened" deformation due to the limitation of channel height (corresponding to...). Figure 4 The deformation design of the sample and the contact area of ​​the recognition molecule fixation part 6 are significantly larger than those of the non-pressurized control group (near-spherical cells). Capture specificity: In the blank control group, no obvious cells were observed in the adherent capture area 3 and the capture components (corresponding to...). Figure 5 (b) Specific binding logic), demonstrating the capture-dependent specific interaction between EGFR antibody and cell; Cell viability: The viable cell rate of captured cells in both the experimental group and the control group without pressure remained at a high level (verified by trypan blue staining), proving that neither channel design damaged cell viability.

[0035] 3.3 Conclusion The "geometric confinement-controlled mild compression" design significantly increases the contact area between cells and the chip surface by inducing reversible cell deformation (deformation rate <20%), which is a core factor in improving the capture efficiency of EGFR-positive cells, while also ensuring low cell damage and meeting the needs of downstream experiments (corresponding to...). Figure 4 Deformation safety, Figure 5 (binding effectiveness).

[0036] The above-described embodiments 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 defined by the claims of the present invention.

Claims

1. A microfluidic chip for cell screening and capture, characterized by: The application relates to a cell-specific recognition microfluidic chip, which is composed of a flow channel part and an identification molecule fixed part, and is bonded to form a closed structure with an internal closed space, wherein a liquid inlet, a flow distribution area, a wall-adhesion capture area and a liquid outlet are integrated in the internal closed space, the flow distribution area is composed of a plurality of parallel microchannels, the liquid inlet, the flow distribution area, the wall-adhesion capture area and the liquid outlet are sequentially connected in a fluid flow direction and are arranged on the flow channel part, the identification molecule fixed part serves as a substrate of the wall-adhesion capture area and is a carrier for constructing a cell-specific recognition functional layer, the vertical distance between the flow channel part and the identification molecule fixed part in the wall-adhesion capture area is taken as a channel height, and the channel height is 80%-95% of the equivalent diameter of target cell suspension; the equivalent diameter of the target cell suspension is the equivalent diameter of spherical / elliptical cells or the long axis diameter of rod-shaped cells.

2. The microfluidic chip for cell screening and capturing according to claim 1, wherein: The width of a single parallel microchannel in the flow distribution area is taken as a channel width, the flow distribution area is composed of 64-256 parallel microchannels, and the channel width is 50-200 mu m.

3. The microfluidic chip for cell screening and capturing according to claim 1, wherein: The extension dimension of the wall-adhesion capture area in the fluid flow direction is taken as a channel length, and the channel length is 500-2000 mu m.

4. The microfluidic chip for cell screening and capturing according to claim 1 or 2, wherein: The flow distribution area is composed of 128 parallel microchannels.

5. The microfluidic chip for cell screening and capture of claim 1, wherein: The wall-adhesion capture area is a rectangular cavity structure and is located in the center of the parallel microchannels.

6. The microfluidic chip for cell screening and capture of claim 1, wherein: The material of the flow channel part is PDMS, a thermoplastic polymer, silicon dioxide or a silicon wafer.

7. The microfluidic chip for cell screening and capture of claim 1, wherein: The material of the identification molecule fixed part is silicon dioxide or glass.

8. The microfluidic chip for cell screening and capture of claim 1, wherein: The method for constructing a cell-specific recognition functional layer on the surface of the flow channel part and the identification molecule fixed part is as follows: (1) substrate amination pretreatment: the identification molecule fixed part is sequentially washed with ultrapure water and isopropanol and is dried by nitrogen blowing; oxygen plasma treatment: the flow channel part and the identification molecule fixed part are subjected to oxygen plasma treatment together, and the closed channel is bonded immediately after the treatment; APTES modification: 1%-10% APTES anhydrous ethanol solution is introduced into the channel through the liquid inlet, the solution is ensured to fill the wall-adhesion capture area, and a dense amino layer is formed on the surface of the identification molecule fixed part; post-treatment: the channel is sequentially washed with anhydrous ethanol and deionized water through the liquid inlet, and is dried by nitrogen blowing and baked to promote the condensation of the silicon-oxygen bond; (2) aldehyde group modification solution preparation: 1%-5% glutaraldehyde aqueous solution is prepared by using deionized water; aldehyde group modification: the above glutaraldehyde aqueous solution is introduced into the channel through the liquid inlet, the wall-adhesion capture area is filled, and incubation is carried out at room temperature in the dark; the aldehyde group at one end of the glutaraldehyde reacts with the -NH2 on the surface of the identification molecule fixed part to form a Schiff base, and the other end retains a free aldehyde group, thereby forming an "substrate-APTES-glutaraldehyde-CHO" intermediate layer; reaction termination: PBS buffer is introduced into the channel through the liquid inlet to remove the unreacted glutaraldehyde; (3) EGFR antibody fixation antibody dilution: the anti-human EGFR monoclonal antibody is diluted with PBS to a final concentration of 1-5 mu g / mL; Antibody immobilization: Place the flow channel part horizontally with the recognition molecule immobilization part, inject the diluted EGFR antibody solution through the liquid inlet, and make sure that the whole wall-adhesion capture area is filled; seal the liquid inlet and liquid outlet, and incubate the wet box overnight to form covalent Schiff base bonds between the free amino groups of the antibody and the aldehyde groups of the substrate, thus realizing the directional immobilization of the antibody. Washing free antibody: Inject PBS into the channel through the liquid inlet to thoroughly wash the channel and remove the free antibody that is not covalently combined.

9. Use of the microfluidic chip for cell screening and capture according to any one of claims 1-8 in screening and capturing cells.

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