Disease diagnosis method based on original cadherin 7 antibody detection and diagnosis system thereof

Abstract

The invention discloses a disease diagnosis system based on an original cadherin 7 antibody and a diagnosis method thereof. The system comprises a micro-fluidic device and a micro-fluidic chip matched with the micro-fluidic device for use. The surface of a substrate of the micro-fluidic chip is sequentially coated with a coating A and a coating B, the coating A is one or more polypeptides, and the coating B is an original cadherin 7 antigen solution. A detection cell monolayer is formed on the surface of the coating B, cells in a sample to be detected are adhered to the surface, and after incubation and washing, known force is applied through the microfluidic device to detect the adhesion force of the cells. And further performing disease diagnosis on the sample by statistically analyzing the adhesion performance of the cells. The system further comprises a detection unit and a control unit which are used for counting the cell detachment condition and cooperatively working for detection. The diagnostic method can be applied to diagnostic analysis of multiple diseases, in particular to disease monitoring based on cell adhesion change.

Description

Technical Field

[0001] The present invention belongs to the field of medical detection technology, and in particular relates to a disease diagnosis method and a diagnosis system thereof based on protocadherin 7 antibody detection. Background Art

[0002] Protocadherin 7 (PCDH7) is a cell adhesion molecule that is widely involved in cell-to-cell adhesion and signal transduction. In many diseases (such as cancer, immune diseases, etc.), the expression and function of PCDH7 may be abnormal. By detecting the adhesion of cells expressing PCDH7 antibodies, it is possible to evaluate whether the antibodies effectively interact with PCDH7 and affect the adhesion behavior of cells. Changes in cell adhesion can reflect the intervention effect of antibodies on cell adhesion properties in specific disease states and help understand the potential biological functions of PCDH7 antibodies.

[0003] PCDH7 antibodies can affect cell adhesion. Therefore, by measuring changes in cell adhesion, it can provide important clues for the application of PCDH7 antibodies in disease diagnosis. For example, some tumor cells or immune cells may show abnormal cell adhesion due to changes in PCDH7 expression, which provides an effective detection method for disease diagnosis based on PCDH7 antibodies. By comparing with normal cells, detecting the effect of antibodies on cell adhesion under pathological conditions can help doctors determine the type or severity of the disease.

[0004] According to the disclosed technical solutions, the technical solution with publication number US07935790B2 proposes a reagent for detecting protein phosphorylation in the T cell receptor signaling pathway, including a component for detecting proto-cadherin. The technical solution with publication number CN103153331A proposes a humanized antibody targeting the EC1 domain of Cadherin-11 and related compositions and methods, realizing a method of administering a therapeutically effective amount of the humanized antibody of the present invention to treat Cadherin-11-mediated diseases in mammalian subjects.

[0005] The above technical solutions all propose a variety of technical solutions for realizing and optimizing the detection of proto-cadherin antibodies. With the deepening of research in related fields, more efficient antibody detection methods and supporting detection systems can be further proposed.

[0006] The foregoing discussion of the background art is intended only to facilitate an understanding of the present invention. This discussion does not acknowledge or admit that any of the material referred to is part of the common general knowledge. Summary of the invention

[0007] The purpose of the present invention is to disclose a disease diagnosis system based on protocadherin 7 antibody and a diagnosis method thereof. The system includes a microfluidic device and a microfluidic chip used in conjunction therewith. The substrate surface of the microfluidic chip is coated with a coating A and a coating B in sequence, wherein the coating A is one or more polypeptides and the coating B is a protocadherin 7 antigen solution. A detection cell monolayer is formed on the surface of the coating B, and the cells in the sample to be detected adhere to the surface. After incubation and washing, a known force is applied to the cells through the microfluidic device to detect the adhesion force. By statistically analyzing the adhesion force performance of the cells, the disease diagnosis of the sample is further performed. The system also includes a detection unit and a control unit, which are used to count the cell detachment and work together for detection. The diagnostic method can be applied to the diagnosis and analysis of multiple diseases, especially disease monitoring based on changes in cell adhesion.

[0008] The present invention adopts the following technical scheme: a disease diagnosis system based on protocadherin 7 antibody detection; the diagnosis system comprises a microfluidic device and a microfluidic chip applied to the microfluidic device; wherein the following coatings are sequentially prepared on the substrate of the microfluidic chip: A coating, which is one or more polypeptides or a mixture of polypeptides; Coating B, protocadherin 7 antigen solution; The detection cell monolayer is formed by using cells in the sample to be detected and adhering to the surface of the B coating to form a monolayer of cells; The diagnostic system applies a known force to the detection cell monolayer on the microfluidic chip by using the microfluidic device to detect the adhesion of multiple cells on the detection cell monolayer, and calculates the adhesion performance of the cells in the detection cell monolayer by statistical means.

[0009] Preferably, the diagnostic system further comprises a detection unit and a control unit; The detection unit is used to count the cells shed from the B coating; The control unit is used to control the microfluidic device and the detection unit to work in coordination.

[0010] Preferably, the detection unit also includes counting the cells that fall off from the coating B and express protocadherin 7 antibodies.

[0011] Preferably, the microfluidic chip comprises a plurality of flow channels, and each flow channel allows for independent detection of the adhesion of different cell types for performing multiple diagnostic analyses of diseases.

[0012] At the same time, a disease diagnosis method based on the detection of protocadherin 7 antibodies is proposed, and the diagnosis method is used in the diagnosis system; the diagnosis method comprises the following steps: S100: preparing a microfluidic chip having a coating A and a coating B, wherein the microfluidic chip after preparation comprises a microfluidic chip substrate, a coating A and a coating B stacked in sequence; S200: diluting the sample to be tested to a suitable concentration, coating it on the surface of the coating, and then incubating and washing it to form a test cell monolayer with a monolayer cell structure; S300: adding a labeled secondary antibody to the surface of the microfluidic chip and incubating the chip, and then observing the binding between the sample and the secondary antibody; S400: Apply the microfluidic device to the microfluidic chip to detect cell adhesion and record the cell detachment ratio; S500: Calculate the cell adhesion strength and the ratio of cell adhesion to cell detachment to perform disease analysis on the donor of the sample.

[0013] Preferably, in step S400, the magnitude of the applied force is controllable in at least one direction.

[0014] Preferably, in step S400, the applied force is directed through different flow channel configurations of the microfluidic device to achieve different mechanical conditions in different areas, thereby accurately detecting cell adhesion on the microfluidic chip.

[0015] The beneficial effects achieved by the present invention are: The diagnostic system of this technical solution can accurately detect the interaction between cells and surface coatings at the microscopic level by combining microfluidic chip technology with cell adhesion detection, especially in the detection of disease markers involving proto-cadherin 7 antibodies. After obtaining information through the first cell adhesion test, the post-experiment can adjust the cell ratio according to the pre-test results to further improve the sensitivity and accuracy of the test. This can not only quickly and reliably detect the potential risk of the disease, but also provide more valuable quantitative data for the biological characteristics of the sample cells; The microfluidic chip design used in this technical solution allows multiple flow channels to work in parallel, and can detect the adhesion of different cell types in the same experiment and perform multiple disease analyses. The chip surface is precisely prepared through coating A and coating B to ensure the stability and controllability of cell adhesion conditions, so that cell adhesion under different experimental conditions can be accurately measured. The structural flexibility and multi-channel design of the chip greatly improve the efficiency and throughput of detection, providing an ideal solution for clinical diagnosis and large-scale screening; The microfluidic chip of this technical solution uses materials with high chemical and mechanical stability, and is designed to support dual modes of single-use and recycling and cleaning, which is more economical and environmentally friendly. In addition, the preparation process of coating A and coating B is highly customizable, and the coating material and concentration can be adjusted according to different disease markers or cell types, further enhancing the wide application of the technology and adapting to the detection needs of different fields. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] The present invention can be further understood from the following description in conjunction with the accompanying drawings. The components in the figures are not necessarily drawn to scale, but the emphasis is placed on illustrating the principles of the embodiments. In different views, the same reference numerals designate corresponding parts.

[0017] Description of serial numbers: 10-microfluidic device; 20-microfluidic chip; 22-substrate; 24-coating A; 26-coating B; 28-detection cell monolayer; 30-detection unit; 40-control unit; 500-computer system; 502-bus; 504-processor; 506-main memory; 508-read-only memory; 510-storage device; 512-display; 514-input device; 516-cursor control device; 518-network device; Figure 1 Schematic diagram of the architecture of the diagnostic system described in an embodiment of the present invention Figure 2 Schematic diagram of each layer in the microfluidic chip in an embodiment of the present invention; Figure 3 Schematic diagram of various directions of applying force to a microfluidic chip in an embodiment of the present invention; Figure 4 A schematic diagram of the steps of the diagnostic method described in an embodiment of the present invention; Figure 5 Schematic diagram of a computer system used in a diagnostic system according to an embodiment of the present invention. DETAILED DESCRIPTION

[0018] In order to make the purpose, technical solutions and advantages of the present invention clearer, the present invention is further described in detail below in conjunction with its embodiments; it should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention. For those skilled in the art, other systems, methods and / or features of this embodiment will become apparent after reviewing the following detailed description. It is intended that all such additional systems, methods, features and advantages are included in this specification. Included within the scope of the present invention and protected by the appended claims. Additional features of the disclosed embodiments are described in the following detailed description, and these features will be apparent from the following detailed description.

[0019] The same or similar reference numerals in the drawings of the embodiments of the present invention correspond to the same or similar components; in the description of the present invention, it should be understood that if there are terms such as "upper", "lower", "left", "right" and the like indicating the orientation or positional relationship based on the orientation or positional relationship shown in the drawings, it is only for the convenience of describing the present invention and simplifying the description, rather than indicating or implying that the device or component referred to must have a specific orientation. The invention is constructed and operated in a specific orientation, so the terms describing the positional relationship in the drawings are only used for exemplary description and cannot be understood as a limitation of this patent. For ordinary technicians in this field, the specific meanings of the above terms can be understood according to the specific circumstances.

[0020] Example 1: Exemplary, as shown in the attached Figure 1 As shown in FIG. 1 , a disease diagnosis system based on protocadherin 7 antibody detection is proposed; the diagnosis system comprises a microfluidic device 10 and a microfluidic chip 20 applied to the microfluidic device; wherein, as shown in FIG. Figure 2 As shown, the following coatings are sequentially prepared on the substrate 22 of the microfluidic chip: A coating layer 24, which is one or more polypeptides or a mixture of polypeptides; B coating 26, is a protocadherin 7 antigen solution; The detection cell monolayer 28 is formed by using cells in the sample to be detected and adhering to the surface of the coating layer as a monolayer of cells; The diagnostic system applies a known force to the detection cell monolayer on the microfluidic chip by using the microfluidic device to detect the adhesion of multiple cells on the detection cell monolayer, and calculates the adhesion performance of the cells in the detection cell monolayer by statistical means.

[0021] Preferably, the diagnostic system further comprises a detection unit 30 and a control unit 40; The detection unit 30 is used to count the cells shed from the coating layer B; The control unit 40 is used to control the microfluidic device 10 and the detection unit 30 to work in coordination.

[0022] Preferably, the detection unit also includes counting the cells that fall off from the coating B and express protocadherin 7 antibodies.

[0023] Preferably, the microfluidic chip comprises a plurality of flow channels, and each flow channel allows for independent detection of the adhesion of different cell types for performing multiple diagnostic analyses of diseases.

[0024] At the same time, a disease diagnosis method based on the detection of protocadherin 7 antibodies is proposed, and the diagnosis method is used in the diagnosis system; the diagnosis method comprises the following steps: S100: preparing a microfluidic chip having a coating A and a coating B, wherein the microfluidic chip after preparation comprises a microfluidic chip substrate, a coating A and a coating B stacked in sequence; S200: diluting the sample to be tested to a suitable concentration, coating it on the surface of the coating, and then incubating and washing it to form a test cell monolayer with a monolayer cell structure; S300: adding a labeled secondary antibody to the surface of the microfluidic chip and incubating the chip, and then observing the binding between the sample and the secondary antibody; S400: Apply the microfluidic device to the microfluidic chip to detect cell adhesion and record the cell detachment ratio; S500: Calculate the cell adhesion strength and the ratio of cell adhesion to cell detachment to perform disease analysis on the donor of the sample.

[0025] Preferably, in step S400, the magnitude of the applied force is controllable in at least one direction.

[0026] Preferably, in step S400, the applied force is directed through different flow channel configurations of the microfluidic device to achieve different mechanical conditions in different areas, thereby accurately detecting cell adhesion on the microfluidic chip.

[0027] The interior of the microfluidic chip includes one or more internal cavities, and the internal cavities are usually in the form of flow channels to support the flow of fluids inside; and one or more interruptions can be set between the flow channels, and the one or more interruptions are configured to support one or more fluids to flow independently in multiple flow channels without interfering with each other; and, preferably, the microfluidic chip can also include one or more input and output ends for connecting to the destination connector, and allow the upstream delivery setting to deliver the fluid into the microfluidic chip, and after the fluid flows out of the microfluidic chip, it is received in the downstream device.

[0028] Preferably, the microfluidic chip is selected to have high chemical and mechanical stability; preferably, the microfluidic chip can be made of high-transmittance glass, or more preferably, can be made of polydimethylsiloxane (PDMS), polymethyl methacrylate (PMMA), polycarbonate (PC) and other materials; and the microfluidic chip can be disposable, or can be recycled and then completely cleaned for reuse.

[0029] Preferably, the microfluidic chip is pretreated by the following steps before coating: Clean the substrate: Use ultrapure water, ethanol, isopropanol or 0.1% SDS solution to immerse the microfluidic chip in an ultrasonic cleaner for cleaning and air-dry.

[0030] Surface activation: Surface activation treatment can enhance the hydrophilicity of the chip surface and the bonding force of the coating; preferably, the microfluidic chip is immersed in a 5% silanization reagent solution and incubated for 10 minutes; then the surface is rinsed with ultrapure water and dried at 60°C for 30 minutes; then the microfluidic chip is placed in a plasma chamber, the oxygen flow rate is set to 40-50W, and after a treatment time of 3 minutes, the armor coating is immediately applied.

[0031] In a preferred embodiment, the A coating is used to form a stable base layer on the surface of the microfluidic chip substrate, providing surface properties that are conducive to subsequent B coating and detection of cell monolayer attachment. Preferably, the first coating may be one or more of the following polypeptides or polypeptide mixtures: poly-L-lysine, poly-D-lysine, poly-L-ornithine, concanavalin A, active polypeptide domain; the first coating provides a positively charged surface, which helps to improve the binding stability of the subsequent coating (second coating) with the substrate surface and enhance the surface's ability to adhere to cells; Furthermore, the material selection of the armor coating is based on the cells selected for the detection cell monolayer, wherein, for example, neuronal cells are more dependent on poly-L-lysine, while immune cells may be more dependent on other biomolecule polypeptides; In step S100, the armor coating is prepared so that the armor coating is evenly coated on the substrate surface of the microfluidic chip. Preferably, the parameters of the armor coating are: commonly used concentration range 50-100 μg / mL; incubation time 30-60 minutes; temperature control from room temperature to 37.5°C; after coating, washing with a buffer solution (such as PBS) to remove unbound polypeptides, and finally blowing the surface with sterile air for drying.

[0032] Furthermore, the initially applied first coating layer is surface activated; the surface activation preferably uses NHS ester or glutaraldehyde to enhance the bonding stability between the first coating layer and the chip substrate.

[0033] In a preferred embodiment, a coating layer B is continuously prepared on the upper layer of the coating layer A; the coating layer B is mainly composed of specific proteins including protocadherin 7 (PCDH7) antigen or extracellular matrix protein, and may also contain other active proteins, such as collagen, fibronectin, laminin, etc.

[0034] Preferably, the specific protein concentration is greater than 95% to reduce the interference of impurities on cell attachment.

[0035] Preferably, the specific protein for B coating is formulated using DPBS or sterile PBS as the solution medium.

[0036] Preferably, the solution preparation of the coating layer may include the following steps: Select a specific protein of proto-cadherin 7 antigen or extracellular matrix protein of appropriate purity, prepare it into a solution of 2-10 µg / mL, and dissolve it in a solution medium; filter the mixed solution through a 0.22 µm filter membrane before use to ensure sterility, and slightly mix the mixed solution to avoid protein aggregation or denaturation.

[0037] The mixed solution is evenly dripped onto the surface of the microfluidic chip. Optionally, the amount used for each microfluidic chip is about 10 to 50 µL, which can be increased or decreased according to the actual size of the microfluidic chip; or the solution is injected into the chip and microchannel through a micro syringe. And to ensure uniform coating, it is preferred to observe whether the surface is fully covered under a microscope without bubbles or blank areas. After coating, place the chip in a humid environment and incubate at 36±1℃ for 30 to 60 minutes to ensure that the protein can fully bind to the surface of the first coating.

[0038] Furthermore, after the incubation is completed, the chip surface is gently rinsed 2 to 3 times with sterile PBS to remove unbound protein substances, and then blown dry with sterile nitrogen.

[0039] To verify the quality of the coating, the uniformity and integrity of the surface coating can be further observed under a microscope, and the biological activity of the protein can be confirmed by antibody detection or cell adhesion experiments. In addition, the ability of the coating surface to support the cell monolayer needs to be tested to ensure that it can form a uniform and stable cell attachment surface. Preferably, the test cell monolayer can be one of the target cells, effector cells, tumor cells or neuronal cells; such cells form a uniform monolayer structure on the coating B for subsequent cell adhesion detection and antibody binding.

[0040] Then, the samples to be tested for this technical solution are processed. Samples refer to biological samples from patients or experimental subjects. Samples may include multiple cell components such as blood, tissues or tumor cells. The samples themselves are not cells, but a collection of cells and other components (such as proteins, DNA, etc.). In these samples, there may be different types of cells, and some cells may express protocadherin 7 on their surfaces, which are the target cells to be tested in this technical solution.

[0041] Protocadherin 7 (PCDH7) is a cell surface adhesion molecule that is usually involved in cell-to-cell adhesion and shows abnormal expression patterns in some diseases (such as cancer). Protocadherin 7 antibodies can specifically identify and bind to protocadherin 7 on the cell surface. Through the binding of antibodies to protocadherin 7 on the surface of target cells, markers are used to generate detectable signals such as fluorescent signals or enzyme-labeled signals to help confirm whether cells show abnormal protocadherin 7 expression and continue further testing of these target cells.

[0042] In a preferred embodiment, the sample to be tested is pretreated and the test cell monolayer is prepared. The steps include: digesting the adherent cells with trypsin; centrifuging the sample and diluting it to a suitable concentration, the time can be 2 to 5 minutes. Then, the suspended cells or immune cells are collected by centrifugation, the centrifuge is set at 200xg, for 5 minutes, and resuspended in an appropriate amount of culture medium, the digestion enzyme or centrifugation buffer is removed, and the cell concentration in the sample is finally adjusted to 80 to 100×10 6 cells / mL.

[0043] Further, the cells in the sample are planted on the surface of the B coating. Optionally, 10 to 50 µL of the cell suspension is evenly dripped onto the B coating each time using a pipette. The microfluidic chip is then placed in an incubator for 30 to 60 minutes to allow the cells to fully contact and adhere to the B coating, while ensuring that the surface of the microfluidic chip remains moist to avoid cell dehydration or coating damage. The microfluidic chip is placed in an incubator at 36 ± 1 ° C and 5% CO 2 The cells are cultured in a culture incubator for 24 to 48 hours, and growth factors or specific culture additives are appropriately supplemented to complete the culture of the test cell monolayer.

[0044] Preferably, the cells of the detection cell monolayer are substantially evenly distributed in a monolayer manner on the surface of the B coating; and preferably, the cells of the detection cell monolayer can be distributed on the surface of the B coating at a relatively low density. The cells of the detection cell monolayer can reach a desired distribution density by adjusting the volume of the cell suspension transferred into the microfluidic chip each time, changing the culture temperature, culture time, etc.

[0045] If the cells are too dense, it may interfere with the interaction between cells and make it impossible to effectively observe the differences in cell behavior. Therefore, a reasonable cell density can ensure that each cell can effectively interact with the target cells in the sample.

[0046] Finally, the test cell monolayer is subjected to quality control. Preferably, the cell morphology is observed under a microscope to ensure that the cells are evenly distributed, well adherent to the wall and free of aggregation, and that the cell arrangement density is moderate to avoid excessive density causing the monolayer cell to become thick or uneven; at the same time, trypan blue staining or other active staining methods can be used to assess the cell survival rate to confirm that the cell survival rate is greater than 90%.

[0047] Through the above settings, the A coating provides a chemically modified material, which serves to enhance the hydrophilicity and cell adhesion of the chip surface and ensure that the cells can be firmly attached to the chip surface. The B coating is generally composed of a specific receptor molecule, namely the protocadherin 7 antigen, which can provide a specific biological recognition interface for the cell, so that the interaction between the cell and the target molecule, namely the protocadherin 7 antibody, is more efficient and specific. The B coating can simulate the biological environment of the cell surface and provide ideal biochemical support for the cultivation and experiment of the detection cell monolayer. Thereafter, the detection cell monolayer is a living cell layer cultured on the basis of the A coating and the B coating. The cell monolayer not only serves as a direct target for the antibody detection in the sample added later, but also forms a stable cell-to-cell interaction and environmental conditions in the microfluidic chip, thereby effectively reflecting the cell's response to antibodies or other exogenous molecules, and serving as a basic condition for the subsequent detection of cell adhesion.

[0048] In a preferred embodiment, a pretreated sample containing an antibody is added to a channel of a microfluidic chip. The pretreatment of the sample includes centrifugal extraction of the sample and dilution to an appropriate concentration. The sample concentration can be selected based on the ELISA experimental standard curve, and a dilution of 1:10 to 1:100 can be optionally selected. The specific concentration needs to be optimized according to the affinity of the antibody and the experimental requirements.

[0049] Furthermore, the microfluidic chip containing the sample is placed in a temperature-controlled box or a greenhouse at 36±1°C for 60 minutes of incubation. The specific incubation time can be optimized according to the experimental requirements to ensure that the antibody is fully bound to the PCDH7 protein antigen. During the incubation process, the binding of the antibody molecule to the PCDH7 antigen on the cell surface is achieved through non-covalent bonds (such as hydrogen bonds, van der Waals forces, etc.).

[0050] Furthermore, the microfluidic chip after incubation is washed with PBS buffer to remove unbound antibody molecules and ensure that only antibodies bound to the PCDH7 antigen on the cell surface remain on the surface. PBS washing can be performed by manual dripping, flushing or using the pump system in the microfluidic system to ensure uniform and thorough flushing. At the same time, it is necessary to use a slowly flowing liquid to flush during cleaning to avoid affecting the bound antibodies. After each cleaning, ensure that the PBS in the microfluidic chip is completely emptied.

[0051] In a preferred embodiment, a specific secondary antibody suitable for the target protocadherin 7 antibody is selected. The secondary antibody is usually an antibody against the Fc region of the primary antibody (i.e., the protocadherin 7 antibody in the present technical solution), and the secondary antibody carries a detectable marker, such as a fluorescent marker, an enzyme marker (e.g., horseradish peroxidase, HRP) or gold nanoparticles.

[0052] Further, after cleaning the surface of the microfluidic chip, the prepared secondary antibody solution is added through the inlet of the microfluidic chip to ensure that the secondary antibody can react with the previously bound primary antibody (i.e., PCDH7 antibody) to form a stable secondary antibody-primary antibody complex. The addition of the secondary antibody solution usually needs to be controlled by the flow rate of the microfluidic system to ensure that it is evenly distributed on the surface of the chip. Then the binding of the secondary antibody and the primary antibody complex is incubated. The incubation time is usually 30 to 60 minutes, during which time the secondary antibody will bind to the Fc region on the primary antibody to form a stable antibody-antigen complex. By adjusting the incubation time, the signal intensity and background noise can be optimized. After the incubation is completed, the microfluidic chip is thoroughly washed again with PBS buffer to remove the unbound secondary antibody and ensure that only the secondary antibody-primary antibody complex that has been bound to the target antigen is retained.

[0053] Furthermore, the microfluidic chip after binding with the secondary antibody is subjected to signal amplification processing. For example, if the secondary antibody carries an enzyme label, a substrate can be further added to develop color or emit light, thereby significantly amplifying the signal; if the secondary antibody carries a fluorescent molecule, an appropriate fluorescence microscope or flow cytometer can be used for detection. During the signal detection process, the intensity of the target signal is recorded to verify the degree of binding between the antibody and the antigen. By adopting a corresponding detection platform, such as a fluorescence microscope, a multifunctional microplate reader, etc., the signal is quantitatively or qualitatively analyzed, and the antibody binding is evaluated by measuring light absorption, fluorescence emission or reflection at a specific wavelength. A stronger signal indicates that the antibody is highly bound to the target antigen, and subsequent cell adhesion detection can be continued.

[0054] The cell adhesion force refers to the binding strength between the target cells on the test cell monolayer and the coating layer. In the cell adhesion test described below, the forces applied to the target cells mentioned may be uniform or not completely uniform, and may show differences on the contact surface.

[0055] Preferably, the cell binding adhesion force is determined by applying a force of known strength to the target cells on the coating B. It should be noted when understanding this step that the applied force may be perpendicular to the surface (for example, perpendicular to the z-axis direction as shown in the figure), that is, the applied force is perpendicular to the xy plane of the cells attached to the coating B. This type of force can be a centrifugal force or an acoustic force. In other embodiments, the applied force can also be lateral, such as in the x-axis or y-axis direction, and this type of force can be a shear force.

[0056] Preferably, the forces applied are controllable and should be approximately equal across multiple cells in the test cell monolayer. The forces applied to the cells to be tested can be detected by optical means, such as by microscopy or other means. Cell shedding events can be monitored and counted.

[0057] Preferably, the method for calculating the cell adhesion force may further include collecting and counting cells that are detached due to the force of known value applied. In this embodiment, the cells to be detected may be provided with a light-activated label, and the light-activated label may be a fluorescent dye that is activated by irradiation with light of appropriate wavelength in a specific interaction area of ​​the device (e.g., the central area below the acoustic force sensor). Subsequently, the cells may be sorted using fluorescence activated cell sorting, and only those cells that are fluorescently activated will be collected, thereby obtaining cells that have been subjected to the force of known value.

[0058] It should be understood that the known force values ​​described above are nominally known values, not the exact force applied to each cell. Since it is difficult to accurately predict the average size, density, compressibility and other parameters of cells, the applied force may only be based on theoretical calculations or calibrated by using test particles with specific properties.

[0059] In a preferred embodiment, various types of forces can be applied to the microfluidic chip through a microfluidic device, including shear force, centrifugal force, acoustic force, electric field force or other constant mechanical force.

[0060] In an exemplary embodiment, the application of shear force mainly depends on fluid flow. The microfluidic chip can be provided with an elongated flow channel to promote fluid flow. The size and shape of the flow channel (such as width, length and curvature) determine the fluid flow rate and the magnitude of the shear force. The flow rate of the fluid is controlled by an external pump or a pressure difference. The higher the fluid flow rate, the greater the shear force flowing through the cells. The magnitude of the shear force can be calculated by the flow rate of the fluid and the geometry of the flow channel. A pressure sensor or a flow rate sensor is used to monitor the velocity of the fluid, thereby indirectly evaluating the applied shear force. Among them, the shear force τ can be calculated by the fluid dynamics formula, that is: ; In the above formula, μ is the viscosity of the fluid, du / dy is the velocity gradient of the fluid, and dy is the distance in the vertical direction.

[0061] In an exemplary embodiment, the centrifugal force generated by rotating the microfluidic chip is often used to separate or apply force to cells by rotation. When the chip is placed in a centrifuge, a corresponding centrifugal force can be generated. In this embodiment, the microfluidic chip is configured to have multiple separated flow channels and integrated microchambers. A sample containing cells to be tested is added to the flow channel of the microfluidic chip. The microfluidic chip is placed in a centrifuge and the speed of the centrifuge is adjusted. The centrifugal force is calculated based on the speed and the radius of the chip, and the magnitude of the centrifugal force can be calculated by the formula: ; In the above formula, m is the mass of the cell, ω is the angular velocity, and r is the radius of rotation.

[0062] In an exemplary embodiment, the acoustic force is a force generated in a microfluidic chip by the propagation of ultrasound or sound waves. The microfluidic chip is configured to be a material and structure capable of transmitting sound waves. Typically, the microchannel or microchamber in the chip needs to be connected to an ultrasonic generator in order to apply the acoustic force. The ultrasonic generator generates sound waves of a specific frequency, which are transmitted through the microchannel and interact with the liquid and cells in the chip. Sound waves can propagate in the liquid to generate pressure waves. According to the frequency and power of the sound waves, the acoustic force applied to the cell can be adjusted. The magnitude of the acoustic force can be controlled by adjusting the ultrasonic frequency, intensity and propagation time. Typically, the magnitude of the acoustic force is proportional to the frequency and power. The propagation of the sound waves is monitored by an acoustic wave sensor or a vibration sensor, thereby indirectly monitoring the acoustic force applied to the cell.

[0063] In an exemplary embodiment, the electric field force is a force generated by applying an external electric field to charged particles or cells. In this embodiment, the microfluidic chip is configured as a conductive structure to apply an electric field in the flow channel. It is common to install electrodes at both ends of the flow channel. A voltage is applied to the electrodes by an external power supply to form an electric field. The strength of the electric field can be adjusted by the voltage and the distance between the electrodes. Charged cells will move along the electric field lines under the action of the electric field. The applied electric field force can be monitored by current monitoring or electric field strength measurement. The electric field force F e It can be calculated by the formula: ; In the above formula, q is the charge of the cell and E is the electric field strength.

[0064] Embodiment 2: This embodiment should be understood to include at least all the features of any of the above embodiments, and further improve on the basis thereof: In a preferred embodiment, control cells are added to the detection cell monolayer for detection comparison with the target cells. Control cells are cell types used for comparison in experiments, usually as reference objects for experiments. The role of control cells is to help determine the basic assumptions and effects of the test, to compare the behavior of the target cells, and to ensure that any changes under experimental conditions are caused by specific properties of the target cells, rather than other external factors.

[0065] Preferably, the control cells can be healthy cells or untreated cells, which are usually cells of the same type as the target cells but have not been subjected to any experimental treatment. Alternatively, the control cells can be invalid labeled cells, such as cells that do not express the target antigen or specific biomarker, to exclude the non-specific effects of the marker. Optionally, the control cells are other types of cells, such as normal epithelial cells, non-tumor cells, etc., for comparison with target cells (such as tumor cells).

[0066] Furthermore, the density of the control cells is kept substantially the same as that of the target cells.

[0067] In a preferred embodiment, at least two adhesion test processes are performed, and based on the basic results of the previous adhesion test, the approximate number of control cells to be added in the subsequent adhesion test, that is, the ratio of control cells to target cells, is determined.

[0068] Preferably, in the pre-detection, the adhesion value of a target is calculated, that is, in the detection of a predefined applied force value, the following is calculated: A target = number of shed cells / total number of cells; Adhesion value A target It can be used to infer the adhesion ability of target cells and serve as a basis for adjusting the ratio of control cells to target cells in subsequent tests.

[0069] Adjust the ratio of control cells to target cells in post-detection. Preferably, the ratio adjustment rule can be designed through the following strategies: The ratio range is set. When the adhesion of the target cells is high, the ratio of the control cells can be appropriately reduced to improve the sensitivity and accuracy of the detection. On the contrary, when the adhesion of the target cells is low, the ratio of the control cells is appropriately increased to ensure the effectiveness of the comparison effect of the control group cells.

[0070] Preferably, the following rules can be used: When A target >0.7, the proportion of control cells is 10% to 20%; When 0.3≤A target ≤0.7, the proportion of control cells is 20% to 40%; When A target <0.3, the proportion of control cells is 40% to 60%.

[0071] The ratio adjustment rules are further optimized through machine learning or regression models, and the relationship between adhesion and cell ratio is learned based on a large amount of experimental data. For example, support vector machines (SVM), decision trees, or linear regression methods are used to predict the adhesion of target cells and optimize the cell ratio setting based on historical experimental data.

[0072] Embodiment 3: This embodiment should be understood to include at least all the features of any of the above embodiments, and further improve on the basis thereof: For example, Figure 5A schematic diagram of a computer system 500 in which the diagnostic system described in this article can be implemented is depicted; the computer system 500 can control the operation of each working unit, module, and component in the system according to the current control program; and also includes collecting, storing, and processing the working data and detection data generated during the operation of the system, so as to ultimately achieve the expected effect of the fluorescent labeling system.

[0073] The computer system 500 includes a bus 502 or other communication mechanism for transmitting information, and one or more processors 504 coupled to the bus 502 for processing information; the processor 504 may be, for example, one or more general-purpose microprocessors; The computer system 500 also includes a main memory 506, such as a random access memory (RAM), a cache, and / or other dynamic storage device, coupled to the bus 502 for storing information and instructions to be executed by the processor 504; the main memory 506 may also be used to store temporary variables or other intermediate information during the execution of instructions to be executed by the processor 504; these instructions, when stored in a storage medium accessible to the processor 504, present the computer system 500 as a special-purpose machine customized to perform the operations specified in the instructions; The computer system 500 may also include a read-only memory (ROM) 508 or other static storage device coupled to the bus 502 for storing static information and instructions for the processor 504; a storage device 510 such as a disk, an optical disk, or a USB drive (flash drive) will be coupled to the bus 502 for storing information and instructions; And further, coupled to the bus 502 may also include a display 512 for displaying various information, data, media, etc., an input device 514 for allowing a user of the computer system 500 to control, manipulate, and / or interact with the computer system 500; A preferred way of interacting with the management system may be through a cursor control device 516, such as a computer mouse or similar control / navigation mechanism; Furthermore, the computer system 500 may also include a network device 518 coupled to the bus 502; wherein the network device 518 may include, for example, a wired network card, a wireless network card, a switching chip, a router, a switch, and other components; In general, the terms "engine", "component", "system", "database", etc., as used herein, may refer to logic embodied in hardware or firmware, or to a collection of software instructions, possibly with entry and exit points, written in a programming language such as Java, C, or C++; software components may be compiled and linked into executable programs, installed in a dynamic link library, or may be written in an interpreted programming language (e.g., BASIC, Perl, or Python); it should be understood that software components may be called from other components or from themselves, and / or may be called in response to detected events or interrupts; Software components configured to execute on a computing device may be provided on a computer-readable medium, such as a compact disc, digital video disc, flash drive, diskette, or any other tangible medium, or as a digital download (and may be initially stored) in a compressed or installable format that requires installation, decompression, or decryption prior to execution); such software code may be stored in part or in whole on a memory device of the executing computing device for execution by the computing device; software instructions may be embedded in firmware, such as an EPROM. It should also be understood that hardware components may consist of connected logic units (such as gates and flip-flops), and / or may consist of programmable units (such as programmable gate arrays or processors); Computer system 500 includes the techniques described herein that may be implemented using custom hardwired logic, one or more ASICs or FPGAs, firmware, and / or program logic that, in combination with the computer system, renders computer system 500 a special-purpose computing device; According to one or more embodiments, the techniques herein may be performed by computer system 500 in response to processor 504 executing one or more sequences of one or more instructions contained in main memory 506; such instructions may be read into main memory 506 from another storage medium, such as storage device 510; execution of the sequences of instructions contained in main memory 506 causes processor 504 to perform the process steps described herein; in alternative embodiments, hard-wired circuitry may be used in place of or in combination with software instructions; As used herein, the term "non-transitory media" and similar terms refer to any media that store data and / or instructions that cause a machine to operate in a specific manner; such non-transitory media may include non-volatile media and / or volatile media; non-volatile media include, for example, optical or magnetic disks, such as storage device 510; volatile media include dynamic memory, such as main memory 506; Among them, common forms of non-transitory media include, for example, floppy disks, diskettes, hard disks, solid-state drives, magnetic tapes or any other magnetic data storage medium, CD-ROMs, any other optical data storage medium, any physical medium having a pattern of holes, RAM, PROM and EPROM, FLASH-EPROM, NVRAM, any other memory chip or cartridge, and networked versions thereof; Non-transient media are distinct from transmission media but may be used in conjunction with transmission media; transmission media participate in the transmission of information between non-transient media; for example, transmission media include coaxial cables, copper wires, and optical fibers, including the wires that make up bus 502; transmission media may also take the form of sound waves or light waves, such as radio waves and infrared data communications.

[0074] Although the present application has been described above with reference to various embodiments, it should be understood that many changes and modifications may be made without departing from the scope of the present application. That is, the methods, systems and devices discussed above are examples. Various configurations may appropriately omit, replace or add various processes or components. For example, in alternative configurations, the method may be performed in an order different from the order described, and / or various components may be added, omitted and / or combined. Moreover, the features described with respect to certain configurations may be combined in various other configurations, such as different aspects and elements of the configuration may be combined in a similar manner. In addition, as the technology develops, the elements therein may be updated, i.e., many elements are examples and do not limit the scope of the present disclosure or claims.

[0075] Specific details are given in the specification to provide a thorough understanding of the exemplary configurations including implementations. However, the configurations may be practiced without these specific details. For example, well-known circuits, processes, algorithms, structures, and techniques have been shown without unnecessary details to avoid obscuring the configurations. This description provides only example configurations and does not limit the scope, applicability, or configurations of the claims. On the contrary, the foregoing description of the configurations will provide those skilled in the art with an enabling description for implementing the described techniques. Various changes may be made to the functions and arrangements of the elements without departing from the spirit or scope of the present disclosure.

[0076] In summary, it is intended that the above detailed description is considered to be illustrative rather than restrictive, and it should be understood that the above embodiments should be understood to be only used to illustrate the present invention and not to limit the scope of protection of the present invention. After reading the contents of the present invention, the technician can make various changes or modifications to the present invention, and these equivalent changes and modifications also fall within the scope defined by the claims of the present invention.

Claims

1. A disease diagnosis system based on protocadherin 7 antibody detection, characterized in that: The diagnostic system comprises a microfluidic device and a microfluidic chip applied to the microfluidic device; wherein the following coatings are sequentially prepared on the substrate of the microfluidic chip: A coating, which is one or more polypeptides or a mixture of polypeptides; Coating B, protocadherin 7 antigen solution; The detection cell monolayer is formed by using cells in the sample to be detected and adhering to the surface of the B coating to form a monolayer of cells; The diagnostic system applies a known force to the detection cell monolayer on the microfluidic chip by using the microfluidic device to detect the adhesion of multiple cells on the detection cell monolayer, and calculates the adhesion performance of the cells in the detection cell monolayer by statistical means.

2. The diagnostic system according to claim 1, characterized in that: The diagnostic system also includes a detection unit and a control unit; The detection unit is used to count the cells shed from the B coating; The control unit is used to control the microfluidic device and the detection unit to work in coordination.

3. The diagnostic system according to claim 2, characterized in that: The detection unit also includes counting the cells that fall off from the B coating and express protocadherin 7 antibodies.

4. The diagnostic system according to claim 3, characterized in that: The microfluidic chip comprises a plurality of flow channels, and each flow channel allows the adhesion of different cell types to be independently detected for multiplex diagnosis analysis of diseases.

5. A disease diagnosis method based on protocadherin 7 antibody detection, characterized in that: The diagnostic method is used in the diagnostic system as claimed in claim 4; the diagnostic method comprises the following steps: S100: preparing a microfluidic chip having a coating A and a coating B, wherein the microfluidic chip after preparation comprises a microfluidic chip substrate, a coating A and a coating B stacked in sequence; S200: diluting the sample to be tested to a suitable concentration, coating it on the surface of the coating, and then incubating and washing it to form a test cell monolayer with a monolayer cell structure; S300: adding a labeled secondary antibody to the surface of the microfluidic chip and incubating the chip, and then observing the binding between the sample and the secondary antibody; S400: Apply the microfluidic device to the microfluidic chip to detect cell adhesion and record the cell detachment ratio; S500: Calculate the cell adhesion strength and the ratio of cell adhesion to cell detachment to perform disease analysis on the donor of the sample.

6. The diagnostic method according to claim 5, characterized in that: In step S400, shear force, centrifugal force, acoustic force, electric field force or other forms of force are used to detect cell adhesion force.

7. The diagnostic method according to claim 6, characterized in that: In step S400, the magnitude of the applied force is controllable in at least one direction.

8. The diagnostic method according to claim 7, characterized in that: In step S400, the applied force is directed through different flow channel configurations of the microfluidic device to achieve different mechanical conditions in different areas, thereby accurately detecting cell adhesion on the microfluidic chip.

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