A disease diagnosis method based on protocadherin 7 antibody detection and a diagnosis system thereof

Abstract

The application discloses a disease diagnosis system based on protocadherin 7 antibody and a diagnosis method thereof. The system comprises a microfluidic device and a microfluidic chip used in cooperation. The base surface of the microfluidic chip is coated with an alpha coating and a beta coating in sequence, wherein the alpha coating is one or more polypeptides, and the beta coating is a protocadherin 7 antigen solution. A detection cell monolayer is formed on the surface of the beta coating, and cells in a sample to be detected adhere to the surface. After incubation and washing, the cells are subjected to adhesion force detection by the microfluidic device. The adhesion force performance of the cells is statistically analyzed, and the sample is further subjected to disease diagnosis. The system further comprises a detection unit and a control unit, which are used for statistically analyzing the cell detachment and cooperatively detecting. The diagnosis method can be applied to the diagnosis and analysis of multiple diseases, especially the disease monitoring based on the change of cell adhesion force.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of medical detection, and particularly relates to a disease diagnosis method based on protocadherin 7 antibody detection and a diagnosis system thereof. BACKGROUND

[0002] Protocadherin 7 (PCDH7) is a cell adhesion molecule that widely participates in cell adhesion and signal transmission between cells. In many diseases (such as cancer, immune diseases, etc.), the expression and function of PCDH7 may be abnormal. By detecting the adhesion force of cells with PCDH7 antibody expression, it can be evaluated whether the antibody effectively interacts with PCDH7 and has an impact on the adhesion behavior of cells. Changes in cell adhesion force can reflect the intervention of antibodies on the adhesion properties of cells in a specific disease state, helping to understand the potential biological functions of PCDH7 antibodies.

[0003] PCDH7 antibodies can affect cell adhesion force, so by measuring changes in cell adhesion force, important clues can be provided for the application of PCDH7 antibodies in disease diagnosis. For example, certain tumor cells or immune cells may exhibit abnormal cell adhesion force due to changes in PCDH7 expression, which provides an effective detection means for disease diagnosis based on PCDH7 antibodies. By comparing with normal cells, the effect of antibodies on cell adhesion force in pathological conditions can help doctors judge the type or severity of the disease.

[0004] According to the disclosed technical solution, the technical solution with publication number US07935790B2 proposes a reagent for detecting protein phosphorylation in the T cell receptor signaling pathway, which includes a component for detecting protocadherin. The technical solution with publication number CN103153331A proposes a humanized antibody targeting the EC1 domain of cadherin-11 and related compositions and methods, which realizes a method of administering a therapeutically effective amount of the humanized antibody of the present application to treat a cadherin-11 mediated disorder in a mammalian subject.

[0005] The above technical solutions all propose various technical solutions for realizing and optimizing protocadherin antibody detection. 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 application. It is not admitted that any of the material referred to is part of the public knowledge. SUMMARY

[0007] The present application discloses a disease diagnosis system based on protocadherin 7 antibody and a diagnosis method thereof. The system comprises a microfluidic device and a microfluidic chip used in cooperation with the microfluidic device. The substrate surface of the microfluidic chip is coated with an alpha coating and a beta coating in sequence, wherein the alpha coating is one or more polypeptides, and the beta coating is a protocadherin 7 antigen solution. A detection cell monolayer is formed on the surface of the beta coating, and cells in a sample to be detected adhere to the surface. After incubation and washing, the cells are subjected to adhesion force detection by the microfluidic device. The adhesion force performance of the cells is statistically analyzed, and the sample is further subjected to disease diagnosis. The system further comprises a detection unit and a control unit for statistical analysis of cell detachment and cooperative detection. The diagnosis method can be applied to the diagnosis and analysis of multiple diseases, especially diseases based on changes in cell adhesion force.

[0008] The present application adopts the following technical solution: 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 prepared on the substrate of the microfluidic chip in sequence:

[0009] The alpha coating is one or more polypeptides or a mixture of polypeptides;

[0010] The beta coating is a protocadherin 7 antigen solution;

[0011] The detection cell monolayer is formed by adhering cells in a sample to be detected to the surface of the beta coating to form a monolayer of cells;

[0012] The diagnosis system uses the microfluidic device to apply a known force to the detection cell monolayer on the microfluidic chip to detect the adhesion of multiple cells in the detection cell monolayer, and calculates the adhesion force performance of the cells in the detection cell monolayer by statistical methods.

[0013] Preferably, the diagnosis system further comprises a detection unit and a control unit;

[0014] The detection unit is used to statistically analyze the cells detached from the beta coating;

[0015] The control unit is used to control the microfluidic device and the detection unit to work cooperatively.

[0016] Preferably, the detection unit further comprises cells with protocadherin 7 antibody performance that are detached from the beta coating.

[0017] Preferably, the microfluidic chip comprises multiple flow channels, and each flow channel allows independent detection of the adhesion force of different cell types for multiple disease diagnosis and analysis.

[0018] Meanwhile, a disease diagnosis method based on the original calcium adhesion protein 7 antibody detection is provided, and the diagnosis method is used for the diagnosis system.

[0019] S100: a microfluidic chip with a coating layer A and a coating layer B is prepared, and the microfluidic chip after preparation is a microfluidic chip substrate, the coating layer A and the coating layer B stacked in sequence;

[0020] S200: after diluting the sample to be detected to a suitable concentration, the sample is coated on the surface of the coating layer B, and then incubation and washing are performed to form a detection cell monolayer with a single-layer cell structure;

[0021] S300: the labeled secondary antibody is added to the surface of the microfluidic chip and incubated, and then the binding of the sample and the secondary antibody is observed;

[0022] S400: the cell adhesion force is detected by applying a microfluidic device to the microfluidic chip, and the cell detachment ratio is recorded;

[0023] S500: the cell adhesion strength and the detachment ratio after adhesion are calculated to analyze the disease of the sample donor.

[0024] Preferably, in step S400, the size of the applied force is controllable in at least one direction.

[0025] Preferably, in step S400, the applied force is oriented by different flow channel configurations of the microfluidic device to achieve different mechanical conditions in different areas, so as to accurately detect the cell adhesion force on the microfluidic chip.

[0026] The present application has the following beneficial effects:

[0027] The diagnosis system of the present application can accurately detect the interaction between cells and the surface coating at the micro level by combining microfluidic chip technology and cell adhesion force detection, especially in the detection of disease markers involving the original calcium adhesion protein 7 antibody. After obtaining information through the detection of cell adhesion force for the first time, the post-experiment can adjust the cell proportion according to the pre-detection result to further improve the sensitivity and accuracy of the detection. This not only can quickly and reliably detect the potential risk of the disease, but also provides more valuable quantitative data for the biological characteristics of the sample cells.

[0028] The microfluidic chip design adopted in the technical solution allows multiple flow channels to work in parallel, enabling the detection of adhesion forces of different cell types in the same experiment and multiple disease analysis. The precise preparation of the chip surface through the A coating layer and the B coating layer ensures the stability and controllability of the cell adhesion conditions, so that the cell adhesion force under different experimental conditions can be accurately measured. The 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.

[0029] The microfluidic chip of the technical solution adopts materials with high chemical stability and mechanical stability, and supports both disposable use and cleaning use after recovery in design, which has higher economic efficiency and environmental friendliness. In addition, the preparation process of the A coating layer and the B coating layer has high customizability, which can adjust the coating material and concentration according to different disease markers or cell types, further enhancing the wide applicability of the technology and adapting to the detection needs of different fields. BRIEF DESCRIPTION OF DRAWINGS

[0030] The present application can be further understood from the following description in conjunction with the accompanying drawings. The components in the drawings are not necessarily drawn to scale, but emphasis is placed on showing the principles of the embodiments. In different views, the same reference numerals designate corresponding parts.

[0031] Sequence number explanation: 10-microfluidic device; 20-microfluidic chip; 22-substrate; 24-A coating layer; 26-B coating layer; 28-detection cell monolayer; 30-detection unit; 40-control unit; 500-computer system; 502-bus; 504-processor; 506-main memory; 508-readonly memory; 510-storage device; 512-display; 514-input device; 516-cursor control device; 518-network device;

[0032] Figure 1 The architecture of the diagnostic system described in the embodiments of the present application

[0033] Figure 2 The schematic diagram of each layer in the microfluidic chip in the embodiments of the present application

[0034] Figure 3 The schematic diagram of each direction of the force applied to the microfluidic chip in the embodiments of the present application

[0035] Figure 4 The schematic diagram of the steps of the diagnostic method described in the embodiments of the present application

[0036] Figure 5 The schematic diagram of the computer system used by the diagnostic system in the embodiments of the present application DETAILED DESCRIPTION

[0037] In order to make the technical solutions and advantages of the present application more clear, the present application will be further described in detail below in combination with its embodiments; it should be understood that the specific embodiments described herein are only used to explain the present application and not to limit the present application. Other systems, methods and / or features of the embodiments will become apparent to those skilled in the art upon inspection of the following detailed description. It is intended that all such additional systems, methods, features and advantages be included within this description. They are included within the scope of the present application and are protected by the accompanying claims. Additional features of the disclosed embodiments are described in the following detailed description, and will be apparent to one of ordinary skill in the art upon examination of the following figures and detailed description.

[0038] The same or similar reference numerals in the drawings of the embodiments of the present application correspond to the same or similar components; in the description of the present application, it should be understood that if the orientations or positional relationships indicated by the terms "upper", "lower", "left", "right" and the like are based on the orientations or positional relationships shown in the drawings, they are only for the convenience of describing the present application and simplifying the description, and do not indicate or imply that the devices or components referred to must have a particular orientation. The orientation and operation are constructed in a particular orientation, therefore the terms describing the positional relationship in the drawings are only used for exemplary illustration, and cannot be understood as a limitation on the present patent, and for those of ordinary skill in the art, the specific meanings of the above terms can be understood according to the specific circumstances.

[0039] Embodiment one: as shown in the accompanying Figure 1 Fig. 1, a disease diagnosis system based on N-cadherin 7 antibody detection is proposed; the diagnosis system comprises a microfluidic device 10 and a microfluidic chip 20 applied to the microfluidic device; as shown in the accompanying Figure 2 Fig. 2, the following coatings are prepared in sequence on the substrate 22 of the microfluidic chip:

[0040] The A coating 24 is one or more polypeptides or a mixture of polypeptides;

[0041] The B coating 26 is a N-cadherin 7 antigen solution;

[0042] The detection cell monolayer 28 is formed by using cells in the sample to be detected and adhering to the surface of the B coating as a monolayer of cells;

[0043] The diagnosis system uses the microfluidic device to apply a known force to the detection cell monolayer on the microfluidic chip to detect the adhesion of multiple cells in the detection cell monolayer, and calculates the adhesion force of the cells in the detection cell monolayer by statistical means.

[0044] Preferably, the diagnosis system further comprises a detection unit 30 and a control unit 40;

[0045] The detection unit 30 is used to count the cells falling off from the second coating layer;

[0046] The control unit 40 is used to control the microfluidic device 10 and the detection unit 30 to work cooperatively.

[0047] Preferably, the detection unit further comprises counting the cells falling off from the second coating layer and having the expression of the anti-osteocalcin 7 antibody.

[0048] Preferably, the microfluidic chip comprises a plurality of flow channels, and each flow channel allows independent detection of the adhesion force of different cell types, for multiple diagnostic analysis of diseases.

[0049] Meanwhile, a disease diagnosis method based on the detection of the anti-osteocalcin 7 antibody is provided, and the diagnosis method is used for the diagnosis system; the diagnosis method comprises the following steps:

[0050] S100: preparing a microfluidic chip with a first coating layer and a second coating layer, wherein the microfluidic chip is a microfluidic chip substrate, a first coating layer and a second coating layer stacked in sequence after preparation;

[0051] S200: diluting the sample to be detected to a suitable concentration, coating the surface of the second coating layer, then incubating and washing, and forming a detection cell monolayer with a single cell structure;

[0052] S300: adding a labeled secondary antibody to the surface of the microfluidic chip and incubating, then observing the binding of the sample and the secondary antibody;

[0053] S400: applying a microfluidic device to the microfluidic chip for cell adhesion force detection and recording the cell detachment ratio;

[0054] S500: calculating the cell adhesion strength and the detachment ratio after adhesion to analyze the donor of the sample for diseases.

[0055] Preferably, in step S400, the size of the applied force is controllable in at least one direction.

[0056] Preferably, in step S400, the applied force is directional through different flow channel configurations of the microfluidic device, so as to realize different mechanical conditions in different areas, thereby accurately detecting the cell adhesion force on the microfluidic chip.

[0057] The internal part of the microfluidic chip comprises one or more internal cavities, and the internal cavities are usually in the form of flow channels to support the flow of fluid inside; and one or more discontinuities can be arranged between the flow channels, and the one or more discontinuities are configured to support the independent flow of one or more fluids in the flow channels without interfering with each other; and, preferably, the microfluidic chip can further comprise one or more input and output connectors for connection purposes, and allow the fluid to be transported into the microfluidic chip by upstream transport, and received in the downstream device after the fluid flows out of the microfluidic chip.

[0058] Preferably, the microfluidic chip is selected to have high chemical and mechanical stability; preferably, the microfluidic chip can be made of high-transparency glass, or more preferably, made of polydimethylsiloxane (PDMS), polymethyl methacrylate (PMMA), polycarbonate (PC) and the like; and the microfluidic chip can be disposable or can be reused by being completely cleaned after being recovered.

[0059] Preferably, the microfluidic chip is pretreated before coating with the following steps:

[0060] Cleaning the substrate: using ultrapure water, ethanol, isopropanol or 0.1% SDS solution, immerse the microfluidic chip in an ultrasonic cleaner for cleaning and air dry.

[0061] Surface activation: surface activation treatment can enhance the hydrophilicity of the chip surface and the adhesion of the coating; preferably, immerse the microfluidic chip in a 5% silanization reagent solution for 10 minutes; then rinse the surface with ultrapure water and dry at 60°C for 30 minutes; then place the microfluidic chip in a plasma chamber, set the oxygen flow rate to 40-50W, and process for 3 minutes, then immediately apply the A coating.

[0062] 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 facilitate the subsequent B coating and the attachment of the cell monolayer for detection

[0063] Preferably, the A coating can 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 A coating provides a positively charged surface, which helps to improve the stability of the subsequent coating (B coating) on the substrate surface, and enhances the adhesion of the surface to cells;

[0064] And the material of the A coating is selected based on the selected cells of the cell monolayer for detection, for example, neuron cells are more dependent on poly-L-lysine, while immune cells can be more dependent on other biomolecule polypeptides;

[0065] The first coating is prepared in step S100 to uniformly coat the surface of the microfluidic chip substrate. Preferably, the parameters of the first coating are as follows: a common concentration range of 50-100 μg / mL; an incubation time of 30-60 minutes; a temperature control of room temperature to 37.5°C; after the coating is completed, a buffer solution (e.g., PBS) is used to clean the surface to remove unbound polypeptides, and finally sterile air is used to gently blow the surface to dry it.

[0066] Further, the first coating is surface-activated. Preferably, NHS ester or glutaraldehyde is used to achieve enhanced stability of the first coating and the chip substrate.

[0067] In a preferred embodiment, a second coating is prepared on the first coating. The second coating is mainly composed of specific proteins including protocadherin 7 (PCDH7) antigens or extracellular matrix proteins, and can also include other active proteins such as collagen, fibronectin, and laminin.

[0068] Preferably, the concentration of the specific proteins is greater than 95% to reduce the interference of impurities on cell adhesion.

[0069] Preferably, the specific proteins of the second coating are prepared using DPBS or sterile PBS as a solution medium.

[0070] Preferably, the solution preparation of the second coating can include the following steps:

[0071] The specific proteins of protocadherin 7 antigens or extracellular matrix proteins with appropriate purity are prepared into a solution with a concentration of 2-10 μg / mL and dissolved in a solution medium. The mixed solution is filtered through a 0.22 μm filter before use to ensure sterility, and the mixed solution is gently mixed to avoid protein aggregation or denaturation.

[0072] The mixed solution is uniformly dropped onto the surface of the microfluidic chip. Optionally, about 10-50 μL of the mixed solution is used per microfluidic chip, which can be adjusted according to the actual size of the microfluidic chip; or the solution is injected into the chip and microchannels through a microsyringe. To ensure uniform coating, the surface is observed under a microscope to determine whether it is completely covered without bubbles or blank areas. After the coating is completed, the chip is placed in a humid environment and incubated at 36±1°C for 30-60 minutes to ensure that the proteins are fully bound to the surface of the first coating.

[0073] Further, after the incubation is completed, the chip surface is gently rinsed with sterile PBS for 2-3 times to remove unbound protein substances, and then dried with sterile nitrogen.

[0074] To verify the quality of the coating, the uniformity and integrity of the surface coating can be observed by microscopy, and the biological activity of the protein can be confirmed by antibody detection or cell adhesion experiments. In addition, the support ability of the coating surface for cell monolayer needs to be tested to ensure that it can form a uniform and stable cell adhesion surface. Preferably, the detection cell monolayer can be one of target cells, effector cells, tumor cells or neuronal cells; such cells form a uniform monolayer structure on the coating for subsequent cell adhesion detection and antibody binding.

[0075] Then the sample to be detected for the present technical solution is processed. The sample refers to a biological sample derived from a patient or experimental subject. The sample can include various cell components such as blood, tissue or tumor cells. The sample itself is not a cell, but a collection of cells and other components such as proteins, DNA, etc. In these samples, there can be different types of cells, some of which can express protocadherin 7 on their surface, i.e. as target cells that need to be detected in the present technical solution.

[0076] Protocadherin 7 (PCDH7) is a cell surface adhesion molecule that is usually involved in cell-cell adhesion and exhibits abnormal expression patterns in some diseases such as cancer. By protocadherin 7 antibody, protocadherin 7 on the surface of cells can be specifically recognized and bound. By binding of the antibody to protocadherin 7 on the surface of the target cell, a detectable signal such as a fluorescent signal or an enzyme-labeled signal is generated, which helps to confirm whether the cell exhibits abnormal protocadherin 7 expression and further detects the target cells.

[0077] In a preferred embodiment, the sample to be detected is pretreated and the detection cell monolayer is prepared. The steps include: using trypsin to digest adherent cells; centrifuging the sample and diluting it to the appropriate concentration, which can be 2-5 minutes. Then the suspension cells or immune cells are collected by centrifugation, the centrifuge is set at 200xg for 5 minutes, and the cells are resuspended in an appropriate amount of culture medium. The digestive enzyme or centrifugation buffer is removed, and finally the concentration of cells in the sample is adjusted to 80-100 x 10 6 cells / mL.

[0078] Further, the cells in the sample are seeded on the surface of the second coating. Optionally, a pipette is used to drop 10-50 μL of cell suspension on the surface of the second coating each time. Then the microfluidic chip is placed in an incubator for 30-60 minutes to allow the cells to contact and adhere to the second coating and to ensure that the surface of the microfluidic chip remains wet to avoid dehydration of the cells or damage to the coating. The microfluidic chip is placed in an incubator at 36±1°C and 5% CO2 concentration for 24-48 hours, and growth factors or specific culture additives are added as needed to complete the culture of the detection cell monolayer.

[0079] Preferably, the cells of the detection cell monolayer are distributed on the surface of the second coating in a substantially uniform monolayer. Preferably, the cells of the detection cell monolayer can be distributed on the surface of the second coating at a lower density. The desired distribution density of the cells of the detection cell monolayer can be achieved by adjusting the volume of cell suspension introduced into the microfluidic chip each time, changing the culture temperature, the culture time, etc.

[0080] If the cells are too dense, the interaction between the cells can be disturbed, and the difference in cell behavior cannot be effectively observed. Therefore, a reasonable cell density can ensure that each cell can effectively interact with the target cells in the sample.

[0081] Finally, the quality of the detection cell monolayer is controlled. Preferably, a microscope is used to observe the morphology of the cells to ensure that the cells are uniformly distributed, well adhered, and not aggregated, and the arrangement density of the cells is moderate to avoid excessive density causing the monolayer cells to be thick or uneven. Trypan blue staining or other vital staining methods can be used to evaluate the survival rate of the cells to confirm that the survival rate of the cells is greater than 90%.

[0082] Through the above settings, the first coating provides a chemical modification material to enhance the hydrophilicity and cell adhesion of the surface of the chip to ensure that the cells can stably adhere to the surface of the chip. The second coating is generally composed of specific receptor molecules, i.e., N-cadherin 7 antigens, which can provide a specific biological recognition interface for the cells to make the interaction between the cells and the target molecules, i.e., N-cadherin 7 antibodies, more efficient and specific. The second coating can simulate the biological environment of the cell surface to provide ideal biochemical support for the culture and experiment of the detection cell monolayer. Then, the detection cell monolayer is a layer of living cells cultured on the basis of the first coating and the second coating. The cell monolayer not only serves as a direct target for the detection of antibodies in the sample added subsequently, but also forms stable intercellular interactions and environmental conditions in the microfluidic chip to effectively reflect the response of the cells to the antibodies or other exogenous molecules and serve as a basic condition for the subsequent detection of cell adhesion.

[0083] In preferred embodiments, the pre-processed sample containing the antibody is added to the channel of the microfluidic chip. The pre-processing of the sample includes centrifugal extraction and dilution to a suitable concentration. The sample concentration can be selected according to the standard curve of the ELISA experiment, and the dilution ratio can be selected as 1:10-1:100. The specific concentration can be optimized according to the affinity of the antibody and the experimental requirements.

[0084] Further, the microfluidic chip containing the sample is placed in a temperature-controlled box or greenhouse at 36±1℃ for 60 minutes of incubation. The specific incubation time can be optimized according to the experimental requirements to ensure that the antibody is fully combined with the PCDH7 protein antigen. During the incubation process, the combination of antibody molecules and cell surface PCDH7 antigen is realized through non-covalent bonds (such as hydrogen bonds, van der Waals forces, etc.).

[0085] Further, the microfluidic chip after incubation is washed with PBS buffer to remove unbound antibody molecules, ensuring that only the antibody combined with the cell surface PCDH7 antigen remains on the surface. PBS washing can be performed by manual dripping, flushing, or using a pump system in the microfluidic system to ensure uniform and thorough flushing. Slowly flowing liquid is used during washing to avoid affecting the bound antibody. After each washing, the PBS in the microfluidic chip is completely emptied.

[0086] In preferred embodiments, 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 label such as a fluorescent label, an enzyme label (e.g., horseradish peroxidase, HRP), or gold nanoparticles, etc.

[0087] Further, after washing 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 combined 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 uniform distribution on the chip surface. Then the secondary antibody and the primary antibody complex are incubated. The incubation time is usually 30-60 minutes, during which the secondary antibody binds to the Fc region of 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 incubation, the microfluidic chip is again thoroughly washed with PBS buffer to remove unbound secondary antibodies, ensuring that only the secondary antibody-primary antibody complex that has been combined with the target antigen is retained.

[0088] Further, the microfluidic chip after binding with the secondary antibody is subjected to signal amplification processing, for example, the secondary antibody carries enzyme labels, and substrates can be further added for color development or luminescence, thereby significantly amplifying the signal; if the secondary antibody carries fluorescent molecules, appropriate fluorescence microscopes or flow cytometers can be used for detection. In the signal detection process, the intensity of the target signal is recorded to verify the binding degree between the antibody and the antigen, and by using corresponding detection platforms such as fluorescence microscopes and multifunctional enzyme labelers, the signal is quantitatively or qualitatively analyzed, and the binding of the antibody is evaluated by measuring the light absorption, fluorescence emission or reflection at a specific wavelength. A stronger signal indicates that the antibody has a higher degree of binding with the target antigen, and subsequent cell adhesion detection can be continued.

[0089] The cell adhesion force refers to the binding strength of the target cells on the detection cell monolayer to the coating layer, and in the following cell adhesion force detection, the forces applied to the target cells can be uniform or not completely uniform, and can exhibit differences on the contact surface.

[0090] Preferably, the cell adhesion force is determined by applying a known strength of force to the target cells on the coating layer. It should be noted that in understanding this step, the applied force can be perpendicular to the surface (for example, perpendicular to the z-axis direction as shown in the drawings), that is, the applied force is perpendicular to the x-y plane of the cells attached to the coating layer, and this type of force can be a centrifugal force or an acoustic force. In other embodiments, the applied force can also be transverse, for example, in the x-axis or y-axis direction, and this type of force can be a shear force.

[0091] Preferably, the applied force is controllable, and the force applied to the cells in the detection cell monolayer should be approximately equal. The force applied to the cells to be detected can be detected by optical means, such as by a microscope or other means. Cell shedding events can be monitored and counted.

[0092] Preferably, the calculation of the cell adhesion force can further include counting by collecting cells shed by the applied known value of force. In this embodiment, the cells to be detected can be provided with a light-activated tag, and the light-activated tag can be activated by irradiation with light of an appropriate wavelength, and the fluorescent dye in the specific interaction area of the device (for example, the central area under the acoustic force sensor) is activated. Subsequently, the cells can be sorted using fluorescence-activated cell sorting, and only those cells activated by fluorescence will be collected, thereby obtaining cells subjected to a known value of force.

[0093] It is to be understood that the above-mentioned known values of force are nominal known values and not the exact force applied to each cell. Due to the difficulty in accurately predicting the average size, density, compressibility, and other parameters of the cells, the force applied can be based only on theoretical calculations or calibrated by using test particles with specific properties.

[0094] In preferred embodiments, various types of forces can be applied to the microfluidic chip by the microfluidic device, including shear force, centrifugal force, acoustic force, electric field force, or other constant mechanical force.

[0095] In exemplary embodiments, the shear force is applied mainly by fluid flow. The microfluidic chip can be provided with an elongated flow channel to push the 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 fluid flow rate is controlled by an external pump or pressure difference. The higher the fluid flow rate, the greater the shear force on the cells. The magnitude of the shear force can be calculated by the fluid flow rate and the geometry of the flow channel. A pressure sensor or flow rate sensor is used to monitor the fluid flow rate, thereby indirectly evaluating the applied shear force. The shear force τ can be calculated by the fluid dynamics formula:

[0096] ;

[0097] 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.

[0098] In exemplary embodiments, the centrifugal force generated by rotating the microfluidic chip is commonly used to separate or apply force to the 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 provided with multiple separate flow channels and integrated microchambers. The sample containing the cells to be detected is added to the flow channel of the microfluidic chip. The microfluidic chip is placed in the centrifuge, and the speed of the centrifuge is adjusted. The centrifugal force is calculated according to the speed and the radius of the chip, and the magnitude of the centrifugal force can be calculated by the formula:

[0099] ;

[0100] In the above formula, m is the mass of the cells, ω is the angular velocity, and r is the radius of rotation.

[0101] In exemplary embodiments, the acoustic force is a force generated in the microfluidic chip by the propagation of ultrasonic or acoustic waves. The microfluidic chip is configured with materials and structures that are capable of transmitting acoustic waves. Typically, microchannels or microchambers in the chip need to be connected to an ultrasonic wave generator in order to apply acoustic force. A specific frequency of acoustic wave is generated by the ultrasonic wave generator, and the acoustic wave is transmitted through the microchannels and interacts with the liquid and cells in the chip. The acoustic wave can propagate in the liquid and generate pressure waves. Depending on the frequency and power of the acoustic wave, the acoustic force applied on the cells can be adjusted. The magnitude of the acoustic force can be controlled by adjusting the frequency, intensity, and propagation time of the ultrasonic wave. Typically, the magnitude of the acoustic force is proportional to the frequency and power. The propagation of the acoustic wave is monitored by an acoustic or vibration sensor, thereby indirectly monitoring the acoustic force applied on the cells.

[0102] In exemplary embodiments, the electric field force is a force generated by an applied electric field acting on charged particles or cells. In such embodiments, the microfluidic chip is configured as an electrically conductive structure to apply an electric field in the flow channel. It is common to install electrodes at both ends of the flow channel. An external power source is used to apply a voltage to the electrodes 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. e It can be calculated by the formula:

[0103] ;

[0104] In the above formula, q is the charge of the cell, and E is the electric field strength.

[0105] Embodiment two: This embodiment should be understood as at least containing all the features of any one of the preceding embodiments, and further improving on the basis thereof:

[0106] In preferred embodiments, control cells for comparison with the target cells are added in the detection cell monolayer. The control cells are cell types used for comparison in the experiment, and are usually used as reference objects in the experiment. The role of the control cells is to help determine the basic hypothesis and effect of the detection, and to compare the behavior of the target cells to ensure that any changes under the experimental conditions are caused by the specific properties of the target cells, rather than other external factors.

[0107] Preferably, the control cells can be healthy cells or untreated cells, usually the same kind of cells as the target cells, but not subjected to any experimental treatment. Alternatively, the control cells can be null marker cells, for example, cells not expressing the target antigen or specific biomarker, for excluding 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 the target cells (e.g., tumor cells).

[0108] Also, the density of the control cells is kept approximately the same as the target cells.

[0109] In preferred embodiments, at least two times of adhesion force detection processes are implemented. Also, based on the preliminary results of the adhesion force detection, the approximate amount of the control cells added in the subsequent adhesion force detection, i.e., the ratio of the control cells to the target cells, is determined.

[0110] Preferably, in the preliminary detection, an adhesion force value of the target is calculated, i.e., in a detection at a predefined applied force value, the adhesion force value A is calculated:

[0111] A target = number of exfoliated cells / total number of cells;

[0112] The adhesion force value A target can be used to infer the adhesion ability of the target cells, and as a basis for adjusting the ratio of the control cells to the target cells in subsequent detections.

[0113] The ratio of the control cells to the target cells in the subsequent detection is adjusted. Preferably, the ratio adjustment rules can be designed by the following strategies:

[0114] The ratio range setting, when the adhesion force of the target cells is high, the ratio of the control cells can be appropriately reduced, thereby improving the sensitivity and accuracy of the detection. Conversely, when the adhesion force of the target cells is low, the ratio of the control cells is appropriately increased to ensure the effectiveness of the control group cell contrast effect.

[0115] Preferably, the following rules can be used:

[0116] When A target > 0.7, the ratio of the control cells is 10% to 20%;

[0117] When 0.3 ≤ A target ≤ 0.7, the ratio of the control cells is 20% to 40%;

[0118] When A target < 0.3, the ratio of the control cells is 40% to 60%.

[0119] The proportion adjustment rule is further optimized by machine learning or regression model to learn the relationship between adhesion force and cell proportion based on a large amount of experimental data. For example, support vector machine (SVM), decision tree or linear regression method is used to predict the adhesion force of target cells, and the cell proportion setting is optimized according to historical experimental data.

[0120] Embodiment three: this embodiment should be understood as at least containing all the features of any one of the preceding embodiments, and further improving on the basis thereof:

[0121] Exemplarily, the attached drawings Figure 5 A schematic diagram of a computer system 500 in which the diagnostic system described herein is implemented is depicted; the computer system 500 can implement the working control of various working units, modules, components in the system according to the current control program; and further includes collecting, storing and processing the working data, detection data generated in the system working, to finally realize the expected effect of the fluorescence labeling system.

[0122] Among them, the computer system 500 includes a bus 502 or other communication mechanism for transmitting information, one or more processors 504 coupled to the bus 502 for processing information; the processor 504 can be, for example, one or more general-purpose microprocessors;

[0123] The computer system 500 also includes a main memory 506, such as a random access memory (RAM), cache and / or other dynamic storage devices, coupled to the bus 502 for storing information and instructions to be executed by the processor 504; the main memory 506 can also be used for storing temporary variables or other intermediate information during execution of instructions by the processor 504; these instructions, when stored in a storage medium accessible to the processor 504, render the computer system 500 into a special-purpose machine customized to perform the operations specified in the instructions;

[0124] The computer system 500 can 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 magnetic disk, optical disk or USB drive (flash drive) will be coupled to the bus 502 for storing information and instructions;

[0125] And further, coupled to the bus 502 can 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;

[0126] One preferred manner of interacting with the management system can be through a cursor control device 516, such as a computer mouse, or similar control / navigational mechanism;

[0127] Further, computer system 500 can also include a network interface device 518 coupled to bus 502; where network interface device 518 can comprise, for example, a wired network adapter, a wireless network adapter, a switching chip, a router, a switch, etc.;

[0128] Generally, the terms "engine," "component," "system," "database," and the like, as used herein, can refer to either hardware or firmware embodied in one or more computer- readable media, or to a collection of software instructions, possibly having entry and exit points, written in a preferably object-oriented programming language, such as Java, C or C++, for example; software components can be compiled into executable programs that are executable on a processor, installed in dynamic link libraries, or can be written in interpreted languages such as BASIC, Perl, or Python, for example; it will be appreciated that software components can be callable from other components or from themselves, and / or can be invoked in response to detected events or interrupts;

[0129] Software components configured to execute on computing devices can be provided on computer-readable media, such as optical, digital, or analog magnetic media, flash memory, or any other tangible media, or as a digital download (and can initially be stored) in a compressed or installable format, requiring installation, decompression or decryption, prior to execution); such software code can be stored partially or entirely on memory devices of the executing computing device, for execution by the computing device; the software instructions can be embedded in firmware, such as an EPROM; it will be appreciated that hardware components can be comprised of connected logic elements (for example, transistors, gates, and flip-flops), and / or can be comprised of programmable logic elements (for example, an FPGA or a processor), connected by a hierarchy of wires;

[0130] Computer system 500 includes a hierarchy of wires connected by a hierarchy of wires, and / or can be comprised of programmable logic elements (for example, an FPGA or a processor), connected by a hierarchy of wires;

[0131] According to one or more embodiments, the techniques herein are 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 can 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 can be used in place of or in combination with software instructions;

[0132] The terms "non-transitory medium" and similar terms as used herein refer to any medium that stores the data and / or instructions that cause a machine to operate in a specific manner; such a non-transitory medium can include non-volatile media and / or volatile media; non-volatile media includes, for example, optical or magnetic disks, such as storage device 510; volatile media includes dynamic memory, such as main memory 506;

[0133] Common forms of non-transitory media include, for example, a floppy disk, flexible disk, hard disk, solid-state drive, magnetic tape, or any other magnetic data storage medium, a CD-ROM, any other optical data storage medium, any physical medium with patterns of holes, a RAM, a PROM, and EPROM, a FLASH-EPROM, NVRAM, any other memory chip or cartridge, and a networked version of any of the above;

[0134] Non-transitory media differentiate from transmission media, but can be used in combination with transmission media; transmission media participate in transferring information between non-transitory media; for example, transmission media includes coaxial cables, copper wire, and optical fibers, including wires that comprise bus 502; transmission media can also take the form of acoustic or light waves, such as those generated during radio frequency and infrared data communications.

[0135] While the present application has been described above with reference to various embodiments, it should be understood that many changes can be made in the details without departing from the scope of the application. That is, the methods, systems and devices discussed above are examples. Various configurations can omit, substitute, or add various procedures or components as appropriate. For instance, the methods described can be performed in an order different from that described, and / or various steps can be added, omitted, and / or combined. Also, features described with respect to certain configurations can be combined in various other configurations, for instance, different aspects and elements of the configurations can be combined in a similar manner. Also, technology evolves and, thus, many of the elements are examples and do not limit the scope of the disclosure or claims. One of ordinary skill in the art will readily recognize a variety of ways to implement the methods and systems described herein.

[0136] In the description, numerous specific details are set forth to provide a thorough understanding of example implementations. However, implementations can be practiced without the specific details. For example, well-known circuits, processes, algorithms, structures, and techniques have not been described in detail as not to unnecessarily obscure the configurations. This description provides example configurations only, and does not limit the scope, applicability, or configurations of the claims. Rather, the preceding description of the configurations will provide those skilled in the art with an enabling description that can be used in conjunction with the appended claims. Numerous modifications can be made to the elements and arrangements described without departing from the spirit and scope of the disclosure.

[0137] In light of the above, it is to be understood that the above description is intended to be illustrative and not restrictive. Many embodiments of the application will be apparent to those of skill in the art upon reading the above description. The scope of the application should, therefore, be determined not with reference to the above description, but should instead be determined with reference to the appended claims, along with their full scope of equivalents.

Claims

1. A disease diagnostic system based on detection of an anti-protocadherin 7 antibody, characterized by comprising: a protocadherin 7 gene; and a protocadherin 7 protein. The diagnostic system comprises a microfluidic device and a microfluidic chip applied to the microfluidic device; wherein the following coatings are prepared on the substrate of the microfluidic chip in sequence: The first coating is one or more polypeptides or a mixture of polypeptides; The second coating is an original calnexin 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 second coating as a monolayer of cells; The diagnostic system detects the adhesion of cells in the detection cell monolayer on the microfluidic chip by applying a known force to the detection cell monolayer on the microfluidic chip using the microfluidic device, and calculates the adhesion force of the cells in the detection cell monolayer by statistical means; The diagnostic system further comprises a detection unit and a control unit; The detection unit is used to count the cells falling off from the second coating; The control unit is used to control the microfluidic device and the detection unit to work cooperatively; The detection unit further comprises a counting unit for counting the cells falling off from the second coating and having an original calnexin 7 antibody The microfluidic chip comprises a plurality of flow channels, and each flow channel allows independent detection of the adhesion force of different cell types for multiple diagnostic analysis of diseases; The microfluidic chip further comprises one or more input and output terminals for connecting the purpose connector, and allows the fluid to be transported into the microfluidic chip by the upstream transport device and received in the downstream device after the fluid flows out of the microfluidic chip; the microfluidic chip is configured of a material and structure capable of transmitting sound waves; the microfluidic chip is configured as an electrically conductive structure so as to apply an electric field in the flow channel. ​

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