Microfluidic system for rapid quantitative detection of breast cancer tissue markers and detection method

Abstract

The invention relates to a microfluidic system for rapid quantitative detection of breast cancer tissue markers and a detection method, and belongs to the technical field of biology, the microfluidic system comprises an acrylic cover plate, a pressure-sensitive adhesive layer and a glass substrate; the acrylic cover plate, the pressure-sensitive adhesive layer and the glass substrate are bonded together from top to bottom, and the reaction cavity of the micro pressure-sensitive adhesive layer is filled with antibody microbeads. And the micro reaction cavity, the inlet channel and the outlet channel of the pressure-sensitive adhesive layer form a micro flow path structure. According to the invention, quantification and standardization of the whole process from a tissue sample to a molecular typing result are realized, and the qualitative / semi-quantitative dependence problem is solved; an innovative tissue protein rapid preparation method, a special microfluidic system and a data analysis process based on fluorescence quantification are integrated into a whole; the whole process thoroughly gets rid of dependence on traditional tissue slicing, dyeing and manual microscopic examination.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biotechnology, and particularly relates to a microfluidic system and a detection method for rapid quantitative detection of breast cancer tissue markers. The key biomarkers of breast cancer are ER, PR, HER2 or Ki-67. BACKGROUND

[0002] Breast cancer shows high heterogeneity at the molecular level; at present, it is mainly divided into Luminal A type, Luminal B type, HER2 positive type and triple negative breast cancer molecular subtypes according to the expression levels of biomarkers such as estrogen receptor (ER), progesterone receptor (PR), human epidermal growth factor receptor 2 (HER2) and proliferation index Ki-67. These subtypes have significant differences in epidemiological characteristics, treatment strategies, prognosis and imaging manifestations. Therefore, accurate and rapid molecular typing of breast cancer has key clinical significance for developing individualized treatment plans, improving patient prognosis and reducing mortality.

[0003] At present, pathological examination based on tissue biopsy is still the gold standard for diagnosis and typing of breast cancer. Among them, puncture biopsy is the main means to obtain tumor samples. After the obtained tissue is fixed, sectioned, stained and other treatments, the pathologist evaluates and immunohistochemical (IHC) analyzes under a microscope to complete the typing. Although this method has been widely used in clinical practice, it still has the following obvious limitations: first, the typing result is highly dependent on the subjective experience and interpretation level of the pathologist, and lacks a standardized and quantitative objective evaluation system, which may lead to insufficient typing accuracy and low result consistency; second, the whole process including sample collection, preparation, manual reading and report issuing takes a long time, which may lead to delayed diagnosis in the case of a large number of patients, exacerbate the shortage of medical resources and affect the treatment opportunity, causing physical trauma and psychological pressure to patients.

[0004] In recent years, with the progress of digital scanning and computational analysis technology, digital pathology provides a new technical path for quantitative analysis and objective typing of breast cancer. The existing technical solutions mainly focus on image segmentation, feature extraction and pattern recognition using whole slide scanning images, in order to assist or realize typing diagnosis[2]. Representative existing technologies are as follows: Linnea et al. proposed a method for quantifying the tumor microenvironment based on digital histological images. This approach first involves high-resolution scanning of breast cancer tissue sections to obtain digital images; then, machine learning or image processing algorithms are used to classify the image pixels and quantify the proportions of different components in the tissue (including epithelial cells, stroma, fat, and lymphocytes); finally, by analyzing the statistical correlation between these quantitative characteristics and different breast cancer molecular subtypes, an attempt is made to establish a histological morphology-based subtyping auxiliary model.

[0005] Shachi et al.'s approach is based on conventional hematoxylin-eosin (HE) stained sections and utilizes deep learning technology. The core of this method is to train a convolutional neural network (CNN) to automatically identify and segment breast epithelial cells in digital pathology images. Based on the identification of epithelial regions, morphological features related to matrix-epithelial interactions are further extracted. Finally, a classifier is constructed to classify breast cancer subtypes based on these extracted deep features. This approach aims to achieve subtyping directly using HE section images without relying on immunohistochemistry (IHC).

[0006] Minseok et al. proposed an innovative solution from the perspective of detection technology platform. This technology designs a microfluidic chip platform that can integrate thin-layer tissue slices into the chip. Through the microfluidic channel system, fluid control of various solutions is realized on this platform, thereby simultaneously detecting and quantifying four key biomarkers (such as ER, PR, HER2, and Ki-67) in breast cancer cells and tissue samples, in order to improve detection efficiency and throughput and provide multi-parameter quantitative data for typing.

[0007] Although the aforementioned existing technologies have made some progress in automation and quantitative analysis, they still have the following inherent defects and key problems that urgently need to be solved: 1. Detection capabilities are limited to qualitative or semi-quantitative levels: Most existing methods are based on secondary analysis of the morphology of stained sections, and the output results are statistical values ​​or probability scores of image features (such as image grayscale, stained area ratio, cell morphology parameters, etc.), rather than the absolute concentration values ​​of the biomarkers themselves. This indirect analysis method cannot achieve accurate quantitative detection of key targets such as ER, PR, HER2, Ki-67, resulting in insufficient accuracy and standardization of the results.

[0008] The front-end sample preparation process is complex and difficult to standardize: the effectiveness of existing technologies relies heavily on the complex pretreatment procedures of traditional histopathology. These steps are cumbersome, time-consuming, and prone to introducing human error and batch-to-batch variability, becoming the main sources of error affecting the accuracy and reliability of results. Summary of the Invention

[0009] The purpose of this invention is to address the problems existing in the prior art and to provide a microfluidic system and detection method for rapid quantitative detection of breast cancer tissue markers. This invention combines immunoassay technology with microfluidic technology to achieve highly sensitive, high-throughput, and automated quantitative detection of multiple target proteins in tissue protein samples.

[0010] A microfluidic system for rapid quantitative detection of breast cancer tissue markers includes an acrylic cover plate, a pressure-sensitive adhesive layer, and a glass substrate. The acrylic cover plate, the pressure-sensitive adhesive layer, and the glass substrate are bonded together from top to bottom, with the pressure-sensitive adhesive layer located between the acrylic cover plate and the glass substrate. The acrylic cover plate has an inlet and an outlet, both with a diameter of 1 mm and a center-to-center distance of 8 mm. The function of the acrylic cover plate is to seal the flow path and provide a fluid interface.

[0011] The pressure-sensitive adhesive layer has a micro-reaction chamber, an inlet channel, and an outlet channel. The inlet channel and outlet channel are located on both sides of the micro-reaction chamber and are connected to the micro-reaction chamber. The inlet channel is connected to the sample inlet of the acrylic cover plate, and the outlet channel is connected to the sample outlet of the acrylic cover plate. The inlet channel and outlet channel are triangular, and the connection between the inlet channel and outlet channel and the micro-reaction chamber is an acute angle. The micro-reaction chamber is filled with antibody microbeads, and the thickness of the pressure-sensitive adhesive layer is 90 μm.

[0012] The connection points between the inlet and outlet channels and the microreaction chamber are acute angles, which effectively prevents the antibody beads filled in the microreaction chamber from escaping under fluid impact, thus ensuring the long-term stability of the microfluidic system.

[0013] The micro-reaction chamber, inlet channel, and outlet channel constitute a microfluidic path structure.

[0014] The antibody microbeads are glass beads with a diameter of 75-90 μm.

[0015] The glass substrate provides mechanical support for the microfluidic system and forms a sealed bottom surface for the microfluidic path structure. The optical transparency of the glass substrate facilitates subsequent fluorescence microscopy observation.

[0016] A method for fabricating a microfluidic system for rapid quantitative detection of breast cancer tissue markers, employing a pressure-sensitive adhesive bonding method, is described below: Step 1: Microchannel fabrication: The pressure-sensitive adhesive layer is bonded to the glass substrate, and the micro-reaction chamber, inlet channel, and outlet channel are precisely etched on the pressure-sensitive adhesive layer through precision cutting.

[0017] Step 2, Filling of antibody microbeads: Pre-treated antibody microbeads with a diameter of 75-90 μm are filled into the microreaction chamber. Since the thickness of the pressure-sensitive adhesive layer is 90 μm, which matches the diameter of the antibody microbeads, the antibody microbeads are arranged in a single layer and are tightly packed in the microreaction chamber. This structure greatly increases the solid-phase surface area and forces the flowing liquid sample to diffuse through the narrow gaps between the antibody microbeads, thereby significantly improving the efficiency of antigen-antibody binding.

[0018] Step 3: Bonding: Remove the protective film from the etched pressure-sensitive adhesive layer, align it with the upper acrylic cover plate and the lower glass substrate, and press them together to complete the irreversible bonding, forming a closed microfluidic system. This method does not require expensive etching machines or high-temperature and high-pressure equipment, is simple to operate, low in cost, and suitable for large-scale production.

[0019] A rapid quantitative detection method for breast cancer tissue markers, using the microfluidic system described in claim 1; Step 1: Preparation of tissue protein samples: (1) Take a breast cancer tissue sample stored at -80°C, weigh it, and then cut it into pieces with sterile scissors to form fragmented breast cancer tissue.

[0020] (2) Transfer the fragmented breast cancer tissue to a 5 mL or 10 mL centrifuge tube, and add lysis buffer at a ratio of 1 mL of pre-cooled lysis buffer per 0.1 g of tissue; the lysis buffer is composed of RIPA lysis buffer and protease inhibitor mixture at a volume ratio of 100:16.

[0021] The protease inhibitor mentioned is a commercially available complete protease inhibitor.

[0022] (3) Under low temperature conditions, use a tissue homogenizer to perform intermittent homogenization for at least 5 minutes until there are no obvious lumps of solid in the solution.

[0023] The intermittent homogenization process involves working for 15 seconds followed by a 15-second pause.

[0024] The low-temperature environment mentioned is an ice bath.

[0025] (4) After homogenization, the product is left to stand at a low temperature for 20 minutes to allow the protein to fully break down and release; the low temperature environment is an ice bath.

[0026] (5) Then perform two-stage centrifugation: first, centrifuge at 4°C and 1200 rpm for 5 minutes, and transfer the supernatant to a 1.5 mL centrifuge tube; then centrifuge at 4°C and 12000 rpm for 30 minutes.

[0027] (6) The final supernatant is the tissue protein extract, which can be used immediately for detection or aliquoted and stored at -20°C / -80°C. Short-term storage is allowed at -20°C, and long-term storage at -80°C. Step 2: Immunofluorescence detection and quantitative analysis on the microfluidic system: (e.g.) Figure 2 As shown; (1) Antibody immobilization: Before the microfluidic system is fabricated, capture antibodies targeting specific breast cancer markers (such as anti-ER antibodies) are immobilized on the surface of glass microbeads using standard coupling chemical methods to form antibody microbeads, which are then filled into the microreaction chamber during the fabrication of the microfluidic system.

[0028] (2) Sample injection and antigen capture: 50µL of the prepared tissue protein sample is injected through the injection port and flows through the microreaction chamber at a low flow rate of 4µL / min controlled by a high-precision injection pump. During this process, the target antigen in the sample is specifically bound and fixed by the capture antibody on the surface of the antibody microbeads.

[0029] The target antigen is ER, PR, HER2, or Ki-67.

[0030] (3) Rinsing: After the sample is injected, PBS buffer is pumped into the microfluidic system at a flow rate of 20µL / min and rinsed continuously for 15 minutes to thoroughly remove non-specifically bound proteins and other matrix components.

[0031] (4) Fluorescent labeling: First, introduce the polyclonal primary antibody diluted with PBS buffer and incubate for binding: After rinsing with PBS buffer, fluorescently labeled secondary antibody diluted with PBS buffer is introduced to bind with the primary antibody. The fluorescently labeled secondary antibody is FITC-labeled goat anti-rabbit IgG.

[0032] After each antibody injection, the sample must be thoroughly rinsed with PBS buffer to remove any unbound antibodies.

[0033] The PBS buffer is a prior art product, and its main components are Na2HPO4, KH2PO4, NaCl and KCl.

[0034] (5) Signal acquisition and quantification: After completing all labeling and rinsing steps, place the microfluidic system on the stage of the inverted fluorescence microscope, select an appropriate excitation / emission wavelength, and perform fluorescence imaging on the antibody microbead array in the microreaction chamber. Quantitatively analyze the average fluorescence intensity of the microreaction chamber or a specific area using image analysis software. The fluorescence intensity value is proportional to the concentration of the captured target antigen. By using a standard curve with a known concentration of standard protein, absolute quantification of the target protein in the unknown sample can be achieved.

[0035] The image analysis software mentioned is ImageJ.

[0036] Beneficial technical effects of the present invention: 1. The microfluidic system of the present invention provides a rapid detection device for rapid quantitative detection of breast cancer tissue markers.

[0037] This invention achieves full quantification and standardization from tissue samples to molecular typing results, solving the problem of qualitative / semi-quantitative dependence: It integrates an innovative rapid tissue protein preparation method, a dedicated microfluidic system, and a data analysis process based on fluorescence quantification into a whole. This integrated process completely eliminates the dependence on traditional tissue sections, staining, and manual microscopic examination. Because this invention uses the immunofluorescence detection principle based on antibody microbeads, the absolute concentration of target proteins (such as ER and PR) can be directly calculated by measuring the fluorescence signal intensity and comparing it with a standard curve. The results do not depend on manual interpretation, thus fundamentally overcoming the core shortcomings of traditional immunohistochemistry (IHC) which is highly subjective and semi-quantitative, providing objective and quantifiable accurate data for molecular typing of breast cancer.

[0038] The method significantly simplifies the sample pretreatment process and improves detection efficiency and standardization: The tissue samples required by the detection method of this invention only need to undergo routine operations such as tissue homogenization and protein extraction centrifugation, avoiding a series of complicated steps such as tissue paraffin embedding, sectioning, and dewaxing in traditional methods; this not only greatly shortens the sample preparation time from several hours or even several days and reduces human error, but also reduces sample loss, making the entire detection process easier to standardize and automate, which is conducive to rapid promotion in the clinical environment.

[0039] This invention provides a precise diagnostic method that can be directly applied in clinical practice, and has strong versatility and scalability: the detection performance of the microfluidic system of this invention is not affected by variables such as different pathologists, different slide batches, or different scanning equipment; the design of monolayer antibody microbeads in the microreaction chamber greatly increases the specific surface area, improves antibody capture efficiency, and focuses the fluorescence signal. Experimental verification shows that the detection limit (LOD) for the target protein is as low as 0.48 ng / mL, which has extremely high sensitivity. Attached Figure Description

[0040] Figure 1 This is a three-dimensional schematic diagram of an embodiment of the microfluidic system of the present invention; Figure 2 This is a flowchart of the measurement method of the present invention. Detailed Implementation

[0041] like Figure 1As shown, a microfluidic system for rapid quantitative detection of breast cancer tissue markers includes an acrylic cover plate 1, a pressure-sensitive adhesive layer 2, and a glass substrate 3; the acrylic cover plate 1, the pressure-sensitive adhesive layer 2, and the glass substrate 3 are bonded together from top to bottom, with the pressure-sensitive adhesive layer 2 located between the acrylic cover plate 1 and the glass substrate 3; The acrylic cover plate 1 has an inlet 11 and an outlet 12, both with a diameter of 1 mm and a center-to-center distance of 8 mm. The function of the acrylic cover plate 1 is to seal the flow path and provide a fluid interface.

[0042] The pressure-sensitive adhesive layer 2 has a micro-reaction chamber 21, an inlet channel 22, and an outlet channel 23. The inlet channel 22 and the outlet channel 23 are located on both sides of the micro-reaction chamber 21 and are connected to the micro-reaction chamber 21. The inlet channel 22 is connected to the sample inlet 11 of the acrylic cover plate 1, and the outlet channel 23 is connected to the sample outlet 12 of the acrylic cover plate 1. The inlet channel 22 and the outlet channel 23 are triangular, and the connection between the inlet channel 22 and the outlet channel 23 and the micro-reaction chamber 21 is an acute angle. The micro-reaction chamber 21 is filled with antibody microbeads 4, and the thickness of the pressure-sensitive adhesive layer 2 is 90 μm.

[0043] The connection points between the inlet channel 22 and the outlet channel 23 and the microreaction chamber 21 are acute angles, which can effectively prevent the antibody microbeads 4 filled in the microreaction chamber 21 from escaping under fluid impact, thus ensuring the long-term stability of the microfluidic system.

[0044] The micro-reaction chamber 21, inlet channel 22 and outlet channel 23 constitute a microflow path structure.

[0045] The antibody microbeads 4 are glass beads with a diameter of 75-90 μm.

[0046] The glass substrate 3 provides mechanical support for the microfluidic system and forms a sealed bottom surface for the microfluidic path structure. The optical transparency of the glass substrate 3 facilitates subsequent fluorescence microscopy observation.

[0047] A method for fabricating a microfluidic system for rapid quantitative detection of breast cancer tissue markers, employing a pressure-sensitive adhesive bonding method, is described below: Step 1, Microchannel fabrication: The pressure-sensitive adhesive layer 2 is bonded to the glass substrate 3, and the micro-reaction chamber 21, the inlet channel 22 and the outlet channel 23 are precisely etched on the pressure-sensitive adhesive layer 2 by precision cutting.

[0048] Step 2, Filling of antibody microbeads: Pre-treated antibody microbeads 4 with a diameter of 75-90 μm are filled into the microreaction chamber 21. Since the thickness of the pressure-sensitive adhesive layer 2 is 90 μm, which matches the diameter of the antibody microbeads 4, the antibody microbeads 4 are arranged in a single layer and are tightly packed in the microreaction chamber 21. This structure greatly increases the solid-phase surface area and forces the flowing liquid sample to diffuse through the narrow gaps between the antibody microbeads 4, thereby significantly improving the efficiency of antigen-antibody binding.

[0049] Step 3: Bonding: Remove the protective film from the etched pressure-sensitive adhesive layer 2, align it with the upper acrylic cover plate 1 and the lower glass substrate 3, and press them together to complete the irreversible bonding, forming a closed microfluidic system. This method does not require expensive etching machines or high-temperature and high-pressure equipment, is simple to operate, low in cost, and suitable for large-scale production.

[0050] A rapid quantitative detection method for breast cancer tissue markers, using the microfluidic system described in claim 1; as follows: Figure 2 As shown; Step 1: Preparation of tissue protein samples: (1) Take a breast cancer tissue sample stored at -80°C, weigh it, and then cut it into pieces with sterile scissors to form fragmented breast cancer tissue.

[0051] (2) Transfer the fragmented breast cancer tissue to a 5 mL or 10 mL centrifuge tube, and add lysis buffer at a ratio of 1 mL of pre-cooled lysis buffer per 0.1 g of tissue; the lysis buffer is composed of RIPA lysis buffer and protease inhibitor mixture at a volume ratio of 100:16.

[0052] The protease inhibitor mentioned is a commercially available complete protease inhibitor.

[0053] The RIPA lysis buffer is a traditional rapid cell and tissue lysis buffer.

[0054] The protease inhibitors mentioned are common preparations, such as leucine, analgesic, chymotrypsin inhibitor, elastase inhibitor, pepsin inhibitor, and phosphatidylcholine, which can inhibit various proteases such as trypsin, papain, chymotrypsin, elastase, pepsin, and metalloproteinases, respectively.

[0055] (3) Under low temperature conditions, use a tissue homogenizer to perform intermittent homogenization for at least 5 minutes until there are no obvious lumps of solid in the solution.

[0056] The intermittent homogenization process involves working for 15 seconds followed by a 15-second pause.

[0057] The low-temperature environment mentioned is an ice bath.

[0058] (4) After homogenization, the product is left to stand at a low temperature for 20 minutes to allow the protein to fully break down and release; the low temperature environment is an ice bath.

[0059] (5) Then perform two-stage centrifugation: first, centrifuge at 4°C and 1200 rpm for 5 minutes, and transfer the supernatant to a 1.5 mL centrifuge tube; then centrifuge at 4°C and 12000 rpm for 30 minutes.

[0060] (6) The final supernatant is the tissue protein extract, which can be used immediately for detection or aliquoted and stored at -20°C / -80°C. Short-term storage is allowed at -20°C, and long-term storage at -80°C. Step 2: Immunofluorescence detection and quantitative analysis on the microfluidic system: (1) Antibody immobilization: Before the microfluidic system is fabricated, the capture antibody targeting a specific breast cancer marker (such as anti-ER antibody) is immobilized on the surface of glass microbeads by a standard coupling chemical method to form antibody microbeads 4, which are then filled into the microreaction chamber 21 during the fabrication of the microfluidic system.

[0061] (2) Sample injection and antigen capture: 50µL of the prepared tissue protein sample is injected through the injection port 11 and flows through the microreaction chamber 21 at a low flow rate of 4µL / min controlled by a high-precision injection pump. During this process, the target antigen in the sample is specifically bound and fixed by the capture antibody on the surface of the antibody microbeads 4.

[0062] The target antigen is ER, PR, HER2, or Ki-67.

[0063] (3) Rinsing: After the sample is injected, PBS buffer is pumped into the microfluidic system at a flow rate of 20µL / min and rinsed continuously for 15 minutes to thoroughly remove non-specifically bound proteins and other matrix components.

[0064] (4) Fluorescent labeling: First, polyclonal primary antibody diluted with PBS buffer is introduced and incubated for binding; after rinsing with PBS buffer, fluorescently labeled secondary antibody diluted with PBS buffer is introduced to bind with the primary antibody; the fluorescently labeled secondary antibody is FITC-labeled goat anti-rabbit IgG. After each antibody injection, it is necessary to rinse thoroughly with PBS buffer to remove unbound antibodies.

[0065] The PBS buffer is a prior art product, and its main components are Na2HPO4, KH2PO4, NaCl and KCl.

[0066] (5) Signal acquisition and quantification: After completing all labeling and rinsing steps, place the microfluidic system on the stage of the inverted fluorescence microscope, select an appropriate excitation / emission wavelength, and perform fluorescence imaging on the antibody microbead 4 array in the microreaction chamber 21. Quantitatively analyze the average fluorescence intensity of the microreaction chamber 21 or a specific area using image analysis software. The fluorescence intensity value is proportional to the concentration of the captured target antigen. By using a standard curve with a known concentration of standard protein, absolute quantification of the target protein in the unknown sample can be achieved.

[0067] The image analysis software mentioned is ImageJ.

Claims

1. A microfluidic system for rapid quantitative detection of breast cancer tissue markers, characterized in that: It includes an acrylic cover plate (1), a pressure-sensitive adhesive layer (2), and a glass substrate (3); the acrylic cover plate (1), the pressure-sensitive adhesive layer (2), and the glass substrate (3) are bonded together from top to bottom, with the pressure-sensitive adhesive layer (2) located between the acrylic cover plate (1) and the glass substrate (3); the acrylic cover plate (1) has an inlet (11) and an outlet (12), and the pressure-sensitive adhesive layer (2) has a micro-reaction chamber (21), an inlet channel (22), and an outlet channel (23), and the inlet channel (22) and outlet channel (23) are connected together. The inlet channel (23) is located on both sides of the microreaction chamber (21) and is connected to the microreaction chamber (21). The inlet channel (22) is connected to the sample inlet (11) of the acrylic cover plate (1), and the outlet channel (23) is connected to the sample outlet (12) of the acrylic cover plate (1). The inlet channel (22) and the outlet channel (23) are triangular, and the connection between the inlet channel (22) and the outlet channel (23) and the microreaction chamber (21) is an acute angle. The microreaction chamber (21) is filled with antibody microbeads (4). The micro-reaction chamber (21), inlet channel (22) and outlet channel (23) constitute a microflow path structure.

2. The microfluidic system for rapid quantitative detection of breast cancer tissue markers according to claim 1, characterized in that: The diameter of the inlet (11) and outlet (12) is 1 mm, and the center distance between the inlet (11) and outlet (12) is 8 mm; the thickness of the pressure-sensitive adhesive layer (2) is 90 μm; the antibody microbeads (4) are glass beads, and the diameter of the antibody microbeads (4) is 75-90 μm.

3. A method for fabricating a microfluidic system for rapid quantitative detection of breast cancer tissue markers as described in claim 1, characterized in that: The specific steps are as follows: Step 1: Microchannel fabrication: The pressure-sensitive adhesive layer (2) is bonded to the glass substrate (3), and the micro-reaction chamber (21), inlet channel (22) and outlet channel (23) are precisely etched on the pressure-sensitive adhesive layer (2) by precision cutting. Step 2, Filling of antibody microbeads: Pre-treated antibody microbeads (4) with a diameter of 75-90 μm are filled into the microreaction chamber (21); Since the thickness of the pressure-sensitive adhesive layer (2) is 90 μm, it matches the diameter of the antibody microbeads (4), and the antibody microbeads (4) are arranged in a single layer in the microreaction chamber (21); This structure increases the solid surface area and forces the liquid sample flowing through to diffuse through only the narrow gaps between the antibody microbeads (4); Step 3, Bonding: Remove the protective film from the etched pressure-sensitive adhesive layer (2), align it with the upper acrylic cover plate (1) and the lower glass substrate (3) and press it together to complete the irreversible bonding and form a closed microfluidic system.

4. A rapid quantitative detection method for breast cancer tissue markers, using the microfluidic system described in claim 1, characterized in that: Includes the following steps: Step 1: Preparation of tissue protein samples: 1) Take a breast cancer tissue sample stored at -80°C, weigh it, and then use sterile scissors to cut it into small pieces to form breast cancer tissue fragments; 2) Transfer the fragmented breast cancer tissue to a 5 mL or 10 mL centrifuge tube, and add lysis buffer at a ratio of 1 mL of pre-cooled lysis buffer per 0.1 g of tissue; the lysis buffer consists of a freshly prepared mixture of RIPA lysis buffer and protease inhibitor at a volume ratio of 100:

16. 3) Under low temperature conditions, use a tissue homogenizer to perform intermittent homogenization for at least 5 minutes until there are no obvious lumps of solids in the solution; 4) After homogenization, the product is allowed to stand at a low temperature for 20 minutes to allow the protein to fully break down and release. 5) Then perform two-stage centrifugation: First, centrifuge at 4°C and 1200 rpm for 5 minutes, and transfer the supernatant to a 1.5 mL centrifuge tube; then centrifuge at 4°C and 12000 rpm for 30 minutes. 6) The final supernatant is the tissue protein extract, which can be used immediately for detection or aliquoted and stored at -20°C / -80°C; short-term storage at -20°C and long-term storage at -80°C. Step 2: Immunofluorescence detection and quantitative analysis on the microfluidic system: 1) Antibody immobilization: Before fabricating the microfluidic system, capture antibodies targeting specific breast cancer markers are immobilized on the surface of glass microbeads using standard coupling chemical methods to form antibody microbeads (4), which are then filled into the microreaction chamber (21) during the fabrication of the microfluidic system. 2) Sample injection and antigen capture: 50µL of the prepared tissue protein sample is injected through the injection port (11) and flows through the microreaction chamber (21) at a low flow rate of 4µL / min controlled by a high-precision injection pump. During this process, the target antigen in the sample is specifically bound and fixed by the capture antibody on the surface of the antibody microbeads (4). 3) Rinsing: After sample injection, pump PBS buffer into the microfluidic system at a flow rate of 20 µL / min and rinse continuously for 15 minutes to thoroughly remove unspecifically bound proteins and other matrix components. 4) Fluorescent labeling: First, pass in polyclonal primary antibody diluted with PBS buffer and incubate for binding. After rinsing with PBS buffer, pass in fluorescently labeled secondary antibody diluted with PBS buffer to bind with the primary antibody. After each antibody injection, rinse thoroughly with PBS buffer to remove unbound antibodies. 5) Signal acquisition and quantification: After completing all labeling and rinsing steps, place the microfluidic system on the stage of the inverted fluorescence microscope, select an appropriate excitation / emission wavelength, and perform fluorescence imaging on the antibody microbead (4) array in the microreaction chamber (21). Quantitatively analyze the average fluorescence intensity of the microreaction chamber (21) or a specific area using image analysis software. The fluorescence intensity value is proportional to the concentration of the captured target antigen. By using a standard curve drawn with a known concentration of standard protein, absolute quantification of the target protein in the unknown sample can be achieved.

5. The rapid quantitative detection method for breast cancer tissue markers according to claim 4, characterized in that: In step 1, 3), the intermittent homogenization is a process of working for 15 seconds and then pausing for 15 seconds. In steps 3) and 4) of step one, the low-temperature environment is an ice bath.

6. The rapid quantitative detection method for breast cancer tissue markers according to claim 4, characterized in that: In step 2), the target antigen is ER, PR, HER2, or Ki-67. In step 2, 4), the fluorescently labeled secondary antibody is FITC-labeled goat anti-rabbit IgG; In step 2, 5), the image analysis software is ImageJ.

Images