A test strip for simultaneously detecting MMP-9 and Occludin in blood and a preparation method thereof

Abstract

The present invention discloses a test strip for simultaneously detecting MMP-9 and Occludin in blood and a preparation method thereof, wherein the test strip comprises a base plate, and a sample pad layer, a binding pad layer, a nitrocellulose membrane layer and a water-absorbing paperboard layer fixed on the base plate in sequence, and two adjacent layers are staggered and overlapped; the nitrocellulose membrane layer is located at the bottom layer, and the bottom of one end of the water-absorbing paperboard layer and the binding pad layer close to the nitrocellulose membrane layer are in contact with the top of the nitrocellulose membrane layer, and the bottom of one end of the sample pad layer close to the binding pad layer is in contact with the top of the binding pad layer; the binding pad layer contains anti-MMP-9 immunomagnetic beads and anti-Occludin immunomagnetic beads, and the cellulose membrane layer is provided with two detection lines and a quality control line, and the two detection lines are respectively coated with MMP-9 antibodies and Occludin antibodies, and the quality control line is coated with rabbit anti-mouse IgG antibodies. The present invention can simultaneously detect MMP-9 and Occludin in blood samples, and assist in the early diagnosis and treatment of stroke patients.

Description

Technical Field

[0001] The present invention relates to the field of medical detection technology, and in particular to a test strip for simultaneously detecting MMP-9 and Occludin in blood and a preparation method thereof. Background Art

[0002] Stroke is an acute cerebrovascular disease caused by hemorrhage or ischemic damage to brain tissue due to cerebral blood circulation disorder. It is the second leading cause of death in humans and the main cause of long-term disability and death. Clinically, ischemic stroke is the most common, accounting for about 70%-80% of all cerebrovascular diseases. The incidence of hemorrhagic stroke is lower than that of ischemic stroke, but its disability rate and disease prognosis are poor. According to the data published in the 2016 Global Burden of Disease Study (GDB), although the prevalence and standardized mortality rate of stroke have declined in recent years, the overall burden of stroke is still heavy. The high disability rate, high mortality rate and high recurrence rate of stroke have led to huge long-term care costs for patients, which has brought heavy burdens to patients' families, health care systems and society. It has become a global public health problem that needs to be solved urgently. Therefore, rapid and accurate early diagnosis can help to formulate the best treatment strategy, thereby reducing the disability rate and mortality rate of stroke and improving the prognosis of patients.

[0003] Neuroimaging diagnosis (including CT and MRI, etc.) is currently a widely used technical means for the diagnosis of stroke. It can effectively distinguish ischemic stroke from hemorrhagic stroke, but there are still many shortcomings, such as a certain lag in the examination, high examination costs, unclear diagnosis when the amount of bleeding is small, moving the patient during the examination may cause the condition to worsen, and there are certain contraindications for MRI. In recent years, studies have shown that after a stroke occurs, in the early stage of brain damage, a large number of cytokines and inflammatory factors will be released into the cerebrospinal fluid and peripheral blood, and can exist stably. Therefore, these elevated body fluid markers can play a certain indicative role in the diagnosis and prognosis of stroke, and achieve early diagnosis and treatment of stroke patients. In addition, the dynamic monitoring of these markers can also reflect the patient's disease progression to a certain extent. Therefore, the detection of stroke-related body fluid markers is extremely important for the diagnosis, treatment and prognosis of stroke.

[0004] MMP-9 is a member of the matrix metalloproteinase family (MMPs) and is the enzyme most closely associated with acute ischemic stroke. It destroys the blood-brain barrier by degrading the extracellular matrix and is believed to play an important role in the inflammatory pathology of ischemic stroke. Studies have shown that the level of MMP-9 in the body's serum after a stroke can reflect the severity and extent of the patient's acute ischemic stroke, and this enzyme is also associated with late hemorrhagic infarction around the 7th day after a stroke. The higher the MMP-9 level in patients with acute ischemic stroke, the greater the incidence of disability and death within 3 months of follow-up. It is an important prognostic indicator that appears early in patients with acute ischemic stroke.

[0005] Occludin is one of the most representative proteins among tight junction-related proteins. It is an important protein that constitutes tight junctions and is crucial to maintaining the integrity of the blood-brain barrier. Its decreased expression can cause damage to the blood-brain barrier and is closely related to the occurrence and development of stroke. Studies have shown that occludin on the blood vessel wall will be degraded after acute cerebral ischemia, and these degraded fragments will enter the peripheral blood circulation with the destruction of the blood-brain barrier, so that the presence of occludin can be detected in the early stage of ischemia. Therefore, occludin is an important blood marker for evaluating the degree of blood-brain barrier damage after stroke. Summary of the invention

[0006] In view of the above-mentioned problems, the present invention aims to provide a test strip for simultaneously detecting MMP-9 and Occludin in blood and a preparation method thereof, which can simultaneously and quantitatively detect MMP-9 and Occludin in blood samples, and assist in the early diagnosis and treatment of stroke patients.

[0007] In order to achieve the above object, the technical solution adopted by the present invention is as follows:

[0008] A test strip for simultaneously detecting MMP-9 and Occludin in blood, characterized in that: it comprises a bottom plate, a water-absorbing paperboard layer, a sample pad layer, a binding pad layer and a nitrocellulose membrane layer, wherein the sample pad layer, the binding pad layer, the nitrocellulose membrane layer and the water-absorbing paperboard layer are fixed in sequence from one end to the other end above the bottom plate, and two adjacent layers are staggered and overlapped; the nitrocellulose membrane layer is located at the bottom layer, the bottoms of one end of the water-absorbing paperboard layer and the binding pad layer close to the nitrocellulose membrane layer are both in contact with the top of the nitrocellulose membrane layer, and the bottom of one end of the sample pad layer close to the binding pad layer is in contact with the top of the binding pad layer;

[0009] The binding pad layer contains anti-MMP-9 immunomagnetic beads and anti-Occludin immunomagnetic beads, and the nitrocellulose membrane layer is provided with two detection lines and a quality control line. The two detection lines are coated with MMP-9 antibodies and Occludin antibodies respectively, and the quality control line is coated with rabbit anti-mouse IgG antibodies.

[0010] Furthermore, a method for preparing a test strip for simultaneously detecting MMP-9 and Occludin in blood is characterized by comprising the following steps:

[0011] S1: Take the prepared base plate and stick the treated nitrocellulose membrane layer on the base plate according to the marked boundary line;

[0012] S2: Pasting a water-absorbing paperboard layer within the boundary line of one side of the nitrocellulose membrane layer, wherein the bottom of one end of the water-absorbing paperboard layer close to the nitrocellulose membrane layer is in contact and fixed with the top of the nitrocellulose membrane layer;

[0013] S3: According to the size of the bottom plate, the pretreated sample pad layer and the binding pad layer containing anti-MMP-9 immunomagnetic beads and anti-occludin immunomagnetic beads are sequentially pasted on the other side of the nitrocellulose membrane layer; the bottom of one end of the binding pad layer close to the nitrocellulose membrane layer is in contact with and fixed to the top of the nitrocellulose membrane layer, and the bottom of one end of the sample pad layer close to the binding pad layer is in contact with and fixed to the top of the binding pad layer;

[0014] S4: After pasting is completed, the large board structure is cut into test strips of the same width.

[0015] Furthermore, the method for treating the nitrocellulose membrane layer in step S1 comprises the following steps:

[0016] S101: diluting MMP-9 antibody, Occludin antibody and rabbit anti-mouse IgG antibody into antibody solutions with the same concentration using antibody diluent;

[0017] S102: using the diluted MMP-9 antibody solution and the Occludin antibody solution to draw two test lines at a volume of 1 μL / cm on the nitrocellulose membrane layer (5), and using the diluted rabbit anti-mouse IgG antibody solution to draw a quality control line at a volume of 1 μL / cm on the nitrocellulose membrane layer (5), the distance between each two adjacent lines is maintained between 0.3 and 1 cm;

[0018] S103: Dry the nitrocellulose membrane layer with the test line and the quality control line at room temperature, add desiccant and seal it for later use.

[0019] Furthermore, the antibody diluent in step S101 is a phosphate buffer containing 1-2 wt% sucrose.

[0020] Furthermore, the method for processing the sample pad layer in step S3 is: soaking the sample pad layer in BS buffer containing 2wt% NaCl, 2wt% TritonX-100, 0.5wt% BSA and 0.5wt% PVP, and then drying it for later use.

[0021] Furthermore, the method for preparing the binding pad layer containing anti-MMP-9 immunomagnetic beads and anti-occludin immunomagnetic beads in step S3 comprises the following steps:

[0022] S301: activating the magnetic nanoparticles, and then coupling them with MMP-9 antibodies and Occludin antibodies, respectively, to prepare anti-MMP-9 immunomagnetic beads and anti-Occludin immunomagnetic beads;

[0023] S302: soaking the bonding pad layer with a bonding pad layer treatment liquid and then drying it;

[0024] S303: The anti-MMP-9 immunomagnetic beads and anti-occludin immunomagnetic beads prepared in step S301 are respectively and evenly sprayed on different positions on the treated binding pad layer, and then dried.

[0025] Furthermore, the specific operation of step S301 includes the following steps:

[0026] S3011: preparing magnetic nanoparticles by chemical reaction, and performing carboxyl modification on the magnetic nanoparticles by using succinic anhydride to obtain carboxylated magnetic nanoparticles with a particle size of 100-300 nm;

[0027] S3012: fully activating the magnetic beads prepared in step S101 with an activation buffer, drying at 60° C., and washing once with PBS after drying;

[0028] S3013: adding activators EDC and NHS to the washed magnetic beads, and performing activation reaction at room temperature for 1 hour. After the reaction, washing with PBS is performed for later use; wherein the molar ratio of EDC, NHS to magnetic beads is 2-5:2-5:1;

[0029] S3014: adding the activated magnetic beads to different disease marker antibodies respectively, and reacting at room temperature for 2 hours; wherein the mass ratio of the magnetic beads to the antibodies is 1:20-40;

[0030] S3015: Using blocking buffer to block the non-specific binding sites of the antibody at 37°C to obtain different immunomagnetic beads; wherein the blocking time is 30 min, and the pH of the boric acid-borax solution is 9;

[0031] S3016: Re-disperse the immunomagnetic beads obtained after blocking in the storage buffer and store at 4°C for future use.

[0032] Furthermore, the bonding pad layer treatment solution in step S302 is a boric acid-borax solution containing 5-10wt% sucrose and 0.05wt% TritonX-100.

[0033] Furthermore, in step S303, the spraying amount of the anti-MMP-9 immunomagnetic beads and the anti-occludin immunomagnetic beads is 30 μL / cm, and the drying temperature is 37°C.

[0034] The beneficial effects of the present invention are:

[0035] 1. The present invention combines the test strip with the magnetic immunochromatography technology to simultaneously detect MMP-9 and Occludin in the blood sample. It not only has the advantages of simplicity, rapidity, and simple operation of the traditional immunochromatography method, but also has higher detection sensitivity and accuracy, short detection time, and is suitable for on-site detection. It can achieve rapid qualitative and quantitative (the SERS signal intensity-marker concentration standard curve of the relevant marker can be drawn by the test strip first, and then the SERS intensity of the relevant marker is detected on the sample, and then the obtained results are compared with the standard curve to obtain the marker concentration in the measured sample) dual index project detection, thereby greatly improving the accuracy of stroke detection. When the MMP-9 content increases and the Occludin content decreases in the clinic, it can play a certain predictive role in the onset and prognosis of stroke, thereby facilitating the early diagnosis and prognosis evaluation of stroke, and has good clinical application value. In addition, the present detection invention can complete relevant clinical tests with only a small amount of blood samples, thereby further reducing medical expenses.

[0036] 2. The test strip of the present invention is combined with a magnetic signal detection system to further improve the sensitivity of stroke marker detection, thereby achieving trace detection of related markers, and the sensitivity can be increased by 100 times compared with traditional immunochromatography technology.

[0037] 3. The test strips of the present invention are simple to operate, convenient and practical, can be mass-produced, and the relevant equipment required for quantitative detection is now on the market. Therefore, they can be further applied to large-volume units such as hospitals and physical examination centers, small-volume units such as some grassroots hospitals and clinics, single-person testing, blood collection sites, and some remote areas with relatively scarce medical resources. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 It is a schematic diagram of the structure of the test strip of the present invention.

[0039] Among them: 1-bottom plate, 2-absorbent paperboard layer, 3-sample pad layer, 4-binding pad layer, 5-nitrocellulose membrane layer. DETAILED DESCRIPTION

[0040] In order to enable those skilled in the art to better understand the technical solution of the present invention, the technical solution of the present invention is further described below in conjunction with the accompanying drawings and embodiments.

[0041] As attached Figure 1 As shown, a test strip for simultaneously detecting MMP-9 and Occludin in blood comprises a bottom plate 1, an absorbent paperboard layer 2, a sample pad layer 3, a binding pad layer 4 and a nitrocellulose membrane layer 5, wherein the sample pad layer 3, the binding pad layer 4, the nitrocellulose membrane layer 5 and the absorbent paperboard layer 2 are fixed in sequence from one end to the other end above the bottom plate 1, and two adjacent layers are staggered and overlapped; the nitrocellulose membrane layer 5 is located at the bottom layer, and the bottoms of one end of the absorbent paperboard layer 2 and the binding pad layer 4 close to the nitrocellulose membrane layer 5 are both in contact with the top of the nitrocellulose membrane layer 5, and the bottom of one end of the sample pad layer 3 close to the binding pad layer 4 is in contact with the top of the binding pad layer 4;

[0042] The binding pad layer 4 contains anti-MMP-9 immunomagnetic beads and anti-Occludin immunomagnetic beads, and the nitrocellulose membrane layer 5 is provided with two detection lines and one quality control line, the two detection lines are coated with MMP-9 antibodies and Occludin antibodies respectively, and the quality control line is coated with rabbit anti-mouse IgG antibodies.

[0043] Furthermore, a method for preparing a test strip for simultaneously detecting MMP-9 and Occludin in blood comprises the following steps:

[0044] S1: Take the prepared base plate 1 and stick the processed nitrocellulose membrane layer 5 on the base plate 1 according to the marked boundary line;

[0045] The method for treating the nitrocellulose membrane layer 5 comprises the following steps:

[0046] S101: diluting MMP-9 antibody, Occludin antibody and rabbit anti-mouse IgG antibody into an antibody solution with a concentration of 1.5 mg / mL using an antibody diluent; the antibody diluent is a phosphate buffer containing 1 wt% sucrose;

[0047] S102: using a three-dimensional film-spraying gold spraying instrument to draw two test lines at a volume of 1 μL / cm on the nitrocellulose membrane layer 5 with the diluted MMP-9 antibody solution and the Occludin antibody solution, and using a three-dimensional film-spraying gold spraying instrument to draw a quality control line at a volume of 1 μL / cm on the nitrocellulose membrane layer 5 with the diluted rabbit anti-mouse IgG antibody solution, and the distance between each two adjacent lines is maintained at 0.4 cm;

[0048] S103: Dry the nitrocellulose membrane layer 5 with the detection line and the quality control line at room temperature, add a desiccant and seal it for later use.

[0049] Further, S2: pasting a water-absorbing paperboard layer 2 within the boundary line of one side of the nitrocellulose membrane layer 5, wherein the bottom of one end of the water-absorbing paperboard layer 2 close to the nitrocellulose membrane layer 5 contacts and is bonded and fixed to the top of the nitrocellulose membrane layer 5;

[0050] Further, S3: according to the size of the bottom plate 1, the pretreated sample pad layer 3 and the binding pad layer 4 containing anti-MMP-9 immunomagnetic beads and anti-Occludin immunomagnetic beads are sequentially pasted on the other side of the nitrocellulose membrane layer 5; the bottom of one end of the binding pad layer 4 close to the nitrocellulose membrane layer 5 is in contact with the top of the nitrocellulose membrane layer 5 and is bonded and fixed, and the bottom of one end of the sample pad layer 3 close to the binding pad layer 4 is in contact with the top of the binding pad layer 4 and is bonded and fixed;

[0051] Specifically, the method for treating the sample pad layer 3 is as follows: soaking the sample pad layer 3 in a BS buffer solution containing 2wt% NaCl, 2wt% TritonX-100, 0.5wt% BSA and 0.5wt% PVP, and then drying the sample pad layer 3 for later use.

[0052] The preparation method of the binding pad layer (4) containing anti-MMP-9 immunomagnetic beads and anti-occludin immunomagnetic beads comprises the following steps:

[0053] S301: activating the magnetic nanoparticles, and then coupling them with MMP-9 antibodies and Occludin antibodies, respectively, to prepare anti-MMP-9 immunomagnetic beads and anti-Occludin immunomagnetic beads;

[0054] Specifically, S3011: preparing magnetic nanoparticles by chemical reaction, and performing carboxyl modification on the magnetic nanoparticles by using succinic anhydride to obtain carboxylated magnetic nanoparticles with a particle size of 100-300 nm;

[0055] S3012: fully activate the magnetic beads prepared in step S101 using APTES or MPTES activation buffer, dry at 60°C, and then wash once with PBS after drying;

[0056] The preparation method of the APTES or MPTES activation buffer is as follows: adding an appropriate amount of Tween 20 to a 0.01M APTES or MPTES buffer to prepare an activation buffer with a final Tween 20 concentration of 0.05wt%, adjusting the pH of the activation buffer to control the pH value between 5.0 and 6.0, filtering the activation buffer with a 0.22μm microporous filter membrane and removing bacteria in the activation buffer, and storing it at 4°C for standby use. The activation buffer has a validity period of 14 days.

[0057] S3013: adding activators EDC and NHS to the washed magnetic beads, and performing activation reaction at room temperature for 1 hour. After the reaction, washing the magnetic beads with boric acid-borax (BS) coupling buffer; wherein the molar ratio of EDC, NHS to magnetic beads is 2:2:1;

[0058] The boric acid-borax (BS) coupling buffer solution is prepared by using 0.05M borax and 0.2M boric acid in a ratio of 4:1 (v:v) to prepare a BS buffer solution (0.2M pH=9.0), diluting it to 0.005M with ultrapure water, adding Tween 20 to a final concentration of 0.05%, filtering the BS coupling buffer solution with a 0.22μm microporous filter membrane and removing bacteria in the buffer solution, and storing it at 4°C for future use. The activation buffer solution has a validity period of 14 days.

[0059] S3014: The activated and fully washed magnetic beads were used to prepare anti-MMP-9 immunomagnetic beads at an amount of 20-40 μg anti-MMP-9-mAb / mg magnetic beads, and the same amount of anti-S100B-mAb was used to prepare anti-S100B immunomagnetic beads, and the reaction was carried out at room temperature for 2 hours; specifically, the mass ratio of magnetic beads to antibodies was 1:30;

[0060] S3015: Wash the magnetic beads with boric acid-borax (BS) blocking buffer to fully wash the magnetic beads, block the non-specific binding sites of the antibody, and fully block for half an hour at 37°C to obtain different immunomagnetic beads;

[0061] The preparation method of the boric acid-borax (BS) blocking buffer is as follows: a BS coupling buffer with a concentration of 0.005M is prepared by a boric acid-borax (BS) coupling buffer preparation method, BSA and Tween 20 are added to make the final concentrations of BSA and Tween 20 1wt% and 0.05wt% respectively, the BS blocking buffer is filtered with a 0.22μm microporous filter membrane and bacteria in the buffer are removed, and the buffer is stored at 4°C for standby use. The effective period of the activation buffer is 7 days.

[0062] S3016: The immunomagnetic beads obtained after blocking were treated with 0.05wt% Tween 20, 1wt% BSA, 0.05wt% NaN 3 The immunomagnetic beads were resuspended in boric acid-borax (BS) storage buffer and stored at 4°C for later use.

[0063] Preparation of boric acid-borax (BS) storage buffer: Prepare 0.005M BS buffer using the method for preparing BS buffer in 1 above, then add BSA, Tween 20, NaN 3 The final concentrations are 1wt%, 0.05wt%, and 0.05wt%. The BS storage buffer is filtered with a 0.22μm microporous filter membrane and the bacteria in the buffer are removed. It is stored at 4°C for use. The activation buffer is valid for 7 days.

[0064] Further, S302: soaking the bonding pad layer (4) with a bonding pad layer treatment solution and then drying; the bonding pad layer treatment solution is a boric acid-borax solution containing 5-10wt% sucrose and 0.05wt% TritonX-100.

[0065] S303: The anti-MMP-9 immunomagnetic beads and anti-occludin immunomagnetic beads prepared in step S301 are respectively and evenly sprayed on different positions on the treated binding pad layer 4, and then dried.

[0066] Specifically, the spraying amount of anti-MMP-9 immunomagnetic beads and anti-occludin immunomagnetic beads is 30 μL / cm, and the drying temperature is 37°C.

[0067] S4: After pasting is completed, the large board structure is cut into test strips of the same width.

[0068] Specifically, the pasted test paper board is cut into finished test paper strips with a width of 5 mm by an automatic cutting machine, which are sealed with an aluminum foil bag and stored with a desiccant for later use.

[0069] The working principle of the present invention is as follows: the present invention adopts monoclonal antibody technology to prepare MMP-9 paired antibodies and S100B paired antibodies respectively, and uses the double antibody sandwich principle to realize the rapid detection of MMP-9 and Occludin double antigens at the same time. When the blood sample to be tested contains two antigens, MMP-9 and Occludin, the two will first be coupled and combined with the anti-MMP-9 immunomagnetic beads and anti-Occludin immunomagnetic beads on the binding pad layer 4, and after the combination, they will move toward the side of the absorbent paper layer 2 under the action of capillary force to reach the detection line coated with Occludin antibody, and stay at the detection line in the form of a double antibody sandwich complex; the conjugate further moves toward the absorbent paper to reach the detection line coated with MMP-9 antibody, forming a double antibody sandwich complex and staying at the detection line coated with MMP-9 antibody; in addition, the unbound anti-MMP-9 immunomagnetic beads and anti-Occludin immunomagnetic beads continue to move forward to the quality control line, and are coupled and combined with rabbit anti-mouse IgG antibodies to form a magnetic bead aggregation phenomenon. The total reaction time of the above reaction is within 30 minutes. Generally, a magnetic signal detector can be used to detect the magnetic signals at T1, T2 and C lines within 20 minutes, and the obtained values ​​can be substituted into the fitting curve for calculation to obtain quantitative results.

[0070] The method of using the present invention is as follows: take out the magnetic immune test strip from the packaging box, tear open the aluminum foil packaging bag, and place it on a clean and flat desktop, use a pipette to draw about 50 μL of the blood sample to be tested and add it to the sample addition well, and let it stand for a reaction for about 20 minutes; after the sample addition reaction of the test strip is completed, use a magnetic immune chromatographic analyzer to automatically read the corresponding bar code information on the test card for detection, and the measured data results can be displayed.

[0071] The above shows and describes the basic principles, main features and advantages of the present invention. It should be understood by those skilled in the art that the present invention is not limited to the above embodiments, and the above embodiments and descriptions are only for explaining the principles of the present invention. Without departing from the spirit and scope of the present invention, the present invention may have various changes and improvements, which fall within the scope of the present invention to be protected. The scope of protection of the present invention is defined by the attached claims and their equivalents.

Claims

1. A test strip for simultaneously detecting MMP-9 and Occludin in blood, characterized in that: It comprises a bottom plate (1), a water-absorbing paperboard layer (2), a sample pad layer (3), a binding pad layer (4) and a nitrocellulose membrane layer (5); the sample pad layer (3), the binding pad layer (4), the nitrocellulose membrane layer (5) and the water-absorbing paperboard layer (2) are fixed in sequence from one end to the other end on the top of the bottom plate (1); two adjacent layers are staggered and overlapped; the nitrocellulose membrane layer (5) is located at the bottom layer; the bottoms of one end of the water-absorbing paperboard layer (2) and the binding pad layer (4) close to the nitrocellulose membrane layer (5) are in contact with the top of the nitrocellulose membrane layer (5); the bottom of one end of the sample pad layer (3) close to the binding pad layer (4) is in contact with the top of the binding pad layer (4); The binding pad layer (4) contains anti-MMP-9 immunomagnetic beads and anti-occludin immunomagnetic beads, and the nitrocellulose membrane layer (5) is provided with two detection lines and one quality control line, the two detection lines are coated with MMP-9 antibodies and Occludin antibodies respectively, and the quality control line is coated with rabbit anti-mouse IgG antibodies; The preparation method of the test strip, The following steps are included: S1: Take the prepared base plate (1), and stick the processed nitrocellulose membrane layer (5) on the base plate (1) according to the marked boundary line; S2: Pasting a water-absorbing paperboard layer (2) within the boundary line of one side of the nitrocellulose membrane layer (5), wherein the bottom of one end of the water-absorbing paperboard layer (2) close to the nitrocellulose membrane layer (5) is in contact and fixed with the top of the nitrocellulose membrane layer (5); S3: According to the size of the bottom plate (1), the pretreated sample pad layer (3) and the binding pad layer (4) containing anti-MMP-9 immunomagnetic beads and anti-occludin immunomagnetic beads are sequentially pasted on the other side of the nitrocellulose membrane layer (5); the bottom of one end of the binding pad layer (4) close to the nitrocellulose membrane layer (5) is in contact with and fixed to the top of the nitrocellulose membrane layer (5), and the bottom of one end of the sample pad layer (3) close to the binding pad layer (4) is in contact with and fixed to the top of the binding pad layer (4); the sample pad layer (3) is treated by: soaking the sample pad layer (3) in a BS buffer solution containing 2wt% NaCl, 2wt% TritonX-100, 0.5wt% BSA and 0.5wt% PVP, and then drying it for use; S4: After pasting is completed, the large board structure is cut into test strips of the same width; The method for treating the nitrocellulose membrane layer (5) in step S1 comprises the following steps: S101: diluting MMP-9 antibody, Occludin antibody and rabbit anti-mouse IgG antibody into antibody solutions with the same concentration using an antibody diluent; the antibody diluent is a phosphate buffer containing 1-2 wt% sucrose; S102: using the diluted MMP-9 antibody solution and the Occludin antibody solution to draw two test lines at a volume of 1 μL / cm on the nitrocellulose membrane layer (5), and using the diluted rabbit anti-mouse IgG antibody solution to draw a quality control line at a volume of 1 μL / cm on the nitrocellulose membrane layer (5), the distance between each two adjacent lines is maintained between 0.3 and 1 cm; S103: drying the nitrocellulose membrane layer (5) marked with the detection line and the quality control line at room temperature, adding a desiccant and sealing it for later use; The method for preparing the binding pad layer (4) containing anti-MMP-9 immunomagnetic beads and anti-occludin immunomagnetic beads in step S3 comprises the following steps: S301: activating the magnetic nanoparticles, and then coupling them with MMP-9 antibodies and Occludin antibodies, respectively, to prepare anti-MMP-9 immunomagnetic beads and anti-Occludin immunomagnetic beads; S302: soaking the bonding pad layer (4) with a bonding pad layer treatment solution and then drying it; the bonding pad layer treatment solution is a boric acid-borax solution containing 5-10wt% sucrose and 0.05wt% TritonX-100; S303: spraying the anti-MMP-9 immunomagnetic beads and anti-occludin immunomagnetic beads prepared in step S301 uniformly on different positions on the treated binding pad layer (4), and drying them; the spraying amount is 30 μL / cm, and the drying temperature is 37° C.; The specific operation of step S301 includes the following steps: S3011: preparing magnetic nanoparticles by chemical reaction, and performing carboxyl modification on the magnetic nanoparticles by using succinic anhydride to obtain carboxylated magnetic nanoparticles with a particle size of 100-300 nm; S3012: fully activate the magnetic beads prepared in step S101 with an activation buffer, dry them at 60° C., and then wash them once with PBS after drying; S3013: adding activators EDC and NHS to the washed magnetic beads, and performing activation reaction at room temperature for 1 hour. After the reaction, washing with PBS is performed for later use; wherein the molar ratio of EDC, NHS to magnetic beads is 2-5:2-5:1; S3014: adding the activated magnetic beads to different disease marker antibodies respectively, and reacting at room temperature for 2 hours; wherein the mass ratio of the magnetic beads to the antibodies is 1:20-40; S3015: Using blocking buffer to block the non-specific binding sites of the antibody at 37°C to obtain different immunomagnetic beads; wherein the blocking time is 30 min, and the pH of the boric acid-borax solution is 9; S3016: Re-disperse the immunomagnetic beads obtained after blocking in the storage buffer and store at 4°C for future use.

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