A rapid detection device for disease markers

Abstract

The present invention discloses a disease marker rapid detection device, including a microfluidic device and a detection device; the microfluidic device includes a detection disc, a driving component for driving the detection disc to rotate, and a main control circuit, the detection disc is fixed on the top of the driving component, the center of the detection disc is provided with a sample loading chamber, the outer periphery of the sample loading chamber is connected with a plurality of flow channels, each of the flow channels is provided with a phase change valve, and the end of each of the flow channels is connected with a reaction chamber, the detection device can be detachably installed in the reaction chamber; the detection device is a magnetic immune test strip that can quantitatively detect different disease markers in the blood at the same time. The present invention can simultaneously perform rapid, accurate, quantitative and automated detection of one or more markers of different diseases, the device cost is low, and the detection operation is convenient, thereby reducing the cost of patients.

Description

Technical Field

[0001] The present invention relates to the technical field of combining microfluidics technology with medical detection technology, and in particular to a disease marker rapid detection device. Background Art

[0002] Stroke, commonly known as stroke, has become one of the main causes of death in the world. Stroke has a high mortality and disability rate. Globally, with the aging of the population, the prevalence of stroke has been increasing year by year. In middle- and low-income countries, young people are also affected by stroke. Stroke seriously endangers people's health and life safety. It not only causes huge psychological and mental trauma to patients, but also causes serious economic burdens on families and the entire society. Stroke types include ischemic stroke and hemorrhagic stroke. Among them, 60-80% of stroke patients are diagnosed with ischemic stroke. The number of hemorrhagic stroke patients is second only to ischemic stroke, but hemorrhagic stroke has a more rapid onset, poor prognosis, and a high risk of disability. Therefore, it is of great significance to develop a method that can quickly identify and accurately diagnose related diseases in the early stage of the disease. It helps us to effectively prevent patients before they become ill and formulate scientific clinical treatment measures in the early stage of the disease to reduce the occurrence of stroke and improve the prognosis of stroke.

[0003] Clinically, the most widely used neuroimaging technologies for the diagnosis of neurological diseases include CT, MRI, TCD and PET, which can distinguish ischemic stroke from hemorrhagic stroke to a certain extent, but cannot achieve early rapid diagnosis and there is a lag; especially for patients with a small lesion range and mild injury, various imaging technologies are difficult to develop, so it is impossible to achieve ultra-early accurate diagnosis of patients. After a stroke occurs, it will cause brain tissue damage immediately. This stage is called early brain damage. Early brain tissue damage is also accompanied by blood-brain barrier damage. Due to the damage of the blood-brain barrier, the body's related inflammatory cells and immune cells reach the damaged brain tissue under chemotaxis to perform immune and inflammatory responses. In this process, inflammatory cells and immune cells will release a large number of cytokines and biochemical molecules into the extracellular fluid, and then pass through the blood-brain barrier again through the body fluid circulation to enter the cerebrospinal fluid and blood. These high levels of cytokines and biochemical molecules released into the cerebrospinal fluid and blood can be used as stroke-related markers. It can be used for the early diagnosis of stroke and is of great clinical significance. By detecting stroke markers, it is possible to quickly and early diagnose stroke patients. By analyzing stroke markers, it is possible to guide the formulation of early treatment decisions for stroke patients, thereby improving the prognosis of stroke and reducing the risk of disability. In addition, during the treatment process, it is necessary to grasp the condition as a whole, predict the progression of the condition, and formulate and implement relevant intervention measures. This can be achieved through dynamic monitoring of relevant disease markers and overall data analysis. Therefore, it is extremely important to apply the detection of disease-related markers in the clinical diagnosis and treatment process.

[0004] In clinical practice, relevant testing instruments are expensive, resulting in high costs for patients. At the same time, the testing operation is relatively complicated and requires a high level of professionalism from the testing personnel, which hinders improving the diagnosis rate of stroke patients. Summary of the invention

[0005] In view of the above-mentioned problems, the present invention aims to provide a disease marker rapid detection device, which can simultaneously perform rapid, accurate, quantitative and automated detection of relevant markers of different diseases. The device has low cost and convenient detection operation, thereby reducing patients' expenses.

[0006] In order to achieve the above object, the technical solution adopted by the present invention is as follows:

[0007] A disease marker rapid detection device, characterized in that: it comprises a microfluidic device and a detection device;

[0008] The microfluidic device includes a detection disc, a driving component for driving the detection disc to rotate, and a main control circuit. The detection disc is fixed on the top of the driving component. A sample loading chamber is provided in the center of the detection disc. A plurality of flow channels are connected to the periphery of the sample loading chamber. A phase change valve is provided in each of the flow channels. A reaction chamber is connected to the end of each of the flow channels. The detection device can be detachably installed in the reaction chamber.

[0009] Furthermore, the driving assembly includes a driving motor and a rotating shaft, wherein the rotating shaft is installed at the output end of the driving motor; and the detection disc is fixedly arranged on the top of the rotating shaft.

[0010] Furthermore, the phase change valve is a structure filled with a temperature-controlled material, which is in an open state when the material is heated to form two hemispherical structures facing each other, and is in a closed state when the entire square structure is formed after cooling and solidification;

[0011] A heating resistor and a temperature-sensitive resistor are arranged in each of the hemispherical structures, and the heating resistor, the temperature-sensitive resistor and the driving component are all connected to the main control circuit.

[0012] Furthermore, the detection device is a magnetic immunoassay strip that can simultaneously and quantitatively detect different disease markers in the blood.

[0013] Furthermore, the magnetic immunoassay strip comprises a bottom plate, an absorbent paperboard layer, a sample pad layer, a binding pad layer and a nitrocellulose membrane layer; the sample pad layer, the binding pad layer, the nitrocellulose membrane layer and the absorbent 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, and the bottoms of one end of the absorbent 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;

[0014] The binding pad layer is provided with a plurality of immunomagnetic beads activated with magnetic beads as the substrate and coupled with disease marker antibodies, the nitrocellulose membrane layer is provided with a plurality of detection lines and a quality control line, each of the detection lines is coated with the disease marker antibody corresponding thereto, and the quality control line is coated with rabbit anti-mouse IgG antibody.

[0015] Furthermore, the method for preparing the magnetic immunoassay strip comprises the following steps:

[0016] S1: Activate the magnetic nanoparticles and couple them with antibodies of different disease markers to form different immunomagnetic beads;

[0017] S2: soaking the sample pad layer (6) and the binding pad layer (7) in different treatment solutions and then drying them; wherein the treatment solution for soaking the sample pad layer (6) is a BS buffer solution containing 2wt% NaCl, 2wt% TritonX-100, 0.5wt% BSA and 0.5wt% PVP; and the treatment solution for soaking the binding pad layer (7) is a BS buffer solution containing 5-10wt% sucrose, 2wt% trehalose and 0.05wt% TritonX-100;

[0018] S3: spraying the different immunomagnetic beads prepared in step S1 evenly on different positions on the treated binding pad layer, and drying them;

[0019] S4: drawing a plurality of test lines and a quality control line on the nitrocellulose membrane layer, wherein each of the test lines is coated with a disease marker antibody corresponding thereto, and the quality control line is coated with a rabbit anti-mouse IgG antibody;

[0020] S5: Paste the absorbent paperboard layer, sample pad layer, binding pad layer and nitrocellulose membrane layer on the bottom plate in order, and keep some overlapping between adjacent layers;

[0021] S6: Use an automatic cutting machine to cut the test paper board assembled in step S5 into finished test paper strips with a width of 3 mm, seal them with aluminum foil bags, add desiccant and store them for later use.

[0022] Furthermore, the specific operation of step S1 includes the following steps:

[0023] S101: 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;

[0024] S102: fully activating the magnetic beads prepared in step S101 with an activation buffer, drying at 60° C., and then washing once with PBS after drying;

[0025] S103: 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 the magnetic beads is 2 to 5:2 to 5:1;

[0026] S104: 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;

[0027] S105: using a 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;

[0028] S106: The immunomagnetic beads obtained after blocking are redispersed in a storage buffer and stored at 4°C for future use.

[0029] Furthermore, the specific operation of step S3 includes the following steps:

[0030] S301: using a quantitative spraying device or a micropipette to absorb different immunomagnetic beads of the same mass, and applying them evenly at different positions on the treated binding pad layer (7) at a volume of 30 μL / cm;

[0031] S302: Dry the binding pad layer sprayed with the immunomagnetic beads at 25-37°C, add a desiccant after the treatment, and seal it for later use.

[0032] Furthermore, the specific operation of step S4 includes the following steps:

[0033] S401: dilute the disease marker antibody used for coating the test line to 1.5 mg / mL with antibody diluent. Similarly, dilute the rabbit anti-mouse IgG antibody used for coating the quality control line to 0.5 mg / mL with antibody diluent.

[0034] S402: Use a three-dimensional film spraying instrument to draw the required test lines and quality control lines on the nitrocellulose membrane layer at a volume of 1 μL / cm, so that the distance between each two adjacent lines is maintained between 0.3 and 1 cm. After drying at room temperature, add desiccant and seal for later use.

[0035] Furthermore, the antibody diluent in step S401 is a phosphate buffer containing 1-2 wt% sucrose.

[0036] The beneficial effects of the present invention are:

[0037] 1. The disease marker rapid detection device of the present invention controls the centrifugal force at the center of the detection disk by adjusting the rotation speed of the driving motor, thereby controlling the amount of sample passing through the flow channel, and then controls the properties of the hot-melt material of the phase change valve by controlling the working state of the heating resistor to adjust the closing state of the phase change valve, so that the sample can quantitatively pass through the flow channel and reach the magnetic immunoassay strip in the reaction chamber, and the disease marker is quantitatively detected by the magnetic immunoassay strip. The device has low cost and convenient detection operation, thereby reducing the cost of patients.

[0038] 2. The disease marker rapid detection device of the present invention is provided with multiple flow channels on the periphery of the sample loading chamber, which can be used to place multiple magnetic immune test strips for detecting different diseases, and can simultaneously perform quantitative detection of single or multiple related markers of different diseases in the blood, significantly improving the accuracy of disease detection, and having more clinical use value. For example, when the stroke-related markers MMP-9 and S100B are detected simultaneously, on the basis of double positivity, not only can the occurrence of stroke be more accurately judged, thereby improving the early diagnosis rate of the disease, but also the prognosis of stroke can be scientifically predicted and effective intervention can be formulated, which is of great practical value in clinical practice.

[0039] 3. The magnetic immunoassay paper strip of the present invention uses nano-scale magnetic beads as a marker substance, and a magnetic signal detector can be used to read relevant data and analyze it. Compared with traditional immunochromatography (such as colloidal gold, latex particles, etc.), it overcomes the problem of difficulty in quantitative sample detection, further improves the accuracy and sensitivity of sample detection, and shortens the time required for sample detection. It is convenient, practical, specific and sensitive. Compared with traditional immunochromatography technology, the detection sensitivity of the magnetic immunoassay paper strip of the present invention is increased by 100 times.

[0040] 4. The present invention combines microfluidics technology with current automatic control technology to simplify the operation procedure. Compared with traditional detection instruments, the device has a simple manufacturing process, simple operation, high integration, can achieve accurate quantitative detection of samples and reduce the cost of detection, and is suitable for large-scale production. It can be used not only in large hospitals for large-scale detection, but also in grassroots clinics or areas with insufficient medical care for small batches and single-person use. Therefore, the device is small and portable and can be used for free clinics and physical examinations. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] Figure 1 This is a schematic diagram of the structure of the disease marker rapid detection device of the present invention.

[0042] Figure 2 This is a top view of the structure of the disease marker rapid detection device of the present invention.

[0043] Figure 3 It is a front view of the structure of the magnetic immunoassay paper strip of the present invention.

[0044] Figure 4 It is a top view of the structure of the magnetic immunoassay paper strip of the present invention.

[0045] Figure 5 This is a circuit diagram between the heating resistor, the temperature-sensitive resistor and the drive motor of the present invention.

[0046] Wherein: 100-detection device, 1-detection disc, 101-sample loading chamber, 102-flow channel, 103-phase change valve, 1031-heating resistor, 1032-thermistor, 104-reaction chamber, 2-driving motor, 3-rotating shaft, 4-bottom plate, 5-absorbent paperboard layer, 6-sample pad layer, 7-binding pad layer, 8-nitrocellulose membrane layer, 801-detection line, 802-quality control line. DETAILED DESCRIPTION

[0047] 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.

[0048] Embodiment 1:

[0049] As attached Figure 1-5 As shown, a disease marker rapid detection device includes a microfluidic device and a detection device 100;

[0050] The microfluidic device includes a detection disc 1, a driving component for driving the detection disc 1 to rotate, and a main control circuit. The detection disc 1 is fixed on the top of the driving component. A sample loading chamber 101 is provided in the center of the detection disc 1. The sample loading chamber 101 is provided with a scale and a corresponding volume value. The periphery of the sample loading chamber 101 is connected to a plurality of flow channels 102 (in the present invention, the number of the flow channels 102 is 4, which are evenly and symmetrically distributed on the periphery of the sample loading chamber 101). A phase change valve 103 is provided in each of the flow channels 102, and the ends of each of the flow channels 102 are connected to a reaction chamber 104. The detection device 100 can be detachably installed in the reaction chamber 104.

[0051] Specifically, the driving assembly includes a driving motor 2 and a rotating shaft 3 . The rotating shaft 3 is installed at the output end of the driving motor 2 ; and the detection disc 1 is fixed on the top of the rotating shaft 3 .

[0052] The phase change valve 103 is filled with temperature-controlling material, and is composed of two hemispherical structures facing each other in the open state, and is a whole square structure in the closed state. The two hemispherical structures can close the corresponding flow channel 102 in the closed state, and can open the corresponding flow channel 102 in the open state.

[0053] A heating resistor 1031 and a temperature-sensitive resistor 1032 are provided in each of the hemispherical structures. The heating resistor 1031 , the temperature-sensitive resistor 1032 and the driving component are all connected to the main control circuit.

[0054] In the initial state, the phase change valve 103 is in the closed state. After the power is turned on, the circuit supplies power to the heating resistor 1031 in the phase change valve 103. The heating resistor 1031 starts working immediately, and the temperature rises rapidly to the melting point of the hot-melt material. The hot-melt material changes from a solid square structure (i.e., a closed state) to a liquid double semicircular structure (i.e., an open state). The sample in the sample adding chamber 101 opens the phase change valve 103 under the action of centrifugal force, enters the reaction chamber 104, and reacts with the detection device 100. At this time, the temperature-sensitive resistor 1032 in the phase change valve measures the melting point of the hot-melt material, and feeds back to the electrical module through the PID algorithm. The electrical module issues an instruction, and the power supply of the heating resistor 1031 is cut off. The detection disc 1 is circular, the speed of the driving motor 2 is constant during operation, and the centrifugal force generated is constant, thereby controlling the volume of the blood sample flowing into the reaction chamber 104, and realizing quantitative and qualitative measurement of the detection sample. By using the temperature control technology of the phase change valve 103, the sample can be loaded sequentially when the flow channel 102 is in the closed and open states alternately. Different detection devices 100, that is, different detection chips are used to detect different disease markers of the same sample, and different samples can also be detected in sequence.

[0055] Furthermore, the detection device 100 is a magnetic immunoassay strip that can simultaneously and quantitatively detect different disease markers in the blood; in this embodiment, the stroke-related markers MMP-9 and S100B antibodies are selected to prepare the magnetic immunoassay strip, and the prepared magnetic immunoassay strip is used to simultaneously detect the stroke markers MMP-9 and S100B.

[0056] Specifically, the magnetic immunoassay paper strip comprises a bottom plate 4, an absorbent paperboard layer 5, a sample pad layer 6, a binding pad layer 7 and a nitrocellulose membrane layer 8; the sample pad layer 6, the binding pad layer 7, the nitrocellulose membrane layer 8 and the absorbent paperboard layer 5 are fixed in sequence from one end to the other end above the bottom plate 4, and two adjacent layers are staggered and overlapped; the nitrocellulose membrane layer 8 is located at the bottom layer, and the bottoms of one end of the absorbent paperboard layer 5 and the binding pad layer 7 close to the nitrocellulose membrane layer 8 are in contact with the top of the nitrocellulose membrane layer 8, and the bottom of one end of the sample pad layer 6 close to the binding pad layer 7 is in contact with the top of the binding pad layer 7; as shown in the attached Figure 3 shown.

[0057] The binding pad layer 7 is provided with a variety of immunomagnetic beads activated with magnetic beads as the substrate and coupled with disease marker antibodies (MMP-9 and S100B antibodies), and the nitrocellulose membrane layer 8 is provided with two detection lines 801 and a quality control line 802. The two detection lines 801 are coated with MMP-9 antibodies and S100B antibodies respectively, and the quality control line 802 is coated with rabbit anti-mouse IgG antibodies.

[0058] Specifically, the method for preparing the magnetic immunoassay strip comprises the following steps:

[0059] S1: Activate the magnetic nanoparticles and couple them with antibodies of different disease markers to form different immunomagnetic beads;

[0060] Specifically, S101: 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;

[0061] S102: fully activate the magnetic beads prepared in step S101 using APTES or MPTES activation buffer, dry them at 60° C., and then wash them once with PBS after drying;

[0062] 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.

[0063] S103: 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;

[0064] 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.

[0065] S104: The activated and fully washed magnetic beads are 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 is used to prepare anti-S100B immunomagnetic beads, and the reaction is carried out at room temperature for 2 hours; specifically, the mass ratio of magnetic beads to antibodies is 1:20-40;

[0066] S105: 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;

[0067] 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.

[0068] S106: The immunomagnetic beads obtained after blocking were treated with a solution containing 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.

[0069] 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.

[0070] Further, S2: soak the sample pad layer 6 and the bonding pad layer 7 in different treatment liquids respectively for 2 hours, and dry them after soaking. The oven temperature for baking the sample pad layer 6 is set between 25°C and 37°C, and the oven temperature for baking the bonding pad layer 7 is set at 30°C, and the baking time is 12 hours; the length of the sample pad layer 6 is 15mm, and the length of the bonding pad layer 7 is 10mm.

[0071] The treatment solution for soaking the sample pad layer 6 is a BS buffer solution (0.005M, pH=9.0) containing 2wt% NaCl, 2wt% TritonX-100, 0.5wt% BSA and 0.5wt% PVP;

[0072] The treatment solution for soaking the binding pad layer 7 is BS buffer (0.005M, pH=9.0) containing 5wt% sucrose, 2wt% trehalose, and 0.05wt% TritonX-100.

[0073] Further, S3: the different immunomagnetic beads prepared in step S1 are evenly sprayed on different positions on the processed binding pad layer 7, and dried;

[0074] Specifically, use a quantitative spray device or a micropipette to absorb different immunomagnetic beads of the same mass, and apply them evenly at different positions on the treated binding pad layer 7 at an amount of 30 μL / cm; dry the binding pad layer 7 sprayed with the immunomagnetic beads at 25-37°C, add a desiccant after the treatment, and seal it for later use.

[0075] Further, S4: two detection lines 801 and a quality control line 802 are drawn on the nitrocellulose membrane layer 8, the two detection lines 801 are coated with MMP-9 antibody and S100β antibody respectively, and the quality control line 802 is coated with rabbit anti-mouse IgG antibody;

[0076] Specifically, the MMP-9 antibody was diluted to 1.5 mg / mL using an antibody diluent (a phosphate buffer containing 1 wt% sucrose), the S100β antibody was also diluted to 1.5 mg / mL using an antibody diluent (a phosphate buffer containing 1 wt% sucrose), and the rabbit anti-mouse IgG antibody was diluted to 0.5 mg / mL using an antibody diluent (a phosphate buffer containing 1 wt% sucrose); two detection lines 801 and a quality control line 802 were drawn on the nitrocellulose membrane layer 8 at a volume of 1 μL / cm using a three-dimensional film spraying instrument, and the distance between the two adjacent lines was 0.4.cm; the nitrocellulose membrane layer 8 with the detection line 801 and the quality control line 802 was dried at room temperature, and then a desiccant was added and sealed at 4°C for later use.

[0077] The preparation method of phosphate buffer containing 1wt% sucrose is as follows: sucrose is added to 0.01M, pH=7.4 phosphate (PBS) buffer to finally prepare an antibody diluent with a sucrose concentration of 1wt%, the antibody diluent is filtered with a 0.22μm microporous filter membrane and bacteria in the buffer are removed, and the antibody diluent is stored at 4°C for future use. The antibody diluent is valid for 7 days.

[0078] Further, S5: the absorbent paperboard layer 5, the sample pad layer 6, the binding pad layer 7 and the nitrocellulose membrane layer 8 are sequentially pasted on the bottom plate 4, and two adjacent plate layers are partially staggered and overlapped.

[0079] Paste the absorbent paperboard layer 5 (25 mm long), the sample pad layer 6 (15 mm long), the binding pad layer 7 (10 mm long) and the nitrocellulose membrane layer 8 (25 mm long) on ​​the bottom plate 4 in order, and keep the adjacent two layers partially overlapped, with an overlap length of 2 mm;

[0080] Further, S6: using an automatic cutting machine to cut the test paper board assembled in step S5 into finished test paper strips with a width of 3 mm, sealing them with aluminum foil bags, adding desiccant and storing them for later use.

[0081] The working principle of the present invention: MMP-9 is a matrix metalloproteinase that plays an important role in maintaining the dynamic balance of extracellular matrix degradation and remodeling. After a stroke, the expression of MMP-9 by neurons and oligodendrocytes in damaged brain tissue will increase significantly, and the expression of MMP-9 by cells involved in the inflammatory response of the damaged site (reactive astrocytes and activated microglia) will also be significantly enhanced. After MMP-9 is released into the extracellular space, it is activated by cleavage of the propeptide and participates in the decomposition of the extracellular matrix, thereby promoting cerebrovascular damage. At the same time, the release of free radicals in the ischemic site increases the activity of MMP-9, further aggravating the brain damage after stroke. The increase of MMP-9 is also related to various post-stroke complications, including excitotoxicity, neuronal damage, apoptosis, oxidative stress, interference with the oxidative DNA repair mechanism, and the opening of the BBB leading to the influence of brain edema and hemorrhagic transformation after cerebral ischemia. Therefore, MMP-9 can indicate the degree of stroke damage to a certain extent, and MMP-9 can be regarded as a possible marker of cerebral ischemia and a predictor of bleeding in patients treated with tPA.

[0082] S100β protein is mainly present in the cytosol of central nervous system glial cells. When brain tissue is damaged, glial cells are activated, cell membrane permeability increases, and a large amount of synthesized S100β protein is secreted into the cerebrospinal fluid. Due to the change in the permeability of the blood-brain barrier, S100β enters the peripheral blood from the cerebrospinal fluid. Therefore, the content of S100β protein in peripheral blood can indicate the integrity of the blood-brain barrier to a certain extent. At the same time, S100β protein is related to the apoptosis and lysis of neurons and glial cells. Some studies have also shown that glial cells will respond accordingly by increasing the synthesis and secretion of S100β during brain injury. Therefore, the level of S100β in peripheral blood is of great significance in judging the severity of stroke and inferring the prognosis of stroke. In different types of stroke, the time when S100β in peripheral blood reaches its peak also varies significantly. Therefore, S100β can not only be used as a marker for early auxiliary diagnosis of stroke, but also can be used to distinguish the severity and type of stroke.

[0083] The present invention adopts monoclonal antibody technology to prepare MMP-9 paired monoclonal antibodies and S100β paired monoclonal antibodies respectively, and uses the double antibody sandwich principle to detect related antigen specimens. First, the successfully prepared anti-MMP-9 immunomagnetic beads and anti-S100B immunomagnetic beads are combined with MMP-9 and S100β antigens in the specimen to be tested on the binding pad layer 7 to form an immune complex. Due to the chromatography effect, the complex will all reach the first detection line (S100β antibody detection line), and the S100β immune complex will combine with the S100B antibody in the detection line again to form a double antibody sandwich complex and stay at the first detection line. The MMP-9 immune complex in the complex will continue to move forward to reach the second detection line (MMP-9 antibody detection line) and combine with the MMP-9 antibody in this detection line to form a double antibody sandwich complex and gather at the second quality control line. The magnetic beads that are not coupled with MMP-9 antibody and S100β antibody will move forward to the quality control line and combine with the rabbit anti-mouse IgG antibody in the quality control line to aggregate and stay at the quality control line. The entire reaction process takes 30 minutes. The magnetic signal detector can generally detect after 20 minutes of reaction. At this time, the corresponding magnetic signal values ​​of the three lines can be obtained. Then, the measured magnetic signal values ​​are brought into the fitting curve for calculation to obtain the quantitative results.

[0084] 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, insert the magnetic immune test strip into the reaction chamber 104 of the detection disc 1, use a micropipette to absorb an appropriate amount of serum sample and add it into the sample adding chamber 101 of the detection disc 1, turn on the switch of the main control circuit, and wait for 1 minute; take out the magnetic immune test strip with complete reaction and insert it into the card insertion port of the magnetic immune chromatographic analyzer, run the instrument, and the instrument will automatically read the bar code information of the detection card, perform detection and display the measurement results in real time.

[0085] 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 rapid detection device for disease markers, characterized in that: Including a microfluidic device and a detection device (100); The microfluidic device comprises a detection disc (1), a driving component for driving the detection disc (1) to rotate, and a main control circuit; the detection disc (1) is fixedly mounted on the top of the driving component; a sample loading chamber (101) is provided at the center of the detection disc (1); a plurality of flow channels (102) are connected to the periphery of the sample loading chamber (101); each of the flow channels (102) is provided with a phase change valve (103); and the end of each of the flow channels (102) is connected to a reaction chamber (104); the detection device (100) is detachably mounted in the reaction chamber (104); The driving assembly comprises a driving motor (2) and a rotating shaft (3), wherein the rotating shaft (3) is mounted on the output end of the driving motor (2); the detection disc (1) is fixedly mounted on the top of the rotating shaft (3); The phase change valve (103) is a structure filled with a temperature-controlled material, which is in an open state when the material is heated to form two hemispherical structures facing each other, and is in a closed state when the material is cooled and solidified to form a whole square structure; A heating resistor (1031) and a temperature-sensitive resistor (1032) are provided in each of the hemispherical structures, and the heating resistor (1031), the temperature-sensitive resistor (1032) and the driving component are all connected to the main control circuit; The detection device (100) comprises a magnetic immunoassay strip capable of simultaneously and quantitatively detecting the stroke disease markers MMP-9 and S100B in the blood; The magnetic immunoassay paper strip comprises a bottom plate (4), a water-absorbing paperboard layer (5), a sample pad layer (6), a binding pad layer (7) and a nitrocellulose membrane layer (8); the sample pad layer (6), the binding pad layer (7), the nitrocellulose membrane layer (8) and the water-absorbing paperboard layer (5) are fixed in sequence from one end to the other end above the bottom plate (4), and two adjacent layers are staggered and overlapped; the nitrocellulose membrane layer (8) is located at the bottom layer, and the bottoms of one end of the water-absorbing paperboard layer (5) and the binding pad layer (7) close to the nitrocellulose membrane layer (8) are in contact with the top of the nitrocellulose membrane layer (8), and the bottom of one end of the sample pad layer (6) close to the binding pad layer (7) is in contact with the top of the binding pad layer (7); The binding pad layer (7) is provided with a plurality of immunomagnetic beads activated with magnetic beads as a substrate and coupled with disease marker antibodies, the nitrocellulose membrane layer (8) is provided with a plurality of detection lines (801) and a quality control line (802), each of the detection lines (801) is coated with a disease marker antibody corresponding thereto, and the quality control line (802) is coated with a rabbit anti-mouse IgG antibody; The method for preparing the magnetic immune test strip comprises the following steps: S1: Activate the magnetic nanoparticles and couple them with antibodies of different disease markers to form different immunomagnetic beads; S2: soaking the sample pad layer (6) and the binding pad layer (7) in different treatment solutions and then drying them; wherein the treatment solution for soaking the sample pad layer (6) is a BS buffer solution containing 2 wt% NaCl, 2 wt% TritonX-100, 0.5 wt% BSA and 0.5 wt% PVP; and the treatment solution for soaking the binding pad layer (7) is a BS buffer solution containing 5-10 wt% sucrose, 2 wt% trehalose and 0.05 wt% TritonX-100; S3: spraying the different immunomagnetic beads prepared in step S1 evenly on different positions on the processed binding pad layer (7), and performing drying treatment; S4: drawing a plurality of detection lines (801) and a quality control line (802) on the nitrocellulose membrane layer (8), wherein each detection line (801) is coated with a disease marker antibody corresponding thereto, and the quality control line (802) is coated with a rabbit anti-mouse IgG antibody; S5: Paste the absorbent paperboard layer (5), the sample pad layer (6), the binding pad layer (7) and the nitrocellulose membrane layer (8) on the bottom plate (4) in order, and keep the adjacent two layers partially staggered and overlapped; S6: using an automatic cutting machine to cut the test paper sheet assembled in step S5 into finished test paper strips with a width of 3 mm, sealing them with aluminum foil bags, adding desiccant and storing them for later use; The specific operation of step S1 includes the following steps: S101: 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; S102: fully activating the magnetic beads prepared in step S101 with an activation buffer, drying at 60° C., and then washing once with PBS after drying; S103: 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; S104: 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; S105: 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; S106: Re-disperse the immunomagnetic beads obtained after blocking in the storage buffer and store them at 4°C for future use.

2. A disease marker rapid detection device according to claim 1, characterized in that: The specific operation of step S3 includes the following steps: S301: Using a quantitative spraying device or a micropipette, different immunomagnetic beads of the same mass are respectively absorbed and evenly applied to different positions on the treated binding pad layer (7) at a volume of 30 μL / cm; S302: Dry the binding pad layer (7) sprayed with the immunomagnetic beads at 25-37°C, add a desiccant after the drying, and seal it for later use.

3. A disease marker rapid detection device according to claim 1, characterized in that: The specific operation of step S4 includes the following steps: S401: dilute the disease marker antibody used for coating the test line (801) to 1.5 mg / mL with antibody diluent. Similarly, dilute the rabbit anti-mouse IgG antibody used for coating the quality control line (802) to 0.5 mg / mL with antibody diluent. S402: Use a three-dimensional film spraying instrument to draw the required test line (801) and quality control line (802) on the nitrocellulose membrane layer (8) at a volume of 1 μL / cm, so that the distance between each two adjacent lines is maintained between 0.3 and 1 cm. After drying at room temperature, add a desiccant and seal for later use.

4. A disease marker rapid detection device according to claim 3, characterized in that: The antibody diluent described in step S401 is a phosphate buffer containing 1-2 wt% sucrose.

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