Flow A / flow B visual quantum dot joint inspection device and application thereof

Abstract

The invention provides an A-flow / B-flow visual quantum dot joint detection device and application thereof. The A-flow / B-flow visual quantum dot joint detection device structurally comprises a quantum dot detection test strip and a composite quantum dot magnetic microsphere probe, a detection line and a control line are arranged on the quantum dot detection test strip; the number of the detection line is one, the detection line is coated with an A-flow capture monoclonal antibody and a B-flow capture monoclonal antibody at the same time, and the A-flow capture monoclonal antibody and the B-flow capture monoclonal antibody are mixed together and located at the same position of the detection line; the control line is coated with chicken egg yolk immunoglobulin; the composite quantum dot magnetic microsphere probe comprises a water-soluble red quantum dot magnetic microsphere detection probe, a water-soluble green quantum dot magnetic microsphere detection probe, a water-soluble red quantum dot magnetic microsphere control probe and a water-soluble green quantum dot magnetic microsphere control probe; according to the invention, joint detection of the A flow and the B flow can be realized.

Description

Technical Field

[0001] The present invention relates to a visual quantum dot joint detection device for influenza A / influenza B and its application, belonging to the technical field of in vitro fluorescence detection. Background Art

[0002] Since influenza A (H1N1) and influenza B (H3N2) in epidemic viruses have similar infection symptoms and infected individuals should be treated differently, it is crucial to quickly and accurately detect and distinguish between influenza A and influenza B during the influenza season; currently, the detection of influenza viruses includes the real-time reverse transcription polymerase chain reaction (RT-PCR) method for detecting influenza virus RNA and the lateral flow immunoassay (LFIA) method for detecting specific antibodies; although the common RT-PCR method has high sensitivity, its detection takes a long time, reducing the detection efficiency and having certain limitations in dealing with emergencies; while the LFIA method has good safety, portability, time-saving, easy operation and high detection efficiency in diagnosis; for the LFIA method, the most commonly used labeling material is colloidal gold, which identifies target substances through colorimetric signals and is commonly used for qualitative and semi-quantitative detection; the LFIA developed based on colorimetric signals has limited sensitivity and is prone to the risk of missed diagnosis, and the fluorescence signal is more sensitive than the traditional colloidal gold colorimetric signal; considering the detection sensitivity and personal recognition error, it is necessary to develop a high-sensitivity quantitative detection scheme for LFIA based on high-performance fluorescent nanolabels.

[0003] Quantum dot materials have characteristics such as tunable emission spectra, strong anti-photobleaching ability, high fluorescence intensity, and multi-color fluorescence excitation by a single light source, making quantum dot materials an indispensable new generation of fluorescent nanolabels with great potential for biomedical applications; in recent years, the lateral flow immunoassay (QD-LFIA) method developed with quantum dots as fluorescent probes has been widely applied to the qualitative and quantitative detection of disease markers, toxic substances in food, etc.; QD-LFIA has advantages such as good stability, high sensitivity, low cost, fast and convenient, and is the most mainstream technology for point-of-care testing (POCT); however, existing test strips for the joint detection of influenza A and influenza B developed based on the QD-LFIA method usually identify the target substances of influenza A and influenza B according to two test lines (T lines) respectively, resulting in different flow reaction times on the test strip and affecting the detection accuracy; and it is based on quantum dots of a single wavelength as fluorescent probes, which is prone to cross-interference between different detection items, and at the same time, the recognition range of a single fluorescent signal is limited, resulting in limited sensitivity; therefore, how to improve the detection sensitivity and accuracy of the QD-LFIA method is very important. Summary of the Invention

[0004] The present invention provides a visual quantum dot joint detection device for influenza A / influenza B and its application, aiming to provide a joint detection device capable of jointly detecting influenza A and influenza B.

[0005] Technical solution of the present invention: A visualization quantum dot joint detection device for influenza A and influenza B, the structure of which includes a quantum dot detection test strip and a composite quantum dot magnetic microsphere probe; a detection line and a control line are arranged on the quantum dot detection test strip; there is 1 detection line, and both influenza A capture monoclonal antibody and influenza B capture monoclonal antibody are coated on the detection line at the same time, and the influenza A capture monoclonal antibody and the influenza B capture monoclonal antibody are mixed together and in the same position on the detection line; chicken egg yolk immunoglobulin is coated on the control line; the composite quantum dot magnetic microsphere probe includes a water-soluble red quantum dot magnetic microsphere detection probe, a water-soluble green quantum dot magnetic microsphere detection probe, a water-soluble red quantum dot magnetic microsphere control probe, and a water-soluble green quantum dot magnetic microsphere control probe.

[0006] Further, the water-soluble red quantum dot magnetic microsphere detection probe includes a water-soluble red quantum dot magnetic microsphere and an influenza B labeled monoclonal antibody; the water-soluble red quantum dot magnetic microsphere control probe includes a water-soluble red quantum dot magnetic microsphere and a goat anti-chicken lgY polyclonal antibody; the water-soluble red quantum dot magnetic microsphere includes magnetic nanoparticles, a silica layer, red fluorescent quantum dots, and a polymer polyacrylic acid from the inside to the outside, wherein the red fluorescent quantum dots are coated on the surface of the magnetic nanoparticles, there is a certain thickness of silica layer between the red fluorescent quantum dots and the surface of the magnetic nanoparticles, and the outer layer of the red fluorescent quantum dots is polymer polyacrylic acid; The water-soluble green quantum dot magnetic microsphere detection probe includes a water-soluble green quantum dot magnetic microsphere and an influenza A labeled monoclonal antibody; the water-soluble green quantum dot magnetic microsphere control probe includes a water-soluble green quantum dot magnetic microsphere and a goat anti-chicken lgY polyclonal antibody; the water-soluble green quantum dot magnetic microsphere includes magnetic nanoparticles, a silica layer, green fluorescent quantum dots, and a polymer polyacrylic acid from the inside to the outside, wherein the green fluorescent quantum dots are coated on the surface of the magnetic nanoparticles, there is a certain thickness of silica layer between the green fluorescent quantum dots and the surface of the magnetic nanoparticles, and the outer layer of the green fluorescent quantum dots is polymer polyacrylic acid.

[0007] Furthermore, the preparation methods of the water-soluble red quantum dot magnetic microsphere detection probe and the water-soluble red quantum dot magnetic microsphere control probe include: mixing a sulfo-NHS solution, an EDC solution with a water-soluble red quantum dot magnetic microsphere BS solution, and performing ultrasonic activation treatment to obtain the activated water-soluble red quantum dot magnetic microspheres; mixing the activated water-soluble red quantum dot magnetic microspheres with an influenza B-labeled monoclonal antibody for conjugation, and then performing centrifugal separation to obtain the precipitate, the water-soluble red quantum dot magnetic microsphere detection probe; mixing the activated water-soluble red quantum dot magnetic microspheres with a goat anti-chicken lgY polyclonal antibody for conjugation, and then performing centrifugal separation to obtain the precipitate, the water-soluble red quantum dot magnetic microsphere control probe; The preparation methods of the water-soluble green quantum dot magnetic microsphere detection probe and the water-soluble green quantum dot magnetic microsphere control probe include: mixing a sulfo-NHS solution, an EDC solution with a water-soluble green quantum dot magnetic microsphere BS solution, and performing ultrasonic activation treatment to obtain the activated water-soluble green quantum dot magnetic microspheres; mixing the activated water-soluble green quantum dot magnetic microspheres with an influenza A-labeled monoclonal antibody for conjugation, and then performing centrifugal separation to obtain the precipitate, the water-soluble green quantum dot magnetic microsphere detection probe; mixing the activated water-soluble green quantum dot magnetic microspheres with a goat anti-chicken lgY polyclonal antibody for conjugation, and then performing centrifugal separation to obtain the precipitate, the water-soluble green quantum dot magnetic microsphere control probe.

[0008] Furthermore, the concentration of the water-soluble red quantum dot magnetic microsphere BS solution is 0.3 - 0.5 mg / mL; the concentration of the sulfo-NHS solution is 40 - 60 mg / mL; the concentration of the EDC solution is 10 - 20 mg / mL; the volume ratio of the water-soluble red quantum dot magnetic microsphere BS solution, the sulfo-NHS solution, and the EDC solution is (16 - 20):(0.5 - 2):(0.5 - 2); the concentration of the water-soluble green quantum dot magnetic microsphere BS solution is 0.3 - 0.5 mg / mL; the concentration of the sulfo-NHS solution is 40 - 60 mg / mL; the concentration of the EDC solution is 10 - 20 mg / mL; the volume ratio of the water-soluble green quantum dot magnetic microsphere BS solution, the sulfo-NHS solution, and the EDC solution is (16 - 20):(0.5 - 2):(0.5 - 2); the ultrasonic activation treatment time is 5 min - 20 min; the ultrasonic activation temperature is 0°C - 10°C; The mixing of the activated water-soluble red quantum dot magnetic microspheres with the influenza B-labeled monoclonal antibody for conjugation is carried out at 0°C - 8°C; the conjugation time is 4 hours - 6 hours; The coupling of the activated water-soluble red quantum dot magnetic microspheres with goat anti-chicken lgY polyclonal antibody is carried out at 0°C to 8°C; the coupling time is 4 hours to 6 hours; The coupling of the activated water-soluble green quantum dot magnetic microspheres with influenza A-labeled monoclonal antibody is preferably carried out at 0°C to 8°C; the coupling time is 4 hours to 6 hours; The coupling of the activated water-soluble green quantum dot magnetic microspheres with goat anti-chicken lgY polyclonal antibody is carried out at 0°C to 8°C; the coupling time is 4 hours to 6 hours.

[0009] Furthermore, the peak emission wavelengths of the red fluorescent quantum dots and the green fluorescent quantum dots are both within 500 nm to 700 nm, and the difference between the peak emission wavelength of the red fluorescent quantum dots and the peak emission wavelength of the green fluorescent quantum dots is more than 30 nm; the sizes of the red fluorescent quantum dots and the green fluorescent quantum dots are both greater than 10 nm; the red quantum dots are any one of CdSe / ZnS quantum dots, ZnCdSeS / ZnS quantum dots, ZnxCd1-xSe quantum dots, ZnSe quantum dots, CuInZnS quantum dots, InP quantum dots, perovskite quantum dots or a combination of two or more; the green quantum dots are any one of CdSe / ZnS quantum dots, ZnCdSeS / ZnS quantum dots, ZnxCd1-xSe quantum dots, ZnSe quantum dots, CuInZnS quantum dots, InP quantum dots, perovskite quantum dots or a combination of two or more; The magnetic nanoparticles are all Fe3O4 magnetic nanoparticles; the size of the Fe3O4 magnetic nanoparticles is 50 nm to 300 nm; the saturation magnetization intensity of the Fe3O4 magnetic nanoparticles is greater than 30 emu / g.

[0010] Furthermore, the preparation method of the water-soluble red quantum dot magnetic microspheres includes: Step 1) Coating the Fe3O4 magnetic nanoparticles with a silica layer; the coating of the Fe3O4 magnetic nanoparticles with a silica layer specifically includes: sequentially adding ultrapure water, tetraethyl orthosilicate, and ammonia water to an ethanol solution of Fe3O4 magnetic nanoparticles for reaction, performing ultrasonic treatment, washing with an ethanol solution, performing solid-liquid separation, and collecting the precipitate; Step 2) After dissolving the precipitate collected in step 1) with ethanol, adding 3-mercaptopropyltrimethoxysilane reagent and ammonia water for reaction, performing ultrasonic treatment, washing with an ethanol solution, performing solid-liquid separation, and collecting the precipitate; Step 3) After dissolving the precipitate collected in step 2) with ethanol, adding a chloroform solution of red fluorescent quantum dots for reaction, performing ultrasonic treatment, performing solid-liquid separation, and collecting the precipitate; Step 4) After dissolving the precipitate collected in Step 3) with ethanol, successively add 3-mercaptopropyltrimethoxysilane reagent and ammonia water, perform ultrasonic treatment, wash with an ethanol solution, separate the solid from the liquid, and collect the precipitate; Step 5) After dissolving the precipitate collected in Step 4) with ethanol, add an aqueous solution of the polymer polyacrylic acid, perform ultrasonic treatment, separate the solid from the liquid, and collect the precipitate. The precipitate collected here is a water-soluble red quantum dot magnetic microsphere; The preparation method of the water-soluble green quantum dot magnetic microsphere is the same as that of the water-soluble red quantum dot magnetic microsphere, except that the red fluorescent quantum dots in the preparation process are replaced with green fluorescent quantum dots.

[0011] Furthermore, the concentration of Fe3O4 magnetic nanoparticles in the ethanol solution of Fe3O4 magnetic nanoparticles is 30 mg / mL to 80 mg / mL; in Step 1), the volume ratio of the ethanol solution of Fe3O4 magnetic nanoparticles, ultrapure water, tetraethyl orthosilicate, and ammonia water is (1 to 3):(0.5 to 1):(0.02 to 0.1):(0.02 to 0.1); in Step 1), the ultrasonic treatment time is 30 min to 120 min; In the process of dissolving the precipitate collected in Step 1) with ethanol, the mass-volume ratio of the collected precipitate to ethanol is (50 to 200) mg:(3 to 8) mL; in Step 2), the volume ratio of ethanol, 3-mercaptopropyltrimethoxysilane reagent, and ammonia water in the process of dissolving the collected precipitate with ethanol is (3 to 8):(0.1 to 0.2):(0.1 to 0.2); in Step 2), the ultrasonic treatment time is 30 min to 120 min; In Step 3), the concentration of red fluorescent quantum dots in the chloroform solution of red fluorescent quantum dots is 5 mg / mL to 20 mg / mL; in the process of dissolving the precipitate collected in Step 2) with ethanol, the mass-volume ratio of the collected precipitate to ethanol is (50 to 200) mg:(3 to 8) mL; in Step 3), the volume ratio of ethanol and the chloroform solution of red fluorescent quantum dots in the process of dissolving the collected precipitate with ethanol is (0.5 to 2):(0.005 to 0.02); in Step 3), the ultrasonic treatment time is 10 min to 30 min; In the process of dissolving the precipitate collected in Step 3) with ethanol, the mass-volume ratio of the collected precipitate to ethanol is (50 to 200) mg:(3 to 8) mL; in Step 4), the volume ratio of ethanol, 3-mercaptopropyltrimethoxysilane reagent, and ammonia water in the process of dissolving the collected precipitate with ethanol is (3 to 8):(0.05 to 0.1):(0.05 to 0.1); in Step 4), the ultrasonic treatment time is 30 min to 120 min; The concentration of polyacrylic acid in the aqueous solution of the polymer polyacrylic acid is 0.01 wt% to 0.05 wt%; during the process of dissolving the precipitate collected in step 4) with ethanol, the mass-volume ratio of the collected precipitate to ethanol is (50 - 200) mg:(3 - 8) mL; in step 5), the volume ratio of ethanol to the aqueous solution of polyacrylic acid during the process of dissolving the collected precipitate with ethanol is (3 - 8):(1 - 5); the ultrasonic treatment time in step 5) is 10 min to 60 min.

[0012] Furthermore, the quantum dot test strip includes a PVC bottom plate, and a sample pad, a conjugate pad, a nitrocellulose membrane, and an absorbent pad are sequentially mounted on the PVC bottom plate from one end to the other end; the sample pad, the conjugate pad, the nitrocellulose membrane, and the absorbent pad are sequentially mounted on the PVC bottom plate from one end to the other end; One detection line and one control line are drawn on the nitrocellulose membrane; the membrane-drawing concentration of the monoclonal antibody for capturing influenza A virus on the detection line is 0.5 mg / mL to 2 mg / mL; the membrane-drawing concentration of the monoclonal antibody for capturing influenza B virus on the detection line is 0.5 mg / mL to 2 mg / mL; the membrane-drawing concentration ratio of the monoclonal antibody for capturing influenza A virus to the monoclonal antibody for capturing influenza B virus is 1:1; the membrane-drawing concentration of chicken egg yolk immunoglobulin on the control line is 0.5 mg / mL to 2 mg / mL.

[0013] A method for detecting influenza A and influenza B viruses using the influenza A / influenza B visual quantum dot combined detection device, the method comprising: Step 1) Prepare a composite quantum dot magnetic microsphere probe; the composite quantum dot magnetic microsphere probe includes a water-soluble red quantum dot magnetic microsphere detection probe, a water-soluble green quantum dot magnetic microsphere detection probe, a water-soluble red quantum dot magnetic microsphere control probe, and a water-soluble green quantum dot magnetic microsphere control probe; the water-soluble red quantum dot magnetic microsphere detection probe, the water-soluble red quantum dot magnetic microsphere control probe, the water-soluble green quantum dot magnetic microsphere detection probe, and the water-soluble green quantum dot magnetic microsphere control probe are respectively added to a probe diluent in a certain proportion and diluted to form a composite quantum dot magnetic microsphere probe mixture; the water-soluble red quantum dot magnetic microsphere detection probe, the water-soluble red quantum dot magnetic microsphere control probe, the water-soluble green quantum dot magnetic microsphere detection probe, and the water-soluble green quantum dot magnetic microsphere control probe are dispersed and mixed with each other in the composite quantum dot magnetic microsphere probe mixture to jointly form a composite quantum dot magnetic microsphere probe; Step (2) Add a diluted sample obtained by diluting a test sample with a sample diluent to the composite quantum dot magnetic microsphere probe mixture to form a sample mixture, and incubate the sample mixture for a period of time to form a detection mixture; Step (3): Magnetically separate the detection mixture to form a precipitated analyte, disperse the precipitated analyte in a suspension buffer to form a detection sample, and drop the detection sample onto a quantum dot test strip. After standing, a test result is obtained.

[0014] Furthermore, the probe diluent is a phosphate buffer solution containing Triton X-100, bovine serum albumin, and polyethylene glycol 4000; the concentration of phosphate in the probe diluent is 0.005 mol / L to 0.2 mol / L; the volume of Triton X-100 in the probe diluent accounts for 0.05% to 0.3% of the total volume of the probe diluent; the mass percentage of bovine serum albumin in the probe diluent is 0.3% to 0.8%; the mass percentage of polyethylene glycol 4000 in the probe diluent is 0.3% to 0.8%; the pH value of the probe diluent is 7 to 8; The concentration of the water-soluble red quantum dot magnetic microsphere detection probe in the composite quantum dot magnetic microsphere probe mixture is 0.1 mg / mL to 0.2 mg / mL; the concentration of the water-soluble red quantum dot magnetic microsphere control probe in the composite quantum dot magnetic microsphere probe mixture is 0.05 mg / mL to 0.1 mg / mL; the concentration of the water-soluble green quantum dot magnetic microsphere detection probe in the composite quantum dot magnetic microsphere probe mixture is 0.2 mg / mL to 0.3 mg / mL; the concentration of the water-soluble green quantum dot magnetic microsphere control probe in the composite quantum dot magnetic microsphere probe mixture is 0.05 mg / mL to 0.2 mg / mL; The volume ratio of the composite quantum dot magnetic microsphere probe mixture to the diluted sample is 1:60 to 1:100, and the sample mixture is incubated for 2 min to 14 min to form a detection mixture; the sample diluent is a phosphate buffer solution containing NP-40 and calf serum; the concentration of phosphate in the sample diluent is 0.005 mol / L to 0.2 mol / L; the volume of NP-40 in the sample diluent accounts for 0.5% to 2% of the total volume of the sample diluent; the volume of calf serum in the sample diluent accounts for 5% to 15% of the total volume of the sample diluent; the pH value of the sample diluent is 7 to 8; The mass-volume ratio of the precipitated analyte to the suspension buffer is (0.5 - 1.5) μg : (100 - 300) μL; the suspension buffer is a phosphate buffer solution, and the concentration of phosphate in the suspension buffer is 0.005 mol / L to 0.2 mol / L; the pH value of the suspension buffer is 7 to 8.

[0015] Advantages of the present invention: 1) The present invention uses water-soluble red fluorescent quantum dot magnetic microspheres and water-soluble green fluorescent quantum dot magnetic microspheres as fluorescent probes to achieve the combined detection of influenza A and influenza B, with high sensitivity, time-saving and high efficiency; 2) By using the quantum dot test strip for the combined detection of influenza A and influenza B prepared by the present invention, dual-mode detection under daylight lamp / ultraviolet light can be realized, and the influenza A and influenza B viruses can be judged by interpreting the colorimetric signal and fluorescent signal; 3) In the present invention, the influenza A capture monoclonal antibody and the influenza B capture monoclonal antibody are simultaneously coated on the same detection line, and it can avoid the signal interference between the two detection items of influenza A virus detection and influenza B virus detection. The influenza A virus and the influenza B virus are simultaneously detected at the same detection line, and there is no interference between the two indicators, avoiding the immune reaction time difference caused by different detection positions of the two indicators, thereby solving the problems affecting the binding efficiency, signal accuracy and sensitivity; 4) The present invention improves the sensitivity and accuracy of the detection of influenza A and influenza B, which is conducive to realizing visual and combined detection. Description of the Drawings

[0016] Att Figure 1 is a schematic diagram of the working principle of the influenza A / influenza B visual quantum dot combined detection device of the present invention.

[0017] Att Figure 2 is an example display of the fluorescence spectra of the water-soluble green quantum dot magnetic microspheres and the water-soluble red fluorescent quantum dot magnetic microspheres used in the present invention.

[0018] Att Figure 3 is the saturation magnetization intensity diagram of the magnetic nanoparticles used in the present invention.

[0019] Att Figure 4 is the transmission electron microscope image of the water-soluble red quantum dot magnetic microspheres and the water-soluble green quantum dot magnetic microspheres.

[0020] Att Figure 5 is the zeta potential diagram of the water-soluble red quantum dot magnetic microspheres and the water-soluble green quantum dot magnetic microspheres before and after being modified by PAA.

[0021] Att Figure 6 is the fluorescence spectrum of the composite quantum dot magnetic microsphere probe mixture.

[0022] Att Figure 7 is the hydrated particle size and the corresponding photo of the composite quantum dot magnetic microsphere probe mixture.

[0023] Att Figure 8 is a schematic diagram showing the detection results of the quantum dot test strip for the combined detection of influenza A / influenza B (including colorimetric signal and fluorescent signal).

[0024] Appendix Figure 9 It is a visualization photo of the combined detection of influenza A and influenza B by the influenza A / B visualization quantum dot combined detection device.

[0025] Appendix Figure 10 It is the standard curve of influenza A for the combined detection of influenza A and influenza B by the influenza A / B visualization quantum dot combined detection device.

[0026] Appendix Figure 11 It is the standard curve of influenza B for the combined detection of influenza A and influenza B by the influenza A / B visualization quantum dot combined detection device. Detailed implementation mode

[0027] An influenza A / B visualization quantum dot combined detection device, the structure of which includes a quantum dot test strip and a composite quantum dot magnetic microsphere probe; a test line (T line) and a control line (C line) are arranged on the quantum dot test strip; there is 1 test line, and both an influenza A capture monoclonal antibody and an influenza B capture monoclonal antibody are coated on the test line, and the influenza A capture monoclonal antibody and the influenza B capture monoclonal antibody are mixed together and in the same position on the test line; chicken egg yolk immunoglobulin (IgY) protein is coated on the control line; the composite quantum dot magnetic microsphere probe includes a water-soluble red quantum dot magnetic microsphere detection probe (the detection probe is also called the T probe), a water-soluble green quantum dot magnetic microsphere detection probe (the detection probe is also called the T probe), a water-soluble red quantum dot magnetic microsphere control probe (the control probe is also called the C probe), and a water-soluble green quantum dot magnetic microsphere control probe (the control probe is also called the C probe).

[0028] The water-soluble red quantum dot magnetic microsphere detection probe includes a water-soluble red quantum dot magnetic microsphere and an influenza B labeled monoclonal antibody; the water-soluble red quantum dot magnetic microsphere control probe includes a water-soluble red quantum dot magnetic microsphere and a goat anti-chicken IgY polyclonal antibody; the water-soluble red quantum dot magnetic microsphere includes a magnetic nanoparticle, a silica layer, a red fluorescent quantum dot, and a polymer polyacrylic acid (PAA) from the inside to the outside, wherein the red fluorescent quantum dot is coated on the surface of the magnetic nanoparticle, there is a silica layer with a certain thickness between the red fluorescent quantum dot and the surface of the magnetic nanoparticle, and the outer layer of the red fluorescent quantum dot is a polymer polyacrylic acid (PAA).

[0029] The water-soluble green quantum dot magnetic microsphere detection probe includes water-soluble green quantum dot magnetic microspheres and monoclonal antibodies labeled with influenza A; the water-soluble green quantum dot magnetic microsphere control probe includes water-soluble green quantum dot magnetic microspheres and polyclonal antibodies of goat anti-chicken IgY; the water-soluble green quantum dot magnetic microspheres include magnetic nanoparticles, a silica layer, green fluorescent quantum dots, and polymer polyacrylic acid (PAA) from the inside to the outside. Among them, the green fluorescent quantum dots are coated on the surface of the magnetic nanoparticles, and there is a silica layer with a certain thickness between the green fluorescent quantum dots and the surface of the magnetic nanoparticles, and the outer layer of the green fluorescent quantum dots is polymer polyacrylic acid (PAA).

[0030] The peak emission wavelengths of the red fluorescent quantum dots and the green fluorescent quantum dots are both within 500 nm to 700 nm, and the difference between the peak emission wavelength of the red fluorescent quantum dots and the peak emission wavelength of the green fluorescent quantum dots is more than 30 nm, as shown in the appendix Figure 2 shown; the sizes of the red fluorescent quantum dots and the green fluorescent quantum dots are both greater than 10 nm, and there is no obvious fluorescence resonance energy transfer (FRET) when the red fluorescent quantum dots and the green fluorescent quantum dots are mixed together; the red quantum dots are any one of CdSe / ZnS quantum dots, ZnCdSeS / ZnS quantum dots, ZnxCd1-xSe quantum dots, ZnSe quantum dots, CuInZnS quantum dots, InP quantum dots, perovskite quantum dots or a combination of two or more; the green quantum dots are any one of CdSe / ZnS quantum dots, ZnCdSeS / ZnS quantum dots, ZnxCd1-xSe quantum dots, ZnSe quantum dots, CuInZnS quantum dots, InP quantum dots, perovskite quantum dots or a combination of two or more.

[0031] The magnetic nanoparticles are all Fe3O4 magnetic nanoparticles; the size of the Fe3O4 magnetic nanoparticles is preferably 50 nm to 300 nm; the saturation magnetization intensity of the Fe3O4 magnetic nanoparticles is greater than 30 emu / g, as shown in the appendix Figure 3 shown; the present invention has no special limitation on the source of the magnetic nanoparticles, and both conventional commercially available products and self-preparation can be used.

[0032] The preparation method of the water-soluble red quantum dot magnetic microspheres includes: Step 1) Coating Fe3O4 magnetic nanoparticles with a silica layer; the specific process of coating Fe3O4 magnetic nanoparticles with a silica layer includes: successively adding ultrapure water, tetraethyl orthosilicate (TEOS), and ammonia water to an ethanol solution of Fe3O4 magnetic nanoparticles for reaction, performing ultrasonic treatment, washing with an ethanol solution, separating solid from liquid, and collecting the precipitate; the concentration of Fe3O4 magnetic nanoparticles in the ethanol solution of Fe3O4 magnetic nanoparticles is preferably 30 mg / mL to 80 mg / mL, and more preferably 50 mg / mL; in this step, the volume ratio of the ethanol solution of Fe3O4 magnetic nanoparticles, ultrapure water, tetraethyl orthosilicate, and ammonia water is preferably (1 - 3):(0.5 - 1):(0.02 - 0.1):(0.02 - 0.1); the volume ratio of the ethanol solution of Fe3O4 magnetic nanoparticles, ultrapure water, tetraethyl orthosilicate, and ammonia water is more preferably 2:0.85:0.05:0.05; in this step, the ultrasonic treatment time is preferably 30 min to 120 min, and more preferably 90 min; Step 2) After dissolving the precipitate collected in Step 1) with ethanol, adding 3-mercaptopropyltrimethoxysilane reagent (MPS) and ammonia water for reaction, performing ultrasonic treatment, washing with an ethanol solution, separating solid from liquid, and collecting the precipitate; in this step, during the process of dissolving the precipitate collected in Step 1) with ethanol, the mass-volume ratio of the collected precipitate to ethanol is (50 - 200) mg:(3 - 8) mL, and more preferably 100 mg:5 mL; the volume ratio of ethanol, 3-mercaptopropyltrimethoxysilane reagent, and ammonia water during the process of dissolving the collected precipitate with ethanol is preferably (3 - 8):(0.1 - 0.2):(0.1 - 0.2); the volume ratio of ethanol, 3-mercaptopropyltrimethoxysilane reagent, and ammonia water during the process of dissolving the precipitate with ethanol is more preferably 5:0.15:0.15; the ammonia water can be commercially available ammonia water; in this step, the ultrasonic treatment time is preferably 30 min to 120 min, and more preferably 60 min; Step 3) After dissolving the precipitate collected in step 2) with ethanol, add a chloroform solution of red fluorescent quantum dots for reaction, perform ultrasonic treatment, separate the solid from the liquid, and collect the precipitate; the concentration of red fluorescent quantum dots in the chloroform solution of red fluorescent quantum dots is preferably 5 mg / mL to 20 mg / mL, and more preferably 10 mg / mL; in this step, during the process of dissolving the precipitate collected in step 2) with ethanol, the mass-volume ratio of the collected precipitate to ethanol is (50 - 200) mg:(3 - 8) mL, and more preferably 100 mg:5 mL; the volume ratio of ethanol to the chloroform solution of red fluorescent quantum dots during the process of dissolving the collected precipitate with ethanol is preferably (0.5 - 2):(0.005 - 0.02); the volume ratio of ethanol to the chloroform solution of red fluorescent quantum dots during the process of dissolving the collected precipitate with ethanol is more preferably 1:0.01; in this step, the ultrasonic treatment time is preferably 10 min to 30 min, and more preferably 20 min; Step 4) After dissolving the precipitate collected in step 3) with ethanol, successively add 3-mercaptopropyltrimethoxysilane reagent (MPS) and ammonia water for ultrasonic treatment, wash with an ethanol solution, separate the solid from the liquid, and collect the precipitate; the ammonia water can be commercially available ammonia water; in this step, during the process of dissolving the precipitate collected in step 3) with ethanol, the mass-volume ratio of the collected precipitate to ethanol is (50 - 200) mg:(3 - 8) mL, and more preferably 100 mg:5 mL; the volume ratio of ethanol, 3-mercaptopropyltrimethoxysilane reagent, and ammonia water during the process of dissolving the collected precipitate with ethanol is preferably (3 - 8):(0.05 - 0.1):(0.05 - 0.1); the volume ratio of ethanol, 3-mercaptopropyltrimethoxysilane reagent, and ammonia water during the process of dissolving the collected precipitate with ethanol is more preferably 5:0.07:0.07; in this step, the ultrasonic treatment time is preferably 30 min to 120 min, and more preferably 60 min; Step 5) After dissolving the precipitate collected in step 4) with ethanol, add an aqueous solution of the polymer polyacrylic acid, perform ultrasonic treatment, carry out solid-liquid separation, and collect the precipitate. The precipitate collected here is a water-soluble red quantum dot magnetic microsphere; the concentration of the polymer polyacrylic acid in the aqueous solution of the polymer polyacrylic acid is preferably 0.01 wt% to 0.05 wt%, and more preferably 0.025 wt%; in this step, during the process of dissolving the precipitate collected in step 4) with ethanol, the mass-volume ratio of the precipitate collected to ethanol is (50 - 200) mg : (3 - 8) mL, and more preferably 100 mg : 5 mL; the volume ratio of ethanol to the aqueous solution of the polymer polyacrylic acid during the process of dissolving the precipitate collected with ethanol is preferably (3 - 8) : (1 - 5); the volume ratio of ethanol to the aqueous solution of the polymer polyacrylic acid during the process of dissolving the precipitate collected with ethanol is more preferably 5 : 3; in this step, the ultrasonic treatment time is preferably 10 min to 60 min, and more preferably 30 min.

[0033] In the present invention, the solid-liquid separation method described in the above step 1) to step 5) is preferably magnetic separation; the magnetic separation time is 1 to 5 minutes, and more preferably 3 minutes.

[0034] The prepared water-soluble red quantum dot magnetic microspheres are as shown in the left figure in the appendix Figure 4 There is a silica layer with a certain thickness between the surface of the red fluorescent quantum dots and the magnetic nanoparticles, and the thickness of the silica layer is 30 nm to 50 nm; as shown in the appendix Figure 5 As shown, before and after being modified with PAA, the zeta potential of the water-soluble red quantum dot magnetic microspheres decreases from -26.8 mV to -49.1 mV. The greater the absolute value of the zeta potential, the more stable the dispersion system and the less likely to agglomerate.

[0035] The preparation method of the water-soluble green quantum dot magnetic microspheres is the same as that of the water-soluble red quantum dot magnetic microspheres, except that the red fluorescent quantum dots in the preparation process are replaced with green fluorescent quantum dots.

[0036] The prepared water-soluble green quantum dot magnetic microspheres are as shown in the right figure in the appendix Figure 4 There is a silica layer with a certain thickness between the surface of the green fluorescent quantum dots and the magnetic nanoparticles, and the thickness of the silica layer is 30 nm to 50 nm; as shown in the appendix Figure 5 As shown, before and after being modified with PAA, the zeta potential of the water-soluble green quantum dot magnetic microspheres decreases from -27.7 mV to -48.5 mV. The greater the absolute value of the zeta potential, the more stable the dispersion system and the less likely to agglomerate.

[0037] A method for detecting influenza A and influenza B viruses using an influenza A / B visual quantum dot multiplex detection device, the method comprising: Step 1) Prepare a composite quantum dot magnetic microsphere probe; the composite quantum dot magnetic microsphere probe includes a water-soluble red quantum dot magnetic microsphere detection probe (the detection probe is also called the T probe), a water-soluble green quantum dot magnetic microsphere detection probe (the detection probe is also called the T probe), a water-soluble red quantum dot magnetic microsphere control probe (the control probe is also called the C probe), and a water-soluble green quantum dot magnetic microsphere control probe (the control probe is also called the C probe); the water-soluble red quantum dot magnetic microsphere detection probe, the water-soluble red quantum dot magnetic microsphere control probe, the water-soluble green quantum dot magnetic microsphere detection probe, and the water-soluble green quantum dot magnetic microsphere control probe are respectively added to a probe diluent according to a certain ratio and diluted to form a composite quantum dot magnetic microsphere probe mixture; preferably, the concentration of the water-soluble red quantum dot magnetic microsphere detection probe in the composite quantum dot magnetic microsphere probe mixture is 0.1 mg / mL to 0.2 mg / mL, and more preferably 0.167 mg / mL; preferably, the concentration of the water-soluble red quantum dot magnetic microsphere control probe in the composite quantum dot magnetic microsphere probe mixture is 0.05 mg / mL to 0.1 mg / mL, and more preferably 0.067 mg / mL; preferably, the concentration of the water-soluble green quantum dot magnetic microsphere detection probe in the composite quantum dot magnetic microsphere probe mixture is 0.2 mg / mL to 0.3 mg / mL, and more preferably 0.25 mg / mL; preferably, the concentration of the water-soluble green quantum dot magnetic microsphere control probe in the composite quantum dot magnetic microsphere probe mixture is 0.05 mg / mL to 0.2 mg / mL, and more preferably 0.1 mg / mL; the water-soluble red quantum dot magnetic microsphere detection probe, the water-soluble red quantum dot magnetic microsphere control probe, the water-soluble green quantum dot magnetic microsphere detection probe, and the water-soluble green quantum dot magnetic microsphere control probe are mutually dispersed and mixed in the composite quantum dot magnetic microsphere probe mixture to jointly form a composite quantum dot magnetic microsphere probe; Attached Figure 6 , Attached Figure 7 is the fluorescence spectrum of the composite quantum dot magnetic microsphere probe and its particle size distribution diagram in aqueous solution. Its polydispersity index (PDI) is 0.202. A value less than 0.3 indicates good dispersion in aqueous solution; Attached Figure 7 The inset in it is a photo of the composite quantum dot magnetic microsphere probe in aqueous solution, and it can be observed that its dispersion is good and no obvious aggregation occurs; The probe diluent is preferably a phosphate buffer solution containing Triton-100, bovine serum albumin, and polyethylene glycol-4000; the concentration of phosphate in the probe diluent is preferably 0.005 mol / L to 0.2 mol / L, more preferably 0.01 mol / L; the volume of Triton-100 in the probe diluent accounts for 0.05% to 0.3% of the total volume of the probe diluent, and more preferably the volume of Triton-100 in the probe diluent accounts for 0.2% of the total volume of the probe diluent; the mass percentage of bovine serum albumin in the probe diluent is preferably 0.3% to 0.8%, and the mass percentage of bovine serum albumin in the probe diluent is more preferably 0.5%; the mass percentage of polyethylene glycol-4000 in the probe diluent is preferably 0.3% to 0.8%, and the mass percentage of polyethylene glycol-4000 in the probe diluent is more preferably 0.4%; the pH value of the probe diluent is preferably 7 to 8, and the pH value of the probe diluent is more preferably 7.4; In step (2), a diluted sample obtained by diluting the test sample with the sample diluent is added to the composite quantum dot magnetic microsphere probe mixture to form a sample mixture. The volume ratio between the composite quantum dot magnetic microsphere probe mixture and the diluted sample is preferably 1:60 to 1:100, more preferably 1:80. After the sample mixture is incubated for a period of time, a detection mixture is formed. Preferably, the incubation time is 2 min to 14 min, more preferably 6 min; if the test sample contains influenza A virus and / or influenza B virus, the influenza A antigen in the influenza A virus and the influenza B antigen in the influenza B virus will respectively react with the corresponding magnetic nanosphere fluorescent probes to form corresponding magnetic nanosphere fluorescent probe-antigen complexes; the magnetic nanosphere fluorescent probes are water-soluble red quantum dot magnetic microsphere detection probes or water-soluble red quantum dot magnetic microsphere control probes or water-soluble green quantum dot magnetic microsphere detection probes or water-soluble green quantum dot magnetic microsphere control probes; the sample diluent is preferably a phosphate buffer solution containing NP-40 (ethylphenyl polyethylene glycol) and calf serum; the concentration of phosphate in the sample diluent is preferably 0.005 mol / L to 0.2 mol / L, more preferably 0.01 mol / L; the volume of NP-40 in the sample diluent accounts for 0.5% to 2% of the total volume of the sample diluent, and more preferably the volume of NP-40 in the sample diluent accounts for 1% of the total volume of the sample diluent; the volume of calf serum in the sample diluent accounts for 5% to 15% of the total volume of the sample diluent, and more preferably the volume of calf serum in the sample diluent accounts for 10% of the total volume of the sample diluent; the pH value of the sample diluent is preferably 7 to 8, and the pH value of the sample diluent is more preferably 7.4; Step (3): Magnetically separate the detection mixture to form a precipitated analyte, retain the precipitate, and disperse the precipitated analyte in a suspension buffer to form a detection sample. The mass-to-volume ratio of the precipitated analyte to the suspension buffer is preferably (0.5 - 1.5) μg : (100 - 300) μL, more preferably 1 μg : 200 μL. The suspension buffer is preferably a phosphate buffer, and the concentration of phosphate in the suspension buffer is preferably 0.005 mol / L - 0.2 mol / L, more preferably 0.01 mol / L. The pH value of the suspension buffer is preferably 7 - 8, more preferably 7.4. Take the detection sample and drop it onto the sample pad of the quantum dot test strip. After standing at room temperature, the test result can be obtained. Each time of detection, preferably take 60 μL - 100 μL of the detection sample, more preferably 80 μL. The standing time at room temperature is preferably 10 min - 20 min, more preferably 15 min. Step (4): The reading method of the test result can be selected as follows: First, visually read the colorimetric signal under a daylight lamp, and then irradiate with a 365 nm ultraviolet lamp to read the fluorescence, so that the result can be visually read or the spectral collection of the NC membrane can be performed using a test strip reader. As Figure 8 shown, under a daylight lamp, if brown bands appear on the test line and the control line (i.e., the T line and the C line), it indicates that the test strip is effective and there may be an infection with influenza A or influenza B virus. Under a 365 nm ultraviolet lamp irradiation, if a red fluorescence appears on the test line (i.e., the T line), it indicates an infection with influenza B virus. Under a 365 nm ultraviolet lamp irradiation, if a green fluorescence appears on the test line (i.e., the T line), it indicates an infection with influenza A virus. Under a 365 nm ultraviolet lamp irradiation, if an orange fluorescence appears on the T line, it indicates an infection with both influenza A and influenza B viruses.

[0038] The water-soluble red quantum dot magnetic microsphere detection probe described above includes water-soluble red quantum dot magnetic microspheres and influenza B-labeled monoclonal antibodies; the water-soluble red quantum dot magnetic microsphere control probe includes water-soluble red quantum dot magnetic microspheres and goat anti-chicken lgY polyclonal antibodies. The reaction is carried out by the method of coupling antibodies, that is, a solution of N-hydroxysulfosuccinimide (sulfo-NHS), a solution of 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC), and a borate buffer solution (BS) of water-soluble red quantum dot magnetic microspheres are mixed and ultrasonically activated to obtain activated water-soluble red quantum dot magnetic microspheres. The activated water-soluble red quantum dot magnetic microspheres are mixed with influenza B-labeled monoclonal antibodies for coupling, and then centrifuged to separate to obtain the precipitate water-soluble red quantum dot magnetic microsphere detection probe (T probe); the activated water-soluble red quantum dot magnetic microspheres are mixed with goat anti-chicken lgY polyclonal antibodies for coupling, and then centrifuged to separate to obtain the precipitate water-soluble red quantum dot magnetic microsphere control probe (C probe). The concentration of the water-soluble red quantum dot magnetic microsphere BS solution is preferably 0.3-0.5 mg / mL, more preferably 0.4 mg / mL; the concentration of the sulfo-NHS solution is preferably 40-60 mg / mL, more preferably 50 mg / mL; the concentration of the EDC solution is preferably 10-20 mg / mL, more preferably 15 mg / mL; the volume ratio of the water-soluble red quantum dot magnetic microsphere BS solution, the sulfo-NHS solution, and the EDC solution is preferably (16-20):(0.5-2):(0.5-2), more preferably 18:1:1; the ultrasonic activation treatment time is preferably 5 min-20 min, more preferably 10 min; the ultrasonic activation temperature is preferably 0°C-10°C, more preferably 4°C; The coupling of the activated water-soluble red quantum dot magnetic microspheres with the monoclonal antibody labeled with ethylamine is preferably carried out at 0°C-8°C, more preferably at 4°C; the coupling time is preferably 4 hours-6 hours, more preferably 5 hours; the coupling is preferably carried out in a rotary shaker; after the coupling is completed, a terminator and a blocking agent can also be added to terminate the reaction first and then centrifuged to obtain a precipitate, the water-soluble red fluorescent quantum dot magnetic microsphere detection probe. The present invention has no special limitation on the terminator and the blocking agent, and conventional terminators and blocking agents in the art can be used; The coupling of the activated water-soluble red quantum dot magnetic microspheres with the goat anti-chicken lgY polyclonal antibody is preferably carried out at 0°C-8°C, more preferably at 4°C; the coupling time is preferably 4 hours-6 hours, more preferably 5 hours; the coupling is preferably carried out in a rotary shaker; after the coupling is completed, a terminator and a blocking agent can also be added to terminate the reaction first and then centrifuged to obtain a precipitate, the water-soluble red fluorescent quantum dot magnetic microsphere control probe. The present invention has no special limitation on the terminator and the blocking agent, and conventional terminators and blocking agents in the art can be used; The water-soluble green quantum dot magnetic microsphere detection probe includes water-soluble green quantum dot magnetic microspheres and monoclonal antibodies labeled with influenza A virus; the water-soluble green quantum dot magnetic microsphere control probe includes water-soluble green quantum dot magnetic microspheres and polyclonal antibodies of goat anti-chicken IgY; the coupling antibody preparation method is used for the reaction, that is, a solution of N-hydroxysulfosuccinimide (sulfo-NHS), a solution of 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC) and a BS solution of water-soluble green quantum dot magnetic microspheres are mixed and ultrasonically activated to obtain activated water-soluble green quantum dot magnetic microspheres; the activated water-soluble green quantum dot magnetic microspheres are mixed with monoclonal antibodies labeled with influenza A virus for coupling, and then centrifuged to obtain the precipitate, the water-soluble green quantum dot magnetic microsphere detection probe; the activated water-soluble green quantum dot magnetic microspheres are mixed with polyclonal antibodies of goat anti-chicken IgY for coupling, and then centrifuged to obtain the precipitate, the water-soluble green quantum dot magnetic microsphere control probe; The concentration of the BS solution of the water-soluble green quantum dot magnetic microspheres is preferably 0.3-0.5 mg / mL, more preferably 0.4 mg / mL; the concentration of the sulfo-NHS solution is preferably 40-60 mg / mL, more preferably 50 mg / mL; the concentration of the EDC solution is preferably 10-20 mg / mL, more preferably 15 mg / mL; the volume ratio of the BS solution, sulfo-NHS solution and EDC solution of the water-soluble green quantum dot magnetic microspheres is preferably (16-20):(0.5-2):(0.5-2), further preferably 18:1:1; the ultrasonic activation treatment time is preferably 5 min-20 min, further preferably 10 min; the ultrasonic activation temperature is preferably 0°C-10°C, further preferably 4°C; The coupling of the activated water-soluble green quantum dot magnetic microspheres with monoclonal antibodies labeled with influenza A virus is preferably carried out at 0°C-8°C, more preferably at 4°C; the coupling time is preferably 4 hours-6 hours, more preferably 5 hours; the coupling is preferably carried out in a rotary shaker; after the coupling is completed, a terminator and a blocking agent can also be added to terminate the reaction and then centrifuged to obtain the precipitate, the water-soluble green fluorescent quantum dot magnetic microsphere detection probe. The present invention has no special limitation on the terminator and the blocking agent, and conventional terminators and blocking agents in the art can be used; The coupling is preferably carried out in a rotary incubator; the coupling of the activated water-soluble green quantum dot magnetic microspheres with goat anti-chicken lgY polyclonal antibody is preferably carried out at 0 °C to 8 °C, more preferably at 4 °C; the coupling time is preferably 4 to 6 hours, more preferably 5 hours; the coupling is preferably carried out in a rotary incubator; after the coupling is completed, a terminator and a blocking agent can also be added to terminate the reaction first and then centrifuged to obtain a precipitate of water-soluble green fluorescent quantum dot magnetic microsphere control probe. The present invention has no special limitation on the terminator and the blocking agent, and conventional terminators and blocking agents in the art can be used.

[0039] The present invention is applicable to the detection of influenza A and influenza B viruses by magnetic enrichment-dispersion method.

[0040] The quantum dot test strip includes a PVC (Polyvinyl chloride, Polyvinyl chloride ) bottom plate, and a sample pad, a conjugate pad, a nitrocellulose membrane (NC membrane), and an absorbent pad are sequentially mounted on the PVC bottom plate from one end to the other end; the sample pad, the conjugate pad, the nitrocellulose membrane, and the absorbent pad are sequentially mounted (such as Figure 1 from left to right in the attachment) on the PVC bottom plate to obtain the quantum dot test strip.

[0041] One detection line and one control line are drawn on the nitrocellulose membrane (NC membrane), and the detection line is coated with influenza A capture monoclonal antibody and influenza B capture monoclonal antibody; the control line is coated with chicken lgY protein (chicken egg yolk immunoglobulin).

[0042] The membrane coating concentration of the influenza A capture monoclonal antibody on the detection line is 0.5 mg / mL to 2 mg / mL, preferably 1 mg / mL; the membrane coating concentration of the influenza B capture monoclonal antibody on the detection line is 0.5 mg / mL to 2 mg / mL, preferably 1 mg / mL; the ratio of the membrane coating concentrations of the influenza A capture monoclonal antibody to the influenza B capture monoclonal antibody is 1:1; the membrane coating concentration of the chicken lgY protein (chicken egg yolk immunoglobulin) on the control line is 0.5 mg / mL to 2 mg / mL, preferably 1 mg / mL.

[0043] The sample pad is a sample pad dried after being treated with a sample pad treatment solution; the sample pad treatment solution is a borate buffer solution containing sucrose, casein, and Triton X-100; the concentration of borate in the sample pad treatment solution is preferably 0.01 mol / L to 0.05 mol / L, more preferably 0.02 mol / L; the mass percentage of sucrose in the sample pad treatment solution is preferably 2% to 8%, and the mass percentage of sucrose in the sample pad treatment solution is more preferably 5%; the mass percentage of casein in the sample pad treatment solution is preferably 0.1% to 0.5%, and the mass percentage of casein in the sample pad treatment solution is more preferably 0.25%; the volume of Triton X-100 in the sample pad treatment solution accounts for 0.1% to 0.5% of the total volume of the sample pad treatment solution, and further preferably, the volume of Triton X-100 in the sample pad treatment solution accounts for 0.25% of the total volume of the sample pad treatment solution; the pH value of the sample pad treatment solution is preferably 7.4 to 9.0, and the pH value of the sample pad treatment solution is more preferably 8.0.

[0044] To verify the detection effect of the present invention, the present invention is used to detect a test sample containing influenza A antigen and / or influenza B antigen; the test sample containing influenza A antigen is diluted with a sample diluent to obtain influenza A antigen-diluted samples with different concentrations, and the concentration of the influenza A antigen in the influenza A antigen-diluted sample is 0 ng / mL to 200 ng / mL; the test sample containing influenza B antigen is diluted with a sample diluent to obtain influenza B antigen-diluted samples with different concentrations, and the concentration of the influenza B antigen in the influenza B antigen-diluted sample is 0 ng / mL to 200 ng / mL; the mixed test sample containing the influenza A antigen test sample and the influenza B antigen test sample is diluted with a sample diluent to obtain influenza A antigen and influenza B antigen mixed-diluted samples with different concentrations, the concentration of the influenza A antigen in the influenza A antigen and influenza B antigen mixed-diluted sample is 0 ng / mL to 200 ng / mL, and the concentration of the influenza B antigen in the influenza A antigen and influenza B antigen mixed-diluted sample is also 0 ng / mL to 200 ng / mL; attached Figure 9 The results of the influenza A antigen and influenza B antigen mixed-diluted samples obtained by diluting the influenza A and influenza B test samples with the sample diluent shown in the figure, where the concentrations of the influenza A antigen test sample and the influenza B antigen test sample are 0, 0.2, 0.4, 0.8, 1.6, 3.1, 6.3, 12.5, 25, 50, 100, 200 ng / mL, etc., are detected and compared; the results are as attached Figure 9 As shown, under a fluorescent lamp, brown bands appear on the test line and the control line (i.e., the T line and the C line), and the brown band signal at the T line gradually deepens as the concentration of the test sample increases; under ultraviolet light at 365 nm, an orange fluorescence appears on the test line (i.e., the T line), and the orange fluorescence at the T line gradually becomes brighter as the concentration of the test sample increases; attached Figure 10 and attachedFigure 11 The results of detecting influenza A and influenza B detection samples by using the influenza A / B visual quantum dot joint detection device are shown, and the fluorescence intensity of the detection signal on the T line is linearly regressed with the concentrations of the influenza A and influenza B detection samples.

Claims

1. A visualization quantum dot joint detection device for influenza A / B, characterized in that It includes a quantum dot detection test strip and a composite quantum dot magnetic microsphere probe; on the quantum dot detection test strip, a detection line and a control line are provided; there is 1 detection line, and both the influenza A capture monoclonal antibody and the influenza B capture monoclonal antibody are coated on the detection line, and the influenza A capture monoclonal antibody and the influenza B capture monoclonal antibody are mixed together and are at the same position on the detection line; the control line is coated with chicken egg yolk immunoglobulin; the composite quantum dot magnetic microsphere probe includes a water-soluble red quantum dot magnetic microsphere detection probe, a water-soluble green quantum dot magnetic microsphere detection probe, a water-soluble red quantum dot magnetic microsphere control probe, and a water-soluble green quantum dot magnetic microsphere control probe.

2. The a H1N1 / Influenza B visual quantum dot joint detection device according to claim 1, characterized in that The water-soluble red quantum dot magnetic microsphere detection probe includes a water-soluble red quantum dot magnetic microsphere and an influenza B-labeled monoclonal antibody; the water-soluble red quantum dot magnetic microsphere control probe includes a water-soluble red quantum dot magnetic microsphere and a goat anti-chicken lgY polyclonal antibody; the water-soluble red quantum dot magnetic microsphere from the inside to the outside includes a magnetic nanoparticle, a silica layer, a red fluorescent quantum dot, and a polymer polyacrylic acid, wherein the red fluorescent quantum dot is coated on the surface of the magnetic nanoparticle, there is a certain thickness of silica layer between the red fluorescent quantum dot and the surface of the magnetic nanoparticle, and the outer layer of the red fluorescent quantum dot is polymer polyacrylic acid; The water-soluble green quantum dot magnetic microsphere detection probe includes a water-soluble green quantum dot magnetic microsphere and an influenza A-labeled monoclonal antibody; the water-soluble green quantum dot magnetic microsphere control probe includes a water-soluble green quantum dot magnetic microsphere and a goat anti-chicken lgY polyclonal antibody; the water-soluble green quantum dot magnetic microsphere from the inside to the outside includes a magnetic nanoparticle, a silica layer, a green fluorescent quantum dot, and a polymer polyacrylic acid, wherein the green fluorescent quantum dot is coated on the surface of the magnetic nanoparticle, there is a certain thickness of silica layer between the green fluorescent quantum dot and the surface of the magnetic nanoparticle, and the outer layer of the green fluorescent quantum dot is polymer polyacrylic acid.

3. The visualization quantum dot joint detection device for influenza A and B according to claim 1, characterized in that The preparation method of the water-soluble red quantum dot magnetic microsphere detection probe and the water-soluble red quantum dot magnetic microsphere control probe includes: mixing a sulfo-NHS solution, an EDC solution with a water-soluble red quantum dot magnetic microsphere BS solution, and performing ultrasonic activation treatment to obtain the activated water-soluble red quantum dot magnetic microsphere; mixing the activated water-soluble red quantum dot magnetic microsphere with an influenza B-labeled monoclonal antibody for coupling, and then performing centrifugal separation to obtain the precipitate water-soluble red quantum dot magnetic microsphere detection probe; mixing the activated water-soluble red quantum dot magnetic microsphere with a goat anti-chicken lgY polyclonal antibody for coupling, and then performing centrifugal separation to obtain the precipitate water-soluble red quantum dot magnetic microsphere control probe; The preparation methods of the water-soluble green quantum dot magnetic microsphere detection probe and the water-soluble green quantum dot magnetic microsphere control probe include: mixing a sulfo-NHS solution, an EDC solution and a water-soluble green quantum dot magnetic microsphere BS solution, and performing ultrasonic activation treatment to obtain the activated water-soluble green quantum dot magnetic microspheres; mixing the activated water-soluble green quantum dot magnetic microspheres with the monoclonal antibody labeled with influenza A for coupling, and then performing centrifugal separation to obtain the precipitate, the water-soluble green quantum dot magnetic microsphere detection probe; mixing the activated water-soluble green quantum dot magnetic microspheres with the polyclonal antibody of goat anti-chicken lgY for coupling, and then performing centrifugal separation to obtain the precipitate, the water-soluble green quantum dot magnetic microsphere control probe.

4. The visualization quantum dot joint detection device for influenza A / influenza B according to claim 3, characterized in that The concentration of the water-soluble red quantum dot magnetic microsphere BS solution is 0.3 - 0.5 mg / mL; the concentration of the sulfo-NHS solution is 40 - 60 mg / mL; the concentration of the EDC solution is 10 - 20 mg / mL; the volume ratio of the water-soluble red quantum dot magnetic microsphere BS solution, the sulfo-NHS solution and the EDC solution is (16 - 20):(0.5 - 2):(0.5 - 2); the concentration of the water-soluble green quantum dot magnetic microsphere BS solution is 0.3 - 0.5 mg / mL; the concentration of the sulfo-NHS solution is 40 - 60 mg / mL; the concentration of the EDC solution is 10 - 20 mg / mL; the volume ratio of the water-soluble green quantum dot magnetic microsphere BS solution, the sulfo-NHS solution and the EDC solution is (16 - 20):(0.5 - 2):(0.5 - 2); the ultrasonic activation treatment time is 5 min - 20 min; the ultrasonic activation temperature is 0°C - 10°C; The coupling of the activated water-soluble red quantum dot magnetic microspheres with the monoclonal antibody labeled with influenza B is carried out at 0°C - 8°C; the coupling time is 4 - 6 hours; The coupling of the activated water-soluble red quantum dot magnetic microspheres with the polyclonal antibody of goat anti-chicken lgY is carried out at 0°C - 8°C; the coupling time is 4 - 6 hours; The coupling of the activated water-soluble green quantum dot magnetic microspheres with the monoclonal antibody labeled with influenza A is preferably carried out at 0°C - 8°C; the coupling time is 4 - 6 hours; The coupling of the activated water-soluble green quantum dot magnetic microspheres with the polyclonal antibody of goat anti-chicken lgY is carried out at 0°C - 8°C; the coupling time is 4 - 6 hours.

5. The a H1N1 / Influenza B visual quantum dot joint detection device according to claim 2, characterized in that The peak emission wavelengths of the red fluorescent quantum dots and the green fluorescent quantum dots are both within 500 nm to 700 nm, and the difference between the peak emission wavelength of the red fluorescent quantum dots and the peak emission wavelength of the green fluorescent quantum dots is more than 30 nm; the sizes of the red fluorescent quantum dots and the green fluorescent quantum dots are both greater than 10 nm; the red quantum dots are any one of CdSe / ZnS quantum dots, ZnCdSeS / ZnS quantum dots, ZnxCd1-xSe quantum dots, ZnSe quantum dots, CuInZnS quantum dots, InP quantum dots, perovskite quantum dots or a combination of two or more; the green quantum dots are any one of CdSe / ZnS quantum dots, ZnCdSeS / ZnS quantum dots, ZnxCd1-xSe quantum dots, ZnSe quantum dots, CuInZnS quantum dots, InP quantum dots, perovskite quantum dots or a combination of two or more; The magnetic nanoparticles are all Fe3O4 magnetic nanoparticles; the size of the Fe3O4 magnetic nanoparticles is 50 nm to 300 nm; the saturation magnetization intensity of the Fe3O4 magnetic nanoparticles is greater than 30 emu / g.

6. The visual quantum dot joint detection device for influenza A and B according to claim 2 or 3, characterized in that The preparation method of the water-soluble red quantum dot magnetic microspheres includes: Step 1) Coating the Fe3O4 magnetic nanoparticles with a silica layer; the coating of the Fe3O4 magnetic nanoparticles with a silica layer specifically includes: sequentially adding ultrapure water, tetraethyl orthosilicate, and ammonia water to the ethanol solution of the Fe3O4 magnetic nanoparticles for reaction, ultrasonic treatment, washing with an ethanol solution, solid-liquid separation, and collecting the precipitate; Step 2) After dissolving the precipitate collected in Step 1) with ethanol, adding 3-mercaptopropyltrimethoxysilane reagent and ammonia water for reaction, ultrasonic treatment, washing with an ethanol solution, solid-liquid separation, and collecting the precipitate; Step 3) After dissolving the precipitate collected in Step 2) with ethanol, adding a chloroform solution of red fluorescent quantum dots for reaction, ultrasonic treatment, solid-liquid separation, and collecting the precipitate; Step 4) After dissolving the precipitate collected in Step 3) with ethanol, sequentially adding 3-mercaptopropyltrimethoxysilane reagent and ammonia water for ultrasonic treatment, washing with an ethanol solution, solid-liquid separation, and collecting the precipitate; Step 5) After dissolving the precipitate collected in Step 4) with ethanol, adding an aqueous solution of the polymer polyacrylic acid, ultrasonic treatment, solid-liquid separation, and collecting the precipitate. The precipitate collected here is the water-soluble red quantum dot magnetic microspheres; The preparation method of the water-soluble green quantum dot magnetic microspheres is the same as that of the water-soluble red quantum dot magnetic microspheres, except that the red fluorescent quantum dots in the preparation process are replaced with green fluorescent quantum dots.

7. The visualization quantum dot joint detection device for influenza A / influenza B according to claim 6, characterized in that The concentration of Fe3O4 magnetic nanoparticles in the ethanol solution of Fe3O4 magnetic nanoparticles is 30 mg / mL to 80 mg / mL; in step 1), the volume ratio of the ethanol solution of Fe3O4 magnetic nanoparticles, ultrapure water, tetraethyl orthosilicate, and ammonia water is (1 to 3):(0.5 to 1):(0.02 to 0.1):(0.02 to 0.1); in step 1), the ultrasonic treatment time is 30 min to 120 min; In the process of dissolving the precipitate collected in step 1) with ethanol, the mass-volume ratio of the collected precipitate to ethanol is (50 to 200) mg:(3 to 8) mL; in step 2), the volume ratio of ethanol, 3-mercaptopropyltrimethoxysilane reagent, and ammonia water in the process of dissolving the collected precipitate with ethanol is (3 to 8):(0.1 to 0.2):(0.1 to 0.2); in step 2), the ultrasonic treatment time is 30 min to 120 min; In step 3), the concentration of red fluorescent quantum dots in the chloroform solution of red fluorescent quantum dots is 5 mg / mL to 20 mg / mL; in the process of dissolving the precipitate collected in step 2) with ethanol, the mass-volume ratio of the collected precipitate to ethanol is (50 to 200) mg:(3 to 8) mL; in step 3), the volume ratio of ethanol to the chloroform solution of red fluorescent quantum dots in the process of dissolving the collected precipitate with ethanol is (0.5 to 2):(0.005 to 0.02); in step 3), the ultrasonic treatment time is 10 min to 30 min; In the process of dissolving the precipitate collected in step 3) with ethanol, the mass-volume ratio of the collected precipitate to ethanol is (50 to 200) mg:(3 to 8) mL; in step 4), the volume ratio of ethanol, 3-mercaptopropyltrimethoxysilane reagent, and ammonia water in the process of dissolving the collected precipitate with ethanol is (3 to 8):(0.05 to 0.1):(0.05 to 0.1); in step 4), the ultrasonic treatment time is 30 min to 120 min; The concentration of polyacrylic acid in the aqueous solution of the polymer polyacrylic acid is 0.01 wt% to 0.05 wt%; in the process of dissolving the precipitate collected in step 4) with ethanol, the mass-volume ratio of the collected precipitate to ethanol is (50 to 200) mg:(3 to 8) mL; in step 5), the volume ratio of ethanol to the aqueous solution of the polymer polyacrylic acid in the process of dissolving the collected precipitate with ethanol is (3 to 8):(1 to 5); in step 5), the ultrasonic treatment time is 10 min to 60 min.

8. The visual quantum dot joint detection device for influenza A and influenza B according to claim 1, characterized in that The quantum dot test strip includes a PVC bottom plate, and a sample pad, a conjugate pad, a nitrocellulose membrane, and an absorbent pad are sequentially mounted on the PVC bottom plate from one end to the other end; the sample pad, the conjugate pad, the nitrocellulose membrane, and the absorbent pad are sequentially mounted on the PVC bottom plate from one end to the other end; One detection line and one control line are drawn on the nitrocellulose membrane; the membrane coating concentration of the influenza A capture monoclonal antibody on the detection line is 0.5 mg / mL to 2 mg / mL; the membrane coating concentration of the influenza B capture monoclonal antibody on the detection line is 0.5 mg / mL to 2 mg / mL; the ratio of the membrane coating concentration of the influenza A capture monoclonal antibody to that of the influenza B capture monoclonal antibody is 1:1; the membrane coating concentration of the chicken egg yolk immunoglobulin on the control line is 0.5 mg / mL to 2 mg / mL.

9. A method for detecting influenza A and influenza B viruses using the influenza A / influenza B visual quantum dot joint detection device according to any one of claims 1-8, characterized in that Including: Step 1) Prepare a composite quantum dot magnetic microsphere probe; The composite quantum dot magnetic microsphere probe includes a water-soluble red quantum dot magnetic microsphere detection probe, a water-soluble green quantum dot magnetic microsphere detection probe, a water-soluble red quantum dot magnetic microsphere control probe, and a water-soluble green quantum dot magnetic microsphere control probe; The water-soluble red quantum dot magnetic microsphere detection probe, the water-soluble red quantum dot magnetic microsphere control probe, the water-soluble green quantum dot magnetic microsphere detection probe, and the water-soluble green quantum dot magnetic microsphere control probe are respectively added to the probe diluent in a certain proportion and diluted to form a composite quantum dot magnetic microsphere probe mixture; the water-soluble red quantum dot magnetic microsphere detection probe, the water-soluble red quantum dot magnetic microsphere control probe, the water-soluble green quantum dot magnetic microsphere detection probe, and the water-soluble green quantum dot magnetic microsphere control probe are dispersed and mixed with each other in the composite quantum dot magnetic microsphere probe mixture to jointly form a composite quantum dot magnetic microsphere probe; Step (2) Add a diluted sample obtained by diluting the test sample with the sample diluent to the composite quantum dot magnetic microsphere probe mixture to form a sample mixture, and after incubating the sample mixture for a period of time, a detection mixture is formed; Step (3) Magnetically separate the detection mixture with a magnet to form a precipitated analyte, disperse the precipitated analyte into a suspension buffer to form a test sample, and drop the test sample onto the quantum dot test strip, and the test result is obtained after standing.

10. The method for detecting influenza A and influenza B viruses according to claim 9, characterized in that The probe diluent is a phosphate buffer solution containing Triton-100, bovine serum albumin, and polyethylene glycol-4000; the concentration of phosphate in the probe diluent is 0.005 mol / L to 0.2 mol / L; the volume of Triton-100 in the probe diluent accounts for 0.05% to 0.3% of the total volume of the probe diluent; the mass percentage of bovine serum albumin in the probe diluent is 0.3% to 0.8%; the mass percentage of polyethylene glycol-4000 in the probe diluent is 0.3% to 0.8%; the pH value of the probe diluent is 7 to 8; The concentration of the water-soluble red quantum dot magnetic microsphere detection probe in the composite quantum dot magnetic microsphere probe mixture is 0.1 mg / mL to 0.2 mg / mL; the concentration of the water-soluble red quantum dot magnetic microsphere control probe in the composite quantum dot magnetic microsphere probe mixture is 0.05 mg / mL to 0.1 mg / mL; the concentration of the water-soluble green quantum dot magnetic microsphere detection probe in the composite quantum dot magnetic microsphere probe mixture is 0.2 mg / mL to 0.3 mg / mL; the concentration of the water-soluble green quantum dot magnetic microsphere control probe in the composite quantum dot magnetic microsphere probe mixture is 0.05 mg / mL to 0.2 mg / mL; The volume ratio of the composite quantum dot magnetic microsphere probe mixture to the diluted sample is 1:60 to 1:100, and the sample mixture is incubated for 2 min to 14 min to form a detection mixture; the sample diluent is a phosphate buffer solution containing NP-40 and calf serum; the concentration of phosphate in the sample diluent is 0.005 mol / L to 0.2 mol / L; the volume of NP-40 in the sample diluent accounts for 0.5% to 2% of the total volume of the sample diluent; the volume of calf serum in the sample diluent accounts for 5% to 15% of the total volume of the sample diluent; the pH value of the sample diluent is 7 to 8; The mass-volume ratio of the precipitated analyte to the suspension buffer is (0.5 to 1.5) μg:(100 to 300) μL; the suspension buffer is a phosphate buffer solution, and the concentration of phosphate in the suspension buffer is 0.005 mol / L to 0.2 mol / L; the pH value of the suspension buffer is 7 to 8.

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