Trace human leukemia virus detection kit

Abstract

The invention discloses a trace human leukemia virus detection kit, which comprises a detection liquid, the detection liquid contains a perovskite quantum dot probe, and the perovskite quantum dot probe is a water-soluble perovskite quantum dot labeled biological material; the water-soluble perovskite quantum dot comprises a perovskite quantum dot and 6-bromohexanoic acid encapsulated on the surface of the perovskite quantum dot; the biological material is selected from one of a target HTLV-1 virus detection antibody, an aptamer or a polypeptide. The human leukemia virus detection kit can be used for detecting trace human leukemia viruses.

Description

Technical Field

[0001] The invention belongs to the technical field of immune detection and relates to a trace human leukemia virus detection kit. Background Art

[0002] Human T-cell Leukemia Virus (HTLV-1) is a type of retrovirus that can cause serious blood system diseases and neurological lesions. At present, the clinical diagnosis of HTLV-1 infection mainly relies on serological antibody detection. However, patients infected with HTLV-1 in the early stage have a latent "window period", and existing detection technologies are difficult to achieve rapid detection of ultra-low concentrations of HTLV-1 virus. Therefore, the development of an ultra-high sensitivity rapid detection technology for trace markers and the study of high-sensitivity rapid differential diagnosis of human leukemia virus are key breakthroughs in solving clinical missed diagnosis and misdiagnosis, and have important research significance in improving patient prognosis and blocking the transmission chain. Perovskite quantum dots are considered to be the next generation of fluorescent materials because their luminescence efficiency can reach nearly 100%. However, their luminescence can only exist stably in the organic phase, and the perovskite structure in the water environment will decompose and cause fluorescence quenching. Adding effective ligands to improve the chemical environment of the perovskite interface can effectively block water and oxygen attacks and realize the preparation of water-soluble perovskite fluorescent materials, which has a significant driving significance for its application in biomedicine. Summary of the invention

[0003] In order to overcome the limitations of existing detection technologies for trace detection, the present invention provides a trace human leukemia virus detection kit, which is a highly sensitive, rapid and convenient diagnosis of trace markers. The kit uses a trace amount of perovskite quantum dot material with high luminescence efficiency (>90%) as a fluorescent marker and combines it with a commercialized targeted HTLV-1 virus detection antibody to achieve rapid and highly sensitive fluorescent immunochromatographic detection.

[0004] The technical solution of the present invention is as follows: A trace human leukemia virus detection kit comprises a detection solution, wherein the detection solution contains a water-soluble perovskite quantum dot probe, and the water-soluble perovskite quantum dot probe is a biological material labeled with a perovskite quantum dot; The water-soluble perovskite quantum dots include perovskite quantum dots and 6-bromohexanoic acid encapsulated on the surface of the perovskite quantum dots; The biological material is selected from one of an antibody, an aptamer or a polypeptide targeting HTLV-1 virus detection.

[0005] According to an embodiment of the present invention, the spectral range of the water-soluble perovskite quantum dots is 500-530 nm, for example, 500 nm, 510 nm, 520 nm, 530 nm, and preferably, the spectral position of the perovskite quantum dots is 520 nm.

[0006] According to an embodiment of the present invention, the particle size of the water-soluble perovskite quantum dots is 30-50 nm, for example, 30 nm, 35 nm, 40 nm or 50 nm. Preferably, the particle size of the water-soluble perovskite quantum dots is 35 nm.

[0007] According to an embodiment of the present invention, the luminescence efficiency of the water-soluble perovskite quantum dots can reach 90% to 100%, for example, 90%, 95%, 98% or 100%. Preferably, the luminescence efficiency of the water-soluble perovskite quantum dots is 95%.

[0008] According to an embodiment of the present invention, the average particle size of the perovskite quantum dots is 5 to 20 nm, for example, 10 to 15 nm; illustratively, the average particle size of the perovskite quantum dots is 5 nm, 8 nm, 10 nm, 12 nm, 15 nm or 20 nm.

[0009] According to an embodiment of the present invention, the mass ratio (mg:mg) of the perovskite quantum dots to 6-bromohexanoic acid is 116:(40-120); for example, it can be 116:(40-80). For example, it can be 116:40, 116:50, 116:60, 116:70 or 116:80.

[0010] According to an embodiment of the present invention, the perovskite quantum dots are all-inorganic CsPbBr3 perovskite quantum dots.

[0011] According to an embodiment of the present invention, the method for preparing water-soluble CsPbBr3 perovskite quantum dots specifically comprises the following steps: (A1) mixing raw materials for preparing perovskite quantum dots with 6-bromohexanoic acid in an organic solvent N,N-dimethylformamide, heating, stirring and dissolving, and then adding oleic acid ligand to form a precursor solution; (A2) adding the precursor solution in step (A1) into anti-solvent toluene, heating and stirring to react, precipitating water-soluble perovskite nanocrystals by anti-solvent supersaturation method, centrifuging, dispersing the precipitate into aqueous solution, and preparing water-soluble perovskite nanocrystals.

[0012] According to an embodiment of the present invention, the perovskite quantum dots are CsPbBr3 perovskite quantum dots, and the raw materials for preparing the perovskite quantum dots are, for example, CsBr and PbBr2.

[0013] The CsPbBr3 perovskite quantum dots can be prepared by methods known in the art.

[0014] According to an embodiment of the present invention, in step (A1), there is no limitation on the mixing order of the raw materials for preparing perovskite quantum dots and 6-bromohexanoic acid. For example, the raw materials for preparing perovskite quantum dots and 6-bromohexanoic acid can be added to the solvent at the same time, or the raw materials for preparing perovskite quantum dots can be added to the solvent first, and then 6-bromohexanoic acid is added to the solvent.

[0015] According to an embodiment of the present invention, in step (A1), the ratio (mg:mg) of the sum of the masses of the raw materials for preparing the perovskite quantum dots to the mass of 6-bromohexanoic acid is 116:(40-120), preferably 116:(40-80).

[0016] According to an embodiment of the present invention, in step (A1), the mass volume ratio of 6-bromohexanoic acid to the solvent is (8~24) mg:1mL, for example, 8 mg:1mL, 10 mg:1mL, 15 mg:1mL, 18 mg:1mL, 20 mg:1mL or 24 mg:1mL.

[0017] According to an embodiment of the present invention, in step (A1), the volume ratio of the organic ligand oleylamine to the organic solvent is (0.5-2):10, for example (1-2):10, exemplified by 1:10, 1.5:10 or 2:10.

[0018] According to an embodiment of the present invention, in step (A2), the volume ratio of the precursor solution to the anti-solvent toluene is (0.1-5):10, for example (0.5-3):10.

[0019] Preferably, the dripping is slow dripping, for example, the dripping speed is 6-12 mL / s, exemplarily 6 mL / s, 8 mL / s, 10 mL / s, 12 L / s.

[0020] Preferably, the dropwise addition is performed under the condition of vigorous stirring of the anti-solvent.

[0021] For example, the stirring time is 1-4 hours, such as 1 hour, 2 hours, 3 hours, 4 hours. For example, the reaction temperature is 45-60°C, such as 45-50°C, such as 45°C or 50°C.

[0022] According to an embodiment of the present invention, the preparation environment of the water-soluble CsPbBr3 perovskite quantum dots is an atmospheric environment.

[0023] According to an embodiment of the present invention, the kit further comprises an ultraviolet lamp, such as a handheld ultraviolet lamp; the ultraviolet lamp is used to excite the water-soluble perovskite quantum dots.

[0024] Furthermore, the wavelength range of the ultraviolet lamp is 320-450 nm, preferably 365 nm.

[0025] Furthermore, the power of the ultraviolet lamp is 10-50W, preferably 10W.

[0026] According to an embodiment of the present invention, the method for preparing the detection solution comprises the following steps: (1) Mix water-soluble perovskite quantum dots with 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC) and sodium N-hydroxysuccinimide sulfonate (sulfo-NHS) and activate them at room temperature; (2) Add the biological material in step (1) and mix, perform ultrafiltration and centrifugation, and collect the supernatant to prepare the test solution.

[0027] According to an embodiment of the present invention, the molar ratio of water-soluble perovskite quantum dots to EDC is 1:(2-5), exemplified by 1:2, 1:3, 1:4 or 1:5.

[0028] According to an embodiment of the present invention, the molar ratio of EDC to sulfo-NHS is (2-5):1, exemplified by 2:1, 3:1, 4:1 and 5:1.

[0029] According to an embodiment of the present invention, the concentration ratio of water-soluble perovskite quantum dots to biomaterials is 1:(1-5), exemplified by 1:2, 1:3, 1:4 or 1:5.

[0030] According to an embodiment of the present invention, the detection solution is stored in a diluent; the diluent is ultrapure water containing 0.01-0.1 wt % Tween-20. Exemplarily, the pH of the ultrapure water is 6.2-6.8.

[0031] According to an embodiment of the present invention, the obtained detection solution can also be stored in a refrigerator. For example, the storage temperature is 1-5°C, such as 1°C, 2°C, 3°C, 4°C or 5°C.

[0032] According to an embodiment of the present invention, in step (1), the activation time is 15 to 60 minutes.

[0033] According to an embodiment of the present invention, in step (2), after mixing, the solution is concentrated; illustratively, it is concentrated to 50-100 μL.

[0034] According to an embodiment of the present invention, the kit includes an immunochromatographic test strip containing a capture antibody, wherein the capture antibody is an antibody that targets the HTLV-1 virus and does not cross-react with a detection antibody that targets the HTLV-1 virus.

[0035] According to an embodiment of the present invention, the kit further comprises a storage medium, and the storage medium comprises at least one of an RFID tag, an IC chip, a magnetic code, and a bar code.

[0036] According to an embodiment of the present invention, the kit further comprises a pipette.

[0037] According to an embodiment of the present invention, the outer packaging of the kit is a hard support, which has no fluorescence under ultraviolet irradiation and has the functions of loading and supporting.

[0038] In the present invention, the test kit includes the test liquid batch number, the test liquid validity period, the storage medium password, the clinical index reference value, the manufacturer information, and can read the identity information of the subject, the tester information, the sample name, the sample number, the test date, and the test result information.

[0039] The present invention also provides the use of the above-mentioned kit in the fields of medical detection, medical diagnosis and treatment, and preferably, for human leukemia virus detection.

[0040] The present invention also provides a method for using a human leukemia virus detection kit, the method comprising: Take the above test solution and mix it with the sample, add it dropwise onto the immunochromatographic test strip and react for 5 to 10 minutes. Irradiate with ultraviolet light to detect whether fluorescence appears on the T line.

[0041] According to an embodiment of the present invention, the amount of the detection solution added is 200-300 μL.

[0042] The evaluation standard of the test kit of the present invention is: use an ordinary handheld ultraviolet lamp with a light source range of 320-450 nm to excite the water-soluble perovskite quantum dots to irradiate the C line and T line area of ​​the test strip. According to the principle of antibody-antigen specific recognition, when the sample contains human leukemia virus-related antigens, the T line will show high-intensity fluorescence; conversely, when the sample does not contain human leukemia virus-related antigens, the T line will almost have no fluorescence. The C line is the quality control line. When the C line is not displayed, it means that the test strip has deteriorated.

[0043] The higher the fluorescence intensity, the higher the concentration of human leukemia virus-related antigen in the sample. Conversely, the lower the fluorescence intensity, the lower the concentration of human leukemia virus-related antigen.

[0044] Beneficial effects of the present invention: 1. The present invention prepares water-soluble perovskite quantum dots with a luminescence efficiency of 95%; at the same concentration, compared with the traditional fluorescent label FITC, the luminescence intensity of the water-soluble perovskite quantum dots is 108 times that of FITC.

[0045] 2. The water-soluble perovskite quantum dots prepared by the present invention can be used as fluorescent labels for rapid detection of low-concentration human leukemia viruses.

[0046] 3. The water-soluble perovskite nanocrystals prepared by the present invention have high fluorescence intensity, can detect low-concentration target molecules, and have the advantages of simple and rapid operation, short detection time, easy result interpretation, etc. It has important medical significance for the rapid screening, diagnosis, prediction and prognosis of human leukemia virus, evaluation of treatment effect and follow-up observation of high-risk population. BRIEF DESCRIPTION OF THE DRAWINGS

[0047] Figure 1 This is a transmission electron microscope image of the perovskite quantum dots in Comparative Example 1.

[0048] Figure 2 This is a transmission electron microscope image of the water-soluble perovskite quantum dots in Example 1.

[0049] Figure 3 This is a diagram of the quantum yield of water-soluble perovskite quantum dots in Example 1.

[0050] Figure 4 This is the PL spectrum of the water-soluble perovskite quantum dots in Example 1.

[0051] Figure 5 This is a physical picture of the detection solution, i.e., the water-soluble perovskite quantum dot probe, in Example 2. The left is an optical image under sunlight, and the right is a fluorescent image irradiated by a 365nm, 10W handheld UV lamp.

[0052] Figure 6 This is a comparison chart of the luminescence intensity of water-soluble perovskite quantum dots and traditional fluorescent label FITC at the same concentration in Example 3. DETAILED DESCRIPTION

[0053] The technical scheme of the present invention will be further described in detail below in conjunction with specific embodiments. It should be understood that the following embodiments are only exemplary descriptions and explanations of the present invention and should not be construed as limiting the scope of protection of the present invention. All technologies implemented based on the above content of the present invention are included in the scope that the present invention is intended to protect.

[0054] Unless otherwise specified, the raw materials and reagents used in the following examples are commercially available or can be prepared by known methods.

[0055] Comparative Example 1 The steps for preparing perovskite quantum dots are as follows: (1) At room temperature and atmospheric environment, 42.5 mg CsBr and 73.4 mg PbBr2 were dissolved in 5 mL N,N-dimethylformamide (DMF) solvent. After complete dissolution, 0.5 mL oleylamine was added to form a stable precursor solution. (2) Take 500 μL of the above precursor solution and slowly add it dropwise into 10 mL of vigorously stirred toluene. Stir for 300 hrs and react for 4 h to obtain perovskite quantum dots (concentration of 1 mg / mL).

[0056] Figure 1 This is a transmission electron microscope image of the water-soluble perovskite quantum dots of comparative example 1.

[0057] Example 1 The preparation steps of water-soluble perovskite quantum dots are as follows: (1) At room temperature and atmospheric environment, 42.5 mg CsBr, 73.4 mg PbBr2 and 40 mg 6-bromohexanoic acid were dissolved in 5 mL of N,N-dimethylformamide (DMF) solvent. After complete dissolution, 0.5 mL of oleylamine was added to form a stable precursor solution. (2) Take 500 μL of the above precursor solution and slowly add it dropwise into 10 mL of vigorously stirred toluene at a stirring speed of 300 rpm. React for 4 h to obtain a perovskite quantum dot solution.

[0058] (3) The perovskite quantum dot solution in (2) was centrifuged at 7000 r for 8 min to obtain a precipitate which was light yellow after drying and turned into green powder under ultraviolet light.

[0059] (4) Weigh 10 mg of the above powder and ultrasonically disperse it in 10 mL of ultrapure water (pH = 6.24) to obtain a water-soluble perovskite quantum dot solution (concentration of 1 mg / mL), which was stored at room temperature.

[0060] Figure 2 is a transmission electron microscope image of the water-soluble perovskite quantum dots of Example 1. Figure 2 It can be seen that the particle size of water-soluble perovskite nanocrystals is about 35 nm.

[0061] Figure 3 This is a diagram of the quantum yield of the water-soluble perovskite quantum dots in Example 1. The quantum yield value of the substance can be directly measured by the FLS980 instrument and is 95%.

[0062] Figure 4 is the PL spectrum of the water-soluble perovskite quantum dots in Example 1; Figure 4 It can be seen from the PL spectrum that the emission peak is at 520nm.

[0063] Example 2 A trace human leukemia virus detection kit and preparation method thereof 1. Kit Structure 1.1 Kit composition A trace human leukemia virus detection kit comprises a handheld ultraviolet lamp, a detection solution, a flushing solution, a dropper and a storage medium. The storage medium is an RFID tag, an IC chip, a magnetic code or a bar code.

[0064] 2. Preparation method of test solution A. Take 1 mL of the water-soluble perovskite quantum dot solution (concentration of 1 mg / mL) in Example 1, add EDC and sulfo-NHS (the molar ratio of water-soluble perovskite quantum dots to EDC is 1:2; the molar ratio of EDC: sulfo-NHS is 4:1), vortex evenly, and activate at room temperature for 30 min; B. Add human leukemia virus detection antibody (HTLV-1 Taxantibody, purchased from Shenzhen Xinbosheng Biotechnology Co., Ltd., catalog number: CVL-PAB0865-P) to the solution activated in step A, shake at 37°C, and react for 30 minutes; C. After the reaction is completed, use an ultrafiltration centrifuge tube with a molecular cutoff of 150 KDa to concentrate to 100 μL and store at 4°C for later use to obtain the detection solution.

[0065] Figure 5 The water-soluble perovskite quantum dot probe is a test solution (left) and the fluorescence under 365nm, 10W handheld UV lamp (right). Figure 5 It can be seen that the water-soluble perovskite quantum dot probe solution exhibits strong green fluorescence.

[0066] Example 3 The traditional fluorescent label FITC was selected as the control dye. The two fluorescent materials were diluted to 1 μg / mL using PBS buffer solution, and the luminescence intensity values ​​of water-soluble perovskite quantum dots and FITC were tested at a temperature of 25°C and a humidity of 20%.

[0067] Figure 6 This is a comparison of the luminescence intensity of water-soluble perovskite quantum dots and traditional fluorescent label FITC at the same concentration in Example 3. Figure 6 It can be seen that the luminescence intensity of water-soluble perovskite quantum dots is 108 times that of FITC.

[0068] Example 4 Method for using the kit of the present invention and evaluation criteria for human leukemia virus detection Usage: Take 200~300 μL of the sample and test solution mixture and add it dropwise to the immunochromatographic test strip. Let it react at room temperature for 5~10 minutes and observe the image with a handheld UV lamp.

[0069] The evaluation standard for the test kit of the present invention is: use an ordinary handheld ultraviolet lamp with a light source range of 320~450 nm to excite the water-soluble perovskite quantum dots to irradiate the C line and T line areas of the test strip. According to the antibody-antigen specific recognition principle, when the sample contains human leukemia virus-related antigens, the T line will show high-intensity fluorescence; conversely, when the sample does not contain human leukemia virus-related antigens, the T line will almost have no fluorescence. The C line is the quality control line. When the C line is not displayed, it means that the test strip has deteriorated. The higher the fluorescence intensity, the higher the concentration of human leukemia virus-related antigens in the sample. Conversely, the lower the fluorescence intensity, the lower the concentration of human leukemia virus-related antigens.

[0070] The above is an exemplary description of the embodiments of the present invention. However, the protection scope of the present invention is not limited to the above embodiments. Any modification, equivalent substitution, improvement, etc. made by those skilled in the art within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A human leukemia virus detection kit, characterized in that: It includes a detection liquid, wherein the detection liquid contains a perovskite quantum dot probe, and the perovskite quantum dot probe is a biological material marked with a water-soluble perovskite quantum dot; The water-soluble perovskite quantum dots include perovskite quantum dots and 6-bromohexanoic acid encapsulated on the surface of the perovskite quantum dots; The biological material is selected from any one of an antibody, an aptamer or a polypeptide targeting HTLV-1 virus detection.

2. The human leukemia virus detection kit according to claim 1, characterized in that: The spectral range of the water-soluble perovskite quantum dots is 500-530 nm; The particle size of the water-soluble perovskite quantum dots is 30-50 nm.

3. The human leukemia virus detection kit according to claim 1, characterized in that: The perovskite quantum dots are CsPbBr3 perovskite quantum dots.

4. The human leukemia virus detection kit according to claim 1 or 2, characterized in that: The method for preparing the water-soluble perovskite quantum dots comprises the following steps: (A1) Lead bromide, cesium bromide and 6-bromohexanoic acid for preparing perovskite quantum dots are mixed in N,N-dimethylformamide, heated, stirred and dissolved, and then oleylamine ligand is added to form a precursor solution; (A2) adding the precursor solution in step (A1) to toluene, heating and stirring to react, precipitating perovskite quantum dots by anti-solvent supersaturation method, centrifuging and drying, dispersing the precipitate into an aqueous solution, and preparing water-soluble perovskite quantum dots; The mass ratio of the total mass of the lead bromide and cesium bromide to 6-bromohexanoic acid is 116:(40-120), preferably 116:(50-80); the mass ratio of the lead bromide and cesium bromide is (85:147); The mass ratio of the 6-bromohexanoic acid to the volume ratio of N,N-dimethylformamide is (8-24) mg:1 mL; The volume ratio of oleylamine to N,N-dimethylformamide is (0.5-2):10; The volume ratio of the precursor solution to toluene is (0.1-5):10; The stirring reaction time is 1 to 4 hours; The temperature of the stirring reaction is 45-60°C.

5. The human leukemia virus detection kit according to claim 1, characterized in that: The preparation method of the detection solution comprises the following steps: (1) mixing the water-soluble perovskite quantum dots with 1-ethyl-(3-dimethylaminopropyl)carbodiimide and sodium salt of N-hydroxysuccinimide sulfonate, and activating them at room temperature; (2) adding biological materials to step (1), mixing, ultrafiltration and centrifugation, and collecting the supernatant to prepare a test solution; The molar ratio of the water-soluble perovskite quantum dots to EDC is 1:(2-5); The molar ratio of 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC) to sodium salt of N-hydroxysuccinimide sulfonate (sulfo-NHS) is (2-5):1; The concentration ratio of the water-soluble perovskite quantum dots to the biomaterial is 1:(1-5); The activation time is 15 to 60 minutes.

6. The human leukemia virus detection kit according to any one of claims 1 to 5, characterized in that: The kit also includes a storage medium, and the storage medium includes at least one of an RFID tag, an IC chip, a magnetic code, and a bar code.

7. The human leukemia virus detection kit according to claim 6, characterized in that: The kit also includes an immunochromatographic test strip containing a capture antibody; The capture antibody is an antibody that targets the HTLV-1 virus and does not cross-react with the detection antibody that targets the HTLV-1 virus.

8. Use of the human leukemia virus detection kit according to any one of claims 1 to 7 in the preparation of trace human leukemia virus detection products.

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