Phosphorylated Tau 217 protein detection card and kit and application thereof

Abstract

The invention discloses a phosphorylated Tau 217 protein detection card as well as a kit and application thereof. A sample pad of a detection test strip of the detection card is sprayed with a microsphere line; the microsphere line is a P-Tau217 monoclonal antibody and a rabbit IgG (Immunoglobulin G) antibody which are marked by an up-conversion enhanced luminescence colorimetric nano probe microsphere; and a detection line and a quality control line of the detection card are respectively coated with P-Tau2172 and a goat anti-rabbit antibody. According to the phosphorylated Tau 217 protein detection card disclosed by the invention, an up-conversion enhanced luminescence colorimetric nanoprobe microsphere is used as a P-Tau 217 monoclonal antibody and rabbit IgG antibody marker, and the nanoprobe has the characteristics of strong specificity, high sensitivity and capability of multi-mode output, and can generate visible light emission of 520-550 nm under the excitation of near-infrared light of 980 nm; the probe is simple in structure and convenient to operate, background fluorescence interference is reduced, the detection sensitivity is improved, the probe has two signal reading modes of up-conversion luminescence and colorimetry, and the test result can be verified in different modes.

Description

Technical Field

[0001] The invention relates to a phosphorylated Tau217 protein detection card and a kit and application thereof, belonging to the technical field of nanometer detection medicine. Background Art

[0002] Alzheimer's disease (AD) is a common neurodegenerative disease in the elderly. The diagnosis and intervention of prodromal AD (pAD) is the focus of clinical and medical research. The current methods used to solve this problem are mainly cerebrospinal fluid or PET-CT examinations. This method is cumbersome, invasive, long-term and extremely expensive, which makes it difficult to popularize the technology. Alzheimer's disease is highly hidden. In fact, brain lesions usually occur 10 to 15 years before symptoms appear. Early screening is helpful to delay the onset of AD, but there is no method on the market that can achieve rapid, accurate and highly sensitive detection. The existing detection technologies generally use single molecule immunoassay SIMOA, chemiluminescence CLIA and enzyme-linked immunosorbent assay. These methods are cumbersome to operate, take a long time to get results, and are not sensitive and fast enough.

[0003] Typical AD-related blood biomarkers include blood Aβ and phosphorylated tau protein, which can be rapidly detected with high sensitivity by detecting p-tau in plasma. However, how to improve the sensitivity of detection and enable low-cost home testing is still not feasible. Summary of the invention

[0004] In order to solve the problems in the above-mentioned background technology, the present invention provides a phosphorylated Tau217 protein detection card.

[0005] The technical scheme for achieving the purpose of the present invention is: a phosphorylated Tau 217 protein detection card, comprising a card shell and a detection test strip arranged in the card shell; the detection test strip comprises a substrate and a sample pad, a coating film and a blotting paper which are sequentially overlapped and pasted on the substrate along the length direction of the substrate; the sample pad is sprayed with a microsphere line; the microsphere line is a P-Tau217 monoclonal antibody and a rabbit IgG antibody labeled with upconversion enhanced luminescence colorimetric nanoprobe microspheres; the coating film comprises a nitrocellulose membrane and a detection line and a quality control line which are sequentially and parallelly arranged on the nitrocellulose membrane along the length direction; the detection line and the quality control line are respectively coated with P-Tau2172 and a goat anti-rabbit antibody, wherein the detection line is close to the sample pad and the quality control line is far away from the sample pad.

[0006] The above-mentioned phosphorylated Tau217 protein detection card uses upconversion enhanced luminescence colorimetric nanoprobe microspheres as P-Tau217 monoclonal antibody and rabbit IgG antibody markers. The nanoprobe has the characteristics of strong specificity, high sensitivity and multi-mode output. It can produce visible light emission (520-550nm) under near-infrared light (980nm) excitation, reduce background fluorescence interference, and improve detection sensitivity. The probe has two signal reading methods: upconversion luminescence and colorimetry, and the test results can be verified in different ways.

[0007] Furthermore, the dual-modal nanoprobe has a gold rod coated with silica nanoparticles as a core, and the core is coated with an upconversion nanocrystal layer and an outer silicon layer. The upconversion nanocrystal layer is composed of a core-shell structure of ytterbium and erbium co-doped sodium yttrium fluoride coated with a sodium yttrium fluoride shell layer NaYF4:Yb,Er@NaYF4.

[0008] In the above dual-mode nanoprobe structure, the nanometer-scale gold rod can effectively amplify the incident light signal, and has a fluorescence enhancement effect. The local electromagnetic field around the metal nanoparticles is enhanced, resulting in the interaction of the fluorophore and the nanogold plasma resonance. Under the resonance, the Er3+ element in the upconversion nanocrystal layer has both excitation and emission enhancements in the green part around 530nm. It can effectively enhance the emission light efficiency near 530nm, and at the same time, the radiation decay rate of the fluorophore is also enhanced. When the above structure is applied to the phosphorylated Tau 217 protein detection card, the detection line on the test strip can be visible to the naked eye at low concentrations, achieving effective colorimetry.

[0009] Further or optionally, in order to control the color development effect under the test state and cooperate with the detection capability of the detection equipment, the microsphere line sprayed on the sample pad is P-Tau217 monoclonal antibody and rabbit IgG antibody labeled with upconversion enhanced luminescence colorimetric nanoprobe microspheres, and the content of both is 20 to 200 μg antibody / 200 μl fluorescent microspheres.

[0010] Further or optionally, in order to control the color development effect of the upconversion enhanced luminescence colorimetric nanoprobe, the particle size of the upconversion enhanced luminescence colorimetric nanoprobe is 190 nm.

[0011] Furthermore, in order to control the luminescence enhancement amplitude of the upconversion enhanced luminescence colorimetric nanoprobe, the inner layer of the upconversion enhanced luminescence colorimetric nanoprobe has a diameter of 80 to 120 nm, an inner silicon layer thickness of 10 to 30 nm, an upconversion nanocrystal layer thickness of 30 to 50 nm, and an outer silicon layer thickness of 10 to 30 nm.

[0012] In the above structure, the thickness of the inner silicon layer affects the amplifying effect of the inner nanogold on the luminescence intensity of the doped elements. The outer silicon layer can protect the inner structure to prevent the test sample from damaging the pointer structure. On the other hand, it can perform interference filtering with the inner structure to ensure effective entry of incident light within the range of 10 to 30 nm, while filtering the blue and red light emitted by the up-conversion nanocrystal layer to enhance its signal-to-noise ratio in the green light range.

[0013] Further or optionally, in order to ensure the color development effect in the colorimetric mode, the coating concentration of the P-Tau2172 monoclonal antibody on the nitrocellulose membrane is 0.1-2 mg / ml, and the amount is 0.5-1.5 μl coating liquid / cm membrane, and the coating concentration of the goat anti-rabbit IgG antibody is 0.5-2 mg / ml, and the amount is 0.5-1.5 μl coating liquid / cm membrane.

[0014] Further or optionally, in order to improve the accuracy of fluorescence detection, the present application also provides a phosphorylated Tau 217 protein detection kit, including the above-mentioned phosphorylated Tau 217 protein detection card and an ID card containing a calibration curve, wherein the calibration curve ID card has a standard concentration curve, wherein the standard concentration curve uses the measured gradient concentration of the test strip as the calibrator, uses the calibrator concentration as the horizontal axis, and uses the fluorescence signal ratio as the vertical axis. When performing the test, the data obtained by the test can be placed on the standard for comparison and can be read.

[0015] In addition, the present application also provides an application scheme of the above-mentioned phosphorylated Tau 217 protein detection kit in the detection of Alzheimer's disease, which specifically includes the following steps:

[0016] Colorimetric test: obtain a blood sample, place the sample on the sample pad, dilute it with sample diluent, and then let it stand for a specified time. After the diluted sample reaches the observation window and the quality control line changes color, confirm the status of the test line and quality control line in the sample observation window;

[0017] When the detection line develops color, fluorescence analysis is performed; the steps of the fluorescence analysis are as follows:

[0018] Step 1: Place the test kit and sample at room temperature and use them after returning to room temperature;

[0019] Step 2: Turn on the rare earth nano fluorescent immunoassay analyzer, preheat for 5 minutes and insert the corresponding ID card;

[0020] Step 3: Wipe the fingertips with an alcohol cotton pad, draw 10 μL of blood with a capillary tube and add it to the sample well of the test card, then add two drops of diluent;

[0021] Step 4: Insert the test card into the test slot, test after 10 minutes, and read and print the test results.

[0022] The above-mentioned detection process for Tau 217 protein can complete the preliminary detection of AD in the colorimetric stage, and the color development effect of the detection line can be used for preliminary screening of AD. This process supports users to conduct self-detection, greatly reducing the difficulty of preliminary screening of AD, and can realize the effective promotion of AD autonomous screening technology.

[0023] When the test line is positive, fluorescence analysis can achieve accurate measurement of P-Tau217. This test method uses an incident wavelength of 980nm and a detection wavelength of 540nm. It has the characteristics of low background noise and high sensitivity. In the actual test process, the minimum detection limit reaches 0.411pg / ml, which is very forward-looking in AD prevention.

[0024] By adopting the above technical solution, the present invention has the following beneficial effects:

[0025] (1) A phosphorylated Tau 217 protein detection card of the present invention uses upconversion enhanced luminescence colorimetric nanoprobe microspheres as markers for P-Tau217 monoclonal antibodies and rabbit IgG antibodies. The nanoprobe has the characteristics of strong specificity, high sensitivity and multi-mode output. It can generate visible light emission (520-550nm) under near-infrared light (980nm) excitation, reduce background fluorescence interference, and improve detection sensitivity. The probe has two signal reading methods: upconversion luminescence and colorimetry, and the test results can be verified in different ways.

[0026] (2) A phosphorylated Tau 217 protein detection card of the present application uses a gold rod coated with silica nanoparticles as the core, and the upconversion nanocrystal layer is doped with NaYF4:Yb, Er@NaYF4. The gold rod can effectively amplify the incident light signal and has a fluorescence enhancement effect. The local electromagnetic field around the metal nanoparticles is enhanced, resulting in the interaction of the fluorophore and the nanogold plasma resonance. Under the resonance, the Er3+ element in the upconversion nanocrystal layer has an enhancement in both excitation and emission in the green part around 530nm, which can effectively enhance the emission light efficiency near 530nm, and at the same time, the radiation decay rate of the fluorophore is also enhanced. When the above structure is applied to the phosphorylated Tau 217 protein detection card, the detection line on the test strip can be made visible to the naked eye at low concentrations, achieving effective colorimetry.

[0027] (3) The present invention controls the content of upconversion enhanced luminescence colorimetric nanoprobe microspheres and the particle size of the nanoprobes, thereby effectively controlling the color development effect.

[0028] (4) The present application controls the thickness parameters of each layer of the upconversion enhanced luminescence colorimetric nanoprobe, thereby effectively preventing impurities in the test environment from damaging the probe structure and achieving interference filtering, thereby increasing the signal-to-noise ratio within the test wavelength range and improving the test accuracy.

[0029] (5) The present application sets limits on the monoclonal antibody coating concentration, coating solution dosage, antibody coating concentration and coating solution dosage, thereby achieving a better color development effect.

[0030] (6) The present application also provides a phosphorylated Tau 217 protein detection kit, which can adopt two testing methods: colorimetric testing and fluorescence testing, and can realize AD home screening with extremely high testing accuracy.

[0031] (7) The present application also provides an application scheme of a phosphorylated Tau 217 protein detection kit in the detection of Alzheimer's disease. The scheme is divided into two stages: a colorimetric stage and a fluorescence analysis stage. In the screening process, hierarchical diversion can be effectively achieved. The fluorescence colorimetric stage can be compared with a standard concentration curve, and the detection accuracy is high. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] In order to make the content of the present invention more clearly understood, the present invention is further described in detail below according to specific embodiments and in conjunction with the accompanying drawings, wherein

[0033] Figure 1 It is a schematic diagram of the structure of the phosphorylated Tau 217 protein detection card of the present invention.

[0034] Figure 2 It is a schematic diagram of the structure of the test strip of the phosphorylated Tau 217 protein detection card of the present invention.

[0035] Figure 3 This is a transmission electron microscope image of the upconversion enhanced luminescence colorimetric nanoprobe of the present invention.

[0036] Figure 4 The standard curve diagram of the invented phosphorylated Tau 217 protein detection kit is drawn based on the p-Tau 217 quality control concentration and the sample signal T / C average value.

[0037] Figure 5 The graph is a comparison of the detection results of the phosphorylated Tau 217 protein detection kit of the present invention and the p-Tau217 detection kit of Ipnocon using the chemiluminescence method for the same sample.

[0038] The reference numerals in the accompanying drawings are:

[0039] Card shell 1, sample adding hole 11, observation window 12;

[0040] Test strip 2, backing 21, sample pad 22, coating film 23, absorbent paper 24. DETAILED DESCRIPTION

[0041] In order to better understand the above technical solution, the above technical solution will be described in detail below in conjunction with the accompanying drawings and specific implementation methods.

[0042] In order to make the purpose, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Generally, the components of the embodiments of the present invention described and shown in the drawings here can be arranged and designed in various different configurations.

[0043] Therefore, the following detailed description of the embodiments of the present invention provided in the accompanying drawings is not intended to limit the scope of the invention claimed for protection, but merely represents selected embodiments of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0044] It should be noted that similar reference numerals and letters denote similar items in the following drawings, and therefore, once an item is defined in one drawing, further definition and explanation thereof is not required in subsequent drawings.

[0045] In the description of the embodiments of the present invention, it should be understood that the terms "center", "up", "down", "left", "right", "vertical", "horizontal", "inside", "outside", etc. indicate orientations or positional relationships based on the orientations or positional relationships shown in the accompanying drawings, or are the orientations or positional relationships in which the inventive product is conventionally placed when used, or are the orientations or positional relationships conventionally understood by those skilled in the art. They are only for the convenience of describing the present invention and simplifying the description, and do not indicate or imply that the device or element referred to must have a specific orientation, be constructed and operated in a specific orientation, and therefore should not be understood as a limitation on the present invention.

[0046] In the description of the embodiments of the present invention, it is also necessary to explain that, unless otherwise clearly specified and limited, the terms "set", "installed", "connected" and "connected" should be understood in a broad sense. For example, it can be a fixed connection, a detachable connection, or an integral connection; it can be directly connected, or indirectly connected through an intermediate medium, or it can be the internal connection of two components. For ordinary technicians in this field, the specific meanings of the above terms in the present invention can be understood according to specific circumstances. The present invention is further described below in conjunction with the accompanying drawings. The following embodiments are only used to more clearly illustrate the technical solution of the present invention, and cannot be used to limit the scope of protection of the present invention.

[0047] (Example 1)

[0048] See Figure 1 and Figure 2 The phosphorylated Tau 217 protein detection kit of this embodiment includes a phosphorylated Tau 217 protein detection card and an ID card containing a calibration curve.

[0049] The card shell 1 includes a plastic lower shell and a plastic upper shell connected by snaps; the test paper strip 2 is fixed on the plastic lower shell, and the surface of the test paper strip 2 is pressed by the plastic upper shell. The positions of the plastic upper shell corresponding to the sample pad 22 and the coating film 23 are respectively provided with a sample addition hole 11 and an observation window 12.

[0050] The phosphorylated Tau 217 protein detection card includes a card shell 1 and a test paper strip 2 arranged in the card shell 1. The test paper strip 2 includes a substrate 21 and a sample pad 22, a coating film 23 and a blotting paper 24 which are sequentially overlapped and pasted on the substrate 21 along the length direction of the substrate 21; the sample pad 22 is a sample addition area for absorbing the blood test sample to be tested. A microsphere line is sprayed on the sample pad 22; the microsphere line is a P-Tau217 monoclonal antibody and a rabbit IgG antibody (the content is 100 μg antibody / 200 μl fluorescent microspheres) labeled with a dual-modal nanoprobe. The dual-modal nanoprobe is that the nanoprobe includes a nanogold core, and an inner silicon layer, an upconversion nanocrystal layer and an outer silicon layer sequentially coated on the surface of the core; the average particle size of the nanogold is 100 nm, the thickness of the inner silicon layer is 20 nm, the thickness of the outer silicon layer is 20 nm, and the average particle size of NaYF4:Yb / Er@NaYF4 is 40 nm.

[0051] The up-conversion nanocrystalline layer is a core-shell structure composed of sodium yttrium fluoride co-doped with ytterbium and erbium and coated with a sodium yttrium fluoride shell, namely NaYF4:Yb,Er@NaYF4, with an average particle size of 30-50nm.

[0052] The dual-mode nanoprobe has a particle size of 200 nm and a morphology of Figure 3 As shown in the figure (100nm is a scale mark). Figure 3 It can be seen that the probe has good monodispersity and uniformity.

[0053] The maximum value of the ultraviolet absorption peak of the dual-modal nanoprobe is located at 550nm; under 980nm excitation light, the fluorescence spectrum emission peak of the dual-modal nanoprobe is within the range of 520-550nm, with a peak value of 540nm.

[0054] The coating membrane 23 includes a nitrocellulose membrane and a detection line and a quality control line arranged in parallel and spaced order on the nitrocellulose membrane along the length direction. The spacing between the detection line and the quality control line is 2 to 4 mm. The detection line and the quality control line are coated with P-Tau2172 and goat anti-rabbit antibody, respectively, wherein the detection line is close to the sample pad 22, and the quality control line is far away from the sample pad 22. The coating concentration of P-Tau2172 monoclonal antibody is 1 mg / ml, and the amount is 1 μl of coating liquid / cm membrane. The coating concentration of goat anti-rabbit IgG antibody is 1 mg / ml, and the amount is 1 μl of coating liquid / cm membrane.

[0055] The preparation method of the test strip 2 comprises the following steps:

[0056] Step 1: synthesize the sodium yttrium fluoride core structure; add oleic acid and 1-octadecene in a volume ratio of 3 to 6:7 to 14 in a container, and then add 1 part of nitrate or acetate or chloride of yttrium, ytterbium and erbium in a molar ratio of 0.78:0.2:0.02; mix and stir at room temperature, evacuate, then heat to 100 to 120°C, react for 20 to 30 minutes, and then heat to 150 to 160°C, react for 10 to 15 minutes to obtain a transparent solution; Then cool to 40-50°C, release the vacuum, add a mixed solution of NaOH and ammonium fluoride methanol with a molar concentration ratio of 1-2:1.6-3.4, and react for 20-30 minutes; heat to 90-100°C, evacuate and ventilate 3-4 times, introduce nitrogen, heat to 290-310°C, react for 1-2 hours, centrifuge, then wash with a cyclohexane ethanol mixed solution 3-4 times, disperse in cyclohexane, and obtain a NaYF4:Yb,Er nanoprobe cyclohexane solution;

[0057] Step 2: Preparation of core-shell structure nanoprobes: In a container, add oleic acid and 1-octadecene in a volume ratio of 3-6:7-14, add yttrium acetate, mix and stir, mix and stir at room temperature, evacuate, then heat to 120°C, react for 20 minutes, and then heat to 160°C, react for 10 minutes to obtain a transparent solution; naturally cool to 50°C, release the vacuum, add a mixed solution of NaOH and ammonium fluoride methanol, and a NaYF4:Yb,Er nanoprobe cyclohexane solution in a volume ratio of 3-6:2-4 with oleic acid, mix and stir, React for 20 to 30 minutes; heat to 90 to 100° C., evacuate and ventilate 3 to 4 times, introduce nitrogen, heat to 290 to 310° C., react for 1 to 2 hours, centrifuge at 8000 rpm, wash with a cyclohexane-ethanol mixture 3 to 4 times, and disperse in cyclohexane; the molar ratio of yttrium acetate, NaOH, and ammonium fluoride is 0.2 to 0.4: 0.5 to 1: 0.8 to 1.6; then transfer the upconversion nanocrystals to ethanol by an acid washing method to obtain an ethanol solution containing upconversion nanocrystals with a concentration of 20 mg / mL;

[0058] Step 3: Preparation of silica-coated nanogold: Synthesis of gold nanorods: In a container, add 250 μL of 10 mM chloroauric acid to 9.75 mL of 0.1 M hexadecyltrimethylammonium bromide (CTAB) under magnetic stirring, then add 0.6 mL of 10 mM sodium borohydride solution, stir for 2 min and place at 28 ° C for 30 min. Growth solution: 7.2 g of CTAB and 0.987 g of sodium oleate are added to 400 ml of ultrapure water to dissolve, then 20 mL of 10 mM chloroauric acid is injected into the solution, and when the solution is colorless, 8 mL of 10 mM silver nitrate solution is added, and after stirring for 5 min, 1.2 mL of hydrochloric acid and 600 μL of 0.1 M ascorbic acid are added. After reacting for about 30 s, 1 mL of seed solution is added, and the reaction mixture is left to stand at 30 ° C for 12 hours to complete the synthesis of gold nanorods. After synthesis, centrifuge at 9000rpm for 15min, wash twice by centrifugation, and disperse in 20mL of ultrapure water. Take 5ml of gold rod solution, mix with 250μL1mg / ml, mPEG-SH solution, and stir slowly at room temperature for 1hr. Then centrifuge the sample at 8000r for 10min. After removing the supernatant, resuspend it in a mixture of ethanol and water (ethanol: water = 9:2), add 50μ of concentrated ammonia and 15μL10% TEOS solution under stirring, seal and stir for 12hr, centrifuge three times, and resuspend it in ethanol solution to obtain silica-coated nanogold;

[0059] Step 4: Disperse the silica-coated nanogold in ethanol, add the ethanol solution containing upconversion nanocrystals obtained in step 2, the volume ratio of the two is 10:1, stir for 8 hours to fully react, centrifuge to remove excess upconversion nanocrystals, disperse the obtained material in 10 mL of ethanol, and add water, 10% tetraethoxysilane ethanol solution and ammonia water in sequence, wherein the volume ratio of ethanol, water, 10% tetraethoxysilane ethanol solution and ammonia water is 10:1:0.05:0.2, stir for 9 hours and then centrifuge and wash to obtain an upconversion enhanced luminescence colorimetric nanoprobe;

[0060] Step 5: Mix the upconversion enhanced luminescence colorimetric nanoprobe and aminopropyltriethoxysilane at 60°C and stir for 6 hours, the volume ratio of the two is 1:0.003, and disperse them in N,N-dimethylformamide (DMF) solvent by centrifugation to obtain a dispersion, and then add 50 mg / mL of succinic anhydride DMF solution, the volume ratio of the dispersion to the succinic anhydride DMF solution is 1:0.05, and stir for 10 hours, wash and disperse in water to obtain a carboxyl-modified upconversion enhanced luminescence colorimetric nanoprobe.

[0061] Step six: Activating the bimodal nanoprobe: subjecting the bimodal nanoprobe of step five to ultrasonic treatment and centrifugation, washing the precipitate with a 10-100 mM MES solution with a pH of 5.0-7.0; adding carbodiimide and N-hydroxysulfosuccinimide, mixing well and centrifuging at high speed, washing the precipitate with a MES solution with a pH of 5.0-7.0 to obtain an activated bimodal nanoprobe.

[0062] Step seven, prepare dual-modal nanoprobe labeled with P-Tau217 monoclonal antibody: ultrasonically treat the fluorescent microspheres activated in step six for 2 minutes, then add P-Tau217 monoclonal antibody and rabbit IgG antibody at 100 μg / 200 μl, mix for 2 hours, block with 50 mM, pH 7.4 PBS blocking solution containing 0.5% BSA for 1 hour, and then centrifuge at 14000 rpm for 15 minutes. Wash twice with 50 mM, pH 7.4 PBS storage solution containing 1% (w / w) Nacl, 0.5% (w / w) BSA, 0.1% (w / w) Tween-20, and ultrasonically resuspend to 200 μl and store at 4°C in the dark.

[0063] Step 8: Prepare the sample pad 22: Use the sample pad treatment solution (20mM PBS containing 0.5% NaCl, 0.5% S17, 0.5% BSA, 1mg / ml anti-RBC antibody, pH 7.4) to spray two lines evenly and parallelly on the side of the substrate 21 close to the sample pad, with a dosage of 4μl liquid / cm sample pad, and dilute the P-Tau217 monoclonal antibody and rabbit IgG antibody labeled with the dual-modal nanoprobe 20 times with microsphere diluent (20mM PBS buffer containing 0.5% (w / w) BSA and 20% (w / w) sucrose) to spray one line evenly, with a dosage of 4μl liquid / cm sample pad. Place in an oven and dry overnight at 37°C.

[0064] Step 9: Preparation of coating membrane 23: Adjust the concentration of P-Tau2172 monoclonal antibody and goat anti-rabbit IgG antibody to 1 mg / ml with coating buffer (20 mM pH 7.4 PBS buffer containing 2.5% (w / w) sucrose), and use 1 μl coating liquid / cm membrane, and draw them on nitrocellulose membrane in parallel as test line and quality control line, respectively, with a spacing of 4 mm between the quality control line and the test line, dry them in an oven at a humidity of <30% and a temperature of 37°C for 10 hours, seal the bag, and set aside

[0065] Step 10: On the backing 21 (size 80*300mm), the sample pad 22 (size 30*300mm, made of glass fiber cotton), the coating film 23 (size 25*300mm, made of nitrocellulose) and the absorbent paper 24 (size 28*300mm) are sequentially overlapped to obtain a test paper board, which is cut into 4mm wide test paper strips 2 as required.

[0066] After the preparation of each part is completed, the base 21, sample pad 22, coating membrane 23, and absorbent paper 24 are loaded into the card shell 1, and then a standard curve is drawn, and the generated two-dimensional code information is retained in the ID card, wherein the ID card can be in the form of a chip or in the form of paper printing for scanning.

[0067] The ID card containing the standard curve is obtained by measuring the gradient concentration of the calibration product by detecting the test strip 2, and writing and generating the corresponding two-dimensional code information and storing it in the ID card.

[0068] The standard curve is drawn by using the concentration of the calibrator as the horizontal axis and the ratio of the fluorescence signal as the vertical axis. The standard curve of the ID card containing the standard curve in the same batch of kits is the same.

[0069] The method for quantitatively detecting P-Tau217 by the phosphorylated Tau 217 protein detection kit of this embodiment comprises the following steps:

[0070] Step 1: Place the test kit and sample at room temperature and use them after returning to room temperature;

[0071] Step 2: Turn on the rare earth nano fluorescent immunoassay analyzer, preheat for 5 minutes and insert the corresponding ID card;

[0072] Step 3: Wipe the fingertips with an alcohol cotton pad, draw 10 μL of blood with a capillary tube and add it to the sample well of the test card, then add two drops of diluent;

[0073] Step 4: Insert the test card into the test slot, test after 10 minutes, and read and print the test results.

[0074] During the test by dry fluorescent immunoassay analyzer, the corresponding QR code information on the rare earth nanofluorescence detection card is read and the corresponding concentration is measured, and then the data is compared.

[0075] The detection principle of the phosphorylated Tau 217 protein detection kit of this embodiment is the double antibody sandwich method, which detects the content of P-Tau217 in human serum, plasma and whole blood samples. The blood sample dilution containing P-Tau217 is dripped into the sample addition area, chromatographed to the sample pad by capillary action, and combined with P-Tau217 labeled with the dual-modal nanoprobe to form a microsphere antibody-antigen complex, which is chromatographed to the detection area on the nitrocellulose membrane. The reaction complex is captured by the P-Tau2172 antibody coated with the detection line, and the excess dual-modal nanoprobe marker continues to be chromatographed forward, and the rabbit IgG is fixed on the quality control line. The detection area is scanned and detected with a light source (980nm), and the nanoprobes on the detection line and the quality control line emit fluorescence (540nm). Because the upconversion process does not occur in nature, the interference of sample autofluorescence is greatly reduced. The concentration of the analyte in the sample can be analyzed by the strength and ratio of the fluorescence intensity of the detection line and the quality control line. At the same time, blood samples containing P-Tau217 will form a visible red band on the corresponding T line; when testing negative samples, there is no red band on the test line (T line). Regardless of whether P-Tau217 is present in the sample, a red band will appear on the quality control line (C line).

[0076] The standard curve of the phosphorylated Tau 217 protein detection kit of this embodiment is drawn as follows: different concentrations of P-Tau217 antigen quality control products are added to the prepared dual-modal nanoprobe reagent card (three replicates are set for each concentration, all of which are obtained by diluting the P-Tau217 antigen with 20% calf serum), and after 10 minutes of sample chromatography, the C and T line fluorescence signals and C / T values ​​are read by a rare earth nano-fluorescence immunoassay with excitation light (980nm) / emission light (540nm). The experimental results and analysis are shown in Table 1:

[0077]

[0078] The standard curve was drawn based on the concentration of P-Tau217 quality control product and the average T / C value of the sample signal. The curve data is shown in Table 1. The standard curve is shown in Figure 4 As shown. The P-Tau217 R value is 0.9998. The concentration of P-Tau217 in the sample is quantitatively determined by this line.

[0079] The performance test of the dual-modal nanoprobe test paper card of the phosphorylated Tau 217 protein detection kit of this embodiment includes:

[0080] (1) Minimum detection limit: The measurement was repeated 20 times with a zero-value sample, and the mean M and standard deviation SD of the 20 results were calculated. The detection limit of the method was reported as the blank mean plus twice the standard deviation (M+2SD). The P-Tau217 results were 0.411 pg / ml, respectively, which met the sensitivity standard of 0.5 pg / ml.

[0081] (2) Linear range: Seven concentration values ​​of p-Tau 217 between 0.5 and 50 pg / ml were taken, and each concentration was measured three times. The average value of the measured concentration was subjected to linear analysis with the theoretical concentration, and the p-Tau 217 linear equation y=0.9615x+0.2311 was obtained, and r=0.9994, indicating that the phosphorylated Tau 217 protein detection kit of the present invention has a good correlation within the linear range.

[0082] (3) Precision: Three batches of the kit of this example were used to test the intra-batch CV of three batches of duplicate quality control products. Each batch of the kit was tested 10 times in parallel with duplicate quality control products. Among them, the intra-batch CV of p-Tau 2175pg / ml was 6.32%, 7.66%, and 8.70%, respectively, and the inter-batch CV was 7.38%. The intra-batch CV of three batches of 20pg / ml was 5.36%, 4.82%, and 8.25%, respectively, and the inter-batch CV was 6.35%, all within 10%.

[0083] The clinical sample detection of the phosphorylated Tau 217 protein detection kit of this example is as follows:

[0084] 50 blood samples were collected from the hospital for testing P-Tau217, and the kit of the present invention was used to test and compare with the kit of Ipnocon for detecting p-Tau 217 by chemiluminescence. In the kit of the present invention, 10 μl of blood sample was added to the sample well of the test card, 2 drops of diluent were added, and the concentration was read by the rare earth nano fluorescent immunoassay after chromatography for 10 minutes. The same sample was tested by the comparison system thermal scene chemiluminescence detection kit for p-Tau 217 for concentration. The test results were linearly analyzed, and p-Tau 217r=0.9881, P>0.05, and the average relative deviation was less than 10%. The results met the requirements of clinical analysis and were suitable for clinical testing.

[0085] The specific embodiments described above further illustrate the objectives, technical solutions and beneficial effects of the present invention in detail. It should be understood that the above description is only a specific embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A phosphorylated Tau 217 protein detection card, characterized in that: The invention comprises a cartridge (1) and a test paper strip (2) arranged in the cartridge (1); the test paper strip (2) comprises a substrate (21) and a sample pad (22), a coating film (23) and absorbent paper (24) which are overlapped and attached to the substrate in sequence along the length direction of the substrate; the sample pad (22) is sprayed with a microsphere line; the microsphere line is a P-Tau217 monoclonal antibody and a rabbit IgG antibody labeled with up-conversion enhanced luminescence colorimetric nanoprobe microspheres; the coating film (23) comprises a nitrocellulose membrane and a detection line and a quality control line which are arranged in parallel and spaced on the nitrocellulose membrane in sequence along the length direction; the detection line and the quality control line are coated with P-Tau217 2 and a goat anti-rabbit antibody respectively, wherein the detection line is close to the sample pad (22) and the quality control line is far from the sample pad (22).

2. A phosphorylated Tau 217 protein detection card according to claim 1, characterized in that: The dual-modal nanoprobe has a gold rod coated with silicon dioxide nanoparticles as a core, and the core is coated with an upconversion nanocrystal layer and an outer silicon layer. The upconversion nanocrystal layer is composed of a core-shell structure of ytterbium and erbium co-doped sodium yttrium fluoride coated with a sodium yttrium fluoride shell NaYF4:Yb,Er@NaYF4.

3. A phosphorylated Tau 217 protein detection card according to claim 1, characterized in that: The microsphere line sprayed on the sample pad (22) is a P-Tau217 monoclonal antibody and a rabbit IgG antibody labeled with upconversion enhanced luminescence colorimetric nanoprobe microspheres, and the content of both is 20-200 μg antibody / 200 μl fluorescent microspheres.

4. A phosphorylated Tau 217 protein detection card according to claim 1, characterized in that: The particle size of the up-conversion enhanced luminescence colorimetric nanoprobe is 190 nm.

5. A phosphorylated Tau 217 protein detection card according to claim 1, characterized in that: The inner layer of the upconversion enhanced luminescence colorimetric nanoprobe has a diameter of 80-120 nm, an inner silicon layer thickness of 10-30 nm, an upconversion nanocrystal layer thickness of 30-50 nm, and an outer silicon layer thickness of 10-30 nm.

6. A phosphorylated Tau 217 protein detection card according to claim 1, characterized in that: The coating concentration of the P-Tau217 2 monoclonal antibody on the nitrocellulose membrane (23) is 0.1-2 mg / ml, and the amount used is 0.5-1.5 µl of coating liquid / cm of membrane. The coating concentration of the goat anti-rabbit IgG antibody is 0.5-2 mg / ml, and the amount used is 0.5-1.5 µl of coating liquid / cm of membrane.

7. A phosphorylated Tau 217 protein detection kit, characterized in that: The invention comprises a phosphorylated Tau 217 protein detection card as described in any one of claims 1 to 7 and an ID card containing a calibration curve, wherein the calibration curve ID card has a standard concentration curve, wherein the standard concentration curve uses the measured gradient concentration of the test strip as the calibrator, uses the calibrator concentration as the horizontal axis, and uses the fluorescence signal ratio as the vertical axis.

8. A phosphorylated Tau 217 protein detection kit according to claim 8, wherein the phosphorylated Tau 217 protein detection card comprises a buckle 1, a sample loading hole 11 and an observation window 12, wherein the buckle 1 comprises a connected plastic lower shell and a plastic upper shell; the test strip is fixed on the plastic lower shell, and the surface of the test strip is pressed by the plastic upper shell; the plastic upper shell is provided with a sample loading hole 11 and an observation window 12 at positions corresponding to the sample pad and the coating membrane.

9. Use of a phosphorylated Tau 217 protein detection kit according to claim 8 in the detection of Alzheimer's disease, characterized in that The following steps are involved: Colorimetric test: obtain a blood sample, place the sample on a sample pad (22), dilute it with a sample diluent, and then let it stand for a specified time. After the diluted sample reaches the observation window and the quality control line changes color, confirm the status of the detection line and the quality control line in the sample observation window; When the detection line develops color, fluorescence analysis is performed, and the steps of the fluorescence analysis are as follows: Place the test kit and samples at room temperature and use them after they return to room temperature; Turn on the rare earth nano fluorescent immunoassay analyzer, preheat for 5 minutes, and then insert the corresponding ID card; Wipe the fingertips with an alcohol pad, draw 10µL of blood with a capillary tube and add it to the sample well of the test card, then add two drops of diluent; Insert the test card into the test slot, test after 10 minutes, and read and print the test results.

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