Phosphorylated Tau 181 protein detection card and application thereof, and detection kit containing phosphorylated Tau 181 protein detection card

Abstract

The invention relates to the technical field of protein detection, in particular to a phosphorylated Tau 181 protein detection card, application of the phosphorylated Tau 181 protein detection card and a detection kit comprising the phosphorylated Tau 181 protein detection card. The phosphorylated Tau 181 protein detection card comprises a card shell and a detection test strip arranged in the card shell, the detection test strip comprises a bottom lining, and a sample pad, a coating film and water absorption paper which are sequentially overlapped and adhered to the bottom lining along the length direction of the bottom lining; a microsphere line is sprayed on the sample pad; and the microsphere line is a phosphorylated Tau 181 monoclonal antibody and a goat anti-rabbit IgG (Immunoglobulin G) antibody which are marked by bimodal nano probe microspheres. The detection kit comprises a phosphorylated Tau 181 protein detection card and an ID card containing a calibration curve. The phosphorylated Tau 181 protein detection kit provided by the invention has the characteristics of wide detection range and high sensitivity, and can quickly and accurately detect the content of phosphorylated Tau 181 in human blood.

Description

Technical Field

[0001] This application relates to the technical field of protein detection, and particularly relates to a phosphorylated Tau 181 protein detection card and its application, and a detection kit containing the same. Background Art

[0002] Alzheimer's disease (AD) is a common senile neurodegenerative disease. The diagnosis and intervention of prodromal AD (pAD) are currently the focus of clinical and medical research. This stage includes cognitively normal individuals positive for β-amyloid protein (Aβ), as well as patients with AD-derived mild cognitive impairment (MCI). Currently, the cerebrospinal fluid or PET-CT examinations, which are the gold standards for diagnosing Alzheimer's disease, are not routinely used in clinical practice due to reasons such as cumbersome operation, invasiveness, high cost, and long waiting time, and are difficult to popularize. Since the onset of Alzheimer's disease is insidious and slow, changes in the brain begin 10 - 15 years before obvious "dementia" symptoms occur. If the detection window can be advanced and home detection can be achieved, it is expected to popularize the early screening of AD and delay or terminate the disease progression. Therefore, it is of great significance to develop a simple, effective, low-cost, and non-invasive pAD screening method.

[0003] The diagnosis of pAD depends on the detection of biomarkers. Most early AD detections are based on a large number of biomarkers, requiring invasive sampling, high cost, and non-specificity. Blood Aβ and phosphorylated tau proteins are currently the most potentially valuable AD-related blood biomarkers. In the plasma of AD patients, a decrease in the Aβ42 / Aβ40 ratio can be observed, and plasma p-tau has high sensitivity and accuracy as a biomarker for diagnosing AD.

[0004] However, the concentrations of peripheral blood biomarkers are generally low and the variation range is relatively small, requiring high detection precision. At the same time, they are affected by peripheral metabolism and systemic diseases. Therefore, an extremely sensitive detection method is needed. It is necessary to achieve a detection limit level of pg and below, which is a common development bottleneck and technical problem in the industry.

[0005] Currently, there are mainly methods such as single molecule immunoassay SIMOA, chemiluminescence immunoassay CLIA, and enzyme-linked immunosorbent assay. However, the above methods are generally cumbersome to operate and require a long time to obtain results. Therefore, there is currently no highly sensitive and rapid immunochromatographic detection method.

[0006] Therefore, it is necessary to develop highly sensitive detection probes, efficiently conjugate them with p-tau and Aβ antibodies, and use them for the detection of p-tau and Aβ biomarkers in blood, and be equipped with a rapid portable fluorescence detection instrument for rapid, accurate, and highly sensitive detection to achieve the purpose of diagnosing and intervening in pAD. Summary of the Invention

[0007] The present application provides a phosphorylated Tau 181 protein detection card, its application, and a detection kit containing the same.

[0008] The phosphorylated Tau 181 protein detection kit provided by the present application has the characteristics of a wide detection range and high sensitivity, and can quickly and accurately detect the content of Tau 181 protein in human blood. The phosphorylated Tau 181 protein detection kit provided by the present application can be used for self-testing of fingertip blood at home. The result can be judged by colorimetry with the naked eye, or can be read through a supporting luminescence device, and can detect patients with early Alzheimer's disease, providing an effective technical means for the early diagnosis and disease monitoring of clinical Alzheimer's disease.

[0009] In the first aspect, the present application provides a phosphorylated Tau 181 protein detection card, adopting the following technical solution:

[0010] A phosphorylated Tau 181 protein detection card, the detection card includes a card shell (1) and a test strip (2) arranged in the card shell (1); a microsphere line is sprayed on the sample pad (22) of the test strip (2); the microsphere line is a phosphorylated Tau 181 monoclonal antibody and a goat anti-rabbit IgG antibody labeled with a dual-modal nanosensor microsphere.

[0011] Optionally, the dual-modal nanosensor includes a gold nanocore and an inner silicon layer, an upconversion nanocrystal layer, and an outer silicon layer sequentially coated on the surface of the gold nanocore; the upconversion nanocrystal layer is a core-shell structure, and the composition is ytterbium and erbium co-doped sodium yttrium fluoride coated with a sodium yttrium fluoride shell layer.

[0012] Optionally, the average particle size of the dual-modal nanosensor is 150-250 nm; the average particle size of the gold nanocore is 80-120 nm; the thickness of the inner silicon layer is 10-40 nm; the thickness of the outer silicon layer is 10-40 nm; wherein, the upconversion nanocrystal layer is a core-shell structure, and the composition is ytterbium and erbium co-doped sodium yttrium fluoride coated with a sodium yttrium fluoride shell layer, and the thickness is 30-50 nm.

[0013] Rare earth elements have unique photophysical and photochemical properties and are the most important strategic resources in China. Doping different rare earth ions into inorganic nanomaterials (nanocrystals) will endow the nanomaterials with excellent luminescence properties, which makes the research on rare earth luminescent nanobiomedical materials become a hot spot for a new generation of technological breakthroughs in the biomedical field. For example, by using the optical properties of rare earth nanosensors such as long fluorescence lifetime, very narrow emission peak full width at half maximum, and large Stokes-like shift, and applying them to medical disease diagnosis, the sensitivity of traditional biofluorescent probe diagnosis can be increased by 2-3 orders of magnitude, and the accuracy of detecting trace Alzheimer's disease markers in blood can be greatly improved.

[0014] This application utilizes the unique optical properties of rare earth nanoprobes. By applying highly sensitive rare earth nanoprobes to the detection of AD blood markers and using a rare earth upconversion immunofluorescence analyzer as the detection equipment, the detection sensitivity of AD blood markers is increased to the level of 0.5 - 1 pg / mL, and the performance reaches or exceeds that of related detection products on the current market. Furthermore, a highly sensitive and rapid detection kit is developed, which can expand the convenience of detection, enable in-depth home screening detection, assist in screening the early stage of AD, and at the same time has a certain predictive value for the progression of the disease.

[0015] In this application, the dual-modal nanoprobe exhibits many advantages, such as low toxicity, high chemical stability, narrow-band emission, large anti-Stokes shift, deep light penetration depth and spatial resolution, no damage to biological tissues, etc. In addition, rare earth upconversion luminescence only requires a low-power density near-infrared continuous laser (typical excitation wavelength is 980 nm). At the same time, gold nanoparticles are introduced for metal-enhanced fluorescence, which results from the interaction between the fluorophore and the surface plasmon resonance of gold nanoparticles, leading to the enhancement of the local electromagnetic field around the metal nanoparticles, thereby enhancing the excitation efficiency. At the same time, the radiative decay rate of the fluorophore is also enhanced. These two aspects work together to enhance the fluorescence intensity of the fluorophore. In addition, the introduction of gold nanoparticles can also increase the visualization of the detection.

[0016] This application provides an upconversion enhanced luminescence colorimetric nanoprobe with high upconversion fluorescence intensity, low background interference, high sensitivity, good monodispersity, and capable of realizing colorimetric / fluorescent dual-modal detection. And a detection kit and a detection method are developed, which can realize home self-testing, have a wide detection range, high sensitivity, and can quickly determine the content of Tau 181 protein in blood.

[0017] Optionally, the dual-modal nanoprobe is an upconversion enhanced luminescence colorimetric nanoprobe.

[0018] Optionally, under 980 nm excitation light, the fluorescence spectrum emission peak of the dual-modal nanoprobe is in the range of 520 - 550 nm.

[0019] Optionally, the test strip (2) includes a backing (21) and a sample pad (22), a coating membrane (23), and a blotting paper (24) that are sequentially lap-pasted on the backing (21) along its length direction.

[0020] Optionally, the coating membrane (23) includes a nitrocellulose membrane and a test line and a quality control line that are sequentially and parallelly spaced along its length direction on the nitrocellulose membrane.

[0021] Optionally, the test line and the quality control line are respectively coated with phosphorylated Tau 181 and goat anti-rabbit IgG antibodies; the test line is close to the sample pad (22), and the quality control line is far from the sample pad (22).

[0022] Optionally, the interval distance between the detection line and the quality control line is 2-4 mm.

[0023] Optionally, the contents of the phosphorylated Tau 181 monoclonal antibody and the goat anti-rabbit IgG antibody labeled with the bimodal nanoprobe microspheres are both 20-200 μg antibody / 200 μl fluorescent microspheres.

[0024] Optionally, on the coating film (23), the coating concentration of the phosphorylated Tau 181 monoclonal antibody is 0.1-2 mg / ml, and the dosage is 0.5-1.5 μl coating solution amount / cm.

[0025] Optionally, the coating concentration of the goat anti-rabbit IgG antibody is 0.5-2 mg / ml, and the dosage is 0.5-1.5 μl coating solution amount / cm.

[0026] In a specific embodiment, a preparation method of a test strip (2) specifically includes the following steps:

[0027] (1) Synthesize a sodium yttrium fluoride core structure - an upconversion nanocrystal layer core structure

[0028] In a container, add oleic acid; 1-octadecene; nitrates or acetates or chlorides of yttrium, ytterbium, and erbium; a NaOH and ammonium fluoride methanol mixed solution, and carry out a reaction; subsequently wash with a cyclohexane ethanol mixed solution and disperse in cyclohexane to obtain a NaYF4:Yb,Er nanoprobe cyclohexane solution.

[0029] Specifically, this step can be: in a container, add oleic acid and 1-octadecene with a volume ratio of (3-6):(7-15), and then add nitrates or acetates or chlorides of yttrium, ytterbium, and erbium with a molar ratio of 0.78:0.2:0.02 according to the molar ratio; mix and stir at room temperature, evacuate, then raise the temperature to 100-120 °C, react for 20-30 min, then raise the temperature to 140-160 °C, react for 10-15 min to obtain a transparent solution; naturally cool to 40-50 °C, release the vacuum, add a NaOH and ammonium fluoride methanol mixed solution, and react for 20-30 min; raise the temperature to 90-100 °C, evacuate and replace the gas 3-4 times, introduce nitrogen, raise the temperature to 290-310 °C, react for 1-2 h, centrifuge at 8000 rpm, then wash with a cyclohexane ethanol mixed solution 3-4 times, and disperse in cyclohexane to obtain a NaYF4:Yb,Er nanoprobe cyclohexane solution.

[0030] Among them, the molar ratio of the nitrates or acetates or chlorides of yttrium, ytterbium, and erbium, and NaOH and ammonium fluoride is 1:(1-2.5):(2-4).

[0031] (2) Preparation of upconversion nanocrystals

[0032] In a container, add oleic acid, 1-octadecene, yttrium acetate, NaOH, ammonium fluoride methanol mixed solution, and the cyclohexane solution of the NaYF4:Yb,Er nanoprobe prepared in step (1), and carry out the reaction; then wash with a cyclohexane-ethanol mixture and disperse in cyclohexane; then transfer the upconversion nanocrystals to ethanol by acid washing to obtain an ethanol solution containing upconversion nanocrystals.

[0033] Specifically, this step can be: In a container, add oleic acid and 1-octadecene with a volume ratio of (3-6):(7-15), add yttrium acetate, mix and stir, stir at room temperature, evacuate, then heat to 100-120 °C, react for 20-30 min, then heat to 140-160 °C, react for 10-15 min to obtain a transparent solution; naturally cool to 40-50 °C, release the vacuum, add the NaOH and ammonium fluoride methanol mixed solution, and the cyclohexane solution of the NaYF4:Yb,Er nanoprobe prepared in step (1), mix and stir, react for 20-30 min; heat to 90-100 °C, evacuate and replace the gas 3-4 times, introduce nitrogen, heat to 290-310 °C, react for 1-2 h, centrifuge at 8000 rpm, then wash 3-4 times with a cyclohexane-ethanol mixture and disperse in cyclohexane.

[0034] Among them, the molar ratio of yttrium acetate, NaOH, and ammonium fluoride is 1:(1-2.5):(2-4).; then transfer the upconversion nanocrystals to ethanol by acid washing to obtain an ethanol solution containing upconversion nanocrystals with a concentration of 20 mg / mL.

[0035] (3) Preparation of nanogold coated with an inner silicon layer

[0036] Synthesis of gold nanorods: In a container, add chloroauric acid to cetyltrimethylammonium bromide (CTAB) under magnetic stirring, then add sodium borohydride solution, stir and let stand for 30 min.

[0037] Growth solution: Dissolve CTAB and sodium oleate in ultrapure water, then inject chloroauric acid into the solution. When the solution is colorless, add silver nitrate solution, stir and then add hydrochloric acid and ascorbic acid. After the reaction, add the seed solution, and let the reaction mixture stand for 12 h to complete the synthesis of gold nanorods.

[0038] After synthesis, centrifuge and wash, and disperse in ultrapure water. Take the gold rod solution, mix it with the mPEG-SH solution, and stir slowly at room temperature. Then centrifuge to remove the supernatant and resuspend in a mixture of ethanol and water. Under stirring conditions, add concentrated ammonia water and TEOS solution, seal and stir, then centrifuge three times and resuspend in ethanol solution to obtain nanogold coated with an inner silicon layer.

[0039] (4) Preparation of a nanoprobe encapsulated with an upconversion nanocrystal layer

[0040] Disperse the gold nanoparticles encapsulated with the inner silicon layer obtained in step (3) in ethanol, add the ethanol solution containing upconversion nanocrystals obtained in step (2), stir to react fully, and after centrifuging to remove the excessive upconversion nanocrystals, disperse the obtained material in ethanol, and successively add water, 10% tetraethoxysilane ethanol solution and ammonia water. After stirring, centrifuge and wash to obtain a nanoprobe encapsulated with an upconversion nanocrystal layer.

[0041] (5) Preparation of a dual-modal nanoprobe

[0042] Mix and stir the nanoprobe encapsulated with an upconversion nanocrystal layer obtained in step (4) and 3-aminopropyltriethoxysilane, centrifuge and disperse it in N,N-dimethylformamide (DMF) solvent to obtain a dispersion, then add succinic anhydride DMF solution and stir, wash and disperse it in water to obtain a dual-modal nanoprobe.

[0043] (6) Activation of the dual-modal nanoprobe

[0044] Perform ultrasonic treatment and centrifugation on the dual-modal nanoprobe obtained in step (5), wash the precipitate with MES solution; add carbodiimide and N-hydroxysuccinimide, mix well and then centrifuge at high speed, wash the precipitate with MES solution with a pH of 5.0 - 7.0 to obtain the activated dual-modal nanoprobe.

[0045] (7) Preparation of a Tau 181 monoclonal antibody labeled with a dual-modal nanoprobe

[0046] After ultrasonic treatment of the activated dual-modal nanoprobe obtained in step (6), add Tau 181 monoclonal antibody and rabbit IgG antibody, mix well, use a blocking solution, then centrifuge at high speed, wash gently twice with PBS storage solution, and resuspend by ultrasonic treatment and store in the dark at 4°C.

[0047] (8) Preparation of sample pad 22

[0048] Use the sample pad treatment solution to spray two parallel and uniform lines on one side of the substrate 21 close to the sample pad 22, and dilute the Tau 181 monoclonal antibody labeled with the dual-modal nanoprobe and rabbit IgG antibody 20 times with the microsphere diluent and spray one uniform line on the side of the sample pad 22 close to the coating film 23, and place it in an oven and dry at 37°C overnight.

[0049] (9) Preparation of coating film 23

[0050] Adjust the concentrations of Tau 181 monoclonal antibody and goat anti-rabbit IgG antibody to 0.5 - 2 mg / ml with coating buffer respectively, and the dosage is 0.5 - 1.5 μl coating solution per cm² of membrane. Coat them parallelly on the nitrocellulose membrane as the test line and the quality control line respectively. The distance between the quality control line and the test line is 2 - 4 mm, and then dry for standby.

[0051] (10) Prepare the test strip 2

[0052] Paste the sample pad 22, the coated membrane 23 and the absorbent paper 24 on the backing 21 in sequence and overlapping each other to obtain the test strip board, and cut it according to the requirements to get the test strip 2.

[0053] In the second aspect, the present application also provides a kit for detecting phosphorylated Tau 181 protein, adopting the following technical scheme:

[0054] A kit for detecting phosphorylated Tau 181 protein, the detection kit includes the above-mentioned detection card for phosphorylated Tau 181 protein.

[0055] Optionally, the detection kit further includes an ID card containing a calibration curve.

[0056] Optionally, the ID card containing the calibration curve is obtained by measuring calibration products with gradient concentrations through the test strip (2), taking the calibration product concentration as the abscissa and the fluorescence signal ratio as the ordinate, plotting a standard curve, writing and generating corresponding two-dimensional code information and storing it in the ID card.

[0057] The corresponding two-dimensional code information on the detection card for phosphorylated Tau 181 protein can be read by a dry fluorescence immunoassay analyzer and the corresponding concentration can be measured.

[0058] In the third aspect, the present application also provides an application of the above-mentioned detection card for phosphorylated Tau 181 protein or the above-mentioned kit for detecting phosphorylated Tau 181 protein in the preparation of a composition for Alzheimer's disease detection.

[0059] In a specific embodiment, a method for quantitatively detecting phosphorylated Tau 181 by a kit for detecting phosphorylated Tau 181 protein specifically includes the following steps:

[0060] (1) Place the detection kit and the sample at room temperature and use them after returning to room temperature;

[0061] (2) Turn on the rare earth nano-fluorescence immunoassay analyzer, insert the corresponding ID card after preheating for 5 min;

[0062] (3) Wipe the fingertip with an alcohol cotton swab, suck 10 μL of blood with a capillary and add it to the sample adding hole of the detection card, and then add two drops of diluent;

[0063] (4) Insert the test card into the test slot, and after 10 minutes, detect, read, and print the test results.

[0064] The detection principle of the above-mentioned phosphorylated Tau 181 protein detection kit is the double antibody sandwich method, which is used to detect the content of phosphorylated Tau 181 in human serum, plasma, and whole blood samples. The blood sample diluent containing phosphorylated Tau 181 is dropped on the sample loading area, and through capillary action, it chromatographs to the sample pad and binds to the phosphorylated Tau 181 antibody labeled with a dual-modal nanoprobe to form a microsphere antibody-antigen complex. Then it chromatographs to the detection area on the nitrocellulose membrane, and the complex is captured by the Tau 181 antibody coated on the test line. The excess dual-modal nanoprobe label continues to chromatograph forward, and the goat anti-rabbit IgG antibody binds to the goat anti-rabbit fixed on the quality control line.

[0065] Use a light source (980 nm) to scan and detect the detection area. The nanoprobe on the test line and the quality control line emits fluorescence (540 nm). Since the upconversion process does not occur in nature, the interference of sample autofluorescence is greatly reduced. By detecting the fluorescence intensity and its ratio of the test line and the quality control line, the concentration of the analyte in the sample can be analyzed. At the same time, a visible red band will be formed on the corresponding T line for the blood sample containing phosphorylated Tau 181; when detecting a negative sample, there is no red band on the test line (T line). Whether or not phosphorylated Tau 181 exists in the sample, a red band will appear on the quality control line (C line).

[0066] In summary, the present application includes at least one of the following beneficial technical effects:

[0067] The present application provides an upconversion enhanced luminescence colorimetric nanoprobe with high upconversion fluorescence intensity, low background interference, high sensitivity, good monodispersity, and capable of realizing colorimetric / fluorescence dual-modal detection.

[0068] The upconversion enhanced luminescence colorimetric nanoprobe provided by the present application combines the advantages of the high absorbance visible colorimetric effect of gold nanoparticles and the low background noise and high sensitivity of upconversion luminescence.

[0069] At the same time, gold nanoparticles are introduced for metal-enhanced fluorescence, which results from the interaction between the fluorophore and the surface plasmon resonance of gold nanoparticles, leading to the enhancement of the local electromagnetic field around the metal nanoparticles, thereby enhancing the excitation efficiency. At the same time, the radiative decay rate of the fluorophore is also enhanced. Both of these aspects work together to enhance the fluorescence intensity of the fluorophore.

[0070] The phosphorylated Tau 181 protein detection kit provided by the present application has the characteristics of a wide detection range and high sensitivity, and can quickly and accurately detect the content of Tau 181 protein in human blood.

[0071] The phosphorylated Tau 181 protein detection kit provided by this application can be used for self-testing of fingertip blood at home. The results can be judged visually by colorimetry or read through a supporting luminescence device, enabling the detection of patients with early Alzheimer's disease and providing an effective technical means for the early diagnosis and disease monitoring of clinical Alzheimer's disease. BRIEF DESCRIPTION OF THE DRAWINGS

[0072] Figure 1 It is a schematic structural diagram of the phosphorylated Tau 181 protein detection card provided by this application.

[0073] Figure 2 It is a schematic structural diagram of the test strip of the phosphorylated Tau 181 protein detection card provided by this application.

[0074] Figure 3 It is a transmission electron microscope image of the dual-modal nanoprobe provided by this application.

[0075] Figure 4 It is a standard curve graph drawn with the concentration of the Tau 181 protein control product and the average T / C value of the sample signal of the phosphorylated Tau 181 protein detection kit provided by this application.

[0076] Figure 5 It is a comparison curve graph of the detection results of the same sample by the phosphorylated Tau 181 protein detection kit provided by this application and the Novizan chemiluminescence method for detecting Tau181 protein kit.

[0077] Reference numerals: 1. Card shell; 11. Sampling hole; 12. Observation window; 2. Test strip; 21. Backing; 22. Sample pad; 23. Coated membrane; 24. Absorbent paper. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0078] Before describing the embodiments of this application in detail, it should be understood that the terms used herein are only for the purpose of describing specific embodiments. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which this term belongs.

[0079] It should be noted that the terms "first" and "second" are only used for descriptive purposes and cannot be understood as indicating or implying relative importance or implicitly indicating the quantity of the indicated technical features. Thus, features defined with "first" and "second" may explicitly or implicitly include one or more of such features. Further, in the description of this application, unless otherwise stated, the meaning of "a plurality" is two or more.

[0080] In the ranges disclosed in this application, the endpoints and any values of the ranges are not limited to the exact ranges or values. These ranges or values should be understood to include values close to these ranges or values. For numerical ranges, the endpoint values of each range, between the endpoint values of each range and individual point values, and between individual point values can be combined with each other to obtain one or more new numerical ranges, and these numerical ranges should be regarded as specifically disclosed herein.

[0081] In this application, the term "comprising" or "including" is an open expression, that is, it includes the content specified in this application, but does not exclude other aspects of the content.

[0082] To make the objectives, technical solutions, and advantages of this application clearer, the technical solutions in the embodiments of this application will be clearly and completely described below. Based on the embodiments of this application, all other embodiments obtained by those skilled in the art without creative efforts shall fall within the scope of protection of this application. The embodiments described below are exemplary and are only used to explain this application and should not be construed as a limitation of this application.

[0083] For those not specifying specific techniques or conditions in the embodiments, follow the techniques or conditions described in the literature in this field or follow the product specifications. For reagents or instruments not indicating the manufacturer, they are all conventional products that can be obtained through commercial purchase.

[0084] The following further details this application in conjunction with embodiments and test results.

[0085] Embodiment

[0086] Embodiment 1

[0087] This embodiment provides a kit for detecting phosphorylated Tau 181 protein. The kit includes a detection card for phosphorylated Tau 181 protein and an ID card containing a calibration curve.

[0088] As Figure 1 shown, the detection card for phosphorylated Tau 181 protein includes a card shell 1 and a test strip 2 arranged in the card shell 1. The card shell 1 includes a plastic lower shell and a plastic upper shell connected by a buckle. The test strip 2 is fixed on the plastic lower shell, and the surface of the test strip 2 is pressed by the plastic upper shell.

[0089] The test strip 2 includes a backing 21 and a sample pad 22, a coating film 23, and a blotting paper 24 that are sequentially overlapped and pasted on the backing 21 along the length direction of the substrate. Among them, the sample pad 22 is the sample addition area for sucking the blood test sample to be detected. The plastic upper shell is respectively provided with a sample addition hole 11 and an observation window 12 corresponding to the positions of the sample pad 22 and the coating film 23.

[0090] Among them, a microsphere line is sprayed on the sample pad 22. The microsphere line is a phosphorylated Tau181 monoclonal antibody and a goat anti-rabbit IgG antibody labeled with a bimodal nanoprobe (both with a content of 100 μg antibody / 200 μl fluorescent microspheres).

[0091] The bimodal nanoprobe includes a gold nanocore and an inner silicon layer, an upconversion nanocrystal layer, and an outer silicon layer that are sequentially coated on the surface of the gold nanocore. Among them, the average particle size of the gold nanocore is 80 - 120 nm, the thickness of the inner silicon layer is 10 - 40 nm, the thickness of the outer silicon layer is 10 - 40 nm, and the thickness of the upconversion nanocrystal layer (NaYF4:Yb / ,r@NaYF4) is 30 - 50 nm. In this embodiment, the average particle size of the gold nanocore 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 thickness of the upconversion nanocrystal layer (NaYF4:Yb / ,r@NaYF4) is 40 nm.

[0092] Among them, the upconversion nanocrystal layer has a core-shell structure, and its composition is: ytterbium and erbium co-doped sodium yttrium fluoride coated with a sodium yttrium fluoride shell layer, that is, NaYF4:Yb,Er@NaYF4, with an average particle size of 30 - 50 nm. As Figure 3 shown (100 nm in the figure is the scale mark), the average particle size of the bimodal nanoprobe is 200 nm. It can be Figure 3 seen that the monodispersity and uniformity of the bimodal nanoprobe are better. The maximum value of the ultraviolet absorption peak of the bimodal nanoprobe is located at 550 nm; under 980 nm excitation light, the fluorescence spectrum emission peak of the bimodal nanoprobe is in the range of 520 - 550 nm, and the peak value is 540 nm.

[0093] Among them, the coating film 23 includes a nitrocellulose membrane, and a detection line and a quality control line are sequentially arranged on the nitrocellulose membrane in parallel at intervals along the length direction. The interval distance between the detection line and the quality control line is 2 - 4 mm. The detection line and the quality control line are respectively coated with a phosphorylated Tau 181 monoclonal antibody and a goat anti-rabbit IgG antibody. The detection line is close to the sample pad 22, and the quality control line is far from the sample pad 22. The coating concentration of the phosphorylated Tau 181 monoclonal antibody (purchased from Beijing Aibosheng Biotechnology Co., Ltd.) is 1 mg / ml, and the dosage is 1 μl coating solution volume / cm membrane. The coating concentration of the goat anti-rabbit IgG antibody (purchased from Luoyang BioTong Experimental Materials Center) is 1 mg / ml, and the dosage is 1 μl coating solution volume / cm membrane.

[0094] The ID card containing the calibration curve measures calibration products with gradient concentrations through the test strip 2. Using the calibration product concentration as the abscissa and the fluorescence signal ratio as the ordinate, a standard curve is plotted, written, and the corresponding QR code information is generated and stored in the ID card. The dry fluorescence immunoassay analyzer can read the corresponding QR code information on the rare earth nano-fluorescent test card and measure the corresponding concentration. The standard curves of the ID cards containing the calibration curve in the same batch of reagent kits are the same.

[0095] Example 2

[0096] This example provides the test strip 2 mentioned in Example 1.

[0097] The preparation method of the above-mentioned test strip 2 specifically includes the following steps:

[0098] (1) Synthesize the sodium yttrium fluoride core structure - the core structure of the upconversion nanocrystal layer

[0099] In a container, add oleic acid and 1-octadecene with a volume ratio of 6:15, and then add nitrates or acetates or chlorides 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 120 °C and react for 20 min, then heat to 140 °C and react for 15 min to obtain a transparent solution; naturally cool to 50 °C, release the vacuum, add a mixed solution of NaOH and ammonium fluoride in methanol, and react for 30 min; heat to 100 °C, evacuate and replace the gas 3 - 4 times, introduce nitrogen, heat to 300 °C, react for 1.5 h, centrifuge at 8000 rpm, then wash 3 - 4 times with a mixed solution of cyclohexane and ethanol, and disperse in cyclohexane to obtain a cyclohexane solution of NaYF4:Yb,Er nanoprobes.

[0100] Among them, the molar ratio of nitrates or acetates or chlorides of yttrium, ytterbium, and erbium, and NaOH and ammonium fluoride is 1:2.5:4.

[0101] (2) Prepare the upconversion nanocrystals

[0102] In a container, add oleic acid and 1-octadecene with a volume ratio of 6:15, add yttrium acetate, mix and stir, mix and stir at room temperature, evacuate, then heat to 120 °C and react for 20 min, then heat to 160 °C and react for 10 min to obtain a transparent solution; naturally cool to 50 °C, release the vacuum, add a mixed solution of NaOH and ammonium fluoride in methanol, and the cyclohexane solution of NaYF4:Yb,Er nanoprobes prepared in step (1), mix and stir, and react for 30 min; heat to 100 °C, evacuate and replace the gas 3 - 4 times, introduce nitrogen, heat to 300 °C, react for 1.5 h, centrifuge at 8000 rpm, then wash 3 - 4 times with a mixed solution of cyclohexane and ethanol, and disperse in cyclohexane.

[0103] Among them, the molar ratio of yttrium acetate, NaOH, and ammonium fluoride is 1:2.5:4; then, the upconversion nanocrystals are transferred into ethanol by pickling to obtain an ethanol solution containing upconversion nanocrystals with a concentration of 20 mg / mL.

[0104] (3) Preparation of silica-coated gold nanoparticles

[0105] Synthesis of gold nanorods: In a container, 250 μL of 10 mM chloroauric acid was added to 9.75 mL of 0.1 M cetyltrimethylammonium bromide (CTAB) under magnetic stirring, and then 0.6 mL of 10 mM sodium borohydride solution was added. After stirring for 2 min, it was left at 28 °C for 30 min.

[0106] Growth solution: 7.2 g of CTAB and 0.987 g of sodium oleate were added to 400 ml of ultrapure water and dissolved. Then, 20 mL of 10 mM chloroauric acid was injected into the solution. When the solution became colorless, 8 mL of 10 mM silver nitrate solution was added. After stirring for 5 min, 1.2 mL of hydrochloric acid and 600 μL of 0.1 M ascorbic acid were added. After reacting for about 30 s, 1 mL of seed solution was added, and the reaction mixture was left standing at 30 °C for 12 h to complete the synthesis of gold nanorods.

[0107] After synthesis, it was centrifuged at 9000 rpm for 15 min, centrifuged and washed twice, and then dispersed in 20 mL of ultrapure water. Take 5 ml of the gold nanorod solution and mix it with 250 μL of 1 mg / ml mPEG-SH solution, and stir slowly at room temperature for 1 h. Then, it was centrifuged at 8000 rpm for 10 min. After removing the supernatant, it was resuspended in a mixed solution of ethanol and water (ethanol:water = 9:2). Under stirring conditions, 50 μL of concentrated ammonia water and 15 μL of 10% TEOS solution were added. After sealing and stirring for 12 h, it was centrifuged three times and resuspended in ethanol solution to obtain silica-coated gold nanoparticles.

[0108] (4) Preparation of nanoprobes encapsulated with upconversion nanocrystal layers

[0109] The silica-coated gold nanoparticles obtained in step (3) were dispersed in ethanol, and the ethanol solution containing upconversion nanocrystals obtained in step (2) was added. The volume ratio of the two was 10:1. After stirring for 8 h for full reaction, after centrifuging to remove the excessive upconversion nanocrystals, the obtained material was dispersed in 10 mL of ethanol, and water, 10% tetraethoxysilane ethanol solution, and ammonia water were added in sequence. After stirring for 9 h, it was centrifuged and washed to obtain nanoprobes encapsulated with upconversion nanocrystal layers. Among them, the volume ratio of ethanol, water, 10% tetraethoxysilane ethanol solution, and ammonia water is 10:1:0.05:0.2.

[0110] (5) Preparation of dual-modal nanoprobes

[0111] The upconversion nanocrystal layer-coated nanoprobes obtained in step (4) and 3-aminopropyltriethoxysilane were mixed and stirred at 60 °C for 6 h, and the volume ratio of the two was 1:0.003. They were centrifuged and dispersed in N,N-dimethylformamide (DMF) solvent to obtain a dispersion. Then, a 50 mg / mL succinic anhydride DMF solution was added and stirred for 10 h, and then washed and dispersed in water to obtain bimodal nanoprobes. Among them, the volume ratio of the dispersion to the succinic anhydride DMF solution was 1:0.05.

[0112] (6) Activate the bimodal nanoprobes

[0113] The bimodal nanoprobes obtained in step (5) were ultrasonically treated and centrifuged, and the precipitate was washed with a 50 mM MES solution with a pH of 5.0 - 7.0; carbodiimide and N-hydroxysulfosuccinimide were added, and after mixing, they were centrifuged at high speed. The precipitate was washed with a MES solution with a pH of 5.0 - 7.0, and the activated bimodal nanoprobes were obtained.

[0114] (7) Prepare the Tau 181 monoclonal antibody labeled with bimodal nanoprobes

[0115] The activated bimodal nanoprobes obtained in step (6) were ultrasonically treated for 2 min and then added to the Tau181 monoclonal antibody and rabbit IgG antibody at 100 μg / 200 μl, and mixed for 2 h. After blocking with a 50 mM, pH 7.4 PBS blocking solution containing 0.5% BSA for 1 h, they were centrifuged at 14000 rpm for 15 min, and gently washed twice with a 50 mM, pH 7.4 PBS storage solution containing 1% (w / w) NaCl, 0.5% (w / w) BSA, and 0.1% (w / w) Tween-20, and then ultrasonically treated and resuspended to 200 μl and stored in the dark at 4 °C.

[0116] (8) Prepare the sample pad 22

[0117] Two lines were evenly sprayed in parallel on one side of the substrate 21 close to the sample pad 22 with a sample pad treatment solution (20 mM, pH 7.4 PBS containing 0.5% NaCl, 0.5% S17, 0.5% BSA, and 1 mg / ml anti-RBC antibody), and the dosage was 4 μl liquid volume / cm sample pad 22. On the side of the sample pad 22 close to the coating film 23, the Tau 181 monoclonal antibody labeled with bimodal nanoprobes and rabbit IgG antibody were diluted 20 times with a microsphere diluent (20 mM PBS buffer containing 0.5% (w / w) BSA and 20% (w / w) sucrose) and evenly sprayed in one line, and the dosage was 4 μl liquid volume / cm sample pad. It was placed in an oven and dried at 37 °C overnight.

[0118] (9) Prepare the coating film 23

[0119] The concentrations of Tau 181 monoclonal antibody and goat anti-rabbit IgG antibody were adjusted to 1 mg / ml with coating buffer (PBS buffer at 20 mM pH 7.4 containing 2.5% (w / w) sucrose), and the amount used was 1 μl of coating solution per cm of membrane. They were respectively drawn in parallel on the nitrocellulose membrane as the test line and the control line for coating. The distance between the control line and the test line was 4 mm. It was air-dried in an oven at a humidity < 30% and a temperature of 37°C for 10 h, sealed in a bag, and reserved for use.

[0120] (10) Preparation of the test strip 2

[0121] On the backing 21 (sized 80 * 300 mm), the sample pad 22 (sized 30 * 300 mm, made of glass fiber cotton), the coated membrane 23 (sized 25 * 300 mm, made of nitrocellulose), and the absorbent paper 24 (sized 28 * 300 mm) were sequentially and mutually overlapped and pasted to obtain a test strip board, which was cut into test strips 2 with a width of 4 mm according to requirements.

[0122] Example 3

[0123] This example provides a method for quantitatively detecting phosphorylated Tau 181 using the detection kit provided in Example 1.

[0124] The method for quantitatively detecting phosphorylated Tau 181 using the above detection kit specifically includes the following steps:

[0125] (1) Place the detection kit and the sample at room temperature and use them after returning to room temperature.

[0126] (2) Turn on the rare earth nanophosphorescence immunoassay analyzer, insert the corresponding ID card after preheating for 5 min.

[0127] (3) Wipe the fingertip with an alcohol cotton swab, suck 10 μL of blood with a capillary tube and add it to the sample addition hole of the test card, and then add two drops of diluent.

[0128] (4) Insert the test card into the detection slot, detect after 10 min, and read and print the detection result.

[0129] In this embodiment, the detection principle of the phosphorylated Tau 181 detection kit is the double antibody sandwich method, which is used to detect the content of phosphorylated Tau 181 in human serum, plasma and whole blood samples. The blood sample diluent containing phosphorylated Tau 181 is dropped into the sample loading area, and through capillary action, it chromatographs to the sample pad and binds to the phosphorylated Tau 181 antibody labeled with the dual-modal nanoprobe to form a microsphere antibody-antigen complex. Then it chromatographs to the detection area on the nitrocellulose membrane, and the complex is captured by the Tau181 antibody coated on the test line. The excess dual-modal nanoprobe label continues to chromatograph forward, and the goat anti-rabbit IgG antibody binds to the goat anti-rabbit fixed on the quality control line.

[0130] The detection area is scanned and detected with a light source (980nm). The nanoprobe on the test line and the quality control line emits fluorescence (540nm). Since the up-conversion process does not occur in nature, the interference of sample autofluorescence is greatly reduced. By detecting the fluorescence intensity and its ratio of the test line and the quality control line, the concentration of the analyte in the sample can be analyzed. At the same time, a visible red band will form on the corresponding T line for the blood sample containing phosphorylated Tau 181; when detecting a negative sample, there is no red band on the test line (T line). Whether or not phosphorylated Tau 181 exists in the sample, a red band will appear on the quality control line (C line).

[0131] The process of drawing the standard curve of the phosphorylated Tau 181 protein detection kit in this embodiment is as follows: Different concentrations of phosphorylated Tau 181 antigen quality control products (each concentration has three replicates, all diluted from phosphorylated Tau 181 antigen with 20% calf serum) are added to the prepared phosphorylated Tau 181 protein detection card. After 10 minutes of sample loading and chromatography, the fluorescence signals of the C and T lines and the C / T value are read by a rare earth nanophosphorescence immunoassay analyzer with excitation light (980nm) / emission light (540nm).

[0132] The analysis results are shown in Table 1.

[0133] Table 1 Analysis results of the standard curve

[0134]

[0135]

[0136] The standard curve is drawn with the concentration of the phosphorylated Tau 181 quality control product and the average value of the sample signal T / C. The curve data is shown in Table 1, and the standard curve is as Figure 4 shown. Among them, the R value of phosphorylated Tau 181 is 0.9957, and the concentration of phosphorylated Tau 181 contained in the sample is quantitatively determined through this calibration curve.

[0137] Example 4

[0138] The test strips in the kit provided in this application were subjected to performance testing. The specific contents are as follows:

[0139] (1) Minimum detection limit

[0140] The zero-value samples were repeatedly measured 20 times, 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 detection results of phosphorylated Tau 181 were 0.775 pg / ml respectively, meeting the sensitivity standard of 1 pg / ml respectively.

[0141] (2) Linear range

[0142] Seven concentration values between 1 - 500 pg / ml of phosphorylated Tau 181 were taken respectively, and each concentration was measured three times repeatedly. The average measured concentration was linearly analyzed with the theoretical concentration to obtain the linear equation of phosphorylated Tau 181.

[0143] The obtained linear equation was: y = 0.9177x + 3.2184, r = 0.9996. It indicates that the phosphorylated Tau 181 protein detection kit provided in this application has a very good correlation within the linear range.

[0144] (3) Precision

[0145] Three batches of the kits of this example were taken, and the within-batch CVs of the repeated quality control products were detected respectively. Each batch of kits was used to detect the repeated quality control products in parallel 10 times.

[0146] The within-batch CVs of phosphorylated Tau 181 at 25 pg / ml for the three batches were 5.93%, 6.08%, and 6.01% respectively, and the between-batch CV was 6.53%. The within-batch CVs of phosphorylated Tau 181 at 100 pg / ml for the three batches were 4.81%, 8.23%, and 5.32% respectively, and the between-batch CV was 6.32%. All were within 10%.

[0147] Example 5

[0148] In this example, the kit provided in Example 1 was used to detect clinical samples.

[0149] The detection process and results are as follows:

[0150] 100 blood samples for detecting phosphorylated Tau 181 were collected from the hospital, and the kits of this application and the Ipunuo Kang chemiluminescence method for detecting phosphorylated Tau 181 kit were used for detection and comparison respectively.

[0151] In the kit of the present application, 10 μl of blood sample is taken and added into the sample adding hole of the test strip, 2 drops of diluent are added, and after chromatography for 10 min, the concentration is read by a rare earth nanophosphorescence immunoassay analyzer. The same sample is respectively detected for concentration by using the comparative system, the IpunuoKang chemiluminescence method for detecting the phosphorylated Tau 181 kit. Linear analysis is performed on the detection results. As Figure 5 shown, the correlation is very good. For phosphorylated Tau 181, r = 0.9843, P>0.05, and the average relative deviation is less than 10%. The results meet the requirements of clinical analysis and are suitable for clinical detection.

[0152] In the description of this specification, the description with reference to terms such as "one embodiment", "some embodiments", "example", "specific example" or "some examples" means that the specific features, structures, materials or characteristics described in connection with the embodiment or example are included in at least one embodiment or example of the present application. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described can be combined in any one or more embodiments or examples in a suitable manner. In addition, without contradiction, those skilled in the art can combine and combine the different embodiments or examples described in this specification and the features of different embodiments or examples.

[0153] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present application, rather than to limit them; although the present application has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that they can still modify the technical solutions recorded in the foregoing embodiments, or perform equivalent replacements on some of the technical features; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the spirit and scope of the technical solutions of the embodiments of the present application.

Claims

1. A phosphorylated Tau 181 protein detection card, characterized in that: The detection card comprises a card shell (1) and a detection test strip (2) arranged in the card shell (1); a microsphere line is sprayed on the sample pad (22) of the detection test strip (2); the microsphere line is a phosphorylated Tau 181 monoclonal antibody and a goat anti-rabbit IgG antibody labeled with dual-modal nanoprobe microspheres.

2. The phosphorylated Tau 181 protein detection card according to claim 1, characterized in that: The dual-mode nanoprobe comprises a nanogold core and an inner silicon layer, an upconversion nanocrystal layer and an outer silicon layer sequentially coated on the surface of the nanogold core; the upconversion nanocrystal layer is a core-shell structure, consisting of sodium yttrium fluoride coated with sodium yttrium fluoride co-doped with ytterbium and erbium.

3. The phosphorylated Tau 181 protein detection card according to claim 2, characterized in that: The average particle size of the dual-mode nanoprobe is 150-250nm; the average particle size of the nanogold core is 80-120nm; the thickness of the inner silicon layer is 10-40nm; the thickness of the outer silicon layer is 10-40nm; The up-conversion nanocrystalline layer is a core-shell structure, consisting of a sodium yttrium fluoride shell layer coated with sodium yttrium fluoride co-doped with ytterbium and erbium, with a thickness of 30-50nm.

4. The phosphorylated Tau 181 protein detection card according to claim 1, characterized in that: The dual-mode nanoprobe is an up-conversion enhanced luminescence colorimetric nanoprobe; Optionally, under 980nm excitation light, the fluorescence spectrum emission peak of the dual-modal nanoprobe is within the range of 520-550nm.

5. The phosphorylated Tau 181 protein detection card according to claim 1, characterized in that: The test paper strip (2) comprises a base liner (21) and a sample pad (22), a coating film (23) and absorbent paper (24) which are sequentially overlapped and pasted on the base liner (21) along its length direction; Optionally, the coating membrane (23) comprises a nitrocellulose membrane and a detection line and a quality control line which are sequentially arranged in parallel and spaced apart on the nitrocellulose membrane along its length direction; Optionally, the detection line and the quality control line are coated with phosphorylated Tau 181 and goat anti-rabbit IgG antibody, respectively; the detection line is close to the sample pad (22), and the quality control line is far away from the sample pad (22); Optionally, the detection line and the quality control line are spaced apart by a distance of 2-4 mm.

6. The phosphorylated Tau 181 protein detection card according to claim 1, characterized in that: The content of phosphorylated Tau 181 monoclonal antibody and goat anti-rabbit IgG antibody labeled by the dual-modal nanoprobe microspheres is 20-200μg antibody / 200μl fluorescent microspheres.

7. The phosphorylated Tau 181 protein detection card according to claim 5, characterized in that: On the coating membrane (23), the coating concentration of the phosphorylated Tau 181 monoclonal antibody is 0.1-2 mg / ml, and the amount used is 0.5-1.5 µl coating liquid / cm; Optionally, the coating concentration of the goat anti-rabbit IgG antibody is 0.5-2 mg / ml, and the dosage is 0.5-1.5 µl coating liquid / cm.

8. A phosphorylated Tau 181 protein detection kit, characterized in that: The detection kit comprises the phosphorylated Tau 181 protein detection card according to any one of claims 1 to 7.

9. The phosphorylated Tau 181 protein detection kit according to claim 8, characterized in that: The detection kit also includes an ID card containing a calibration curve; Optionally, the ID card containing the calibration curve measures the gradient concentration of the calibrator by using the test strip (2), plots a standard curve with the calibrator concentration as the horizontal axis and the fluorescence signal ratio as the vertical axis, writes and generates corresponding QR code information and stores it in the ID card.

10. Use of the phosphorylated Tau 181 protein detection card according to any one of claims 1 to 7 or the phosphorylated Tau 181 protein detection kit according to any one of claims 8 to 9 in preparing a composition for detecting Alzheimer's disease.

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