Human beta amyloid protein 1-42 detection card and application thereof, and detection kit containing human beta amyloid protein 1-42 detection card

Abstract

The invention relates to the technical field of protein detection, in particular to a human beta amyloid protein 1-42 detection card, application of the human beta amyloid protein 1-42 detection card and a detection kit comprising the human beta amyloid protein 1-42 detection card. The human beta amyloid protein 1-42 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 an A beta 1-42 monoclonal antibody and a goat anti-rabbit IgG antibody which are marked by bimodal nano probe microspheres. The detection kit comprises a human beta amyloid protein 1-42 detection card and an ID card containing a calibration curve. The human beta amyloid protein 1-42 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 A beta 1-42 in human blood.

Description

Technical Field

[0001] This application relates to the technical field of protein detection, and particularly relates to a human β-amyloid 1-42 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 (Aβ), as well as patients with AD-source mild cognitive impairment (MCI). Currently, the cerebrospinal fluid or PET-CT examinations, which are the gold standards for the diagnosis of 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, brain changes begin 10-15 years before the 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 onset process of AD. 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, which require invasive sampling, are costly, and are non-specific. Plasma Aβ and phosphorylated tau protein 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 the diagnosis of AD.

[0004] However, the concentrations of peripheral blood biomarkers are generally low and the variation range is relatively small, which requires high detection precision. At the same time, it is 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, which are efficiently conjugated with p-tau and Aβ antibodies for the detection of p-tau and Aβ biomarkers in blood, and a rapid portable fluorescence detection instrument is supported 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 human β-amyloid 1-42 detection card, its application, and a detection kit containing the same.

[0008] The human β-amyloid 1-42 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 Aβ1-42 in human blood. The human β-amyloid 1-42 detection kit provided by the present application can be used for self-testing fingertip blood at home. The result can be judged by colorimetry with the naked eye, or can be read through a supporting luminescence device. It can detect patients with early Alzheimer's disease, and provides 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 dual-modal nanoprobe, adopting the following technical solution:

[0010] A dual-modal nanoprobe, the dual-modal nanoprobe 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;

[0011] The average particle size of the dual-modal nanoprobe is 150-250 nm; the average particle size of the gold nanocore is 80-120 nm; the thickness of the inner silicon layer is 10-30 nm; the thickness of the outer silicon layer is 10-30 nm;

[0012] Among them, the upconversion nanocrystal layer is a core-shell structure, composed of 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 of rare earth luminescent nano-biomedical materials become a hot spot for new technological breakthroughs in the biomedical field. For example, by using the optical properties of rare earth nanoprobes 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] The present 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 pg / mL, and the performance reaches or exceeds the relevant detection products on the current market. Further, a highly sensitive and rapid detection kit is developed, which can expand the convenience of detection, deeply carry out home screening detection, can 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 an 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 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 Aβ1-42 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 average particle size of the dual-modal nanoprobe is 180 - 220 nm.

[0020] In a second aspect, this application provides a human β-amyloid 1-42 detection card, adopting the following technical solution:

[0021] A human β-amyloid 1-42 detection card, the detection card includes a card shell (1) and a test strip (2) arranged in the card shell (1);

[0022] 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 lapped and pasted on the backing (21) along its length direction;

[0023] The sample pad (22) is sprayed with a microsphere line; the microsphere line is the Aβ1-42 monoclonal antibody and goat anti-rabbit IgG antibody labeled with the above dual-modal nanoprobe microspheres.

[0024] Optionally, the contents of both the Aβ1-42 monoclonal antibody and goat anti-rabbit IgG antibody labeled with the dual-modal nanoprobe microspheres are 20 - 200 μg antibody / 200 μl fluorescent microspheres.

[0025] Optionally, the coating film (23) includes a nitrocellulose membrane, and a detection line and a quality control line which are sequentially arranged in parallel at intervals along the length direction thereof on the nitrocellulose membrane.

[0026] Optionally, the detection line and the quality control line are respectively coated with an Aβ1-42 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).

[0027] Optionally, the interval distance between the detection line and the quality control line is 2-4 mm.

[0028] Optionally, on the coating film (23), the coating concentration of the Aβ1-42 monoclonal antibody is 0.1-2 mg / ml, and the dosage is 0.5-1.5 μl coating solution amount / cm.

[0029] 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.

[0030] In a specific embodiment, a preparation method of a test strip (2) specifically includes the following steps:

[0031] (1) Synthesize a sodium yttrium fluoride core structure - an upconversion nanocrystal layer core structure

[0032] 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; then wash with a cyclohexane ethanol mixed solution and disperse in cyclohexane to obtain a NaYF4:Yb,Er nanoprobe cyclohexane solution.

[0033] Specifically, this step may 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 heat up to 100-120 °C, react for 20-30 min, then heat up 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; heat up to 90-100 °C, pump air and change gas 3-4 times, introduce nitrogen, heat up 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.

[0034] Among them, the molar ratio of nitrate or acetate or chloride of yttrium, ytterbium and erbium to NaOH and ammonium fluoride is 1:(1 - 2.5):(2 - 4).

[0035] (2) Preparation of upconversion nanocrystals

[0036] 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 nanosensor 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 pickling to obtain an ethanol solution containing upconversion nanocrystals.

[0037] 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, mix and stir at room temperature, evacuate, then heat up to 100 - 120 °C, react for 20 - 30 min, then heat up 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 nanosensor prepared in step (1), mix and stir, react for 20 - 30 min; heat up to 90 - 100 °C, evacuate and change the gas 3 - 4 times, introduce nitrogen, heat up 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.

[0038] 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 pickling to obtain an ethanol solution containing upconversion nanocrystals with a concentration of 20 mg / mL.

[0039] (3) Preparation of gold nanoparticles coated with an inner silicon layer

[0040] Synthesis of gold nanorods: In a container, under magnetic stirring, add chloroauric acid to cetyltrimethylammonium bromide (CTAB), then add sodium borohydride solution, and stir and let stand for 30 min.

[0041] 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.

[0042] After synthesis, centrifuge and wash, then disperse in ultrapure water. Take the gold nanorod solution, mix it with the mPEG-SH solution, and stir slowly at room temperature. Then centrifuge to remove the supernatant and resuspend in a mixed solution of ethanol and water. Under stirring conditions, add concentrated ammonia water and TEOS solution. After sealing and stirring, centrifuge three times and resuspend in ethanol solution to obtain gold nanoparticles coated with an inner silica layer.

[0043] (4) Prepare the nanosensor encapsulated with an upconversion nanocrystal layer

[0044] Disperse the gold nanoparticles coated with an inner silica layer obtained in step (3) in ethanol, add the ethanol solution containing upconversion nanocrystals obtained in step (2), stir for a sufficient reaction, centrifuge to remove the excess upconversion nanocrystals, then disperse the obtained material in ethanol, and successively add water, 10% tetraethoxysilane ethanol solution and ammonia water. After stirring, centrifuge and wash to obtain the nanosensor encapsulated with an upconversion nanocrystal layer.

[0045] (5) Prepare the dual-modal nanosensor

[0046] Mix and stir the nanosensor encapsulated with an upconversion nanocrystal layer obtained in step (4) and aminopropyltriethoxysilane, centrifuge and disperse in N,N-dimethylformamide (DMF) solvent to obtain a dispersion. Then add succinic anhydride DMF solution and stir, wash and disperse in water to obtain the dual-modal nanosensor.

[0047] (6) Activate the dual-modal nanosensor

[0048] Perform ultrasonic treatment and centrifugation on the dual-modal nanosensor obtained in step (5), wash the precipitate with MES solution; add carbodiimide and N-hydroxysulfosuccinimide, mix well and centrifuge at high speed, wash the precipitate with MES solution with a pH of 5.0 - 7.0 to obtain the activated dual-modal nanosensor.

[0049] (7) Prepare the Aβ1-42 monoclonal antibody labeled with the dual-modal nanosensor

[0050] Perform ultrasonic treatment on the activated dual-modal nanosensor obtained in step (6), then add Aβ1-42 monoclonal antibody and rabbit IgG antibody, mix well, after using the blocking solution, centrifuge at high speed, wash twice gently with PBS storage solution, and perform ultrasonic treatment and resuspend for storage in the dark at 4℃.

[0051] (8) Prepare the sample pad 22

[0052] 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. Dilute the Aβ1-42 monoclonal antibody labeled with the dual-modal nanosensor 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. Place it in an oven and dry at 37℃ overnight.

[0053] (9) Prepare the coating film 23

[0054] Adjust the concentrations of the anti-Aβ1-42 monoclonal antibody and the goat anti-rabbit IgG antibody to 0.5-2 mg / ml with the coating buffer respectively, and the dosage is 0.5-1.5 μl of coating solution per cm 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 it for standby.

[0055] (10) Prepare the test strip 2

[0056] Paste the sample pad 22, the coating film 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 obtain the test strip 2.

[0057] In a third aspect, the present application also provides a kit for detecting human β-amyloid 1-42, adopting the following technical solution:

[0058] A kit for detecting human β-amyloid 1-42, the detection kit includes the above-mentioned detection card for human β-amyloid 1-42.

[0059] Optionally, the detection kit further includes an ID card containing a calibration curve.

[0060] Optionally, the ID card containing the calibration curve is obtained by measuring calibration products with gradient concentrations by 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.

[0061] The corresponding two-dimensional code information on the detection card for human β-amyloid 1-42 can be read by a dry fluorescence immunoassay analyzer and the corresponding concentration can be measured.

[0062] In a fourth aspect, the present application also provides an application of the above-mentioned detection card for human β-amyloid 1-42 or the above-mentioned kit for detecting human β-amyloid 1-42 in the preparation of a composition for Alzheimer's disease detection.

[0063] In a specific embodiment, a method for quantitatively detecting Aβ1-42 by a kit for detecting human β-amyloid 1-42 specifically includes the following steps:

[0064] (1) Place the detection kit and the sample at room temperature and use them after returning to room temperature;

[0065] (2) Turn on the rare earth nanometer fluorescence immunoassay analyzer, insert the corresponding ID card after preheating for 5 min;

[0066] (3) Wipe the fingertip with an alcohol swab, aspirate 10 μL of blood with a capillary tube and add it to the sample loading well of the test card, and then add two drops of diluent.

[0067] (4) Insert the test card into the test slot, and after 10 min, detect and read and print the test result.

[0068] The detection principle of the above-mentioned human β-amyloid 1-42 detection kit is the double antibody sandwich method, which is used to detect the content of Aβ1-42 in human serum, plasma and whole blood samples. The diluent of the blood sample containing Aβ1-42 is dropped on the sample loading area, and through capillary action, it chromatographs to the sample pad and binds to the Aβ1-42 antibody labeled with a dual-modal nanoprobe to form a microsphere antibody-antigen complex, which chromatographs to the detection area on the nitrocellulose membrane. The complex is captured by the Aβ1-42 antibody coated on the test line, and 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.

[0069] The detection area is scanned and detected with a light source (980 nm), and the nanoprobe on the test line and the quality control line emits fluorescence (540 nm). Because 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 Aβ1-42; when detecting a negative sample, there is no red band on the test line (T line). Whether or not Aβ1-42 exists in the sample, a red band will appear on the quality control line (C line).

[0070] In summary, the present application includes at least one of the following beneficial technical effects:

[0071] 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 / fluorescent dual-modal detection.

[0072] 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.

[0073] 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, and both of these aspects act together to enhance the fluorescence intensity of the fluorophore.

[0074] The human β-amyloid 1-42 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 Aβ1-42 in human blood.

[0075] The human β-amyloid 1-42 detection kit provided by this application can be used for self-testing fingertip blood at home. The result can be judged by colorimetry with the naked eye or read through the supporting luminescence device. It can detect patients with early Alzheimer's disease and provide an effective technical means for the early diagnosis and disease monitoring of clinical Alzheimer's disease. Description of the Drawings

[0076] Figure 1 It is a schematic structural diagram of the human β-amyloid 1-42 detection card provided by this application.

[0077] Figure 2 It is a schematic structural diagram of the test strip of the human β-amyloid 1-42 detection card provided by this application.

[0078] Figure 3 It is a transmission electron microscope image of the dual-modal nanoprobe provided by this application.

[0079] Figure 4 It is a standard curve graph drawn with the Aβ1-42 quality control product concentration and the average T / C value of the sample signal of the human β-amyloid 1-42 detection kit provided by this application.

[0080] Figure 5 It is a comparison curve graph of the detection results of the human β-amyloid 1-42 detection kit provided by this application and the Novizan chemiluminescence method for detecting Aβ1-42 kit for the same sample.

[0081] Reference Signs: 1. Card case; 11. Sampling hole; 12. Observation window; 2. Test strip; 21. Backing; 22. Sample pad; 23. Coated membrane; 24. Absorbent paper. Detailed Embodiments

[0082] 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.

[0083] 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.

[0084] 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.

[0085] 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.

[0086] To make the purpose, 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 belong to 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.

[0087] For those not specified in the embodiments in terms of specific technologies or conditions, they shall be carried out according to the technologies or conditions described in the literature in this field or according to the product instructions. For reagents or instruments not specified by the manufacturer, they are all conventional products that can be obtained through commercial purchase.

[0088] The following further describes this application in detail in combination with embodiments and test results.

[0089] Embodiment

[0090] Embodiment 1

[0091] This embodiment provides a kit for detecting human β-amyloid 1-42. The kit includes a detection card for human β-amyloid 1-42 and an ID card containing a calibration curve.

[0092] As Figure 1 and Figure 2 shown, the detection card for human β-amyloid 1-42 includes a card shell 1 and a detection 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 detection test strip 2 is fixed on the plastic lower shell, and the surface of the detection test strip 2 is pressed by the plastic upper shell.

[0093] The detection test strip 2 includes a bottom liner 21 and a sample pad 22, a coating film 23 and a blotting paper 24 that are sequentially lapped and pasted on the bottom liner 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.

[0094] Among them, a microsphere line is sprayed on the sample pad 22. The microsphere line is a monoclonal antibody of Aβ1-42 and a goat anti-rabbit IgG antibody labeled with a bimodal nanoprobe (both with a content of 100 μg antibody / 200 μl fluorescent microspheres).

[0095] 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.

[0096] Among them, the upconversion nanocrystal layer is 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. As Figure 3 shown (100 nm in the figure is the scale mark), the average particle size of the bimodal nanoprobe is 200 nm. From Figure 3 this, it can be 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.

[0097] 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 monoclonal antibody of Aβ1-42 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 monoclonal antibody of Aβ1-42 (purchased from Nanjing Zhonghongtai Biotechnology Co., Ltd.) is 1 mg / ml, and the dosage is 1 μl coating solution amount / cm membrane. The coating concentration of the goat anti-rabbit IgG antibody (purchased from Luoyang Bai'aotong Experimental Materials Center) is 1 mg / ml, and the dosage is 1 μl coating solution amount / cm membrane.

[0098] The ID card containing the calibration curve measures 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, a standard curve is drawn, written, and the corresponding two-dimensional code information is generated and stored in the ID card. The corresponding two-dimensional code information on the rare earth nano-fluorescence detection card can be read by a dry fluorescence immunoassay analyzer, and the corresponding concentration can be measured. The standard curves of the ID cards containing the calibration curve in the same batch of kits are the same.

[0099] Example 2

[0100] This example provides the test strip 2 mentioned in Example 1.

[0101] The preparation method of the above test strip 2 specifically includes the following steps:

[0102] (1) Synthesize the sodium yttrium fluoride core structure - the core structure of the upconversion nanocrystal layer

[0103] 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 up to 120 °C, react for 20 min, then heat up to 140 °C, react for 15 min to obtain a transparent solution; naturally cool to 50 °C, release the vacuum, add a NaOH and ammonium fluoride methanol mixed solution, and react for 30 min; heat up to 100 °C, evacuate and replace the gas 3 - 4 times, introduce nitrogen, heat up to 300 °C, react for 1.5 h, centrifuge at 8000 rpm, then wash 3 - 4 times with a cyclohexane ethanol mixed solution, and disperse in cyclohexane to obtain a NaYF4:Yb,Er nanosensor cyclohexane solution.

[0104] Among them, the molar ratio of the nitrates or acetates or chlorides of yttrium, ytterbium, and erbium, as well as NaOH and ammonium fluoride, is 1:2.5:4.

[0105] (2) Prepare the upconversion nanocrystals

[0106] 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 up to 120 °C, react for 20 min, then heat up to 160 °C, react for 10 min to obtain a transparent solution; naturally cool to 50 °C, release the vacuum, add a NaOH and ammonium fluoride methanol mixed solution, and the NaYF4:Yb,Er nanosensor cyclohexane solution prepared in step (1), mix and stir, and react for 30 min; heat up to 100 °C, evacuate and replace the gas 3 - 4 times, introduce nitrogen, heat up to 300 °C, react for 1.5 h, centrifuge at 8000 rpm, then wash 3 - 4 times with a cyclohexane ethanol mixed solution, and disperse in cyclohexane.

[0107] Among them, the molar ratio of yttrium acetate, NaOH, and ammonium fluoride is 1:2.5:4; then transfer the upconversion nanocrystals to ethanol by pickling to obtain an ethanol solution containing upconversion nanocrystals with a concentration of 20 mg / mL.

[0108] (3) Prepare the silica-coated gold nanoparticles

[0109] 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. Subsequently, 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.

[0110] 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. The reaction mixture was left stationary at 30 °C for 12 h to complete the synthesis of gold nanorods.

[0111] After synthesis, it was centrifuged at 9000 rpm for 15 min, centrifuged and washed twice, and then dispersed in 20 mL of ultrapure water. 5 ml of the gold rod solution was taken and mixed with 250 μL of 1 mg / ml mPEG-SH solution, and slowly stirred 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.

[0112] (4) Preparation of nanoprobes encapsulated with upconversion nanocrystals

[0113] 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, and the volume ratio of the two was 10:1. After stirring for 8 h to fully react, after centrifuging to remove the excess 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 nanocrystals. Among them, the volume ratio of ethanol, water, 10% tetraethoxysilane ethanol solution, and ammonia water is 10:1:0.05:0.2.

[0114] (5) Preparation of dual-modal nanoprobes

[0115] 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 the dual-modal nanoprobes. Among them, the volume ratio of the dispersion to the succinic anhydride DMF solution was 1:0.05.

[0116] (6) Activate the dual-modal nanoprobes

[0117] The dual-modal nanoprobes obtained in step (5) were subjected to ultrasonic treatment and centrifugation. 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 dual-modal nanoprobes were obtained.

[0118] (7) Prepare the Aβ1-42 monoclonal antibody labeled with dual-modal nanoprobes

[0119] The dual-modal nanoprobes activated in step (6) were ultrasonically treated for 2 min and then added with the Aβ1-42 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.

[0120] (8) Prepare the sample pad 22

[0121] The 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) was sprayed evenly in two parallel lines on one side of the substrate 21 close to the sample pad 22, and the dosage was 4 μl liquid volume / cm of the sample pad 22. On the side of the sample pad 22 close to the coating film 23, the Aβ1-42 monoclonal antibody labeled with dual-modal 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 sprayed evenly in one line, and the dosage was 4 μl liquid volume / cm of the sample pad. It was placed in an oven and dried at 37 °C overnight.

[0122] (9) Prepare the coating film 23

[0123] Adjust the concentrations of the Aβ1-42 monoclonal antibody and the goat anti-rabbit IgG antibody to 1 mg / ml with the coating buffer (PBS buffer at 20 mM, pH 7.4 containing 2.5% (w / w) sucrose), and use 1 μl of the coating solution per cm² of the membrane. Coat them in parallel 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 4 mm. Dry them in an oven at a humidity <30% and a temperature of 37°C for 10 h, then seal them in bags for later use.

[0124] (10) Prepare the test strip 2

[0125] Sequentially and overlappingly paste 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) on the backing 21 (sized 80 * 300 mm) to obtain the test strip board, and cut it into test strips 2 with a width of 4 mm as required.

[0126] Example 3

[0127] This example provides a method for quantitatively detecting Aβ1-42 using the detection kit provided in Example 1.

[0128] The method for quantitatively detecting Aβ1-42 using the above detection kit specifically includes the following steps:

[0129] (1) Place the detection kit and the sample at room temperature and use them after returning to room temperature.

[0130] (2) Turn on the rare earth nano-fluorescence immunoassay analyzer, insert the corresponding ID card after preheating for 5 min.

[0131] (3) Wipe the fingertip with an alcohol cotton swab, draw 10 μL of blood with a capillary and add it to the sample addition hole of the test card, and then add two drops of the diluent.

[0132] (4) Insert the test card into the detection slot, detect and read and print the test results after 10 min.

[0133] In this example, the detection principle of the Aβ1-42 detection kit is the double antibody sandwich method, which is used to detect the content of Aβ1-42 in human serum, plasma and whole blood samples. The diluted blood sample containing Aβ1-42 is dropped on the sample addition area, and through capillary action, it chromatographs to the sample pad, binds to the Aβ1-42 antibody labeled with the dual-modal nanoprobe to form a microsphere antibody-antigen complex, and chromatographs to the detection area on the nitrocellulose membrane. The complex is captured by the Aβ1-42 antibody coated on the test line, and 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.

[0134] The detection area is scanned and detected with a light source (980 nm). The nano-probes on the test line and the quality control line emit fluorescence (540 nm). 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 blood samples containing Aβ1-42; when detecting negative samples, there is no red band on the test line (T line). Whether or not Aβ1-42 is present in the sample, a red band will appear on the quality control line (C line).

[0135] The process of drawing the standard curve of the human β-amyloid 1-42 detection kit in this example is as follows: Different concentrations of Aβ1-42 antigen quality control products (each concentration is set with three replicates, all diluted from Aβ1-42 antigen with 20% calf serum) are added to the prepared human β-amyloid 1-42 detection cards. After 10 minutes of sample addition and chromatography, the fluorescence signals of the C and T lines and the C / T value are read by a rare earth nano-fluorescence immunoassay analyzer with excitation light (980 nm) / emission light (540 nm).

[0136] The analysis results are shown in Table 1.

[0137] Table 1 Analysis results of the standard curve

[0138]

[0139]

[0140] A standard curve is drawn with the concentration of the Aβ1-42 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 Aβ1-42 R value is 0.9996, and the concentration of Aβ1-42 contained in the sample is quantitatively determined through this calibration curve.

[0141] Example 4

[0142] The performance of the test strip in the kit provided by this application is detected. The specific contents include the following:

[0143] (1) Minimum detection limit

[0144] The zero-value sample is repeatedly measured 20 times, and the mean M and standard deviation SD of the 20 results are calculated. The detection limit of the method is reported as the blank mean plus twice the standard deviation (M + 2SD). The Aβ1-42 detection results are 0.775 pg / ml respectively, meeting the sensitivity standard of 1 pg / ml respectively.

[0145] (2) Linear range

[0146] Seven concentration values between 1 - 500 pg / ml of Aβ1-42 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 Aβ1-42.

[0147] The obtained linear equation was: y = 0.9177x + 3.2184, r = 0.9996. It shows that the human β-amyloid 1-42 detection kit provided by this application has a very good correlation within the linear range.

[0148] (3) Precision

[0149] Three batches of the kits of this example were taken, and the within-batch CVs of three batches of repeated quality control products were detected respectively. Each batch of kits was used to detect the repeated quality control products in parallel 10 times.

[0150] The obtained within-batch CVs of Aβ1-42 at 25 pg / ml for three batches were 6.03%, 5.91%, and 6.11% respectively, and the between-batch CV was 6.55%. The within-batch CVs of Aβ1-42 at 100 pg / ml for three batches were 5.36%, 4.82%, and 8.25% respectively, and the between-batch CV was 6.35%. All were within 10%.

[0151] Example 5

[0152] In this example, the kit provided in Example 1 was used to detect clinical samples.

[0153] The detection process and results are as follows:

[0154] 100 blood samples for detecting Aβ1-42 were collected from the hospital, and the kit of this application and the Ipunocare chemiluminescence method for detecting Aβ1-42 kit were used for detection and comparison respectively.

[0155] In the kit of this application, 10 μl of blood sample was added to the sample addition hole of the test strip, 2 drops of diluent were added, and after chromatography for 10 min, the concentration was read by a rare earth nanophosphorescence immunoassay analyzer. The same sample was detected for concentration by the comparative system Ipunocare chemiluminescence method for detecting Aβ1-42 kit. Linear analysis was performed on the detection results. As Figure 5 shown, its correlation is very good, Aβ1-42 r = 0.9878, 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.

[0156] In the description of this specification, the descriptions referring to terms such as "one embodiment", "some embodiments", "examples", "specific examples", or "some examples" mean 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 this 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 may 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.

[0157] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of this application, rather than to limit them; although this 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 cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of this application.

Claims

1. A dual-mode nanoprobe, 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 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.

2. The dual-modal nanoprobe according to claim 1, characterized in that: The dual-mode nanoprobe is an up-conversion enhanced luminescence colorimetric nanoprobe.

3. The dual-modal nanoprobe according to claim 1, characterized in that: The dual-mode nanoprobe has a fluorescence spectrum emission peak within the range of 520-550 nm under 980 nm excitation light.

4. The dual-modal nanoprobe according to claim 1, characterized in that: The average particle size of the dual-mode nanoprobe is 180-220 nm.

5. A human amyloid β-protein 1-42 detection card, characterized in that: The detection card comprises a card housing (1) and a detection test strip (2) arranged in the card housing (1); The test paper strip (2) comprises a backing (21) and a sample pad (22), a coating film (23) and absorbent paper (24) which are sequentially overlapped and pasted on the backing (21) along its length direction; The sample pad (22) is sprayed with a microsphere line; the microsphere line is the dual-modal nanoprobe microsphere labeled with Aβ 1-42 monoclonal antibody and goat anti-rabbit IgG antibody as described in claim 1.

6. The human amyloid β-protein 1-42 detection card according to claim 5, characterized in that: The content of Aβ 1-42 monoclonal antibody and goat anti-rabbit IgG antibody labeled by the dual-modal nanoprobe microspheres is 20-200 μg antibody / 200 μl fluorescent microspheres; 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 Aβ 1-42 monoclonal antibody 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.

7. The human amyloid β-protein 1-42 detection card according to claim 5, characterized in that: On the coating membrane (23), the coating concentration of the Aβ 1-42 monoclonal antibody is 0.1-2 mg / ml, and the amount used is 0.5-1.5 µl coating liquid volume / 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 human amyloid β-protein 1-42 detection kit, characterized in that: The detection kit comprises the human amyloid β-protein 1-42 detection card according to any one of claims 5 to 7.

9. The human amyloid β-protein 1-42 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 human β-amyloid protein 1-42 detection card according to any one of claims 5 to 7 or the human β-amyloid protein 1-42 detection kit according to any one of claims 8 to 9 in preparing a composition for detecting Alzheimer's disease.

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