Three-dimensional homogeneous filling type magnetic-noble metal composite nanoscale enzyme, novel crown antigen immunochromatography test paper and application thereof

Abstract

The application discloses a three-dimensional homogeneous filling type magnetic-noble metal composite nano-enzyme, a new crown antigen immunochromatography test paper thereof and application, the magnetic-noble metal composite nano-enzyme is with dendritic mesoporous silica dSiO2 as carrier, the carrier is modified and grafted polyethylene imine PEI as intermediate medium layer, the coordination of the amino group of PEI on the dSiO2 carrier is used to load magnetic nanoparticles Fe3O4 and nanoparticles Pt in turn, and finally the dendritic mesoporous dSiO2 / Fe3O4 / Pt-PEI composite structure obtained by carboxylation modification.The magnetic-noble metal composite nano-enzyme of the application has SiO2 / Fe3O4 / Pt-polyethylene imine PEI composite structure, the multi-layer porous structure has good permeability from inside to outside, ensures the efficient contact of Fe3O4 and Pt with catalytic color developing substrate, and the structure has magnetism, can be repeatedly and efficiently used, is used in test paper strip immunochromatography, and through enzyme catalysis signal amplification effect, novel coronavirus S protein antigen is rapidly and high-sensitivity detected.

Description

TECHNICAL FIELD

[0001] The present application relates to a three-dimensional homogeneous filling type magnetic-noble metal composite nanoscale enzyme, a new crown antigen immunochromatography test paper and application thereof. BACKGROUND

[0002] At present, the diagnosis methods of novel coronavirus can be divided into two categories. The first category is molecular testing to detect viral RNA sequences through nucleic acid amplification testing, such as the most commonly used real-time reverse transcription polymerase chain reaction (RT-PCR), but it requires long processing time, special experimental equipment and well-trained technicians, and is not suitable for on-site rapid diagnosis of patients. The second category is immunological detection method, including antibody detection and antigen detection. This method is simple to operate and can be completed in a short time, but the early detection rate of novel coronavirus antibody in the body is low, while the antigen can be detected before the appearance of clinical symptoms and can be used for the diagnosis of early infection. Because of the excellent immunogenicity and specificity of the S protein of novel coronavirus, it is considered to be an ideal detection target antigen of SARS-cOV-2. The World Health Organization pointed out that in order to effectively diagnose SARS-CoV-2 infection, the antigen diagnostic method should have at least 80% sensitivity and 97% specificity. And lateral flow immunochromatography technology (LFIA) as a popular POCT diagnostic platform has played an important role in controlling the COVID-19 pandemic in industrialized countries and resource-limited environments.

[0003] At present, the lateral flow immunochromatography technology for detecting SARS-CoV-2 antigen in commercial detection mainly uses colloidal gold and fluorescent materials as markers for qualitative or semi-quantitative detection. Colloidal gold nanoparticles have the characteristics of naked eye visualization and good biocompatibility. Colloidal gold chromatography test paper is rapid, simple, does not require the use of complex laboratory equipment and professional training personnel, and has a wide range of applications. However, the existing colloidal gold immunochromatography method has low sensitivity and is easy to cause missed diagnosis.

[0004] While fluorescent materials have a higher detection signal-to-noise ratio, compared with colloidal gold-based LFIA, they improve the detection performance and to some extent solve the problem of low sensitivity. For example, Guo et al. invented a lateral flow immunochromatography with mesoporous silica-supported upconversion fluorescent nanomaterials to detect SARS-CoV-2 S and N proteins, with detection limits (LOD) of 1.6 ng / mL and 2.2 ng / mL, respectively. Zhao, Xiao et al. developed a colorimetric and fluorescent dual-functional lateral flow immunoassay biosensor for rapid and sensitive detection of SARS-CoV-2 S protein, with detection limits of 1 and 0.033 ng / mL for S1 protein using the colorimetric and fluorescent functions of the biosensor, respectively. However, LFIA based on fluorescent markers requires an additional excitation light source to observe the signal and complex quantitative detection instruments to realize signal readout, with problems such as high detection cost, limited application scenarios, etc. At the same time, the degree of signal amplification with fluorescent probes is limited, and the problem of false negatives in colloidal gold test strips still cannot be effectively solved. Therefore, it is still necessary to explore efficient signal amplification principles and convenient, de-instrumented immunochromatographic modes to achieve high sensitivity and visual interpretation, in order to meet the current real-time diagnostic application needs of new crown detection, etc.

[0005] Enzyme-linked immunosorbent assay (ELISA) is one of the most common serological immunoassay methods based on the catalytic color reaction of enzyme-labeled antibody. It has the characteristics of strong specificity, high sensitivity, simple operation, etc. It uses the catalytic color reaction of horseradish peroxidase (HRP) and color substrate to achieve signal amplification effect, thereby improving sensitivity. However, the preparation and detection processes of ELISA kit are complex and lengthy, and large laboratory instruments are required for detection, which is not suitable for real-time detection in the field. Lateral flow immunoassay (LFIA) can provide a low-cost visual transmission and instant diagnosis strategy. If the catalytic color reaction of enzyme activity label and color substrate (e.g., 3,3',5,5'-tetramethylbenzidine (TMB)) is used in LFIA, signal amplification effect can be achieved, which is expected to become an effective tool for SARS-CoV-2 detection, helping to detect in the field, providing information for regional disease control, and preventing further spread of the virus. However, the number of HRP molecules that can be labeled on the antibody is very limited, and if HRP molecules are directly used as labels, the amplification effect of enzyme catalysis in a single immune reaction will be limited. At the same time, natural enzymes are fragile biological molecules that will lose catalytic activity due to denaturation even under normal detection conditions, so they are not suitable for POC applications. Nanoenzymes based on inorganic nanomaterials are an attractive alternative because they exhibit enzyme-like catalytic activity without denaturation and can be stored and used in a wide range of pH and temperature. Therefore, the development of nanoenzymes with various catalytic properties inherent to enzymes, while allowing fine structural regulation, has attracted great interest from researchers.

[0006] So far, a variety of nano-materials with enzyme-like activity have been found, such as noble metal nanoparticles and metal oxides, and have been applied to immunodiagnosis. Among them, platinum (Pt) nanoparticles and ferroferric oxide (Fe3O4) nanoparticles have excellent catalytic activity, strong light absorption color development characteristics and excellent biocompatibility, which are suitable for constructing visual color development probes. At the same time, Fe3O4 has good liquid phase suspension and magnetic separation performance, which can be used for homogeneous immunoassay and antigen enrichment and concentration, providing an effective way for high-sensitivity detection of antigens. Liu et al. prepared PS-Pt nanoscale enzyme by growing platinum nanoparticles (Pt) on the surface of carboxyl functionalized polymer nanospheres (PS), and established a simple and sensitive colorimetric method for detecting Salmonella typhimurium; Doh et al. integrated the mimetic enzyme Fe3O4-Pt core-shell nanoparticles into LFIA, and developed a simple and sensitive biological detection system, which achieved higher sensitivity than traditional LFIA. Yan et al. optimized the activity of Fe3O4 nanoscale enzyme, providing a new theoretical basis for the design and efficiency improvement of enzyme-like activity nanomaterial catalysts. Compared with single functional component nanoscale enzyme, composite component nanoscale enzyme such as mixed metal nanostructure and metal-oxide composite structure can effectively improve the catalytic reaction rate of the catalytic active center due to the synergistic effect between functional units, and is an excellent material for constructing enzyme catalytic signal amplification probes. Wang et al. developed a LFIA test strip for detecting gastrin-17 based on noble metal-metal oxide probes, which effectively improved the detection sensitivity. Yan et al. introduced in the book "Nanozymology Connecting Biology and Nanotechnology" that the modification of biological enzymes or the replacement of part of the enzyme with nanomaterials, and the final combination shows stronger catalytic performance than single functional component in many cases. However, the enzyme-like activity metal particles themselves are easy to condense or fuse, and the surface cannot be effectively protected, resulting in insufficient stability and not achieving effective dispersion state in the reaction process. From the existing research, how to homogeneously and closely fill single catalytic functional units in three-dimensional space to achieve performance enhancement, and how to integrate different types of enzyme catalytic functional units in colloidal scale to achieve synergistic catalytic effect, and finally obtain high-performance color development probes with colloidal and liquid dispersion, are the key problems to be solved in the field of nanoscale enzyme catalysts and biological marker detection. SUMMARY

[0007] In view of the problems of low sensitivity of existing commercial detection SARS-CoV-2 antigen lateral flow immunochromatography technology (LFIA), complex and long process of enzyme-labeled immunochromatography technology, and unsuitability for real-time detection, the purpose of the present application is to provide a three-dimensional homogeneous filling type magnetic-precious metal composite nano-enzyme for visual detection of novel coronavirus antigen, a novel coronavirus antigen immunochromatography test paper thereof and application. The magnetic-precious metal composite nano-enzyme provided by the present application utilizes the superposition and synergistic effect of the absorbance of Fe3O4 and Pt to realize a direct naked-eye interpretation mode; utilizes the synergistic catalytic effect between Fe3O4 and Pt to realize high-efficiency enzyme catalytic activity and signal amplification strategy, thereby improving sensitivity; and utilizes the magnetic separation and enrichment function to realize homogeneous immunoreaction and enrichment and concentration of antigens, thereby improving the detection performance of antigens.

[0008] In order to improve the immunodetection effect, how to effectively assemble single nanometer units while controllably integrating different enzyme catalytic functional units in colloidal scale is a difficulty in the construction of nano-enzyme composite materials. Dendritic mesoporous silica is an ideal nanometer unit carrier, which has high pore volume, adjustable mesopore-macropore pore size, and especially highly open pore structure, and can effectively load nanometer units. Therefore, the present application uses dendritic mesoporous silica (dSiO2) as a carrier, utilizes high-density filling and layered assembly of the carrier pores to different functional units, and prepares an enzyme catalytic color developing immunolabeling probe with high loading and functional synergy. The dSiO2 carrier can provide support and protection, strictly limit the size and uniformity of the material, and the prepared catalyst has highly open pores and numerous accessible enzyme catalytic active sites, and still has typical central-radial pore structure and enzyme catalytic sites after loading multiple layers of nanoparticles, so as to enhance visual chroma, improve sensitivity and expand the detection range.

[0009] The present application uses dendritic mesoporous silica as a carrier to provide larger specific surface area, pore size and pore volume, and uses polyethyleneimine (PEI) as an intermediate medium layer to realize deposition of magnetic nanoparticles and platinum nanoparticles, realize high-density filling, and realize integration and synergistic effect of iron oxide and platinum nanoparticles, thereby improving enzyme catalysis and color developing activity. The dendritic mesoporous SiO2 / Fe3O4 / Pt-polyethyleneimine PEI composite structure is prepared, the multi-layered porous structure has good permeability from inside to outside, ensures efficient contact of Fe3O4 and Pt with catalytic color developing substrates, and simultaneously, the structure has superparamagnetism, can perform liquid suspension reaction and magnetic separation and enrichment, and is used in immunochromatography of test paper strips, realizes rapid and high-sensitivity detection of novel coronavirus S protein antigen through enzyme catalytic signal amplification effect.

[0010] The magnetic-noble metal composite nanoscale enzyme for visual detection of SARS-CoV-2 antigen is characterized in that it is a dendritic mesoporous silica dSiO2 carrier, which is modified with polyethyleneimine PEI as an intermediate medium layer, and magnetic nanoparticles Fe3O4 and platinum nanoparticles are sequentially loaded on the PEI amino group of the dSiO2 carrier, and finally a dendritic mesoporous dSiO2 / Fe3O4 / Pt-PEI composite structure is obtained through carboxyl modification.

[0011] The magnetic-noble metal composite nanoscale enzyme for visual detection of SARS-CoV-2 antigen is characterized in that the preparation method of the dendritic mesoporous silica dSiO2 is as follows: cetyltrimethylammonium bromide CTAB is used as a template, sodium salicylate is used as a structure directing agent, triethanolamine is used as a catalyst, water is used as a solvent, and the mixture is stirred at 70-90°C for 0.5-2h, then tetraethyl orthosilicate is added, and the stirring is continued for 2-5h to obtain the dendritic mesoporous silica template with an ultra-large pore size; wherein the mass ratio of cetyltrimethylammonium bromide, sodium salicylate and triethanolamine is 1:0.4-0.6:0.1-0.3, preferably 1:0.55-0.6:0.16-0.2; the mass ratio of cetyltrimethylammonium bromide to tetraethyl orthosilicate is (0.08-0.1)g:1mL.

[0012] The magnetic-noble metal composite nanoscale enzyme for visual detection of SARS-CoV-2 antigen is characterized in that the specific preparation method of the magnetic-noble metal composite nanoscale enzyme comprises the following steps:

[0013] 1) Preparation of dendritic mesoporous silica dSi / IO-PEI composite microspheres loaded with magnetic magnetite

[0014] The dendritic mesoporous silica dSiO2 is dispersed in anhydrous ethanol, iron salt and polyethyleneimine PEI are added, and triethylene glycol is ultrasonically homogenized; under an argon atmosphere, vigorous stirring is carried out at 200-220°C for 1.5-2.5h, and then the temperature is continuously increased to 280-290°C, and stirring is carried out for 0.5-1.5h, and the product is obtained by magnetic separation after cooling to room temperature, and washed with ethanol several times to obtain dendritic mesoporous silica composite microspheres loaded with magnetic magnetite, which is marked as dSi / IO-PEI;

[0015] 2) Preparation of dendritic mesoporous silica microspheres / magnetite / platinum nanomicrospheres dSi / IO / Pt-PEI

[0016] The dSi / IO-PEI microspheres obtained in step 1) are added to ultrapure water, mixed uniformly, then platinum salt and polyvinylpyrrolidone PVP are added and ultrasonically mixed uniformly, and then stirred in an ice water bath for 5-20 min, so that the platinum salt is loaded on the dSi / IO-PEI microspheres; then sodium borohydride solution is added and stirred for 1-3 h, so that the platinum salt loaded on the dSi / IO-PEI microspheres is reduced to platinum nanoparticles, and the product is obtained by magnetic separation after the reaction is completed, and the product is washed with ultrapure water several times to obtain dendritic mesoporous silica microspheres / ferroferric oxide / platinum nanomicrospheres, which are marked as dSi / IO / Pt-PEI;

[0017] 3) Preparation of carboxylated dSi / IO / Pt-PEI microspheres

[0018] The dSi / IO / Pt-PEI microspheres obtained in step 2) are dispersed in anhydrous DMF, then succinic anhydride is added and stirred at room temperature for 16-20 h, and the product is obtained by magnetic separation after the reaction is completed, and the product is washed with ultrapure water several times to obtain dendritic mesoporous dSiO2 / Fe3O4 / Pt-PEI composite microspheres modified by carboxylation, which are marked as dSi / IO / Pt-PEI-COOH.

[0019] The magnetic-noble metal composite nanoscale enzyme for visual detection of SARS-CoV-2 antigen by enzyme amplification, characterized in that in step 1), the mass ratio of dendritic mesoporous silica dSiO2, iron salt and polyethyleneimine PEI is 60-70: 350-380: 35-45, and the iron salt is triacetylacetone iron.

[0020] The magnetic-noble metal composite nanoscale enzyme for visual detection of SARS-CoV-2 antigen by enzyme amplification, characterized in that in step 1), the mass ratio of dSiO2 to platinum salt and polyvinylpyrrolidone PVP in step 2) is 60-70: 20-22: 45-55, and the platinum salt is potassium tetrachloroplatinate; the mass ratio of platinum salt to sodium borohydride is 1.4-1.5:1.

[0021] The magnetic-noble metal composite nanoscale enzyme for visual detection of SARS-CoV-2 antigen by enzyme amplification, characterized in that in step 1), the mass ratio of dSiO2 to succinic anhydride in step 3) is 40-42:50.

[0022] A test strip for rapid detection and analysis of SARS-CoV-2 S protein antigen by immunochromatography, comprising the following assembly steps:

[0023] 1) Preparation of dSi / IOI / Pt-PEI-COOH signal probe: The magnetic-noble metal composite nanozyme was dispersed in PB buffer, EDC and Sulfo-NHS were added and the mixture was stirred for 20-40 min to activate the carboxyl groups on the surface of the magnetic-noble metal composite nanozyme in the buffer. The activated magnetic-noble metal composite nanozyme was centrifuged to remove the supernatant and dispersed in PB buffer. Then, a monoclonal antibody for detecting the COVID-19 S protein was added and reacted at room temperature for 2-3 h. Then, BSA was added to block the reaction for 1-3 h. After the reaction, the product was washed several times with PB buffer to obtain the dSi / IOI / Pt-PEI-COOH signal probe. The probe was then prepared in PBS buffer containing 0.05-0.2% BSA at a concentration of 3-5 mg / mL to obtain the signal probe solution.

[0024] 2) Preparation of the test strips includes the following processes:

[0025] S1: Before use, both the sample pad and the conjugate pad should be soaked in a treatment solution containing 0.5% to 2% BSA. After being fully soaked, they should be removed and dried to complete the pretreatment.

[0026] S2: After spraying the signal probe solution onto the conjugate pad, dry it.

[0027] S3: The T line of the NC membrane is coated with monoclonal capture antibody against the SARS-CoV-2 S protein, and the C line of the NC membrane is coated with goat anti-mouse antibody IgG. The membrane is then dried.

[0028] S4: Overlap the conjugation pad onto the end of the NC membrane near the T line, overlap the absorption pad onto the end of the NC membrane near the C line, and then overlap the sample pad onto the end of the conjugation pad away from the NC membrane. Cut to form an immunochromatographic test strip.

[0029] The application of the test strip in the rapid immunochromatographic detection and analysis of the COVID-19 S protein antigen is characterized by the following steps in the application method:

[0030] 1) standard curve drawing: a series of different concentrations of solutions containing new coronavirus S protein antigens are added dropwise to the sample pad of the immunochromatography test strip, the solution is gradually chromatographed to the NC membrane of the immunochromatography test strip under capillary action, after 15-30 min, the color developing solution containing 3,3',5,5'-tetramethylbenzidine TMB and hydrogen peroxide H2O2 is added dropwise on the NC membrane, after 1-3 min, the signal amplification effect is achieved through the catalytic reaction of the signal probe and TMB, then whether the new coronavirus S protein antigen exists is determined by naked eye, the photo of the T line on the NC membrane is taken, the blue signal in the T line area is displayed, the T line gray value is read by color recognition software to obtain the blue signal intensity gray value of the T line area, the blue signal gray value is taken as the vertical coordinate, and the concentration of the new coronavirus S protein antigen is taken as the horizontal coordinate to draw a standard curve, and a linear regression equation is calculated;

[0031] 2) actual sample detection: the solution containing the sample is added dropwise to the sample pad of the immunochromatography test strip, the solution is gradually chromatographed to the NC membrane of the immunochromatography test strip under capillary action, after 15-30 min, the color developing solution containing 3,3',5,5'-tetramethylbenzidine TMB and hydrogen peroxide H2O2 is added dropwise on the NC membrane, after 1-3 min, the signal amplification effect is achieved through the catalytic reaction of the signal probe and TMB, then whether the new coronavirus S protein antigen exists is determined by naked eye, the photo of the T line on the NC membrane is taken, the blue signal in the T line area is displayed, the T line gray value is read by color recognition software to obtain the blue signal intensity gray value of the T line area, the linear regression equation obtained in step 1) is substituted, and the content of the new coronavirus S protein antigen in the sample can be inferred.

[0032] Compared with the prior art, the beneficial effects obtained by the present application are:

[0033] 1. The present application provides a preparation method of a nano-enzyme based on magnetic iron oxide nanoparticles, polyethyleneimine (PEI), platinum nanoparticles and dendritic mesoporous silica microspheres. The method has simple steps and easy-to-control conditions. The structure realizes high-density filling, and further realizes the integration and synergistic effect of iron oxide and platinum nanoparticles, thereby improving the enzyme catalytic activity. In the preparation method of the magnetic-noble metal composite nano-enzyme, PEI is used as a ligand to connect metal ions to prepare dSi / IO-PEI / Pt-PEI, and the surface of the material contains amino groups provided by PEI, which is easy to functionalize and modify. A large number of carboxyl groups provided by the surface grafted succinic anhydride are used for covalent coupling of biological macromolecules, and the antibody coupling mode by electrostatic adsorption has better stability.

[0034] 2、The application prepares a dSiO2 / Fe3O4 / Pt-PEI composite structure, the multi-layered porous structure has good inside-out permeability, ensures efficient contact of Fe3O4 and Pt with catalytic color developing substrates, and the structure has magnetism, can be repeatedly and efficiently used, is used in test paper strip immunochromatography, and through enzyme catalytic signal amplification, novel coronavirus S protein antigens are rapidly and highly sensitively detected.

[0035] 3、The application provides a nano enzyme with high enzyme catalytic activity, high sensitivity and magnetism to overcome the problems of low sensitivity and the need for an additional excitation light source in lateral flow immunological analysis based on colloidal gold and fluorescence.

[0036] 4、The application provides a dSi / IO / Pt-PEI nanomicrosphere as a probe, which realizes direct naked-eye interpretation mode by using superposition of absorbance of Fe3O4 and Pt, realizes super-high catalytic activity by using the synergistic effect between Fe3O4 and Pt, and further improves sensitivity, and can realize homogeneous immunoreaction and enrichment and concentration of antigens by using magnetic separation and enrichment functions, and further improve sensitivity. BRIEF DESCRIPTION OF DRAWINGS

[0037] Figure 1 The dSi / IO / Pt-PEI-COOH microsphere is a process flow diagram for assembly and synthesis of the magnetic-gold composite nano enzyme of the application.

[0038] Figure 2 It is a principle diagram for detecting new crown S protein antigens by using the dSi / IO / Pt-PEI LFIA test paper strip of the application.

[0039] Figure 3 It is a transmission electron microscope image and a scanning electron microscope image result of materials synthesized in different steps in embodiment 1 of the application.

[0040] Fig. 4 is a test result of testing a catalytic color developing reaction in embodiment 1 of the application.

[0041] Figure 5 It is a photograph of an NC membrane after a solution of a new crown S protein antigen is detected by an immunochromatography test paper strip in embodiment 1 of the application, a photograph of the NC membrane after a TMB catalytic reaction, and a standard curve diagram. DETAILED DESCRIPTION

[0042] The application will be further described in conjunction with specific examples. However, the scope of the application is not limited to the examples.

[0043] Example 1

[0044] 1. Synthesis of dendritic mesoporous silica (dSiO2) template

[0045] 0.068 g of triethanolamine (TEA) was dissolved in ultrapure water, and the reaction was stirred magnetically in an 80 °C oil bath for 0.5 h. Then, 0.38 g of cetyltrimethylammonium bromide (CTAB) and 0.218 g of sodium salicylate (NaSal) were added, and the mixture was stirred for 1 h. Then, 4 ml of tetraethyl orthosilicate (TEOS) was added, and the reaction was allowed to proceed for 3 h. After the reaction was completed, the supernatant was removed by centrifugation, and the precipitate was washed with anhydrous ethanol three times. Then, the precipitate was dissolved in 50 ml of a mixed solution of hydrochloric acid and methanol at a volume ratio of 1:1, and the organic template was extracted by stirring at 60 °C for 6 h. The process of adding hydrochloric acid and methanol was repeated once. The product was washed with anhydrous ethanol three times, and then redispersed in anhydrous ethanol to obtain a dSiO2 template dispersion.

[0046] The transmission electron microscope image and the scanning electron microscope image of the dSiO2 template are shown in Figs. a and d of Figure 3 , respectively.

[0047] 2. Preparation of magnetic iron oxide-loaded dendritic mesoporous silica (dSi / IO-PEI) composite microspheres

[0048] The dSiO2 template dispersion 15 mL (containing 68 mg of dSiO2 template), 360 mg of iron trisacetylacetonate, and 40 mg of polyethyleneimine (PEI) with a molecular weight of 1800 obtained in step 1 were placed in a three-necked flask, and 30 mL of triethylene glycol (TEG) was added and uniformly ultrasonicated. The flask was filled with argon in a Schlenk pipeline, and the reaction was allowed to proceed at a vigorous stirring speed at 210 °C for 2 h, and then the temperature was continuously increased to 285 °C, and the reaction was allowed to proceed at a vigorous stirring speed for 1 h. After the reaction was cooled to room temperature, 80 mL of acetone was added, and the product was magnetically separated. The precipitate was washed with ethanol three times, and the final product was dispersed in 40 mL of ethanol to obtain magnetic iron oxide-loaded dSi / IO-PEI microspheres.

[0049] The transmission electron microscope image and the scanning electron microscope image of the dSi / IO-PEI microspheres are shown in Figs. b and e of Figure 3 , respectively. Figure 3 Figs. b and e of show that all the radial channels of the dSiO2 template are occupied by a dense layer of IOs (i.e., ferroferric oxide). Although a dense layer of IOs has been deposited, the dSi / IO-PEI nanostructure still has a considerable pore volume to facilitate the next step of assembly.

[0050] 3. Preparation of dendritic mesoporous silica microspheres / ferroferric oxide / platinum nanomicrospheres (dSi / IO / Pt-PEI)

[0051] The dSi / IO-PEI microspheres obtained above were centrifuged to remove the supernatant, 8 mL ultrapure water was added to the solid precipitate, oscillated for 30 s, stirred at room temperature for 10 min, 0.0208 g of potassium tetrachloroplatinate (K2PtCL4) and 50 mg of polyvinylpyrrolidone (PVP) were uniformly ultrasonicated and then stirred in an ice bath for 10 min. 0.5 mL of sodium borohydride solution (containing 14.56 mg of sodium borohydride) was added, and stirred for 2 h. After the reaction was completed, the product was obtained by magnetic separation, washed with ultrapure water for 3 times, and finally dispersed in 10 mL of ultrapure water to obtain dSi / IO / Pt-PEI microspheres.

[0052] The transmission electron microscope image and the scanning electron microscope image of the dSi / IO / Pt-PEI microspheres are shown in sub-figures c and f of FIG. 3, respectively. Figure 3 Figure 3 Sub-figures c and f of FIG. 3 show that the platinum nanoparticles are tightly fixed in the pores of the dSi / IO-PEI template, and although the pore size of the dSi / IO / Pt-PEI nanostructure is significantly reduced, the particle size is almost not increased.

[0053] 4. Preparation of dSi / IO / Pt-PEI-COOH microspheres

[0054] 6 mL of the dSi / IO / Pt-PEI microspheres prepared above were centrifuged to remove the supernatant, washed with anhydrous N,N-dimethylformamide (DMF) for 3 times, then dissolved in 5 mL of anhydrous DMF, 50 mg of succinic anhydride was added and slowly stirred at room temperature for 18 h. After the reaction was completed, the product dSi / IO / Pt-PEI-COOH microspheres were obtained by magnetic separation, washed with ultrapure water for 3 times, and then dispersed in a PB buffer (0.01 M, pH 6.0) for further use.

[0055] The dSi / IO / Pt-PEI-COOH microspheres prepared in step 4 are the magnetic-noble metal composite nanoscale enzyme of the present application, and the process flow diagram of the assembly and synthesis is shown in FIG. 4. Figure 1

[0056] 5. Preparation of dSi / IOI / Pt-PEI-COOH signal probe

[0057] ​​First, take the PB buffer solution containing 2 mg of dSi / IO / Pt-PEI-COOH microspheres in step 4, add 1.25 mg of 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) and 1.25 mg of N-hydroxysulfosuccinimide (Sulfo-NHS), ultrasonic homogenization, then shake at room temperature for 30 min. Centrifuge the activated microspheres to remove the supernatant and re-disperse them in 1 mL of 10 mM phosphate (PB) buffer. Then add 40 μg of monoclonal antibody against SARS-CoV-2 S protein (RBD5313, Haipet) to the solution and react at room temperature for 2.5 h. Then add BSA to a final concentration of 1% and block for 2 h. Wash the product with PB buffer (0.01 M, pH 7.4) twice, collect by centrifugation, and disperse in protein storage solution (10 mM phosphate buffer (PBS) pH 7.4 containing 0.1% bovine serum albumin (BSA), 0.01% Tween 20) at a concentration of 4 mg / mL and store at 4°C.

[0058] 6. Preparation and detection of immunochromatographic test strip based on dSi / IO / Pt-PEI signal probe

[0059] First, treat the conjugate pad and sample pad with conjugate pad treatment solution (20 mM PB 7.4 containing 1% Tween 20, 0.1% tris(hydroxymethyl) aminomethane (Tris), 2.5% sucrose, 1% BSA, 0.1% sodium caseinate) and dry at 37°C. Spray the protein storage solution containing dSi / IOI / Pt-PEI-COOH signal probe from step 5 evenly on the conjugate pad and dry at 37°C. Fix monoclonal antibody against SARS-CoV-2 S protein (RBD5308, Haipet) (1 mg / mL) and goat anti-mouse antibody (IgG) (1 mg / mL) on the test line (C) and control line (T) of nitrocellulose membrane (NC membrane) respectively with a membrane sputtering instrument at a dosage of 1 μL / cm, and the distance between the test line and the control line is 5 mm. Then dry at 37°C. Then assemble the treated sample pad, conjugate pad, NC membrane and absorbent paper in order, cut into 3.8 mm wide strips with a strip cutter, and store under dry conditions, to obtain the immunochromatographic test strip (i.e. dSi / IO / Pt-PEI-based LFIA test strip). The principle of detecting SARS-CoV-2 S protein antigen using dSi / IO / Pt-PEI-based LFIA test strip is shown in Figure 2 .

[0060] 7. Catalytic color reaction test

[0061] A) 1.5 ml of 1.66 mM TMB solution and 1.5 ml of 5.8 mM H2O2 solution were mixed, then 10 μl of dSi / IO / Pt-PEI solution (1.5 mg / ml) was added to conduct catalytic color reaction, and UV absorption spectrum was tested. The UV absorption spectrum of dSi / IO / Pt-PEI catalytic color reaction with TMB / H2O2 over time is shown in Figure 4a , Figure 4a The photo of color change of catalytic color reaction is shown in the inset of Figure 4a The absorbance curves in

[0062] B) 1 ml of 1.2 mM H2O2 solution was added with 1 ml of TMB solution of different concentrations, and 10 μl of dSi / IO / Pt-PEI solution (1.5 mg / ml) was added to conduct catalytic color reaction, and UV absorption spectrum was tested. The relationship between different TMB substrate concentrations and the absorbance at 650 nm wavelength after dSi / IO / Pt-PEI catalytic color reaction over time is shown in Figure 4b , Figure 4b The inset of Figure I , Ⅱ , Ⅲ, Ⅳ, Ⅴ are photos of color reaction of 0.2 mM, 0.6 mM, 0.8 mM, 1.0 mM, 1.2 mM TMB solution with dSi / IO / Pt-PEI, respectively.

[0063] C) According to the test results of step B), the corresponding reaction rate v of each TMB concentration C TMB , the corresponding relationship curve between concentration C TMB and reaction rate v was drawn, the results are shown in Figure 4c Then according to the reaction rate v and the concentration C TMB of TMB, the double reciprocal curve of 1 / v and 1 / C TMB was drawn, the intercept was 1 / Vmax (Vmax was the maximum speed of enzymatic reaction), and the slope was Km / Vmax (Km was Michaelis constant), the results are shown in the inset of Figure 4c .

[0064] 8. Detection and standard curve drawing

[0065] A) A series of different concentrations of solutions containing new crown S protein antigens, the concentrations are 0, 0.001, 0.005, 0.008, 0.01, 0.05, 0.1, 0.5, 1, 10, 100 ng / ml of new crown S protein antigen solution are tested, 80 μL of solution containing different concentrations of new crown S protein antigens is gradually chromatographed to the NC membrane and the absorption pad layer of the immunochromatography test strip under the capillary action, after 15 min, the NC membrane is photographed using a smart phone, the photo results are as shown in Figure 5 As shown in the lower part of the figure a, it can be seen that the test strip does not change color. Then the prepared 3,3',5,5'-tetramethylbenzidine (TMB) and hydrogen peroxide (H2O2) developing solution (the developing solution is mixed by 10 μL of 10 mM TMB solution and 60 μL of 10 mM H2O2 solution) is added to the NC membrane, after 2 min, the signal probe and TMB catalytic reaction achieve the effect of signal amplification, then naked eye interpretation and mobile phone quantitative analysis are used, the NC membrane is photographed using a smart phone, the photo results are as shown in the lower part of the figure b of FIG. 5, it can be seen that the T line of the photographed NC membrane shows blue color. The gray value of the blue signal of the T line region of the NC membrane of the lower part of the figure b is read by the color recognition software Color picker. Figure 5

[0066] The standard curve is plotted with the concentration of new crown S protein antigen as the abscissa and the gray value obtained in the T line region of the NC membrane as the ordinate, the results are shown in the lower part of the figure c. Figure 5 Figure 5 The insert of the lower part of the figure c shows the linear range area of the dSi / IO / Pt-PEI based LFIA in the high sensitivity mode. The experimental results show that the detection limit of the new crown S protein antigen is 0.807 pg / mL, and the method can be conveniently used for accurate and ultra-sensitive detection of new crown S protein antigens.

[0067] The content described in the specification is only a list of forms of the inventive concept, and the protection scope of the present application should not be regarded as limited to the specific forms stated in the embodiments.​​

Claims

1. A magnetic-noble metal composite nanozyme for enzyme amplification and visualization detection of SARS-CoV-2 antigen, characterized in that... The specific preparation method of this magnetic-noble metal composite nanozyme includes the following steps: 1) Preparation of dendritic mesoporous silica (dSi / IO-PEI) composite microspheres supported by magnetic iron oxide (Fe3O4). Dendritic mesoporous silica (dSiO2) was dispersed in anhydrous ethanol, iron salt and polyethyleneimine (PEI) were added, and then triethylene glycol was added and ultrasonically homogenized. Under an argon atmosphere, the mixture was vigorously stirred at 200-220°C for 1.5-2.5 h, and then heated to 280-290°C and stirred for 0.5-1.5 h. After cooling to room temperature, the product was obtained by magnetic separation and washed several times with ethanol to obtain magnetic iron oxide-supported dendritic mesoporous silica composite microspheres, which were labeled as dSi / IO-PEI. 2) Preparation of dendritic mesoporous silica microspheres / iron tetroxide / platinum nanospheres dSi / IO / Pt-PEI The dSi / IO-PEI microspheres obtained in step 1) were added to ultrapure water and mixed evenly. Then, platinum salt and polyvinylpyrrolidone (PVP) were added and ultrasonically homogenized. The mixture was stirred in an ice-water bath for 5-20 min to load the platinum salt onto the dSi / IO-PEI microspheres. Then, sodium borohydride solution was added and the mixture was stirred for 1-3 h to reduce the platinum salt loaded on the dSi / IO-PEI microspheres into platinum nanoparticles. After the reaction was completed, the product was obtained by magnetic separation. The product was washed several times with ultrapure water to obtain dendritic mesoporous silica microspheres / iron oxide / platinum nanospheres, which were labeled as dSi / IO / Pt-PEI. 3) Finally, dSi / IO / Pt-PEI was modified by carboxylation to obtain dendritic mesoporous dSiO2 / Fe3O4 / Pt-PEI composite microspheres, which were labeled as dSi / IO / Pt-PEI-COOH.

2. The magnetic-noble metal composite nanozyme for enzyme amplification and visualization detection of SARS-CoV-2 antigen as described in claim 1, characterized in that... The method for preparing the dendritic mesoporous silica dSiO2 is as follows: using hexadecyltrimethylammonium bromide (CTAB) as a template, sodium salicylate as a structure directing agent, triethanolamine as a catalyst, and water as a solvent, the reaction is carried out at 70-90℃ with stirring for 0.5-2 hours. Then, tetraethyl silicate, a silicon source, is added, and the reaction is continued with stirring for 2-5 hours to obtain a dendritic mesoporous silica template with ultra-large pore size. The mass ratio of hexadecyltrimethylammonium bromide, sodium salicylate, and triethanolamine is 1:0.4-0.6:0.1-0.3, and the mass ratio of hexadecyltrimethylammonium bromide to the volume ratio of tetraethyl silicate is (0.08-0.1) g: 1 mL.

3. The magnetic-noble metal composite nanozyme for enzyme amplification and visualization detection of SARS-CoV-2 antigen as described in claim 2, characterized in that... The mass ratio of hexadecyltrimethylammonium bromide, sodium salicylate, and triethanolamine is 1:0.55~0.6:0.16~0.

2.

4. The magnetic-noble metal composite nanozyme for enzyme amplification and visualization detection of SARS-CoV-2 antigen as described in claim 1, characterized in that... Step 3) The preparation steps of carboxylated dSi / IO / Pt-PEI microspheres are as follows: The dSi / IO / Pt-PEI microspheres obtained in step 2) were dispersed in anhydrous DMF, and then succinic anhydride was added. The mixture was stirred at room temperature for 16-20 h. After the reaction was completed, the product was obtained by magnetic separation. The product was washed several times with ultrapure water to obtain the dendritic mesoporous dSiO2 / Fe3O4 / Pt-PEI composite microspheres modified by carboxylation, namely dSi / IO / Pt-PEI-COOH.

5. The magnetic-noble metal composite nanozyme for enzyme amplification and visualization detection of SARS-CoV-2 antigen as described in claim 1, characterized in that... In step 1), the mass ratio of dendritic mesoporous silica (dSiO2), iron salt, and polyethyleneimine (PEI) is 60-70:350-380:35-45, and the iron salt is iron triacetylacetone.

6. The magnetic-noble metal composite nanozyme for enzyme amplification and visualization detection of SARS-CoV-2 antigen as described in claim 1, characterized in that... In step 1), the mass ratio of dSiO2 to platinum salt and polyvinylpyrrolidone (PVP) in step 2) is 60~70:20~22:45~55, and the platinum salt is potassium tetrachloroplatinate; the mass ratio of platinum salt to sodium borohydride is 1.4~1.5:

1.

7. The magnetic-noble metal composite nanozyme for enzyme amplification and visualization detection of SARS-CoV-2 antigen as described in claim 4, characterized in that... In step 1), the mass ratio of dSiO2 to succinic anhydride in step 3) is 40~42:

50.

8. A test strip for rapid immunochromatographic detection of SARS-CoV-2 S protein antigen, using the magnetic-noble metal composite nanozyme for enzyme-amplified visualization detection of SARS-CoV-2 antigen as described in any one of claims 1-7, the test strip comprising the following assembly steps: 1) Preparation of dSi / IO / Pt-PEI-COOH signal probe: The magnetic-noble metal composite nanozyme was dispersed in PB buffer, EDC and Sulfo-NHS were added and the mixture was stirred for 20-40 min to activate the carboxyl groups on the surface of the magnetic-noble metal composite nanozyme in the buffer. The activated magnetic-noble metal composite nanozyme was centrifuged to remove the supernatant and dispersed in PB buffer. Then, a monoclonal antibody for detecting the COVID-19 S protein was added and reacted at room temperature for 2-3 h. Then, BSA was added to block the reaction for 1-3 h. After the reaction, the product was washed several times with PB buffer to obtain the dSi / IO / Pt-PEI-COOH signal probe. The probe was then prepared in PBS buffer containing 0.05-0.2% BSA at a concentration of 3-5 mg / mL to obtain the signal probe solution. 2) Preparation of the test strips, including the following processes: S1: Before use, both the sample pad and the conjugate pad should be soaked in a treatment solution containing 0.5%~2% BSA. After being fully soaked, they should be taken out and dried to complete the pretreatment. S2: After spraying the signal probe solution onto the conjugate pad, dry it. S3: The T line of the NC membrane is coated with monoclonal capture antibody against the SARS-CoV-2 S protein, and the C line of the NC membrane is coated with goat anti-mouse antibody IgG. The membrane is then dried. S4: Overlap the conjugation pad onto the end of the NC membrane near the T line, overlap the absorption pad onto the end of the NC membrane near the C line, and then overlap the sample pad onto the end of the conjugation pad away from the NC membrane. Cut to form an immunochromatographic test strip.

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