CN115060893A - Three-dimensional homogeneous phase filling type magnetic-precious metal composite nano enzyme, neocorona antigen immunochromatographic test paper and application thereof - Google Patents

Three-dimensional homogeneous phase filling type magnetic-precious metal composite nano enzyme, neocorona antigen immunochromatographic test paper and application thereof Download PDF

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CN115060893A
CN115060893A CN202210593486.9A CN202210593486A CN115060893A CN 115060893 A CN115060893 A CN 115060893A CN 202210593486 A CN202210593486 A CN 202210593486A CN 115060893 A CN115060893 A CN 115060893A
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黄亮
刘鑫月
汪晶
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Zhejiang University of Technology ZJUT
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Abstract

The invention discloses a three-dimensional homogeneous phase filling type magnetic-precious metal composite nano enzyme, a neocorona antigen immunochromatographic test paper and application thereof, wherein the magnetic-precious metal composite nano enzyme is dendritic mesoporous silica dSiO 2 Is a carrier, the carrier is modified with grafted polyethyleneimine PEI as an intermediate medium layer, and dSiO is utilized 2 Magnetic nano-particle Fe is sequentially loaded on the coordination function of amino groups of PEI on carrier 3 O 4 And nano-particle Pt, finally obtaining the dendriform mesoporous dSiO through carboxylation modification 2 /Fe 3 O 4 the/Pt-PEI composite structure. The magnetic-precious metal composite nano enzyme provided by the invention has SiO 2 /Fe 3 O 4 The Pt-polyethyleneimine PEI composite structure has good permeability from inside to outside, and ensures that Fe 3 O 4 And high efficiency contact of Pt with the catalytic chromogenic substrateThe structure has magnetism, can be repeatedly and efficiently used, and can be used for quickly and highly sensitively detecting a novel coronavirus S protein antigen through the amplification effect of an enzyme catalytic signal in the immunochromatography of a test strip.

Description

Three-dimensional homogeneous phase filling type magnetic-precious metal composite nano enzyme, neocorona antigen immunochromatographic test paper and application thereof
Technical Field
The invention relates to a three-dimensional homogeneous phase filling type magnetic-precious metal composite nano enzyme, a neocorona antigen immunochromatographic test paper and application thereof.
Background
Currently, novel methods for coronavirus diagnosis can be divided into two broad categories. The first is a molecular test for detecting viral RNA sequences by nucleic acid amplification tests, such as the most commonly used real-time reverse transcription polymerase chain reaction (RT-PCR), but which requires long processing times, specialized laboratory equipment and highly trained technicians, and is not suitable for rapid on-site diagnosis of patients. The second category is immunological detection methods, including antibody detection and antigen detection. The method is simple to operate and can be completed in a short time, but the early detection rate of the novel coronavirus antibody in vivo is low, and the antigen can be detected before clinical symptoms appear, so that the method can be used for diagnosing early infection. The novel coronavirus S protein is considered to be an ideal target antigen for detecting SARS-cOV-2 due to the excellent immunogenicity and specificity. The world health organization states that for efficient diagnosis of SARS-CoV-2 infection, antigen diagnostic methods have a sensitivity of at least 80% and a specificity of 97%. Lateral Flow Immunochromatography (LFIA) is taken as a popular POCT diagnosis platform, and plays an important role in controlling the pandemic of new coronary pneumonia in industrialized countries and resource-limited environments.
The present commercial side-stream immunochromatography technology for detecting SARS-CoV-2 antigen mainly uses colloidal gold and fluorescent material as markers for qualitative or semi-quantitative detection. The colloidal gold nanoparticles have the characteristics of naked eye visual interpretation, good biocompatibility and the like. The colloidal gold chromatography test paper is quick and simple, does not need complex laboratory equipment and professional training personnel, and has wide application range. However, the existing colloidal gold immunochromatography method has low sensitivity and is easy to cause missed diagnosis.
The fluorescent material has higher detection signal-to-noise ratio, improves the detection performance compared with the colloidal gold-based LFIA, and solves the problem of low sensitivity to a certain extent. For example, Guo et al invented a lateral flow immunochromatography of mesoporous silica-loaded up-conversion fluorescent nanomaterial to detect S protein and N protein of SARS-CoV-2, with a limit of detection (LOD) of 1.6ng/mL and 2.2ng/mL, respectively. Zhao, Xiao et al developed a colorimetric and fluorescent bifunctional lateral flow immunoassay biosensor for rapidly and sensitively detecting the S protein of SARS-CoV-2, and the detection limits of detecting the S1 protein by using the colorimetric and fluorescent functions of the biosensor are 1 and 0.033ng/mL, respectively. However, LFIAs based on fluorescent markers all require additional excitation light sources to observe signals and complex quantitative detection instruments to realize signal readout, and have the problems of high detection cost, limited applicable scenes and the like. Meanwhile, the degree of amplification of detection signals by using a fluorescent probe is limited, and the problem of false negative existing in the colloidal gold test strip still cannot be effectively solved. Therefore, an efficient signal amplification principle and a convenient and fast instrumentation-free immunochromatographic mode still need to be explored to realize high-sensitivity and visual interpretation so as to meet the practical requirements in current new crown detection and other real-time diagnosis applications.
Enzyme-linked immunosorbent assay (ELISA) is one of the most commonly used serological immunoassay methods, and has the characteristics of strong specificity, high sensitivity, simple operation and the like. The effect of signal amplification is achieved by using the catalytic chromogenic reaction of horseradish peroxidase (HRP) and a chromogenic substrate, so that the sensitivity is improved. However, the preparation process and the detection process of the ELISA kit are complicated and long, the detection time is long, and the ELISA kit needs to be matched with a large-scale laboratory instrument, and is not suitable for field real-time detection. Lateral flow immunochromatography provides a low-cost visual conduction and point-of-care diagnostic strategy, and if a catalytic chromogenic reaction of an enzyme activity marker and a chromogenic substrate (e.g., 3',5,5' -Tetramethylbenzidine (TMB)) is utilized and applied to LFIA to realize a signal amplification effect, the lateral flow immunochromatography is expected to become a practical and effective SARS-CoV-2 detection tool, contribute to on-site rapid detection, provide information for regional disease control work, and prevent further spread of viruses. However, the number of HRP molecules capable of being labeled on the antibody is very limited, and if the HRP molecules are directly used as labels, the effect of enzyme-catalyzed amplification in a single immune reaction is limited. Meanwhile, natural enzymes are fragile biomolecules that lose catalytic activity due to denaturation even under normal detection conditions, and thus are not suitable for POC applications. Whereas nanoenzymes based on inorganic nanomaterials are an attractive alternative since they exhibit enzyme-like catalytic activity without denaturation and can be stored and used over a wide range of pH and temperature. Therefore, the development of enzyme-like active nanomaterials which have various catalytic properties inherent to enzymes and can perform fine structure regulation has attracted great interest of researchers.
Various nanomaterials having enzyme-like activity, such as noble metal nanoparticles and metal oxides, have been discovered so far and used in immunodiagnosis. Wherein the platinum (Pt) nanoparticles and ferroferric oxide (Fe) 3 O 4 ) The nano particles have excellent catalytic activity, stronger light absorption color development characteristic and excellent biocompatibility, and are suitable for constructing a visual color development probe. While Fe 3 O 4 The magnetic separation device has good liquid phase suspension and magnetic separation performance, can be used for homogeneous immunoreaction and antigen enrichment and concentration, and provides an effective idea for high-sensitivity detection of antigens. Liu et al prepared PS-Pt nanoenzyme by growing platinum nanoparticles (Pt) on the surface of a carboxyl functionalized polymer nanosphere (PS), and established a simple, convenient and sensitive colorimetric method for detecting salmonella typhimurium; doh et al mimic the enzyme Fe 3 O 4 The integration of-Pt core-shell nanoparticles into LFIA has developed a simple and sensitive biological detection system, achieving higher sensitivity than traditional LFIA. Yan et al optimize Fe 3 O 4 The activity of the nano enzyme provides a new theoretical basis for the design of the enzyme-like active nano material catalyst and the improvement of the efficiency of the enzyme-like active nano material catalyst. Compared with the nano enzyme with single functional component, the nano enzyme with composite component, such as mixed metal nano structure and metal-oxide composite structure, can effectively improve the catalytic reaction rate of catalytic active center due to the synergistic effect between functional units, and is an excellent material for constructing an enzyme catalytic signal amplification probe. Wang et al developed an LFIA test strip based on noble metal-metal oxide probes for detecting gastrin-17, effectively improving detection sensitivity. Yan et al in Nanozology Connecting Biology and nanotechnology a book of modification of biological enzymes or substitution of part of enzymes with nanomaterials, the final combination in many cases showing stronger catalytic performance than the single functional component. However, the enzyme-like active metal particles are liable to agglomerate or fuse with themselves, and the surface is not effectively protected, resulting in insufficient stability and failure to attain an effective dispersion state during the reaction. FromThe existing research shows that how to perform homogeneous and compact filling on a single catalytic functional element in a three-dimensional space to realize performance enhancement and simultaneously integrate different types of enzymatic catalytic functional elements on a colloidal scale to realize a synergistic catalytic effect, and finally obtain a colloidal and liquid-phase dispersed high-performance chromogenic probe is a key problem to be solved urgently in the field of nano enzyme catalysts and biomarker detection thereof.
Disclosure of Invention
Aiming at the problems that the existing commercial lateral flow immunochromatographic technology (LFIA) for detecting SARS-CoV-2 antigen is low in sensitivity, the immunochromatographic technology based on enzyme labeling is complex and long in process flow and is not suitable for real-time detection and the like, the invention aims to provide the three-dimensional homogeneous phase filling type magnetic-precious metal composite nano enzyme for enzyme amplification visual detection of the neocoronary antigen, the neocoronary antigen immunochromatographic test paper and the application thereof. The magnetic-precious metal composite nano enzyme provided by the invention utilizes Fe 3 O 4 And the absorbance of the two components of Pt are superposed and cooperated to realize a direct naked eye interpretation mode; using Fe 3 O 4 And Pt, high-efficiency enzyme catalytic activity and a signal amplification strategy are realized, and the sensitivity is further improved; the magnetic separation and enrichment function is expected to realize homogeneous immunoreaction and enrichment and concentration of antigen, and the detection performance of the antigen is improved.
In order to improve the immunodetection effect, how to effectively assemble a single nanometer element and controllably integrate different enzyme catalysis functional units in a colloidal scale is a difficult point in the construction of a nanometer enzyme composite material. The dendritic mesoporous silica is an ideal nano-element carrier, has high pore volume, adjustable mesoporous-macroporous pore diameter and particularly a highly open pore channel structure, and can effectively load nano-elements. Therefore, the invention uses the dendritic mesoporous silica (dSiO) 2 ) The enzyme-catalyzed chromogenic immune marker probe with high load and synergistic functions is prepared by using a carrier pore passage to carry out high-density filling and layered assembly on different functional units. dSiO 2 2 The carrier can provide support and protection, the size and uniformity of the material are strictly limited, and the prepared catalyst has highly open pore channels and a plurality of accessible enzyme catalytic active sitesAfter the multi-layer nano particles are loaded, the nano particles still have a typical central-radial pore channel structure and an enzyme catalysis site, so that the visual chromaticity is enhanced, the sensitivity is improved, and the detection range is widened.
The invention takes the dendritic mesoporous silica as a carrier providing larger specific surface area, aperture and pore volume, takes Polyethyleneimine (PEI) as an intermediate medium layer, realizes the deposition of magnetic nanoparticles and platinum nanoparticles, realizes high-density filling, further realizes the integration and synergistic effect of iron oxide and platinum nanoparticles, and further improves the enzyme catalysis and color development activity. To prepare the dendriform mesoporous SiO 2 /Fe 3 O 4 The Pt-polyethyleneimine PEI composite structure has good permeability from inside to outside, and ensures that Fe 3 O 4 The structure has superparamagnetism, can perform liquid phase suspension reaction and magnetic separation enrichment, is used in test strip immunochromatography, and can rapidly and highly sensitively detect novel coronavirus S protein antigen through the amplification effect of enzyme catalytic signals.
The magnetic-precious metal composite nano enzyme for enzyme amplification visual detection of neocorona antigen is characterized in that the magnetic-precious metal composite nano enzyme is dendritic mesoporous silica dSiO 2 Is a carrier, the carrier is modified with grafted polyethyleneimine PEI as an intermediate medium layer, and dSiO is utilized 2 Magnetic nano-particle Fe is sequentially loaded on the carrier through coordination of amino groups of PEI 3 O 4 And nano-particle Pt, finally obtaining the dendriform mesoporous dSiO by carboxylation modification 2 /Fe 3 O 4 A Pt-PEI composite structure.
The magnetic-precious metal composite nanoenzyme for enzyme amplification visual detection of neocorona antigen is characterized in that the dendritic mesoporous silica dSiO 2 The preparation method comprises the following steps: cetyl Trimethyl Ammonium 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, stirring and reacting are carried out at 70-90 ℃ for 0.5-2 h, then silicon source tetraethyl silicate is added, stirring and reacting are carried out continuously for 2-5 h, and the ultra-large-aperture dendritic mesoporous silica template is prepared; wherein, the hexadecyl trimethylThe mass ratio of the ammonium bromide to the sodium salicylate to the 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 the hexadecyl trimethyl ammonium bromide to the tetraethyl silicate is (0.08-0.1) g:1 mL.
The magnetic-precious metal composite nanoenzyme for enzyme amplification visual detection of neocorona antigen is characterized in that the specific preparation method of the magnetic-precious metal composite nanoenzyme comprises the following steps:
1) preparation of magnetic ferroferric oxide loaded dendritic mesoporous silica dSi/IO-PEI composite microsphere
Making dendritic mesoporous silicon dioxide dSiO 2 Dispersing into absolute ethyl alcohol, adding ferric salt and polyethyleneimine PEI, and adding triethylene glycol for ultrasonic homogenization; violently stirring and reacting for 1.5-2.5 h at 200-220 ℃ under argon atmosphere, continuously heating to 280-290 ℃, stirring and reacting for 0.5-1.5 h, cooling to room temperature, carrying out magnetic separation to obtain a product, washing with ethanol for several times to obtain the magnetic ferroferric oxide loaded dendritic mesoporous silica composite microspheres, and marking the magnetic ferroferric oxide loaded dendritic mesoporous silica composite microspheres as dSi/IO-PEI;
2) preparation of dendritic mesoporous silica microsphere/ferroferric oxide/platinum nano microsphere dSi/IO/Pt-PEI
Adding the dSi/IO-PEI microspheres obtained in the step 1) into ultrapure water, uniformly mixing, adding platinum salt and polyvinylpyrrolidone PVP (polyvinylpyrrolidone), ultrasonically uniformly stirring for 5-20 min in an ice water bath, and loading the platinum salt on the dSi/IO-PEI microspheres; then adding a sodium borohydride solution, stirring and reacting for 1-3 h to reduce platinum salt loaded on the dSi/IO-PEI microsphere into platinum nanoparticles, carrying out magnetic separation after the reaction is finished to obtain a product, washing the product with ultrapure water for a plurality of times to obtain a dendritic mesoporous silica microsphere/ferroferric oxide/platinum nanoparticle, and marking the dendritic mesoporous silica microsphere/ferroferric oxide/platinum nanoparticle as dSi/IO/Pt-PEI;
3) preparation of carboxylated dSi/IO/Pt-PEI microsphere
Dispersing the dSi/IO/Pt-PEI microspheres obtained in the step 2) in anhydrous DMF, adding succinic anhydride, stirring and reacting at room temperature for 16-20 h, obtaining a product through magnetic separation after the reaction is finished, and washing the product with ultrapure water for several times to obtain the carboxylated modified treeMesoporous dSiO 2 /Fe 3 O 4 the/Pt-PEI composite microsphere is marked as dSi/IO/Pt-PEI-COOH.
The magnetic-precious metal composite nanoenzyme for enzyme amplification visual detection of neocorona antigen is characterized in that in the step 1), dendritic mesoporous silica dSiO 2 The feeding mass ratio of the ferric salt to the polyethyleneimine PEI is 60-70: 350-380: 35-45, and the iron salt is ferric triacetylacetone.
The magnetic-noble metal composite nano enzyme for enzyme amplification visual detection of neocorona antigen is characterized in that dSiO in step 1) 2 The feeding mass ratio of the platinum salt to the polyvinylpyrrolidone PVP in the step 2) is 60-70: 20-22: 45-55, wherein the platinum salt is potassium tetrachloroplatinate; the feeding mass ratio of the platinum salt to the sodium borohydride is 1.4-1.5: 1.
The magnetic-noble metal composite nano enzyme for enzyme amplification visual detection of neocorona antigen is characterized in that dSiO in step 1) 2 The mass ratio of the succinic anhydride to the feed of the succinic anhydride in the step 3) is 40-42: 50.
a test strip for immunochromatography rapid detection and analysis of a novel crown S protein antigen comprises the following assembly steps:
1) preparation of a dSi/IOI/Pt-PEI-COOH signal probe: dispersing the magnetic-precious metal composite nano enzyme into a PB buffer solution, adding EDC and Sulfo-NHS, and stirring for reacting for 20-40min to activate the surface carboxyl of the magnetic-precious metal composite nano enzyme in the buffer solution; centrifuging the activated magnetic-precious metal composite nano enzyme, removing the supernatant, dispersing the supernatant into a PB buffer solution, adding a new crown S protein monoclonal detection antibody, reacting at room temperature for 2-3 h, adding BSA (bovine serum albumin) with the final concentration of 0.5-2%, and sealing for 1-3 h; after the reaction is finished, washing the product for several times by using a PB buffer solution to obtain a dSi/IOI/Pt-PEI-COOH signal probe, and preparing the dSi/IOI/Pt-PEI-COOH signal probe into a PBS buffer solution containing 0.05-0.2% BSA at the concentration of 3-5 mg/mL to obtain a signal probe solution;
2) the preparation of the test strip comprises the following steps:
s1: the sample pad and the combined pad are soaked in a treating solution containing 0.5-2% BSA before use, and are taken out and dried after full soaking, so that the pretreatment is completed;
s2: spraying a signal probe solution on the bonding pad, and drying;
s3: coating a new crown S protein monoclonal capture antibody on a T line of the NC membrane, coating a goat anti-mouse antibody IgG on a C line of the NC membrane, and drying;
s4: the combination pad is lapped at one end, close to the T line, of the NC membrane, the absorption pad is lapped at one end, close to the C line, of the NC membrane, the sample pad is lapped at one end, far away from the NC membrane, of the combination pad, and the immunochromatography test strip is formed through cutting.
The test strip is applied to immunochromatography rapid detection and analysis of the novel crown S protein antigen, and is characterized in that the application method comprises the following steps:
1) drawing a standard curve: dripping a series of solutions containing new crown S protein antigen with different concentrations on the sample pad of the immunochromatographic test strip, gradually chromatographing the solutions on the NC membrane of the immunochromatographic test strip under capillary action, and after 15-30min, adding a solution containing 3,3',5,5' -tetramethylbenzidine TMB and hydrogen peroxide H 2 O 2 The color developing solution is dripped on an NC membrane, the effect of signal amplification is achieved through the catalytic reaction of a signal probe and TMB after 1-3 min, then whether new crown S protein antigen exists or not is judged rapidly and qualitatively by naked eyes, a picture of a T line on the NC membrane is shot, a blue signal is displayed on a T line area, the gray value of the T line is read by color identification software to obtain the gray value of the intensity of the blue signal of the T line area, a standard curve is drawn by taking the gray value of the blue signal as the ordinate and the concentration of the new crown S protein antigen as the abscissa, and a linear regression equation is calculated;
2) detection of the actual sample: dripping the solution containing the sample on the sample pad of the immunochromatographic test strip, gradually carrying out chromatography on the solution on an NC membrane of the immunochromatographic test strip under the capillary action, and after 15-30min, adding the solution containing 3,3',5,5' -tetramethylbenzidine TMB and hydrogen peroxide H 2 O 2 The developing solution is dripped on an NC membrane, the effect of signal amplification is achieved through the catalytic reaction of a signal probe and TMB after 1-3 min, then whether new crown S protein antigen exists is rapidly and qualitatively judged by naked eyes, a picture of a T line on the NC membrane is shot, a blue signal is displayed on a T line area,reading the gray value of the T line by using color identification software to obtain the gray value of the intensity of the blue signal of the T line area, substituting the gray value into the linear regression equation obtained in the step 1), and deducing the content of the new crown S protein antigen in the sample.
Compared with the prior art, the invention has the following beneficial effects:
1. the invention provides a preparation method of a nano enzyme based on magnetic iron oxide nanoparticles, Polyethyleneimine (PEI), platinum nanoparticles and dendritic mesoporous silica microspheres, which is simple in steps and easy to control conditions. In the preparation method of the magnetic-precious metal composite nanoenzyme, the dSi/IO-PEI/Pt-PEI is prepared by using PEI as a ligand for connecting metal ions, and the surface of the material contains amino provided by PEI, so that the amino is easy to functionalize and modify. A large amount of carboxyl provided by the succinic anhydride grafted on the surface is used for covalent coupling of biomacromolecules, and has better stability compared with an antibody coupling mode of electrostatic adsorption.
2. The invention prepares dSiO 2 /Fe 3 O 4 The multi-level porous structure has good permeability from inside to outside, and ensures Fe 3 O 4 The structure has magnetism, can be repeatedly and efficiently used, and can be used for quickly and highly sensitively detecting novel coronavirus S protein antigen through the amplification effect of an enzyme catalytic signal in immunochromatography of a test strip. In the magnetic-precious metal composite nano enzyme provided by the invention, dendritic mesoporous silica microspheres with vertical radial pore channels are used as carriers, and the functional units are integrated and coordinated by high-density filling and layering of the carrier pore channels on different functional units to prepare enzyme-catalyzed chromogenic signals.
3. The invention provides a magnetic nano enzyme with high enzyme catalytic activity and high sensitivity, aiming at overcoming the problems of low sensitivity, additional excitation light source requirement and the like of the lateral flow immunoassay based on colloidal gold and fluorescence.
4. The inventionProvides a dSi/IO/Pt-PEI nano microsphere as a probe, which utilizes Fe 3 O 4 And the superposition of two absorbances of Pt realize the direct naked eye interpretation mode; and also use Fe 3 O 4 The catalyst and Pt have synergistic effect, so that ultrahigh catalytic activity is realized, and the sensitivity is further improved; but also can realize homogeneous immunoreaction and enrichment and concentration of antigen by utilizing the magnetic separation and enrichment function, thereby improving the sensitivity.
Drawings
FIG. 1 is a schematic diagram of the process flow of the assembly synthesis of dSi/IO/Pt-PEI-COOH microspheres, namely the magnetic-noble metal composite nanoenzyme of the invention;
FIG. 2 is a schematic diagram of the detection of a novel corona S protein antigen using the dSi/IO/Pt-PEI based LFIA test strip of the present invention;
FIG. 3 shows the transmission electron microscope image and the scanning electron microscope image of the material synthesized in the different steps of example 1;
FIG. 4 is a graph showing the results of the test for the catalytic color reaction in example 1 of the present invention;
FIG. 5 is a photograph of an NC membrane after the immunochromatographic test strip of example 1 of the present invention detects a novel crown S protein antigen solution, a photograph of an NC membrane after a catalytic reaction with TMB, and a plotted standard curve.
Detailed Description
The present invention is further illustrated by the following examples, which should not be construed as limiting the scope of the invention.
Example 1:
1. dendritic mesoporous silica (dSiO) 2 ) Synthesis of the template
Dissolving 0.068g of Triethanolamine (TEA) in ultrapure water, carrying out magnetic stirring reaction in an oil bath kettle at 80 ℃ for 0.5h, adding 0.38g of cetyltrimethylammonium bromide (CTAB) and 0.218g of sodium salicylate (NaSaI), stirring for 1h, then adding 4ml of tetraethyl silicate (TEOS), reacting for 3h, centrifuging after the reaction is finished, removing supernatant, and washing the precipitate with absolute ethyl alcohol for 3 times; dissolving the precipitate in 50ml of mixed solution of hydrochloric acid and methanol at a volume ratio of 1:1, stirring in water bath at 60 deg.C for 6 hr, extracting to remove organic template, adding hydrochloric acid and methanol repeatedly, and stirringThe stirring process is carried out once; washing the product with anhydrous ethanol for 3 times, and re-dispersing in anhydrous ethanol to obtain dSiO 2 A template dispersion.
dSiO 2 Transmission electron microscope images and scanning electron microscope images of the template are shown in panels a and d, respectively, in fig. 3.
2. Preparation of magnetic iron oxide loaded dendritic mesoporous silica (dSi/IO-PEI) composite microsphere
Mixing the dSiO obtained in step 1 2 Template Dispersion 15mL (containing dSiO) 2 68mg of template), 360mg of ferric triacetylacetone and 40mg of Polyethyleneimine (PEI) (molecular weight 1800) are placed in a three-neck flask, 30mL of triethylene glycol (TEG) is added, and the mixture is subjected to uniform ultrasonic treatment. And (2) filling argon into a flask on a Schlenk production line, violently stirring at 210 ℃ for reacting for 2 hours, continuously heating to 285 ℃, stirring for reacting for 1 hour, cooling to room temperature, adding 80mL of acetone, carrying out magnetic separation to obtain a product, washing the precipitate with ethanol for three times, and dispersing the final product in 40mL of ethanol to obtain the dSi/IO-PEI microsphere loaded with magnetic iron oxide.
Transmission electron microscopy images and scanning electron microscopy images of dSi/IO-PEI microspheres are shown in panels b and e, respectively, of FIG. 3. Panels b and e of FIG. 3 show dSiO 2 All radial channels of the template are occupied by a dense IOs layer (i.e. magnetite), and although a dense IOs layer has been deposited, the dSi/IO-PEI nanostructure still has a considerable pore volume for further assembly.
3. Preparation of dendritic mesoporous silica microsphere/ferroferric oxide/platinum nano microsphere (dSi/IO/Pt-PEI)
Centrifuging the dSi/IO-PEI microspheres obtained above, removing the supernatant, adding 8mL of ultrapure water into the solid precipitate, oscillating for 30s, stirring at room temperature for 10min, adding 0.0208g of potassium tetrachloroplatinate (K) 2 PtCL 4 ) And 50mg of polyvinylpyrrolidone (PVP) were ultrasonically homogenized and then stirred for 10min in an ice bath. Adding 0.5mL of sodium borohydride solution (containing 14.56mg of sodium borohydride), stirring for 2h, carrying out magnetic separation after the reaction is finished to obtain a product, washing the product for 3 times by using ultrapure water, and finally dispersing the product in 10mL of ultrapure water to obtain the dSi/IO/Pt-PEI microsphere.
Transmission electron microscopy images and scanning electron microscopy images of dSi/IO/Pt-PEI microspheres are shown in panels c and f, respectively, of FIG. 3. Panels c and f of fig. 3 illustrate that the platinum nanoparticles are tightly fixed in the pores of the dSi/IO-PEI template, and the size of the dSi/IO/Pt-PEI nanostructure is hardly increased although the pore size is significantly reduced.
4. Preparation of dSi/IO/Pt-PEI-COOH microspheres
6mL of the prepared dSi/IO/Pt-PEI microsphere is centrifuged to remove the supernatant, washed with anhydrous N, N-Dimethylformamide (DMF) for three times, dissolved in 5mL of anhydrous DMF, added with 50mg of succinic anhydride and slowly stirred at room temperature for 18h, after the reaction is completed, the product dSi/IO/Pt-PEI-COOH microsphere is obtained through magnetic separation, washed with ultrapure water for three times and then dispersed in a PB buffer (0.01M, pH 6.0) for further use.
The dSi/IO/Pt-PEI-COOH microspheres prepared in the step 4 are the magnetic-precious metal composite nanoenzyme, and the schematic process flow diagram of the assembly synthesis of the magnetic-precious metal composite nanoenzyme is shown in figure 1.
5. Preparation of dSi/IOI/Pt-PEI-COOH signal probe
Firstly, taking a PB buffer solution containing 2mg dSi/IO/Pt-PEI-COOH microspheres in the step 4, adding 1.25mg of 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) and 1.25mg of N-hydroxy thiosuccinimide (Sulfo-NHS), uniformly performing ultrasonic treatment, shaking at room temperature for 30min, centrifuging the activated microspheres, removing supernatant, re-dispersing in 1mL of 10mM phosphoric acid (PB) buffer solution, adding 40 mu g of neocoronal S protein monoclonal antibody (RBD5313, sea peptide) into the solution, and reacting at room temperature for 2.5 h; blocking was then performed by adding 1% final concentration BSA to the solution for 2 h. The product was washed 2 times with PB buffer (0.01M, pH 7.4), collected by centrifugation, and dispersed in protein preservation solution (10mM Phosphate Buffer Solution (PBS) pH 7.4, containing 0.1% Bovine Serum Albumin (BSA), 0.01% Tween 20(Tween 20)) at a preservation concentration of 4mg/mL, and stored at 4 ℃.
6. Preparation and detection of immunochromatographic test paper based on dSi/IO/Pt-PEI signal probe
The conjugate pad and sample pad were first treated with conjugate pad treatment solution (20mM PB 7.4, containing 1% Tween 20, 0.1% Tris (hydroxymethyl) aminomethane (Tris), 2.5% sucrose, 1% BSA, 0.1% sodium caseinate) and dried at 37 ℃. And (4) uniformly spraying the protein preservation solution preserved with the dSi/IOI/Pt-PEI-COOH signal probe in the step (5) on the bonding pad, and drying at 37 ℃. The monoclonal antibody of the novel crown S protein (RBD5308, sea peptide) (1mg/mL) and the goat anti-mouse antibody (IgG) (1mg/mL) are respectively fixed on a test line (C) and a quality control line (T) of a nitrocellulose membrane (NC membrane) by a film-cutting gold spraying instrument in an amount of 1 mu L/cm, and the distance between the test line and the quality control line is 5 mm. Then dried at 37 ℃. And then sequentially assembling the treated sample pad, the combined pad, the NC membrane and the absorbent paper, cutting the sample pad, the combined pad, the NC membrane and the absorbent paper into strips with the width of 3.8mm by using a slitter, and sealing and storing the strips under a dry condition to obtain the immunochromatographic test strip (namely the dSi/IO/Pt-PEI-based LFIA test strip). The schematic diagram of detecting the novel crown S protein antigen by using the dSi/IO/Pt-PEI based LFIA test strip is shown in figure 2.
7. Test for catalytic color reaction
A) 1.5ml of 1.66mM TMB solution and 5.8mM H 2 O 2 After mixing 1.5ml of the solution, 10. mu.l of dSi/IO/Pt-PEI solution (1.5mg/ml) was added to conduct a catalytic color reaction, and the ultraviolet absorption spectrum was measured. dSi/IO/Pt-PEI and TMB/H 2 O 2 The ultraviolet absorption spectrum of the catalytic color reaction with time is shown in FIG. 4a, and the inset in FIG. 4a is a photograph taken of the color change of the catalytic color reaction. Wherein the absorbance curves in FIG. 4a are the results at the color development times of 30, 27, 24, 21, 18, 15, 12, 9, 6, 3, and 0min in sequence from top to bottom.
B)1.2mM H 2 O 2 1ml of the solution, 1ml of TMB solution of different concentrations and 10. mu.l of dSi/IO/Pt-PEI solution (1.5mg/ml) were added to conduct a catalytic color reaction, and the ultraviolet absorption spectrum was measured. The relationship between the substrate concentrations of different TMB and the change of the absorbance at 650nm wavelength after the dSi/IO/Pt-PEI catalyzed color reaction with time is shown in FIG. 4b, and the photographs taken by the color reaction of TMB solution with the concentrations of 0.2mM, 0.6mM, 0.8mM, 1.0mM and 1.2mM and dSi/IO/Pt-PEI in FIGS. 4b are inserted.
C) Each TMB concentration C according to the test results of step B) TMB The corresponding reaction velocity v of the catalytic color reaction, and the concentration C TMB The result is shown in FIG. 4c, which corresponds to the reaction speed v. Then according to the reaction velocity v and the concentration C of TMB TMB As 1/v and 1/C TMB The double reciprocal plot of (A) has an intercept of 1/Vmax (Vmax is the maximum speed of the enzymatic reaction) and a slope of Km/Vmax (Km is the Michaelis constant), the results being shown in the inset of FIG. 4 c.
8. Detection and standard curve drawing
A) A series of solutions containing the new crown S protein antigen with different concentrations are tested by using the new crown S protein antigen solutions with the concentrations of 0, 0.001, 0.005, 0.008, 0.01, 0.05, 0.1, 0.5, 1, 10 and 100ng/ml in sequence, 80 mu L of the solutions containing the new crown S protein antigen with different concentrations are gradually chromatographed to an NC membrane and an absorption pad of the immunochromatographic test paper under the capillary action, after 15min, the NC membrane is photographed by using a smart phone, and the photo result is shown in a graph a in figure 5, so that the test paper strip is not discolored. Then the prepared 3,3',5,5' -tetramethyl benzidine (TMB) and hydrogen peroxide (H) 2 O 2 ) Color developing solution (10 microliter of 10mM TMB solution and 10mM H solution) 2 O 2 60 microliter of solution) is dripped on the NC membrane, the effect of signal amplification is achieved through the catalytic reaction of the signal probe and TMB after 2min, then the solution is used for naked eye interpretation and quantitative analysis of the mobile phone, the NC membrane is photographed by using the smart phone, the photograph result is shown as a panel b in figure 5, and the blue color is shown on the T line of the photographed NC membrane. The gray values of the blue signals of the T-line area on the NC film of panel b in fig. 5 were read with the Color recognition software Color picker.
The concentration of the new crown S protein antigen was plotted on the abscissa and the gray value obtained for the T-line region on the NC membrane was plotted on the ordinate to prepare a calibration curve, the result of which is shown in panel c of FIG. 5. The inset of panel c in FIG. 5 shows the linear range region of the dSi/IO/Pt-PEI based LFIA in the high sensitivity mode. The experimental result shows that the detection limit of the new crown S protein antigen is 0.807pg/mL, and the method can be conveniently used for the accurate and ultrasensitive detection of the new crown S protein antigen.
The statements in this specification merely set forth a list of implementations of the inventive concept and the scope of the present invention should not be construed as limited to the particular forms set forth in the examples.

Claims (8)

1. The magnetic-noble metal composite nano enzyme for enzyme amplification visual detection of neocorona antigen is characterized in that the nano enzyme is dendritic mesoporous silica dSiO 2 Is a carrier, the carrier is modified with grafted polyethyleneimine PEI as an intermediate medium layer, and dSiO is utilized 2 Magnetic nano-particle Fe is sequentially loaded on the coordination function of amino groups of PEI on carrier 3 O 4 And nano-particle Pt, finally obtaining the dendriform mesoporous dSiO through carboxylation modification 2 /Fe 3 O 4 the/Pt-PEI composite structure.
2. The magnetic-noble metal composite nanoenzyme for enzyme-amplified visual detection of neocorona antigen as claimed in claim 1, wherein the dendritic mesoporous silica dSiO is 2 The preparation method comprises the following steps: cetyl Trimethyl Ammonium 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, stirring and reacting are carried out at 70-90 ℃ for 0.5-2 h, then silicon source tetraethyl silicate is added, stirring and reacting are carried out continuously for 2-5 h, and the ultra-large-aperture dendritic mesoporous silica template is prepared; wherein the mass ratio of the cetyl trimethyl ammonium bromide to the sodium salicylate to the 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 the hexadecyl trimethyl ammonium bromide to the tetraethyl silicate is (0.08-0.1) g:1 mL.
3. The magnetic-noble metal composite nanoenzyme for enzyme-amplified visual detection of neocorona antigen as claimed in claim 1, wherein the specific preparation method of the magnetic-noble metal composite nanoenzyme comprises the following steps:
1) preparation of magnetic ferroferric oxide loaded dendritic mesoporous silica dSi/IO-PEI composite microsphere
Making dendritic mesoporous silicon dioxide dSiO 2 Dispersing into absolute ethyl alcohol, adding iron salt and polyethyleneimine PEI, adding triethylene glycol and performing ultrasonic treatmentHomogenizing; violently stirring and reacting for 1.5-2.5 h at 200-220 ℃ under argon atmosphere, continuously heating to 280-290 ℃, stirring and reacting for 0.5-1.5 h, cooling to room temperature, carrying out magnetic separation to obtain a product, washing with ethanol for several times to obtain the magnetic ferroferric oxide loaded dendritic mesoporous silica composite microspheres, and marking the magnetic ferroferric oxide loaded dendritic mesoporous silica composite microspheres as dSi/IO-PEI;
2) preparation of dendritic mesoporous silica microsphere/ferroferric oxide/platinum nano microsphere dSi/IO/Pt-PEI
Adding the dSi/IO-PEI microspheres obtained in the step 1) into ultrapure water, uniformly mixing, adding platinum salt and polyvinylpyrrolidone PVP (polyvinylpyrrolidone), ultrasonically uniformly stirring for 5-20 min in an ice water bath, and loading the platinum salt on the dSi/IO-PEI microspheres; then adding a sodium borohydride solution, stirring and reacting for 1-3 h to reduce platinum salt loaded on the dSi/IO-PEI microsphere into platinum nanoparticles, carrying out magnetic separation after the reaction is finished to obtain a product, washing the product with ultrapure water for a plurality of times to obtain a dendritic mesoporous silica microsphere/ferroferric oxide/platinum nanoparticle, and marking the dendritic mesoporous silica microsphere/ferroferric oxide/platinum nanoparticle as dSi/IO/Pt-PEI;
3) preparation of carboxylated dSi/IO/Pt-PEI microsphere
Dispersing the dSi/IO/Pt-PEI microspheres obtained in the step 2) in anhydrous DMF, adding succinic anhydride, stirring and reacting at room temperature for 16-20 h, obtaining a product through magnetic separation after the reaction is finished, and washing the product with ultrapure water for several times to obtain the dendriform mesoporous dSiO through carboxylation modification 2 /Fe 3 O 4 the/Pt-PEI composite microsphere is marked as dSi/IO/Pt-PEI-COOH.
4. The magnetic-noble metal composite nanoenzyme for the enzyme-amplified visual detection of neocorona antigen as claimed in claim 3, wherein in step 1), the dendritic mesoporous silica dSiO is 2 The feeding mass ratio of the ferric salt to the polyethyleneimine PEI is 60-70: 350-380: 35-45, and the iron salt is ferric triacetylacetone.
5. The magnetic-noble metal composite nanoenzyme for the enzyme-amplified visual detection of neocorona antigen as claimed in claim 3, wherein dSiO in step 1) is 2 And step (d)2) The feeding mass ratio of the medium platinum salt to the polyvinylpyrrolidone PVP is 60-70: 20-22: 45-55, wherein the platinum salt is potassium tetrachloroplatinate; the feeding mass ratio of the platinum salt to the sodium borohydride is 1.4-1.5: 1.
6. The magnetic-noble metal composite nanoenzyme for the enzyme-amplified visual detection of neocorona antigen as claimed in claim 3, wherein dSiO in step 1) is 2 And the feeding mass ratio of the succinic anhydride in the step 3) is 40-42: 50.
7. a test strip for immunochromatography rapid detection and analysis of neocorona S protein antigen, which uses the magnetic-precious metal composite nanoenzyme for enzyme amplification visual detection of neocorona antigen of any one of claims 1 to 6, the test strip comprises the following assembly steps:
1) preparation of a dSi/IO/Pt-PEI-COOH signal probe: dispersing the magnetic-precious metal composite nano enzyme into a PB buffer solution, adding EDC and Sulfo-NHS, and stirring for reacting for 20-40min to activate the surface carboxyl of the magnetic-precious metal composite nano enzyme in the buffer solution; centrifuging the activated magnetic-precious metal composite nano enzyme, removing the supernatant, dispersing the supernatant into a PB buffer solution, adding a new crown S protein monoclonal detection antibody, reacting at room temperature for 2-3 h, adding BSA (bovine serum albumin) with the final concentration of 0.5-2%, and sealing for 1-3 h; after the reaction is finished, washing the product for several times by using a PB buffer solution to obtain a dSi/IO/Pt-PEI-COOH signal probe, and preparing the dSi/IO/Pt-PEI-COOH signal probe into a PBS buffer solution containing 0.05-0.2% BSA at the concentration of 3-5 mg/mL to obtain a signal probe solution;
2) the preparation of the test strip comprises the following steps:
s1: the sample pad and the combined pad are soaked in a treating solution containing 0.5-2% BSA before use, and are taken out and dried after full soaking, so that the pretreatment is completed;
s2: spraying a signal probe solution on the bonding pad, and drying;
s3: coating a new crown S protein monoclonal capture antibody on a T line of the NC membrane, coating a goat anti-mouse antibody IgG on a C line of the NC membrane, and drying;
s4: the combination pad is lapped at one end, close to the T line, of the NC membrane, the absorption pad is lapped at one end, close to the C line, of the NC membrane, the sample pad is lapped at one end, far away from the NC membrane, of the combination pad, and the immunochromatography test strip is formed through cutting.
8. The use of the test strip of claim 7 in immunochromatographic rapid assay of the neocoronary S protein antigen, characterized in that the application method comprises the following steps:
1) drawing a standard curve: dripping a series of solutions containing new crown S protein antigen with different concentrations on the sample pad of the immunochromatographic test strip, gradually chromatographing the solutions on the NC membrane of the immunochromatographic test strip under capillary action, and after 15-30min, adding a solution containing 3,3',5,5' -tetramethylbenzidine TMB and hydrogen peroxide H 2 O 2 The color developing solution is dripped on an NC membrane, the effect of signal amplification is achieved through the catalytic reaction of a signal probe and TMB after 1-3 min, then whether new crown S protein antigen exists or not is judged rapidly and qualitatively by naked eyes, a picture of a T line on the NC membrane is shot, a blue signal is displayed on a T line area, the gray value of the T line is read by color identification software to obtain the gray value of the intensity of the blue signal of the T line area, a standard curve is drawn by taking the gray value of the blue signal as the ordinate and the concentration of the new crown S protein antigen as the abscissa, and a linear regression equation is calculated;
2) detection of the actual sample: dripping the solution containing the sample on the sample pad of the immunochromatographic test strip, gradually carrying out chromatography on the solution on an NC membrane of the immunochromatographic test strip under the capillary action, and after 15-30min, adding the solution containing 3,3',5,5' -tetramethylbenzidine TMB and hydrogen peroxide H 2 O 2 The developing solution is dripped on an NC membrane, the effect of signal amplification is achieved through the catalytic reaction of a signal probe and TMB after 1-3 min, then whether new crown S protein antigen exists is rapidly and qualitatively judged by naked eyes, a picture of a T line on the NC membrane is shot, a blue signal is displayed on a T line area, the gray value of the T line is read by color recognition software to obtain the gray value of the intensity of the blue signal of the T line area, and the gray value is substituted into the linear regression equation obtained in the step 1), so that the content of the new crown S protein antigen in the sample can be deduced.
CN202210593486.9A 2022-05-28 2022-05-28 Three-dimensional homogeneous phase filling type magnetic-precious metal composite nano enzyme, neocorona antigen immunochromatographic test paper and application thereof Pending CN115060893A (en)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN115532221A (en) * 2022-09-29 2022-12-30 山东博科生物产业有限公司 Fe for efficiently extracting novel coronavirus nucleic acid 3 O 4 -SiO 2 Magnetic bead

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN115532221A (en) * 2022-09-29 2022-12-30 山东博科生物产业有限公司 Fe for efficiently extracting novel coronavirus nucleic acid 3 O 4 -SiO 2 Magnetic bead
CN115532221B (en) * 2022-09-29 2024-04-16 山东博科生物产业有限公司 Fe for extracting novel coronavirus nucleic acid3O4-SiO2Magnetic bead

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