Universal magnetic nano-enzyme probe for detecting bacteria and viruses, preparation method, reagent strip and application

Abstract

The invention discloses a universal magnetic nano-enzyme probe for detecting bacteria and viruses, a preparation method, a reagent strip and application, the universal magnetic nano-enzyme probe comprises magnetic Fe3O4 nano-particles and multiple layers of cationic polymer strong positive electricity interlayers loaded on the surfaces of the magnetic Fe3O4 nano-particles, the magnetic Fe3O4 nano-particles serve as inner cores, and the magnetic Fe3O4 nano-particles serve as outer cores. A cationic polymer is used as a connecting layer to adsorb multi-layer small gold iridium alloy nanoparticles (Au (at) Ir), the surfaces of the multi-layer small gold iridium alloy nanoparticles are coupled with wheat germ agglutinin protein molecules, Fe3O4 nanoparticles provide magnetic response ability, and the multi-layer Au (at) Ir provides colorimetric signals, catalytic sites and binding sites. Wheat germ agglutinin (WGA) provides the ability of a label to capture pathogens in a broad spectrum, the Fe-DAu-Ir-WGA probe is combined with an LFA technology, colorimetric signal reading is carried out through ImageJ software, and efficient and ultra-sensitive quantitative detection of multiple pathogens is achieved.

Description

Technical Field

[0001] The present invention relates to the technical field of immunochromatographic detection, and in particular to a universal magnetic nanoenzyme probe for detecting bacteria and viruses, a preparation method thereof, a reagent strip and applications thereof. Background Art

[0002] Acute respiratory infections (ARIs) are a global health problem caused by a variety of respiratory viruses and bacteria. Their morbidity and mortality rates rank first among infectious diseases worldwide, resulting in more than 5 million deaths each year. Common respiratory pathogens mainly include bacteria and viruses (such as: novel coronavirus (severe acute respiratory syndrome coronavirus 2, SARS-CoV-2), influenza virus A (influenza virus A), influenza virus B (influenza virus B), Streptococcus pneumoniae (S.pneumoniae), Legionella pneumophila (L.pneumophila), etc. These pathogens often cause similar symptoms such as dyspnea, fever and cough, which are difficult to accurately judge clinically, and can easily lead to misdiagnosis and missed diagnosis. In addition, their transmission methods and epidemic trends also show similar characteristics. Therefore, early, rapid and accurate diagnosis of respiratory pathogens can effectively cut off the spread of pathogens and help guide clinicians to take targeted medical measures. It is the key to preventing and controlling ARIs.

[0003] Colorimetric lateral flow assay (LFA) is considered one of the most popular point-of-care (POCT) methods due to its ease of use, low cost, portability, and user-friendliness. It is widely used in areas such as food safety control, disease screening, and environmental monitoring. However, in practical applications for pathogen detection, current LFA methods still face two major challenges: i) Inadequate nanoprobe performance: Traditional gold nanoparticle (Au NP)-based colorimetric nanomaterials have low sensitivity (at the ng / mL level). While novel signaling materials (such as fluorescent probes, chemiluminescent signals, and Raman tags) can provide more sensitive signals, they require instrument-assisted readout, which not only increases costs but also significantly reduces the convenience of LFA probes. ii) LFA is highly dependent on specific antibodies. The type and properties of antibodies significantly influence the detection performance of LFA. The preparation of specific antibodies for emerging pathogens is time-consuming (typically 3-6 months) and costly. Furthermore, the antibodies modified on the nanoprobe surface are prone to variability and poor reproducibility, resulting in reduced stability and accuracy of LFA detection. Therefore, developing a nanoprobe with high sensitivity and the ability to simultaneously identify bacteria and viruses is key to effectively detecting pathogens.

[0004] In recent years, strategies based on non-antibody labeled probes have received widespread attention to address the difficulties of antibody pairing and labeling. Wheat germ agglutinin (WGA) is a widely studied plant lectin that can effectively bind to specific groups (n-acetylglucosamine) of certain carbohydrates, such as glycoproteins, glycosides, and glycolipids. Compared with other commonly used biorecognition agents (such as antibodies, aptamers, phages, and peptides), WGA has obvious advantages such as good stability, low cost, and broad-spectrum binding ability to pathogens. Therefore, we believe that WGA-modified probes can provide the broad-spectrum detection ability of LFA for bacteria and viruses.

[0005] The invention patent with patent application number CN202211430177 in the prior art provides a nanozyme-based immunochromatographic detection method and its application. The method comprises the following steps: (1) coupling a single-atom FeN3P peroxidase nanozyme with a first recognition antibody of a target molecule to obtain a nanozyme probe; streaking a second recognition antibody solution on a detection line on a nitrocellulose membrane, streaking a quality control antibody solution on a quality control line on a nitrocellulose membrane, drying, and sealing after drying to obtain a test strip; (2) mixing the nanozyme probe, a sample to be tested, and a buffer solution to obtain a reaction system, inserting the test strip into the reaction system for incubation; (3) taking out the incubated test strip, inserting it into a nanozyme chromogenic substrate solution for incubation, terminating the color reaction, and recording the color change result of the test line. The detection method has high stability and low cost, and is of great significance in the field of immunochromatographic detection, etc. However, the patent has a single detection target, a narrow detection range, insufficient sensitivity, and limited application scenarios.

[0006] The invention patent with patent application number CN202410854917 discloses a magnetic nanozyme particle, including a magnetic ZIF-8 metal organic framework porous nanomaterial and horseradish peroxidase; the magnetic metal organic framework porous nanomaterial is a spherical magnetic nanoparticle synthesized with ferrous sulfate, zinc acetate, and dimethylimidazole as precursors; the magnetic nanoparticle has a mesoporous structure with a pore size of 2-30nm; due to electrostatic attraction, the magnetic nanoparticles aggregate on the surface of the horseradish peroxidase to form magnetic porous nanozyme particles with horseradish peroxidase as the core. The magnetic porous nanozyme particles can be used for specific immunodetection of bacterial concentration, specifically including: specific pathogenic bacteria immunofluorescence detection in water bodies, colorimetric detection of total bacterial concentration in water bodies, and broad-spectrum testing of pathogenic bacteria concentration in water bodies. However, this patent is applicable to the detection of water environments and is not suitable for medical detection.

[0007] The invention patent with patent application number CN202210585695 discloses a lateral flow chromatography detection method for nanozymes of foodborne pathogens based on phage recognition. Primers are designed based on the gene encoding the tail fiber protein of the pathogenic bacteriophage, and the phage tail fiber protein is expressed. The nanozyme probe with dual functions of capture and catalytic activity is combined to assemble a visualized paper-based chromatography sensor to establish a lateral flow chromatography detection method for nanozymes of foodborne pathogens based on phage recognition. This invention has good sensitivity and specificity in detecting foodborne pathogens, and can successfully detect pathogens in food with a detection limit of up to 10 CFU / m. However, there are problems such as insufficient stability of biological materials, significant interference from complex food matrices, and complex probe preparation process, which may affect the convenience and reliability of its practical application.

[0008] Nanozymes are a class of metal-based nanomaterials with peroxidase-like activity. They are excellent signal reporter molecules and have attracted widespread attention. Compared with natural enzymes, they have higher stability, lower cost, and are easy to modify and functionalize. These characteristics make nanozymes promising to become dual-signal colorimetric nanolabels, thereby improving the detection sensitivity of traditional immunochromatography. However, first of all, the current nanozymes have a small active surface area, few and uncontrollable catalytic sites, which limits the maximum peroxidase-like activity of a single nanostructure. In addition, the current nanozymes are mainly colloidal materials, which are unstable in complex environments (such as salt ions and matrix interference). This characteristic limits the application of nanozymes in immunochromatography.

[0009] In view of this, the present invention is proposed. Summary of the Invention

[0010] In view of the shortcomings of the prior art, the purpose of the present invention is to provide a universal magnetic nanozyme probe for detecting bacteria and viruses, a preparation method, a reagent strip and its application. It is a magnetic multifunctional nanozyme probe (Fe-DAu@Ir-WGA) based on wheat germ agglutinin. Two layers of dense Au@Ir nanoparticles are adsorbed on the surface of Fe3O4 nanoparticles using poly(ethyleneimine) (PEI). Fe3O4 provides magnetic response capability, and the double-layer Au@Ir provides a strong colorimetric signal while providing a large number of catalytic sites and binding sites. The surface-coupled wheat germ agglutinin solves the limitation of the commonly used LFA in screening antibody pairing difficulties, and can effectively capture viruses and bacteria in a wide spectrum. The present invention also combines the Fe-DAu@Ir-WGA probe with LFA technology, and reads the colorimetric signal through ImageJ software to achieve efficient and ultra-sensitive quantitative detection of multiple pathogens.

[0011] In order to achieve the above object, the present invention adopts the following technical solutions:

[0012] A universal magnetic nanoenzyme probe (Fe-DAu@Ir-WGA) for detecting bacteria and viruses includes magnetic Fe3O4 nanoparticles and a multilayer cationic polymer strong positively charged interlayer loaded on the surface of the magnetic Fe3O4 nanoparticles. The magnetic Fe3O4 nanoparticles serve as the core, and the cationic polymer is used as a connecting layer to adsorb multilayer small gold-iridium alloy nanoparticles (Au@Ir). The surface of the multilayer small gold-iridium alloy nanoparticles is coupled with wheat germ agglutinin protein molecules. The Fe3O4 nanoparticles provide magnetic response capability, the multilayer small gold-iridium alloy nanoparticles (Au@Ir) provide colorimetric signals as well as catalytic sites and binding sites, and wheat germ agglutinin (WGA) provides the labeling ability to capture a broad spectrum of pathogens.

[0013] Preferably, the particle size of the Fe3O4 magnetic particles is 80 to 280 nm, and the particle size of the Fe3O4 magnetic particles is any value within the range of 80 to 280 nm, such as 80 nm, 100 nm, 120 nm, 140 nm, 160 nm, 180 nm, 200 nm, 220 nm, 240 nm, 260 nm, 280 nm, preferably 160 nm.

[0014] In any of the above schemes, it is preferred that the particle size of the small gold-iridium alloy nanoparticles is 2 to 10 nm, and specifically can be any value within the range of 2 to 10 nm, such as 2 nm, 4 nm, 5 nm, 8 nm, 10 nm, and preferably 5 nm.

[0015] In any of the above solutions, it is preferred that the number of layers of the wrapped Au@Ir nanoparticles is 1-3 layers, specifically 1 layer, 2 layers, 3 layers, and preferably 2 layers.

[0016] In any of the above solutions, preferably, the cationic polymer is polyacetimide, and the multilayer small gold-iridium alloy nanoparticles are wrapped on the surface of the Fe3O4 nanoparticles using polyacetimide.

[0017] A method for preparing a universal magnetic nanozyme probe for detecting bacteria and viruses, the method comprising the following steps:

[0018] (1) Synthesize Fe3O4 magnetic particles according to the classical solvothermal reaction;

[0019] (2) adding the Fe3O4 magnetic particles obtained in step (1) to a PEI aqueous solution to form Fe3O4@PEI nanospheres under ultrasonic action, and then adding the Fe3O4@PEI solution to form Fe-Au@Ir magnetic nanospheres by intense ultrasonic treatment.

[0020] (3), the Fe-Au@Ir magnetic nanomicrosphere obtained in step (2) is added to the PEI aqueous solution, under the action of ultrasonic, Fe-Au@Ir-PEI nanomicrosphere is formed, the obtained Fe-Au@Ir-PEI nanomicrosphere is added to the Au@Ir solution again, and through the action of ultrasonic, the magnetic nanoscale enzyme Fe-DAu@Ir with high performance is obtained;

[0021] (4), the magnetic nanoscale enzyme Fe-DAu@Ir obtained in step (3) is mixed with a DTNB ethanol solution, and is modified under the condition of intense ultrasonic to obtain a nanoscale enzyme material with rich carboxyl groups;

[0022] (5), the three-dimensional flexible film nanoscale enzyme material with rich carboxyl groups obtained in step (4) is mixed with a morpholine ethanesulfonic acid solution containing N-hydroxy succinimide and 1-ethyl-(3-dimethylaminopropyl) carbonyldiimidazole under the action of intense ultrasonic, and is incubated with wheat germ agglutinin to prepare a multifunctional magnetic nanoscale enzyme probe with spectral capture capacity.

[0023] Preferably in any of the above schemes, in step (1), the Fe3O4 magnetic particle has a particle size of 80-280 nm, and the Fe3O4 magnetic particle has a particle size of any value in the range of 80-280 nm, such as 80 nm, 100 nm, 120 nm, 140 nm, 160 nm, 180 nm, 200 nm, 220 nm, 240 nm, 260 nm, 280 nm, preferably 160 nm; the addition amount is 1-3.5 mg, preferably 1 mg. The Fe3O4 magnetic particle has a particle size of any value in the range of 150-180, such as 150 nm, 160 nm, 180 nm, preferably 160 nm.

[0024] Preferably in any of the above schemes, in step (2), the PEI has a concentration of 5-10 mg / mL, which can be 5 mg / mL, 6 mg / mL, 8 mg / mL, 10 mg / mL, preferably 10 mg / mL, and the addition amount of the nanoparticles relative to 100 mL of the PEI aqueous solution is any value in the range of 1.0-3.5 mg, which can be 1 mg, 2 mg, 3 mg, 3.5 mg; the concentration is the mass concentration of the Au@Ir nanoparticle aqueous dispersion, which is 1.5 mg / mL, and the addition amount of the Fe3O4@PEI nanomicrosphere relative to 100 mL of the Au@Ir nanoparticle aqueous dispersion is 1 mg; the Au@Ir nanoparticle has a particle size of any value in the range of 2-10 nm, such as 2 nm, 5 nm, 8 nm, 10 nm, preferably 5 nm.

[0025] Preferably in any of the above-mentioned solutions, in step (2) and step (3), the ultrasonic time in step (2) and (3) is any value in the range of 10-30 min, such as 10 min, 15 min, 20 min, 25 min, 30 min, preferably 20 min; the ultrasonic frequency involved in step (2) and (3) is 60-100 Hz, specifically any value in the range of 60-100 Hz, such as 60 Hz, 80 Hz, 100 Hz, preferably 80 Hz; the ultrasonic temperature is 15-25℃, which can be any value in the range of 15-25℃, such as 15℃, 18℃, 20℃, 25℃, preferably 20℃.

[0026] Preferably in any of the above-mentioned solutions, in step (2), the number of layers of the wrapped Au@Ir is 1-3 layers, specifically 1 layer, 2 layers, 3 layers, preferably 2 layers.

[0027] Preferably in any of the above-mentioned solutions, in step (5), the wheat germ agglutinin is added in an amount of 10-40 μg, specifically 10 μg, 15 μg, 20 μg, 25 μg, 30 μg, 35 μg, 40 μg, preferably 15 μg.

[0028] The application also discloses a colorimetric lateral chromatographic reagent strip for universal detection of bacteria and viruses, which comprises the magnetic nanoscale enzyme probe described above, and the pathogenic bacteria are at least one of SARS-CoV-2, L. pneumophila (Legionella pneumophila) and S. pneumoniae (Streptococcus pneumoniae).

[0029] Preferably, the immunochromatographic test strip further comprises a sample pad, a chromatographic membrane and an absorbent pad connected in sequence, the chromatographic membrane is provided with three detection lines and one quality control line, anti-SARS-CoV-2 antibody, anti-L. pneumophila (Legionella pneumophila) antibody, anti-S. pneumoniae (Streptococcus pneumoniae) and anti-WGA antibody are respectively sprayed on the detection lines T1, T2, T3 and the quality control line, and the nitrocellulose membrane, the sample pad, the water absorption pad and the bottom plate are assembled into the immunochromatographic test strip.

[0030] The present invention also discloses a detection method for the above-mentioned fluorescent immunochromatographic reagent strip, which comprises adding a magnetic nanoenzyme probe (Fe-DAu@Ir-WGA) to a sample solution to be tested, and shaking the mixture with an oscillator at room temperature for 5-25 minutes; using a magnet to quickly separate the Fe-DAu@Ir-WGA-pathogen complex from the sample solution, and resuspending it in a running buffer, and then dripping it onto the sample pad of the immunochromatographic test strip. After 10 to 20 minutes, the colorimetric signals at the three detection lines of the immunochromatographic test strip are read, and then the AEC substrate is dripped, and the catalytic signals on the three detection lines of the test strip are read.

[0031] Preferably, the concentration of the anti-SARS-CoV-2 antibody is any value in the range of 1-3 mg / mL, such as 1 mg / mL, 2 mg / mL, 3 mg / mL, preferably 2 mg / mL, and the concentration of the anti-Legionella pneumophila antibody is any value in the range of 1.0-2.0 mg / mL, such as 1 mg / mL, 1.5 mg / mL, 2 mg / mL, preferably 2.0 mg / mL; the concentration of the anti-Streptococcus pneumoniae antibody is 0.8-1.4 mg / mL, such as 0.8 mg / mL, 1 mg / mL, 1.2 mg / mL, 1.4 mg / mL, preferably 1.2 mg / mL; the concentration of the anti-WGA antibody is 0.5-2 mg / mL, specifically 0.5 mg / mL, 1 mg / mL, 1.5 mg / mL, 2 mg / mL, preferably 1.5 mg / mL.

[0032] The present invention also discloses a method for modifying the nitrocellulose membrane in the above-mentioned fluorescent immunochromatographic reagent strip, wherein anti-SARS-CoV-2 spike protein, Streptococcus pneumoniae, Legionella pneumophila, and anti-WGA antibodies are sprayed on the surface of the nitrocellulose membrane respectively, and then the modified NC membrane is placed in a constant temperature drying oven to obtain the nitrocellulose membrane in the DAu@Ir-WGA-LFA test strip.

[0033] Preferably, the concentration of the anti-SARS-CoV-2 antibody is 1-3 mg / mL, preferably 2 mg / mL, the concentration of the anti-Legionella pneumophila antibody is 1.0-2.0 mg / mL, preferably 2.0 mg / mL, the concentration of the anti-Streptococcus pneumoniae antibody is 0.8-1.4 mg / mL, preferably 1.2 mg / mL, and the concentration of the anti-WGA antibody is 0.5-2 mg / mL, preferably 1.5 mg / mL.

[0034] Preferably, in any of the above solutions, the temperature of the constant temperature drying oven is preferably set to 35°C.

[0035] Beneficial effects

[0036] The present invention provides a universal magnetic nanozyme probe for detecting bacteria and viruses, comprising magnetic Fe3O4 nanoparticles and a multilayer cationic polymer strong positively charged interlayer loaded on the surface of the magnetic Fe3O4 nanoparticles. The magnetic Fe3O4 nanoparticles serve as the core, and the cationic polymer is used as a connecting layer to adsorb multilayer small gold-iridium alloy nanoparticles. The surface of the multilayer small gold-iridium alloy nanoparticles is coupled with wheat germ agglutinin protein molecules. The Fe3O4 nanoparticles provide magnetic response capability, the multilayer Au@Ir provides colorimetric signals as well as catalytic sites and binding sites, and the wheat germ agglutinin provides the labeling capability of broad-spectrum capture of pathogens.

[0037] Structurally, the magnetic multifunctional nanozyme probe material of this application has a magnetic core that can quickly enrich the target substance in a complex environment; the double-layer Au@Ir shell not only provides a strong colorimetric signal, but also provides a large number of catalytic sites and recognition molecule binding sites;

[0038] The proposed magnetic multifunctional nanozyme probe possesses broad-spectrum pathogen recognition capabilities and can be used to detect a wide range of bacteria, fungi, and viruses. Furthermore, the use of magnetic enrichment eliminates the need for complex sample pretreatment, further improving detection sensitivity and reducing analysis time.

[0039] The magnetic multifunctional nanozyme proposed in the present invention is used as a dual-signal label for immunochromatographic detection, outputting both colorimetric and catalytic signals, and can be flexibly applied under different environmental conditions.

[0040] The method for preparing a novel core-satellite structured magnetic nanoenzyme material proposed in the present invention is simple in preparation and can be produced in batches.

[0041] The Fe-DAu@Ir-WGA nanozyme-based immunochromatographic technology proposed in this paper simultaneously achieves highly sensitive detection of respiratory viruses and bacteria (SARS-CoV-2SP, Streptococcus pneumoniae, and Legionella pneumophila). The detection sensitivity is greatly improved, 40 times higher than that of traditional enzyme-linked immunosorbent assay (ELISA) and 185 to 1000 times higher than that of gold nanoparticle (Au NP)-based LFA, respectively. The Fe-DAu@Ir-WGA nanozyme exhibits excellent stability, specificity, and accuracy in actual nasopharyngeal swab samples, and has great potential in the clinical diagnosis of respiratory infections.

[0042] Compared to traditional colloidal gold colorimetric immunochromatography, the nanoenzyme immunochromatography technology of the present invention has higher sensitivity. This technology is applicable to different types of target analytes (including viruses and bacteria) and has broad application prospects in fields such as clinical diagnosis.

[0043] In summary, this application proposes the Fe-DAu@Ir-WGA-LFA technology, which can efficiently capture and detect viruses and bacteria in complex samples. Using a WGA-based magnetic nanozyme multifunctional probe, it can efficiently capture and detect viruses and bacteria in complex samples. The magnetic nanozyme probe consists of three main components: large Fe₃O₄ particles provide magnetic responsiveness; a double-layer Au@Ir nanoparticle provides not only a strong colorimetric signal but also a large number of binding and catalytic sites, ensuring high catalytic activity; and a surface-conjugated wheat germ agglutinin, which overcomes the limitations of conventional LFAs, which often make it difficult to screen for antibody pairings, enabling effective broad-spectrum capture of viruses and bacteria. Fe-DAu@Ir-WGA-based LFA results can be read directly by the naked eye or with the aid of ImageJ software for colorimetric readings. The Fe-DAu@Ir-WGA technology proposed in this application has great potential and can be expanded to detect other viruses or bacteria by using Fe-DAu@Ir-WGA to modify the detection antibodies on the T line. It has broad application prospects in the detection field. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] Figure 1 Schematic diagram of the preparation of the Fe-DAu@Ir-WGA probe of Example 1 of the present invention;

[0045] Figure 2 TEM image and elemental characterization image of the Fe-DAu@Ir-WGA probe of Example 1 of the present invention;

[0046] Figure 3 The fluorescence, colorimetric, and magnetic properties of the Fe-DAu@Ir-WGAA probe of Example 1 of the present invention were verified;

[0047] Figure 4 Verification of the viral protein capture performance of Fe-DAu@Ir-WGA in Example 2 of the present invention;

[0048] Figure 5 is the viral protein capture efficiency of Fe-DAu@Ir-WGA in Example 2 of the present invention;

[0049] Figure 6 This is a flow chart of Fe-DAu@Ir-WGA-LFA detection of target viral protein in Example 2 of the present invention;

[0050] Figure 7 The antibody spraying concentration of Fe-DAu@Ir-WGA-LFA of Example 2 of the present invention is optimized;

[0051] Figure 8 The results of Fe-DAu@Ir-WGA-LFA in Example 3 of the present invention for detecting SARS-CoV-2 spike protein, L.pneumophila, and S.pneumoniae;

[0052] Figure 9 The results of detecting SARS-CoV-2 spike protein using colloidal gold and a commercial ELISA kit according to Example 3 of the present invention are as follows;

[0053] Figure 10 This is the specificity verification of Fe-DAu@Ir-WGA-LFA in Example 3 of the present invention;

[0054] Figure 11 This is the repeatability verification of Fe-DAu@Ir-WGA-LFA in Example 3 of the present invention;

[0055] Figure 12 This is the analysis result of Fe-DAu@Ir-WGA-LFA in Example 3 of the present invention on actual samples. DETAILED DESCRIPTION

[0056] The following describes the embodiments of the present invention, but the present invention is not limited thereto. The present invention is not limited to the various structures described below, and various modifications can be made within the scope of the invention. Embodiments and examples obtained by appropriately combining the technical means disclosed in different embodiments and examples are also included in the technical scope of the present invention.

[0057] In the present invention, unless otherwise specified, all raw materials / components are commercially available products known to those skilled in the art. In the examples of the present invention, any experimental conditions not specified are based on conventional conditions known to those skilled in the art or conditions recommended by the manufacturer.

[0058] Example 1

[0059] A universal magnetic nanozyme probe (Fe-DAu@Ir-WGA) for detecting bacteria and viruses consists of a well-dispersed magnetic Fe₃O₄ core, a cationic polymer as a linker layer, and two layers of Au@Ir nanoparticles. The outer layer is modified with DTNB molecules to provide abundant carboxyl groups and conjugated to wheat germ agglutinin, which can broadly recognize pathogens.

[0060] Specifically, the method includes magnetic Fe3O4 nanoparticles and a multilayer cationic polymer strong positively charged interlayer loaded on the surface of the magnetic Fe3O4 nanoparticles. The magnetic Fe3O4 nanoparticles serve as the core, and the cationic polymer is used as a connecting layer to adsorb multilayer small gold-iridium alloy nanoparticles (Au@Ir). The surface of the multilayer small gold-iridium alloy nanoparticles is coupled with wheat germ agglutinin protein molecules. The Fe3O4 nanoparticles provide magnetic response ability, the multilayer Au@Ir provides colorimetric signals as well as catalytic sites and binding sites, and the wheat germ agglutinin (WGA) provides the label with the ability to capture a broad spectrum of pathogens.

[0061] A further optimized technical solution of this embodiment is that the particle size of the Fe3O4 magnetic particles is 80 to 280 nm, preferably 160 nm.

[0062] A further optimized technical solution of this embodiment is that the particle size of the small gold-iridium alloy nanoparticles (Au@Ir) is 2 to 10 nm, preferably 5 nm.

[0063] A further optimized technical solution of this embodiment is that the number of layers of the wrapped Au@Ir nanoparticles is 1-3 layers, preferably 2 layers.

[0064] The preparation method of the above-mentioned magnetic multifunctional nanozyme probe for universal detection of bacteria and viruses based on wheat germ agglutinin is as follows: Figure 1 As shown, the following steps are included:

[0065] (1) Synthesis of magnetic Fe3O4 nanoparticles based on a classical solvothermal reaction;

[0066] (2) The Fe3O4 magnetic particles (1 mg) obtained in step (1) were added to a PEI aqueous solution (5 mg / mL) to form Fe3O4@PEI nanospheres with positive surface charge; the obtained Fe3O4@PEI nanospheres (1 mg) were added to an Au@Ir solution (20 mL), and after intense ultrasonic treatment, the negatively charged Au@Ir was adsorbed on the surface of the Fe3O4@PEI nanospheres due to electrostatic action to form Fe-Au@Ir magnetic nanospheres;

[0067] (3) The Fe-Au@Ir magnetic nanospheres (1 mg) obtained in step (2) were added to a PEI aqueous solution (5 mg / mL) to form Fe-Au@Ir-PEI nanospheres with positive surface charge; the obtained Fe-Au@Ir-PEI (1 mg) was again added to a negatively charged Au@Ir solution (20 mL), and ultrasonic treatment was performed to obtain a high-performance magnetic nanozyme Fe-DAu@Ir;

[0068] (4) The magnetic nanozyme Fe-DAu@Ir (1 mg) was mixed with DTNB ethanol solution (10 μM) and mixed and modified under intense ultrasonic conditions (80 Hz, 2 h) to obtain a nanozyme material with abundant carboxyl groups;

[0069] (5) Under intense ultrasound, the carboxyl-rich nanozyme material was mixed with a morpholinoethanesulfonic acid (MES) solution containing N-hydroxysuccinimide (NHS) and 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC) for activation. The activation treatment included: mixing 1 mg of Fe-DAu@Ir with 10 mL of activation solution and ultrasonicating at 60-100 Hz and 15-25°C for 10-20 min; wherein the activation solution contained 10 mmol / L of 2-(N-nitro)ethanesulfonic acid (MES), 1 mmol / L of 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC), and 1 mmol / L of N-hydroxysuccinimide (NHS). The activated Fe-DAu@Ir was incubated with WGA at 25°C for 3 h to couple the carboxyl end of Fe-DAu@Ir with the amino end of WGA to form a peptide bond, thereby obtaining a WGA-modified nanoprobe.

[0070] A further optimized technical solution of this embodiment is that in steps (2) and (3), the ultrasonic time is 10-30 minutes, preferably 20 minutes.

[0071] A further optimized technical solution of this embodiment is that in step (5), the added concentration of WGA is 10-40 μg, preferably 15 μg.

[0072] Figure 2 (af) are transmission electron micrographs (TEM) of Fe3O4 in step (1), transmission electron micrographs (TEM) of Fe-Au@Ir nanoparticles in step (2), transmission electron micrographs (TEM) of Fe-Au@Ir nanoparticles in step (3), and transmission electron micrographs (TEM) of Fe-Au@Ir nanoparticles in step (4). The above images show that the synthesized Fe-Au@Ir is a core-shell structure with Fe3O4 nanoparticles as the core and colloidal gold and quantum dots as the shell.

[0073] Figure 2 (gh) is the energy dispersive X-ray spectroscopy (EDS) spectrum of Fe-DAu@Ir obtained in step (3) of this embodiment. The above image shows that the synthesized Fe-DAu@Ir contains O, Fe, Ir, and Au, with Fe (red) and O (green) concentrated in the core region. Au (blue) and Ir (purple) atoms are evenly distributed in the outer layer of Fe-DAu@Ir. These characterization results indicate the successful synthesis of the designed multilayer Fe-DAu@Ir.

[0074] Figure 3 This is a diagram showing the verification results of the catalytic and magnetic properties of Fe-DAu@Ir in step (3) of this implementation case. Figure 2a The peroxidase catalytic activities of Fe3O4, Au@Ir, Fe-Au@Ir, Fe-, and DAu@Ir were compared using TMB as a chromogenic substrate. The color and external absorption spectra of the post-reaction solutions showed that in the presence of H2O2 and TMB, the colors of the four nanoparticle solutions changed from colorless to light blue and dark blue, respectively, within 10 seconds. Au@Ir nanoparticles exhibited stronger catalytic activity than Fe3O4 nanoparticles. Furthermore, as Au@Ir was adsorbed layer by layer on the Fe3O4 surface, the catalytic activity gradually increased, with Fe-DAu@Ir nanoparticles exhibiting the highest catalytic activity. Figure 2 b-2c) Enzyme kinetics experiments were used to evaluate the peroxidase activity of Fe3O4, Au@Ir, Fe-Au@Ir, and Fe-DAu@Ir nanoparticles by varying TMB and H2O2 concentrations. For H2O2, the maximum reaction rate and Michaelis constant of Fe-DAu@Ir were 11.478 Ms⁻¹ and 2.973 mM, respectively. Similarly, for TMB, the values ​​were 11.138 Ms⁻¹ and 2.698 mM, respectively. Compared with the other three nanoparticles, Fe-DAu@Ir exhibited a higher Vmax and lower Km, further demonstrating its superior peroxidase catalytic ability. Fig. 2i) DFT simulations were used to calculate the energy changes during substrate oxidation. The results indicate that loading Au@Ir NPs onto the Fe3O4 surface enhances its peroxidase activity. In addition, Fig.2d-2e evaluated the effect of pH on enzyme activity and found that the prepared Fe-DAu@Ir nanozyme can still maintain stable colorimetric reaction effect and catalytic activity within the pH range of 2 to 13. Figure 2 As shown in Figures g-2h, even after being coated with two layers of Au@Ir nanoparticles, the saturation magnetization (MS) of Fe-DAu@Ir remained high at 426 emu / mg. This excellent MS ensured that Fe-DAu@Ir could be completely separated from complex samples such as a 1 mL swab, lake water, and PBS within 1 minute. In actual separation experiments, under the influence of an external magnetic field, Fe-DAu@Ir could be completely separated from a 1 mL PBS solution within 1 minute.

[0075] Figure 4 This is the verification result of whether Fe-DAu@Ir-WGA can capture viruses and bacteria in step (5) of this implementation case. First, the immunofluorescence method was used to observe the binding ability of Fe-DAu@Ir-WGA to the virus (SARS-CoV-2SP). Figure 4The fluorescence pictures of a-b, when the nanomaterials were modified with WGA, the complexes showed green fluorescence under the excitation wavelength of 488 nm. These results indicated that only the WGA-modified Fe-DAu@Ir could capture the target virus antigens. Then, the SEMD electron microscopy was used to verify the capture ability of Fe-DAu@Ir-WGA to L. pneumophila and S. pneumoniae. According to the results of Figure 4 The electron microscopy results of C showed that the Fe-DAu@Ir-WGA labels could be combined to the surface of L. pneumophila and S. pneumoniae bacteria to form bacterial-label complexes. The complexes contained multiple Fe-DAu@Ir NPs. In addition, the capture efficiency of Fe-DAu@Ir-WGA to one virus and two bacteria was determined by plate capture and BCA protein kit, according to the results of Figure 5 The capture efficiency of Fe-DAu@Ir-WGA to the antigens of the three pathogens was all above 90%.

[0076] Example 2

[0077] Figure 6 The flow chart of detecting SARS-CoV-2 SP, L. pneumophila and S. pneumoniae based on Fe-DAu@Ir-WGA-LFA. After the Fe-DAu@Ir-WGA-LFA in experimental example 1 was incubated with SARS-CoV-2 SP, L. pneumophila and S. pneumoniae in the incubation buffer, the sample was enriched, resuspended in the running buffer and loaded on the sample pad of the immunochromatographic strip. The solution containing the Fe-DAu@Ir-WGA nanoszyme-virus / bacteria complex migrated to the T line and the C line through the capillary action of the absorption pad, and formed the nanoszyme probe-virus / bacteria complex through the antigen-antibody reaction with the capture antibodies on the T1 / T2 / T3 lines. Therefore, when the concentration of the target virus / bacteria in the sample solution was higher, the more Fe-DAu@Ir-WGA-bacteria / virus complexes were formed, and the stronger the colorimetric signal on the three T lines was. When there was no target virus / bacteria in the sample, the Fe-DAu@Ir-WGA nanoszyme probe directly passed through the three T lines and was captured by the anti-goat WGA antibody on the C line. Then, the catalytic substrate solution was dropped on the NC membrane for colorimetric signal amplification, and after 2 min of catalysis, the results on the test strip could be observed by the naked eye for rapid screening, and the colorimetric intensity on the T line could be quantitatively detected by Image J. Figure 7 The optimization results of the spraying concentration of the anti-SARS-CoV-2 SP, L. pneumophila and S. pneumoniae antibodies on the T line. When the concentration of the anti-SARS-CoV-2 SP antibody on the detection line reached 1.2 mg / mL, the concentration of the anti-L. pneumophila antibody reached 2 mg / mL, and the concentration of the anti-S. pneumoniae antibody was 1.2 mg / mL, the Fe-DAu@Ir-WGA-LFA had the highest signal-to-noise ratio for the detection of SARS-CoV-2 SP, L. pneumophila and S. pneumoniae antibodies.

[0078] Example 3

[0079] This example uses different concentrations (10-0 ng / mL / 10 5 ~ 0 cells / mL / 10 5 ~ 0 cells) of SARS-CoV-2 SP, Legionella pneumophila and Streptococcus pneumoniae antibodies to verify the detection performance of Fe-DAu@Ir-WGA-LFA. Figure 8 a(I) shows the picture of LFA test strip for simultaneous detection of one virus and two pathogenic bacteria before catalysis. Fe-DAu@Ir nanoszyme fixed on T line presents dark brown at high bacterial concentration, and the dark brown on the three T lines gradually lightens and finally becomes colorless as the concentration of SARS-CoV-2 SP / Legionella pneumophila / Streptococcus pneumoniae decreases. Before catalysis, the naked-eye visual signal of SARS-CoV-2 SP, Legionella pneumophila, Streptococcus pneumoniae is 0.5 ng / mL, 5x103 cells / mL, 5x103 cells / mL, respectively. After catalysis by AEC / H2O2, the color signal intensity on the T line is greatly amplified, and the detection range for low virus and bacterial concentration is obviously expanded. After catalysis, the visual signal of SARS-CoV-2 SP, Legionella pneumophila, Streptococcus pneumoniae is increased to 0.01 ng / mL, 10 cells / mL, 10 cells / mL, respectively. These results show that the sensitivity and detection range of WGA-based spherical nanoszyme LFA are increased by 50-150 times after catalysis. Then the colorimetric signal of the test strip in the image is directly measured by using Image J, and the signal intensity is also significantly enhanced after catalysis Figure 8 b~8c). The S-type calibration curves of SARS-CoV-2 SP, Legionella pneumophila, Streptococcus pneumoniae are shown in fig 4d~4f in direct / catalytic mode, which are established by plotting the colorimetric signal measured for each T line against the virus / bacterial concentration. The LOD of Fe-DAu@Ir-WGA-LFA detection platform is determined according to the IUPAC standard protocol (LOD = average intensity of blank control + 3x standard deviation of blank measurement). Before catalysis, the LODs of Fe-DAu@Ir-WGA-LFA for SARS-CoV-2 SP, Legionella pneumophila, Streptococcus pneumoniae are 0.1099 ng / mL, 1218 cell / mL and 1009 cell / mL, respectively. After catalysis, the LODs of the three targets are reduced to 0.0018 ng / mL, 4 cells / mL and 4 cells / mL, respectively. The dynamic range of quantitative analysis of the three detection targets is 5 orders of magnitude (0.001-10 ng / mL and 10 5 ~ 10 cells / mL), and the correlation coefficient R of SARS-CoV-2 SP, Legionella pneumophila, Streptococcus pneumoniae is 0.9999, 0.9999 and 0.9999, respectively.2 were 0.982, 0.958, and 0.985, respectively. Next, we compared the detection performance of Fe-DAu@Ir-WGA-LFA with existing POCT technologies, including Au NPs-based colorimetric LFA and ELISA kits. Figure 9 a As can be seen, the LOD of ELISA for detecting SARS-CoV-2SP is 0.21 ng / mL. In comparison, the sensitivity of our Fe-DAu@Ir-WGA-LFA in catalytic mode is at least 42 times higher than that of ELISA. In addition, Figure 9 b shows that the visual sensitivity of LFA based on gold nanoparticles (Au NP) to SARS-CoV-2SP, Legionella pneumophila, and Streptococcus pneumoniae is approximately 1 ng / mL, 5×10 3 cells / mL, 10 4 Therefore, the visualization sensitivity of Fe-DAu@Ir-WGA-LFA in direct and catalytic modes is about 2 times and 185-1000 times lower than that of the traditional AuNP-based LFA method, respectively.

[0080] At the same time, the specificity of Fe-DAu@Ir-WGA-LFA for target viruses and bacteria was also evaluated. Two common respiratory virus antigens, including influenza A virus (FluA) particles and influenza B virus (FluB) particles, as well as four important pathogenic bacteria, including Salmonella typhi (S.typhi), Staphylococcus aureus (S.aureus), Escherichia coli: O157 (E.coli: O157), and Listeria monocytogenes (L.mono) were used to test the specificity of Fe-DAu@Ir-WGA-LFA. The concentration of added pathogens was 10 5 cells / mL, and the added viral protein was 10ng / mL. Figure 10 As shown in the figure, the colorimetric and catalytic signals of all bands for non-detection targets are negligible. This result shows that the Fe-DAu@Ir-WGA nanozyme probe with broad-spectrum capture ability has high specificity. Under the same conditions, the Fe-DAu@Ir-WGA-LFA platform was used to detect mixed samples with high (10 ng / mL, 105 cells / mL, 105 cells / mL) and low (0.05 ng / mL, 5×102 cells / mL, 5×102 cells / mL) concentrations. The results are shown in Figure 5. Figure 11 In nine independent tests, the colorimetric results of the test strips before catalysis were uniform, while the signals remained stable after catalysis with a relative standard deviation (RSD < 12.4%), confirming that the established Fe-DAu@Ir-WGA-LFA-based system has good stability.

[0081] We first evaluated the stability of the established broad-spectrum capture nanoszyme LFA in real sample detection by directly adding target viruses or bacteria to the actual samples. By adding different concentrations of SARS-CoV-2 SP (10, 1, 0.1 ng / mL), Legionella pneumophila (10 5 , 10 4 , 10 3 cells / mL), Streptococcus pneumoniae (10 5 , 10 4 , 10 3 cells / mL) to healthy subjects and untreated lake water, and using the established nanoszyme LFA for detection. Figure 12 The results showed that the colorimetric signal on the T line and the intensity of the catalytic signal in the throat swab and lake water samples were consistent with the results in the PBS samples. The recovery rates of the three different targets were calculated based on the intensity of the catalytic signal on the T line. The SARS-CoV-2 SP was 82.40% to 123.42%, Legionella pneumophila was 98.38% to 104.25%, and Streptococcus pneumoniae was 99.31% to 104.95%, with RSD values less than 13.8%. These results demonstrate that the established Fe-DAu@Ir-WGA-LFA detection method has good accuracy and reliability in the detection of real samples.

[0082] In summary, a magnetic multifunctional nanoprobes (Fe-DAu@Ir) with core-dual satellite structure was proposed in the present application, and a catalytic enhanced lateral flow assay (LFA) with ultra-sensitivity and universal detection of viruses and bacteria was successfully developed by surface modification with wheat germ agglutinin (WGA). The probe has strong magnetic response and excellent peroxidase catalytic activity by loading two layers of Au@Ir NPs on 160 nm Fe3O4 NPs. In the present application, the wheat germ agglutinin modified Fe-DAu@Ir has the ability to capture viruses and bacteria with broad spectrum and high efficiency, and can efficiently enrich a variety of viruses and bacteria in complex samples (clinical samples and environmental samples) in a short time, and quantitatively detect target pathogens on the immunochromatographic detection line through specific antibodies. In the present application, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), Streptococcus pneumoniae (S. pneumoniae) and Legionella pneumophila (L. pneumophila) were used as models to test the detection performance of Fe-DAu@Ir-WGA mediated immunochromatography, which proved that the sensitivity of the method for detecting the three pathogens reached 0.0018 ng / mL, 4 cells / mL and 4 cells / mL, respectively. The sensitivity of the technology for detecting pathogens is 40 times and at least 185 times higher than that of traditional enzyme-linked immunosorbent assay (ELISA) and gold nanoparticle (Au NP) based colorimetric LFA, respectively. By testing 32 real clinical respiratory samples, the clinical application potential of Fe-DAu@Ir-WGA-LFA was verified, which showed the significant potential of the method in ultra-sensitivity and universal pathogen detection.

[0083] The preferred embodiments of the present application are described in detail above with reference to the drawings, but the present application is not limited to the specific details of the above-described embodiments. Various simple modifications can be made to the technical solutions of the present application within the technical concept of the present application, and these simple modifications all belong to the protection scope of the present application. In addition, any combination of various embodiments of the present application can also be made as long as it does not deviate from the idea of the present application, and it should also be considered as disclosed by the present application. The above is only the preferred specific implementation of the present application, but the protection scope of the present application is not limited to this. Any modification made by any person skilled in the art according to the technical solutions and inventive concept of the present application within the technical range disclosed by the present application should be covered within the protection scope of the present application.

Claims

1. A universal magnetic nanozyme probe for detecting bacteria and viruses, characterized in that: The invention comprises magnetic Fe3O4 nanoparticles and a multilayer cationic polymer strong positively charged interlayer loaded on the surface of the magnetic Fe3O4 nanoparticles. The magnetic Fe3O4 nanoparticles serve as the core, and the cationic polymer is used as a connecting layer to adsorb multilayer small gold-iridium alloy nanoparticles. The surface of the multilayer small gold-iridium alloy nanoparticles is coupled with wheat germ agglutinin protein molecules. The Fe3O4 nanoparticles provide magnetic response capability, the multilayer small gold-iridium alloy nanoparticles provide colorimetric signals as well as catalytic sites and binding sites, and the wheat germ agglutinin provides the label with the ability to capture a broad spectrum of pathogens.

2. The universal magnetic nanozyme probe for detecting bacteria and viruses according to claim 1, characterized in that: The Fe3O4 magnetic particles have a particle size of 80 to 280 nm.

3. The universal magnetic nanozyme probe for detecting bacteria and viruses according to claim 1, characterized in that: The particle size of the small gold-iridium alloy nanoparticles is 2 to 10 nm.

4. The universal magnetic nanozyme probe for detecting bacteria and viruses according to claim 1, characterized in that: The number of layers of the wrapped Au@Ir nanoparticles is 1-3.

5. The universal magnetic nanozyme probe for detecting bacteria and viruses according to claim 1, characterized in that: The cationic polymer is polyacetimide, and the polyacetimide is used to wrap multiple layers of small gold-iridium alloy nanoparticles on the surface of Fe3O4 nanoparticles.

6. A method for preparing a universal magnetic nanozyme probe for detecting bacteria and viruses, characterized in that: The method comprises the following steps: (1) Synthesize Fe3O4 magnetic particles according to the classical solvothermal reaction; (2) adding the Fe3O4 magnetic particles obtained in step (1) to a PEI aqueous solution to form Fe3O4@PEI nanospheres under ultrasonic action, and then adding the Fe3O4@PEI nanospheres to an Au@Ir solution. Through ultrasonic treatment, Au@Ir is wrapped and adsorbed on the surface of the Fe3O4@PEI nanospheres to form Fe-Au@Ir magnetic nanospheres; (3) adding the Fe-Au@Ir magnetic nanospheres obtained in step (2) into the PEI aqueous solution to form Fe-Au@Ir-PEI nanospheres under the action of ultrasound, and adding the obtained Fe-Au@Ir-PEI nanospheres into the Au@Ir solution again to obtain the high-performance magnetic nanozyme Fe-DAu@Ir through the action of ultrasound; (4) mixing the magnetic nanozyme Fe-DAu@Ir obtained in step (3) with a DTNB ethanol solution, and performing mixing modification under intense ultrasonic conditions to obtain a nanozyme material having abundant carboxyl groups; (5) Under intense ultrasonic action, the three-dimensional flexible thin film nanozyme material with abundant carboxyl groups obtained in step (4) is mixed and activated with a morpholineethanesulfonic acid solution containing N-hydroxysuccinimide and 1-ethyl-(3-dimethylaminopropyl)carbodiimide, and then incubated with wheat germ agglutinin to prepare a multifunctional magnetic nanozyme probe with spectral capture capability.

7. The method for preparing the magnetic nanozyme probe according to claim 6, wherein: In step (2), the average particle size of the Fe3O4 is 80 to 280 nm, the mass concentration of the PEI aqueous solution is 5 to 10 mg / mL, and the amount of the nanoparticles added is 1.0 to 3.5 mg per 100 mL of the PEI aqueous solution.

8. A colorimetric lateral flow reagent strip containing magnetic nanozyme probes for universal detection of bacteria and viruses, characterized in that: The kit comprises the magnetic nanozyme probe according to any one of claims 1 to 5, and the pathogen is at least one of SARS-CoV-2, L.pneumophila and S.pneumoniae.

9. The fluorescent immunochromatographic reagent strip according to claim 8, characterized in that: It also includes an immunochromatographic test strip, which includes a sample pad, a chromatographic membrane and an absorbent pad connected in sequence. Three test lines and a quality control line are provided on the chromatographic membrane. Anti-SARS-CoV-2 antibodies, anti-L.pneumophila antibodies, anti-S.pneumoniae and anti-WGA antibodies are sprayed on the test lines T1, T2, T3 and the quality control line respectively. The nitrocellulose membrane, the sample pad, the absorbent pad and the bottom plate are assembled into the immunochromatographic test strip.

10. The detection method of the fluorescent immunochromatographic reagent strip according to claim 8 or 9, characterized in that: The magnetic nanozyme probe is added to the sample solution to be tested, and the mixture is shaken with an oscillator at room temperature for 5-25 minutes; the Fe-DAu@Ir-WGA-pathogen complex is quickly separated from the sample solution with a magnet, resuspended in running buffer, and added dropwise to the sample pad of the immunochromatographic test strip. After 10 to 20 minutes, the colorimetric signals at the three detection lines of the immunochromatographic test strip are read, and then the AEC substrate is added dropwise to read the catalytic signals on the three detection lines of the test strip.

Images