Three-dimensional magnetic multi-metal nanozyme and its preparation method, immune label and detection product

Abstract

The present invention provides a three-dimensional magnetic multi-metal nanozyme and a preparation method, an immune label and a detection product, and specifically relates to the field of nanomaterial technology. The three-dimensional magnetic multi-metal nanozyme is a MoS2@Fe3O4 / Ag@AP nanosheet; the MoS2@Fe3O4 / Ag@AP nanosheet includes a MoS2 substrate layer, a first polymer layer, a Fe3O4 / Ag particle layer, a second polymer layer and an Au@Pt nanoflower layer from the bottom layer to the top layer. The three-dimensional magnetic multi-metal nanozyme has excellent superparamagnetism and peroxidase activity, and is used as a catalytic amplification sensor for detecting influenza A virus (Flu A). The purpose of the present invention is also to provide an immune label and a detection product based on the three-dimensional magnetic multi-metal nanozyme to improve the detection method and detection product based on lateral flow immunochromatography.

Description

Technical Field

[0001] The present invention relates to the technical field of nanomaterials, and in particular to a three-dimensional magnetic multi-metal nanozyme and a preparation method thereof, an immune label and a detection product. Background Art

[0002] Influenza viruses usually cause upper respiratory tract infections in humans and are transmitted through direct contact or droplets through the respiratory tract, including coughing, sneezing, talking, etc. Typical symptoms of an infected person are high fever, cough, headache, and other respiratory symptoms. Based on the antigenicity of the nucleoprotein and matrix protein, influenza viruses can be divided into four types: A, B, C, and D, but currently only influenza A (Flu A) is known to cause a global pandemic. Early and accurate diagnosis of Flu A is essential for timely treatment of patients and effective control of the epidemic.

[0003] Recently, lateral flow immunochromatography (LFIA) has attracted increasing attention in the field of point-of-care testing (POCT). Due to its advantages of rapidity, low cost, simple operation, and low sample processing volume, LFIA is considered to be the optimal detection method, especially in resource-limited areas. However, qualitative or semi-quantitative detection and limited sensitivity have greatly hindered the widespread application of LFIA.

[0004] To solve these problems, technologies such as fluorescence, surface-enhanced Raman scattering, and electrochemistry have been introduced into LFIA. Although the sensitivity has been greatly improved, the demand for detection equipment and professional technicians still limits the application of LFIA. In addition, new or improved nanomaterials have been prepared to improve the sensitivity of LFIA by increasing adsorption capacity and enhancing the signal of the test strip.

[0005] Sample pretreatment is also necessary to achieve accurate diagnosis, especially for complex clinical samples (such as serum, urine, oral / nasopharyngeal swabs), which require separation and enrichment of the target to eliminate the interference of impurities. Other reasons for the low sensitivity of LFIA detection are mainly the limited target antigens in the sample and the limited immune labels captured by the detection line of the LFIA test strip.

[0006] In view of this, the present invention is proposed. Summary of the invention

[0007] The purpose of the present invention is to provide a three-dimensional magnetic multi-metal nanozyme with excellent superparamagnetism and peroxidase activity, which is used as a catalytic amplification sensor for detecting influenza A virus (Flu A). The purpose of the present invention is also to provide an immune label and detection product based on the three-dimensional magnetic multi-metal nanozyme to improve the detection method and detection product based on lateral flow immunochromatography.

[0008] In order to achieve the above-mentioned purpose of the present invention, the following technical solutions are particularly adopted:

[0009] The first aspect of the present invention provides a three-dimensional magnetic multi-metal nanozyme, wherein the three-dimensional magnetic multi-metal nanozyme is a MoS2@Fe3O4 / Ag@AP nanosheet.

[0010] The MoS2@Fe3O4 / Ag@AP nanosheets include a MoS2 substrate layer, a first polymer layer, a Fe3O4 / Ag particle layer, a second polymer layer and an Au@Pt nanoflower layer from the bottom layer to the top layer.

[0011] Furthermore, the material of the first polymer layer and / or the second polymer layer includes polyethyleneimine.

[0012] Furthermore, in the Au@Pt nanoflower layer, the Au@Pt nanoflower has a core-shell structure, the core is made of Au, and the shell is made of Pt.

[0013] The second aspect of the present invention provides a method for preparing the three-dimensional magnetic multi-metal nanozyme, comprising the following steps:

[0014] A. Disperse MoS2 nanosheets in an aqueous solution of a polymer, and obtain MoS2 / a first polymer after a first ultrasonic treatment; wash the MoS2 / a first polymer and resuspend it, then add Fe3O4 / Ag particles, and perform a second ultrasonic treatment to electrostatically adsorb the Fe3O4 / Ag particles on the surface of the MoS2 / a first polymer to obtain MoS2@Fe3O4 / Ag.

[0015] B. Continue to disperse the MoS2@Fe3O4 / Ag in the aqueous solution of the polymer, and obtain MoS2@Fe3O4 / Ag / second polymer after the third ultrasound; wash the MoS2@Fe3O4 / Ag / second polymer and resuspend it, then add Au@Pt nanoflowers, and use the fourth ultrasound to electrostatically adsorb the Au@Pt nanoflowers on the surface of MoS2@Fe3O4 / Ag / second polymer to obtain MoS2@Fe3O4 / Ag@AP nanosheets.

[0016] Furthermore, the concentration of the aqueous solution of the polymer is 0.8-1.2 mg / mL.

[0017] The mass ratio of the MoS2 nanosheets to the polymer is 1:1.5~2.5.

[0018] The time of the first ultrasound and / or the third ultrasound is 30 to 50 minutes.

[0019] The mass ratio of the MoS2 nanosheets to the Fe3O4 / Ag particles is 1:0.4-0.8.

[0020] The time of the second ultrasound and / or the fourth ultrasound is 20 to 40 minutes.

[0021] The mass ratio of the MoS2@Fe3O4 / Ag to the polymer is 1:1.5~2.5.

[0022] The volume ratio of the MoS2@Fe3O4 / Ag to the Au@Pt nanoflower is 1:0.4~0.8.

[0023] The third aspect of the present invention provides a three-dimensional magnetic multi-metal nanozyme immune label, including one of the members of the specific binding pair and a three-dimensional magnetic multi-metal nanozyme labeled therewith;

[0024] The specific binding pairs include antigens and antibodies, enzyme inhibitors and enzymes, complementary nucleotide sequences, biotin and avidin, or cofactors and enzymes.

[0025] Furthermore, the specific binding pair is Flu A antigen and anti-Flu A antibody.

[0026] The three-dimensional magnetic multi-metal nanozyme immune label includes an anti-Flu A antibody coupled to the surface of the three-dimensional magnetic multi-metal nanozyme through an amide bond.

[0027] The fourth aspect of the present invention provides a method for preparing the three-dimensional magnetic multi-metal nanoenzyme immune label, comprising the following steps:

[0028] a. Add MoS2@Fe3O4 / Ag@AP nanosheets to the carboxyl donor solution for the fifth ultrasound treatment, and then separate by magnetic enrichment to obtain MoS2@Fe3O4 / Ag@AP-carboxyl donor.

[0029] b. Add MES buffer to the MoS2@Fe3O4 / Ag@AP-carboxyl donor to resuspend and disperse, and continue to add an activator to activate it to obtain an activated MoS2@Fe3O4 / Ag@AP-carboxyl donor.

[0030] c. The activated MoS2@Fe3O4 / Ag@AP-carboxyl donor was resuspended in PBST, and then anti-Flu A antibody was added and incubated with shaking to obtain a three-dimensional magnetic multi-metal nanozyme immunolabel.

[0031] Wherein, the carboxyl donor includes MUA.

[0032] The fifth aspect of the present invention provides a detection kit comprising the three-dimensional magnetic multi-metal nanozyme or the three-dimensional magnetic multi-metal nanozyme immune label.

[0033] A sixth aspect of the present invention provides a use of a detection kit in detecting Flu A.

[0034] Compared with the prior art, the present invention has at least the following beneficial effects:

[0035] The present invention provides a three-dimensional magnetic multi-metal nanozyme with MoS2 nanosheets as the substrate. The MoS2 nanosheets have excellent physicochemical properties and a large specific surface area, and can load more functional nanoparticles and molecules. With the assistance of polymers, Fe3O4 / Ag nanoparticles and Au@Pt nanoflowers (AP NFs) are sequentially self-assembled on the MoS2 nanosheets. Fe3O4 / Ag nanoparticles not only provide magnetism for the 3D nanozyme, but also Ag can further improve the catalytic performance of APNFs due to the LSPR between Ag and Pt. The shell (i.e., platinum) of AP NFs can provide POD enzyme-like catalytic activity in LFIA based on three-dimensional magnetic multi-metal nanozymes. Therefore, MoS2 nanosheets containing AP NFs can produce stronger colorimetric signals and POD enzyme-like activity. In addition, AP NFs loaded on the three-dimensional magnetic multi-metal nanozyme can also accelerate the oxidation of substrate TMB with H2O2 to form blue oxTMB. The three-dimensional magnetic multi-metal nanozyme combines excellent superparamagnetism, enhanced colorimetric signal and excellent POD-like enzyme activity, avoiding the interference of impurities in complex samples on the catalytic performance of the nanozyme.

[0036] The metals (gold and silver) contained in the three-dimensional magnetic multimetallic nanozyme further enhance the peroxidase-like activity of the platinum shell due to local surface plasmon resonance. Based on the magnetic separation and catalytic amplification system, the three-dimensional magnetic multimetallic nanozyme LFIA can detect Flu A nucleoprotein as low as 0.8 pg / mL in 26 minutes, with a sensitivity 125 times lower than that of commercial colloidal gold immunochromatographic test strips. In addition, the method has a detection accuracy of 100% for 20 clinical throat swab samples infected with Flu A, and a sensitivity of 140 copies / mL for inactivated Flu A virus. This multifunctional LFIA integrates magnetic separation, colorimetric analysis, and catalytic amplification, and shows great potential in rapid and accurate diagnostic methods without pretreatment and equipment. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] In order to more clearly illustrate the specific implementation methods of the present invention or the technical solutions in the prior art, the drawings required for use in the specific implementation methods or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some implementation methods of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0038] Figure 1 a in the figure is the preparation based on MoS2@Fe3O4 / Ag@AP immunolabeling. Figure 1b in the figure is the detection principle of magnetic separation and catalytic amplification system;

[0039] Figure 2 a in the figure is an electron microscope image of MoS2 nanosheets. Figure 2 b is the electron microscope image of MoS2@Fe3O4 / Ag. Figure 2 c in the figure is the electron microscope image of MoS2@Fe3O4 / Ag@AP. Figure 2 Figure d is a local magnified electron microscope image of MoS2@Fe3O4 / Ag. Figure 2 e in the figure is a local magnified electron microscope image of MoS2@Fe3O4 / Ag@AP. Figure 2 f in the figure is the Zeta potential of MoS2@Fe3O4 / Ag@AP at different preparation stages. Figure 2 g in it is the EDS spectrum of MoS2@Fe3O4 / Ag@AP;

[0040] Figure 3 a in the figure is the hysteresis curve of MoS2@Fe3O4 / Ag and MoS2@Fe3O4 / Ag@AP. Figure 3 b is the XRD diagram of MoS2@Fe3O4 / Ag and MoS2@Fe3O4 / Ag@AP. Figure 3 c in the figure is the XPS graph of MoS2@Fe3O4 / Ag@AP. Figure 3 d in the figure is the high-resolution XPS image corresponding to Fe2p in MoS2@Fe3O4 / Ag@AP. Figure 3 The e in the figure is the high-resolution XPS corresponding to Au 4f in MoS2@Fe3O4 / Ag@AP. Figure 3 f in the figure is the high-resolution XPS image corresponding to Pt 4f in MoS2@Fe3O4 / Ag@AP;

[0041] Figure 4 a in the figure is the absorption value of AP NFs prepared with different concentrations (1.3, 2.8, 4.3, 5.8 and 7.3 μM) of H2PtCl6 at 652 nm. Figure 4 b is the absorption value of MoS2@Fe3O4 / Ag@AP prepared by different AP in a at 652nm. Figure 4 c is the absorption value of MoS2@Fe3O4 / Ag@AP measured at 652nm in the colorimetric solution with different pH values. Figure 4 The d in the figure is the EPR spectra of APNFs, MoS2@Fe3O4 / Ag and MoS2@Fe3O4 / Ag@AP detected in the DMPO system. Figure 4 e in the figure is the kinetic curve of MoS2@Fe3O4 / Ag@AP measured at different H2O2 concentrations. Figure 4f in the figure is the kinetic curve of MoS2@Fe3O4 / Ag@AP measured at different TMB concentrations;

[0042] Figure 5 a in the figure is a photo of a three-dimensional magnetic multi-metal nanozyme multifunctional LFIA test strip before and after detecting the catalytic reaction of different concentrations of Flu A nucleoprotein. Figure 5 b in Figure 5 The calibration curve corresponding to a in Figure 5 c in Figure 5 d and Figure 5 Figure e in the figure is a diagram of the repeatability test results of the multifunctional LFIA test strip based on three-dimensional magnetic multi-metal nanozymes;

[0043] Figure 6 Figure a shows the results of the three-dimensional magnetic multi-metal nanozyme multifunctional LFIA test strip detecting different concentrations of inactivated Flu A virus in throat swab samples. Figure 6 Figure b is a graph showing the specific detection results of the multifunctional LFIA test strip based on three-dimensional magnetic multi-metal nanozymes. Figure 6 c in the figure is the result of testing clinical throat swab samples based on the three-dimensional magnetic multi-metal nanozyme multifunctional LFIA test strip;

[0044] Figure 7 a in the figure is the electron microscope image of AP NFs (i), MoS2@AP (ii) and MoS2@Fe3O4 / Ag@AP (iii). Figure 7 b is a photo of the LFIA test strips based on AP NFs (i), MoS2@AP (ii) and MoS2@Fe3O4 / Ag@AP (iii) for detecting Flu A. Figure 7 c in the figure is the gray value on the corresponding detection line of Flu A detected by LFIA test strips based on AP NFs (i), MoS2@AP (ii) and MoS2@Fe3O4 / Ag@AP (iii);

[0045] Figure 8 The a in the figure is the result of Biolong's commercial colloidal gold immunochromatographic test strips detecting Flu A. Figure 8 The b in the figure is the result of the commercial colloidal gold immunochromatographic test strip of Jianke Technology for detecting Flu A. Figure 8 Figure c is the result of the commercial colloidal gold immunochromatographic test strip of Runboford for detecting Flu A;

[0046] Fig. 9 This is the ROC curve of 25 clinical throat swab samples detected based on the three-dimensional magnetic multi-metal nanozyme multifunctional LFIA test strip. DETAILED DESCRIPTION

[0047] In order to make the purpose, technical solutions and advantages of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below in combination with the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all the embodiments.

[0048] Hereinafter, the terms "including", "having" and their cognates, which may be used in various embodiments of the present invention, are intended only to indicate specific features, numbers, steps, operations, elements, components or combinations of the foregoing items, and should not be understood as first excluding the existence of one or more other features, numbers, steps, operations, elements, components or combinations of the foregoing items or adding the possibility of one or more features, numbers, steps, operations, elements, components or combinations of the foregoing items.

[0049] The first aspect of the present invention provides a three-dimensional magnetic multi-metal nanozyme, wherein the three-dimensional magnetic multi-metal nanozyme is a MoS2@Fe3O4 / Ag@AP nanosheet.

[0050] The MoS2@Fe3O4 / Ag@AP nanosheets include a MoS2 substrate layer, a first polymer layer, a Fe3O4 / Ag particle layer, a second polymer layer and an Au@Pt nanoflower layer from the bottom layer to the top layer.

[0051] Compared with the prior art, the present invention has at least the following beneficial effects:

[0052] The present invention provides a three-dimensional magnetic multi-metal nanozyme with MoS2 nanosheets as the substrate. The MoS2 nanosheets have excellent physicochemical properties and a large specific surface area, and can load more functional nanoparticles and molecules. With the assistance of polymers, Fe3O4 / Ag nanoparticles and Au@Pt nanoflowers (AP NFs) are sequentially self-assembled on the MoS2 nanosheets. Fe3O4 / Ag nanoparticles not only provide magnetism for the 3D nanozyme, but also Ag can further improve the catalytic performance of APNFs due to the LSPR between Ag and Pt. The shell (i.e., platinum) of AP NFs can provide POD enzyme-like catalytic activity in LFIA based on three-dimensional magnetic multi-metal nanozymes. Therefore, MoS2 nanosheets containing AP NFs can produce stronger colorimetric signals and POD enzyme-like activity. In addition, AP NFs loaded on the three-dimensional magnetic multi-metal nanozyme can also accelerate the oxidation of substrate TMB with H2O2 to form blue oxTMB. The three-dimensional magnetic multi-metal nanozyme combines excellent superparamagnetism, enhanced colorimetric signal and excellent POD-like enzyme activity, avoiding the interference of impurities in complex samples on the catalytic performance of the nanozyme.

[0053] Nanozymes are artificial enzymes based on nanomaterials, and are a method of amplifying signals on LFIA test strips using enzyme-catalyzed reactions. The application of nanozymes has greatly improved the situation of insufficient LFIA colorimetric signals of nanomaterials, which depends on their inherent optical properties. Compared with natural enzymes, nanozymes have similar catalytic properties, but are more chemically stable, have better biocompatibility, and are less expensive.

[0054] Nanozymes can be roughly divided into two categories: 1) catalysts or enzymes fixed on nanomaterials; 2) inorganic nanomaterials with autocatalytic properties. The three-dimensional magnetic multi-metal nanozyme of the present invention belongs to the above-mentioned category 2). As a POD-like nanozyme, it can produce hydroxyl radicals (-OH) and catalyze the conversion of chromogenic substrates into oxidized colored products. It can be used for colorimetric and instrument-free detection of bacteria, viruses, toxins, proteins, etc.

[0055] Nanomaterials used to synthesize nanozymes mainly include metals (such as platinum, palladium), transition metal oxides (such as Fe3O4), covalent organic frameworks, metal organic frameworks, etc. In addition, different chromogenic substrates can be oxidized into different colors by nanozymes in solution, such as 3,3′,5,5′-tetramethylbenzidine (TMB), 3,3'-diaminobenzidine (DAB) and 3-amino-9-ethylcarbazole (AEC). Among them, TMB is usually used for colorimetric detection and can be rapidly oxidized into a blue product (oxTMB) by POD-like nanozymes in the presence of H2O2, which has the advantages of low cost, non-toxicity, and large molar extinction coefficient. Therefore, the combination of POD-like nanozymes with LFIA further improves its colorimetric sensitivity, because the color of the test strip after catalysis becomes significantly darker. In addition, multi-metal nanozymes have higher synergistic catalytic performance than single-metal nanozymes, which is due to the electronic structure of the nanozymes or the local surface plasmon resonance (LSPR) between the constituent metals.

[0056] Furthermore, the material of the first polymer layer and / or the second polymer layer includes polyethyleneimine (PEI). PEI is a positively charged high molecular polymer that can form a complex with negatively charged substances (such as DNA or certain nanoparticles) through electrostatic interaction.

[0057] Furthermore, in the Au@Pt nanoflower layer, the Au@Pt nanoflower has a core-shell structure, the core is made of Au, and the shell is made of Pt.

[0058] The second aspect of the present invention provides a method for preparing the three-dimensional magnetic multi-metal nanozyme, comprising the following steps:

[0059] A. Disperse MoS2 nanosheets in an aqueous solution of a polymer, and obtain MoS2 / a first polymer after a first ultrasonic treatment; wash the MoS2 / a first polymer and resuspend it, then add Fe3O4 / Ag particles, and perform a second ultrasonic treatment to electrostatically adsorb the Fe3O4 / Ag particles on the surface of the MoS2 / a first polymer to obtain MoS2@Fe3O4 / Ag.

[0060] B. Continue to disperse the MoS2@Fe3O4 / Ag in the aqueous solution of the polymer, and obtain MoS2@Fe3O4 / Ag / second polymer after the third ultrasound; wash the MoS2@Fe3O4 / Ag / second polymer and resuspend it, then add Au@Pt nanoflowers, and use the fourth ultrasound to electrostatically adsorb the Au@Pt nanoflowers on the surface of MoS2@Fe3O4 / Ag / second polymer to obtain MoS2@Fe3O4 / Ag@AP nanosheets.

[0061] Furthermore, the concentration of the aqueous solution of the polymer is 0.8-1.2 mg / mL.

[0062] Typically but not limiting, the concentration of the aqueous solution of the polymer may be, for example, 0.8 mg / mL, 0.9 mg / mL, 1.0 mg / mL, 1.1 mg / mL or 1.2 mg / mL, or any value within the range of 0.8 mg / mL to 1.2 mg / mL.

[0063] The mass ratio of the MoS2 nanosheets to the polymer is 1:1.5~2.5.

[0064] Typically but not limiting, the mass ratio of the MoS2 nanosheets to the polymer can be 1:1.5, 1:2, 1:2.1, 1:2.2, 1:2.3, 1:2.4 or 1:2.5, or any ratio within the range of 1:1.5 to 1:2.5.

[0065] The time of the first ultrasound and / or the third ultrasound is 30 to 50 minutes.

[0066] Typically but not limiting, the duration of the first ultrasound and / or the third ultrasound may be 30 min, 35 min, 40 min, 45 min or 50 min, or any time within the range of 30 min to 50 min.

[0067] The mass ratio of the MoS2 nanosheets to the Fe3O4 / Ag particles is 1:0.4-0.8.

[0068] Typically but not limiting, the mass ratio of the MoS2 nanosheets to the Fe3O4 / Ag particles can be 1:0.4, 1:0.5, 1:0.6, 1:0.7 or 1:0.8, or any ratio within the range of 1:0.4 to 1:0.8.

[0069] The time of the second ultrasound and / or the fourth ultrasound is 20 to 40 minutes.

[0070] Typically but not limiting, the duration of the second ultrasound and / or the fourth ultrasound may be 20 min, 25 min, 30 min, 35 min or 40 min, or any time within the range of 20 min to 40 min.

[0071] The mass ratio of the MoS2@Fe3O4 / Ag to the polymer is 1:1.5~2.5.

[0072] Typically but not limiting, the mass ratio of the MoS2@Fe3O4 / Ag to the polymer can be 1:1.5, 1:2, 1:2.1, 1:2.2, 1:2.3, 1:2.4 or 1:2.5, or any ratio within the range of 1:1.5 to 1:2.5.

[0073] The volume ratio of the MoS2@Fe3O4 / Ag to the Au@Pt nanoflower is 1:0.4~0.8.

[0074] Typically but not limiting, the volume ratio of MoS2@Fe3O4 / Ag to Au@Pt nanoflowers can be 1:0.4, 1:0.5, 1:0.6, 1:0.7 or 1:0.8, or any ratio within the range of 1:0.4 to 1:0.8.

[0075] The preparation method of Fe3O4 / Ag adopts one-pot synthesis, and the specific process is as follows:

[0076] The divalent iron salt is dissolved in a mixed solution of PVP-deionized water, and an alkaline aqueous solution is added under heating conditions; then a mixed solution of PVP-deionized water containing a soluble silver salt is added to react to obtain Fe3O4 / Ag.

[0077] Under alkaline conditions, the divalent iron salt (FeCl2·4H2O) reacts with the Ag in AgNO3 + A redox reaction occurs to form Fe3O4 and Ag particles. PVP acts as a stabilizer to help the subsequent formation of dispersed Fe3O4 / Ag nanoparticles. Through this synthesis process, Fe3O4 / Ag nanoparticles with magnetic properties and catalytic activity can be obtained.

[0078] AP NFs were prepared by ascorbic acid (AA)-mediated seed growth method. The specific preparation steps are as follows:

[0079] After the HAuCl4 solution was heated to boiling, trisodium citrate was added thereto for reduction to obtain gold seeds. Then, AA aqueous solution and H2PtCl6 solution were added to the gold seeds prepared above, and the solution was sealed and left to stand at room temperature to grow a platinum shell on the surface of Au NPs to obtain AP NFs.

[0080] The concentration of H2PtCl6 solution is 1.3~7.3μM.

[0081] Typically, but not limiting, the concentration of the H2PtCl6 solution can be 1.3 μM, 1.5 μM, 1.7 μM, 1.9 μM, 2.1 μM, 2.3 μM, 2.5 μM, 2.7 μM, 2.9 μM, 3.1 μM, 3.3 μM, 3.5 μM, 3.7 μM, 3.9 μM, 4.1 μM, 4.3 μM, 4.5 μM, 4.7 μM, 4.9 μM, 5.1 μM, 5.3 μM, 5.5 μM, 5.7 μM, 5.9 μM, 6.1 μM, 6.3 μM, 6.5 μM, 6.7 μM, 6.9 μM or 7.3 μM, or it can be any concentration value in the range of 1.3 μM to 7.3 μM.

[0082] The third aspect of the present invention provides a three-dimensional magnetic multi-metal nanozyme immune label, including one of the members of the specific binding pair and a three-dimensional magnetic multi-metal nanozyme labeled therewith;

[0083] The specific binding pairs include antigens and antibodies, enzyme inhibitors and enzymes, complementary nucleotide sequences, biotin and avidin, or cofactors and enzymes.

[0084] In one embodiment of the invention, one of the members of the specific binding pair is an antibody (fixed on the test strip test line), and a three-dimensional magnetic multi-metal nanozyme immunolabel that labels it, and the antibody can recognize and bind to the antigen. When a sample containing these pathogens comes into contact with the three-dimensional magnetic multi-metal nanozyme immunolabel, the pathogens are captured by the specific antibodies on the test line, and the results are usually indicated by color changes. If the test line changes color, it indicates that the pathogens are present in the sample; if there is no color change, it indicates that these pathogens have not been detected in the sample.

[0085] Furthermore, the specific binding pair is Flu A antigen and anti-Flu A antibody.

[0086] The three-dimensional magnetic multi-metal nanozyme immune label includes an anti-Flu A antibody coupled to the surface of the three-dimensional magnetic multi-metal nanozyme through an amide bond.

[0087] The metals (gold and silver) contained in the three-dimensional magnetic multimetallic nanozyme further enhance the peroxidase-like activity of the platinum shell due to local surface plasmon resonance. Based on the magnetic separation and catalytic amplification system, the three-dimensional magnetic multimetallic nanozyme LFIA can detect Flu A nucleoprotein as low as 0.8 pg / mL in 26 minutes, with a sensitivity 125 times lower than that of commercial colloidal gold immunochromatographic test strips. In addition, the method has a detection accuracy of 100% for 20 clinical throat swab samples infected with Flu A, and a sensitivity of 140 copies / mL for inactivated Flu A virus. This multifunctional LFIA integrates magnetic separation, colorimetric analysis, and catalytic amplification, and shows great potential in rapid and accurate diagnostic methods without pretreatment and equipment.

[0088] The fourth aspect of the present invention provides a method for preparing the three-dimensional magnetic multi-metal nanoenzyme immune label, comprising the following steps:

[0089] a. Add MoS2@Fe3O4 / Ag@AP nanosheets to the carboxyl donor solution for the fifth ultrasound treatment, and then separate by magnetic enrichment to obtain MoS2@Fe3O4 / Ag@AP-carboxyl donor.

[0090] b. Add MES buffer to the MoS2@Fe3O4 / Ag@AP-carboxyl donor to resuspend and disperse, and continue to add an activator to activate it to obtain an activated MoS2@Fe3O4 / Ag@AP-carboxyl donor.

[0091] c. The activated MoS2@Fe3O4 / Ag@AP-carboxyl donor was resuspended in PBST, and then anti-Flu A antibody was added and incubated with shaking to obtain a three-dimensional magnetic multi-metal nanozyme immunolabel.

[0092] Wherein, the carboxyl donor includes MUA.

[0093] The concentration of the carboxyl donor is 8-12 mM.

[0094] Typically but not limiting, the concentration of the carboxyl donor may be 8 mM, 9 mM, 10 mM, 11 mM or 12 mM, or any concentration value within the range of 8 mM to 12 mM.

[0095] The fifth ultrasound lasts for 40 to 80 minutes.

[0096] Typically but not limiting, the fifth ultrasound duration may be 40 min, 45 min, 50 min, 60 min, 70 min or 80 min, or any time within the range of 40 min to 80 min.

[0097] The activation is to enhance the activity of the carboxyl groups on the surface of the three-dimensional magnetic multi-metal nanozyme. The activator of the activator solution is a mixture of EDC and NHS. EDC is 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide, and NHS is N-hydroxysuccinimide. EDC and NHS activate the carboxyl groups to form active NHS esters, which can react with primary amines (-NH2) to form stable amide bonds. After that, the anti-Flu A antibody is covalently bound to the surface of the three-dimensional magnetic multi-metal nanozyme through amide bonds.

[0098] Synthesis of three-dimensional magnetic MoS2@Fe3O4 / Ag@AP Figure 1 In short, Figure 1 As shown in a, MoS2 nanosheets were used as two-dimensional substrates and layer-by-layer self-assembly was performed with the help of PEI. First, Fe3O4 / Ag and AP NFs were prepared by redox method and AA-mediated reduction method, respectively. Under ultrasonic conditions, Fe3O4 / Ag and AP NFs were successively adsorbed on the two-dimensional MoS2 nanosheets. It is worth noting that in the process of preparing Fe3O4 / Ag nanoparticles, AgNO3 was not only used as an oxidant, but also its oxidation product (Ag) also enhanced the catalytic activity of AP NFs in MoS2@Fe3O4 / Ag@AP due to the effect of LSPR. Similarly, the core Au of AP NFs can not only provide growth sites for the platinum shell, but also further improve the POD-like enzyme activity of platinum. Then, anti-Flu A antibody was modified on the three-dimensional magnetic MoS2@Fe3O4 / Ag@AP to prepare functional immune labels.

[0099] The detection process of LFIA magnetic separation and catalytic amplification system based on three-dimensional magnetic multimetallic nanozymes is as follows Figure 1 As shown in b in the figure. Specifically, the prepared tags are added to the collected throat swab samples to specifically identify and capture Flu A in the samples to obtain tags-Flu A immune complex. The antibodies pre-coated on the test line can specifically capture the tags-Flu A complex to form a sandwich structure of tags-virus-antibody. After the chromatography reaction is completed, the color development liquid is dropped on the test line of the test strip. Due to the catalytic properties of MoS2@Fe3O4 / Ag@AP, the color development signal is amplified. The smartphone captures the enhanced colorimetric signal and analyzes it through ImageJ software to quantitatively detect Flu A.

[0100] The fifth aspect of the present invention provides a detection kit comprising the three-dimensional magnetic multi-metal nanozyme or the three-dimensional magnetic multi-metal nanozyme immune label.

[0101] In one embodiment of the present invention, the LFIA test strip consists of four parts: a PVC base plate, an absorbent pad, a nitrocellulose membrane (CN140) and a sample pad. It is worth noting that there are two lines (a quality control line and a test line) on the nitrocellulose membrane. Goat anti-mouse IgG (1 mg / mL) and anti-Flu A antibody (1 mg / mL) were sprayed onto the quality control line and the test line respectively using a Biodot XYZ3050 film streaker at a speed of 1 μL / cm. Finally, the absorbent pad, nitrocellulose membrane and sample pad were assembled on the PVC base plate in sequence at a spacing of 2 mm, and cut into a width of 3 mm for standby use.

[0102] A sixth aspect of the present invention provides a use of a detection kit in detecting Flu A.

[0103] A method for detecting Flu A comprises the following steps:

[0104] The three-dimensional magnetic multi-metal nanozyme immune label is added to the solution to be detected, incubated, and then the product is magnetically enriched to obtain the Flu A nucleoprotein-immune label complex;

[0105] The Flu A nucleoprotein-immunolabel complex was resuspended in a buffer solution and dropped onto the sample pad of the LFIA test strip. After 15 minutes of chromatography reaction, a colorimetric solution (10 μL 50 mM HAc, 1 μL 4 mM H2O2, and 1 μL 2 mM TMB) was added to the detection line, and the colorimetric signal at the detection line of the LFIA test strip was observed;

[0106] The colorimetric signal is converted into a grayscale value through software to achieve quantitative detection of Flu A.

[0107] The technical solution of the present invention will be clearly and completely described below in conjunction with the embodiments. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0108] Example 1

[0109] This embodiment provides a three-dimensional magnetic multi-metal nanozyme, and the synthesis process is as follows:

[0110] 1. Dissolve 3.75 mM FeCl2·4H2O in 150 mL deionized water containing 20 mM PVP. After heating to 90°C, add 10 mL 1M NaOH aqueous solution under stirring. When the temperature is heated to 90°C again, add 40 mL water containing 2.5 mM AgNO3 and 5 mM PVP to the mixed solution and continue heating for 1 hour. The product is washed with deionized water and anhydrous ethanol three times to remove impurities. Finally, the synthesized Fe3O4 / Ag is dried at 60°C and sealed for storage.

[0111] 2. After 100 mL of 0.97 mM HAuCl4 was heated to boiling, 3 mL of 1 wt% trisodium citrate was added thereto, heated and stirred for 15 min, then the heat was turned off and stirred to room temperature to obtain gold seeds. Then, 10 mL of 100 mM AA aqueous solution and 15 mL of 1 wt% H2PtCl6 were added to the gold seeds prepared above, sealed and allowed to stand at room temperature for 1 hour to grow platinum shells on the surface of Au NPs to obtain AP NFs.

[0112] 3. Disperse 5 mg of MoS2 nanosheets in 10 mL of deionized water and sonicate for 10 minutes. Discard the smaller MoS2 nanosheets after centrifugation at 7000 rcf for 8 minutes. Resuspend the precipitate in 10 mL of PEI (1 mg / mL) aqueous solution and sonicate for 40 minutes to coat the first layer of positively charged PEI (MoS2 / PEI) on the surface of MoS2. Wash the synthesized MoS2 / PEI twice with deionized water by centrifugation (7000 rcf, 8 minutes) and resuspend it with 10 mL of deionized water. Then, add 0.5 mL of Fe3O4 / Ag (5 mg / mL) prepared by the above method to the above MoS2 / PEI solution and continue sonication for 30 minutes. Negatively charged Fe3O4 / Ag can be adsorbed on the surface of MoS2 / PEI by electrostatic force to form MoS2@Fe3O4 / Ag. The prepared MoS2@Fe3O4 / Ag was magnetically enriched and washed twice with deionized water. Then, the second layer of PEI was adsorbed on MoS2@Fe3O4 / Ag in the same way as the first layer. After washing with deionized water, the synthesized MoS2@Fe3O4 / Ag / PEI was resuspended in 10 mL of water. Then 4 mL of APNFs was added and ultrasonicated for 30 min. Finally, the prepared MoS2@Fe3O4 / Ag@AP was washed twice with deionized water and stored in 5 mL of ethanol.

[0113] Example 2

[0114] This embodiment provides a three-dimensional magnetic multi-metal nanoenzyme immunolabel, and the preparation process is as follows:

[0115] 1. Add 80 μL of MUA ethanol solution (10 mM) to 1 mL of MoS2@Fe3O4 / Ag@AP obtained in Example 1 and sonicate for 1 hour. After magnetic enrichment, resuspend the precipitate with 500 μL of MES buffer (100 mM, pH 5.5) and sonicate for several seconds to disperse it evenly.

[0116] 2. Add a mixture of EDC and NHS (1mM / 2mM) to the above solution and activate MoS2@Fe3O4 / Ag@AP under ultrasound for 15 minutes. Then separate the activated MoS2@Fe3O4 / Ag@AP with an external magnet and resuspend it in 200 μL of PBST (10 mM, 0.05wt% Tween-20). Then add 10 μg of anti-Flu A antibody and incubate at room temperature with shaking for 2.5 hours. Add 100 μL of 10% BSA and continue to incubate at room temperature with shaking for 1 hour.

[0117] 3. After washing with 1 mL PBST, a three-dimensional magnetic multi-metal nanozyme immunolabel was obtained. The three-dimensional magnetic multi-metal nanozyme immunolabel was stored in 200 μL storage solution (10 mM PBS, containing 1wt% BSA, 0.5wt% sucrose).

[0118] Example 3

[0119] This embodiment provides a LFIA test strip based on a three-dimensional magnetic multi-metal nanoenzyme immunolabel. The LFIA test strip consists of four parts: a PVC base plate, a water-absorbing pad, a nitrocellulose membrane (CN140) and a sample pad. It is worth noting that there are two lines (quality control line and detection line) on the nitrocellulose membrane. Use a Biodot XYZ3050 film stripper to spray goat anti-mouse IgG (1 mg / mL) and anti-Flu A antibody (1 mg / mL) onto the quality control line and the detection line at a speed of 1 μL / cm. Finally, the water-absorbing pad, nitrocellulose membrane and sample pad are assembled on the PVC base plate in sequence at a spacing of 2 mm, and cut into a width of 3 mm for standby use.

[0120] Test Example 1

[0121] In order to analyze the sensitivity of the method, a series of different concentrations (0.001-50 ng / mL) of Flu A nucleoprotein were prepared as standard samples.

[0122] First, 1 mL of Flu A nucleoprotein dilution was added to the test tube, and then 1.2 μL of the three-dimensional magnetic multi-metal nanozyme immunolabel (tags) obtained in Example 2 was added. After incubation for 10 minutes, the Flu A nucleoprotein-immunolabel complex was magnetically enriched and resuspended with 70 μL of loading buffer (10 mM PBS, containing 1wt% Tween-20 and 1wt% BSA).

[0123] Then, the mixture was dripped onto the sample pad of the LFIA test strip prepared in Example 3. After 15 minutes of chromatography reaction, 1 μL of colorimetric solution (10 μL of 50 mM HAc, 1 μL of 4 mM H2O2, and 1 μL of 2 mM TMB) was dripped onto the test line. After 30 seconds, the colorimetric signal of the test line was greatly enhanced.

[0124] It is worth noting that the colorimetric signal can be judged by the naked eye or quantified with the help of a smartphone. In short, a photo of the test strip was taken with a smartphone, and the grayscale value of the test strip was analyzed using ImageJ software. The average grayscale value on the detection line was regarded as the optical intensity of the test strip. Each experiment was performed three times.

[0125] Test Example 2

[0126] This test case tests inactivated Flu A virus and clinical throat swab samples.

[0127] To demonstrate the clinical applicability of the method, inactivated viruses and 20 clinical samples were tested using the 3D magnetic multimetal nanozyme-based LFIA strip. Flu A (H1N1 2009 / A) was cultured in chicken embryos in our laboratory and quantified by PCR, while clinical throat swab samples infected with Flu A were collected from the Capital Institute of Pediatrics with approval from the Ethics Committee of the Capital Institute of Pediatrics (Registration No. SHERLL-2024-033).

[0128] First, add the inactivated Flu A virus to 1 mL of lysis buffer and perform gradient dilutions with the lysis buffer. Then add 1.2 μL of immune tags to capture the nucleoprotein of the virus to form a nucleoprotein-immune tag complex. Similarly, the inactivated virus solution and tags are incubated for 15 minutes, enriched and resuspended with loading buffer. The chromatography and catalytic color development process on the test strip is the same as above.

[0129] The clinical sample was first diluted 10-fold with lysis buffer and then incubated with 1.2 μL tags for 15 minutes. The other detection steps were the same as above. Photos of the test strips were recorded with a smartphone and analyzed with ImageJ software.

[0130] Characterization Example 1

[0131] Transmission electron microscopy (TEM) images of MoS2 nanosheets are shown in Figure 2. Figure 2 As shown in a in Figure 1. First, thin MoS2 nanosheets (400-800 nm) were selected by centrifugation, and then the first layer of PEI was coated to attach Fe3O4 / Ag magnetic nanoparticles. Due to the presence of positively charged PEI, the negatively charged MoS2 nanosheets were transformed into positively charged. Therefore, the positively charged MoS2 / PEI can adsorb the negatively charged Fe3O4 / Ag magnetic nanoparticles (30 nm). Figure 2 Figure b shows that many small Fe3O4 / Ag magnetic nanoparticles are attached to the substrate of MoS2 nanosheets. Then, a second layer of PEI is coated on the surface of MoS2@Fe3O4 / Ag to adsorb AP NFs with nanozyme properties. Figure 2 As shown in (c), the surface of MoS2@Fe3O4 / Ag is filled with negatively charged AP NFs due to electrostatic adsorption. The enlarged TEM image of MoS2@Fe3O4 / Ag@AP shows that AP NFs are not only directly attached to the MoS2 surface, but also attached to the Fe3O4 / Ag nanoparticles ( Figure 2 In addition, the zeta potential of the products at different stages was also recorded, such as Figure 2 As shown in f. It can be seen that the Zeta potentials of MoS2, Fe3O4 / Ag and AP NFs are all negative, namely -38.7, -17.3 and -39.4 mV, respectively. It is worth noting that when the first layer of PEI is coated on MoS2, the zeta potential of MoS2 / PEI soars from -38.7 mV to 43.6 mV, but after the addition of Fe3O4 / Ag, it drops to -21.1 mV. In addition, the zeta potential of MoS2@Fe3O4 / Ag / PEI is 38.1 mV after covering the second layer of PEI, and drops to -36.7 mV after forming MoS2@Fe3O4 / Ag@AP. This regular change in the Zeta potential just confirms the successful synthesis of MoS2@Fe3O4 / Ag@AP. In addition, the energy dispersive spectroscopic analysis (EDS) of MoS2@Fe3O4 / Ag@AP showed the distribution of Mo, S, Fe, O, Ag, Au, and Pt atoms, indicating that Fe3O4 / Ag and AP NFs were successfully assembled on MoS2 ( Figure 2 g in the figure).

[0132] In addition, the magnetic properties of the prepared composites were also measured. Figure 3 As shown in a, the magnetic saturation (MS) value of MoS2@Fe3O4 / Ag is 52.15 emu / g. However, the MS value of MoS2@Fe3O4 / Ag@AP is greatly reduced to only 25.05 emu / g, which is due to the dense adsorption of non-magnetic elements AP NFs. Figure 3 The photo in a shows that the synthesized MoS2@Fe3O4 / Ag@AP can be separated from the solution by an external magnet within 40 s. Figure 3 As shown in (b), the X-ray diffraction (XRD) patterns of MoS2@Fe3O4 / Ag and MoS2@Fe3O4 / Ag@AP were measured to analyze their structures and crystal phases. The diffraction peaks of MoS2@Fe3O4 / Ag and MoS2@Fe3O4 / Ag@AP can be indexed into the corresponding powder diffraction file (PDF) database. Specifically, Figure 3 The main peaks of the green curve (MoS2@Fe3O4 / Ag) at 16.71° and 46.25° in (b) are indexed to the corresponding reflections of the 001 and 103 crystal planes of MoS2 (PDF#37-1492). The main peaks at 35.17°, 41.49° and 74.34° can be respectively corresponded to the reflections of the 220, 222 and 440 crystal planes of Fe3O4 (PDF#88-0315), while the main peak at 44.57° corresponds to the 111 reflection of Ag (PDF#04-0783). In addition, the main peaks of the purple curve (MoS2@Fe3O4 / Ag@AP) at 46.52°, 54.26° and 80.3° correspond to the reflections of the 111, 200 and 220 crystal planes of Pt (PDF#04-0802).

[0133] also, Figure 3 The X-ray photoelectron spectroscopy (XPS) graph shown in Figure c shows that elements such as Mo, S, Fe, O, Ag, Au and Pt coexist in the MoS2@Fe3O4 / Ag@AP composite material. In particular, Figure 3 The high-resolution Fe peaks at 710.29 and 723.56 eV shown in (d) correspond to Fe 2+ 2p 3 / 2 and 2p 1 / 2 (17.93%), the peaks at 711.44 and 725.02 eV correspond to Fe 3+ 2p 3 / 2 and 2p 1 / 2 (82.07%). In addition, the high-resolution Au peaks at 84.02 and 87.65 eV are the 4f 7 / 2 and 4f 5 / 2 Features ( Figure 3 Similarly, Figure 3 The peaks at 71.05 and 74.39 eV shown in f correspond to the 4f of Pt0 7 / 2 and 4f 5 / 2 (100%), which indicates that the catalytic performance of MoS2@Fe3O4 / Ag@AP is excellent because the valence state of platinum in it is 0.

[0134] Test Example 3

[0135] This test example tests the catalytic performance of three-dimensional magnetic multi-metal nanozymes. In order to make the synthesized MoS2@Fe3O4 / Ag@AP have good catalytic performance, platinum shells of different thicknesses coated on gold nanoparticles were first prepared. Figure 4 As shown in a, the absorbance at 652 nm increases with the increase of H2PtCl6 concentration, but when the H2PtCl6 concentration is higher than 4.3 μM, the absorbance decreases. In addition, these AP NFs with different platinum shell thicknesses were used to synthesize MoS2@Fe3O4 / Ag@AP, and the corresponding absorbance at 652 nm is shown in Figure 4 As shown in b. Obviously, when the concentration of H2PtCl6 is 4.3 μM, the absorbance value of MoS2@Fe3O4 / Ag@AP is the highest. Therefore, in the following experiments, we chose 4.3 μM H2PtCl6 to grow the platinum shell on the gold nanoparticles.

[0136] The pH value of the colorimetric solution greatly affects the sensitivity of nanozyme-based detection. Figure 4 Figure c shows that the three-dimensional magnetic MoS2@Fe3O4 / Ag@AP exhibits excellent POD enzyme-like performance in the pH range of 2 to 6, especially at pH 3. In addition, the electron spin resonance (EPR) of AP NFs, MoS2@Fe3O4 / Ag, and MoS2@Fe3O4 / Ag@AP was also studied to analyze their POD enzyme-like performance. Figure 4 As shown in (d), MoS2@Fe3O4 / Ag@AP showed the highest reactive oxygen species (ROS) (-OH) in the 5,5-dimethyl-1-pyrroline oxide (DMPO) system, indicating that MoS2@Fe3O4 / Ag@AP has excellent POD-like enzyme activity. Using the Michaelis-Menten model, the steady-state kinetic analysis of the POD-like enzyme activity of MoS2@Fe3O4 / Ag@AP was also carried out using TMB and H2O2 detection systems, respectively ( Figure 4 e in and f in 4). Specifically, by detecting the absorbance at the corresponding wavelength of 652 nm under different substrate concentrations (H2O2 and TMB), a typical steady-state kinetic curve was drawn. By calculation, the Michaelis-Menten constant (Km) values ​​of MoS2@Fe3O4 / Ag@AP for H2O2 and TMB were 4.48 and 1.11 mM, respectively, indicating that MoS2@Fe3O4 / Ag@AP has a higher affinity for TMB. In addition, the corresponding maximum reaction rates (Vmax) of H2O2 and TMB were 2.25 and 2.04*10 -6M / S. The Km and Vmax values ​​of MoS2@Fe3O4 / Ag@AP, HRP and other related POD-like nanozymes are listed in Table 1. In comparison, MoS2@Fe3O4 / Ag@AP shows stronger catalytic performance than HRP and most reported POD-like nanozymes.

[0137] Table 1 Comparison of kinetic parameters of MoS2@Fe3O4 / Ag@AP, HRP and reported POD-like nanozymes

[0138]

[0139] Test Example 4

[0140] Ultrasensitive detection of Flu A by LFIA based on three-dimensional magnetic multimetallic nanozymes. MoS2@Fe3O4 / Ag@AP has excellent magnetic enrichment and catalytic properties. Therefore, the immunolabel prepared by three-dimensional magnetic MoS2@Fe3O4 / Ag@AP can capture and enrich Flu A virus from clinical throat swab samples without pretreatment. The immunolabel can specifically recognize Flu A virus and form tags-virus complexes. The formed complex can be separated from impurities in the sample by magnetic enrichment. Using the POD-like enzyme activity of MoS2@Fe3O4 / Ag@AP, the colorimetric solution (TMB / H2O2 system) changes from white to blue on the test line of the test strip because TMB is oxidized to oxTMB under the action of H2O2. Therefore, the use of the colorimetric solution can greatly improve the light colorimetric or colorless on the test line, especially when the virus concentration in the sample is low.

[0141] In order to achieve ultrasensitive detection of Flu A using LFIA based on three-dimensional magnetic MoS2@Fe3O4 / Ag@AP, several key parameters in the detection step were optimized, including the composition and content of the colorimetric solution, loading buffer, antibody concentration on the detection line, and the amount of immunolabel added.

[0142] Under optimal conditions, we established a multifunctional LFIA for ultrasensitive detection of Flu A based on three-dimensional magnetic multimetal nanozymes, which integrates magnetism, colorimetric analysis, and catalytic amplification. Then, a series of different concentrations of FluA nucleoprotein were prepared to test the sensitivity and quantitative ability of this multifunctional LFIA test strip. Figure 5As shown in a, before the addition of the colorimetric solution (TMB / H2O2 system), the detection line visible to the naked eye corresponds to a virus concentration of 0.05 ng / mL. Compared with other detection lines, the 50 ng / mL detection line is relatively thin because the immune labels are mostly concentrated at the bottom. It is worth noting that after the addition of the colorimetric solution, the color on the detection line deepened and the visualization sensitivity increased by 10 times (5pg / mL). In addition, the colorimetric signal amplified by the detection line under catalysis was also analyzed using ImageJ software. The grayscale value of the detection line and the corresponding concentration (0.001-50 ng / mL) of the standard antigen (Flu A nucleoprotein) were used to draw the calibration curve of Flu A ( Figure 5 The minimum detection limit (LOD) of the multifunctional LFIA test strip for Flu A nucleoprotein was calculated to be 0.8 pg / mL (defined as y blank +3*SD blank ,y blank is the average intensity value of the blank control group, SD blank is the standard deviation of the blank control group). In addition, the performances of APNFs, MoS2@AP, and multifunctional LFIA strips based on MoS2@Fe3O4 / Ag@AP were evaluated. Figure 7 As shown. Figure 7 As shown in b, when detecting low concentration Flu A nuclear protein, the color of the detection line corresponding to the MoS2@Fe3O4 / Ag@AP immunolabeling is darker than that of the detection line using MoS2@AP and AP NFs immunolabeling. Figure 7 (c), the sensitivity of the LFIA strip based on MoS2@Fe3O4 / Ag@AP is 0.8 pg / mL, which is 10 times and 100 times lower than that of the LFIA based on MoS2@AP (10 pg / mL) and the LFIA based on AP (100 pg / mL), respectively. In addition, we also used three different companies ( Figure 8 The a in it stands for Boaolong, Figure 8 The b in it stands for Simple Technology and Figure 8 The commercial colloidal gold LFIA strips (c in the figure) of Runboford were used to detect the same concentration of Flu A nucleoprotein. The photos show that the visualization sensitivity of these three commercial test strips is 100pg / mL, which is 125 times lower than the multifunctional LFIA test strips we proposed. The above results show that the LFIA based on three-dimensional magnetic multimetallic nanozymes has high sensitivity and quantitative detection performance in detecting Flu A.

[0143] In addition, the stability of the proposed 3D magnetic multimetallic nanozyme-based LFIA strips was also verified. Figure 5 c in Figure 5 d and Figure 5Figure e shows that the grayscale values ​​on each test line are similar. The relative standard deviation (RSD) values ​​of 5, 0.05, and 0.005 ng / mL Flu A nucleoprotein were calculated to be 6.74%, 4.73%, and 4.19%, respectively, indicating that the method has high reproducibility.

[0144] Test Example 5

[0145] To further evaluate the clinical applicability of this method, we used the 3D magnetic multi-metal nanozyme-based LFIA test strip to detect a series of inactivated Flu A virus solutions of different concentrations added to throat swab samples.

[0146] like Figure 6 As shown in a, before the catalytic reaction, the visualization sensitivity of the inactivated Flu A (H1N1) virus was 5*10 3 copies / mL. After the catalytic reaction, the calibration curve was drawn using the grayscale value on the detection line and the corresponding concentration of inactivated Flu A virus. By calculation, the detection limit of the inactivated Flu A virus based on the three-dimensional magnetic multi-metal nanozyme LFIA was 140 copies / mL. In addition, several common respiratory viruses, including H1N1, H7N9, Flu B, SARS-CoV-2, SARS-CoV, MERS, ADV, PIV-I~III and RSV, were selected to verify the specificity of the proposed method. Figure 6 The photo of the relevant test strip in b shows that, except for low-concentration Flu A subtypes (H1N1 and H7N9), the detection lines of high-concentration non-target respiratory viruses are not visible. In addition, the grayscale values ​​on the detection lines of the corresponding test strips show that there is a significant difference between the Flu A subtype and the blank control, indicating that the method has excellent specificity.

[0147] In addition, the LFIA test strip based on three-dimensional magnetic MoS2@Fe3O4 / Ag@AP also tested 20 clinical positive throat swab samples and 5 negative throat swab samples collected from the Capital Institute of Pediatrics. The obtained samples were diluted 10 times with lysis buffer and then tested with the prepared test strip. The photos and grayscale values ​​of the corresponding test strips are shown in Figure 2. Figure 6 In addition, Fig. 9 The corresponding ROC curves of 20 positive samples and 5 negative samples were also drawn, and the results showed that the diagnostic accuracy of influenza A was extremely high, with the area under the curve (AUC) reaching 100%. The results showed that the LFIA test strips could detect all positive samples, indicating that it has extremely high precision and accuracy.

[0148] Finally, it should be noted that the above-described embodiments are only specific implementations of the present invention, which are used to illustrate the technical solutions of the present invention, rather than to limit them. The protection scope of the present invention is not limited thereto. Although the present invention is described in detail with reference to the above-described embodiments, ordinary technicians in the field should understand that any technician familiar with the technical field can still modify the technical solutions recorded in the above-described embodiments within the technical scope disclosed by the present invention, or can easily think of changes, or make equivalent replacements for some of the technical features therein; and these modifications, changes or replacements do not make the essence of the corresponding technical solutions deviate from the spirit and scope of the technical solutions of the embodiments of the present invention, and should be included in the protection scope of the present invention. Therefore, the protection scope of the present invention shall be based on the protection scope of the claims.

Claims

1. A method for preparing a three-dimensional magnetic multi-metal nanozyme, characterized in that: The three-dimensional magnetic multi-metal nanozyme is a MoS2@Fe3O4 / Ag@AP nanosheet; the MoS2@Fe3O4 / Ag@AP nanosheet includes a MoS2 substrate layer, a PEI layer, a Fe3O4 / Ag particle layer, a PEI layer and an Au@Pt nanoflower layer from the bottom to the top; The preparation method comprises the following steps: A. Dispersing MoS2 nanosheets in an aqueous solution of PEI, and obtaining MoS2 / PEI after a first ultrasonic treatment; washing the MoS2 / PEI and resuspending it, and then adding Fe3O4 / Ag particles, and subjecting the Fe3O4 / Ag particles to electrostatic adsorption on the surface of MoS2 / PEI by a second ultrasonic treatment, to obtain MoS2@Fe3O4 / Ag; B. Continue to disperse the MoS2@Fe3O4 / Ag in the aqueous solution of PEI, and obtain MoS2@Fe3O4 / Ag / PEI after the third ultrasound; wash the MoS2@Fe3O4 / Ag / PEI and resuspend it, then add Au@Pt nanoflowers, and use the fourth ultrasound to electrostatically adsorb the Au@Pt nanoflowers on the surface of MoS2@Fe3O4 / Ag / PEI to obtain MoS2@Fe3O4 / Ag@AP nanosheets.

2. The preparation method according to claim 1, characterized in that: In the Au@Pt nanoflower layer, the Au@Pt nanoflower has a core-shell structure, the core is made of Au, and the shell is made of Pt.

3. The preparation method according to claim 1, characterized in that: The concentration of the PEI aqueous solution is 0.8-1.2 mg / mL; The mass ratio of the MoS2 nanosheets to the PEI is 1:1.5-2.5; The duration of the first ultrasound and / or the third ultrasound is 30 to 50 minutes; The mass ratio of the MoS2 nanosheets to the Fe3O4 / Ag particles is 1:0.4-0.8; The duration of the second ultrasound and / or the fourth ultrasound is 20 to 40 minutes; The mass ratio of the MoS2@Fe3O4 / Ag to the PEI is 1:1.5-2.5; The volume ratio of the MoS2@Fe3O4 / Ag to the Au@Pt nanoflower is 1:0.4~0.

8.

4. A three-dimensional magnetic multi-metal nanoenzyme immunolabel, characterized in that: A three-dimensional magnetic multi-metal nanozyme prepared by the preparation method according to any one of claims 1 to 3, comprising one of the members of the specific binding pair and labeling the member; The specific binding pairs include antigens and antibodies, enzyme inhibitors and enzymes, complementary nucleotide sequences, biotin and avidin, or cofactors and enzymes.

5. The three-dimensional magnetic multi-metal nanoenzyme immunolabel according to claim 4, characterized in that: The specific binding pair is Flu A antigen and anti-Flu A antibody; The three-dimensional magnetic multi-metal nanozyme immune label includes an anti-Flu A antibody coupled to the surface of the three-dimensional magnetic multi-metal nanozyme through an amide bond.

6. A method for preparing the three-dimensional magnetic multi-metal nanoenzyme immunolabel according to claim 5, characterized in that: The following steps are involved: a. Add MoS2@Fe3O4 / Ag@AP nanosheets to the carboxyl donor solution for the fifth ultrasonic treatment, and then perform magnetic enrichment and separation to obtain MoS2@Fe3O4 / Ag@AP-carboxyl donor; b. Add MES buffer to the MoS2@Fe3O4 / Ag@AP-carboxyl donor to resuspend and disperse, and continue to add an activator to activate it to obtain an activated MoS2@Fe3O4 / Ag@AP-carboxyl donor; c. The activated MoS2@Fe3O4 / Ag@AP-carboxyl donor was resuspended in PBST, and then anti-Flu A antibody was added and incubated with shaking to obtain a three-dimensional magnetic multi-metal nanozyme immunolabel; Wherein, the carboxyl donor includes MUA.

7. A detection kit, characterized in that: It comprises the three-dimensional magnetic multi-metal nanoenzyme immunolabel as described in claim 4 or 5.

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