Magnetic nano-enzyme probe and preparation method thereof, kit for detecting helicobacter pylori and application

Abstract

The invention discloses a magnetic nano-enzyme probe and a preparation method thereof, a kit for detecting helicobacter pylori and application of the magnetic nano-enzyme probe, the core of the magnetic nano-enzyme probe is Fe3O4 nano-particles, magnetic enrichment capacity is provided, and interference of a complex matrix in a sample is eliminated; a layer of Pt nano-particles is electrostatically adsorbed through the cationic polymer to serve as a colorimetric interlayer, and a strong colorimetric signal is provided; au (at) Pt for modifying 5, 5 '-dithiobis (2-nitrobenzoic acid) is arranged on the surface layer and is used for providing strong catalytic signals and sites coupled with a large number of specific antibodies; meanwhile, the inner and outer layers of nano enzyme particles can synergistically enhance the catalytic action. The magnetic nano-enzyme probe disclosed by the invention has strong magnetic responsiveness, high colorimetric signal, strong catalytic activity and specific bacterium capturing capacity, realizes rapid enrichment and quantitative analysis of helicobacter pylori in a sample to be detected, eliminates matrix interference in practical application, and has high sensitivity and high specificity. The method has great potential in the aspects of high sensitivity and clinical monitoring of bacterial infection or pollution.

Description

Technical Field

[0001] The present invention relates to the field of biological detection technology, and in particular to a magnetic nanozyme probe and a preparation method thereof, a kit for detecting Helicobacter pylori and applications thereof. Background Art

[0002] Although several mature analytical techniques, including microbial culture, PCR, and mass spectrometry, have been developed for accurate detection of bacteria, they cannot support rapid and direct analysis because they require professional experimenters, complex instruments, clean laboratories, and cumbersome experimental steps.

[0003] In POCT, lateral flow immunochromatography (LFA) has been widely used in clinical disease screening and personal health monitoring due to its significant advantages such as real-time, simplicity, rapidity and low cost. However, traditional LFA test strips based on colorimetric signals have obvious defects in bacterial detection, including low sensitivity, limited quantitative ability, and poor detection accuracy of low-concentration targets in complex samples. In the past decade, a class of quantitatively detectable LFA technologies has been established by using quantitative signal nanolabels such as fluorescent probes, quantum dots, bioluminescent labels, upconversion nanoparticles, magnetic nanoparticles, carbon nanoparticles, etc. Among them, nanozymes have been used in bioassays and biosensors due to their excellent biocompatibility, good water solubility, high stability and catalytic activity, which can effectively improve the sensitivity of colorimetric immunochromatography.

[0004] Magnetic nanomaterials are considered to be a promising method to solve the problem of complex matrix interference in samples and are widely used to build stable detection technologies for complex biological samples. This is because they can capture and separate targets, remove impurities, amplify signals, and improve the accuracy and sensitivity of detection systems. By adsorbing one or more layers of nanozyme particles on the surface of Fe3O4, preparing magnetic quantum dots and introducing the colorimetric LFA method, the interference of complex matrices in samples can be removed by magnetic enrichment and the detection sensitivity can be improved.

[0005] The traditional immunochromatographic method has three obvious defects: (1) low sensitivity, difficult to quantify, and difficult to meet the needs of low-concentration Helicobacter pylori detection; (2) poor stability in complex systems: colloidal labels such as gold nanoparticles are easily affected by biological matrices (such as high sugar, high salt, plant fiber, etc.) in fecal samples. They have poor stability when detecting complex samples and are prone to false positives or false negatives, causing trouble for patients infected with Helicobacter pylori. (3) Helicobacter pylori is 2.5 to 5.0 μm long, which is relatively large compared to the pore size (6-15 μm) of the commonly used immunochromatographic nitrocellulose (NC) membrane, which creates obstacles to the construction of a stable detection system on the test strip. Therefore, it is urgent to develop new immunochromatographic technologies with high sensitivity and good stability for rapid screening and accurate detection of Helicobacter pylori in complex samples.

[0006] The invention patent with patent application number 202410885615.0 in the prior art discloses a method for preparing cuprous oxide nanozyme, and the preparation method comprises the following steps: respectively preparing cupric chloride dihydrate solution, polyvinyl pyrrolidone solution, sodium hydroxide solution, and L-ascorbic acid solution for use; dripping the polyvinyl pyrrolidone solution into the cupric chloride dihydrate solution drop by drop; adding sodium hydroxide solution drop by drop into the mixed solution in step A2; adding L-ascorbic acid solution drop by drop into the mixed solution in step A3, and continuing the reaction at a constant temperature for 2.5-3.5 hours; after the reaction of the mixed solution in step A4 is completed, cooling to room temperature, and then centrifuging the mixed solution to obtain a first precipitate, and drying the first precipitate after washing to obtain cuprous oxide nanozyme powder.

[0007] The invention patent with application number 202211611770.0 discloses a colorimetric fluorescent dual-signal nano-microsphere and its preparation method and application. The colorimetric fluorescent dual-signal nano-microsphere of the invention includes an inner core and a first positively charged interlayer, a first shell layer, a second positively charged interlayer and a second shell layer wrapped around the inner core from the inside to the outside; the inner core is SiO2 nanoparticles; the first shell layer is a composite layer of gold nanoparticles and carboxylated quantum dots; the second shell layer is a carboxylated quantum dot layer. The colorimetric fluorescent dual-signal nano-microspheres of the present invention have the advantages of good dispersibility, adjustable particle size, strong colorimetric and fluorescent properties, and easy batch preparation. The carboxyl sites on the surface of the quantum dot shell are easy to modify with antibodies and can be used for qualitative and highly sensitive on-site rapid detection of the new coronavirus. However, the above patent cannot achieve rapid enrichment and quantitative analysis of Helicobacter pylori in the sample to be tested. Summary of the invention

[0008] In view of the shortcomings of the prior art, one of the purposes of the present invention is to provide a magnetic nanozyme probe and a preparation method, a kit for detecting Helicobacter pylori and its application. The magnetic nanozyme probe has the dual capabilities of magnetic enrichment effect and multiple catalytic sites, can achieve rapid enrichment and quantitative analysis of Helicobacter pylori, and has a fast detection speed and high sensitivity.

[0009] In order to achieve the above object, the present invention adopts the following technical solutions:

[0010] A magnetic nanoenzyme probe, comprising a composite nanomaterial and a specific antibody;

[0011] The composite nanomaterial has a core-shell structure, wherein the core of the core-shell structure is a Fe3O4 nanoparticle, and an interlayer and a shell layer are sequentially coated from the outer surface of the core to the outside;

[0012] The interlayer is a multi-layer stacked structure formed by alternating multiple positive interlayers and negative interlayers;

[0013] The positive interlayer is polyethyleneimine, the negative interlayer is nanoparticles, the shell layer is carboxyl-modified Au@Pt, and the carboxyl group of the shell layer is coupled with a specific antibody.

[0014] Preferably, the stacked structure includes at least 3 layers, specifically 3 layers, 4 layers, 5 layers, 6 layers, etc.

[0015] Preferably, in any of the above schemes, the stacked structure has three layers, the first layer along the stacking direction of the stacked structure is a positive interlayer, the second layer is a negative interlayer, and the last layer is a positive interlayer.

[0016] The average particle size of the Fe3O4 nanoparticles is 50-500nm, preferably 100-300nm;

[0017] The thickness of each positive interlayer is ≤5 nm, and can be specifically 5 nm, 4 nm, 3 nm, 2 nm, 1 nm, and preferably 0.5-2 nm.

[0018] Preferably, in any of the above schemes, the nanoparticles are Pt nanoparticles and / or Au@Pt nanoparticles, the average particle size of the Pt nanoparticles is 2-50 nm, preferably 20 nm; the average particle size of the Au@Pt nanoparticles is 5-30 nm, preferably 5 nm.

[0019] The negatively charged interlayer Pt nanoparticles function to provide a colorimetric signal and catalase activity, and the Au@Pt nanoparticles function to provide a stronger catalase activity and synergistically amplify the catalytic signal with the negatively charged interlayer Pt nanoparticles.

[0020] Specifically, the average particle size of the Fe3O4 nanoparticles is any value in the range of 50-500nm, such as 50nm, 100nm, 150nm, 200nm, 250nm, 300nm, 350nm, 400nm, 450nm, 500nm, 550nm, 600nm. The thickness of each positive interlayer is ≤5nm, specifically 5nm, 4.5nm, 4nm, 3.5nm, 3nm, 2.5nm, 2nm, 1.5nm, 1nm, 0.5nm.

[0021] The average particle size of the Pt nanoparticles is any value in the range of 2-50nm, specifically 2nm, 5nm, 10nm, 15nm, 20nm, 25nm, 30nm, 35nm, 40nm, 45nm, 50nm. The average particle size of the Au@Pt nanoparticles is any value in the range of 5-30nm, specifically 5nm, 10nm, 15nm, 20nm, 25nm, 30nm, 35nm, 40nm, 45nm, 50nm.

[0022] In any of the above schemes, preferably, 1 gram of the composite nanomaterial is coupled with ≤1 mg of specific antibody, preferably 0.001-0.01 mg; the specific antibody is a Helicobacter pylori antibody.

[0023] Specifically, 1g of the composite nanomaterial is coupled with ≤1mg of specific antibody, which can be 1mg, 0.9mg, 0.8mg, 0.7mg, 0.6mg, 0.5mg, 0.4mg, 0.3mg, 0.2mg, 0.1mg, 0.01mg, 0.005mg, 0.001mg.

[0024] The present invention also discloses a method for preparing the magnetic nanozyme probe, which comprises the following steps:

[0025] S1, dispersing Fe3O4 nanoparticles in deionized water to obtain a dispersion, then mixing the dispersion with a polyethyleneimine aqueous solution and performing a first ultrasonic treatment to obtain Fe3O4-PEI nanospheres;

[0026] S2, mixing the Fe3O4-PEI nanoparticles obtained in step S1 with the Pt nanoparticle solution and performing a second ultrasonic treatment to obtain Fe-Pt nanoparticles;

[0027] S3, mixing the Fe-Pt nanosphere polyethyleneimine aqueous solution obtained in step S2 and performing a third ultrasonic treatment to obtain Fe-Pt-PEI nanospheres;

[0028] S4, mixing the Fe-Pt-PEI nanoparticles obtained in step S3 with the Au@Pt nanoaqueous solution and performing a fourth ultrasonic treatment to obtain Fe-Pt-Au@Pt nanoparticles;

[0029] S5, mixing the Fe-Pt-Au@Pt microspheres obtained in step S4 with the 5,5'-dithiobis(2-nitrobenzoic acid) ethanol solution and performing a fifth ultrasonic treatment to obtain a magnetic nanozyme with an outer surface modified with 5,5'-dithiobis(2-nitrobenzoic acid);

[0030] S6. After activating the magnetic nanozyme with 5,5'-dithiobis(2-nitrobenzoic acid) modified on the outer surface obtained in step S5, the nanozyme is mixed with a specific antibody for incubation to obtain a magnetic nanozyme probe.

[0031] Preferably, in step S1, the average particle size of the Fe3O4 nanoparticles is 50-500nm, preferably 100-300nm; per 100mL of deionized water, ≤3g of Fe3O4 nanoparticles are dispersed, preferably 0.5-1.5g; the molecular weight of the polyethyleneimine is 5000-80000Da, preferably 25000Da; the mass concentration of the polyethyleneimine aqueous solution is ≤2mg / mL; the volume ratio of the dispersion to the polyethyleneimine aqueous solution is 1-50:1, preferably 40-50:1.

[0032] For every 100 mL of deionized water, there is dispersed ≤3 g of Fe3O4 nanoparticles, specifically 3 g, 2.5 g, 2 g, 1.5 g, 1 g, 0.5 g, preferably 0.5-1.5 g; the molecular weight of the polyethyleneimine is 5000-80000 Da, preferably 25000 Da; the mass concentration of the polyethyleneimine aqueous solution is ≤2 mg / mL; the volume ratio of the dispersion to the polyethyleneimine aqueous solution is 1-50:1, specifically 10:1, 20:1, 30:1, 40:1, 50:1, preferably 40-50:1.

[0033] Preferably in any of the above schemes, in step S2, the Pt nanoaqueous solution is prepared using Pt nanoparticles, the mass concentration of the Pt nanoparticles is ≤10 mg / mL, and the average particle size of the Pt nanoparticles is 10-50 nm, preferably 20 nm; in every 100 mL of Pt nanoaqueous solution, the added amount of Fe3O4-PEI nanospheres is ≤4 g, specifically 4 g, 3 g, 2 g, 1 g, preferably 0.5-1.5 g.

[0034] In any of the above schemes, preferably, in step S4, the average particle size of the Au@Pt nanoparticles is 5-30 nm, preferably 5 nm; and the amount of Fe-Pt-PEI nanospheres added to each 100 mL Au@Pt nanoaqueous solution is ≤ 4 g, preferably 0.5-1.5 g.

[0035] Preferably, in any of the above schemes, in step S5, the mass concentration of the 5,5'-dithiobis(2-nitrobenzoic acid)ethanol solution is ≤2 mg / mL; and the added amount ratio of Fe-Pt-Au@Pt microspheres to 5,5'-dithiobis(2-nitrobenzoic acid)ethanol solution is 18-22:1 (mass ratio).

[0036] The addition ratio of Fe-Pt-Au@Pt microspheres to 5,5'-dithiobis(2-nitrobenzoic acid)ethanol solution is 18-22:1 (mass ratio), and can be any value within the range of 18-22:1, such as 18:1, 19:1, 22:1.

[0037] In any of the above schemes, preferably, the addition ratio of Fe-Pt-Au@Pt microspheres to 5,5'-dithiobis(2-nitrobenzoic acid)ethanol solution is 20:1 (mass ratio).

[0038] Preferably, in any of the above schemes, in step S6, the activation treatment comprises: mixing the magnetic nanozyme with the outer surface modified with 5,5'-dithiobis(2-nitrobenzoic acid) with the activation solution and performing a fifth ultrasonic treatment; in each 100 mL of the activation solution, the amount of the magnetic nanozyme nanomicrospheres with the outer surface modified with 5,5'-dithiobis(2-nitrobenzoic acid) is 0.01-1 g;

[0039] The added amount of the magnetic nanozyme nanoparticles with the outer surface modified with 5,5'-dithiobis(2-nitrobenzoic acid) is any value in the range of 0.01-1g, such as 0.01g, 0.05g, 0.1g, 0.2g, 0.3g, 0.4g, 0.5g, 0.6g, 0.7g, 0.8g, 0.9g, 1g.

[0040] In any of the above schemes, preferably, the activation solution is prepared by adding 0.001-0.1 g of N-hydroxysuccinimide, 0.001-0.1 g of 1-ethyl-(3-dimethylaminopropyl)carbodiimide and 0.001-0.1 g of morpholineethanesulfonic acid to 100 mL of deionized water;

[0041] The activation solution is 0.001-0.1 g of N-hydroxysuccinimide added to 100 mL of deionized water. The specific amount of N-hydroxysuccinimide added can be 0.001 g, 0.002 g, 0.003 g, 0.004 g, 0.005 g, 0.006 g, 0.007 g, 0.008 g, 0.009 g, 0.1 g; the amount of 1-ethyl-(3-dimethylaminopropyl)carbodiimide added is 0.001 g, 0.002 g, 0.003 g, 0.004 g, 0.005 g, 0.006 g, 0.007 g, 0.008 g, 0.009 g, 0.1 g. The specific amount of morpholineethanesulfonic acid can be 0.001g, 0.002g, 0.003g, 0.004g, 0.005g, 0.006g, 0.007g, 0.008g, 0.009g, or 0.1g. The specific amount of morpholineethanesulfonic acid can be 0.001g, 0.002g, 0.003g, 0.004g, 0.005g, 0.006g, 0.007g, 0.008g, 0.009g, or 0.1g.

[0042] For every 1g of the magnetic nanozyme with the outer surface modified with 5,5'-dithiobis(2-nitrobenzoic acid), the added amount of the specific antibody is ≤1mg, preferably 0.001-0.02mg; the specific antibody is a Helicobacter pylori antibody.

[0043] For each 1 g of the magnetic nanozyme with the outer surface modified with 5,5'-dithiobis(2-nitrobenzoic acid), the amount of the specific antibody added can be 0.001 mg, 0.005 mg, 0.01 mg, 0.015 mg, or 0.02 mg.

[0044] The present invention also discloses the use of the magnetic nanozyme probe described above in the preparation of a product for detecting Helicobacter pylori. The product for detecting Helicobacter pylori at least comprises the magnetic nanozyme probe, an immunochromatographic test strip, and a test kit.

[0045] The present invention also discloses a kit for detecting Helicobacter pylori, the kit comprising any one of the magnetic nanozyme probes described above, an immunochromatographic test strip, a catalytic solution and a running buffer.

[0046] Preferably, the immunochromatographic test strip comprises a sample pad, a chromatographic membrane and an absorption pad connected in sequence, and the chromatographic membrane is provided with a detection line and a quality control line; the detection line is loaded with anti-Helicobacter pylori antibodies; the quality control line is loaded with sheep anti-mouse IgG.

[0047] Preferably, in any of the above schemes, the catalyst liquid comprises: sodium acetate solution (pH 2-6, preferably pH 3.5, concentration 1-20 mmol / L), hydrogen peroxide (concentration 0.1-20 mol / L, preferably 10 mol / L), 3-amino-9-ethylcarbazole solution (concentration 10-50 mmol / L, preferably 20 mmol / L), and a thickener (e.g., ethylene-vinyl alcohol copolymer, concentration 10%-60% (V / V), preferably 30%).

[0048] Specifically, the pH of the sodium acetate solution is any value in the range of 2-6, such as 2, 3, 4, 5, 6, preferably 3.5, and the concentration is any value in the range of 1-20mmol / L, such as 1mmol / L, 5mmol / L, 10mmol / L, 15mmol / L, 20mmol / L; the concentration of hydrogen peroxide is any value in the range of 0.1-20mol / L, such as 0.1mol / L, 5mol / L, 10mol / L, 15mol / L, 20mol / L, preferably The concentration is 10 mol / L; the concentration of 3-amino-9-ethylcarbazole solution is any value in the range of 10-50 mmol / L, such as 10 mol / L, 20 mol / L, 30 mol / L, 40 mol / L, 50 mol / L, and the preferred concentration is 20 mmol / L; the concentration of the thickener (e.g., ethylene-vinyl alcohol copolymer) is any value in the range of 10%-60% (V / V), such as 10%, 20%, 30%, 40%, 50%, 60%, and the preferred concentration is 30%.

[0049] Beneficial Effects

[0050] The core of the magnetic nanozyme probe of the present application is Fe3O4 nanoparticles, which provide magnetic enrichment ability and eliminate the interference of complex matrices in the sample; a layer of Pt nanoparticles is electrostatically adsorbed by a cationic polymer as a colorimetric interlayer to provide a strong colorimetric signal; the surface layer is provided with Au@Pt modified with 5,5'-dithiobis(2-nitrobenzoic acid) to provide a strong catalytic signal and a large number of specific antibody coupling sites; at the same time, the inner and outer layers of nanozyme particles can synergistically enhance the catalytic effect. The magnetic nanozyme probe disclosed in the present invention has strong magnetic responsiveness, high colorimetric signal, strong catalytic activity and specific bacterial capture ability, realizes the rapid enrichment and quantitative analysis of Helicobacter pylori in the sample to be tested, realizes the multiple signal amplification of the magnetic enrichment effect and the nanozyme catalytic effect, eliminates the matrix interference in practical applications, and has great potential in high sensitivity and clinical monitoring of bacterial infection or contamination. Through the above technical scheme, the magnetic nanozyme probe disclosed in the present invention has the dual capabilities of magnetic enrichment effect and multiple catalytic sites, which realizes the rapid capture and highly sensitive quantitative detection of Helicobacter pylori in fecal samples, and has great application potential in on-site rapid detection of Helicobacter pylori. BRIEF DESCRIPTION OF THE DRAWINGS

[0051] Figure 1 Schematic diagram of the preparation method for the synthesis of the magnetic nanozyme probe disclosed in the present invention.

[0052] Figure 2 The electron microscopy characterization of the structure of the magnetic nanozyme probe disclosed in the present invention.

[0053] Figure 3 The present invention is a flowchart of the magnetic nanozyme probe used for detecting Helicobacter pylori.

[0054] Figure 4 This is the optimization result of the membrane concentration of the magnetic nanozyme probe disclosed in the present invention.

[0055] Figure 5 The incubation time optimization results of the magnetic nanozyme probe disclosed in the present invention.

[0056] Figure 6 It is the capture efficiency of Helicobacter pylori by the magnetic nanozyme probe disclosed in the present invention.

[0057] Figure 7 This is a graph showing the detection results of Helicobacter pylori using the magnetic nanozyme probe disclosed in the present invention.

[0058] Figure 8 This is the test result of Helicobacter pylori detection using the colloidal gold method.

[0059] Fig. 9 This is a specific verification of the magnetic nanozyme probe disclosed in the present invention.

[0060] Fig.10 This is a repeatability verification of the magnetic nanozyme probe disclosed in the present invention.

[0061] Fig.11 This is the analysis result of the magnetic nanozyme probe disclosed in the present invention in actual samples.

[0062] Fig.12 The results of the analysis of the magnetic nanozyme probe disclosed in the present invention in fecal samples. DETAILED DESCRIPTION

[0063] 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 changes can be made within the scope of the invention claim, and the 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.

[0064] 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 specific experimental conditions not specified are based on conventional conditions known to those skilled in the art or conditions recommended by the manufacturer.

[0065] Example 1

[0066] The present invention discloses a magnetic nanoenzyme probe, wherein the composite nanoprobe comprises a composite nanomaterial and a specific antibody;

[0067] The composite nanomaterial has a core-shell structure; the core of the core-shell structure is Fe3O4 nanoparticles, and the outer surface of the core is sequentially coated with an interlayer and a shell layer.

[0068] The interlayer is a multilayer stacked structure formed by alternating positive interlayers and negative interlayers, the first layer along the stacking direction of the stacked structure is a positive interlayer, the second layer is a negative interlayer, and the last layer is a positive interlayer; the stacked structure includes at least 3 layers, specifically 3 layers, 4 layers, 5 layers, 6 layers, etc., and the present embodiment has 3 layers.

[0069] The positive interlayer is polyethyleneimine; the negative interlayer is Pt nanozyme particles; the shell layer is carboxyl-modified Au@Pt nanoparticles; and the carboxyl group of the shell layer is coupled with a specific antibody.

[0070] In this embodiment, the composite nanomaterial is based on Fe3O4 nanoparticles with superparamagnetism as the core, which provides magnetic enrichment ability and colorimetric signal at the same time; then Pt nanoparticles and Au@Pt nanoparticles are adsorbed on the outer surface of Fe3O4 nanoparticles through positively charged interlayer polyethyleneimine (PEI); the composite nanomaterial has the dual properties of superparamagnetism and strong colorimetric signal; Pt nanoparticles have catalase activity, which is used to enhance the colorimetric signal and improve the catalytic performance; Au@Pt nanoparticles show excellent catalytic performance in redox reactions; the Au@Pt shell is treated with 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) to form a shell containing a large number of carboxyl sites, which can be used for coupling with specific antibodies, and a magnetic nanozyme probe with strong magnetic responsiveness, high colorimetric signal and specific bacterial capture ability is prepared, which realizes the rapid capture of substances to be detected in the sample to be tested, such as Helicobacter pylori, and has great application potential in the on-site rapid detection of Helicobacter pylori.

[0071] The Fe3O4 nanoparticles disclosed in the present invention are nanoparticles with superparamagnetism. A further optimized technical solution of this embodiment is that the average particle size of the Fe3O4 nanoparticles is 50-500nm, specifically, the average particle size of the Fe3O4 nanoparticles is any value within the range of 50-500nm, such as 50nm, 100nm, 150nm, 200nm, 250nm, 300nm, 350nm, 400nm, 450nm, 500nm, 550nm, 600nm, preferably 100-300nm.

[0072] The thickness of each positive interlayer can vary within a large range. The technical solution further optimized in this embodiment is that the thickness of each positive interlayer is ≤5nm, and the thickness of each positive interlayer is ≤5nm, which can be 5nm, 4nm, 3nm, 2nm, 1nm, preferably 0.5-2nm. By controlling the thickness of each positive interlayer, the present invention can efficiently load nanozyme particles without affecting the strong magnetism of Fe3O4, thereby giving the composite nanomaterial strong magnetism and strong catalytic effect.

[0073] The technical solution further optimized in this embodiment is that the nanozyme particles are Pt nanoparticles and Au@Pt nanoparticles; the average particle size of the Pt nanoparticles is 2-50nm, specifically 2nm, 5nm, 10nm, 15nm, 20nm, 25nm, 30nm, 35nm, 40nm, 45nm, 50nm, preferably 20nm; the average particle size of the Au@Pt nanoparticles is 5-30nm, specifically 5nm, 10nm, 15nm, 20nm, 25nm, 30nm, 35nm, 40nm, 45nm, 50nm, preferably 5nm; relative to every 100mA Au@Pt nanoaqueous solution, the added amount of the Fe-Pt-PEI nanospheres is ≤4g, preferably 0.5-1.5g.

[0074] The technical solution further optimized in this embodiment is that, relative to 1g of composite nanomaterial, ≤1mg of specific antibody is coupled, which can be 1mg, 0.9mg, 0.8mg, 0.7mg, 0.6mg, 0.5mg, 0.4mg, 0.3mg, 0.2mg, 0.1mg, 0.01mg, 0.005mg, 0.001mg, preferably 0.001-0.01mg. The specific antibody is preferably an antibody against Helicobacter pylori. The magnetic nanozyme probe of the present invention can efficiently capture, enrich and detect Helicobacter pylori in complex samples.

[0075] The present invention also discloses a method for preparing the magnetic nanozyme probe as described above, Figure 1 As shown, the method comprises the following steps:

[0076] S1, dispersing Fe3O4 nanoparticles in deionized water to obtain a dispersion, then mixing the dispersion with a polyethyleneimine aqueous solution and performing a first ultrasonic treatment to obtain Fe3O4-PEI nanospheres;

[0077] S2, mixing the Fe3O4-PEI nano-microspheres with the Pt nano-water solution and performing a second ultrasonic treatment to obtain Fe-Pt nano-microspheres;

[0078] S3, mixing the Fe-Pt nano-microspheres with a polyethyleneimine aqueous solution and performing a third ultrasonic treatment to obtain Fe-Pt-PEI nano-microspheres;

[0079] S4, mixing the Fe-Pt-PEI nano-microspheres with the Au@Pt nano-water solution and performing a fourth ultrasonic treatment to obtain Fe-Pt-Au@Pt nano-microspheres;

[0080] S5, mixing the Fe-Pt-Au@Pt nanospheres with a 5,5'-dithiobis(2-nitrobenzoic acid) ethanol solution and performing a fifth ultrasonic treatment to obtain a magnetic nanozyme with an outer surface modified with 5,5'-dithiobis(2-nitrobenzoic acid);

[0081] S6. After activating the magnetic nanozyme whose outer surface is modified with 5,5'-dithiobis(2-nitrobenzoic acid), the nanozyme is mixed with a specific antibody for incubation to obtain a magnetic nanozyme composite nanoprobe.

[0082] The technical solution further optimized in this embodiment is that in step S1, the average particle size of the Fe3O4 nanoparticles is 50-500nm, preferably 100-300nm; for every 100mL of deionized water, ≤3g of Fe3O4 nanoparticles are dispersed, preferably 0.5-1.5g; the molecular weight of the polyethyleneimine is 5000-80000Da, preferably 25000Da; the mass concentration of the polyethyleneimine aqueous solution is ≤2mg / mL; the volume ratio of the dispersion to the polyethyleneimine aqueous solution is 1-50:1, preferably 40-50:1.

[0083] For every 100 mL of deionized water, there is dispersed ≤3 g of Fe3O4 nanoparticles, specifically 3 g, 2.5 g, 2 g, 1.5 g, 1 g, 0.5 g, preferably 0.5-1.5 g; the molecular weight of the polyethyleneimine is 5000-80000 Da, preferably 25000 Da; the mass concentration of the polyethyleneimine aqueous solution is ≤2 mg / mL; the volume ratio of the dispersion to the polyethyleneimine aqueous solution is 1-50:1, specifically 10:1, 20:1, 30:1, 40:1, 50:1, preferably 40-50:1.

[0084] The technical solution further optimized in this embodiment is that in step S2, the Pt nano-water solution is prepared by using Pt nano-particles, the mass concentration of the Pt nano-particles is ≤10mg / mL, and the average particle size of the Pt nano-particles is 10-50nm, preferably 20nm; per 100mL of Pt nano-water solution, the amount of Fe3O4-PEI nano-microspheres added is ≤4g, specifically 4g, 3g, 2g, 1g, preferably 0.5-1.5g. The conditions of the second ultrasonic treatment are: ultrasonic frequency of 50-100Hz, temperature of 10-40℃, and time of 10-90min.

[0085] The technical solution further optimized in this embodiment is that in step S3, the molecular weight of the polyethyleneimine is 5000-80000Da, preferably 25000Da; the mass concentration of the polyethyleneimine aqueous solution is ≤2mg / mL; the volume ratio of the dispersion to the polyethyleneimine aqueous solution is 1-50:1, preferably 40-50:1; the conditions of the third ultrasonic treatment are: ultrasonic frequency of 50-100Hz, temperature of 10-40°C, and time of 10-90min.

[0086] The technical solution further optimized in this embodiment is that in step S4, the Au@Pt nano-water solution is prepared by using Au@Pt nanoparticles, the mass concentration of the Au@Pt nanoparticles is ≤10mg / mL, and the average particle size of the Au@Pt nanoparticles is 5-30nm, preferably 5nm; per 100mL Au@Pt nano-water solution, the amount of Fe-Pt-PEI nano-microspheres added is ≤4g, preferably 0.5-1.5g. The conditions of the fourth ultrasonic treatment are: ultrasonic frequency of 50-100Hz, temperature of 10-40℃, and time of 10-90min.

[0087] The technical solution further optimized in this embodiment is that in step S5, the mass concentration of the 5,5'-dithiobis(2-nitrobenzoic acid)ethanol solution is ≤2mg / mL; the addition ratio of Fe-Pt-Au@Pt microspheres to the 5,5'-dithiobis(2-nitrobenzoic acid)ethanol solution is 18-22:1 (mass ratio). The conditions of the fifth ultrasonic treatment are: frequency of 50-100Hz, temperature of 10-40℃, and time of 10-90min.

[0088] The addition ratio of Fe-Pt-Au@Pt microspheres to 5,5'-dithiobis(2-nitrobenzoic acid)ethanol solution is 18-22:1 (mass ratio), and can be any value within the range of 18-22:1, such as 18:1, 19:1, 22:1.

[0089] The technical solution further optimized in this embodiment is that the addition ratio of Fe-Pt-Au@Pt microspheres to 5,5'-dithiobis(2-nitrobenzoic acid)ethanol solution is 20:1 (mass ratio).

[0090] The technical solution further optimized in this embodiment is that in step S6, the activation treatment includes: mixing the magnetic nanozyme with the outer surface modified with 5,5'-dithiobis(2-nitrobenzoic acid) with the activation solution for a fifth ultrasonic treatment; the amount of the magnetic nanozyme nanomicrospheres with the outer surface modified with 5,5'-dithiobis(2-nitrobenzoic acid) is 0.01-1g for each 100mL of the activation solution; wherein the activation solution is 0.001-0.1g of N-hydroxysuccinimide, 0 .001-0.1g of 1-ethyl-(3-dimethylaminopropyl) carbodiimide and 0.001-0.1g of morpholineethanesulfonic acid; for every 1g of the magnetic nanozyme probe, the amount of specific antibody added is ≤1mg, preferably 0.001-0.02mg; the specific antibody is a Helicobacter pylori antibody; the conditions of the sixth ultrasonic treatment are: ultrasonic frequency of 50-100Hz, temperature of 10-40℃, time of 10-90min; the temperature of the incubation treatment is 20-40℃, and the time is 20-180min. Further, this step can also include blocking unreacted carboxyl groups with bovine serum albumin (BSA).

[0091] The technical solution further optimized in this embodiment is that in step S6, the activation treatment comprises: mixing the magnetic nanozyme with the outer surface modified with 5,5'-dithiobis(2-nitrobenzoic acid) with the activation solution and performing a fifth ultrasonic treatment; in every 100 mL of the activation solution, the amount of the magnetic nanozyme nanomicrospheres with the outer surface modified with 5,5'-dithiobis(2-nitrobenzoic acid) is 0.01-1 g;

[0092] The added amount of the magnetic nanozyme nanoparticles with the outer surface modified with 5,5'-dithiobis(2-nitrobenzoic acid) is any value in the range of 0.01-1g, such as 0.01g, 0.05g, 0.1g, 0.2g, 0.3g, 0.4g, 0.5g, 0.6g, 0.7g, 0.8g, 0.9g, 1g.

[0093] The technical solution further optimized in this embodiment is that the activation solution is prepared by adding 0.001-0.1 g of N-hydroxysuccinimide, 0.001-0.1 g of 1-ethyl-(3-dimethylaminopropyl)carbodiimide and 0.001-0.1 g of morpholineethanesulfonic acid to 100 mL of deionized water;

[0094] The activation solution is 0.001-0.1 g of N-hydroxysuccinimide added to 100 mL of deionized water. The specific amount of N-hydroxysuccinimide added can be 0.001 g, 0.002 g, 0.003 g, 0.004 g, 0.005 g, 0.006 g, 0.007 g, 0.008 g, 0.009 g, 0.1 g; the amount of 1-ethyl-(3-dimethylaminopropyl)carbodiimide added is 0.001 g, 0.002 g, 0.003 g, 0.004 g, 0.005 g, 0.006 g, 0.007 g, 0.008 g, 0.009 g, 0.1 g. The specific amount of morpholineethanesulfonic acid can be 0.001g, 0.002g, 0.003g, 0.004g, 0.005g, 0.006g, 0.007g, 0.008g, 0.009g, or 0.1g. The specific amount of morpholineethanesulfonic acid can be 0.001g, 0.002g, 0.003g, 0.004g, 0.005g, 0.006g, 0.007g, 0.008g, 0.009g, or 0.1g.

[0095] For every 1g of the magnetic nanozyme with the outer surface modified with 5,5'-dithiobis(2-nitrobenzoic acid), the added amount of the specific antibody is ≤1mg, preferably 0.001-0.02mg; the specific antibody is a Helicobacter pylori antibody.

[0096] For each 1 g of the magnetic nanozyme with the outer surface modified with 5,5'-dithiobis(2-nitrobenzoic acid), the amount of the specific antibody added can be 0.001 mg, 0.005 mg, 0.01 mg, 0.015 mg, or 0.02 mg.

[0097] The present invention also discloses the use of the magnetic nanozyme probe described above in the preparation of a product for detecting Helicobacter pylori. The product for detecting Helicobacter pylori at least comprises the magnetic nanozyme probe, an immunochromatographic test strip, and a test kit.

[0098] The present invention also discloses a kit for detecting Helicobacter pylori, the kit comprising any one of the magnetic nanozyme probes described above, an immunochromatographic test strip, a catalytic solution and a running buffer.

[0099] Preferably, the immunochromatographic test strip comprises a sample pad, a chromatographic membrane and an absorption pad connected in sequence, and the chromatographic membrane is provided with a detection line and a quality control line; the detection line is loaded with anti-Helicobacter pylori antibodies; the quality control line is loaded with sheep anti-mouse IgG.

[0100] A further optimized technical solution of this embodiment is that the catalytic liquid includes: sodium acetate solution (pH 2-6, preferably pH 3.5, concentration 1-20 mmol / L), hydrogen peroxide (concentration 0.1-20 mol / L, preferably 10 mol / L), 3-amino-9-ethylcarbazole solution (concentration 10-50 mmol / L, preferably 20 mmol / L), and a thickener (e.g. ethylene-vinyl alcohol copolymer, concentration 10%-60% (V / V), preferably 30%).

[0101] Specifically, the pH of the sodium acetate solution is any value in the range of 2-6, such as 2, 3, 4, 5, 6, preferably 3.5, and the concentration is any value in the range of 1-20mmol / L, such as 1mmol / L, 5mmol / L, 10mmol / L, 15mmol / L, 20mmol / L; the concentration of hydrogen peroxide is any value in the range of 0.1-20mol / L, such as 0.1mol / L, 5mol / L, 10mol / L, 15mol / L, 20mol / L, preferably The concentration is 10 mol / L; the concentration of 3-amino-9-ethylcarbazole solution is any value in the range of 10-50 mmol / L, such as 10 mol / L, 20 mol / L, 30 mol / L, 40 mol / L, 50 mol / L, and the preferred concentration is 20 mmol / L; the concentration of the thickener (e.g., ethylene-vinyl alcohol copolymer) is any value in the range of 10%-60% (V / V), such as 10%, 20%, 30%, 40%, 50%, 60%, and the preferred concentration is 30%.

[0102] Example 2

[0103] like Figure 1 As shown, a method for preparing a magnetic nanozyme probe is similar to that of Example 1, except that it includes the following steps:

[0104] (1) 0.5 g of Fe3O4 nanoparticles (particle size 180 nm) were dispersed in 50 mL of deionized water to obtain a dispersion, and then the dispersion was mixed with a polyethyleneimine aqueous solution (2 mg / mL, molecular weight 25000 Da) at a volume ratio of 50:1 and subjected to a first ultrasonic treatment at 100 Hz and 20°C for 30 min to obtain Fe3O4-PEI nanospheres;

[0105] (2) mixing 0.5 g of the Fe3O4-PEI nanospheres obtained in step (1) with 100 mL of Pt nanoaqueous solution (10 mg / mL), and subjecting the mixture to a second ultrasonic treatment (100 Hz, 20° C.) to obtain Fe-Pt nanospheres; the Pt nanoaqueous solution is prepared using Pt nanoparticles, wherein the mass concentration of the Pt nanoparticles is ≤10 mg / mL, and the average particle size of the Pt nanoparticles is 20 nm;

[0106] (3) 0.5 g of the Fe-Pt nanoparticles obtained in step (2) was mixed with 50 mL of a polyethyleneimine aqueous solution (2 mg / mL) and subjected to a third ultrasonic treatment (100 Hz, 20° C.) to obtain Fe-Pt-PEI nanoparticles;

[0107] (4) 0.5 g of the Fe-Pt-PEI nanospheres obtained in step (3) were mixed with 100 mL of Au@Pt nanoaqueous solution (10 mg / mL) and subjected to a fourth ultrasonic treatment (100 Hz, 20° C.) to obtain Fe-Pt-Au@Pt nanospheres;

[0108] (5) 0.5 g of Fe-Pt-Au@Pt nanospheres were mixed with 100 mL of 5,5'-dithiobis(2-nitrobenzoic acid) ethanol solution (10 mM / mL) and subjected to a fifth ultrasonic treatment at 100 Hz and 20 °C for 2 h to obtain a magnetic nanozyme with 5,5'-dithiobis(2-nitrobenzoic acid) modified on the outer surface;

[0109] (6) 0.5 g of the magnetic nanozyme with the outer surface modified with 5,5'-dithiobis(2-nitrobenzoic acid) was mixed with 100 mL of activation solution for activation treatment, and then mixed with 8 μg of specific antibody and incubated at 27°C. The activation solution was 0.02 mg of N-hydroxysuccinimide, 0.01 mg of 1-ethyl-(3-dimethylaminopropyl)carbodiimide and 0.025 mg of morpholineethanesulfonic acid added to 0.5 mL of deionized water. Bovine serum albumin (BSA) was used to block the unreacted carboxyl groups.

[0110] Figure 2 The Fe3O4 nanoparticles and the prepared composite nanomaterial (magnetic nanozyme with 5,5'-dithiobis(2-nitrobenzoic acid) modified on the outer surface) in step (1) of this embodiment, and the transmission electron microscopy (TEM) image of the composite nanozyme material prepared in step (2). The above image shows that the synthesized composite magnetic nanozyme material is a core-shell structure with Fe3O4 nanoparticles as the core, a Pt interlayer and an Au@Pt outer shell as the shell.

[0111] In this embodiment, based on the electrostatic self-assembly method mediated by positively charged interlayer polyethyleneimine, a layer of nanozyme interlayer and a layer of nanozyme shell are assembled layer by layer on the outer surface of superparamagnetic Fe3O4 nanoparticles. The preparation method is efficient and repeatable, and is suitable for industrial production. The specific antibody of the present invention is a Helicobacter pylori antibody, and a magnetic nanozyme probe is obtained.

[0112] In the present invention, "ultrasonic treatment" can be performed using devices conventionally used by those skilled in the art, which will not be described in detail here.

[0113] The present invention also discloses the application of the magnetic nanozyme probe in the preparation of a product for detecting Helicobacter pylori. Specifically, the product for detecting Helicobacter pylori at least comprises the magnetic nanozyme probe, an immunochromatographic test strip, and a test kit.

[0114] The present invention also discloses a kit for detecting Helicobacter pylori, wherein the kit comprises the magnetic nanozyme probe described above, and also comprises an immunochromatographic test strip, a catalytic solution and a running buffer.

[0115] A further optimized technical solution of this embodiment is that the immunochromatographic test strip includes a sample pad, a chromatographic membrane and an absorption pad connected in sequence, and the chromatographic membrane is provided with a detection line and a quality control line; the detection line is loaded with anti-Helicobacter pylori antibodies; the quality control line is loaded with sheep anti-mouse IgG.

[0116] The technical solution further optimized in this embodiment is that the kit includes a catalytic liquid, specifically, the catalytic liquid includes: sodium acetate solution (pH 2-6, preferably pH 3.5, concentration 1-20mmol / L), hydrogen peroxide (concentration 0.1-20mol / L, preferably 10mol / L), 3-amino-9-ethylcarbazole solution (concentration 10-50mmol / L, preferably 20mmol / L), and a thickener (e.g., ethylene-vinyl alcohol copolymer, concentration 10%-60% (V / V), preferably 30%).

[0117] The present invention applies magnetic nanozyme probes in immunochromatography (LFA), which can realize the rapid enrichment and quantitative analysis of Helicobacter pylori in samples, realize the magnetic enrichment effect and multiple signal amplification of multi-layer nanozymes, eliminate matrix interference in practical applications, and further improve the sensitivity of detection. It has great potential in high-sensitivity and on-site monitoring of bacterial infection or contamination.

[0118] The magnetic nanozyme probe disclosed in the present invention provides two detection modes: (1) in the colorimetric mode, the gray colorimetric signal on the front line of catalysis and the reddish-brown colorimetric signal on the back line of catalysis are detected by naked eye for rapid qualitative detection; (2) in the data analysis mode, the change of the colorimetric signal on the detection line is used to achieve highly sensitive quantitative analysis.

[0119] The technical solution further optimized in this embodiment is that the immunochromatographic test strip also includes a base plate, which serves as a backing card for the immunochromatographic test strip, and the material of the base plate is preferably PVC. The sample pad is used for loading the sample solution to be tested. The absorbent pad is used to provide capillary force. The chromatographic membrane can be a nitrocellulose membrane (NC membrane).

[0120] The further optimized technical solution of this embodiment is to spray anti-Helicobacter pylori and goat anti-mouse IgG on the surface of the nitrocellulose membrane respectively; then put the modified NC membrane into a constant temperature drying oven (37°C) to obtain the nitrocellulose membrane in the immunochromatographic test strip. Among them, the concentration of anti-Helicobacter pylori antibody is ≤2mg / mL, preferably 1.5mg / mL; the concentration of goat anti-mouse IgG is ≤3mg / mL, preferably 1mg / mL.

[0121] The technical solution further optimized in this embodiment is that the kit further includes a loading buffer. Specifically, the loading buffer includes: 100% 10 mmol / L PBS buffer, 1% Tween 20 and 3% fetal bovine serum (FBS).

[0122] The method for detecting the sample to be tested using the above kit comprises the following steps:

[0123] First, 1 mL of the sample to be tested was mixed with 2 μL of the composite nanoprobe, and then incubated on an oscillator for 3 minutes. The bacteria-probe immune complex was recovered by magnetic enrichment of the magnetic nanozyme probe. The volume ratio of the test sample to the composite nanoprobe was 500:1. It was resuspended in 80 μL of loading buffer and loaded onto the sample pad of the immunochromatographic test strip, and contacted with the detection line on the immunochromatographic test strip for chromatography reaction. After about 13 minutes, the colorimetric signal on the detection line of the immunochromatographic test strip was read by the naked eye, and the colorimetric signal was read by the immunocolorimetric analyzer.

[0124] Example 3

[0125] The magnetic nanozyme probes prepared in Example 1 and Example 2 were used to detect Helicobacter pylori. The specific detection process is as follows: Figure 3 As shown, after mixing 1 mL of the sample to be tested with 2 μL of the composite nanoprobe, they were incubated together on an oscillator for 3 minutes, and the bacteria-probe immune complex was recovered by magnetic enrichment of the magnetic nanozyme probe. The volume ratio of the test sample to the composite nanoprobe is 500:1. It is resuspended in 80 μL of loading buffer and loaded onto the sample pad of the immunochromatographic test strip, and contacted with the detection line on the immunochromatographic test strip for chromatography reaction. After 13 minutes, the colorimetric signal on the detection line of the immunochromatographic test strip is read by the naked eye, and the colorimetric signal is read by the immunocolorimetric analyzer.

[0126] This example optimizes the operating conditions of the magnetic nanozyme probe immunochromatographic detection platform to achieve the best detection performance of the platform. Figure 4 As shown in the figure, when the concentration of anti-Helicobacter pylori antibodies on the detection line reaches 1.5 mg / mL, the magnetic nanozyme probe has the best detection effect on Helicobacter pylori. Figure 5 This is the optimization result of the incubation time between the magnetic nanozyme probe and the sample. Incubating with the sample for 3 minutes can enable the magnetic nanozyme probe to obtain a stronger colorimetric signal and the highest signal-to-noise ratio when detecting Helicobacter pylori. Among them, the signal-to-noise ratio of the present invention refers to the ratio of the colorimetric intensity of Helicobacter pylori to the colorimetric intensity of other interfering bacteria. The larger the value of the signal-to-noise ratio, the fewer interfering bacteria are captured by the magnetic nanozyme probe of the present invention, and the more Helicobacter pylori is captured, indicating that the magnetic nanozyme probe of the present invention has a stronger ability to capture Helicobacter pylori.

[0127] In summary, the immunochromatographic detection of the present invention is highly dependent on the ability of the magnetic nanozyme probe to capture and bind to the target bacteria. The present invention uses plate culture to determine the capture efficiency of the magnetic nanozyme probe on Helicobacter pylori. The results are as follows Figure 6 As shown in the figure, the plate counting results show that after 3 minutes of incubation, the capture efficiency of the magnetic nanozyme probe on Helicobacter pylori exceeds 89%. The above optimization helps the magnetic nanozyme probe and the bacteria-probe immune complex formed by it to flow smoothly on the NC membrane, ensuring stable operation in the immunochromatographic detection.

[0128] Example 4

[0129] The performance of the magnetic nanozyme probe in detecting different concentrations of Helicobacter pylori was verified in the immunochromatographic test. Figure 7 As shown by Figure 7 a It can be seen that the magnetic nanozyme probe is in the colorimetric mode before catalysis ( Figure 7 The visual limit of detection (vLOD) was 1000 CFU / mL in the colorimetric mode after catalysis ( Figure 7 a(ii)) The vLOD under the colorimetric mode was 50 CFU / mL, and the vLOD of the colorimetric mode after catalysis was 50 times higher than that of the colorimetric mode before catalysis. We used an immunocolorimetric analyzer to measure the colorimetric signal on the detection line and analyze it. Figure 7 The bar graph results in b show that the colorimetric signal on the test line is positively correlated with the concentration of Helicobacter pylori in the sample. The calibration curve ( Figure 7 c), the detection limit (LOD) of Helicobacter pylori can be calculated. The results show that the magnetic nanozyme probe immunochromatographic detection method has high analytical performance for the target Helicobacter pylori, a wide dynamic range (5 orders of magnitude), and a good correlation coefficient (R 2 >0.97), the LOD is low.

[0130] The immunochromatographic detection method containing magnetic nanozyme probes can qualitatively screen Helicobacter pylori with a concentration of more than 1000 CFU / mL by observing the color change on the test line under a fluorescent lamp; it can also be observed under a fluorescent lamp after applying a catalytic liquid, and qualitatively screen Helicobacter pylori with a concentration of more than 50 CFU / mL by observing the color change on the test line, and support the analysis of the concentration range of 10-10 5 CFU / mL of Helicobacter pylori was used for quantitative screening.

[0131] and Figure 8 Compared with the detection results of traditional colloidal gold, the structure of traditional colloidal gold is a monodisperse spherical structure with a particle size of 20-45nm that is easy to agglomerate. The sensitivity of the immunochromatographic detection method using the magnetic nanoenzyme probe in the present invention is increased by 1000 times.

[0132] At the same time, the present invention also selected a variety of common pathogenic bacteria such as Campylobacter jejuni, Listeria monocytogenes, Staphylococcus aureus, Vibrio cholerae, etc. (10 5 cells / mL) as bacterial interferents to test the specificity of the magnetic nanozyme probe. Fig. 9 As shown in Figure 1, the photo of the immunochromatographic test strip and the colorimetric signal on the detection line clearly demonstrate that the magnetic nanozyme probe has good selectivity for the target bacteria. Therefore, the specific bacterial antibodies applied on the detection line of the immunochromatographic test strip are sufficient to ensure the high specificity of the detection platform.

[0133] By adding 5×10 4 and 5×10 2 cells / mL tap water samples were continuously tested and the relative standard deviation (RSD) was calculated to verify the repeatability of the immunochromatographic detection method containing the magnetic nanozyme probe. Fig.10 As shown, the colorimetric signal on the detection line changes slightly, and the RSD value of each test group is less than 3.62%, indicating that the method of the present invention using the immunochromatographic detection containing the magnetic nanozyme probe has good repeatability.

[0134] This example also evaluates the performance of the proposed immunochromatographic detection method containing magnetic nanozyme probes in actual clinical testing and environmental monitoring by testing actual samples with added bacteria. 3 cells / mL) of Helicobacter pylori, the results are as follows Fig.11As shown, the colorimetric signals on the corresponding detection lines in feces and river water samples are stable, and the intensity of the colorimetric signals decreases as the concentration of the corresponding target pathogens in the samples decreases. The recovery rates of the magnetic nanozyme probes in two different environmental samples were calculated based on the colorimetric signals. The average sample recovery rate was 89.65%-105.39%, and the RSD value was 2.14%-5.28%, indicating that the immunochromatographic detection method containing magnetic nanozyme probes established by the present invention has good accuracy and reliability for quantitative detection of bacteria in complex samples.

[0135] This example also tested 81 Helicobacter pylori positive stool samples and 36 Helicobacter pylori negative stool samples to evaluate the performance of the proposed immunochromatographic detection method containing magnetic nanozyme probes in actual clinical applications. Fig.12 As shown, the established immunochromatographic detection method containing magnetic nanozyme probes has good accuracy and reliability in the detection of actual clinical samples.

[0136] The preferred embodiments of the present invention are described in detail above in conjunction with the accompanying drawings. However, the present invention is not limited to the specific details in the above embodiments. Within the technical concept of the present invention, the technical solution of the present invention can be subjected to a variety of simple modifications, and these simple modifications all belong to the protection scope of the present disclosure. In addition, the various embodiments of the present invention can also be arbitrarily combined, as long as they do not violate the concept of the present invention, they should also be regarded as the contents disclosed by the present invention. The above are only preferred specific embodiments of the present invention, but the protection scope of the present invention is not limited thereto. Any modifications made by any technician familiar with the technical field within the technical scope disclosed by the present invention by equivalently replacing or changing the technical solution and the inventive concept of the present invention should be covered within the protection scope of the present invention.

Claims

1. A magnetic nanozyme probe, characterized in that: Including composite nanomaterials and specific antibodies; The composite nanomaterial has a core-shell structure, wherein the core of the core-shell structure is a Fe3O4 nanoparticle, and an interlayer and a shell layer are sequentially coated from the outer surface of the core to the outside; The interlayer comprises a multi-layer stacked structure formed by alternating positive and negative interlayers; The positive interlayer is polyethyleneimine, the negative interlayer is nanoparticles, the shell layer is carboxyl-modified Au@Pt, and the carboxyl group of the shell layer is coupled with a specific antibody.

2. The magnetic nanozyme probe according to claim 1, characterized in that The first layer of the interlayer along the stacking direction of the stacking structure is a positive interlayer, the second layer is a negative interlayer, and the last layer is a positive interlayer.

3. The magnetic nanozyme probe according to claim 1, characterized in that The average particle size of the Fe3O4 nanoparticles is 50-500nm, the thickness of each positive interlayer is ≤5nm, the nanoparticles are Pt nanoparticles and / or Au@Pt nanoparticles, the average particle size of the Pt nanoparticles is 2-50nm, the average particle size of the Au@Pt nanoparticles is 5-30nm, 1g of the composite nanomaterial is coupled with ≤1mg of specific antibodies, and the specific antibodies are Helicobacter pylori antibodies.

4. The method for preparing the magnetic nanozyme probe according to any one of claims 1 to 3, characterized in that: The method comprises the following steps: S1, dispersing Fe3O4 nanoparticles in deionized water to obtain a dispersion, then mixing the dispersion with a polyethyleneimine aqueous solution and performing a first ultrasonic treatment to obtain Fe3O4-PEI nanospheres; S2, mixing the Fe3O4-PEI nanoparticles obtained in step S1 with the Pt nanoparticle solution and performing a second ultrasonic treatment to obtain Fe-Pt nanoparticles; S3, mixing the Fe-Pt nanoparticles obtained in step S2 with a polyethyleneimine aqueous solution and performing a third ultrasonic treatment to obtain Fe-Pt-PEI nanoparticles; S4, mixing the Fe-Pt-PEI nanoparticles obtained in step S3 with the Au@Pt nanoaqueous solution and performing a fourth ultrasonic treatment to obtain Fe-Pt-Au@Pt nanoparticles; S5, mixing the Fe-Pt-Au@Pt microspheres obtained in step S4 with the 5,5'-dithiobis(2-nitrobenzoic acid) ethanol solution and performing a fifth ultrasonic treatment to obtain a magnetic nanozyme with an outer surface modified with 5,5'-dithiobis(2-nitrobenzoic acid); S6. After activating the magnetic nanozyme with 5,5'-dithiobis(2-nitrobenzoic acid) modified on the outer surface obtained in step S5, the nanozyme is mixed with a specific antibody for incubation to obtain a magnetic nanozyme probe.

5. The method for preparing the magnetic nanozyme probe according to claim 4, characterized in that: In step S1, the average particle size of the Fe3O4 nanoparticles is 50-500nm, and ≤3g of Fe3O4 nanoparticles are dispersed in every 100mL of deionized water; the mass concentration of the polyethyleneimine aqueous solution is ≤2mg / mL; and the volume ratio of the dispersion to the polyethyleneimine aqueous solution is 1-50:

1.

6. The method for preparing the magnetic nanozyme probe according to claim 4, characterized in that: In step S2, the average particle size of the Pt nanoparticles is 10-50 nm, the amount of Fe3O4-PEI nanospheres added to each 100 mL Pt nanoaqueous solution is ≤4 g, and the mass concentration of the Pt nanoparticles is ≤10 mg / mL.

7. The method for preparing the magnetic nanozyme probe according to claim 4, characterized in that: In step S4, the average particle size of the Au@Pt nanoparticles is 5-30 nm, and the amount of Fe-Pt-PEI nanospheres added to each 100 mL Au@Pt aqueous solution is ≤ 4 g.

8. The method for preparing the magnetic nanozyme probe according to claim 4, characterized in that: In step S5, the mass concentration of the 5,5'-dithiobis(2-nitrobenzoic acid)ethanol solution is ≤2 mg / mL.

9. The method for preparing the magnetic nanozyme probe according to claim 4, characterized in that: In step S6, the activation treatment includes: mixing the magnetic nanozyme with the outer surface modified with 5,5'-dithiobis(2-nitrobenzoic acid) with the activation solution and performing a fifth ultrasonic treatment; in every 100 mL of the activation solution, the added amount of the magnetic nanozyme nanomicrospheres with the outer surface modified with 5,5'-dithiobis(2-nitrobenzoic acid) is 0.01-1 g; the activation solution is 100 mL of deionized water with 0.001-0.1 g of N-hydroxysuccinimide, 0.001-0.1 g of 1-ethyl-(3-dimethylaminopropyl)carbodiimide and 0.001-0.1 g of morpholineethanesulfonic acid added; in every 1 g of the magnetic nanozyme with the outer surface modified with 5,5'-dithiobis(2-nitrobenzoic acid), the added amount of the specific antibody is ≤1 mg; the specific antibody is a Helicobacter pylori antibody.

10. Use of the magnetic nanozyme probe according to any one of claims 1 to 3 in the preparation of a product for detecting Helicobacter pylori, wherein the product for detecting Helicobacter pylori comprises at least a magnetic nanozyme probe, an immunochromatographic test strip, and a kit.

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