Composite nano-material probe for detecting salmonella typhimurium, preparation method of composite nano-material probe and dual-mode immunochromatography test strip

Abstract

The invention belongs to the technical field of biochemical analysis and detection, and particularly relates to a composite nanomaterial probe for detecting salmonella typhimurium, a preparation method of the composite nanomaterial probe and a dual-mode immunochromatography test strip. According to the present invention, the improved dual-mode reading is achieved by improving the signal label, the BrM (at) Os with the surface modified with the rabbit anti-salmonella typhimurium polyclonal antibody is adopted as the detection probe, and the fluorescence and catalytic colorimetric dual-mode immunochromatography test strip capable of detecting salmonella typhimurium is prepared; the immunochromatographic test strip can realize sensitive immunochromatographic analysis on salmonella typhimurium in both fluorescence and catalytic colorimetric modes.

Description

Technical Field

[0001] The present invention belongs to the technical field of biochemical analysis and detection, and specifically relates to a composite nanomaterial probe for detecting Salmonella typhimurium, a preparation method thereof, and a dual-mode immunochromatographic test strip. Background Art

[0002] The great threat of foodborne pathogenic bacteria to public health and food safety has attracted great attention from the World Health Organization and governments of various countries. Salmonella typhimurium is an important zoonotic Gram-negative pathogen that can cause intestinal infections. It is mainly transmitted through feces, contaminated food or water. After entering the intestine through the stomach, it multiplies, adheres to the intestinal mucosal epithelial cells, and then invades the lamina propria, causing diseases with symptoms such as enteritis, severe sepsis, systemic infection, and visceral damage.

[0003] In recent years, various detection methods for Salmonella typhimurium have emerged in an endless stream, such as colony counting detection method, enzyme-linked immunosorbent assay, polymerase chain reaction, and microfluidic chips. Most of these detection methods are expensive and time-consuming, or require complex sample preparation steps. Therefore, it is necessary to develop rapid, economical, and sensitive detection methods as alternatives. Among them, lateral flow immunoassay (LFIA) has developed into a routine method in the fields of clinical diagnosis, food safety detection, environmental impact assessment, etc. due to its advantages such as visibility, high efficiency, and simple operation.

[0004] In recent years, perovskite quantum dots have the characteristics of simple synthesis, high quantum yield, adjustable emission, and high color purity, and are excellent fluorescent signal tags; nanozymes have been widely used in the field of catalytic colorimetric sensing due to their good catalytic effect, high stability, and low cost. Therefore, it is necessary to develop test strips and kits with catalytic colorimetric and fluorescence dual modes for detecting Salmonella typhimurium to meet the requirements of sensitive, rapid, and visual on-site detection and screening. Summary of the Invention

[0005] In order to solve the above problems, the first object of the present invention is to provide a composite material (BrM@Os) with dual functions of fluorescence and catalytic colorimetry conjugated with rabbit anti-Salmonella typhimurium polyclonal antibody as a labeling probe, which has high sensitivity.

[0006] The second object of the present invention is to provide a preparation method of a composite nanomaterial probe for detecting Salmonella typhimurium.

[0007] Another object of the present invention is to provide a test strip containing the above-mentioned composite nanomaterial probe for detecting Salmonella typhimurium.

[0008] This test strip combines the advantages of fluorescence detection and catalytic colorimetric detection, not only improving the detection sensitivity, but also making the detection results more intuitive and reliable.

[0009] For this reason, the first technical solution provided by the present invention is as follows:

[0010] A composite nanomaterial probe for detecting Salmonella typhimurium, wherein the composite nanomaterial probe is formed by conjugating the composite nanomaterial BrM@Os with rabbit anti-Salmonella typhimurium polyclonal antibody;

[0011] The composite nanomaterial BrM@Os is formed by modifying the surface of BrM nanoparticles with Os nanozyme. The BrM nanoparticles are formed by MSN nanoparticles encapsulating the fluorescent material CsPbBr3 inside; the MSN nanoparticles are porous materials formed from a silicon source.

[0012] Furthermore, for the above-mentioned composite nanomaterial probe for detecting Salmonella typhimurium, the mass ratio of the composite nanomaterial BrM@Os to the rabbit anti-Salmonella typhimurium polyclonal antibody is 20 - 40:1;

[0013] The mass ratio of the MSN nanoparticles, the fluorescent material CsPbBr3, and the Os nanozyme is 24 - 36:8 - 12:1.

[0014] Furthermore, for the above-mentioned composite nanomaterial probe for detecting Salmonella typhimurium, the mass ratio of the composite nanomaterial BrM@Os to the rabbit anti-Salmonella typhimurium polyclonal antibody is 30:1;

[0015] The mass ratio of the MSN nanoparticles, the fluorescent material CsPbBr3, and the Os nanozyme is 30:10:1.

[0016] Furthermore, for the above-mentioned composite nanomaterial probe for detecting Salmonella typhimurium, the silicon source is tetraethyl orthosilicate.

[0017] Furthermore, for the above-mentioned composite nanomaterial probe for detecting Salmonella typhimurium, the composite nanomaterial BrM@Os is prepared by the following method:

[0018] Step 1) Synthesize MSN nanoparticles

[0019] Stir the cetyltrimethylammonium bromide solution, anhydrous ethanol, and triethanolamine evenly, then add tetraethyl orthosilicate and stir. After cooling to room temperature, centrifuge, wash, dry, and heat-treat at 450 - 550 °C for 5 - 6 h under atmospheric conditions to obtain MSN;

[0020] Step 2) Synthesize BrM nanoparticles

[0021] Dissolve cesium bromide and lead bromide in ultrapure water. After ultrasonic treatment, add it to the MSN solution and stir at 70 - 80 °C for 15 - 20 min to obtain a mixed solution; then add a stabilizer to this mixture and continue stirring until the solvent completely evaporates; grind the powder and wash it, centrifuge to collect the product and dry it; then heat-treat at 400 - 500 °C for 20 - 30 min under atmospheric conditions, grind and wash with ultrapure water to obtain BrM nanoparticles;

[0022] Step 3) Synthesize Os nanozyme

[0023] Dissolve trisodium citrate and potassium osmate hexachloride in ultrapure water and stir. Subsequently, add sodium borohydride and stir in the dark for 45 - 60 min to obtain Os nanozyme;

[0024] Step 4) Synthesize composite nanomaterial BrM@Os

[0025] Ultrasonicate the BrM nanoparticles obtained in Step 2 with polyethyleneimine, centrifuge and wash; add the product to the Os nanozyme and ultrasonicate for 30 - 40 min, centrifuge and wash to obtain the composite nanomaterial BrM@Os.

[0026] Furthermore, for the above-mentioned composite nanomaterial probe for detecting Salmonella typhimurium, the mass ratio of tetraethyl orthosilicate, lead bromide, cesium bromide, trisodium citrate, potassium osmate hexachloride, and sodium borohydride is 15000:155:200:15:6:5.

[0027] The stabilizer is potassium carbonate solution. The technical solution of the present application adds potassium carbonate solution, which can generate hydroxide ions during subsequent water washing and react with lead bromide to form Pb(OH), making CsPbBr3 more stable; the template agent is cetyltrimethylammonium bromide.

[0028] Another technical solution of the present invention is to provide a preparation method of the above-mentioned composite nanomaterial probe for detecting Salmonella typhimurium. First, modify and activate carboxyl groups on the surface of BrM@Os, then add rabbit anti-Salmonella typhimurium polyclonal antibody, stir and then add bovine serum albumin solution to block the reaction sites, centrifuge and wash, and redisperse in the complex solution to obtain the composite nanomaterial probe for detecting Salmonella typhimurium.

[0029] Furthermore, for the above-mentioned preparation method of the composite nanomaterial probe for detecting Salmonella typhimurium, the method for modifying and activating carboxyl groups on the surface of BrM@Os is to add 1-ethyl-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide to the composite nanomaterial BrM@Os and ultrasonically activate the carboxyl groups.

[0030] Another technical solution of the present invention is to provide a dual-mode immunochromatographic test strip for detecting Salmonella typhimurium, including the composite nanomaterial probe for detecting Salmonella typhimurium described in the first technical solution.

[0031] Further, the above-mentioned dual-mode immunochromatographic test strip for detecting Salmonella typhimurium includes a bottom plate, on which a sample pad, a conjugate pad, a chromatographic membrane and an absorbent pad are sequentially fixed from left to right. The conjugate pad is sprayed with the composite nanomaterial probe for detecting Salmonella typhimurium; the chromatographic membrane is provided with a detection line and a quality control line arranged in parallel. The detection line is provided with a Salmonella typhimurium monoclonal antibody, and the quality control line is provided with a goat anti-rabbit IgG antibody.

[0032] Preferably, the sample pad is a glass fiber membrane; more preferably: the sample pad: the glass cellulose membrane is immersed in a Tris-HCL solution containing 1% sucrose, 1% bovine serum albumin and 0.5% Tween-20 for 0.5 h, and then dried to obtain.

[0033] Preferably, the conjugate pad is a glass fiber membrane; more preferably: the conjugate pad: the glass cellulose membrane is immersed in a Tris-HCL solution containing 5% sucrose, 1% bovine serum albumin and 0.05% Tween-20 for 0.5 h, and then dried to obtain.

[0034] The above-mentioned dual-mode immunochromatographic test strip for detecting Salmonella typhimurium is prepared by the following method:

[0035] Step 1) Preparation method of the composite nanomaterial probe: Modify the surface of BrM@Os with carboxyl groups and activate the carboxyl groups, add rabbit anti-Salmonella typhimurium polyclonal antibody, stir and then add bovine serum albumin solution, centrifuge and wash, and redisperse in the complex solution for use as the composite nanomaterial probe;

[0036] Step 2) Preparation method of the detection line and the quality control line: Use an XYZ three-dimensional membrane scribing and gold spraying instrument to scribe the Salmonella typhimurium monoclonal antibody and the goat anti-rabbit IgG antibody on the membrane respectively to prepare a chromatographic membrane with a detection line and a quality control line;

[0037] Step 3) Preparation method of the test strip: Use an XYZ three-dimensional membrane scribing and gold spraying instrument to spray the composite nanomaterial probe on the conjugate pad to prepare a conjugate pad with the composite nanomaterial probe. Stick the sample pad, the conjugate pad, the chromatographic membrane and the absorbent pad on the bottom plate to obtain the immunochromatographic test strip.

[0038] Preferably) In the above step 1, the complex solution of the composite nanomaterial probe is a Tris-HCL solution containing sucrose, fructose, polyethylene glycol 20000, bovine serum albumin and Tween-20;

[0039] Preferably, in step 2, the concentrations of both the Salmonella typhimurium monoclonal antibody and the goat anti-rabbit IgG antibody are 1.0 mg / mL, the spotting speed is 1.0 μL / cm, and the distance between the test line and the control line is 5 mm.

[0040] Preferably, in step 3, the spotting speed of the composite nanomaterial probe is 8.5 μL / cm.

[0041] Preferably, for the fluorescence and catalytic colorimetric dual-mode immunochromatographic test strip, the fluorescence mode can be visually judged under an ultraviolet lamp and the values can be read using a fluorescence immunoassay analyzer.

[0042] Preferably, for the catalytic colorimetric mode of the fluorescence and catalytic colorimetric dual-mode immunochromatographic test strip, it can be immersed in a chromogenic solution for catalytic chromogenesis; more preferably, the chromogenic solution is a phosphate-citrate buffer solution containing 3,3',5,5'-tetramethylbenzidine and hydrogen peroxide.

[0043] For the prior art, the advantages of the present invention are as follows:

[0044] 1. The present invention synthesizes BrM nanoparticles with uniform size and good dispersion, excellent optical properties and water stability through an in-situ crystallization growth method; this BrM is obtained by wrapping CsPbBr3 with MSN and then performing high-temperature calcination, resulting in the collapse of mesopores, making CsPbBr3 exhibit excellent fluorescence stability in aqueous solution; the nanozyme is simply synthesized and has excellent steady-state kinetic parameters and specific activities. The synthesized BrM in this application has a negative Zeta potential, which can well combine with polyethyleneimine to form a positive charge, and better combine with the Os nanozyme. Therefore, BrM@Os with dual functions of fluorescence and catalytic colorimetry is formed.

[0045] 2. The technical solution provided by the present invention uses BrM@Os as a labeling probe, first makes it carry carboxyl groups and activates the carboxyl groups, better combines with the rabbit anti-Salmonella typhimurium polyclonal antibody, and integrates it into the lateral flow immunoassay platform. The test strip adopts a double-antibody sandwich method, and shows good sensitivity in detecting Salmonella typhimurium.

[0046] 3. The dual-mode immunochromatographic test strip prepared by the technical solution provided by the present invention can be used to detect Salmonella typhimurium, and the method is simple to operate, time-consuming, fast and accurate in detection, and low in cost. BRIEF DESCRIPTION OF THE DRAWINGS

[0047] Figure 1 It is a transmission electron microscope characterization diagram of BrM prepared in Example 1 of the present invention.

[0048] Figure 2 It is a transmission electron microscope characterization diagram of BrM@Os prepared in Example 1 of the present invention.

[0049] Figure 3 Photographs of MSN, Os, BrM, BrM@PEI, and BrM@Os solutions prepared in Example 1 of the present invention taken under ultraviolet light.

[0050] Figure 4 Steady-state kinetic diagram of BrM@Os nanozyme prepared in Example 1 of the present invention and absorbance value diagrams affected by pH and temperature factors.

[0051] Figure 5 Schematic diagram of the fluorescence and catalytic colorimetric dual-mode immunochromatographic test strip in Example 4 of the present invention.

[0052] Figure 6 Fluorescence and catalytic colorimetric detection performance of the immunochromatographic test strip for Salmonella typhimurium in Example 5 of the present invention. Detailed implementation manners

[0053] To further illustrate the methods and effects of the present invention, the present invention will be further described in detail below in conjunction with specific implementation manners. All technologies implemented based on the above content of the present invention are covered within the scope of protection of the present invention.

[0054] The polyclonal antibody against Salmonella typhimurium used in the present invention comes from the laboratory. The polyclonal antibody against Salmonella typhimurium was prepared by immunizing New Zealand white rabbits with inactivated Salmonella typhimurium bacterial solution as an antigen.

[0055] Example 1

[0056] This example provides a composite nanomaterial probe for detecting Salmonella typhimurium. The composite nanomaterial probe is formed by conjugating the composite nanomaterial BrM@Os with a polyclonal antibody against Salmonella typhimurium. The mass ratio of the composite nanomaterial BrM@Os to the polyclonal antibody against Salmonella typhimurium is 30:1.

[0057] The composite nanomaterial BrM@Os is formed by modifying the outer surface of BrM nanoparticles with Os nanozyme. The BrM nanoparticles are formed by MSN nanoparticles wrapped with the fluorescent material CsPbBr3 inside. The MSN nanoparticles are a porous material formed by tetraethyl orthosilicate. The mass ratio of the MSN nanoparticles, the fluorescent material CsPbBr3, and the Os nanozyme is 30:10:1.

[0058] It is prepared by the following method:

[0059] 1) Synthesis of MSN:

[0060] Dissolve 200 mg of cetyltrimethylammonium bromide in 25 mL of ultrapure water. Subsequently, add 8 mL of ethanol and 50 μL of triethanolamine, and stir in a water bath at 60 °C for 30 min. Quickly add 2 mL of tetraethyl orthosilicate, and stir in a water bath at 60 °C for 2 h. After cooling to room temperature, centrifuge, wash the collected product with ultrapure water three times, and dry at 80 °C. Then calcine the MSN at 450 °C for 5 h under atmospheric conditions to remove the template cetyltrimethylammonium bromide.

[0061] 2) Synthesis of BrM:

[0062] 2.1) Dissolve cesium bromide (12 mmol / L) and lead bromide (12 mmol / L) in 6 mL of ultrapure water, then sonicate for 5 min and stir at 80 °C for 5 min to obtain solution A;

[0063] Dissolve 125 mg of MSN in 2.4 mL of ultrapure water, and sonicate until a homogeneous solution is formed to obtain solution B;

[0064] 2.2) Mix solution A and solution B and stir at 70 °C for another 15 min. While stirring, add 120 μL of potassium carbonate solution (0.3 mol / L) to this mixture until the solvent completely evaporates. Grind the collected powder and wash it several times with ethanol until the solution becomes white. Centrifuge to collect the product and dry it at 80 °C;

[0065] 2.3) Calcine the product obtained in 2.2) at 400 °C for 20 min under atmospheric conditions. After cooling to room temperature, finely grind the product and wash it three times with ultrapure water to obtain BrM nanoparticles.

[0066] 3) Synthesis of Os nanozyme:

[0067] Mix 1 mL of trisodium citrate (40 mmol / L) and 38 mL of ultrapure water, stir for 5 min, add 1 mL of potassium hexachloroiridate (10 mmol / L) and stir for 30 min. Subsequently, dropwise add 200 μL of sodium borohydride (50 mmol / L) and stir in the dark for 45 min (500 rpm). The color gradually changes from yellowish-green to brown to obtain Os nanozyme.

[0068] 4) Synthesis of BrM@PEI:

[0069] Dissolve 20 mg of BrM in 1 mL of ultrapure water, sonicate for 20 min, add 125 mg of polyethyleneimine aqueous solution (50%), sonicate for 30 min, centrifuge and wash twice with ultrapure water to obtain BrM@PEI.

[0070] 5) Synthesis of BrM@Os:

[0071] Add 90 μL of Os nanozyme to 1 mL of BrM@PEI (1 mg / mL), sonicate for 30 min, and centrifuge and wash three times to obtain the nanocomposite BrM@Os.

[0072] 6) Preparation of the composite nanomaterial BrM@Os probe

[0073] Add 15 μL of 1-ethyl-(3-dimethylaminopropyl)carbodiimide (100 mg / mL) and 30 μL of N-hydroxysuccinimide (100 mg / mL) to 1 mL of the composite nanomaterial BrM@Os (2 mg / mL), and sonicate for 10 min to activate the carboxyl group. After activation, centrifuge and collect the precipitate. Resuspend the precipitate in Tris-HCL buffer, add 50 μL of rabbit anti-Salmonella typhimurium polyclonal antibody (2 mg / mL), stir evenly for 2 h, and then add 1 mL of blocking solution (bovine serum albumin solution, 100 mg / mL) and continue to stir and react for 1 h to block the sites on the nanocomposite that are not bound to the antibody. After the reaction, centrifuge to collect the precipitate and wash it once with Tris-HCL buffer. Resuspend the precipitate in 50 μL of the complex solution and store it in the dark at 4 °C for later use.

[0074] Example 2

[0075] This example provides a composite nanomaterial probe for detecting Salmonella typhimurium, which is formed by coupling the composite nanomaterial BrM@Os with rabbit anti-Salmonella typhimurium polyclonal antibody; the mass ratio of the composite nanomaterial BrM@Os to the rabbit anti-Salmonella typhimurium polyclonal antibody is 30:1.

[0076] The composite nanomaterial BrM@Os is formed by modifying the outside of BrM nanoparticles with Os nanozyme. The BrM nanoparticles are formed by MSN nanoparticles wrapped with the fluorescent material CsPbBr3 inside; the MSN nanoparticles are a porous material formed by tetraethyl orthosilicate; the mass ratio of the MSN nanoparticles, the fluorescent material CsPbBr3, and the Os nanozyme is 30:10:1.

[0077] It is prepared by the following method:

[0078] 1) Synthesis of MSN:

[0079] Dissolve 200 mg of cetyltrimethylammonium bromide in 25 mL of ultrapure water. Subsequently, add 8 mL of ethanol and 50 μL of triethanolamine, and stir in a 60 °C water bath for 30 min. Quickly add 2 mL of tetraethyl orthosilicate and stir in a 60 °C water bath for 2 h. After cooling to room temperature, centrifuge, and wash the collected product 3 times with ultrapure water and dry it at 80 °C. Then calcine the MSN at 500 °C for 5.5 h under atmospheric conditions to remove the template cetyltrimethylammonium bromide.

[0080] 2) Synthesis of BrM:

[0081] 2.1) Dissolve cesium bromide (12 mmol / L) and lead bromide (12 mmol / L) in 6 mL of ultrapure water, then sonicate for 5 min and stir at 80 °C for 5 min to obtain solution A;

[0082] Dissolve 125 mg of MSN in 2.4 mL of ultrapure water and sonicate until a homogeneous solution is formed to obtain solution B;

[0083] 2.2) Mix solution A and solution B and stir at 75 °C for another 18 min. While stirring, add 120 μL of potassium carbonate solution (0.3 mol / L) to the mixture, continue stirring at 75 °C until the solvent completely evaporates. Grind the collected powder and wash it several times with ethanol until the solution turns white. Centrifuge to collect the product and dry it at 80 °C;

[0084] 2.3) Calcinate the product obtained in 2.2) at 450 °C for 25 min under atmospheric conditions. After cooling to room temperature, finely grind the product and wash it three times with ultrapure water to obtain BrM nanoparticles.

[0085] 3) Synthesis of Os nanozyme:

[0086] Mix 1 mL of trisodium citrate (40 mmol / L) and 38 mL of ultrapure water, stir for 5 min, add 1 mL of potassium hexachloroiridate (10 mmol / L) and stir for 30 min. Subsequently, add 2 hundred μL of sodium borohydride (50 mmol / L) and stir in the dark for 55 min (500 rpm). The color gradually changes from yellowish-green to brown to obtain the Os nanozyme.

[0087] 4) Synthesis of BrM@PEI:

[0088] Dissolve 20 mg of BrM in 1 mL of ultrapure water, sonicate for 20 min, add 125 mg of polyethyleneimine aqueous solution (50%), sonicate for 30 min, centrifuge and wash twice with ultrapure water to obtain BrM@PEI.

[0089] 5) Synthesis of BrM@Os:

[0090] Add 90 μL of the Os nanozyme to 1 mL of BrM@PEI (1 mg / mL), sonicate for 35 min, centrifuge and wash three times to obtain the nanocomposite BrM@Os.

[0091] 6) Preparation of the composite nanomaterial BrM@Os probe

[0092] Add 15 μL of 1-ethyl-(3-dimethylaminopropyl)carbodiimide (100 mg / mL) and 30 μL of N-hydroxysuccinimide (100 mg / mL) to 1 mL of the composite nanomaterial BrM@Os (2 mg / mL), and ultrasonically activate the carboxyl group for 10 min. After activation, centrifuge to obtain the precipitate, resuspend the precipitate in Tris-HCL buffer, add 50 μL of rabbit anti-Salmonella typhimurium polyclonal antibody (2 mg / mL), stir evenly for 2 h, then add 1 mL of blocking solution (bovine serum albumin solution, 100 mg / mL) and continue to stir and react for 1 h to block the sites on the nanocomposite that are not bound to the antibody. After the reaction, centrifuge to collect the precipitate and wash it once with Tris-HCL buffer, then resuspend the precipitate in 50 μL of the complex solution and store it in the dark at 4 °C for later use.

[0093] Example 3

[0094] This example provides a composite nanomaterial probe for detecting Salmonella typhimurium, which is formed by coupling the composite nanomaterial BrM@Os with rabbit anti-Salmonella typhimurium polyclonal antibody; the mass ratio of the composite nanomaterial BrM@Os to rabbit anti-Salmonella typhimurium polyclonal antibody is 30:1.

[0095] The composite nanomaterial BrM@Os is formed by modifying Os nanozyme on the outside of BrM nanoparticles, and the BrM nanoparticles are formed by MSN nanoparticles wrapped with the fluorescent material CsPbBr3 inside; the MSN nanoparticles are a porous material formed by tetraethyl orthosilicate; the mass ratio of the MSN nanoparticles, the fluorescent material CsPbBr3, and the Os nanozyme is 30:10:1.

[0096] It is prepared by the following method:

[0097] 1) Synthesis of MSN:

[0098] Dissolve 200 mg of cetyltrimethylammonium bromide in 25 mL of ultrapure water. Subsequently, add 8 mL of ethanol and 50 μL of triethanolamine, and stir in a 60 °C water bath for 30 min. Quickly add 2 mL of tetraethyl orthosilicate and stir in a 60 °C water bath for 2 h. After cooling to room temperature, centrifuge, wash the collected product 3 times with ultrapure water, and dry it at 80 °C. Then calcine the MSN at 550 °C for 6 h under atmospheric conditions to remove the template cetyltrimethylammonium bromide.

[0099] 2) Synthesis of BrM:

[0100] 2.1) Dissolve cesium bromide (12 mmol / L) and lead bromide (12 mmol / L) in 6 mL of ultrapure water, then ultrasonically treat for 5 min and stir at 80 °C for 5 min to obtain solution A;

[0101] Dissolve 125 mg of MSN in 2.4 mL of ultrapure water and sonicate until a homogeneous solution B is formed;

[0102] 2.2) Mix solution A and solution B and stir for another 20 min at 80 °C. While stirring, add 120 μL of potassium carbonate solution (0.3 mol / L) to the mixture until the solvent is completely evaporated. Grind the collected powder and wash it several times with ethanol until the solution becomes white. Centrifuge to collect the product and dry it at 80 °C;

[0103] 2.3) Calcinate the product obtained in 2.2) at 500 °C for 30 min under atmospheric conditions. After cooling to room temperature, finely grind the product and wash it three times with ultrapure water to obtain BrM nanoparticles.

[0104] 3) Synthesis of Os nanozyme:

[0105] Mix 1 mL of trisodium citrate (40 mmol / L) and 38 mL of ultrapure water, stir for 5 min, add 1 mL of potassium hexachloroiridate (10 mmol / L) and stir for 30 min. Subsequently, dropwise add 200 μL of sodium borohydride (50 mmol / L) and stir in the dark for 60 min (500 rpm). The color gradually changes from yellow-green to brown to obtain Os nanozyme.

[0106] 4) Synthesis of BrM@PEI:

[0107] Dissolve 20 mg of BrM in 1 mL of ultrapure water, sonicate for 20 min, add 125 mg of polyethyleneimine aqueous solution (50%), sonicate for 30 min, centrifuge and wash twice with ultrapure water to obtain BrM@PEI.

[0108] 5) Synthesis of BrM@Os:

[0109] Add 90 μL of Os nanozyme to 1 mL of BrM@PEI (1 mg / mL), sonicate for 40 min, centrifuge and wash three times to obtain the nanocomposite BrM@Os.

[0110] 5) Preparation of the composite nanomaterial BrM@Os probe

[0111] Add 15 μL of 1-ethyl-(3-dimethylaminopropyl)carbodiimide (100 mg / mL) and 30 μL of N-hydroxysuccinimide (100 mg / mL) to 1 mL of the composite nanomaterial BrM@Os (2 mg / mL), and ultrasonically activate the carboxyl group for 10 min. After activation, centrifuge to obtain the precipitate, resuspend the precipitate in Tris-HCL buffer, add 50 μL of rabbit anti-Salmonella typhimurium polyclonal antibody (2 mg / mL), stir evenly for 2 h, and then add 1 mL of blocking solution (bovine serum albumin solution, 100 mg / mL) and continue to stir and react for 1 h to block the sites on the nanocomposite that are not bound to the antibody. After the reaction, centrifuge to collect the precipitate and wash it once with Tris-HCL buffer, and resuspend the precipitate in 50 μL of the complex solution for standby at 4 °C in the dark.

[0112] As Figure 1 shown, dilute the BrM solution into a clear solution, drop 10 μL onto a copper grid, dry at 80 °C for 2 h, and take a picture on a transmission electron microscope. The transmission electron micrograph of BrM shows that it has a regular spherical shape with black dots inside, and the black dots are CsPbBr3 crystals.

[0113] As Figure 2 shown, dilute the BrM@Os solution into a clear solution, drop 10 μL onto a copper grid, dry at 80 °C for 2 h, and take a picture on a transmission electron microscope. Compared with BrM, the transmission electron micrograph of BrM@Os shows that Os nanozymes are significantly adsorbed on its surface, indicating the successful preparation of BrM@Os.

[0114] As Figure 3 shown, under ultraviolet lamp irradiation, the BrM, BrM@PEI, and BrM@Os solutions produce bright green fluorescence among the MSN, Os, BrM, BrM@PEI, and BrM@Os solutions.

[0115] As Figure 4 shown, the composite nanomaterial BrM@Os nanozyme has peroxidase-like activity and can catalyze TMB to show an obvious blue change. By measuring the absorbance of its blue product oxTMB at 652 nm, the BrM@Os nanozyme has steady-state kinetics, has good enzyme activity in the range of 20-80 °C, and the optimal catalytic condition is pH = 4.

[0116] Example 4

[0117] This embodiment provides a dual-mode immunochromatographic test strip for detecting Salmonella typhimurium, including a bottom plate, on which a sample pad, a conjugate pad, a chromatographic membrane, and an absorbent pad are sequentially fixed from left to right. The conjugate pad is sprayed with the composite nanomaterial probe for detecting Salmonella typhimurium according to any one of Embodiments 1-3. The chromatographic membrane is provided with a detection line and a quality control line arranged in parallel. The detection line is provided with a Salmonella typhimurium monoclonal antibody, and the quality control line is provided with a goat anti-rabbit IgG antibody.

[0118] It is prepared through the following steps:

[0119] The sample pad is pretreated by immersing a glass fiber membrane in a Tris-HCL solution containing 1% sucrose, 1% bovine serum albumin, and 0.5% Tween-20 for 0.5 h, and then drying it.

[0120] The conjugate pad is pretreated by immersing a glass fiber membrane in a Tris-HCL solution containing 5% sucrose, 1% bovine serum albumin, and 0.05% Tween-20 for 0.5 h, and then drying it.

[0121] Using an XYZ three-dimensional membrane scribing and gold spraying instrument, goat anti-rabbit IgG (1 mg / mL) and Salmonella typhimurium monoclonal antibody (1 mg / mL) are scribed and fixed on the chromatographic membrane at a sample loading amount of 1 μL / cm. Goat anti-rabbit IgG serves as the quality control line (C), and the Salmonella typhimurium monoclonal antibody serves as the detection line (T). The distance between the two lines is 5 mm. After scribing, the chromatographic membrane is pasted on the bottom plate and placed in an oven at 37 °C for drying for 2 h.

[0122] Using an XYZ three-dimensional membrane scribing and gold spraying instrument, the composite nanomaterial probe for detecting Salmonella typhimurium prepared according to any one of Embodiments 1-3 is printed on the conjugate pad at a sample loading amount of 8.5 μL / cm, and dried at room temperature.

[0123] Finally, the test strip is assembled: the sample pad, the conjugate pad, the chromatographic membrane, and the absorbent pad are pasted on the bottom plate in sequence from left to right. There is an overlap of about 2 mm between the sample pad, the conjugate pad, the chromatographic membrane, and the absorbent pad. At the same time, the absorbent pad is close to one end of the quality control line on the chromatographic membrane, and the sample pad is close to the end of the detection line. Finally, the test strip is installed in a plastic cartridge and stored in a dry environment at room temperature. For its structure diagram, refer to Figure 5 .

[0124] Experimental Example 5 Detection of Salmonella typhimurium

[0125] At the sample loading position of the dual-mode immunochromatographic test strip for detecting Salmonella typhimurium prepared in Example 4, 10, 10 2 , 103 and 10 4 and 10 5 and 10 6 and 10 7 and 10 8 and 10 9 60 μL each of Salmonella typhimurium at CFU / mL and sterile Tris-HCL buffer solution. After 15 minutes, first observe the fluorescence color development of the test strip band under ultraviolet light (365 nm) with the naked eye, and then insert the plastic cartridge into the dry fluorescence immunoassay analyzer in sequence to read the fluorescence intensity of the test strip band by the instrument. Immerse the test strip in the color development solution and observe the catalytic color development situation with the naked eye. The results are referred to Figure 6 . The T line of the test strip becomes darker with the increase of the bacterial concentration. The fluorescence and catalytic colorimetric dual-mode immunochromatographic test strip has a detection line of 10 5 CFU / mL and has good sensitivity, and can be used to detect Salmonella typhimurium.

[0126] The embodiments of the present invention have been described above in conjunction with the accompanying drawings. However, the present invention is not limited to the above specific embodiments. The above specific embodiments are merely illustrative and not restrictive. Under the inspiration of the present invention, those of ordinary skill in the art can also make many forms without departing from the spirit and scope protected by the claims of the present invention. All of these are within the protection scope of the present invention.

Claims

1. A composite nanomaterial probe for detecting Salmonella typhimurium, characterized in that, The described composite nanomaterial probe is formed by conjugating the composite nanomaterial BrM@Os with rabbit anti-Salmonella typhimurium polyclonal antibody; The described composite nanomaterial BrM@Os is formed by modifying the surface of BrM nanoparticles with Os nanozyme. The described BrM nanoparticles are formed by MSN nanoparticles wrapped with fluorescent material CsPbBr3 inside; The described MSN nanoparticles are porous materials formed from a silicon source.

2. The composite nanomaterial probe for detecting Salmonella typhimurium according to claim 1, wherein, The mass ratio of the described composite nanomaterial BrM@Os to rabbit anti-Salmonella typhimurium polyclonal antibody is 20 - 40:1; The mass ratio of the described MSN nanoparticles, fluorescent material CsPbBr3, and Os nanozyme is 24 - 36:8 - 12:

1.

3. The composite nanomaterial probe for detecting Salmonella typhimurium according to claim 2, wherein, The mass ratio of the described composite nanomaterial BrM@Os to rabbit anti-Salmonella typhimurium polyclonal antibody is 30:1; The mass ratio of the described MSN nanoparticles, fluorescent material CsPbBr3, and Os nanozyme is 30:10:

1.

4. The composite nanomaterial probe for detecting Salmonella typhimurium according to claim 1, wherein The described silicon source is tetraethyl orthosilicate.

5. The composite nanomaterial probe for detecting Salmonella typhimurium according to claims 1-4, characterized in that, The described composite nanomaterial BrM@Os is prepared by the following method: Step 1: Synthesize MSN nanoparticles Stir the cetyltrimethylammonium bromide solution, absolute ethanol, and triethanolamine evenly, then add tetraethyl orthosilicate and stir. After cooling to room temperature, centrifuge, wash, dry, and heat-treat at 450 - 550 °C for 5 - 6 h under atmospheric conditions to obtain MSN; Step 2: Synthesize BrM nanoparticles Dissolve cesium bromide and lead bromide in ultrapure water, add it to the MSN solution after ultrasonic treatment, and stir at 70 - 80 °C for 15 - 20 min to obtain a mixed solution; then add a stabilizer to this mixture and continue stirring until the solvent completely evaporates; Grind the powder and wash it, centrifuge to collect the product and dry it; then heat-treat at 400 - 500 °C for 20 - 30 min under atmospheric conditions, grind and wash with ultrapure water to obtain BrM nanoparticles; Step 3: Synthesize Os nanozyme Dissolve trisodium citrate and potassium hexachloroosmate in ultrapure water and stir, then add sodium borohydride and stir in the dark for 45 - 60 min to obtain Os nanozyme; Step 4: Synthesize the composite nanomaterial BrM@Os Ultrasonically treat the BrM nanoparticles obtained in Step 2 with polyethyleneimine, centrifuge and wash; add the product to Os nanozyme and ultrasonically treat for 30 - 40 min, centrifuge and wash to obtain the composite nanomaterial BrM@Os.

6. The composite nanomaterial probe for detecting Salmonella typhimurium according to claim 5, wherein The mass ratio of tetraethyl orthosilicate, lead bromide, cesium bromide, trisodium citrate, potassium hexachloroosmate, and sodium borohydride is 15000:155:200:15:6:

5. The described stabilizer is a potassium carbonate solution; the described template agent is cetyltrimethylammonium bromide.

7. The preparation method of the composite nanomaterial probe for detecting Salmonella typhimurium according to claim 5, characterized in that, First, modify and activate carboxyl groups on the surface of BrM@Os, then add rabbit anti-Salmonella typhimurium polyclonal antibody, stir, add bovine serum albumin solution to block the reaction sites, centrifuge and wash, and redisperse in the complex solution to obtain the composite nanomaterial probe for detecting Salmonella typhimurium.

8. The preparation method of the composite nanomaterial probe for detecting Salmonella typhimurium according to claim 7, characterized in that, The method for modifying and activating carboxyl groups on the surface of BrM@Os is to add 1-ethyl-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide to the composite nanomaterial BrM@Os and ultrasonically activate the carboxyl groups.

9. A dual-mode immunochromatographic test strip for detecting Salmonella typhimurium, characterized in that, Comprising the composite nanomaterial probe for detecting Salmonella typhimurium as described in claim 1.

10. The dual-mode immunochromatographic test strip for detecting Salmonella typhimurium according to claim 9, comprising a bottom plate, wherein a sample pad, a conjugate pad, a chromatographic membrane and an absorbent pad are sequentially fixed on the bottom plate from left to right, and is characterized in that, The composite nanomaterial probe for detecting Salmonella typhimurium sprayed on the conjugate pad; on the chromatographic membrane, a test line and a quality control line are arranged in parallel, the test line is provided with a Salmonella typhimurium monoclonal antibody as the test line, and the quality control line is provided with a goat anti-rabbit IgG antibody.

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