Compound nanomaterial probe for detecting salmonella typhimurium, preparation method of compound nanomaterial probe and dual-mode immunochromatographic test strip

Abstract

The present application 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 thereof, and a dual-mode immunochromatographic test strip; the present application realizes improved dual-mode reading by improving a signal tag, takes BrM@Os with a rabbit anti-salmonella typhimurium polyclonal antibody on the surface as a detection probe, and prepares a fluorescence and catalytic colorimetric dual-mode immunochromatographic test strip capable of detecting salmonella typhimurium. The immunochromatographic test strip can realize sensitive immunochromatographic analysis of salmonella typhimurium in fluorescence and catalytic colorimetric modes.

Description

Technical Field

[0001] This invention belongs to the field of biochemical analysis and detection technology, specifically relating to a composite nanomaterial probe for detecting Salmonella typhimurium and its preparation method, as well as a dual-mode immunochromatographic test strip. Background Technology

[0002] The significant threat posed by foodborne pathogens to public health and food safety has drawn serious attention from the World Health Organization and governments worldwide. Salmonella Typhimurium is an important zoonotic Gram-negative pathogen that causes intestinal infections. It is primarily transmitted through feces, contaminated food, or water. After entering the intestines via the stomach, it proliferates, adheres to the intestinal mucosal epithelial cells, and subsequently invades the lamina propria, causing diseases characterized by enteritis, severe sepsis, systemic infection, and visceral damage.

[0003] In recent years, numerous methods for detecting Salmonella typhimurium have emerged, such as colony counting, enzyme-linked immunosorbent assay (ELISA), polymerase chain reaction (PCR), and microfluidic chips. However, most of these methods are expensive and time-consuming, or require complex sample preparation steps. Therefore, it is necessary to develop rapid, economical, and sensitive alternatives. Among these, lateral flow immunoassay (LFIA), with its advantages of visibility, high efficiency, and ease of operation, has become a routine method in clinical diagnosis, food safety testing, and environmental impact assessment.

[0004] In recent years, perovskite quantum dots have been recognized as excellent fluorescent signal tags due to their simple synthesis, high quantum yield, tunable emission, and high color purity. Nanozymes, on the other hand, are widely used in catalytic colorimetric sensing due to their good catalytic effect, high stability, and low cost. Therefore, it is crucial to develop test strips and kits with both catalytic colorimetric and fluorescence modes for the detection of Salmonella Typhimurium to meet the needs for sensitive, rapid, and visualized instant detection and screening. Summary of the Invention

[0005] To address the aforementioned problems, the first objective of this invention is to provide a labeling probe that uses a composite material (BrM@Os) with dual functions of fluorescence and catalytic colorimetry coupled with rabbit anti-Salmonella typhimurium polyclonal antibodies as a labeling probe, exhibiting high sensitivity.

[0006] The second objective of this invention is to provide a method for preparing 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, which not only improves the sensitivity of the detection, but also makes the test results more intuitive and reliable.

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

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

[0011] The composite nanomaterial BrM@Os is formed by modifying BrM nanoparticles with Os nanozymes. The BrM nanoparticles are formed by MSN nanoparticles containing the fluorescent material CsPbBr3. The MSN nanoparticles are porous materials formed from silicon sources.

[0012] Furthermore, in the aforementioned 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 MSN nanoparticles, fluorescent material CsPbBr3, and Os nanozyme is 24-36:8-12:1.

[0014] Furthermore, in the aforementioned 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 MSN nanoparticles, fluorescent material CsPbBr3, and Os nanozyme is 30:10:1.

[0016] Furthermore, in the aforementioned composite nanomaterial probe for detecting Salmonella typhimurium, the silicon source is tetraethyl silicate.

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

[0018] Step 1) Synthesis of MSN nanoparticles

[0019] Hexadecyltrimethylammonium bromide solution, anhydrous ethanol and triethanolamine were stirred evenly, then tetraethyl silicate was added and stirred. After cooling to room temperature, the mixture was centrifuged, washed and dried. MSN was obtained by heat treatment at 450-550℃ for 5-6 hours under atmospheric conditions.

[0020] Step 2) Synthesis of BrM nanoparticles

[0021] Cesium bromide and lead bromide were dissolved in ultrapure water, sonicated, and then added to MSN solution. The mixture was stirred at 70–80 °C for 15–20 min to obtain a mixture. A stabilizer was then added to the mixture, and stirring was continued until the solvent was completely evaporated. The powder was ground and washed, and the product was collected by centrifugation and dried. The product was then heat-treated at 400–500 °C for 20–30 min under atmospheric conditions, ground, and washed with ultrapure water to obtain BrM nanoparticles.

[0022] Step 3) Synthesis of Os nanozymes

[0023] Dissolve trisodium citrate and potassium hexachloroosmium tetroxide in ultrapure water and stir. Then add sodium borohydride and stir in the dark for 45-60 minutes to obtain Os nanozyme.

[0024] Step 4) Synthesis of composite nanomaterial BrM@Os

[0025] The BrM nanoparticles obtained in step 2 were ultrasonicated with polyethyleneimine and then washed by centrifugation. The product was then added to Os nanozyme and ultrasonicated for 30-40 min, followed by centrifugation and washing to obtain the composite nanomaterial BrM@Os.

[0026] Furthermore, in the aforementioned composite nanomaterial probe for detecting Salmonella typhimurium, the mass ratio of tetraethyl silicate, lead bromide, cesium bromide, trisodium citrate, potassium hexachloroosmium tetroxide, and sodium borohydride is 15000:155:200:15:6:5.

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

[0028] Another technical solution of the present invention is to provide a method for preparing the above-mentioned composite nanomaterial probe for detecting Salmonella typhimurium. First, the carboxyl group is modified and activated on the surface of BrM@Os, then rabbit anti-Salmonella typhimurium polyclonal antibody is added, and after stirring, bovine serum albumin solution is added to block the reaction site. After centrifugation and washing, it is redispersed in a complex solution to obtain the composite nanomaterial probe for detecting Salmonella typhimurium.

[0029] Furthermore, in the above-mentioned method for preparing the composite nanomaterial probe for detecting Salmonella typhimurium, the method for modifying and activating the carboxyl groups on the BrM@Os surface involves adding 1-ethyl-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide to the composite nanomaterial BrM@Os and then activating the carboxyl groups by ultrasonication.

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

[0031] Furthermore, the aforementioned dual-mode immunochromatographic test strip for detecting Salmonella typhimurium includes a base plate, on which a sample pad, a conjugate pad, a chromatography membrane, and an absorbent pad are fixed sequentially from left to right. The conjugate pad is sprayed with a composite nanomaterial probe for detecting Salmonella typhimurium. The chromatography membrane has parallel detection lines and control lines. The detection lines contain Salmonella typhimurium monoclonal antibodies, and the control lines contain goat anti-rabbit IgG antibodies.

[0032] Preferably, the sample pad is a glass fiber membrane; more preferably, the sample pad is prepared by immersing a glass fiber membrane in a Tris-HCl solution containing 1% by mass of sucrose, 1% by mass of bovine serum albumin and 0.5% by mass of Tween-20 for 0.5 h, and then drying it.

[0033] Preferably, the conjugate pad is a glass fiber membrane; more preferably, the conjugate pad is prepared 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.

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

[0035] Step 1) Preparation method of composite nanomaterial probe: Modify the carboxyl group on the surface of BrM@Os and activate the carboxyl group, add rabbit anti-Salmonella typhimurium polyclonal antibody, stir and add bovine serum albumin solution, centrifuge and wash, and redisperse in the reconstitution solution to be used as composite nanomaterial probe;

[0036] Step 2) Preparation of detection lines and control lines: Use the XYZ three-dimensional streak sprayer to streak Salmonella typhimurium monoclonal antibody and goat anti-rabbit IgG antibody to prepare chromatographic membranes with detection lines and control lines.

[0037] Step 3) Preparation of the test strip: The composite nanomaterial probe is sprayed onto the conjugate pad using an XYZ three-dimensional gold spraying instrument to prepare a conjugate pad with the composite nanomaterial probe. The sample pad, conjugate pad, chromatography membrane, and absorbent pad are then attached to a substrate to obtain the immunochromatographic test strip.

[0038] (Preferred) In step 1, the complexation solution of the composite nanomaterial probe is a Tris-HCl solution containing sucrose, fructose, polyethylene glycol 20000, bovine serum albumin and Tween-20;

[0039] (Preferred) In step 2, the concentrations of Salmonella Typhimurium monoclonal antibody and goat anti-rabbit IgG antibody are both 1.0 mg / mL, the spraying speed is 1.0 μL / cm, and the distance between the detection line and the control line is 5 mm.

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

[0041] Preferably, the fluorescence mode of the dual-mode immunochromatographic test strip, which combines fluorescence and catalytic colorimetric methods, can be visually determined under ultraviolet light and its value can be read using a fluorescence immunoassay analyzer.

[0042] Preferably, the catalytic colorimetric mode of the fluorescence and catalytic colorimetric dual-mode immunochromatographic test strip can be immersed in a colorimetric solution for catalytic color development; more preferably, the colorimetric solution is a phosphoric acid-citric acid buffer solution containing 3,3',5,5'-tetramethylbenzidine and hydrogen peroxide.

[0043] The advantages of the present invention compared to the prior art are as follows:

[0044] 1. This invention synthesizes uniformly sized and well-dispersed BrM nanoparticles with excellent optical properties and water stability via in-situ crystallization growth. The BrM is produced by encapsulating CsPbBr3 with MSN followed by high-temperature calcination, causing mesoporous collapse and resulting in excellent fluorescence stability of CsPbBr3 in aqueous solution. The nanozyme is simple to synthesize and exhibits excellent steady-state kinetic parameters and specific activities. The BrM synthesized in this application has a negative Zeta potential, which allows it to bind well with polyethyleneimine to form a positive potential, thus better binding with Os nanozymes. Therefore, a BrM@Os nanozyme with dual functions of fluorescence and catalytic colorimetric analysis is formed.

[0045] 2. The technical solution provided by this invention uses BrM@Os as a labeled probe, first attaching and activating a carboxyl group to it, allowing for better binding with rabbit anti-Salmonella typhimurium polyclonal antibodies, and then integrating it into a lateral flow immunoassay platform. This test strip employs a double-antibody sandwich method, exhibiting excellent 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. The method is simple to operate, time-saving, rapid and accurate, and low in cost. Attached Figure Description

[0047] Figure 1 This is a transmission electron microscope (TEM) characterization image of BrM prepared in Example 1 of this invention.

[0048] Figure 2 The image shows the transmission electron microscopy characterization of BrM@Os prepared in Example 1 of this invention.

[0049] Figure 3 The photographs taken under a UV lamp show the MSN, Os, BrM, BrM@PEI and BrM@Os solutions prepared for Example 1 of this invention.

[0050] Figure 4 The graph shows the steady-state kinetics of the BrM@Os nanozyme prepared in Example 1 of this invention, as well as the absorbance values ​​affected by pH and temperature.

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

[0052] Figure 6 This invention demonstrates the fluorescence and catalytic colorimetric detection performance of the immunochromatographic test strip for Salmonella typhimurium in Example 5 of this invention. Detailed Implementation

[0053] To further illustrate the method and effects of the present invention, the present invention will be described in further detail below with reference to specific embodiments. All technologies implemented based on the above content of the present invention are covered within the scope of protection intended by the present invention.

[0054] The rabbit anti-Salmonella typhimurium polyclonal antibody used in this invention comes from our laboratory. It was prepared by immunizing New Zealand white rabbits with inactivated Salmonella typhimurium bacterial solution as an antigen.

[0055] Example 1

[0056] This embodiment provides a composite nanomaterial probe for detecting Salmonella typhimurium. The composite nanomaterial probe is formed by coupling composite nanomaterial BrM@Os with rabbit anti-Salmonella typhimurium polyclonal antibody. The mass ratio of composite nanomaterial BrM@Os to rabbit anti-Salmonella typhimurium polyclonal antibody is 30:1.

[0057] The composite nanomaterial BrM@Os is formed by modifying BrM nanoparticles with Os nanozymes. The BrM nanoparticles are MSN nanoparticles containing fluorescent material CsPbBr3. The MSN nanoparticles are porous materials formed of tetraethyl silicate. The mass ratio of MSN nanoparticles, fluorescent material CsPbBr3, and Os nanozymes is 30:10:1.

[0058] It is prepared by the following method:

[0059] 1) MSN Synthesis:

[0060] 200 mg of hexadecyltrimethylammonium bromide was dissolved in 25 mL of ultrapure water. Then, 8 mL of ethanol and 50 μL of triethanolamine were added, and the mixture was stirred in a 60 °C water bath for 30 min. 2 mL of tetraethyl silicate was then rapidly added, and the mixture was stirred in a 60 °C water bath for 2 h. After cooling to room temperature, the mixture was centrifuged, and the collected product was washed three times with ultrapure water and dried at 80 °C. Subsequently, MSN was calcined at 450 °C for 5 h under atmospheric conditions to remove the template hexadecyltrimethylammonium bromide.

[0061] 2) Synthesis of BrM:

[0062] 2.1) Cesium bromide (12 mmol / L) and lead bromide (12 mmol / L) were dissolved in 6 mL of ultrapure water, then sonicated for 5 min, and stirred 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 B is formed;

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

[0065] 2.3) The product obtained in 2.2) was calcined at 400℃ for 20 min under atmospheric conditions. After cooling to room temperature, the product was finely ground and washed three times with ultrapure water to obtain BrM nanoparticles.

[0066] 3) Synthesis of Os nanozymes:

[0067] Mix 1 mL of trisodium citrate (40 mmol / L) with 38 mL of ultrapure water and stir for 5 min. Add 1 mL of potassium hexachloroosmium tetroxide (10 mmol / L) and stir for 30 min. Then add 200 μL of sodium borohydride (50 mmol / L) and stir in the dark for 45 min (500 rpm). The color gradually changes from yellow-green to brown, yielding 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, centrifuge and wash three times to obtain the nanocomposite BrM@Os.

[0072] 6) Preparation of 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 groups. After activation, centrifuge to 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, then add 1 mL of blocking buffer (bovine serum albumin solution, 100 mg / mL), and continue stirring 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, wash once with Tris-HCl buffer, resuspend the precipitate in 50 μL of reconstitution solution, and store at 4 °C in the dark.

[0074] Example 2

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

[0076] The composite nanomaterial BrM@Os is formed by modifying BrM nanoparticles with Os nanozymes. The BrM nanoparticles are MSN nanoparticles containing fluorescent material CsPbBr3. The MSN nanoparticles are porous materials formed of tetraethyl silicate. The mass ratio of MSN nanoparticles, fluorescent material CsPbBr3, and Os nanozymes is 30:10:1.

[0077] It is prepared by the following method:

[0078] 1) MSN Synthesis:

[0079] 200 mg of hexadecyltrimethylammonium bromide was dissolved in 25 mL of ultrapure water. Then, 8 mL of ethanol and 50 μL of triethanolamine were added, and the mixture was stirred in a 60 °C water bath for 30 min. 2 mL of tetraethyl silicate was then rapidly added, and the mixture was stirred in a 60 °C water bath for 2 h. After cooling to room temperature, the mixture was centrifuged, and the collected product was washed three times with ultrapure water and dried at 80 °C. Subsequently, MSN was calcined at 500 °C for 5.5 h under atmospheric conditions to remove the template hexadecyltrimethylammonium bromide.

[0080] 2) Synthesis of BrM:

[0081] 2.1) Cesium bromide (12 mmol / L) and lead bromide (12 mmol / L) were dissolved in 6 mL of ultrapure water, then sonicated for 5 min, and stirred 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 B is formed;

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

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

[0085] 3) Synthesis of Os nanozymes:

[0086] Mix 1 mL of trisodium citrate (40 mmol / L) with 38 mL of ultrapure water and stir for 5 min. Add 1 mL of potassium hexachloroosmium tetroxide (10 mmol / L) and stir for 30 min. Then add 200 μL of sodium borohydride (50 mmol / L) and stir in the dark for 55 min (500 rpm). The color gradually changes from yellow-green to brown, yielding 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 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 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 sonicate for 10 min to activate the carboxyl groups. After activation, centrifuge to 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, then add 1 mL of blocking buffer (bovine serum albumin solution, 100 mg / mL), and continue stirring 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, wash once with Tris-HCl buffer, resuspend the precipitate in 50 μL of reconstitution solution, and store at 4 °C in the dark.

[0093] Example 3

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

[0095] The composite nanomaterial BrM@Os is formed by modifying BrM nanoparticles with Os nanozymes. The BrM nanoparticles are MSN nanoparticles containing fluorescent material CsPbBr3. The MSN nanoparticles are porous materials formed of tetraethyl silicate. The mass ratio of MSN nanoparticles, fluorescent material CsPbBr3, and Os nanozymes is 30:10:1.

[0096] It is prepared by the following method:

[0097] 1) MSN Synthesis:

[0098] 200 mg of hexadecyltrimethylammonium bromide was dissolved in 25 mL of ultrapure water. Then, 8 mL of ethanol and 50 μL of triethanolamine were added, and the mixture was stirred in a 60 °C water bath for 30 min. 2 mL of tetraethyl silicate was then rapidly added, and the mixture was stirred in a 60 °C water bath for 2 h. After cooling to room temperature, the mixture was centrifuged, and the collected product was washed three times with ultrapure water and dried at 80 °C. Subsequently, MSN was calcined at 550 °C for 6 h under atmospheric conditions to remove the template hexadecyltrimethylammonium bromide.

[0099] 2) Synthesis of BrM:

[0100] 2.1) Cesium bromide (12 mmol / L) and lead bromide (12 mmol / L) were dissolved in 6 mL of ultrapure water, then sonicated for 5 min, and stirred 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 solutions A and B and stir at 80°C for 20 min. While stirring, add 120 μL of potassium carbonate solution (0.3 mol / L) to the mixture until the solvent completely evaporates. Grind the collected powder and wash several times with ethanol until the solution turns white. Centrifuge to collect the product and dry at 80°C.

[0103] 2.3) The product obtained in 2.2) was calcined at 500℃ for 30 min under atmospheric conditions. After cooling to room temperature, the product was finely ground and washed three times with ultrapure water to obtain BrM nanoparticles.

[0104] 3) Synthesis of Os nanozymes:

[0105] Mix 1 mL of trisodium citrate (40 mmol / L) with 38 mL of ultrapure water and stir for 5 min. Add 1 mL of potassium hexachloroosmium tetroxide (10 mmol / L) and stir for 30 min. Then 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, yielding 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 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 sonicate for 10 min to activate the carboxyl groups. After activation, centrifuge to 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, then add 1 mL of blocking buffer (bovine serum albumin solution, 100 mg / mL), and continue stirring 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, wash once with Tris-HCl buffer, resuspend the precipitate in 50 μL of reconstitution solution, and store at 4 °C in the dark.

[0112] like Figure 1 As shown, the BrM solution was diluted to a clear solution, and 10 μL was dropped onto a copper grid, dried at 80 °C for 2 h, and then imaged using a transmission electron microscope. The transmission electron microscope image of BrM shows that it has a regular spherical shape and is filled with black dots, which are CsPbBr3 crystals.

[0113] like Figure 2 As shown, the BrM@Os solution was diluted to a clear solution, and 10 μL was dropped onto a copper grid, dried at 80 °C for 2 h, and then imaged using a transmission electron microscope. Compared to BrM, the transmission electron microscope image of BrM@Os shows that its surface is clearly adsorbed with Os nanozymes, indicating the successful preparation of BrM@Os.

[0114] like Figure 3 As shown, MSN, Os, BrM, BrM@PEI and BrM@Os solutions exhibit bright green fluorescence under ultraviolet light irradiation.

[0115] like Figure 4 As shown, the composite nanomaterial BrM@Os nanozyme exhibits peroxidase-like activity, catalyzing TMB to produce a distinct blue color change. By testing the absorbance of its blue product oxTMB at 652 nm, the BrM@Os nanozyme demonstrates steady-state kinetics, exhibiting good enzyme activity within the temperature range of 20–80 °C, with the optimal catalytic condition being pH = 4.

[0116] Example 4

[0117] This embodiment provides a dual-mode immunochromatographic test strip for detecting Salmonella typhimurium, comprising a base plate, on which a sample pad, a conjugate pad, a chromatography membrane, and an absorbent pad are fixed from left to right. The conjugate pad is sprayed with a composite nanomaterial probe for detecting Salmonella typhimurium as described in any of Examples 1-3. The chromatography membrane has parallel detection lines and control lines. The detection lines contain Salmonella typhimurium monoclonal antibodies, and the control lines contain goat anti-rabbit IgG antibodies.

[0118] It is prepared through the following steps:

[0119] The sample pad was pretreated by immersing a glass cellulose 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 was pretreated by immersing a glass cellulose 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] Goat anti-rabbit IgG (1 mg / mL) and Salmonella typhimurium monoclonal antibody (1 mg / mL) were immobilized on a chromatography membrane using an XYZ three-dimensional streaking and gold sputtering apparatus at a loading volume of 1 μL / cm. Goat anti-rabbit IgG served as the control line (C), and Salmonella typhimurium monoclonal antibody served as the detection line (T), with a distance of 5 mm between the two lines. After streaking, the chromatography membrane was adhered to a substrate and dried in an oven at 37°C for 2 hours.

[0122] Using an XYZ three-dimensional etching and gold spraying instrument, the composite nanomaterial probe for detecting Salmonella typhimurium prepared in any of Examples 1-3 was sprayed onto the conjugate pad at a sample loading of 8.5 μL / cm and dried at room temperature.

[0123] Finally, assemble the test strip: Attach the sample pad, conjugate pad, chromatography membrane, and absorbent pad to the base plate from left to right, ensuring an approximately 2mm overlap between each. The absorbent pad should be positioned closer to the control line on the chromatography membrane, and the sample pad closer to the detection line. Finally, install the test strip into the plastic casing and store it in a dry environment at room temperature. Refer to the structural diagram for details. Figure 5 .

[0124] Experiment Example 5: Detection of Salmonella Typhimurium

[0125] At the sample application site of the dual-mode immunochromatographic test strip for detecting Salmonella typhimurium prepared in Example 4, 10,0 ... 2 103 10 4 10 5 10 6 10 7 10 8 10 9 60 μL each of CFU / mL Salmonella Typhimurium and sterile Tris-HCl buffer solution were added. After 15 minutes, the fluorescence of the test strip bands under UV light (365 nm) was observed visually. Then, the plastic cartridges were inserted into the dry fluorescence immunoassay analyzer, and the fluorescence intensity of the test strip bands was read using the instrument. The test strip was then immersed in the colorimetric solution, and the catalytic colorimetric reaction was observed visually. The results are shown in [reference needed]. Figure 6 The T line on the test strip darkens with increasing bacterial concentration. The detection line for this fluorescence and catalytic colorimetric dual-mode immunochromatographic test strip is 10. 5 With a concentration of CFU / mL, it has good sensitivity and can be used to detect Salmonella typhimurium.

[0126] The embodiments of the present invention have been described above with reference to the accompanying drawings. However, the present invention is not limited to the specific embodiments described above. The specific embodiments described above are merely illustrative and not restrictive. Those skilled in the art can make many other forms under the guidance of the present invention without departing from the spirit and scope of the claims. All of these forms are within the protection scope of the present invention.

Claims

1. A composite nanomaterial probe for immunochromatographic detection of Salmonella typhimurium, characterized in that, The aforementioned composite nanomaterial probe is formed by coupling the composite nanomaterial BrM@Os with rabbit anti-Salmonella typhimurium polyclonal antibodies; The composite nanomaterial BrM@Os is formed by modifying BrM nanoparticles with Os nanozymes. The BrM nanoparticles are MSN nanoparticles containing fluorescent material CsPbBr3. The MSN nanoparticles are porous materials formed from silicon sources. The composite nanomaterial BrM@Os was prepared by the following method: Step 1: Synthesis of MSN nanoparticles Hexadecyltrimethylammonium bromide solution, anhydrous ethanol and triethanolamine were stirred evenly, then tetraethyl silicate was added, and the mixture was stirred in a water bath at 60 °C for 2 h. After cooling to room temperature, the mixture was centrifuged, washed and dried, and then heat-treated at 450-550 °C for 5-6 h under atmospheric conditions to obtain MSN. Step 2: Synthesis of BrM nanoparticles Cesium bromide and lead bromide were dissolved in ultrapure water, sonicated, and then added to MSN solution. The mixture was stirred at 70-80 °C for 15-20 min to obtain a mixture. Then, potassium carbonate solution was added to the mixture, and stirring was continued until the solvent was completely evaporated. The powder was ground and washed, and the product was collected by centrifugation and dried. Then, heat-treat at 400-500 ℃ for 20-30 min under atmospheric conditions, grind and wash with ultrapure water to obtain BrM nanoparticles; Step 3: Synthesize Os nanozymes Dissolve trisodium citrate and potassium hexachloroosmium tetroxide in ultrapure water and stir. Then add sodium borohydride and stir in the dark for 45-60 min to obtain Os nanozyme. Step 4: Synthesis of composite nanomaterial BrM@Os The BrM nanoparticles obtained in step 2 were ultrasonicated with polyethyleneimine and then washed by centrifugation. The product was then added to Os nanozyme and ultrasonicated for 30-40 min, followed by centrifugation and washing to obtain the composite nanomaterial BrM@Os. The mass ratio of the composite nanomaterial BrM@Os to the rabbit anti-Salmonella typhimurium polyclonal antibody is 20-40:

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

1.

2. The composite nanomaterial probe for immunochromatographic detection of Salmonella typhimurium according to claim 1, characterized in that, The mass ratio of the composite nanomaterial BrM@Os to the rabbit anti-Salmonella typhimurium polyclonal antibody is 30:

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

1.

3. The composite nanomaterial probe for immunochromatographic detection of Salmonella typhimurium according to claim 1, characterized in that, The mass ratio of tetraethyl silicate, lead bromide, cesium bromide, trisodium citrate, potassium hexachloroosmium tetroxide, and sodium borohydride is 15000:155:200:15:6:

5.

4. The method for preparing the composite nanomaterial probe for immunochromatographic detection of Salmonella typhimurium according to claim 1, characterized in that, First, the carboxyl groups were modified and activated on the surface of BrM@Os. Then, rabbit anti-Salmonella typhimurium polyclonal antibody was added. After stirring, bovine serum albumin solution was added to block the reaction site. After centrifugation and washing, the mixture was redispersed in a complex solution to obtain a composite nanomaterial probe for immunochromatographic detection of Salmonella typhimurium.

5. The method for preparing the composite nanomaterial probe for immunochromatographic detection of Salmonella typhimurium according to claim 4, characterized in that, The method for modifying and activating carboxyl groups on the surface of BrM@Os involves adding 1-ethyl-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide to the composite nanomaterial BrM@Os and then activating the carboxyl groups by ultrasonication.

6. A dual-mode immunochromatographic test strip for the immunochromatographic detection of Salmonella typhimurium, characterized in that, Includes the composite nanomaterial probe for immunochromatographic detection of Salmonella typhimurium as described in claim 1.

7. The dual-mode immunochromatographic test strip for immunochromatographic detection of Salmonella typhimurium according to claim 6, comprising a base plate, wherein a sample pad, a conjugate pad, a chromatography membrane, and an absorbent pad are fixed sequentially from left to right on the base plate, characterized in that, The conjugation pad is sprayed with a composite nanomaterial probe for immunochromatographic detection of Salmonella typhimurium; the chromatographic membrane is provided with parallel detection lines and control lines, the detection lines are provided with Salmonella typhimurium monoclonal antibody as the detection line, and the control lines are provided with goat anti-rabbit IgG antibody.

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