A method and device for simultaneous detection of salmonella typhimurium and staphylococcus aureus in chicken based on transient-state steady-state upconversion fluorescence sensor

Abstract

This invention relates to a method for simultaneously detecting *Salmonella typhimurium* and *Staphylococcus aureus* in chicken meat based on an instantaneous steady-state upconversion fluorescence sensor. First, a water-soluble upconversion material is encapsulated in a hollow cylinder to prepare an upconversion fluorescence cuvette. Next, a fluorescence sensor is constructed, comprising magnetic graphene oxide, aptamers specifically recognizing *Salmonella typhimurium* and *Staphylococcus aureus*, and enzyme modifications. By measuring the fluorescence signals, a detection standard curve for the two target bacteria is established. This method can determine the content of *Salmonella typhimurium* and *Staphylococcus aureus* in chicken meat. This invention improves the reusability of materials and the accuracy of detection results through the prepared upconversion fluorescence cuvette, simplifies the operation process and reduces costs by utilizing magnetic graphene oxide, and enhances the sensitivity of the sensor by utilizing enzyme catalysis. This detection method has a wide linear detection range and a low detection limit, showing promising application prospects.

Description

Technical Field

[0001] This invention belongs to the field of food safety testing technology, specifically relating to a method and apparatus for the simultaneous detection of Salmonella typhimurium and Staphylococcus aureus in chicken meat based on an instantaneous steady-state upconversion fluorescence sensor. Background Technology

[0002] Salmonella and Staphylococcus aureus, two common foodborne pathogens, are widely present in food and the environment. They can enter the body through food, causing various diseases such as typhoid fever, sepsis, and acute gastroenteritis. Chicken is one of the main vectors for Salmonella and Staphylococcus aureus infection in humans. Chicken is an important component of poultry, possessing advantages such as high nutritional value, low cholesterol, low fat, and low price, making it a good source of nutrients. At the same time, chicken also serves as a natural culture medium for microorganisms. Chicken is easily contaminated by foodborne pathogens and putrefactive bacteria during slaughter, processing, packaging, transportation, and sales. Therefore, the detection of Salmonella typhimurium and Staphylococcus aureus in chicken is extremely important.

[0003] Traditional methods for detecting foodborne pathogens include plate counting, immunological methods (such as enzyme-linked immunosorbent assay), and molecular amplification methods (such as PCR, loop-mediated isothermal amplification, and rolling circle amplification). These techniques generally suffer from drawbacks such as long detection times, expensive equipment, high testing costs, low sensitivity, and a high susceptibility to false positives. To address these challenges in detecting foodborne pathogens, rapid detection technologies based on fluorescence sensors have been developed for the rapid detection of microorganisms in food. These methods have demonstrated advantages such as speed, accuracy, high selectivity, and low cost in food safety testing. However, they still have some limitations, such as high background interference and unstable detection signals. Therefore, it is necessary to develop a sensitive and highly selective novel method for detecting foodborne pathogens to further improve the detection technology for foodborne pathogens. Summary of the Invention

[0004] The purpose of this invention is to overcome the technical defects of existing detection technologies, such as low detection sensitivity, low reusability of detection materials, and low detection efficiency. This invention provides a method and apparatus for simultaneously detecting *Salmonella typhimurium* and *Staphylococcus aureus* in chicken meat based on an instantaneous steady-state upconversion fluorescence sensor. Specifically, it utilizes a self-designed upconversion fluorescence cuvette, horseradish peroxidase-labeled aptamer modification, and alkaline phosphatase-labeled aptamer modification of magnetic graphene oxide to construct a highly sensitive fluorescence sensor for detecting *Salmonella typhimurium* and *Staphylococcus aureus*, improving the reusability of detection materials and the accuracy of detection results, thus achieving highly sensitive and specific detection of *Salmonella typhimurium* and *Staphylococcus aureus* in chicken meat.

[0005] To achieve the above objectives, the specific steps of this affray are as follows:

[0006] Step 1, Preparation of oil-soluble upconversion material: Erbium chloride hexahydrate, yttrium chloride hexahydrate, ytterbium chloride hexahydrate, and gadolinium chloride hexahydrate were added to methanol and sonicated. After sonication and dissolution, the solution was transferred to a three-necked flask containing oleic acid and octadecene. The mixture was heated under nitrogen and magnetic stirring to carry out the first reaction. After the reaction, a mixed solution of ammonium fluoride and sodium hydroxide was added dropwise, and the mixture was heated to carry out the second reaction. After the reaction, the mixture was heated again to allow the methanol in the reaction solution to completely evaporate. After the reaction, the mixture was heated under magnetic stirring and nitrogen protection to carry out the third reaction. After the reaction, the mixture was cooled to room temperature to obtain the reaction product. The reaction product was washed with a mixed solution of cyclohexane and ethanol and dried in a vacuum oven to obtain oil-soluble upconversion nanoparticles.

[0007] Step 2: Alendronic acid-modified upconversion synthesis: The upconversion nanomaterials obtained in Step 1 were added to a mixed solution of ethanol and chloroform, and dispersed by sonication. The mixture was then transferred to a centrifuge tube containing alendronic acid solution. Hydrochloric acid solution was added under magnetic stirring to adjust the pH and carry out the reaction. After the reaction was completed, the mixture was washed with a mixed solution of ethanol and water and dried under vacuum to obtain alendronic acid-modified upconversion nanomaterials.

[0008] Step 3: Preparation of upconversion fluorescent cuvette: The upconversion nanoparticles obtained in Step 2 are added into a hollow cylinder and sealed. The solid rectangular cylinder and the sealed cylinder containing the upconversion nanoparticles are then embedded into a standard cuvette to obtain an upconversion fluorescent cuvette.

[0009] Step 4: Fabrication of Magnetic Graphene Oxide: Graphene oxide is added to deionized water and ultrasonically dispersed. Ferric chloride and ferric chloride are then added. The mixed solution is heated to 85°C. Ammonium hydroxide solution is then added and reacted at 85°C for 50 minutes. After the reaction, the solution is washed three times with deionized water to obtain the magnetic graphene oxide material.

[0010] Step 5: Construction of the fluorescence sensor: The magnetic graphene oxide solution obtained in Step 4 was reacted for the first time with horseradish peroxidase-labeled aptamer solutions and alkaline phosphatase-labeled aptamer solutions, respectively. After the reaction, magnetic graphene oxide modified with the two enzyme-labeled aptamers was obtained. Then, TMB chromogenic solution and BCIP / NBT chromogenic solution were added to the magnetic graphene oxide solutions modified with the two enzyme-labeled aptamers, respectively. After the reaction, the solutions were transferred to an upconversion fluorescence cuvette to obtain the upconversion fluorescence sensor.

[0011] Step Six: Establishment of the standard curve for the detection of *Salmonella Typhimurium*: Different concentrations of *Salmonella Typhimurium* solution were added to the upconversion fluorescence sensor obtained in Step Five to obtain the detection solution. The upconversion fluorescence intensity signal value of the detection solution was measured. A standard curve for the detection of *Salmonella Typhimurium* was established with the *Salmonella Typhimurium* concentration as the x-axis and the difference in upconversion fluorescence intensity at 547 nm as the y-axis. Establishment of the standard curve for the detection of *Staphylococcus aureus*: Different concentrations of *Staphylococcus aureus* solution were added to the upconversion fluorescence sensor obtained in Step Five to obtain the detection solution. The upconversion fluorescence intensity signal value of the detection solution was measured. A standard curve for the detection of *Staphylococcus aureus* was established with the *Staphylococcus aureus* concentration as the x-axis and the difference in upconversion fluorescence intensity at 658 nm as the y-axis.

[0012] Step 7: Detection of Salmonella Typhimurium and Staphylococcus aureus in chicken: Place the chicken on a sterile operating table and cut it into small pieces. Then add it to a homogenization bag containing sterile water, mix well, and let it stand. Collect the supernatant to obtain the test solution. Measure the fluorescence intensity of the test solution using a fluorescence sensor. Substitute this into the standard curves for Salmonella Typhimurium and Staphylococcus aureus obtained in Step 6 to calculate the content of Salmonella Typhimurium and Staphylococcus aureus in the chicken.

[0013] Preferably, in step one, the ratio of yttrium chloride hexahydrate, ytterbium chloride hexahydrate, erbium chloride hexahydrate, gadolinium chloride hexahydrate, and methanol A is 0.087g:0.047g:0.005g:0.066g:3mL; the ultrasonic dissolution time is 5-10min; the first heating and stirring reaction is carried out at a temperature of 140-180℃ for 30min; and the second heating and stirring reaction is carried out at a temperature of 280-320℃ for 1h.

[0014] Preferably, in step two, the volume ratio of methanol A, oleic acid, and 1-octadecene is 3:4.5:10.5; the ratio of the amount of the oleic acid-coated upconversion material, water, ethanol, chloroform, and alendronate is 100mg:3mL:2mL:4mL:24.9mg; the concentration of HCl is 1mol / L; and the ultrasonic dispersion time is 10-20min. The volume ratio of the water-ethanol mixed solution is 1:1.

[0015] Preferably, the cylindrical cube in step three has a base radius of 2.5 mm and a height of 12.5 mm, and the upconversion concentration is 1-2 mg / mL; the solid cube has a height of 20 cm and a base side length of 12.5 cm.

[0016] Preferably, in step four, the mass ratio of graphene oxide:ferric chloride:ferric chloride:ammonium hydroxide is 0.8:25.3mg:16.2mg:0.7mg, the ultrasonic time is 30min, and the reaction time is 50min.

[0017] Preferably, in step five, the volume ratio of the magnetic graphene oxide solution, the horseradish peroxidase-labeled aptamer, and the alkaline phosphatase-labeled aptamer is 100:100:100, and the concentration ratio is 0.625 mg / mL:0.4 mM:0.5 mM; the first reaction time is 5 min, the volume of the TM3 colorimetric solution is 100 μL, the volume of the BCIP / NBT colorimetric solution is 100 μL, the second reaction time is 13 min, and the volume of the transfer solution is 400 μL.

[0018] Preferably, the concentration range of Salmonella typhimurium in step six is ​​1.14 × 10⁻⁶. 2 -1.14×10 8 CFU / mL; the volume of the *Salmonella typhimurium* was 0.1 mL; the concentration range of the *Staphylococcus aureus* was 8.6 × 10⁻⁶. 2 -8.6×10 7 CFU / mL; the volume of the Staphylococcus aureus is 0.1 mL; the measured fluorescence intensity signal of the sensor is specifically the fluorescence intensity values ​​at 547 nm and 658 nm under excitation by a 980 nm excitation source, denoted as I. 547nm and I 658nm .

[0019] Preferably, the chicken meat in step seven is 25g; the standing time is 30min; and the solution volume is 100mL.

[0020] Compared with existing detection technologies, the beneficial effects of this invention are as follows:

[0021] 1. This invention discloses a method for detecting Salmonella typhimurium and Staphylococcus aureus in chicken meat based on an instantaneous steady-state upconversion fluorescence sensor. Specifically, it constructs a fluorescence sensor for detecting Salmonella typhimurium and Staphylococcus aureus by using a self-assembled upconversion fluorescence cuvette, horseradish peroxidase-labeled aptamers, and alkaline phosphatase-labeled aptamers-functionalized magnetic graphene oxide. This improves the reusability of detection materials, reduces detection costs, and increases the accuracy of the detection method, achieving high sensitivity and specificity for detecting Salmonella typhimurium and Staphylococcus aureus in chicken meat.

[0022] 2. The upconversion fluorescence sensor constructed in this invention specifically optimizes the upconversion concentration of the cylindrical block in the fluorescence cuvette and the height of the cylinder in the cuvette, resulting in a stable upconversion fluorescence signal. This reduces the error in the detection results and improves the accuracy of detecting Salmonella typhimurium and Staphylococcus aureus.

[0023] 3. The upconversion fluorescence sensor constructed in this invention specifically optimizes the concentrations of magnetic graphene oxide and horseradish peroxidase-labeled aptamers in the sensor, thereby improving the fluorescence response of Salmonella and enhancing the sensitivity of the detection method.

[0024] 4. The linear concentration range established by this invention for the difference between the concentration of Salmonella Typhimurium and the fluorescence intensity at 547 nm of the fluorescence sensor is 1.14 × 10⁻⁶. 2 -1.14×10 8 The detection limit was 33 CFU / mL. A linear range of 8.6 × 10⁻⁶ CFU / mL was established between the concentration of Staphylococcus aureus and the difference in fluorescence intensity at 658 nm using the fluorescence sensor. 2 -8.6×10 7 The detection limit is 14 CFU / mL. It can meet the requirements for rapid and highly sensitive detection of Salmonella typhimurium and Staphylococcus aureus in chicken meat, and has good versatility. Compared with traditional methods, it has a shorter detection time and higher sensitivity. Attached Figure Description

[0025] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0026] Figure 1 The image is an upconversion transmission electron microscope image of the ADA-modified material prepared in Example 1.

[0027] Figure 2 Three-view diagrams and actual image of the fluorescent cuvette prepared in Example 1;

[0028] Figure 3 This is a flowchart of the Salmonella Typhimurium detection based on an upconversion fluorescence sensor in Example 1;

[0029] Figure 4 The difference in fluorescence signal of the fluorescence sensor under different Staphylococcus aureus concentrations in Example 1;

[0030] Figure 5 This is the standard curve for Staphylococcus aureus detection in Example 1;

[0031] Figure 6 The difference in fluorescence signal of the fluorescence sensor under different concentrations of Salmonella typhimurium in Example 1;

[0032] Figure 7 The standard curve for the detection of Salmonella typhimurium in Example 1. Detailed Implementation

[0033] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly described below. Obviously, the described embodiments are some embodiments of the present invention, but not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0034] The specific embodiments of the present invention will be further described in detail below with reference to the accompanying drawings.

[0035] Example 1:

[0036] This invention provides a method and apparatus for detecting Salmonella typhimurium and Staphylococcus aureus in chicken meat based on an instantaneous steady-state upconversion fluorescence sensor. The specific steps are as follows:

[0037] Step 1: Preparation of oil-soluble upconversion material: 0.0052 g of erbium chloride hexahydrate, 0.085 g of yttrium chloride hexahydrate, 0.045 g of ytterbium chloride hexahydrate, and 0.064 g of gadolinium chloride hexahydrate were added to 4 mL of methanol and sonicated. After sonication and dissolution, the solution was transferred to a three-necked flask containing 6 mL of oleic acid and 14 mL of octadecene. The mixture was heated to 160 °C under nitrogen and magnetic stirring. After the reaction was complete, a mixed solution of ammonium fluoride and sodium hydroxide was added dropwise. The temperature was raised to 60 °C and reacted for 0.5 h. After the reaction was complete, the temperature was raised to 80 °C to allow the methanol in the reaction solution to completely evaporate. After the reaction was complete, the temperature was raised to 300 °C under magnetic stirring and nitrogen protection and reacted for 1 h. After the reaction was complete, the mixture was cooled to room temperature to obtain the reaction product. The reaction product was washed with a mixed solution of cyclohexane and ethanol and dried in a vacuum oven at 60 °C to obtain oil-soluble upconversion nanoparticles.

[0038] Step 2: Alendronate-modified upconversion synthesis: 200 mg of the upconversion nanomaterial obtained in Step 1 was added to a mixed solution of 3 mL ethanol and 2 mL chloroform, and sonicated for 30 min to disperse it. The mixture was then transferred to a 50 mL centrifuge tube containing 24.9 mg of alendronate solution. 1 M hydrochloric acid solution was added under magnetic stirring to adjust the pH to 2, and the reaction was carried out for 0.5 h. After the reaction was complete, the nanomaterial was washed with an ethanol-water mixture and vacuum dried to obtain the alendronate-modified upconversion nanomaterial.

[0039] Step 3: Preparation of upconversion fluorescent cuvette: Take 0.25 mL of the upconversion nanoparticle solution with a concentration of 2 mg / mL obtained in Step 2 and add it into a hollow cylinder. Seal the cylinder and embed a solid long cubic column at a height of 23 mm from the bottom of the cuvette. Then embed a solid cubic block into the bottom of the cuvette to obtain the upconversion fluorescent cuvette.

[0040] Step 4: Add 0.8 mg of graphene oxide to deionized water and sonicate to completely disperse it. Then add 25.3 mg of ferric chloride and 16.2 mg of ferric chloride. Heat the mixed solution to 85°C. Then add 0.7 mg of ammonium hydroxide solution and react at 85°C for 50 minutes. After the reaction is complete, wash three times with deionized water to obtain magnetic graphene oxide material.

[0041] Step 5: Construction of the fluorescence sensor: 0.1 mL of the magnetic graphene oxide solution obtained in Step 4 (0.625 mg / mL) was mixed with 0.1 mL of a 0.4 mM horseradish peroxidase-labeled aptamer solution and 0.1 mL of a 0.5 mM alkaline phosphatase-labeled aptamer solution, respectively, and reacted for 5 min. After the reaction, magnetic graphene oxide modified with the two enzyme-labeled aptamers was obtained. Then, 0.1 mL of TMB chromogenic solution and 0.1 mL of BCIP / NBT chromogenic solution were added to the aptamer-modified magnetic graphene oxide solution, respectively. After the reaction, 0.3 mL of the reaction solution was transferred to an upconversion fluorescence cuvette to obtain the upconversion fluorescence sensor.

[0042] Step Six: Establishment of the standard curve for the detection of Salmonella Typhimurium: Add 1.14 × 10⁻⁶ ppm of the standard curve. 2 -1.14×10 8 A CFU / mL solution of *Salmonella typhimurium* was added to the upconversion fluorescence sensor obtained in step five to obtain the detection solution. The upconversion fluorescence intensity signal value of the detection solution was measured. A standard curve for the detection of *Salmonella typhimurium* was established by plotting the concentration of *Salmonella typhimurium* on the x-axis and the difference in upconversion fluorescence intensity at 547 nm on the y-axis. The standard curve for the detection of *Staphylococcus aureus* was established by adding CFU / mL of *Salmonella typhimurium* solution at a concentration of 8.6 × 10⁻⁶ CFU / mL. 2 -8.6×10 7 A CFU / mL solution of Staphylococcus aureus was added to the upconversion fluorescence sensor obtained in step five to obtain a detection solution. The upconversion fluorescence intensity signal value of the detection solution was measured. A standard curve for the detection of Staphylococcus aureus was established with the concentration of Staphylococcus aureus on the x-axis and the difference in upconversion fluorescence intensity at 658 nm on the y-axis.

[0043] Step 7: Detection of Salmonella Typhimurium and Staphylococcus aureus in chicken: Place 25g of chicken on a sterile operating table and cut it into small pieces. Then add it to a homogenization bag containing 100mL of sterile water, mix well, and let it stand for 30min. Take the supernatant to obtain the test solution. Measure the fluorescence intensity of the fluorescence sensor under the test solution. Substitute the standard curves of Salmonella Typhimurium and Staphylococcus aureus obtained in Step 6 to calculate the content of Salmonella Typhimurium and Staphylococcus aureus in the chicken.

[0044] Example 2:

[0045] This invention provides a method and apparatus for detecting Salmonella typhimurium in chicken meat based on an instantaneous steady-state upconversion fluorescence sensor. The specific steps are as follows:

[0046] Step 1: Preparation of oil-soluble upconversion material: 0.0051 g of erbium chloride hexahydrate, 0.082 g of yttrium chloride hexahydrate, 0.048 g of ytterbium chloride hexahydrate, and 0.062 g of gadolinium chloride hexahydrate were added to 4 mL of methanol and sonicated. After sonication and dissolution, the solution was transferred to a three-necked flask containing 6 mL of oleic acid and 14 mL of octadecene. The mixture was heated to 150 °C under nitrogen and magnetic stirring. After the reaction was complete, 20 mL of a mixed solution of ammonium fluoride and sodium hydroxide was added dropwise, and the mixture was heated to 50 °C and reacted for 0.5 h. After the reaction was complete, the temperature was raised to 70 °C to allow the methanol in the reaction solution to completely evaporate. After the reaction was complete, the temperature was raised to 300 °C under magnetic stirring and nitrogen protection and reacted for 1 h. After the reaction was complete, the mixture was cooled to room temperature to obtain the reaction product. The product was washed with a mixed solution of cyclohexane and ethanol and dried in a vacuum oven at 60 °C to obtain oil-soluble upconversion nanoparticles.

[0047] Step 2: Alendronate-modified upconversion synthesis: 200 mg of the upconversion nanomaterial obtained in Step 1 was added to a mixed solution of 3 mL ethanol and 2 mL chloroform, and sonicated for 40 min to disperse it. The mixture was then transferred to a 50 mL centrifuge tube containing 24.5 mg of alendronate solution. 1 M hydrochloric acid solution was added under magnetic stirring to adjust the pH to 3, and the reaction was allowed to proceed for 0.5 h. After the reaction was complete, the nanomaterial was washed with an ethanol-water mixture and vacuum dried to obtain the alendronate-modified upconversion nanomaterial.

[0048] Step 3: Preparation of upconversion fluorescent cuvette: Take 0.25 mL of the upconversion solution obtained in Step 2 with a concentration of 1.5 mg / mL and add it into a hollow cylinder. Seal the cylinder and embed a solid rectangular column into the bottom of the cuvette at a height of 22 mm. Then embed a solid cube block into the bottom of the cuvette to obtain the upconversion fluorescent cuvette.

[0049] Step 4: Add 0.7 mg of graphene oxide to deionized water and sonicate to completely disperse it. Then add 24.3 mg of ferric chloride and 15.3 mg of ferric chloride. Heat the mixed solution to 85°C. Then add 0.7 mg of ammonium hydroxide solution and react at 80°C for 50 minutes. After the reaction is complete, wash three times with deionized water to obtain magnetic graphene oxide material.

[0050] Step 5: Construction of the fluorescence sensor: 0.1 mL of the magnetic graphene oxide solution obtained in Step 4 (0.625 mg / mL) was mixed with 0.1 mL of a 0.35 mM horseradish peroxidase-labeled aptamer solution and 0.1 mL of a 0.45 mM alkaline phosphatase-labeled aptamer solution, respectively, and reacted for 5 min. After the reaction, magnetic graphene oxide modified with the two enzyme-labeled aptamers was obtained. Then, 0.1 mL of TMB chromogenic solution and 0.1 mL of BCIP / NBT chromogenic solution were added to the aptamer-modified magnetic graphene oxide solution, respectively. After the reaction, 0.3 mL of the reaction solution was transferred to an upconversion fluorescence cuvette to obtain the upconversion fluorescence sensor.

[0051] Step Six: Establishment of the standard curve for the detection of Salmonella Typhimurium: Add 1.14 × 10⁻⁶ ppm of the standard curve. 2 -1.14×10 8 A CFU / mL solution of *Salmonella typhimurium* was added to the upconversion fluorescence sensor obtained in step five to obtain the detection solution. The upconversion fluorescence intensity signal value of the detection solution was measured. A standard curve for the detection of *Salmonella typhimurium* was established with the *Salmonella typhimurium* concentration as the x-axis and the difference in upconversion fluorescence intensity at 547 nm as the y-axis. The standard curve for the detection of *Staphylococcus aureus* was established by adding CFU / mL of *Salmonella typhimurium* solution at a concentration of 8.6 × 10⁻⁶ CFU / mL. 2 -8.6×10 7 A CFU / mL solution of Staphylococcus aureus was added to the upconversion fluorescence sensor obtained in step five to obtain the detection solution. The upconversion fluorescence intensity signal value of the detection solution was measured, and a standard curve for Staphylococcus aureus detection was established with the Staphylococcus aureus concentration as the x-axis and the difference in upconversion fluorescence intensity at 658 nm as the y-axis.

[0052] Step 7: Detection of Salmonella Typhimurium and Staphylococcus aureus in chicken: Place 25g of chicken on a sterile operating table and cut it into small pieces. Then add it to a homogenization bag containing 100mL of sterile water, mix well, and let it stand for 30min. Take the supernatant to obtain the test solution. Measure the fluorescence intensity of the fluorescence sensor under the test solution. Substitute the results into the standard curves of Salmonella Typhimurium and Staphylococcus aureus obtained in Step 6 to calculate the content of Salmonella Typhimurium and Staphylococcus aureus in the chicken.

[0053] Example 3:

[0054] This invention provides a method and apparatus for detecting Salmonella typhimurium in chicken meat based on an instantaneous steady-state upconversion fluorescence sensor. The specific steps are as follows:

[0055] Step 1: Preparation of oil-soluble upconversion material: 0.0055 g of erbium chloride hexahydrate, 0.088 g of yttrium chloride hydrate, 0.053 g of ytterbium chloride hexahydrate, and 0.064 g of gadolinium chloride hexahydrate were added to 4 mL of methanol and sonicated. After sonication and dissolution, the solution was transferred to a three-necked flask containing 6 mL of oleic acid and 14 mL of octadecene. The mixture was heated to 160 °C under nitrogen and magnetic stirring. After the reaction was complete, 20 mL of a mixed solution of ammonium fluoride and sodium hydroxide was added dropwise, and the temperature was raised to 60 °C for 0.5 h. After the reaction was complete, the temperature was raised to 80 °C to completely evaporate the methanol in the reaction solution. After the reaction was complete, the temperature was raised to 300 °C under magnetic stirring and nitrogen protection for 1 h. After the reaction was complete, the mixture was cooled to room temperature to obtain the reaction product. The product was washed with a mixed solution of cyclohexane and ethanol and dried in a vacuum oven at 60 °C to obtain oil-soluble upconversion nanoparticles.

[0056] Step 2: Alendronate-modified upconversion synthesis: 200 mg of the upconversion nanomaterial obtained in Step 1 was added to a mixed solution of 3 mL ethanol and 2 mL chloroform, and sonicated for 30 min to disperse it. The mixture was then transferred to a 50 mL centrifuge tube containing 25.2 mg of alendronate solution. 1 M hydrochloric acid solution was added under magnetic stirring to adjust the pH to 2.5, and the reaction was allowed to proceed for 0.5 h. After the reaction was complete, the nanomaterial was washed with an ethanol-water mixture and vacuum dried to obtain the alendronate-modified upconversion nanomaterial.

[0057] Step 3: Preparation of upconversion fluorescent cuvette: Take 0.25 mL of the upconversion obtained in Step 2 with a concentration of 1.5 mg / mL and add it into a hollow cylinder. Seal the cylinder and embed a solid rectangular column into the bottom of the cuvette at a height of 24 mm. Then embed a solid cube into the bottom of the cuvette to obtain the upconversion fluorescent cuvette.

[0058] Step 4: Add 0.85 mg of graphene oxide to deionized water and sonicate to completely disperse it. Then add 25.6 mg of ferric chloride and 16.8 mg of ferric chloride. Heat the mixed solution to 85°C. Then add 0.78 mg of ammonium hydroxide solution and react at 85°C for 50 minutes. After the reaction is complete, wash three times with deionized water to obtain magnetic graphene oxide material.

[0059] Step 5: Construction of the fluorescence sensor: 0.1 mL of the magnetic graphene oxide solution obtained in Step 4 (0.625 mg / mL) was mixed with 0.1 mL of a 0.45 mM horseradish peroxidase-labeled aptamer solution and 0.1 mL of a 0.55 mM alkaline phosphatase-labeled aptamer solution, respectively, and reacted for 5 min. After the reaction, magnetic graphene oxide modified with the two enzyme-labeled aptamers was obtained. Then, 0.1 mL of TMB chromogenic solution and 0.1 mL of BCIP / NBT chromogenic solution were added to the aptamer-modified magnetic graphene oxide solution, respectively. After the reaction, 0.3 mL of the reaction solution was transferred to an upconversion fluorescence cuvette to obtain the upconversion fluorescence sensor.

[0060] Step Six: Establishment of the standard curve for the detection of Salmonella Typhimurium: Add 1.14 × 10⁻⁶ ppm of the standard curve. 2 -1.14×10 8 A CFU / mL solution of *Salmonella typhimurium* was added to the upconversion fluorescence sensor obtained in step five to obtain the detection solution. The upconversion fluorescence intensity signal value of the detection solution was measured. A standard curve for the detection of *Salmonella typhimurium* was established with the *Salmonella typhimurium* concentration as the x-axis and the difference in upconversion fluorescence intensity at 547 nm as the y-axis. The standard curve for the detection of *Staphylococcus aureus* was established by adding CFU / mL of *Salmonella typhimurium* solution at a concentration of 8.6 × 10⁻⁶ CFU / mL. 2 -8.6×10 7 A CFU / mL solution of Staphylococcus aureus is added to the upconversion fluorescence sensor obtained in step five to obtain a detection solution. The upconversion fluorescence intensity signal value of the detection solution is measured. A standard curve for the detection of Staphylococcus aureus is established with the concentration of Staphylococcus aureus on the x-axis and the difference in upconversion fluorescence intensity at 658 nm on the y-axis.

[0061] Step 7: Detection of Salmonella Typhimurium and Staphylococcus aureus in chicken: Place 25g of chicken on a sterile operating table and cut it into small pieces. Then add it to a homogenization bag containing 100mL of sterile water, mix well, and let it stand for 30min. Take the supernatant to obtain the test solution. Measure the fluorescence intensity of the fluorescence sensor under the test solution. Substitute the results into the standard curves of Salmonella Typhimurium and Staphylococcus aureus obtained in Step 6 to calculate the content of Salmonella Typhimurium and Staphylococcus aureus in the chicken.

Claims

1. A method for simultaneously detecting Salmonella typhimurium and Staphylococcus aureus in chicken meat based on an instantaneous steady-state upconversion fluorescence sensor, characterized in that, Includes the following steps: Step 1: Prepare oleic acid-coated upconversion nanomaterials; Step 2: The oleic acid-coated upconversion nanomaterials obtained in Step 1 were added to a mixed solution of ethanol and chloroform, ultrasonically dispersed, and then alendronate solution was added for the first magnetic stirring reaction. Hydrochloric acid was added and the pH was adjusted to 2-3 for the second magnetic stirring reaction. After the reaction was completed, the reaction product was obtained, washed with a water-ethanol mixed solution, and dried in a vacuum oven to obtain alendronate-modified upconversion nanomaterials. Step 3: Fabrication of upconversion fluorescent cuvette: The alendronate-modified upconversion nanomaterials obtained in Step 2 are encapsulated in a hollow cylinder. A solid cubic cylinder and the cylinder containing the upconversion nanomaterials are embedded in the cuvette to create a fluorescent cuvette encapsulating upconversion fluorescent nanoparticles. Step 4: Fabrication of magnetic graphene oxide: Add graphene oxide to deionized water and sonicate it to disperse completely. Then add ferric chloride and ferric chloride and heat the mixed solution to 85°C. Then add ammonium hydroxide solution and react at 85°C. After the reaction is complete, wash three times with deionized water to obtain magnetic graphene oxide material. Step 5: Construction of the fluorescence sensor: The magnetic graphene oxide solution obtained in Step 4 was reacted for the first time with horseradish peroxidase-labeled aptamer and alkaline phosphatase-labeled aptamer solutions. After the reaction, magnetic graphene oxide modified with the two enzyme-labeled aptamers was obtained. Then, TMB chromogenic solution and BCIP / NBT chromogenic solution were added to the aptamer-modified magnetic graphene oxide solution, respectively. After the reaction, the solution was transferred to an upconversion fluorescence cuvette to obtain the upconversion fluorescence sensor. Step Six: Establishment of Standard Curves for the Detection of Salmonella Typhimurium and Staphylococcus aureus: Add different concentrations of Salmonella Typhimurium solution and Staphylococcus aureus solution to the upconversion fluorescence sensor obtained in Step Five to obtain the detection solution. Measure the upconversion fluorescence intensity signal value of the detection solution. Establish the detection standard curves for Salmonella Typhimurium and Staphylococcus aureus by plotting the concentrations of Salmonella Typhimurium and Staphylococcus aureus on the x-axis and the difference in upconversion fluorescence intensity signal value on the y-axis. Step 7: Detection of Salmonella Typhimurium and Staphylococcus aureus in chicken: Place the chicken on a sterile operating table and cut it into pieces. Then add it to a homogenization bag containing sterile water, mix well, and let it stand. Take the supernatant to obtain the test solution. Measure the fluorescence intensity of the fluorescence sensor under the test solution. Substitute the results into the standard curves of Salmonella Typhimurium and Staphylococcus aureus obtained in Step 6 to calculate the content of Salmonella Typhimurium and Staphylococcus aureus in the chicken.

2. The method for simultaneously detecting Salmonella typhimurium and Staphylococcus aureus in chicken meat based on an instantaneous steady-state upconversion fluorescence sensor according to claim 1, characterized in that... In step two, the ratio of the oleic acid-coated upconversion nanomaterial, water, ethanol, chloroform, and alendronate is 100 mg: 3 mL: 2 mL: 4 mL: 24.9 mg; the concentration of hydrochloric acid is 1 mol / L; and the ultrasonic dispersion time is 10-20 min.

3. The method for simultaneously detecting Salmonella typhimurium and Staphylococcus aureus in chicken meat based on an instantaneous steady-state upconversion fluorescence sensor according to claim 1, characterized in that... The first magnetic stirring reaction in step two is carried out at a temperature of 25°C for 5-10 minutes; the second magnetic stirring reaction is carried out at a temperature of 25°C for 30-60 minutes; and the volume ratio of the water-ethanol mixed solution is 1:

1.

4. The method for simultaneously detecting Salmonella typhimurium and Staphylococcus aureus in chicken meat based on an instantaneous steady-state upconversion fluorescence sensor according to claim 1, characterized in that... In step three, the cylinder containing the upconversion nanomaterial has a base radius of 2.5 mm and a height of 12.5 mm, and the concentration of the upconversion nanomaterial is 1-2 mg / mL; the solid cubic column has a height of 20 mm and a base side length of 12.5 mm.

5. The method for simultaneously detecting Salmonella typhimurium and Staphylococcus aureus in chicken meat based on an instantaneous steady-state upconversion fluorescence sensor according to claim 1, characterized in that... In step four, the ratio of graphene oxide: ferric chloride: ferric chloride: ammonium hydroxide is 0.8:25.3:16.2:0.7, the time for complete dispersion by ultrasound is 30 minutes, and the reaction time at 85°C is 50 minutes.

6. The method for simultaneously detecting Salmonella typhimurium and Staphylococcus aureus in chicken meat based on an instantaneous steady-state upconversion fluorescence sensor according to claim 1, characterized in that... In step five, the volume ratio of the magnetic graphene oxide solution, the horseradish peroxidase-labeled aptamer, and the alkaline phosphatase-labeled aptamer is 100:100:100, and the concentration ratio is 0.625 mg / mL:0.4 mM:0.5 mM; the first reaction time is 5 min; the volume of the TMB colorimetric solution is 100 μL; the volume of the BCIP / NBT colorimetric solution is 100 μL; and the second reaction time is 13 min. The volume of the transfer to the upconversion fluorescence cuvette is 400 μL.

7. The method for simultaneously detecting Salmonella typhimurium and Staphylococcus aureus in chicken meat based on an instantaneous steady-state upconversion fluorescence sensor according to claim 1, characterized in that... In step six, the concentration of Salmonella typhimurium ranges from 1.14 x 10 2 CFU / mL; the volume of Salmonella typhimurium is 0.1 mL; the concentration of Staphylococcus aureus ranges from 8.6 x 10 8 CFU / mL; the volume of Staphylococcus aureus is 0.1 mL; the sensor fluorescence intensity signal of the assay: specifically the fluorescence intensity values at 547 nm and 658 nm under 980 nm excitation light source excitation, are recorded as I 2 and I 7 . 547nm and I 658nm .

8. The method for simultaneously detecting Salmonella typhimurium and Staphylococcus aureus in chicken meat based on an instantaneous steady-state upconversion fluorescence sensor according to claim 1, characterized in that... In step seven, the amount of chicken meat is 25g; the time for standing after mixing is 30min; and the volume of the solution is 100mL.

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