A method for detecting salmonella typhimurium based on magnetic covalent organic framework and aptamer modified gold nanoparticles

Abstract

This invention discloses a method for detecting *Salmonella typhimurium* based on magnetic covalent organic frameworks and aptamer-modified gold nanoparticles, belonging to the field of food safety technology. The method uses a magnetic covalent organic framework as a capture probe to capture *Salmonella typhimurium* present in the detection system. After incubation, the magnetic covalent organic framework-*Salmonella typhimurium* complex is collected by magnetic separation. Then, aptamer-modified gold nanoparticles are added, and after incubation, the complex formed by the magnetic covalent organic framework-*Salmonella typhimurium* complex and the aptamer-modified gold nanoparticles is collected by magnetic separation. Finally, a TPE-4A / hydrogen peroxide substrate solution is added, and the absorbance and / or fluorescence intensity are measured. Color parameters are extracted using a smartphone. This method is simple to operate, highly sensitive, and highly specific. Three detection modes work together for self-testing, resulting in accurate and reliable results.

Description

Technical Field

[0001] This invention belongs to the field of food safety technology, and in particular relates to a method for detecting Salmonella typhimurium based on gold nanoparticles modified with magnetic covalent organic frameworks and aptamers. Background Technology

[0002] Salmonella typhimurium is a common foodborne pathogen widely distributed in nature. It can cause gastrointestinal infection in humans through the consumption of contaminated meat, eggs, milk, and other related products, resulting in symptoms such as fever, nausea, vomiting, diarrhea, and abdominal cramps, and can even lead to bacteremia, endangering life. This disease is widespread globally and can cause outbreaks of food poisoning with a high mortality rate. Therefore, rapid and accurate detection of Salmonella typhimurium contamination levels in food is of significant public health importance. Traditional detection methods, such as culture methods, immunological detection methods, and molecular biology-based detection methods, each have their limitations, such as long detection cycles, high costs, and requirements for large-scale equipment and highly skilled personnel, thus restricting their practical application.

[0003] With the development of various new materials, nanomaterials have become one of the most widely used tools in the field of food safety, showing great potential in the detection of foodborne pathogens. Covalent organic frameworks (COFs) are a new type of crystalline porous material composed of organic structural units. COFs possess advantages such as ordered pores, large surface area, and high stability, and are widely used as carriers for molecules such as natural enzymes and nanozymes, exhibiting excellent performance in catalysis and sensing. Nanozymes are a class of nanomaterials that catalyze the conversion of enzyme substrates into products according to enzyme kinetics, possessing advantages such as high catalytic activity, mild reaction conditions, good stability, and ease of large-scale production. Summary of the Invention

[0004] This invention provides a method for detecting Salmonella typhimurium based on magnetic covalent organic frameworks and aptamer-modified gold nanoparticles. The method is characterized by using a magnetic covalent organic framework as a capture probe to capture Salmonella typhimurium present in the detection system. After incubation, the magnetic covalent organic framework-Salmonella typhimurium complex is collected by magnetic separation. Then, aptamer-modified gold nanoparticles are added to the complex, and after incubation, the complex formed by the magnetic covalent organic framework-Salmonella typhimurium complex and the aptamer-modified gold nanoparticles is collected by magnetic separation. Finally, a TPE-4A / hydrogen peroxide substrate solution is added, and the absorbance and / or fluorescence intensity are measured.

[0005] Preferably, the method for preparing the magnetic covalent organic framework includes the following steps:

[0006] (1) Weigh 1.2-1.5g of ferric chloride hexahydrate and 3-4g of anhydrous sodium acetate, dissolve them completely in 30-50mL of ethylene glycol, heat the solution to 180-220℃, react for 14-18 hours, cool to room temperature, separate the black product with a magnet, wash with deionized water and anhydrous ethanol respectively, and obtain Fe3O4 magnetic nanoparticle powder after vacuum drying;

[0007] (2) Weigh 0.07-0.09g of 2,5-dimethylaminobenzene-1,4-dicarboxaldehyde and 0.1-0.11g of 1,3,5-tris(4-aminophenyl)benzene, and dissolve them in a mixed solution of 30-50mL of 1,4-dioxane and n-butanol; take 0.1-0.15g of Fe3O4 magnetic nanoparticle powder and disperse it in the above mixed solution after ultrasonication, slowly add 400-600μL of glacial acetic acid, and suspend and react at room temperature for 2 hours. Then take 4-5mL of acetic acid solution and add it to the mixed solution, and react in a water bath at 60-80℃ in the dark for 45-50 hours. After cooling to room temperature, separate the yellow-green product with a magnet, wash with acetone and tetrahydrofuran respectively, and dry under vacuum to obtain the magnetic covalent organic framework.

[0008] More preferably, in step (1), ferric chloride hexahydrate is 1.35 g, anhydrous sodium acetate is 3.6 g, and ethylene glycol is 40 mL.

[0009] More preferably, in step (1), the solution is heated to 200°C and reacted for 16 hours.

[0010] More preferably, in step (2), the volume ratio of 1,4-dioxane to n-butanol is 1:1.

[0011] More preferably, the concentration of the acetic acid solution in step (2) is 12M.

[0012] More preferably, the method for preparing the aptamer-modified gold nanoparticles includes the following steps:

[0013] (1) Measure 450-550 μL of chloroauric acid solution and 50-55 mL of deionized water into a three-necked flask, stir vigorously with a magnetic force and heat to boiling. Quickly add 0.5-1.5% of 1.5-2.5 mL of trisodium citrate mixture, continue heating and stirring vigorously until the solution turns wine red. After cooling to room temperature, gold nanoparticles are obtained.

[0014] (2) Take 15-25 μL of Salmonella Typhimurium specific nucleic acid aptamer sequence, heat at 94-98℃ for 4-6 minutes, cool at 3-5℃ for 4-6 minutes, add 1-3 μL of TCEP solution, incubate at room temperature in the dark for 50-70 minutes, add 1-2 mL of gold nanoparticle solution, suspend for 14-18 hours, centrifuge to remove unbound aptamer sequence, and resuspend in 0.5-1 mL of deionized water to obtain aptamer-modified gold nanoparticles.

[0015] More preferably, in step (1), the concentration of the chloroauric acid solution is 1%, w / v, and the amount used is 500 μL; the amount of deionized water used is 52 mL; and the concentration of trisodium citrate is 1%, w / v, and the amount used is 2 mL.

[0016] More preferably, in step (2), the amount of Salmonella typhimurium-specific nucleic acid aptamer sequence used is 20 μL, the concentration is 10 μM; the concentration of TCEP solution is 1 mM, the amount used is 2 μL; and the amount of gold nanoparticle solution used is 1.5 mL.

[0017] More preferably, the Salmonella Typhimurium-specific nucleic acid aptamer sequence in step (2) is shown in SEQ ID NO.1.

[0018] Compared with the prior art, the present invention has the following beneficial effects:

[0019] 1) The materials used in this invention are easy to prepare and the operation is simple and convenient;

[0020] 2) This invention simultaneously outputs both color and fluorescence signals, both of which have a good linear relationship with the concentration of the target bacteria. The detection limit is less than 10 CFU / mL, and the sensitivity is high, enabling quantitative detection. It identifies the target bacteria through nucleic acid aptamers, which has good specificity. Using the "gold standard" plate count method as a reference, this invention can obtain relatively accurate results when detecting milk samples with different bacterial concentrations.

[0021] 3) With the help of smartphones and LDA, this invention can effectively identify and distinguish different concentrations of Salmonella typhimurium; through analysis of actual milk samples containing bacteria, the rationality and practicality of this detection mode have been preliminarily verified; in addition, this mode is simple and portable to operate, which is conducive to achieving rapid on-site detection.

[0022] 4) This invention combines colorimetric / fluorescence / smartphone-based LDA multi-mode detection, and the detection results of each mode can be self-checked collaboratively, resulting in more accurate and reliable results. Attached Figure Description

[0023] Figure 1 This is a flowchart of the present invention.

[0024] Figure 2aThis is a standard curve of normalized absorbance versus logarithmic concentration of Salmonella typhimurium in Example 1.

[0025] Figure 2b This is a standard curve of normalized fluorescence intensity versus logarithmic concentration of Salmonella typhimurium in Example 1.

[0026] Figure 3 The diagram shows the detection specificity of Salmonella Typhimurium by colorimetric (a) and fluorescence (b) methods in Example 1. 1-8 represent blank control, Staphylococcus aureus, Listeria monocytogenes, Escherichia coli O157:H7, Vibrio parahaemolyticus, the above four mixed bacteria, Salmonella Typhimurium, and the above five mixed bacteria, respectively.

[0027] Figure 4 The image shows the 2D LDA standard score chart in Example 1, where a represents the 2D LDA standard score chart corresponding to the colorimetric results of different concentrations of Salmonella Typhimurium; bd represents the 2D LDA standard score chart of the actual contaminated milk sample test (actual sample concentration: b, 5.5 × 10⁻⁶). 3 CFU / mL; c, 1.2 × 10 4 CFU / mL; d, 1.1×10 6 (CFU / mL) Detailed Implementation

[0028] Example 1

[0029] 1) A method for preparing a magnetic covalent organic framework, comprising:

[0030] Weigh 1.35 g of ferric chloride hexahydrate and 3.6 g of anhydrous sodium acetate, and dissolve them completely in 40 mL of ethylene glycol. Transfer the solution to a polytetrafluoroethylene-lined autoclave and heat it to 200 °C in a drying oven for 16 hours. After cooling to room temperature, separate the black product using a magnet and wash it three times each with deionized water and anhydrous ethanol. After vacuum drying at 60 °C for 12 hours, obtain Fe3O4 magnetic nanoparticle powder.

[0031] Weigh 0.087 g of 2,5-dimethylaminophenyl-1,4-dicarboxaldehyde (CAS No.: 7310-97-6) and 0.105 g of 1,3,5-tris(4-aminophenyl)benzene (CAS No.: 118727-34-7), and dissolve them completely in 40 mL of a mixed solution of 1,4-dioxane and n-butanol (volume ratio = 1:1). Disperse 0.12 g of Fe3O4 magnetic nanoparticles in the above mixed solution by sonication for 10 minutes. Slowly add 500 μL of glacial acetic acid and suspend the mixture at room temperature for 2 hours. Then add 4.5 mL of acetic acid aqueous solution (12M) to the mixed solution and react in a 70°C water bath in the dark for 48 hours. After cooling to room temperature, magnetically separate the yellow-green product using a magnet, and wash three times each with acetone and tetrahydrofuran. Dry under vacuum at 30°C for 12 hours to obtain the magnetic covalent organic framework.

[0032] 2) A method for preparing aptamer-modified gold nanoparticles, comprising:

[0033] Measure 500 μL of chloroauric acid aqueous solution (1%, w / v) and 52 mL of deionized water into a three-necked flask, stir vigorously with a magnetic stirrer and heat to boiling. Quickly add 2 mL of trisodium citrate mixed aqueous solution (containing 1% trisodium citrate and 0.05% citric acid, w / v), continue heating and stirring vigorously for 5 minutes until the solution turns wine red. After cooling to room temperature, gold nanoparticles are obtained and stored at 4 °C for later use.

[0034] Take 20 μL of an aqueous solution of Salmonella Typhimurium-specific nucleic acid aptamer (10 μM) (the nucleic acid aptamer sequence is SEQ ID NO.1: 5'-SH-C6-AGTAATGCCCGGTAGTTATTCAAAGATGAGTAGGAAAAGA-3'), heat at 95℃ for 5 minutes, cool at 4℃ for 5 minutes, add 2 μL of LTCEP aqueous solution (1 mM), incubate at room temperature in the dark for 60 minutes, add 1.5 mL of the prepared gold nanoparticle solution, suspend for 16 hours, centrifuge at 13000 rpm for 20 minutes to remove unbound aptamer sequences, and resuspend in 0.75 mL of deionized water to obtain aptamer-modified gold nanoparticles.

[0035] 3) A method for preparing an enzyme-catalyzed substrate solution, comprising:

[0036] Weigh 0.0039 g of tetra-(4-aminophenyl)ethylene (TPE-4A) and dissolve it in 1 mL of 1,4-dioxane to prepare a 10 mM TPE-4A solution;

[0037] A chromogenic substrate solution was prepared by mixing 30 μL of TPE-4A solution (10 mM), 10 μL of hydrogen peroxide aqueous solution (1 M), and 70 μL of sodium acetate buffer (pH = 4, 20 mM).

[0038] A luminescent substrate solution was prepared by mixing 10 μL of TPE-4A solution (10 mM), 10 μL of hydrogen peroxide aqueous solution (1 M), and 90 μL of sodium acetate buffer (pH = 7, 20 mM).

[0039] 4) Colorimetric detection methods for Salmonella Typhimurium, including:

[0040] Sensitivity analysis of the detection method:

[0041] 500 μL of different concentrations (1×10) 3 -1×10 8 Salmonella typhimurium (CFU / mL) was incubated with 20 μL of magnetic covalent organic framework (1 mg / mL) dispersed in deionized water at room temperature for 20 minutes. After magnetic separation, 40 μL of aptamer-modified gold nanoparticle solution was added, and the mixture was incubated at room temperature for 30 minutes. After magnetic separation, a chromogenic substrate solution was added, and the mixture was reacted at room temperature in the dark for 10 minutes. The absorbance A of the supernatant was then measured by magnetic separation. 600 A standard curve was plotted with absorbance on the ordinate and the logarithm of Salmonella typhimurium concentration on the abscissa. The results are shown in [reference needed]. Figure 2a .

[0042] Specificity analysis of the detection method:

[0043] Mix 500 μL of a solution containing Staphylococcus aureus, Listeria monocytogenes, Escherichia coli O157:H7, Vibrio parahaemolyticus, a mixture of the above four bacteria, Salmonella typhimurium, and a mixture of the above five bacteria (Salmonella typhimurium concentration 1×10⁻⁶). 5 CFU / mL, the concentration of other strains was 1×10⁻⁶. 6 CFU / mL) was incubated separately with 20 μL of magnetic covalent organic framework (1 mg / mL) dispersed in deionized water at room temperature for 20 minutes. After magnetic separation, 40 μL of aptamer-modified gold nanoparticle solution was added, and the mixture was incubated at room temperature for 30 minutes. After magnetic separation, chromogenic substrate solution was added, and the reaction was carried out at room temperature in the dark for 10 minutes. The absorbance A of the supernatant was measured by magnetic separation. 600 For specificity results of the colorimetric detection method, please refer to [link / reference]. Figure 3 a.

[0044] 5) Fluorescent detection methods for Salmonella Typhimurium, including:

[0045] Sensitivity analysis of the detection method:

[0046] 500 μL of different concentrations (1×10) 1 -1×10 7Salmonella typhimurium (CFU / mL) was incubated with 20 μL of magnetic covalent organic framework (1 mg / mL) dispersed in deionized water at room temperature for 20 minutes. After magnetic separation, 40 μL of aptamer-modified gold nanoparticle solution was added, and the mixture was incubated at room temperature for 30 minutes. After magnetic separation, luminescent substrate solution was added, and the reaction was carried out at room temperature in the dark for 10 minutes. The reaction was terminated by adding 100 μL of deionized water, and the reaction was continued at room temperature in the dark for another 10 minutes. The fluorescence intensity F of the supernatant was detected by magnetic separation. 500 A standard curve was plotted with fluorescence intensity as the ordinate and the logarithm of Salmonella typhimurium concentration as the abscissa. (See [reference needed]). Figure 2b .

[0047] Specificity analysis of the detection method:

[0048] Mix 500 μL of a solution containing Staphylococcus aureus, Listeria monocytogenes, Escherichia coli O157:H7, Vibrio parahaemolyticus, a mixture of the above four bacteria, Salmonella typhimurium, and a mixture of the above five bacteria (Salmonella typhimurium concentration 1×10⁻⁶). 5 CFU / mL, the concentration of other strains was 1×10⁻⁶. 6 CFU / mL) was incubated with 20 μL of magnetic covalent organic framework (1 mg / mL) dispersed in deionized water at room temperature for 20 minutes. After magnetic separation, 40 μL of aptamer-modified gold nanoparticle solution was added, and the mixture was incubated at room temperature for 30 minutes. After magnetic separation, luminescent substrate solution was added, and the reaction was carried out at room temperature in the dark for 10 minutes. The reaction was terminated by adding 100 μL of deionized water, and the reaction was continued at room temperature in the dark for another 10 minutes. The fluorescence intensity F of the supernatant was detected by magnetic separation. 500 For specificity results of the fluorescence detection method, please refer to [link / reference]. Figure 3 b.

[0049] 6) A smartphone-based method for detecting Salmonella typhimurium combined with linear discriminant analysis, which includes:

[0050] Images of the colorimetric samples in step 4) were collected using a smartphone. The red, green, and blue (RGB) parameters in the images were read using a color recognition app on the phone. The data were then imported into SPSS software for linear discriminant analysis. The eigenvectors of the first two linear discriminant functions were used as the first and second factors, respectively. The first factor was used as the abscissa and the second factor as the ordinate to obtain a 2D standard score map of different concentrations of Salmonella typhimurium.

[0051] 7) Testing of actual contaminated milk samples, including:

[0052] Based on the colorimetric and fluorescence detection methods for Salmonella Typhimurium established in the experiment, the absorbance and fluorescence intensity of different Salmonella Typhimurium samples were measured, and the corresponding logarithmic concentration values ​​were calculated. Then, the recovery rate and relative standard deviation were calculated by comparing them with the plate count results. The results are shown in Table 1. The recovery rate in milk samples ranged from 96.89% to 106.45%, and the relative standard deviations were all less than 10%, indicating that the method has high accuracy and is feasible for the detection of actual samples.

[0053] Sample colorimetric images were acquired using a smartphone, and the red, green, and blue (RGB) parameters in the images were read using a color recognition app on the phone. The obtained data was imported into the training set established using the Salmonella typhimurium standard samples in step 6) for linear discriminant analysis to obtain the 2D LDA standard score map of the actual sample test. The results are shown in […]. Figure 4 The concentration range of Salmonella typhimurium predicted by this method is basically consistent with that of the plate count method, indicating that the integrated detection method of smartphone and LDA has certain practicality and potential for on-site detection.

[0054] Table 1. Results of milk sample determination by plate count method and colorimetric and fluorescence method of the present invention.

[0055]

[0056] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A method for detecting Salmonella typhimurium based on gold nanoparticles modified with magnetic covalent organic frameworks and aptamers, characterized in that, The detection method uses a magnetic covalent organic framework as a capture probe to capture Salmonella typhimurium present in the detection system. After incubation, the magnetic covalent organic framework-Salmonella typhimurium complex is collected by magnetic separation. Then, aptamer-modified gold nanoparticles are added to the complex. After incubation, the complex formed by the magnetic covalent organic framework-Salmonella typhimurium complex and the aptamer-modified gold nanoparticles is collected by magnetic separation. Finally, a TPE-4A / hydrogen peroxide substrate solution is added, and the absorbance and / or fluorescence intensity are measured. The TPE-4A is tetra-(4-aminophenyl)ethylene.

2. The method for detecting Salmonella typhimurium according to claim 1, characterized in that, The method for preparing the magnetic covalent organic framework includes the following steps: (1) Weigh 1.2-1.5 g of ferric chloride hexahydrate and 3-4 g of anhydrous sodium acetate, dissolve them completely in 30-50 mL of ethylene glycol, heat the solution to 180-220℃, react for 14-18 hours, cool to room temperature, separate the black product with a magnet, wash with deionized water and anhydrous ethanol respectively, and dry under vacuum to obtain Fe3O4 magnetic nanoparticle powder; (2) Weigh 0.07-0.09 g of 2,5-dimethylaminobenzene-1,4-dicarboxaldehyde and 0.1-0.11 g of 1,3,5-tris(4-aminophenyl)benzene, and dissolve them in a mixed solution of 30-50 mL of 1,4-dioxane and n-butanol; take 0.1-0.15 g of Fe3O4 magnetic nanoparticle powder and disperse it in the above mixed solution after ultrasonication, slowly add 400-600 μL of glacial acetic acid, and suspend and react at room temperature for 2 hours. Then take 4-5 mL of acetic acid solution and add it to the mixed solution, and react in a water bath at 60-80℃ in the dark for 45-50 hours. After cooling to room temperature, separate the yellow-green product with a magnet, wash with acetone and tetrahydrofuran respectively, and dry under vacuum to obtain the magnetic covalent organic framework.

3. The method for detecting Salmonella typhimurium according to claim 2, characterized in that, In step (1), the amount of ferric chloride hexahydrate is 1.35g, the amount of anhydrous sodium acetate is 3.6g, and the amount of ethylene glycol is 40mL.

4. The method for detecting Salmonella typhimurium according to claim 3, characterized in that, In step (1), the solution is heated to 200°C and reacted for 16 hours.

5. The method for detecting Salmonella typhimurium according to claim 4, characterized in that, In step (2), the volume ratio of 1,4-dioxane to n-butanol is 1:

1.

6. The method for detecting Salmonella typhimurium according to claim 5, characterized in that, The concentration of the acetic acid solution in step (2) is 12 M.

7. The method for detecting Salmonella typhimurium according to claim 6, characterized in that, The method for preparing the aptamer-modified gold nanoparticles includes the following steps: (1) Take 450-550 μL of chloroauric acid solution and 50-55 mL of deionized water into a three-necked flask, stir vigorously with a magnetic force and heat to boiling. Quickly add 0.5-1.5% of 1.5-2.5 mL of trisodium citrate mixture, continue heating and stirring vigorously until the solution turns wine red. After cooling to room temperature, gold nanoparticles are obtained. (2) Take 15-25 μL of Salmonella Typhimurium specific nucleic acid aptamer, heat at 94-98℃ for 4-6 minutes, cool at 3-5℃ for 4-6 minutes, add 1-3 μL of TCEP solution, incubate at room temperature in the dark for 50-70 minutes, add 1-2 mL of gold nanoparticle solution, suspend for 14-18 hours, centrifuge to remove unbound aptamers, and resuspend in 0.5-1 mL of deionized water to obtain aptamer-modified gold nanoparticles.

8. The method for detecting Salmonella typhimurium according to claim 7, characterized in that, In step (1), the concentration of chloroauric acid solution is 1%, w / v, and the amount used is 500 μL; the amount of deionized water used is 52 mL; and the concentration of trisodium citrate is 1%, w / v, and the amount used is 2 mL.

9. The method for detecting Salmonella typhimurium according to claim 8, characterized in that, In step (2), the amount of Salmonella Typhimurium-specific nucleic acid aptamer used is 20 μL, and the concentration is 10 μM; the concentration of TCEP solution is 1 mM, and the amount used is 2 μL; the amount of gold nanoparticle solution used is 1.5 mL.

10. The method for detecting Salmonella typhimurium according to claim 9, characterized in that, The specific nucleic acid aptamer sequence of Salmonella typhimurium in step (2) is shown in SEQ ID NO.1.

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