Fluorescent paper chip for detecting staphylococcus aureus as well as preparation method and application of fluorescent paper chip

Abstract

The invention belongs to the field of food analysis, and particularly discloses a fluorescent paper chip for detecting staphylococcus aureus as well as a preparation method and application of the fluorescent paper chip. The preparation method comprises the following steps: firstly, pre-assembling a template bacterial solution, a functional monomer and a fluorescent monomer, then adding a cross-linking agent to carry out polymerization reaction, and after elution, synthesizing the fluorescent molecularly imprinted nanoparticles taking staphylococcus aureus as the template, and immobilizing the fluorescent molecular imprinting nanoparticles on the surface of the paper chip by a covalent immobilization method to obtain the fluorescent paper chip. When bacterial strains are detected, staphylococcus aureus is sensitively and rapidly recognized according to the quenching rate of fluorescence, the method has the advantages of being easy to operate, good in selectivity, low in cost and the like, food can be rapidly and timely detected, and a new thought and a novel strategy are provided for detecting pathogenic bacteria in the food.

Description

Technical Field

[0001] The invention belongs to the field of food analysis, and in particular relates to a fluorescent paper chip for detecting Staphylococcus aureus and a preparation method and application thereof. Background Art

[0002] Staphylococcus aureus (S. aureus) is an aerobic or facultative anaerobic, non-spore Gram-positive bacterium, one of the common zoonotic pathogens in clinical practice. The bacteria are widely present in soil, water sources and breeding environments, and most healthy animals are colonized with the bacteria [J. Agric. Food Chem., 72 (2024) 16569-16582]. Staphylococcus aureus can infect a variety of livestock, poultry and humans, causing a series of diseases and food poisoning, seriously endangering animal and human health. It is of great significance to study the rapid detection method of the bacteria. Traditional methods for detecting Staphylococcus aureus mainly rely on the time-consuming and labor-intensive bacterial count method [GB 4789.10-2016]. Various PCR analysis methods developed in recent years [SN / T 2206.10-2014; SN / T2206.11-2014; SN / T 5364.5-2021; SN / T 5439.2-2022] and mass spectrometry [SN / T 5228.3-2019] all rely on expensive large instruments, and their analysis process requires bacterial growth for more than 8 hours, and professional personnel are required to operate and maintain, making it difficult to achieve rapid on-site screening and analysis. Although there are some rapid detection Staphylococcus aureus test strips [SN / T4546-2017] Staphylococcus aureus test strips), they still require bacterial growth for more than 24 hours. Therefore, researchers are committed to developing faster and more sensitive methods for rapid detection of Staphylococcus aureus, thereby reducing the threat of Staphylococcus aureus infection to medical devices, food, environment, livestock production and other fields.

[0003] Molecular imprinting technique (MIT) is a technology that simulates antigen-antibody specific recognition and establishes artificial recognition sites in polymers. The polymer prepared by this technology is called molecularly imprinted polymer (MIP), and its internal imprinted cavity specifically binds to the template target. Molecularly imprinted polymers have good selectivity, specificity, physical and mechanical properties and biocompatibility, and are easy to synthesize and low cost [Anal.Methods., 13 (2021) 4538-4556]. At present, there are many examples of molecular imprinting in scientific research, mostly with small molecule compounds and protein macromolecules as targets or templates. However, imprinting of microorganisms and other cells is more difficult. The most direct method is whole-cell imprinting, and selective capture and recognition of microorganisms can also be achieved by imprinting specific molecular components on the cell surface.

[0004] The carrier materials of molecular imprinting polymers generally include glass plates, micro-nanoparticles, silicon wafers and some biomass materials. Among them, biocellulose materials are three-dimensional sheets formed by hydrogen bonds between hydroxyl groups in cellulose, and are raw materials made from plant fibers. Not only can it be widely used, it is inexpensive, light, biodegradable, and easy to operate, but cellulose also has a porous structure, good adsorption properties and multiple hydroxyl groups, etc., making it an ideal carrier material for making bacterial molecular imprinting polymers [Carbohydr.Polym., 305(2023)120570]. Summary of the invention

[0005] Staphylococcus aureus is a ubiquitous human pathogen that causes staphylococcal food poisoning, one of the most common foodborne diseases. Traditional methods for detecting Staphylococcus aureus mainly rely on time-consuming and labor-intensive bacterial count methods [GB 4789.10-2016]. Various PCR analysis methods and mass spectrometry methods developed in recent years all rely on expensive large instruments, and their analysis process requires bacterial growth for more than 8 hours, making it difficult to achieve rapid on-site screening and analysis. In order to develop a simple and portable pathogen detection device, the purpose of the present invention is to provide a paper chip for quantitative identification and detection of Staphylococcus aureus. Molecular imprinting polymers are prepared by sol-gel polymerization and magnetic stirring, and then the polymers are immobilized on paper chips by immobilization method for fluorescence detection and analysis of Staphylococcus aureus in actual samples.

[0006] In order to solve the problems of the prior art, the technical solution adopted by the present invention is:

[0007] A method for preparing a fluorescent paper chip for detecting Staphylococcus aureus comprises the following steps ：

[0008] Step 1. Preparation of fluorescent molecularly imprinted nanoparticles:

[0009] Staphylococcus aureus was dispersed in a phosphate buffer solution at pH 7.4, and functional monomers and fluorescent monomers were added for pre-assembly; after being evenly mixed, a cross-linking agent was added and stirred for 12 hours. After the reaction was completed, the nanoparticles were first washed with a phosphate buffer solution, and then the amino groups were modified on the surface of the nanoparticles. Finally, the templates were removed by washing with a phosphate buffer solution and a methanol / water co-solvent of acetic acid, respectively, to obtain fluorescent molecularly imprinted nanoparticles;

[0010] Step 2. Immobilization of fluorescent molecularly imprinted nanoparticles on the surface of paper chip:

[0011] By utilizing the abundant hydroxyl groups on the surface of the paper chip, the surface of the paper chip was modified with amino groups and aldehyde groups in sequence. The paper chip was then immersed in the nanoparticle solution prepared in step 1 and reacted for 12 hours to obtain a fluorescent paper chip for detecting Staphylococcus aureus.

[0012] As an improvement, the functional monomer in step one is obtained by reacting 3-aminopropyltriethoxysilane with 3-formylphenylboronic acid, and the fluorescent monomer is obtained by reacting 3-aminopropyltriethoxysilane with fluorescein isothiocyanate.

[0013] As an improvement, the concentration of the functional monomer in step 1 is 4.2 mmol / L, the concentration of the fluorescent monomer is 0.2 mmol / L, and the concentration of Staphylococcus aureus is 0.5*10 9 CFU / mL.

[0014] As an improvement, in step one, the volume ratio of the functional monomer to the fluorescent monomer is 1:1.

[0015] As an improvement, in step 1, the cross-linking agent is tetraethoxysilane, and the concentration is 351 mmol / L.

[0016] As an improvement, the concentration of the fluorescent nanoparticles in step 2 is 0.1 mg / mL.

[0017] The fluorescent paper chip for detecting Staphylococcus aureus is used for quantitatively detecting Staphylococcus aureus in food testing.

[0018] As an improvement, the quantitative detection and analysis steps are as follows: first use the Jingxin microarray chip scanner to determine the initial fluorescence value of the fluorescent paper chip, add the sample to be tested on the surface of the paper chip, incubate for a period of time, determine its fluorescence signal value, and establish a standard curve based on the fluorescence quenching rate to perform quantitative analysis of Staphylococcus aureus.

[0019] The fluorescent paper chip for detecting Staphylococcus aureus described in the present invention takes Staphylococcus aureus as the research object, designs and synthesizes molecular imprinting fluorescent nanoparticles with Staphylococcus aureus as the template through molecular imprinting technology, and after the molecular imprinting fluorescent nanoparticles are modified on the surface of filter paper, the Staphylococcus aureus template can be sensitively and quickly identified according to the change of fluorescence quenching rate, thereby preparing a paper-based array chip that can specifically identify Staphylococcus aureus, which has the characteristics of simplicity, low cost, real-time, etc.

[0020] Beneficial effects:

[0021] Compared with the prior art, the fluorescent paper chip for detecting Staphylococcus aureus and its preparation method and application in the present invention have the following advantages:

[0022] 1. The present invention adopts a sol-gel polymerization reaction of a silane reagent, and the reaction conditions are mild and carried out at room temperature, thereby ensuring the stability of the molecularly imprinted polymer and facilitating the acquisition of a highly specific polymer.

[0023] 2. The amino groups modified on the surface of the fluorescent molecular imprinted nanoparticles in the present invention have little effect on the imprinting of the target bacteria.

[0024] 3. Molecular imprinting technology for pathogenic bacteria detection does not require sample pretreatment, which simplifies the analysis process, has a fast identification and detection speed, and has good stability of the detection material.

[0025] 4. The fluorescent molecular imprinting paper chip for rapid detection of Staphylococcus aureus prepared by the present invention can sensitively and rapidly identify Staphylococcus aureus according to the change of fluorescence quenching rate, and achieve accurate quantification thereof.

[0026] 5. The paper chip optical sensor is easy to operate, requires less reagents, and has a shorter analysis time, which greatly improves the efficiency and sensitivity of detection and makes it easy to implement on-site sampling and analysis.

[0027] 6. Molecular imprinting technology does not require sample pretreatment for pathogen detection, which simplifies the analysis process, has a fast identification and detection speed, and has good stability of the detection material. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 The schematic diagram of the fluorescent molecular imprinting paper chip of Staphylococcus aureus; (3-aminopropyltriethoxysilane (APTES), triformylphenylboronic acid (PBA), fluorescein isothiocyanate (FITC), functional monomer (PBA-APTES), fluorescent monomer (FITC-APTES));

[0029] Figure 2 Selectivity experiments of fluorescent molecularly imprinted nanoparticles for Staphylococcus aureus;

[0030] Figure 3Specificity experiments of fluorescent molecularly imprinted nanoparticles for Staphylococcus aureus;

[0031] Figure 4 The selectivity experiment of fluorescent molecular imprinted nanoparticles (with amino groups on the surface) for Staphylococcus aureus;

[0032] Figure 5 This is a specific experiment of fluorescent molecular imprinted nanoparticles (with amino groups on the surface) for Staphylococcus aureus;

[0033] Figure 6 This is a specific experiment of Staphylococcus aureus fluorescent molecular imprinted nanoparticles immobilized on the surface of paper chip;

[0034] Figure 7 This is a selectivity experiment of Staphylococcus aureus after the fluorescent molecular imprinted nanoparticles were immobilized on the surface of paper chip;

[0035] Figure 8 This is the standard curve of Staphylococcus aureus fluorescent molecularly imprinted nanoparticles immobilized on the surface of the paper chip. DETAILED DESCRIPTION

[0036] The present invention is further described below in conjunction with specific embodiments and drawings.

[0037] The experimental methods described in the examples are conventional methods unless otherwise specified; Escherichia coli O157:H7 (CICC21530) and Staphylococcus aureus (ATCC25923) used in the examples of the present invention were kindly provided by the Animal Food Control Laboratory of Nanjing Normal University; Salmonella (ATCC14028) and Shigella (CMCC(B)51592) were kindly provided by Nanjing Product Quality Supervision and Inspection Institute. The reagents and materials described above can be obtained from commercial sources unless otherwise specified.

[0038] Example 1

[0039] Preparation of fluorescent monomers:

[0040] Dissolve fluorescein isothiocyanate (FITC, 6.6 mg, 17 μmol) in 1.5 mL of anhydrous ethanol, then add 3-aminopropyltriethoxysilane (APTES, 4 μL, 17 μmol) and stir at room temperature (25°C) for 12 h. After the reaction is completed, remove ethanol to obtain FITC-APTES product. Take 0.2 mL of FITC-APTES solution for later use.

[0041] Preparation of functional monomers:

[0042] 3-Formylphenylboronic acid (3-FPBA, 75 mg, 0.5 mmol) was dissolved in 10 mL of anhydrous ethanol, and then 3-aminopropyltriethoxysilane (APTES, 120 μL, 0.5 mmol) was added and stirred at room temperature for 12 h. After the reaction was completed, ethanol was removed to obtain a light yellow PBA-APTES product. 20 mg of PBA-APTES was dissolved in 0.2 ml of anhydrous ethanol for later use.

[0043] Example 2

[0044] Preparation of molecularly imprinted fluorescent nanoparticles, the specific synthesis route is as follows Figure 1 As shown:

[0045] Put 0.5mL of 10 9 CFU / mL Staphylococcus aureus bacterial solution was evenly dispersed in 6.55mL PBS (10mM, pH=7.4), and mixed with 0.2mL PBA-APTES solution and 0.2mL FITC-APTES solution in a round-bottom flask for 40min. TEOS (675μL, 3.2mmol) was added, followed by 1mL anhydrous ethanol, and stirred at 1000rpm for 12h. After centrifugation, the polymer was washed twice with phosphate buffer, and amino groups were modified on the polymer surface. The template was then washed with acetic acid methanol / water co-solvent solution to obtain Staphylococcus aureus molecularly imprinted fluorescent nanoparticles FMIP-NH2.

[0046] Comparative Example 1

[0047] Non-molecularly imprinted fluorescent nanoparticles (FNIP-NH2) were prepared as a control, and the nanoparticles were generated using the same conditions as those of FMIP-NH2 nanoparticles, with specific reference to the preparation of Example 2, except that the template Staphylococcus aureus was omitted.

[0048] Example 3

[0049] Selectivity test of the Staphylococcus aureus molecularly imprinted fluorescent nanoparticles prepared in Example 2

[0050] Take 100 μL of 1 mg / mL FMIP solution (solvent is PBS, pH 7.4) and FNIP solution (solvent is PBS, pH 7.4), add them to 4 mL fluorescence cuvettes, then add 1.9 mL PBS (10 mM, pH = 7.4), and use a fluorescence spectrophotometer (excitation wavelength 471 nm, emission wavelength 513 nm) to measure the initial fluorescence signal F0. Then, a certain amount of Staphylococcus aureus is added to the cuvette to make the concentration of 10 1 , 10 2 , 10 3 , 10 4 , 105 , 10 6 and 10 7 CFU / mL, stirred at room temperature (25°C) for 5 min, and the fluorescence signal F was measured using a fluorescence spectrophotometer to calculate the fluorescence quenching rate. The selectivity experiment of FMIP-NH2 and FNIP-NH2 solutions was also determined.

[0051] like Figure 2 Shown 10 1 , 10 2 , 10 3 , 10 4 , 10 5 , 10 6 and 10 7 The fluorescence response change rate of FMIP at CFU / mL concentration was 10 higher than that of their corresponding fluorescent nanoparticles. 1 , 10 2 , 10 3 , 10 4 , 10 5 , 10 6 and 10 7 CFU / mL concentration of FNIP, indicating that the molecularly imprinted fluorescent nanoparticles have good response selectivity to Staphylococcus aureus.

[0052] from Figure 4 It can also be seen that the fluorescence quenching rate of FMIP-NH2 is higher than that of FNIP-NH2, indicating that the modification of the amino group has little effect on the fluorescent molecular imprinting nanoparticles, and almost no effect.

[0053] Example 4

[0054] Take 100 μL of 1 mg / mL FMIP solution (solvent is PBS, pH 7.4) and FNIP solution (solvent is PBS, pH 7.4), add them to 4 mL fluorescence cuvettes, then add 1.9 mL PBS (10 mM, pH = 7.4), and use a fluorescence spectrophotometer (excitation wavelength 471 nm, emission wavelength 513 nm) to measure the initial fluorescence signal F0. Then add 20 μL and 10 μL of 1 mg / mL FMIP solution to the cuvettes, respectively. 7 CFU / mL of Staphylococcus aureus, Escherichia coli O157:H7, Salmonella, and Shigella were stirred at room temperature (25°C) for 5 min, and the fluorescence signal F was measured using a fluorescence spectrophotometer to calculate the fluorescence responsiveness ratio. The specificity experiment of FMIP-NH2 and FNIP-NH2 solutions was also determined.

[0055] like Figure 3The imprinting factor shown refers to the ratio of the amount of the target adsorbed by MIP or the signal change caused to the amount adsorbed by NIP or the signal change caused, which can be used to indicate the selectivity. The higher the imprinting factor, the higher the selectivity. It can be seen from the figure that the imprinting factors are 4.5, 3.5, 0.8, and 0.6, respectively. It can be seen that the fluorescent molecular imprinted nanoparticles have good specificity for Staphylococcus aureus.

[0056] like Figure 5 The fluorescent molecular imprinted nanoparticles modified with amino groups still have the largest imprinting factor for Staphylococcus aureus and good specificity.

[0057] Example 5

[0058] Place the filter paper in a 6% APTES ethanol solution and shake it for 12 hours. Take it out, rinse it with ethanol, and dry it. Prepare a 2.5% glutaraldehyde aqueous solution, place the amino-modified filter paper in a centrifuge tube, and shake it for 12 hours. Take it out, rinse it with deionized water, and dry it. Prepare a 1mg / mL FMIP-NH2 solution, soak the filter paper for 12 hours, take it out, and rinse it with PBS.

[0059] Comparative Example 2

[0060] According to the operation of Example 5, a 1 mg / mL FNIP-NH2 solution was prepared, the filter paper was soaked for 12 hours, then taken out and rinsed with PBS.

[0061] Example 6

[0062] Selectivity test of the fluorescent paper chip of Staphylococcus aureus prepared in Example 5 and the chip prepared in Comparative Example 2

[0063] The initial fluorescence signal F0 of the paper chip was measured using a microarray chip scanner. After the measurement, 20 μL of 10 1 , 10 2 , 10 3 , 10 4 , 10 5 , 10 6 , 10 7 , 10 8 and 10 9 CFU / mL of Staphylococcus aureus liquid was incubated for 5 minutes, and the fluorescence signal F was measured to calculate the fluorescence response ratio (blank sample was measured at the same time).

[0064] like Figure 6 Shown 10 1 , 10 2 , 10 3 , 10 4 , 105 , 10 6 , 10 7 , 10 8 and 10 9 The fluorescence response change rate of FMIP@P with CFU / mL concentration was 10 higher than that of their corresponding fluorescent nanoparticles. 1 , 10 2 , 10 3 , 10 4 , 10 5 , 10 6 , 10 7 , 10 8 and 10 9 CFU / mL concentration of FNIP@P, indicating that the molecularly imprinted fluorescent nanoparticles have good response selectivity to Staphylococcus aureus.

[0065] Example 7

[0066] Specificity test of the fluorescent paper chip of Staphylococcus aureus prepared in Example 5 and the filter paper prepared in Comparative Example 2

[0067] The initial fluorescence signal F0 of the filter paper was measured using a microarray chip scanner, and then 20 μL, 10 9 CFU / mL Staphylococcus aureus, Escherichia coli O157:H7, Salmonella, Shigella solution, after incubation for 10 minutes, measure the fluorescence value F to calculate the fluorescence response ratio. (Blank sample is measured at the same time)

[0068] like Figure 7 The imprinting factor shown refers to the ratio of the amount of the target adsorbed by MIP or the signal change caused to the amount adsorbed by NIP or the signal change caused, which can be used to indicate the selectivity. The higher the imprinting factor, the higher the selectivity. It can be seen from the figure that the imprinting factors are 3.1, 0.9, 1.5, and 1.3, respectively. It can be seen that the fluorescent paper chip has good specificity for Staphylococcus aureus.

[0069] Example 8

[0070] Establishment of a standard curve for the fluorescent paper chip for detecting Staphylococcus aureus prepared in Example 5

[0071] The initial fluorescence signal F0 of the fluorescent paper chip was measured using a microarray chip scanner. After the measurement, 20 μL of 10 1 , 10 2 , 10 3 , 10 4 , 10 5 , 10 6 , 10 7 , 108 and 10 9 CFU / mL of Staphylococcus aureus solution was incubated for 5 min (temperature was 15°C) and the fluorescence signal F was measured to calculate the fluorescence response ratio (blank sample was measured at the same time).

[0072] Example 9

[0073] Used for testing actual samples (pure water and Junlebaoyue fresh milk samples).

[0074] The standard curve was prepared according to the steps of Example 8 to determine the recovery rate of spike addition in this method and calculate the recovery rate of spike addition. First, the initial fluorescence signal F0 of the paper chip was measured using a microarray chip scanner. The bacterial solution was diluted to 10% with tap water. 4 , 10 5 , 10 6 CFU / mL, drop the diluted bacterial solution on the surface of the paper chip, incubate for 5 minutes (temperature is 15°C), measure the fluorescence signal F, and calculate the fluorescence response ratio (simultaneously measure the blank sample). Drop 100μL of bacterial solution in 900μL of milk, and then dilute it 10 times with PBS to make the final concentration of the bacterial solution 10 4 , 10 5 , 10 6 CFU / mL. The treated milk was added to the diluted bacterial solution on the surface of the paper chip, incubated for 5 minutes (temperature was 15°C), and the fluorescence signal F was measured to calculate the fluorescence responsiveness ratio (blank sample was measured at the same time). Recovery rate = (sample recovery test value - blank recovery test value) / standard test value * 100%, calculate the recovery rate, the results are shown in Table 1.

[0075] Table 1 Determination of spike recovery

[0076]

[0077] It can be seen from Table 1 that the spiked recovery of the tested samples of the present invention is between 87.49% and 113.15%, which proves the accuracy and reliability of the method.

[0078] The present invention and its embodiments are described schematically above, and the description is not restrictive. The drawings show only one embodiment of the present invention, and the actual structure is not limited thereto. Therefore, if a person skilled in the art is inspired by it and designs a structural method and an embodiment similar to the technical solution without creativity without departing from the purpose of the invention, they shall all fall within the protection scope of the present invention.

Claims

1. A method for preparing a fluorescent paper chip for detecting Staphylococcus aureus, characterized in that: The following steps are involved: Step 1. Preparation of fluorescent molecularly imprinted nanoparticles: Staphylococcus aureus was dispersed in a phosphate buffer solution at pH 7.4, and functional monomers and fluorescent monomers were added for pre-assembly; after being evenly mixed, a cross-linking agent was added and stirred for 12 h. After the reaction was completed, the nanoparticles were first washed with a phosphate buffer solution, and then amino groups were modified on the surface of the nanoparticles. Finally, the templates were removed by washing with a phosphate buffer solution and a methanol / water co-solvent of acetic acid, respectively, to obtain fluorescent molecularly imprinted nanoparticles; Step 2. Immobilization of fluorescent molecularly imprinted nanoparticles on the surface of paper chip: Taking advantage of the abundant hydroxyl groups on the surface of the paper chip, the amino group and the aldehyde group were modified on the surface of the paper chip in sequence. The paper chip was then immersed in the nanoparticle solution prepared in step 1 for 12 h to obtain a fluorescent paper chip for detecting Staphylococcus aureus.

2. The method for preparing a fluorescent paper chip for detecting Staphylococcus aureus according to claim 1, characterized in that: The functional monomer in step 1 is obtained by reacting 3-aminopropyltriethoxysilane with 3-formylphenylboronic acid, and the fluorescent monomer is obtained by reacting 3-aminopropyltriethoxysilane with fluorescein isothiocyanate.

3. The method for preparing a fluorescent paper chip for detecting Staphylococcus aureus according to claim 1, characterized in that: In step 1, the concentration of the functional monomer is 4.2 mmol / L, the concentration of the fluorescent monomer is 0.2 mmol / L, and the concentration of Staphylococcus aureus is 0.5*10 9 CFU / mL.

4. The method for preparing a fluorescent paper chip for detecting Staphylococcus aureus according to claim 1, characterized in that: In the step 1, the volume ratio of the functional monomer to the fluorescent monomer is 1:

1.

5. The method for preparing a fluorescent paper chip for detecting Staphylococcus aureus according to claim 1, characterized in that: In the step 1, the cross-linking agent is tetraethoxysilane, and the concentration is 351 mmol / L.

6. The method for preparing a fluorescent paper chip for detecting Staphylococcus aureus according to claim 1, characterized in that: The concentration of the fluorescent nanoparticles in step 2 was 0.1 mg / mL.

7. Use of the fluorescent paper chip for detecting Staphylococcus aureus according to claim 1 in quantitatively detecting Staphylococcus aureus in food testing.

8. The use according to claim 7, characterized in that: The quantitative detection and analysis steps are as follows: first, the initial fluorescence value of the fluorescent paper chip is determined by using a Jingxin microarray chip scanner, the sample to be tested is dripped on the surface of the paper chip, and after incubation for a period of time, its fluorescence signal value is determined, and a standard curve is established according to the fluorescence quenching rate, so as to perform quantitative analysis on Staphylococcus aureus.

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