Rapid staphylococcus aureus detection method based on nano dual-fluorescent probe
Through the ratiometric fluorescence sensing method of carbon dots and silicon nanoparticles, vancomycin and specific antibodies are used to identify Staphylococcus aureus, which solves the problems of high cost, cumbersome operation and insufficient anti-interference ability of existing detection methods, and achieves rapid and accurate detection results.
Patent Information
- Application Number
- CN202510753796.6
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-06-06
- Publication Date
- 2025-09-19
AI Technical Summary
Existing Staphylococcus aureus detection methods are costly, cumbersome to operate, have long detection cycles, and lack anti-interference capabilities for complex samples, making it difficult to meet the needs of rapid detection and large-scale screening.
A ratiometric fluorescence sensing method using carbon dots and silicon nanoparticles was adopted, and vancomycin and specific antibodies were used to achieve efficient and specific recognition of Staphylococcus aureus. A rapid detection system was constructed by measuring the signal ratio of the two fluorescent materials.
It simplifies the detection process, reduces costs, improves the convenience and accuracy of detection, enhances the selectivity and accuracy in complex food matrices, and reduces errors caused by environmental interference.
Smart Images
Figure CN120668620A_ABST
Abstract
Description
Technical Field
[0001] The present invention belongs to the technical field of food detection, and in particular relates to a method for rapid detection of Staphylococcus aureus in milk based on a nano dual fluorescent probe. Background Art
[0002] my country is a major producer and consumer of dairy products in the world, and milk and its products occupy an important position in the national diet. With the continuous expansion of livestock farming and the improvement of modern production levels, milk production and supply capacity continue to increase. However, dairy products are extremely susceptible to contamination by harmful microorganisms such as Staphylococcus aureus during production, transportation, and storage, which not only affects the edible quality of milk but also poses a threat to the health of consumers. Staphylococcus aureus (S. aureus), a common foodborne pathogen with strong pathogenicity and drug resistance, has become a difficult problem that needs to be solved urgently in the field of food safety.
[0003] Currently, detection methods for Staphylococcus aureus primarily include lateral flow chromatography, polymerase chain reaction, and electrochemical analysis. While these methods offer advantages such as strong specificity and high sensitivity, they suffer from high costs, long cycles, and cumbersome procedures, making them difficult to meet the demands of rapid on-site testing and large-scale sample screening. Therefore, developing a simple, sensitive, accurate, and low-cost method for rapid detection of Staphylococcus aureus is of great practical significance.
[0004] Fluorescence-based biosensing methods have attracted widespread attention in recent years due to their advantages, including rapid detection, high sensitivity, and ease of operation. Ratiometric fluorescence sensing strategies, in particular, effectively mitigate interference from food matrices and environmental factors by measuring the ratio of the signals from two fluorescent materials, significantly improving detection accuracy and stability. However, most traditional fluorescent probes require complex synthesis steps, cumbersome material preparation processes, and limited anti-interference capabilities in complex biological samples, hindering their practical application and widespread adoption.
[0005] Therefore, how to select nanomaterials with simple structure, easy functionalization and excellent fluorescence properties to construct a ratiometric fluorescent biosensor system with high sensitivity, high specificity and strong anti-interference ability to achieve efficient detection of Staphylococcus aureus in complex samples has become an important research direction in the field of food safety testing. Summary of the Invention
[0006] To address the challenges of existing Staphylococcus aureus detection technologies, including high costs, complex procedures, long detection cycles, and insufficient anti-interference capabilities for complex samples, this paper proposes a ratiometric fluorescence sensing method based on carbon dots (CDs) and silicon nanoparticles (SiNPs), enabling rapid, sensitive, and low-cost detection of Staphylococcus aureus in food. This method leverages the synergistic effect of two different nanomaterials to achieve efficient and specific identification of the target bacteria and fluorescent signal output, significantly improving the sensitivity and stability of the detection system.
[0007] In order to achieve the above-mentioned object of the invention, the specific technical solutions of the present invention are as follows:
[0008] A method for rapid detection of Staphylococcus aureus in milk comprises the following steps:
[0009] (1) Synthesis and modification of carbon dots;
[0010] S1. Weigh citric acid and urea, dissolve them in distilled water, and ultrasonically treat them to form a transparent solution. Then, microwave heat the resulting transparent solution, cool it, and then add distilled water again to form a mixed solution. The mixed solution is centrifuged (to remove large particles), and the supernatant is collected and purified using a dialysis membrane to obtain a carbon dot solution.
[0011] S2. Add EDC and NHS to the carbon dot solution, stir at room temperature, then add vancomycin solution, continue stirring at room temperature, and dialyze (to remove unreacted components) to obtain vancomycin-modified carbon dots (denoted as CD@Van);
[0012] (2) Preparation and modification of silicon nanoparticles;
[0013] APTMS (3-aminopropyltrimethoxysilane) and sodium ascorbate were mixed with distilled water, and the mixture was stirred magnetically to obtain a mixed solution. The obtained mixed solution was dialyzed (to remove impurities), and the dialyzed solution was diluted to obtain a silicon nanoparticle (SiNPS) dispersion.
[0014] First, EDC and NHS are mixed to obtain an EDC-NHS mixture; the EDC-NHS mixture is added to the obtained nanoparticle dispersion, and a S. aureus specific antibody solution is added, and after stirring and reacting, antibody-modified silicon nanoparticles (SiNPS-Ab) are obtained;
[0015] (3) Prepare a standard solution of Staphylococcus aureus; the concentration of the solution is 0 to 2×10 6 CFU / mL;
[0016] (4) Construction of ratiometric fluorescence sensing system;
[0017] The vancomycin-modified carbon dots of step (1) are mixed with the antibody-modified silicon nanoparticles of step (2), and then the Staphylococcus aureus standard solution of step (3) is added. After incubation, a reaction system is obtained, and the fluorescence intensity of the reaction system at 492 nm and 529 nm is measured, which are recorded as F492 and F529, respectively; then, the ratio of F492 to F529 is calculated, and a standard curve is established corresponding to the concentration of the Staphylococcus aureus standard solution;
[0018] (5) Rapid detection and quantification of Staphylococcus aureus;
[0019] S1: Preparation of the sample to be tested: grind or blend the sample, add PBS buffer, and shake thoroughly to mix to prepare the sample to be tested.
[0020] S2: Follow the operation of step (4), except that the Staphylococcus aureus standard solution is replaced by the test sample obtained in S1, and the final ratio F492 / F529 is substituted into the standard curve established in step (4), thereby realizing the detection of Staphylococcus aureus in the milk sample.
[0021] Preferably, the amount of citric acid, urea, distilled water, and distilled water added again in S1 of step (1) is 3 g:3 g:10 mL:20 mL, the ultrasonic treatment time is 10 minutes, the microwave heating power is 500 W to 1000 W, and the reaction time is 5-15 minutes (react until the solution changes from transparent to yellow); the centrifugation condition is: 5000 rpm for 5 minutes; the molecular weight cutoff of the dialysis membrane is 3000 Da, and the purification time is 12-36 hours;
[0022] The dosage of EDC, NHS, carbon dot solution, and vancomycin solution in S2 is 10 mg:10 mg:2 mL:2 mL, wherein the concentration of the vancomycin solution is 10 mg / mL. The stirring time at room temperature is 4 hours, and the stirring reaction time is continued for 6 hours. The dialysis uses a dialysis membrane with a molecular weight cutoff of 3000 Da, and the time is 12-36 hours.
[0023] Preferably, the amount of APTMS, sodium ascorbate solution and distilled water in step (2) is 2 mL: 2 mL: 16 mL, wherein the concentration of sodium ascorbate solution is 100 mM; the magnetic stirring reaction time is 60 min; the dialysis uses a dialysis membrane with a molecular weight cutoff of 3000 Da, and the time is 12-36 h; the dilution multiple is 20 times;
[0024] The volume ratio of the EDC to NHS mixture is 1:1, wherein the concentration of EDC is 10 mM and the concentration of NHS is 5 mM; the dosage of the nanoparticle dispersion, EDC-NHS mixture, and S. aureus specific antibody solution is 1 mL: 0.5 mL: 40 μL, wherein the concentration of the S. aureus specific antibody solution is 0-2×10 6 CFU / mL, and the stirring reaction time was 3 h.
[0025] Preferably, in step (4), the volume ratio of vancomycin-modified carbon dots, antibody-modified silicon nanoparticles, and Staphylococcus aureus standard solution is 1:1:1; the incubation temperature is 37° C., and the time is 30-60 minutes.
[0026] Preferably, the sample in S1 of step (5) includes milk, cheese, or milk powder. If the sample itself is in a solution or powder state, it does not need to be crushed or pulverized; the amount of the sample and the PBS buffer solution is 2 g:5 mL.
[0027] Beneficial effects of the present invention:
[0028] The present invention utilizes the ratiometric dual fluorescence signal characteristics of carbon dots and silicon nanoparticles for the rapid detection of Staphylococcus aureus in milk, which simplifies the construction process of the fluorescent biosensor, effectively reduces the detection cost, and improves the convenience and efficiency of the detection. The present invention introduces vancomycin and specific antibodies as recognition elements to achieve high-specificity recognition of Staphylococcus aureus, significantly enhancing the selectivity and accuracy of the sensor in complex food matrices. At the same time, the ratiometric fluorescence detection strategy significantly reduces the detection error caused by environmental interference and insufficient system stability, and improves the reliability and repeatability of the test results. Overall, the present invention provides a new technical means for the rapid, accurate and efficient detection of Staphylococcus aureus in foods such as dairy products.
[0029] The detection method provided by the present invention has the advantages of simple material synthesis, low cost, simple operation steps, fast detection speed, high sensitivity, and strong anti-interference ability. It can meet the needs of rapid screening and quantitative detection of Staphylococcus aureus in foods such as dairy products. BRIEF DESCRIPTION OF THE DRAWINGS
[0030] Figure 1 Constructed as a ratiometric fluorescence biosensor.
[0031] Figure 2 TEM image of CD@Van.
[0032] Figure 3 F 492nm / F 529nm Linear fitting curve. Specific implementation plan
[0033] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.
[0034] It should be understood that the terms described herein are intended only to describe particular embodiments and are not intended to limit the present invention. In addition, for numerical ranges herein, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Each smaller range between any intermediate value within a stated value or stated range and any other stated value or intermediate value within the stated range is also encompassed by the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded within the scope.
[0035] Unless otherwise indicated, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein may also be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of any conflict with any incorporated document, the contents of this specification shall prevail.
[0036] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments described herein without departing from the scope or spirit of the invention. Other embodiments will be apparent to those skilled in the art from the description of the invention. The description and examples are intended to be illustrative only.
[0037] The N-hydroxysuccinimide (NHS) and vancomycin (C 66 H 75 Cl2N9O 24 ; Van), (3-aminopropyl)trimethoxysilane (C6H 17 NO₃Si; APTMS), citric acid (C₆H₂O₇), urea (CH₄N₂O), 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC), and sodium ascorbate (C₆H₂O₆Na) were purchased from MACKLIN (Shanghai, China); LB broth and PBS buffer were from Thermo Fisher Scientific (Suzhou, China). All reagents used in the experiments were of analytical grade and were used directly. Ultrapure water was used as the solvent for all dilutions and preparations.
[0038] S. aureus-specific antibody solution and 20× Tris-buffered saline (TBS buffer) were purchased from Biosun Biotechnology Co., Ltd. (Beijing, China). S. aureus standard bacteria were purchased from Ningbo Mingzhou Biotechnology Co., Ltd. (Ningbo, China). Milk samples used in the actual sample testing were purchased from Kaiyuan Supermarket, Jiangsu University (Zhenjiang, China). Nutrient agar medium was purchased from Haibo Biotechnology (Qingdao, China).
[0039] Example 1:
[0040] A ratiometric fluorescence biosensor for rapid detection of Staphylococcus aureus in milk. Figure 1 As shown, the specific steps include:
[0041] (1) Synthesis of carbon dots and vancomycin modification
[0042] S1. Dissolve 3g of citric acid and 3g of urea in 10mL of distilled water and thoroughly mix them by ultrasonic treatment for 10 minutes to obtain a clear solution. Heat the clear solution in a microwave oven at 600W until the solution turns from transparent to yellow (5 minutes). Cool the solution to room temperature, then add 20mL of distilled water to dissolve the mixture. Transfer the mixture to a centrifuge tube and centrifuge at 5000rpm for 5 minutes to remove large particles. Collect the supernatant. The supernatant is then placed in a dialysis bag with a molecular weight cutoff of 3000Da and dialyzed against distilled water for 36 hours, changing the dialysate three times, to obtain a purified carbon dot (CD) solution.
[0043] S2. To 2 mL of the CDs solution, 10 mg each of EDC and NHS were added and stirred at room temperature for 4 hours to activate the carboxyl groups on the CDs surface. Then, 2 mL of a 10 mg / mL vancomycin solution was added and stirred for another 6 hours to ensure a complete reaction. After the reaction, the resulting mixture was placed in a dialysis bag with a molecular weight cutoff of 3000 Da and dialyzed against distilled water for 36 hours. The dialysate was changed three times during the dialysis process to remove unreacted products and small molecule impurities. Vancomycin-modified carbon dots, designated CD@Van, were obtained. Figure 2 This is the TEM image of CD@Van. It can be observed from the figure that CDs are round particles with a particle diameter of less than 10 nm, indicating that the prepared CD@Van has good dispersibility and nanoscale, providing a good structural basis for its subsequent applications.
[0044] (2) Silicon nanoparticles and antibody modification
[0045] S1. 2 mL of APTMS, 2 mL of sodium ascorbate solution (100 mM concentration) and 16 mL of distilled water were mixed and magnetically stirred for 60 minutes to obtain a mixed solution (orange-yellow). The mixed solution was placed in a dialysis bag with a molecular weight cutoff of 3000 Da and dialyzed to remove impurities. The dialyzed solution was diluted 20-fold to obtain a silicon nanoparticle dispersion, referred to as SiNPS solution.
[0046] S2. First, EDC (concentration 10 mM) and NHS (concentration 5 mM) were mixed in equal volumes to obtain an EDC-NHS mixture. Then, 1 mL of SiNPS solution was added to 0.5 mL of the EDC-NHS mixture and magnetically stirred for 30 minutes. Subsequently, 40 μL of S. aureus-specific antibody solution was added and stirring was continued for 3 hours to obtain antibody-modified silica nanoparticles, designated SiNPS-Ab.
[0047] (3) Preparation of Staphylococcus aureus standard sample
[0048] Prepare Staphylococcus aureus standard solutions of different concentrations as required, with concentrations ranging from 0, 200, and 2×10 3 , 2×10 4 , 2×10 5 , 2×10 6 CFU / mL.
[0049] (4) Construction of a ratiometric fluorescence reaction system
[0050] 1 mL of CD@Van and 1 mL of SiNPS-Ab were respectively mixed evenly; then 1 mL of Staphylococcus aureus standard solution was added and incubated at 37°C for 50 minutes to obtain a reaction system.
[0051] The fluorescence intensity of the reaction system at 492 nm and 529 nm was measured using a fluorescence spectrophotometer at an excitation wavelength of 420 nm, and recorded as F492 and F529, respectively; the ratio of F492 to F529 was then calculated;
[0052] The fitting curve was constructed by the logarithmic relationship between the fluorescence ratio F492 nm / F529 nm of each standard sample and the concentration of Staphylococcus aureus ( Figure 3 ), the equation after fitting is y = -1.28×10 -3 x+0.84722, correlation coefficient R 2 =0.9806. F 492nm / F 529nmA good linear relationship exists between the fluorescence signal and the logarithm of the bacterial concentration, providing a reliable method for the quantitative detection of S. aureus in real samples. The fluorescence signal of the milk sample to be tested was measured according to the above detection system steps and substituted into the standard curve to quickly predict the content of S. aureus in milk.
[0053] (5) Actual testing:
[0054] S1: Take 2.0 g of milk sample in a 10 mL centrifuge tube, add 5 mL of PBS buffer, and shake thoroughly to mix to prepare the sample to be tested.
[0055] S2: Follow the operation of step (4), except that the Staphylococcus aureus standard solution is replaced by the test sample obtained in S1, and the final ratio F492 / F529 is substituted into the standard curve established in step (4) to calculate the actual content. The results are shown in Table 1.
[0056] Each sample was run in triplicate to ensure reliable results. The fluorescence signal ratio after the detection reaction decreased with increasing S. aureus concentration. As the concentration of S. aureus in milk increased, the binding of S. aureus to vancomycin and the antibody increased, and the fluorescence signal ratio in the system gradually decreased. Calculated spike recoveries ranged from 94.0% to 102.9%, with relative standard deviations less than 5%. These results demonstrate that the constructed ratiometric fluorescence sensor has good accuracy and application value for detecting S. aureus in milk and other food samples.
[0057] Table 1 Detection of S. aureus in milk samples
[0058]
[0059] Note: The above embodiments are only used to illustrate the present invention and are not intended to limit the technical solutions described in the present invention. Therefore, although this specification has described the present invention in detail with reference to the above embodiments, it should be understood by those skilled in the art that the present invention may still be modified or replaced by equivalents. All technical solutions and improvements that do not depart from the spirit and scope of the present invention should be included within the scope of the claims of the present invention.
Claims
1. A method for rapid detection of Staphylococcus aureus based on nano dual fluorescent probes, characterized in that: The following steps are involved: (1) Synthesis and modification of carbon dots; S1. Weigh citric acid and urea, dissolve them in distilled water, and ultrasonically treat them to form a transparent solution; then microwave-heat the resulting transparent solution, cool it, and then add distilled water again to form a mixed solution. The mixed solution is centrifuged, the supernatant is collected, and then purified using a dialysis membrane to obtain a carbon dot solution; S2. Add EDC and NHS to the carbon dot solution, stir at room temperature, then add vancomycin solution, continue stirring at room temperature, and dialyze to obtain vancomycin-modified carbon dots. (2) Preparation and modification of silicon nanoparticles; APTMS and sodium ascorbate were mixed with distilled water, and the mixture was stirred magnetically to obtain a mixed solution, and the mixed solution was dialyzed, and the dialyzed solution was diluted to obtain a silicon nanoparticle dispersion. First, EDC and NHS are mixed to obtain an EDC-NHS mixed solution; the EDC-NHS mixed solution is added to the obtained nanoparticle dispersion, and a S. aureus specific antibody solution is added, and after stirring and reacting, antibody-modified silicon nanoparticles are obtained; (3) Prepare a standard solution of Staphylococcus aureus for later use; (4) Construction of ratiometric fluorescence sensing system; The vancomycin-modified carbon dots of step (1) are mixed with the antibody-modified silicon nanoparticles of step (2), and then the Staphylococcus aureus standard solution of step (3) is added. After incubation, a reaction system is obtained, and the fluorescence intensity of the reaction system at 492 nm and 529 nm is measured, which are recorded as F492 and F529, respectively; then, the ratio of F492 to F529 is calculated, and a standard curve is established corresponding to the concentration of the Staphylococcus aureus standard solution; (5) Rapid detection and quantification of Staphylococcus aureus; S1: Preparation of the sample to be tested: grind or blend the sample, add PBS buffer, and shake thoroughly to mix to prepare the sample to be tested. S2: Follow the operation of step (4), except that the Staphylococcus aureus standard solution is replaced by the test sample obtained in S1, and the final ratio F492 / F529 is substituted into the standard curve established in step (4), thereby realizing the detection of Staphylococcus aureus in the milk sample.
2. The method for rapid detection of Staphylococcus aureus based on nano dual fluorescent probes according to claim 1, characterized in that: The amount of citric acid, urea, distilled water, and distilled water added again in S1 of step (1) is 3 g:3 g:10 mL:20 mL, the ultrasonic treatment time is 10 minutes, the microwave heating power is 500 W to 1000 W, and the reaction time is 5-15 minutes; the centrifugation conditions are: 5000 rpm for 5 minutes; the molecular weight cutoff of the dialysis membrane is 3000 Da, and the purification time is 12-36 hours.
3. The method for rapid detection of Staphylococcus aureus based on nano dual fluorescent probes according to claim 1, characterized in that: In step (1), the dosage of EDC, NHS, carbon dot solution, and vancomycin solution in S2 is 10 mg:10 mg:2 mL:2 mL, wherein the concentration of the vancomycin solution is 10 mg / mL, the stirring time at room temperature is 4 hours, and the stirring reaction time is continued for 6 hours; the dialysis uses a dialysis membrane with a molecular weight cutoff of 3000 Da, and the time is 12-36 hours.
4. The method for rapid detection of Staphylococcus aureus based on nano dual fluorescent probes according to claim 3, characterized in that: In step (2), the dosage of APTMS, sodium ascorbate solution, and distilled water is 2 mL: 2 mL: 16 mL, wherein the concentration of the sodium ascorbate solution is 100 mM; the magnetic stirring reaction time is 60 min; the dialysis uses a dialysis membrane with a molecular weight cutoff of 3000 Da, and the time is 12-36 h; and the dilution multiple is 20 times.
5. The method for rapid detection of Staphylococcus aureus based on nano dual fluorescent probes according to claim 4, characterized in that: In step (2), the volume ratio of EDC to NHS is 1:1, wherein the concentration of EDC is 10 mM and the concentration of NHS is 5 mM; the dosage of the nanoparticle dispersion, EDC-NHS mixed solution, and S. aureus specific antibody solution is 1 mL: 0.5 mL: 40 μL, wherein the concentration of the S. aureus specific antibody solution is 0-2×10 6 CFU / mL, and the stirring reaction time was 3 h.
6. The method for rapid detection of Staphylococcus aureus based on nano dual fluorescent probes according to claim 1, characterized in that: The concentration of the Staphylococcus aureus standard solution in step 3 is 0 to 2×10 6 CFU / mL.
7. The method for rapid detection of Staphylococcus aureus based on nano dual fluorescent probes according to claim 1, characterized in that: In step (4), the volume ratio of vancomycin-modified carbon dots, antibody-modified silicon nanoparticles and Staphylococcus aureus standard solution is 1:1:1; the incubation temperature is 37° C. and the time is 30-60 minutes.
8. The method for rapid detection of Staphylococcus aureus based on nano dual fluorescent probes according to claim 1, characterized in that: The sample described in S1 of step (5) includes milk, cheese, and milk powder. If the sample itself is in a solution or powder state, it does not need to be crushed or stirred; the amount of sample and PBS buffer solution is 2g:5mL.