NL2033865B1 - Method for detecting salmonella typhimurium by fluorescent probe based on nitrogen and sulfur co-doped graphene quantum dots - Google Patents

Method for detecting salmonella typhimurium by fluorescent probe based on nitrogen and sulfur co-doped graphene quantum dots Download PDF

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NL2033865B1
NL2033865B1 NL2033865A NL2033865A NL2033865B1 NL 2033865 B1 NL2033865 B1 NL 2033865B1 NL 2033865 A NL2033865 A NL 2033865A NL 2033865 A NL2033865 A NL 2033865A NL 2033865 B1 NL2033865 B1 NL 2033865B1
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Cao Lulu
Zhang Danfeng
Ren Yuwei
Ye Yingwang
Ling Na
Shen Yizhong
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Univ Hefei Technology
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Abstract

Disclosed is a method for detecting Salmonella typhimurium by a fluorescent probe based on nitrogen and sulphur co-doped graphene quantum dots, belonging to the technical field of food safety. Based on the sandwich method strategy, the invention modifies streptavidin magnetic beads and nitrogen and sulphur co-doped graphene quantum dots with aptamers, using them as magnetic separation probes and signal probes, respectively; when the detected substance contains Salmonella typhimurium, the signal probe and the magnetic separation probe synchronously and specifically identify Salmonella typhimurium to form a sandwich structure. The sandwich structure is adsorbed by magnetic separation, and the supernatant is taken, and then its fluorescence intensity at 423 nm is measured to determine the concentration of Salmonella typhimurium in the detected solution. The method realizes synchronous identification of Salmonella typhimurium, is simple and convenient to operate, reduces the influence of sample matrix, has low cost, and has stable fluorescence characteristics; and as nitrogen and sulphur elements are doped in graphene quantum dots, the fluorescence quantum yield of graphene quantum dots is effectively improved, and the detection sensitivity is increased to 11.9 cfu/ml, so that the method has a good application and popularization prospect.

Description

METHOD FOR DETECTING SALMONELLA TYPHIMURIUM BY FLUORESCENT PROBE
BASED ON NITROGEN AND SULFUR CO-DOPED GRAPHENE QUANTUM DOTS
TECHNICAL FIELD
The invention belongs to the technical field of food safety, and in particular to a method for detecting Salmonella typhimurium by a fluorescent probe based on nitrogen and sulphur co- doped graphene quantum dots.
BACKGROUND
Salmonella typhimurium is one of the main pathogens causing acute gastroenteritis worldwide, and its infection symptoms usually include fever, nausea, vomiting and diarrhoea.
Ingesting contaminated dairy products, eggs, meat and poultry is the main way of infection.
Dairy products are a kind of food with rich nutrition and complex contents. However, because they contain protein, carbohydrates and fat, they not only provide important nutrition for human beings, but also provide living conditions for Salmonella typhimurium. Therefore, it is of great significance to establish a method for detecting Salmonella typhimurium in dairy products.
At present, Salmonella typhimurium is mainly detected by traditional detection technique, molecular biological technique and immunological detection technology. Traditional detection technology is regarded as the gold standard of pathogenic bacteria detection because of its high accuracy. However, the traditional culture method is complicated in operation, the detection period is about 5-7 days, and professionals are needed, which can't satisfy the rapid screening and risk detection of Salmonella typhimurium in dairy products. Molecular biological technique includes polymerase chain reaction (PCR), recombinase polymerase amplification (RPA), loop-mediated isothermal amplification (LAMP) and rolling circle amplification (RCA), etc. which can't meet the needs of on-site detection due to the specific primer design, complicated nucleic acid amplification procedures and expensive instruments and equipment.
Immunological detection technology is mainly based on the rapid screening of antigen-antibody recognition technology, which has the advantages of simplicity, rapidness, high sensitivity, high throughput and high specificity. However, the quality of antibodies prepared in different batches is quite different, and the antibodies are expensive and their activity is easily affected by external factors, which limits their popularization and application.
In recent years, optical detection strategy has become the trend of rapid detection.
Traditional organic fluorescent dyes, such as carboxyfluorescein (FAM) and Rhodamine B, are widely used as signal markers in fluorescence detection strategies, but their further popularization is limited by poor fluorescence stability and narrow excitation spectrum.
Therefore, it is urgent to develop efficient and stable fluorescent nanoparticles. As a new nanomaterial, graphene quantum dots have excellent characteristics such as easy synthesis, stable fluorescence characteristics, broad-range light spectrum excitation and response, and good biocompatibility, and have achieved good results in many detection methods. Compared with graphene quantum dots, the doping of nitrogen and sulphur significantly changes the surface properties of graphene quantum dots, and further improves the absolute fluorescence quantum yield and photostability of graphene quantum dots.
The serious interference of milk matrix to the rapid detection strategy will significantly reduce the detection sensitivity and reliability. At present, although the pre-concentration of samples by centrifugation and the microfluidic method of adsorbing and decomposing interfering substances by embedding corresponding molecules in channels can reduce the interference of milk matrix, immunomagnetic separation technology is still the mainstream method to get rid of the interference of milk matrix because of its simple operation and high separation efficiency.
In addition, in view of the limitations of antibody, such as large quality differences between different batches during preparation, high price, and easy to be affected by external factors, aptamer, as a nucleic acid sequence that can specifically bind to the target substance, has successfully replaced antibody for specific and efficient recognition of the target substance due to its advantages of easy synthesis and labelling, low production cost, high stability, and strong specificity. Therefore, by modifying the magnetic beads on the aptamer as a means to get rid of the interference of milk matrix, and combining them with nitrogen and sulphur co-doped graphene quantum dots modified by aptamers with excellent luminescent properties and high- efficiency recognition characteristics, a new simple and highly sensitive technique for rapid detecting Salmonella typhimurium in milk matrix has been provided, and this technique has not been reported.
SUMMARY
In order to realize simple, highly sensitive and rapid detection of Salmonella typhimurium in milk matrix, the invention provides a method for detecting Salmonella typhimurium by a fluorescent probe based on nitrogen and sulphur co-doped graphene quantum dots.
A method for detecting Salmonella typhimurium by a fluorescent probe based on nitrogen and sulphur co-doped graphene quantum dots: the signal probe solution is made of nitrogen and sulphur co-doped graphene quantum dots modified by the amino-functionalized aptamer (1); the DNA sequence of the amino- functionalized aptamer (1) is: 5'-NH2-(CH2)s-TAT GGC GGC GTC ACC CGA CGG GGA CTT
GAC ATT ATG ACA G-3' (SEQ ID NO: 1); the magnetic separation probe solution is made of streptavidin magnetic beads modified by the biotinylated aptamer (2); the DNA sequence of the biotinylated aptamer (2) is 5'-Biotin-GAG
GAA AGT CTA TAG CAG AGG AGA TGT GTG AAC CGA GTA A-3' (SEQ ID NO: 2); the standard curve: the abscissa X is the logarithmic value of Salmonella typhimurium concentration (cfu/ml), and the ordinate Y is lg-I, where lg is the fluorescence intensity at 423 nm when blank control solution is detected, and | is the fluorescence intensity at 423 nm when 102, 103, 10%, 105, 108 and 107 cfu/ml Salmonella typhimurium solutions are detected; the specific detection operation steps are as follows: (1) preparing of liquid to be detected taking 1 ml of liquid milk, centrifuging at 8000 x g for 5 min, discarding the supernatant, resuspending the precipitate with 1 ml of sterile PBS buffer with a concentration of 0.1 M and a pH of 7.4 to obtain the solution to be detected; (2) detecting (2.1) adding 100 HI signal probe solution and 36 pl magnetic separation probe solution into 500 pl solution to be detected, incubating at 37°C for 45 min to obtain the composite solution; (2.2) placing the composite solution on a magnetic frame for magnetic separation, and taking supernatant to obtain the solution to be detected; (2.3) putting the solution to be detected in a quartz colorimetric cuvette, setting the excitation wavelength of the F97Pro fluorescence spectrophotometer to 349 nm, and measuring the fluorescence intensity of the solution to be detected at 423 nm; (3) calculating the detected results substituting lo-I value into the standard curve, and calculating the concentration of
Salmonella typhimurium in the solution to be detected, that is, completing the detection; where
Io is the fluorescence intensity at 423 nm when the blank control solution is detected, and | is the fluorescence intensity at 423 nm when the solution to be detected is detected; when the lo-l value is greater than 104, it indicates that Salmonella typhimurium is detected in the detected substance; when the lg-l value is less than 104, it indicates that Salmonella typhimurium is not detected in the detected substance.
The further technical scheme is as follows:
The operation steps for preparing the signal probe solution are as follows: (1) adding 0.1 ml of nitrogen and sulphur co-doped graphene quantum dot stock solution into 9.9ml of ultrapure water to obtain 10 ml of nitrogen and sulphur co-doped graphene quantum dot aqueous solution; (2) adding 5 mg of N-hydroxysuccinimide and 10 mg of 1-(3-dimethylaminopropyl)-3- ethylcarbodiimide hydrochloride into 10 ml of nitrogen and sulphur co-doped graphene quantum dot aqueous solution, adjusting the pH value to 5.0 with 10 mg/ml of sodium hydroxide (NaOH) solution, and incubating in the dark at room temperature for 30 min to obtain activated nitrogen and sulphur co-doped graphene quantum dot aqueous solution; (3) taking the activated nitrogen and sulphur co-doped graphene quantum dot aqueous solution, adjusting the pH value to 7.4 with 10 mg/ml sodium hydroxide (NaOH) solution, adding 20 pl of 100 uM amino-functionalized aptamer (1) solution, and suspending at room temperature for 2 h to obtain signal probe solution.
The operation steps for preparing the nitrogen and sulphur co-doped graphene quantum dot stock solution are as follows: (1) uniformly mixing 1.0 g citric acid with 0.3 g L-cysteine to obtain a reaction mixture; (2) heating the reaction mixture to 200°C in an oil bath pot to liquefy it, stopping heating when the colour of the reaction mixture liquid changes from light yellow to orange, and naturally cooling to room temperature to obtain an orange product; (3) dissolving the orange product in 100 ml of ultrapure water to obtain the nitrogen and sulphur co-doped graphene quantum dot stock solution.
The operation steps for preparing the magnetic separation probe solution are as follows: (1) taking 18 HI streptavidin magnetic beads with a concentration of 10 mg/ml in a siliconized centrifuge tube, adding 72 HI of 1 x B&W buffer solution, mixing them evenly, and putting them on the magnetic frame to stand for 1min, and discarding the supernatant after the streptavidin magnetic beads are fully adsorbed on the wall of the centrifuge tube; then, adding 72 ul of 1 x B&W buffer to repeat the above operation to obtain the treated streptavidin magnetic beads; (2) resuspending the treated streptavidin magnetic beads in 36 pl of 2 x B&W buffer, adding 36 pl of biotinylated aptamer (2) solution with a concentration of 1.5 uM, and incubating at room temperature for 30 min to obtain a coupling solution; (3) placing the coupling solution on the magnetic frame, standing for 1min, discarding the supernatant, adding 50 pl of 1% bovine serum albumin (BSA) solution, and incubating at room temperature for 30 min to obtain a closed coupling solution; (4) putting the closed coupling solution on the magnetic frame, standing for 1min, discarding the supernatant, and resuspending the precipitate in 36 ul of sterile PBS buffer with a concentration of 0.1 M and a pH value of 7.4 to obtain a magnetic separation probe solution.
The 1 x B&W buffer solution is obtained by diluting 2 x B&W buffer solution with equal volume of ultrapure water; the operation steps for preparing the 2 x B&W buffer solution are as follows: weighing 0.1211 g tris-(hydroxymethyl)-aminomethane (Tris}, 11.7 g sodium chloride (NaCl) and 0.03729 ethylenediaminetetraacetic acid (EDTA), adding deionized water, fully stirring and dissolving, adjusting the pH value to 7.5 with 36.5 mg/ml hydrochloric acid (HCI) solution, stirring evenly, fixing the volume to 100 ml, autoclaving at 121°C for 15 min, cooling to obtain 2 x B&W buffer, and storing in the refrigerator at 4°C for later use.
The detection and analysis principle of the method provided by the invention is as follows:
Based on the sandwich method strategy, nitrogen and sulphur co-doped graphene quantum dots modified by the amino-functionalized aptamer (1) are used as signal probes, and the biotinylated aptamer (2) is coupled to streptavidin magnetic beads to make magnetic separation probes. When the detected substance contains Salmonella typhimurium, the signal probe and the magnetic separation probe synchronously and specifically identify Salmonella typhimurium to form a sandwich structure. The sandwich structure is adsorbed by magnetic separation, and the supernatant is taken, and then its fluorescence intensity at 423 nm is measured to determine the concentration of Salmonella typhimurium in the detected solution.
Therefore, the content of signal probe in the supernatant of the sandwich structure removed by 5 magnetic separation decreases with the increase of Salmonella typhimurium concentration in the system. There is a linear relationship between the logarithmic value of Salmonella typhimurium concentration and the decrease value of fluorescence intensity of the system.
Therefore, a fluorescence probe based on nitrogen and sulphur co-doped graphene quantum dots is established to detect Salmonella typhimurium.
The beneficial technical effects of the invention are as follows: (1) Compared with traditional fluorescent compounds, such as organic dyes and polymers, the nitrogen and sulphur co-doped graphene quantum dots used in the method of the invention have more advantages, because graphene quantum dots have adjustable optical properties, high water solubility, high stability and organic inertness based on quantum confinement and edge effect. Nitrogen and sulphur co-doped graphene quantum dots are prepared by hydrothermal method with citric acid and L- cysteine as raw materials. Using cysteine in the synthesis process can homogenize the surface state of graphene quantum dots by doping heteroatoms and effectively improve the fluorescence quantum yield of graphene quantum dots. On the other hand, compared with most high-performance quantum dots, which are limited by the toxicity of metal elements (such as lead, cadmium and arsenic), graphene quantum dots have the advantages of low toxicity, good biocompatibility and metabolic degradation in biological applications. (2) According to the invention, immune magnetic beads formed by binding aptamers with magnetic beads are used to specifically identify and enrich Salmonella typhimurium in the sample matrix, so that the load of identifying molecules is effectively increased, the interference of milk matrix is reduced, and the detection sensitivity and reliability are improved. (3) The method of the invention uses aptamers instead of antibodies as identification originals, the detection cost is reduced by about 75%. Meanwhile, the synthetic raw materials of nitrogen and sulphur co-doped graphene quantum dots are low in price, high in yield and high in cost performance. (4) According to the invention, two different aptamers are respectively coupled on the nitrogen and sulphur co-doped graphene quantum dots and streptavidin magnetic beads, so that the defect that the same aptamer is used in other sandwich structures to compete for binding sites in target bacteria is avoided. (5) Based on the reverse detection strategy, the invention utilizes aptamer modified streptavidin magnetic beads and nitrogen and sulphur co-doped graphene quantum dots to specifically identify Salmonella typhimurium, and converts target substances into fluorescent signals to realize quantitative detection. In the traditional quantum dots-based immunosensor, the fluorescence signal of "magnetic bead-target substances-quantum dots" immunoconjugate is directly used as the detection reading. This detection method has two obvious problems.
Firstly, due to the blocking effect of magnetic beads, the fluorescence signal of quantum dots cannot be measured effectively. This is because the volume of magnetic beads is 6 orders of magnitude larger than that of quantum dots. When quantum dots are trapped on the surface of magnetic beads to form "magnetic beads-target substances-quantum dots" conjugates, quantum dots that are blocked in space cannot be excited, and the fluorescence signals of corresponding quantum dots are lost and cannot be measured, resulting in only a part of quantum dots being excited to generate fluorescence signals. Secondly, the "magnetic beads- target substances-quantum dots" immunoconjugate needs several washing and resuspension steps, which affects the speed and accuracy of the whole method. In order to solve these problems, the supernatant on the sandwich structure of "magnetic beads-target substances- quantum dots" removed by magnetic separation is used as the signal output in the reverse detection strategy, thus avoiding the interference of magnetic beads and complicated washing steps. Compared with the traditional quantum dots-based immunosensor, the sensitivity of the invention is improved by two orders of magnitude, the detection limit of Salmonella typhimurium is 11.9 cfu/ml, and the whole analysis process can be completed within 50 minutes. (6) According to the invention, the nitrogen and sulphur co-doped graphene quantum dots are combined with magnetic beads for the first time, and the rapid detection of Salmonella typhimurium in dairy products is realized through a sandwich method and a reverse detection strategy. Where, the nitrogen and sulphur co-doped graphene quantum dots in the sandwich structure have high fluorescence emission and photobleaching resistance, which can provide efficient and stable fluorescence signals for detection; immunomagnetic beads react specifically with Salmonella typhimurium, and the target substance is separated from the complex matrix under the action of external magnetic field, which can improve the detection sensitivity and reliability. In addition, the reverse detection strategy avoids the interference of magnetic beads on fluorescent signals and complicated washing steps, and improves the accuracy and speed of the invention.
BRIEF DESCRIPTION OF THE FIGURES
FIG. 1 is a schematic diagram of the detection method of the present invention;
FIG. 2 is a fluorescence spectrum of Salmonella typhimurium with different concentrations in
Example 2 of the present invention;
FIG. 3 is a standard curve diagram of concentration logarithm of Salmonella typhimurium and fluorescence intensity drawn in Example 2 of the present invention;
FIG. 4 is a specificity diagram of the detection method in Example 4 of the present invention.
DESCRIPTION OF THE INVENTION
The following examples will further illustrate the present invention, but not limit the present invention.
Unless otherwise defined, the technical and scientific terms used in the following examples have the same meanings as commonly understood by those skilled in the art to which this invention belongs.
The test reagent consumables used in the following examples, unless otherwise specified, are conventional biochemical reagents; unless otherwise specified, the experimental methods are conventional. For the quantitative experiments in the following examples, three repeated experiments are set, and the results are averaged; % in the following examples, unless otherwise specified, are percentages by mass.
In the following examples, streptavidin magnetic beads and the magnetic frame are purchased from Beyotime Biotech. Inc. (Beyotime); citric acid, L-cysteine, bovine serum albumin, PBS buffer with a concentration of 0.1 M and pH value of 7.4 and amino- functionalized/biotinylated nucleic acid aptamer are purchased from Sangon Biotech (Shanghai)
Co., Ltd.; N-hydroxysuccinimide and 1-(3- dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride are purchased from Sigma-Aldrich, Inc., United States.
Example 1
The preparations of nitrogen and sulphur co-doped graphene quantum dot stock solution, signal probe solution and magnetic separation probe solution used in the detection method of the invention. (1) The operation steps for preparing the nitrogen and sulphur co-doped graphene quantum dot stock solution are as follows: (1.1) uniformly mixing 1.0 g citric acid with 0.3 g L-cysteine to obtain a reaction mixture; (1.2) heating the reaction mixture to 200°C in an oil bath pot to liquefy it, stopping heating when the colour of the reaction mixture liquid changes from light yellow to orange, and naturally cooling to room temperature to obtain an orange product; (1.3) dissolving the orange product in 100 ml of ultrapure water to obtain the nitrogen and sulphur co-doped graphene quantum dot stock solution. (2) The operation steps for preparing the signal probe solution are as follows: (2.1) adding 0.1 ml of nitrogen and sulphur co-doped graphene quantum dot stock solution into 9.9ml of ultrapure water to obtain 10 ml of nitrogen and sulphur co-doped graphene quantum dot aqueous solution; (2.2) adding 5 mg of N-hydroxysuccinimide and 10 mg of 1-(3-dimethylaminopropyl)-3- ethylcarbodiimide hydrochloride into 10 ml of nitrogen and sulphur co-doped graphene quantum dot aqueous solution, adjusting the pH value to 5.0 with 10 mg/ml of sodium hydroxide (NaOH)
solution, and incubating in the dark at room temperature for 30 min to obtain activated nitrogen and sulphur co-doped graphene quantum dot aqueous solution;
(2.3) taking the activated nitrogen and sulphur co-doped graphene quantum dot aqueous solution, adjusting the pH value to 7.4 with 10 mg/ml sodium hydroxide (NaOH) solution, adding
20 plof 100 uM amino-functionalized aptamer (1) solution, and suspending at room temperature for 2 h to obtain signal probe solution;
the DNA sequence of the amino-functionalized aptamer (1) is:
5'-NH2-(CH2)s-TAT GGC GGC GTC ACC CGA CGG GGA CTT GAC ATT ATG ACA G-3'
(SEQ ID NO: 1).
(3) The operation steps for preparing the magnetic separation probe solution are as follows:
(3.1) taking 18 pl streptavidin magnetic beads with a concentration of 10 mg/ml in a siliconized centrifuge tube, adding 72 ul of 1 x B&W buffer solution, mixing them evenly, and putting them on the magnetic frame to stand for 1min, and discarding the supernatant after the streptavidin magnetic beads are fully adsorbed on the wall of the centrifuge tube; then, adding
72 Jl of 1 x B&W buffer to repeat the above operation to obtain the treated streptavidin magnetic beads;
the 1 x B&W buffer solution is obtained by diluting 2 x B&W buffer solution with equal volume of ultrapure water;
the operation steps for preparing the 2 x B&W buffer solution are as follows:
weighing 0.1211 g tris-(hydroxymethyl)-aminomethane (Tris}, 11.7 g sodium chloride (NaCl) and 0.0372 g ethylenediaminetetraacetic acid (EDTA), adding deionized water, fully stirring and dissolving, adjusting the pH value to 7.5 with 36.5 mg/ml hydrochloric acid (HCI) solution, stirring evenly, fixing the volume to 100 ml, autoclaving at 121°C for 15 min, cooling to obtain 2 x B&W buffer, and storing in the refrigerator at 4°C for later use;
(3.2) resuspending the treated streptavidin magnetic beads in 36 pl of 2 x B&W buffer, adding 36 pl of biotinylated aptamer (2) solution with a concentration of 1.5 uM, and incubating at room temperature for 30 min to obtain a coupling solution;
the DNA sequence of the biotinylated aptamer (2) is
5'-Biotin-GAG GAA AGT CTA TAG CAG AGG AGA TGT GTG AAC CGA GTA A-3'
(SEQ ID NO: 2);
(3.3) placing the coupling solution on the magnetic frame, standing for 1min, discarding the supernatant, adding 50 pl of 1% bovine serum albumin (BSA) solution, and incubating at room temperature for 30 min to obtain a closed coupling solution;
(3.4) putting the closed coupling solution on the magnetic frame, standing for 1min,
discarding the supernatant, and resuspending the precipitate in 36 ul of sterile PBS buffer with a concentration of 0.1 M and a pH value of 7.4 to obtain a magnetic separation probe solution.
Example 2
Establishment of detection equation
Referring to FIG. 1, the specific detection operation steps are as follows: (1) taking 1 ml of Salmonella typhimurium stock solution cultured in LB broth for 12 h to the late logarithmic growth stage, transferring it into a sterilized centrifuge tube, centrifuging at 8000 x g for 5 min, discarding the supernatant, and resuspending in 1 ml of sterile PBS buffer with concentration of 0.1 M and pH value of 7.4 to obtain bacterial suspension; diluting the bacteria suspension with sterile PBS buffer with the concentration of 0.1 M and pH value of 7.4, and preparing the Salmonella typhimurium solutions with the concentrations of 102, 10%, 10%, 105, 108, 107 cfu/ml; in addition, taking sterile PBS buffer with a concentration of 0.1 M and a pH value of 7.4 as a blank control solution, and the concentration of Salmonella typhimurium in the blank control solution is 0 cfu/ml; (2) adding the same 100 pl signal probe solution and 36 ul magnetic separation probe solution to 500 pl blank control solution and Salmonella typhimurium solution with concentrations of 102, 103, 10%, 105, 10°, 107 cfu/ml, and incubating at 37°C for 45 min to obtain seven composite solutions; the signal probe solution is prepared by Example 1; the magnetic separation probe solution is prepared by Example 1; (3) placing the seven composite solutions on a magnetic frame for magnetic separation, and taking supernatant to obtain the corresponding seven solutions to be detected; (4) taking seven solutions to be detected in quartz colorimetric cuvettes, and setting the excitation wavelength of F97Pro fluorescence spectrophotometer to 349 nm, the excitation broadband to 10nm, the emission broadband to 10nm, and the detection range of emission wavelength to 370-600nm; determining the fluorescence intensity at 423 nm of seven solutions to be detected with Salmonella typhimurium concentrations of 0, 102, 103, 10%, 105, 108, 107 cfu/ml, the results are shown in FIG. 2; taking lo-l as the ordinate Y, and the logarithmic value of
Salmonella typhimurium concentration (cfu/ml) as the abscissa X to draw the standard curve, the results are shown in FIG. 3, and the detection equation is obtained: Y = 378.30X - 302.45, the correlation coefficient is 0.9976, and the detection limit is 11.9 cfu/ml;
Io is the fluorescence intensity at 423 nm when blank control solution is detected, and | is the fluorescence intensity at 423 nm when Salmonella typhimurium solutions with the concentrations of 10%, 103, 10%, 105, 108, 107 cfu/ml are detected.
Example 3
Detection of Salmonella typhimurium in artificially contaminated milk (1) Preparation of liquid to be detected of artificially contaminated milk
Inoculating Salmonella typhimurium solution with unknown concentration in 1 ml sterile milk, centrifuging at 8000 x g for 5 min, discarding the supernatant, resuspending the precipitate with 1 ml of sterile PBS buffer with a concentration of 0.1 M and a pH of 7.4 to obtain the milk solution to be detected; (2) Sample determination (2.1) adding 100 HI signal probe solution and 36 pl magnetic separation probe solution into 500 ul milk solution to be detected, incubating at 37°C for 45 min to obtain the composite solution; the signal probe solution is prepared by Example 1; the magnetic separation probe solution is prepared by Example 1; (2.2) placing the composite solution on a magnetic frame for magnetic separation, and taking supernatant to obtain the solution to be detected; (2.3) putting the solution to be detected in a quartz colorimetric cuvette, setting the excitation wavelength of the F97Pro fluorescence spectrophotometer to 349 nm, and measuring the fluorescence intensity of the solution to be detected at 423 nm; (3) Calculating the detected results (3.1) calculating the test result according to the test equation, the detection equation is: Y = 378.30X - 302.45, where X represents the logarithmic value of Salmonella typhimurium concentration, and Y is lo-I; where lg is the fluorescence intensity at 423 nm when the blank control solution is detected, and | is the fluorescence intensity at 423 nm when the solution to be detected is detected; the detection equation is obtained by Example 2; the fluorescence intensity lo of the blank control solution at 423 nm is measured by
Example 2; (3.2) the calculated lo-1 value is 1026.13, which indicates that Salmonella typhimurium is detected in artificially contaminated milk; by substituting the lo-I value into the detection equation, the concentration of Salmonella typhimurium is 3.25 x 103 cfu/ml; when the lo-I value is greater than 104, it indicates that Salmonella typhimurium is detected in the detected substance.
Example 4
Specific detection method of Salmonella typhimurium in milk (1) Sample processing
Placing the same 1 ml sterile milk samples in seven sterile centrifuge tubes, and adding 1 ml of Salmonella typhimurium, Listeria monocytogenes, Pseudomonas aeruginosa, Escherichia coli, Enterobacter sakazakii, Staphylococcus aureus and Vibrio parahaemolyticus with the concentration of 10° cfu/ml into the seven centrifuge tubes to obtain seven mixed solutions; centrifuging the seven mixed solutions at 8000 x g for 5 min, discarding the supernatant, resuspending the precipitate with 1 ml sterile PBS buffer with the concentration of 0.1 M and pH value of 7.4 to obtain seven dairy products to be detected containing different pathogenic bacteria; (2) Detection (2.1) adding 500 pl of corresponding dairy products to be detected containing different pathogenic bacteria into seven sterile centrifuge tubes, adding 100 pl of signal probe solution and 36 pl of magnetic separation probe solution, and incubating at 37°C for 45 min to obtain seven composite solutions; the signal probe solution is prepared by Example 1; the magnetic separation probe solution is prepared by Example 1; (2.2) placing the seven composite solutions on a magnetic frame for magnetic separation, and taking supernatant to obtain the corresponding seven solutions to be detected; (2.3) taking seven solutions to be detected in quartz colorimetric cuvettes, and setting the excitation wavelength of F97Pro fluorescence spectrophotometer to 349 nm, and measuring the fluorescence intensity of seven solutions to be detected at 423 nm; (3) Result analysis (3.1) calculating the test result according to the test equation, the detection equation is: Y = 378.30X - 302.45, where X represents the logarithmic value of Salmonella typhimurium concentration, and Y is lo-I; where lg is the fluorescence intensity at 423 nm when the blank control solution is detected, and | is the fluorescence intensity at 423 nm when the solution to be detected is detected; the detection equation is obtained by Example 2; the fluorescence intensity ls of the blank control solution at 423 nm is measured by
Example 2; (3.2) results as shown in FIG. 4, due to the specific recognition of Salmonella typhimurium by aptamers, when Salmonella typhimurium exists in the system, the fluorescence intensity of the solution to be detected at 423 nm is obviously weaker than that of the blank control solution, and the lg-l value is 1958; when Salmonella typhimurium does not exist in the system, the fluorescence intensities of the solution to be detected of Listeria monocytogenes, Pseudomonas aeruginosa, Escherichia coli, Enterobacter sakazakii, Staphylococcus aureus and Vibrio parahaemolyticus at 423 nm have no obvious change compared with the blank control solution, and the corresponding lg-l values are 59, 43, 58, 49, 52 and 43, respectively, which shows that the invention has good specificity for Salmonella typhimurium; when the lo-I value is greater than 104, it indicates that Salmonella typhimurium is detected in the detected substance; when the ls-l value is less than 104, it indicates that Salmonella typhimurium is not detected in the detected substance.
Example 5
Application of the invention in detecting the content of Salmonella typhimurium in milk
(1) Sample processing
Taking 1 ml of commercially available milk, centrifuging at 8000 x g for 5 min, discarding the supernatant, resuspending the precipitate with 1 ml of sterile PBS buffer with a concentration of 0.1 M and a pH of 7.4 to obtain the solution to be detected; (2) Detection (2.1) adding 100 HI signal probe solution and 36 pl magnetic separation probe solution into 500 pl solution to be detected, incubating at 37°C for 45 min to obtain the composite solution; the signal probe solution is prepared by Example 1; the magnetic separation probe solution is prepared by Example 1; (2.2) placing the composite solution on a magnetic frame for magnetic separation, and taking supernatant to obtain the solution to be detected; (2.3) putting the solution to be detected in a quartz colorimetric cuvette, setting the excitation wavelength of the F97Pro fluorescence spectrophotometer to 349 nm, and measuring the fluorescence intensity of the solution to be detected at 423 nm; (3) Result analysis (3.1) calculating the test result according to the test equation, the detection equation is: Y = 378.30X - 302.45, where X represents the logarithmic value of Salmonella typhimurium concentration, and Y is lo-I; where lg is the fluorescence intensity at 423 nm when the blank control solution is detected, and | is the fluorescence intensity at 423 nm when the solution to be detected is detected; the detection equation is obtained by Example 2; the fluorescence intensity ls of the blank control solution at 423 nm is measured by
Example 2; (3.2) compared with the blank control solution, the fluorescence intensity | of the solution to be detected at 423 nm hardly changes, and the Is-I value is 12, which indicates that Salmonella typhimurium has not been detected in milk; when the lo-l value is less than 104, it indicates that Sa/monella typhimurium is not detected in the detected substance.
The embodiments of the present invention have been described in detail above, but the foregoing contents are only embodiments of the present invention and are not intended to limit the present invention. Any modification, equivalent substitution and improvement made within the scope of application of the present invention shall be included in the scope of protection of the present invention.
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Claims (5)

CONCLUSIESCONCLUSIONS 1. Een werkwijze voor het detecteren van Salmonella typhimurium met behulp van een fluorescerende probe op basis van met stikstof en zwavel gedopeerde grafeen- kwantumdots, met het kenmerk dat: — de signaalprobe-oplossing is bereid van stikstof- en zwavel-co-gedopeerde grafeen- kwantumdots die zijn gemodificeerd met het amino-gefunctionaliseerde aptameer (1); — de DNA-sequentie van het amino-gefunctionaliseerde aptameer (1) is: 5'-NH2-(CH2)6- TAT GGC GGC GTC ACC CGA CGG GGA CTT GAC ATT ATG ACA G-3' (SEQ ID NO: 1); — de probe-oplossing voor magnetische scheiding is bereid van magnetische streptavidine korrels die gemodificeerd zijn met het met biotine gemerkte aptameer (2); — de DNA-sequentie van het met biotine gemerkte aptameer (2) is: 5'-Biotine-GAG GAA AGT CTA TAG CAG AGG AGA TGT GTG AAC CGA GTA A-3' (SEQ ID NO: 2); — in de standaardkromme de X-as de logaritmische waarde van de concentratie Salmonella typhimurium aanduidt (kve/ml), en de Y-as lo-l aanduidt, waarbij lo staat voor de fluorescentie-intensiteit bij 423 nm wanneer een blanco controleoplossing wordt gedetecteerd, en | staat voor de fluorescentie-intensiteit bij 423 nm wanneer oplossingen met 102, 103, 10%, 105, 103 and 107 kve/ml Salmonella typhimurium worden gedetecteerd; — de specifieke detectiestappen als volgt zijn: (1) bereiden van de te detecteren vloeistof nemen van 1 ml vloeibare melk, centrifugeren bij 8000 x g gedurende 5 minuten, verwijderen van de bovenstaande vloeistof, weer in suspensie brengen van het neerslag met 1 ml steriele PBS-buffer in een concentratie van 0,1 M en een pH van 7,4 om de te detecteren oplossing te verkrijgen; (2) detecteren1. A method for detecting Salmonella typhimurium using a fluorescent probe based on nitrogen- and sulfur-doped graphene quantum dots, characterized in that : — the signal probe solution is prepared from nitrogen- and sulfur-co-doped graphene - quantum dots modified with the amino-functionalized aptamer (1); — the DNA sequence of the amino-functionalized aptamer (1) is: 5'-NH2-(CH2)6- TAT GGC GGC GTC ACC CGA CGG GGA CTT GAC ATT ATG ACA G-3' (SEQ ID NO: 1) ; — the magnetic separation probe solution is prepared from magnetic streptavidin beads modified with the biotin labeled aptamer (2); — the DNA sequence of the biotin labeled aptamer (2) is: 5'-Biotin-GAG GAA AGT CTA TAG CAG AGG AGA TGT GTG AAC CGA GTA A-3' (SEQ ID NO: 2); — in the standard curve, the X-axis denotes the logarithmic value of the concentration of Salmonella typhimurium (cfu/ml), and the Y-axis denotes lo-1, where lo denotes the fluorescence intensity at 423 nm when a blank control solution is detected , and | represents the fluorescence intensity at 423 nm when solutions containing 102, 103, 10%, 105, 103 and 107 cfu/ml Salmonella typhimurium are detected; — the specific detection steps are as follows: (1) preparing the liquid to be detected taking 1 ml of liquid milk, centrifuging at 8000 x g for 5 minutes, removing the supernatant, resuspending the precipitate with 1 ml of sterile PBS buffer at a concentration of 0.1 M and a pH of 7.4 to obtain the solution to be detected; (2) detect (2.1) toevoegen van 100 pul signaalprobe-oplossing en 36 Jl probe-oplossing voor magnetische scheiding aan 500 ul van te detecteren oplossing, incuberen bij 37 °C gedurende 45 minuten om de samengestelde oplossing te verkrijgen;(2.1) adding 100 µl of signal probe solution and 36 µl of magnetic separation probe solution to 500 µl of the solution to be detected, incubating at 37°C for 45 minutes to obtain the compounded solution; (2.2) plaatsen van de samengestelde oplossing op een magnetisch frame voor magnetische scheiding en het nemen van de bovenstaande vloeistof om de te detecteren oplossing te verkrijgen;(2.2) placing the compound solution on a magnetic frame for magnetic separation and taking the supernatant to obtain the solution to be detected; (2.3) het plaatsen van de te detecteren oplossing in een colorimetrische kwarts cuvette, de het instellen van de excitatiegolflengte van de F97Pro fluorescentiespectrofotometer op 349 nm en het meten van de intensiteit van de fluorescentie van de te detecteren oplossing bij 423 nm; (3) berekenen van de gedetecteerde resultaten substitueren van de lg-I-waarde in de standaardkromme en berekenen van de concentratie van Salmonella typhimurium in de te detecteren oplossing, d.w.z. voltooien van de detectie; waarbij lp staat voor de fluorescentie-intensiteit bij 423 nm wanneer de blanco controleoplossing wordt gedetecteerd, en | staat voor de fluorescentie-intensiteit bij 423 nm wanneer de te detecteren oplossing wordt gedetecteerd; waarbij wanneer de lg-I-waarde groter is dan 104, dit betekent dat Sa/monella typhimurium in de gedetecteerde stof is aangetroffen; en wanneer de lo-I-waarde kleiner is dan 104, dit betekent dat Sa/monella typhimurium niet in de gedetecteerde stof is aangetroffen.(2.3) placing the solution to be detected in a colorimetric quartz cuvette, setting the excitation wavelength of the F97Pro fluorescence spectrophotometer to 349 nm and measuring the fluorescence intensity of the solution to be detected at 423 nm; (3) calculating the detected results substituting the Ig-I value into the standard curve and calculating the concentration of Salmonella typhimurium in the solution to be detected, i.e. completing the detection; where lp is the fluorescence intensity at 423 nm when the blank control solution is detected, and | represents the fluorescence intensity at 423 nm when the solution to be detected is detected; where if the Ig-I value is greater than 104, it means that Sa/monella typhimurium has been found in the detected substance; and when the lo-I value is less than 104, this means that Sa/monella typhimurium was not found in the detected substance. 2. De werkwijze voor het detecteren van Salmonella typhimurium met behulp van een fluorescerende probe op basis van met stikstof en zwavel gedopeerde grafeen- kwantumdots volgens conclusie 1, met het kenmerk dat de stappen voor het bereiden van de signaalprobe-oplossing als volgt zijn: (1) toevoegen van 0,1 ml voorraadoplossing van met stikstof en zwavel gedopeerde grafeen-kwantumdots in 9,9 ml ultrazuiver water om 10 ml waterige oplossing van met stikstof en zwavel gedopeerde grafeen-kwantumdots te verkrijgen; (2) toevoegen van 5 mg N-hydroxysuccinimide en 10 mg 1-(3-dimethylaminopropyl)-3- ethylcarbodiimidehydrochloride in 10 ml waterige oplossing van met stikstof en zwavel gedopeerde grafeen-kwantumdots, waarbij de pH-waarde op 5,0 wordt gebracht met 10 mg/ml natriumhydroxideoplossing (NaOH) en 30 minuten in het donker bij kamertemperatuur wordt geïncubeerd om een geactiveerde met stikstof en zwavel gedopeerde waterige oplossing van grafeen-kwantumdots te verkrijgen; (3) het nemen van de waterige oplossing van de met stikstof en zwavel gedopeerde grafeen-kwantumdots, het aanpassen van de pH-waarde op 7,4 met een oplossing van 10 mg/ml natriumhydroxide (NaOH), het toevoegen van 20 pl van de oplossing van 100 uM amino-gefunctionaliseerd aptamer (1), en het gedurende 2 uur bij kamertemperatuur te suspenderen om signaalprobe-oplossing te verkrijgen.The method for detecting Salmonella typhimurium using a fluorescent probe based on nitrogen- and sulfur-doped graphene quantum dots according to claim 1, characterized in that the steps for preparing the signal probe solution are as follows: ( 1) adding 0.1 ml stock solution of nitrogen and sulfur doped graphene quantum dots in 9.9 ml ultrapure water to obtain 10 ml aqueous solution of nitrogen and sulfur doped graphene quantum dots; (2) adding 5 mg of N-hydroxysuccinimide and 10 mg of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride in 10 ml of an aqueous solution of nitrogen- and sulfur-doped graphene quantum dots, adjusting the pH to 5.0 with 10 mg/ml sodium hydroxide solution (NaOH) and incubated in the dark at room temperature for 30 minutes to obtain an activated nitrogen- and sulfur-doped aqueous solution of graphene quantum dots; (3) taking the aqueous solution of the nitrogen- and sulfur-doped graphene quantum dots, adjusting the pH to 7.4 with a 10 mg/ml solution of sodium hydroxide (NaOH), adding 20 µl of the solution of 100 µM amino-functionalized aptamer (1), and suspending it at room temperature for 2 hours to obtain signal probe solution. 3. De werkwijze voor het detecteren van Salmonella typhimurium met behulp van een fluorescerende probe op basis van met stikstof en zwavel gedopeerde grafeen- kwantumdots volgens conclusie 2, met het kenmerk dat dat de bewerkingsstappen voor het bereiden van voorraadsoplossing van de met stikstof en zwavel gedopeerde grafeen- kwantumdots als volgt zijn: (1) Het gelijkmatig mengen van 1,0 g citroenzuur met 0,3 g L-cysteine om een reactiemengsel te verkrijgen;The method for detecting Salmonella typhimurium using a fluorescent probe based on nitrogen- and sulfur-doped graphene quantum dots according to claim 2, characterized in that the processing steps for preparing stock solution of the nitrogen- and sulfur-doped graphene quantum dots are as follows: (1) Evenly mixing 1.0 g of citric acid with 0.3 g of L-cysteine to obtain a reaction mixture; (2) het in een oliebad tot 200°C verwarmen van het reactiemengsel om het vloeibaar te maken, het stoppen van het verwarmen zodra de kleur van de vloeistof van het reactiemengsel verandert van lichtgeel naar oranje, en het op natuurlijke wijze tot kamertemperatuur laten afkoelen om een oranje voortbrengsel te verkrijgen; (3) het in 100 ml ultrazuiver water oplossen van het oranje voortbrengsel om de voorraadoplossing van de met stikstof en zwavel gedopeerde grafeen-kwantumdots te verkrijgen.(2) heating the reaction mixture to 200°C in an oil bath to liquefy it, stopping the heating once the color of the liquid of the reaction mixture changes from light yellow to orange, and allowing it to cool naturally to room temperature to obtain an orange product; (3) dissolving the orange article in 100 ml of ultrapure water to obtain the stock solution of the nitrogen- and sulfur-doped graphene quantum dots. 4. De werkwijze voor het detecteren van Salmonella typhimurium met behulp van een fluorescerende probe op basis van met stikstof en zwavel gedopeerde grafeen- kwantumdots volgens conclusie 1, met het kenmerk dat de bewerkingsstappen voor het bereiden van de oplossing van de magnetische scheidingsprobe als volgt zijn: — Het nemen van 18 pl magnetische streptavidinekorrels met een concentratie van 10 mg/ml in een gesiliconiseerde centrifugebuis, het toevoegen van 72 ul 1 x B&W bufferoplossing, het gelijkmatig mengen ervan, en het op het magnetische frame plaatsen ervan om 1min te staan, het verwijderen van de bovenstaande vloeistof nadat de magnetische streptavidinekorrels volledig aan de wand van de centrifugebuis zijn geadsorbeerd; vervolgens toevoegen van 72 ul 1 x B&W buffer om de bovenstaande bewerking te herhalen om de behandelde magnetische streptavidinekorrels te verkrijgen; — Het opnieuw suspenderen van de behandelde magnetische streptavidinekorrels in 36 Ml 2 x B&W buffer, het toevoegen van 36 pl oplossing van het gebiotinyleerde aptamer (2) in een concentratie van 1,5 HM, en het bij kamertemperatuur gedurende 30 minuten incuberen om een koppelingsoplossing te verkrijgen; — Het plaatsen van de koppelingsoplossing op het magnetische frame, het gedurende 1 minuut laten staan, het verwijderen van de bovenstaande vloeistof, het toevoegen van 50 pl van een 1%'s oplosssing van runderserumalbumine (BSA), en het incuberen bij kamertemperatuur gedurende 30 minuten om een gesloten koppelingsoplossing te verkrijgen; — het plaatsen van de gesloten koppeloplossing op het magnetische frame plaatsen, het gedurende 1 minuut laten staan, het verwijderen van de bovenstaande vloeistof en het opnieuw laten neerslaan van het neerslag in 36 pl steriele PBS-buffer met een concentratie van 0,1 M en een pH-waarde van 7,4 om een probe-oplossing voor magnetische scheiding te verkrijgen.The method for detecting Salmonella typhimurium using a fluorescent probe based on nitrogen- and sulfur-doped graphene quantum dots according to claim 1, characterized in that the processing steps for preparing the solution of the magnetic separation probe are as follows : — Taking 18 µl of streptavidin magnetic beads at a concentration of 10 mg/ml into a siliconized centrifuge tube, adding 72 µl of 1x B&W buffer solution, mixing it evenly, and placing it on the magnetic frame to stand for 1 min, removing the supernatant after the magnetic streptavidin beads are completely adsorbed to the wall of the centrifuge tube; then adding 72 µl of 1 x B&W buffer to repeat the above operation to obtain the treated magnetic streptavidin beads; — Resuspending the treated magnetic streptavidin beads in 36 µl of 2 x B&W buffer, adding 36 µl of a solution of the biotinylated aptamer (2) at a concentration of 1.5 HM, and incubating at room temperature for 30 minutes to form a coupling solution to obtain; — Placing the coupling solution on the magnetic frame, allowing it to stand for 1 minute, removing the supernatant, adding 50 µl of a 1% bovine serum albumin (BSA) solution, and incubating at room temperature for 30 minutes to obtain a closed coupling solution; — placing the closed coupling solution on the magnetic frame, allowing it to stand for 1 minute, removing the supernatant and reprecipitating the precipitate in 36 µl of sterile PBS buffer at a concentration of 0.1 M and a pH value of 7.4 to obtain a magnetic separation probe solution. 5. De werkwijze voor het detecteren van Salmonella typhimurium met behulp van een fluorescerende probe op basis van met stikstof en zwavel gedopeerde grafeen- kwantumdots volgens conclusie 4, met het kenmerk datThe method for detecting Salmonella typhimurium using a fluorescent probe based on nitrogen- and sulfur-doped graphene quantum dots according to claim 4, characterized in that — de 1 x B&W-bufferoplossing wordt verkregen door een 2 x B&W-bufferoplossing te verdunnen met een gelijk volume ultrapuur water; — de bewerkingsstappen voor de bereiding van de 2 x B&W-bufferoplossing als volgt zijn: het afwegen van 0,1211 g tris-(hydroxymethyl}-aminomethaan (Tris), 11,7 @— the 1 x B&W buffer solution is obtained by diluting a 2 x B&W buffer solution with an equal volume of ultrapure water; — the processing steps for the preparation of the 2 x B&W buffer solution are as follows: weighing 0,1211 g of Tris-(hydroxymethyl}-aminomethane (Tris), 11,7 @ natriumchloride (NaCl) en 0,0372 g ethyleendiaminetetraazijnzuur (EDTA), het toevoegen van gedeioniseerd water, het volledig roeren en laten oplossen, het instellen van de pH-waarde op 7. 5 met een oplossing van 36,5 mg/ml zoutzuur (HCI), het gelijkmatig roeren, het instellen van het volume op 100 ml, het gedurende 15 min onderwerpen aan een autoclaafbehandeling bij 121°C, laten afkoelen om 2 x B&W buffer te verkrijgen, en het bewaren in de koelkast bij 4°C voor later gebruik.sodium chloride (NaCl) and 0.0372 g ethylenediaminetetraacetic acid (EDTA), adding deionized water, stirring and allowing to dissolve completely, adjusting the pH to 7.5 with a solution of 36.5 mg/ml hydrochloric acid ( HCl), stirring uniformly, adjusting the volume to 100 ml, autoclaving at 121°C for 15 min, allowing to cool to obtain 2 x B&W buffer, and refrigerating at 4°C for later use.
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