Detection reagent for detecting escherichia coli and preparation method

Abstract

The invention relates to a detection reagent for detecting escherichia coli and a preparation method, and belongs to the technical field of food safety detection. The detection reagent is a graphene-loaded targeted copper-chlorine co-doped carbon nano-enzyme aptamer compound and is light brown transparent liquid; the copper-chlorine co-doped carbon nano-enzyme / amino-modified aptamer composite material is prepared from magnetic graphene, (3-dimethylaminopropyl)-3-ethylcarbodiimide, N-hydroxysuccinimide, copper-chlorine co-doped carbon nano-enzyme and an amino-modified aptamer according to the molar mass ratio of 15 g: 3 mol: 1.5 mol: 262.5 g: 0.3 mol through a reaction of the magnetic graphene, (3-dimethylaminopropyl)-3-ethylcarbodiimide, N-hydroxysuccinimide, the copper-chlorine co-doped carbon nano-enzyme and the amino-modified aptamer. According to the invention, cascade signal amplification is carried out on targeted copper-chlorine co-doped carbon nanoenzyme based on high-strength signal output and a reverse magnetic attraction strategy; when an object to be detected contains escherichia coli O157: H7, the targeted copper-chlorine co-doped carbon nano enzyme on the magnetic carrier can specifically recognize a target strain to form a composite structure, and interference of the magnetic carrier on signal output is eliminated through a reverse magnetic separation effect; taking supernate, and respectively measuring the fluorescence intensity at 456nm and the ultraviolet absorption intensity at 654nm so as to determine the concentration of the escherichia coli O157: H7.

Description

Technical Field

[0001] The invention belongs to the technical field of food safety detection, and particularly relates to a method for preparing a detection reagent for detecting Escherichia coli. Background Art

[0002] Food contamination by pathogenic bacteria is a major global health concern. Escherichia coli O157:H7, a serotype of E. coli, is a major cause of foodborne illness. Food is a key route of contamination for E. coli O157:H7, causing significant harm to human health and significant losses to the food industry. Therefore, developing highly specific and sensitive detection reagents is a crucial technical prerequisite for the effective prevention and control of E. coli O157:H7.

[0003] Currently, detection reagents for E. coli include microbial culture media, specific recognition element antibodies, and new aptamer sensors. Traditional culture methods based on microbial culture media require multiple steps, including bacterial isolation and culture, morphological observation, and biochemical identification. These are cumbersome and time-consuming, failing to meet market demand for rapid testing. Antibodies, which serve as specific recognition elements in immunological methods, are expensive, require the use of large, sophisticated instruments, and are significantly affected by the external environment. As for new aptamer-based biosensor detection technologies, most of them use a single signal output, resulting in large errors in detection results. Furthermore, when applied to food testing, complex matrices reduce sensitivity, making it impossible to detect trace amounts of E. coli O157:H7. Therefore, the development of simple, rapid, accurate, and highly sensitive detection reagents for the detection of E. coli O157:H7 is urgent.

[0004] To shorten detection time, improve sensitivity, and reduce costs, detection strategies based on catalase activity have been developed and applied. Catalase catalyzes hydrogen peroxide to generate hydroxyl radicals, which oxidize the colorless substrate 3,3′,5,5′-tetramethylbenzidine to form a blue product, exhibiting excellent catalytic activity and substrate specificity. However, stringent storage conditions have prompted researchers to develop novel peroxidase mimics with simpler synthesis and more stable structures. Carbon nanozymes, as a new type of catalyst, have attracted widespread attention due to their simple synthesis steps and environmentally friendly properties. Among them, carbon nanozymes derived from lignin, the second largest renewable resource, have attracted widespread attention. Chloride ions play an important role in the adsorption and reduction of hydrogen peroxide and can also accelerate the electron loss process from hydrogen peroxide to hydroxyl radicals. However, the co-doping of chloride ions with other highly catalytically active metal elements has not been reported. In addition, the detection strategy based on nanozymes is limited by low detection sensitivity and weak anti-interference ability of food matrices. There is an urgent need to combine new high-sensitivity, low-cost nanozyme detection reagents with signal amplification technology and magnetic separation technology to achieve ultra-sensitive detection of Escherichia coli O157:H7 in food. Summary of the Invention

[0005] In order to achieve high-sensitivity, visual and rapid detection of Escherichia coli in food matrices, the present invention provides a method for preparing a detection reagent for detecting Escherichia coli.

[0006] A detection reagent for detecting Escherichia coli is a graphene-loaded targeted copper-chloride co-doped carbon nanozyme aptamer complex, which is a light brown transparent liquid;

[0007] The graphene-loaded targeted copper-chloride co-doped carbon nanozyme aptamer complex is prepared by reacting magnetic graphene, (3-dimethylaminopropyl)-3-ethylcarbodiimide, N-hydroxysuccinimide, copper-chloride co-doped carbon nanozyme and amino aptamer in a molar mass ratio of 15g:3mol:1.5mol:262.5g:0.3mol;

[0008] The copper-chloride co-doped carbon nanozyme is prepared by mixing sodium lignin sulfonate and copper chloride in a mass ratio of 1:1;

[0009] The copper-chloride co-doped carbon nanozyme reacts with the amino aptamer to prepare a targeted copper-chloride co-doped carbon nanozyme aptamer;

[0010] The DNA sequence of the amino-terminated aptamer is shown in SEQ ID No: 1.

[0011] The preparation steps of a detection reagent for detecting Escherichia coli are as follows:

[0012] (1) Preparation of magnetic graphene

[0013] (1.1) Graphene oxide and sodium hydroxide are mixed in ultrapure water and sonicated to obtain a graphene oxide solution;

[0014] (1.2) Mixing ferric salt, ferrous salt, and hydrochloric acid to obtain a reaction solution;

[0015] The mass ratio of the graphene oxide, the iron salt and the ferrous salt is 52:475:250;

[0016] (1.3) The reaction solution was added dropwise to the graphene oxide solution to produce magnetic graphene with a mass volume concentration of 150 μg / mL.

[0017] (2) Preparation of copper-chloride co-doped carbon nanozymes

[0018] Sodium lignin sulfonate and copper chloride were ultrasonically dispersed in deionized water at a mass ratio of 1:1, incubated, cooled to room temperature, centrifuged, purified, dialyzed, concentrated, and freeze-dried to prepare a copper-chloride co-doped carbon nanozyme with a mass volume concentration of 525 μg / mL.

[0019] (3) Preparation of targeted copper-chloride co-doped carbon nanozyme aptamers

[0020] The (3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride solution, N-hydroxysuccinimide solution and copper-chloride co-doped carbon nanozyme were mixed at a molar mass ratio of 3 mol:1.5 mol:262.5 g:0.3 mol, and then added to the amino-modified aptamer solution and incubated to obtain a targeted copper-chloride co-doped carbon nanozyme aptamer;

[0021] (4) Preparation of detection reagents for detecting Escherichia coli

[0022] 1 mL of targeted copper-chloride co-doped carbon nanozyme aptamer was added to 100 μL of magnetic graphene with a mass volume concentration of 150 μg / mL and incubated to obtain a graphene-loaded targeted copper-chloride co-doped carbon nanozyme aptamer complex, which is a detection reagent for detecting Escherichia coli.

[0023] Further technical solutions are as follows:

[0024] The specific operations in step (1) are as follows:

[0025] (1.1) To 26 mL of 2 mg / mL graphene oxide (pH 8.23), add 1.4 g of sodium hydroxide pellets and 5.25 mL of ultrapure water. Mix by oscillation for 10 min and then sonicate for 30 min to obtain a graphene oxide solution.

[0026] (1.2) Take 0.475 g of ferric chloride and 0.25 g of ferrous chloride, add 0.104 mL of 12 M hydrochloric acid and 3.021 mL of ultrapure water, respectively, and shake for 1 min to obtain 3.125 mL of reaction solution.

[0027] (1.3) Slowly add 3.125 mL of the reaction solution to the graphene oxide solution and react at 80°C under nitrogen for 1 h to obtain magnetic graphene with a mass volume concentration of 150 μg / mL.

[0028] The specific operations in step (2) are as follows:

[0029] (2.1) Add 100 mg of sodium lignin sulfonate and 100 mg of copper chloride to 39 mL of deionized water and sonicate for 10 min to obtain the sonicated product.

[0030] (2.2) Incubate the sonicated product in a reaction vessel at 180°C for 8 h and allow to cool naturally to room temperature to obtain a suspension.

[0031] (2.3) Centrifuging the suspension at 6500 rpm for 15 min, and filtering through a 0.22 µm membrane to obtain a filtrate;

[0032] (2.4) The filtrate was dialyzed through a 3500 Da dialysis bag for 12 h, collected and concentrated, and then freeze-dried and dialyzed to obtain a copper-chloride co-doped carbon nanozyme with a mass volume concentration of 525 μg / mL;

[0033] The specific operations in step (3) are as follows:

[0034] (3.1) Add 300 μL of a 10 mM solution of (3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and a 5 mM solution of N-hydroxysuccinimide to 500 μL of a 525 μg / mL copper-chloride co-doped carbon nanozyme and shake at 150 rpm for 30 min.

[0035] (3.2) Add 1 mL of 300 nM amino-modified aptamer solution and incubate at 200 rpm for 3 h to obtain the targeted copper-chloride co-doped carbon nanozyme aptamer.

[0036] In step (4), the incubation conditions are: incubation at 37°C for 10 min.

[0037] The beneficial technical effects of the present invention are embodied in the following aspects:

[0038] (1) The present invention synthesizes non-toxic, environmentally friendly, and highly catalytically active copper-chlorine co-doped lignin carbon nanozymes by mixing lignin with copper and chlorine. The addition of copper promotes a synergistic effect with chlorine, enhancing oxidase activity. This effect is superior to that achieved by doping with potassium chloride and hydrochloric acid, with peroxidase activity increased by 56.6% and 94.0%, respectively, significantly improving the detection reagent developed based on the copper nanozyme.

[0039] (2) This invention utilizes a reverse magnetic separation strategy combined with a copper-chloride co-doped carbon nanozyme with high peroxidase catalytic activity to achieve dual cascade signal amplification, significantly improving the sensitivity of the detection reagent, avoiding detection interference caused by the material itself, and enhancing the specificity of the detection reagent, providing a new method for ultra-precise detection of trace E. coli O157:H7 in food. In addition, the aptamer and the magnetic nanomaterial are directly adsorbed through π-π stacking, effectively avoiding expensive chemical modification and reducing detection costs by 30%.

[0040] (3) It has been verified that the detection reagent of the present invention has a high degree of linear fitting, with a correlation coefficient as high as 0.99821, and exhibits a low detection limit of 57.2 cfu / mL. The current existing technology for Escherichia coli O157:H7 generally has a detection limit of more than 10 2 cfu / mL; while conventional detection times range from 4 hours to 7 days, the present invention can detect bacteria within 1 hour, demonstrating excellent specificity. Furthermore, the cross-calibration of dual-modal detection signals, combining fluorescence and colorimetric signals, ensures excellent accuracy even at high sensitivity. Substituting fluorescence intensity and UV absorbance intensity values ​​into the resulting linear regression equation yielded correlation coefficients of 0.99955 and 0.99993, respectively, with the true value of E. coli O157:H7 in the sample.

[0041] (4) In summary, the copper-chloride co-doped carbon nanozyme of the present invention is the strongest peroxide-like nanozyme with green raw material lignin as its precursor. It cooperates with the copper-chloride co-doped carbon nanozyme with super strong signal output through reverse magnetic attraction to achieve cascade signal amplification, significantly improving the detection speed, accuracy and stability of trace E. coli O157:H7 in food. Through the specific reaction of the aptamer with E. coli O157:H7 and the separation of the magnetic carrier from the signal output system under the action of an external magnetic field, the detection sensitivity and reliability are further improved. In addition, the fluorescence and colorimetric signal detection of the test solution are carried out simultaneously, and the dual-modal signal of fluorescence and colorimetry can be obtained in one experiment, which not only enhances the sensitivity of the detection, but also can avoid the misjudgment caused by operation errors and food matrix by mutual correction of the two data results. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] Figure 1Schematic diagram of the detection method of the present invention.

[0043] Figure 2 This is a scanning electron microscope image of Escherichia coli O157:H7 in Example 2 of the present invention.

[0044] Figure 3 This is the elemental mapping diagram of Escherichia coli O157:H7 in Example 2 of the present invention.

[0045] Figure 4 This is a standard curve of the logarithm of the concentration of Escherichia coli O157:H7 bacteria-fluorescence intensity value in Example 2 of the present invention.

[0046] Figure 5 This is a standard curve of the logarithm of the Escherichia coli O157:H7 bacterial concentration-absorbance intensity value in Example 2 of the present invention.

[0047] Figure 6 Graph showing specific fluorescence intensity values ​​of the detection method in Example 5 of the present invention.

[0048] Figure 7 This is a graph of the specific absorbance intensity values ​​of the detection method in Example 5 of the present invention. DETAILED DESCRIPTION

[0049] The present invention will be further described below with reference to the embodiments, but the present invention is not limited thereto.

[0050] Unless otherwise defined, technical and scientific terms used in the following examples have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0051] Unless otherwise specified, the experimental reagents and consumables used in the following examples are all conventional biochemical reagents; the experimental methods described are all conventional methods unless otherwise specified; the quantitative tests in the following examples are all repeated three times, and the results are averaged; the "%" in the following examples, unless otherwise specified, are all weight percentages.

[0052] In the following examples, 10× enzyme digestion buffer was purchased from Dalian Bao Bioengineering Co., Ltd.; 3,3′,5,5′-tetramethylbenzidine was purchased from Aladdin Reagent Co., Ltd.; 10 M hydrogen peroxide solution was purchased from Shanghai Wokai Biotechnology Co., Ltd.; N-hydroxysuccinimide and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride were purchased from Sigma-Aldrich, USA; sodium lignin sulfonate was purchased from Aladdin Reagent Co., Ltd.; anhydrous ethanol was purchased from Sinopharm Chemical Reagent Co., Ltd.; cupric chloride was purchased from Merck Chemical Reagent Co., Ltd. (Shanghai, China); a filter membrane with a filter size of 0.22 μm was purchased from Jinteng Experimental Equipment Co., Ltd.; and an amino aptamer was purchased from Shanghai Sangon Bioengineering Co., Ltd.

[0053] Among the bacterial species used in the present invention, the number of Escherichia coli O157:H7 is ATCC 43895, the number of Listeria monocytogenes is ATCC 7644, the number of Salmonella typhimurium is ATCC 14028, the number of Pseudomonas aeruginosa is ATCC 25922, the number of Vibrio parahaemolyticus is ATCC 17802, the number of Staphylococcus aureus is ATCC 65389, and the number of Cronobacter sakazakii is ATCC 12806, all of which are provided by Guangdong Institute of Microbiology.

[0054] The instruments, equipment, raw materials, reagents or method steps used in the present invention are all ensured to be processed under sterile conditions.

[0055] Instruments, equipment, raw materials, reagents or method steps not mentioned in the present invention are conventional or well-known technical methods for those skilled in the art and are not described in detail in the present invention.

[0056] Example 1

[0057] The preparation steps of a detection reagent for detecting Escherichia coli are as follows:

[0058] (1) Preparation of magnetic graphene

[0059] (1.1) To 26 mL of 2 mg / mL graphene oxide (pH 8.23), add 1.4 g of sodium hydroxide pellets and 5.25 mL of ultrapure water. Mix by oscillation for 10 min and then sonicate for 30 min to obtain a graphene oxide solution.

[0060] (1.2) Take 0.475 g of ferric chloride and 0.25 g of ferrous chloride, add 0.104 mL of 12 M hydrochloric acid and 3.021 mL of ultrapure water, respectively, and shake and mix for 1 min to obtain 3.125 mL of reaction solution.

[0061] (1.3) Slowly add 3.125 mL of the reaction solution dropwise to the graphene oxide solution. The reaction is carried out at 80°C under nitrogen for 1 h to obtain magnetic graphene with a mass volume concentration of 150 μg / mL.

[0062] (2) Preparation of copper-chloride co-doped carbon nanozymes

[0063] (2.1) Add 100 mg of sodium lignin sulfonate and 100 mg of copper chloride to 39 mL of deionized water and sonicate for 10 min to obtain the sonicated product.

[0064] (2.2) Incubate the sonicated product in a reaction vessel at 180°C for 8 h and allow to cool naturally to room temperature to obtain a suspension.

[0065] (2.3) Centrifuging the suspension at 6500 rpm for 15 min, and filtering through a 0.22 µm membrane to obtain a filtrate;

[0066] (2.4) The filtrate was dialyzed through a 3500 Da dialysis bag for 12 h, collected and concentrated, and then freeze-dried and dialyzed to obtain a copper-chloride co-doped carbon nanozyme with a mass volume concentration of 525 μg / mL;

[0067] (3) Preparation of targeted copper-chloride co-doped carbon nanozyme aptamers

[0068] (3.1) Add 300 μL of a 10 mM solution of (3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and a 5 mM solution of N-hydroxysuccinimide to 500 μL of a 525 μg / mL copper-chloride co-doped carbon nanozyme and shake at 150 rpm for 30 min.

[0069] (3.2) Add 1 mL of 300 nM amino-modified aptamer solution and incubate at 200 rpm for 3 h to obtain a targeted copper-chloride co-doped carbon nanozyme aptamer.

[0070] The DNA sequence of the amino-modified aptamer is shown in SEQ ID No: 1, and the 5' end of the modified aptamer is modified with an amino group;

[0071] (4) Preparation of detection reagents for detecting Escherichia coli

[0072] 1 mL of targeted copper-chloride co-doped carbon nanozyme aptamer was added to 100 μL of magnetic graphene with a mass volume concentration of 150 μg / mL, and incubated at 37°C for 10 minutes to obtain a graphene-loaded targeted copper-chloride co-doped carbon nanozyme aptamer complex, which is a detection reagent for detecting Escherichia coli.

[0073] The detection reagent for detecting Escherichia coli prepared in this Example 1 is a light brown transparent liquid.

[0074] Example 2

[0075] Establishment of detection equation

[0076] (1) Take 1 mL of Escherichia coli O157:H7 stock solution cultured in LB broth for 12 h to the late logarithmic growth stage, transfer it to a sterilized centrifuge tube, centrifuge it at 5000 × g for 5 min, discard the supernatant, and resuspend it in 1 mL of sterile PBS buffer with a concentration of 0.1 M and a pH of 7.4 to obtain a bacterial suspension; dilute the bacterial suspension with a sterile PBS buffer with a concentration of 0.1 M and a pH of 7.4 to prepare a 10 1 , 10 2 , 103 , 10 4 , 10 5 , 10 6 cfu / mL of Escherichia coli O157:H7 solution; a sterile PBS buffer solution with a concentration of 0.1M and a pH value of 7.4 was taken as a blank control solution, and the concentration of Escherichia coli O157:H7 in the blank control solution was 0 cfu / mL;

[0077] (2) In 200 μL of blank control solution and 10 1 , 10 2 , 10 3 , 10 4 , 10 5 , 10 6 cfu / mL of Escherichia coli O157:H7 solution, 50 μL of the same double-stranded probe solution was added, incubated at 37°C for 60 min, centrifuged at 5000 × g for 5 min, and the supernatant was collected to obtain seven composite solutions;

[0078] (3) Seven identical 200 μL graphene-loaded targeted copper-chloride co-doped carbon nanozyme aptamer complexes were added to seven 1 mL composite solutions, and the composite solutions were obtained after incubation at 37 °C for 40 min.

[0079] The graphene-supported targeted copper-chloride co-doped carbon nanozyme aptamer complex is prepared by Example 1;

[0080] (4) Place the seven composite solutions on a magnetic rack and let them stand for 2 min. Take the supernatant solution to obtain seven test solutions.

[0081] (5) First, take seven 50 μL aliquots of supernatant and add them to 450 μL of 1× PBS solution. Measure the fluorescence intensity at 456 nm under an excitation wavelength of 330 nm.

[0082] (6) Take seven 50 μL aliquots of the supernatant solution, add 50 μL of 10 M hydrogen peroxide solution and 50 μL of 3,3′,5,5′-tetramethylbenzidine solution, add 350 μL of acetic acid-sodium acetate buffer solution, react at 35°C in the dark for 10 min, and measure the ultraviolet absorption intensity at 654 nm;

[0083] The 100 mM hydrogen peroxide solution is prepared by adding 10 μL of 10 M hydrogen peroxide to 990 μL of ultrapure water; the 4.8 g / L 3,3′,5,5′-tetramethylbenzidine solution is prepared by adding 0.048 g of 3,3′,5,5′-tetramethylbenzidine to 10 mL of anhydrous ethanol;

[0084] (7) It was observed that the concentration was 10 2 , 10 3 , 10 4 , 10 5 , 10 6 The reaction solution containing E. coli O157:H7 at a concentration of 10 1 The reaction solution of E. coli O157:H7 with 10 cfu / mL was colorless and transparent, and the result was negative. Therefore, the visual detection limit was 10 2 cfu / mL; the fluorescence intensity of the five positive reaction solutions at 456nm is the vertical axis Y, and the logarithm of the concentration of E. coli O157:H7 bacteria (cfu / mL) is the horizontal axis X to draw the standard curve 1. The results are shown in the attached Figure 4 , the detection equation is: Y=0.14843X-0.06199, with a correlation coefficient of 0.99821; take the absorbance intensity of five 200μL positive reaction solutions with a peak at 654nm as the vertical coordinate Y, and use the logarithm of the concentration of E. coli O157:H7 bacteria (cfu / mL) as the horizontal coordinate X to draw the standard curve 2. The results are shown in the attached Figure 5 , the detection equation is: Y=0.26039X-0.32652, and the correlation coefficient is 0.99345.

[0085] Example 3

[0086] Application of the present invention in detecting Escherichia coli O157:H7 content in ham

[0087] (1) Sample processing

[0088] The ham to be tested was cut into 25 g pieces and mixed with 25 mL of PBS solution with a pH value of 7.4 and a concentration of 0.1 M in a sterile bag. The PBS mixture was homogenized for 2 min by a homogenizer to prepare a PBS mixture. Subsequently, 3 × 10 4 cfu / mL of E. coli O157:H7 solution, let the spiked mixture stand for 5 min, and take the supernatant to obtain the test solution;

[0089] (2) Detection

[0090] (2.1) Take 200 μL of the graphene-loaded targeted copper-chloride co-doped carbon nanozyme aptamer complex and add 1 mL of the test solution. Incubate at 37°C for 40 min to obtain a complex solution.

[0091] The graphene-supported targeted copper-chloride co-doped carbon nanozyme aptamer complex is obtained from Example 1;

[0092] (2.2) Magnetic separation is performed on the composite solution. After standing on the magnetic rack for 2 minutes, the supernatant solution is collected to obtain the test solution.

[0093] (2.3) Add 50 μL of the test solution to 450 μL of 1× PBS solution and measure the fluorescence intensity at 456 nm under an excitation wavelength of 330 nm.

[0094] (2.4) Take another 50 μL of the test solution and add 50 μL of 100 mM hydrogen peroxide solution and 50 μL of 4.8 g / L 3,3′,5,5′-tetramethylbenzidine solution, respectively. Then add 350 μL of acetic acid-sodium acetate buffer and react at 35°C in the dark for 10 min. Measure the UV absorbance at 654 nm.

[0095] (3) Calculate the test results

[0096] (3.1) Observe the color of the reaction solution. When the reaction solution is colorless and transparent, the test result is negative; when the reaction solution is blue, the test result is positive. Substitute the fluorescence intensity value I of the reaction solution of the positive sample at 456 nm into the detection equation. The detection equation is: Y=0.14843X-0.06199, where X is the logarithm of the concentration of the Escherichia coli O157:H7 bacterial solution, Y is I, and I is the fluorescence intensity value at 456 nm when the present invention detects the test solution. The concentration of Escherichia coli O157:H7 in the test solution is calculated, and the test is completed. Take another 200 μL of the positive reaction solution and measure its ultraviolet absorption intensity value A at 654 nm. Substitute it into the standard curve 2. The detection equation is Y=0.26039X-0.32652. The concentration of Escherichia coli O157:H7 in the test solution is calculated and calibrated with the target bacteria concentration obtained by fluorescence.

[0097] The detection equation is obtained from Example 2;

[0098] (3.2) The reaction solution was observed to be blue, indicating a positive test result. 200 μL of the positive reaction solution was placed in a cuvette, and its fluorescence value at 456 nm was measured to be 0.599. Substituting this into the test equation 1, the concentration of E. coli O157:H7 in the sample was calculated to be 2.84×10 4 cfu / mL; 200 μL of the positive reaction solution was taken and its UV absorption intensity at 654 nm was measured to be 0.843, indicating that the concentration of E. coli O157:H7 in the analyte was 3.09×10 4 cfu / mL.

[0099] Example 4

[0100] Application of the present invention in detecting Escherichia coli O157:H7 content in milk

[0101] (1) Sample processing

[0102] Take 1 mL of commercially available milk, centrifuge at 5000 × g for 5 min, discard the supernatant, and resuspend the precipitate in 1 mL of sterile PBS buffer with a concentration of 0.1 M and a pH value of 7.4 to obtain the test solution;

[0103] (2) Detection

[0104] (2.1) Take 200 μL of the graphene-loaded targeted copper-chloride co-doped carbon nanozyme aptamer complex and add 1 mL of the test solution. Incubate at 37°C for 40 min to obtain a complex solution.

[0105] The graphene-supported targeted copper-chloride co-doped carbon nanozyme aptamer complex is obtained from Example 1;

[0106] (2.2) Magnetic separation is performed on the composite solution. After standing on the magnetic rack for 2 minutes, the supernatant solution is collected to obtain the test solution.

[0107] (2.3) Add 50 μL of the test solution to 450 μL of 1× PBS solution and measure the fluorescence intensity at 456 nm under an excitation wavelength of 330 nm.

[0108] (2.4) Take another 50 μL of the test solution and add 50 μL of 100 mM hydrogen peroxide solution and 50 μL of 4.8 g / L 3,3′,5,5′-tetramethylbenzidine solution, respectively. Then add 350 μL of acetic acid-sodium acetate buffer and react at 35°C in the dark for 10 min. Measure the UV absorbance at 654 nm.

[0109] (3) Calculate the test results

[0110] (3.1) Observe the color of the reaction solution. When the reaction solution is colorless and transparent, the test result is negative; when the reaction solution is blue, the test result is positive. Substitute the fluorescence intensity value I of the reaction solution of the positive sample at 456 nm into the detection equation. The detection equation is: Y=0.14843X-0.06199, where X is the logarithm of the concentration of the Escherichia coli O157:H7 bacterial solution, Y is I, and I is the fluorescence intensity value at 456 nm when the present invention detects the test solution. Calculate the concentration of Escherichia coli O157:H7 in the test solution, and the test is completed. Take another 200 μL of the positive reaction solution, measure its ultraviolet absorption intensity value A at 654 nm, and substitute it into the standard curve 2 to calculate the concentration of Escherichia coli O157:H7 in the test solution, and calibrate it with the target bacteria concentration obtained by fluorescence.

[0111] The detection equation is obtained from Example 2;

[0112] (3.2) The reaction solution was transparent and the test result was negative, indicating that E. coli O157:H7 was not detected in the test object.

[0113] Example 5

[0114] Application of the present invention in detecting Escherichia coli O157:H7 content in drinking water

[0115] (1) Sample processing

[0116] Drinking water was used directly as the test solution without any treatment.

[0117] (2) Detection

[0118] (2.1) Take 200 μL of the graphene-loaded targeted copper-chloride co-doped carbon nanozyme aptamer complex and add 1 mL of the test solution. Incubate at 37°C for 40 min to obtain a complex solution.

[0119] The graphene-supported targeted copper-chloride co-doped carbon nanozyme aptamer complex is obtained from Example 1;

[0120] (2.2) Magnetic separation is performed on the composite solution. After standing on the magnetic rack for 2 minutes, the supernatant solution is collected to obtain the test solution.

[0121] (2.3) Add 50 μL of the test solution to 450 μL of 1× PBS solution and measure the fluorescence intensity at 456 nm under an excitation wavelength of 330 nm.

[0122] (2.4) Take another 50 μL of the test solution and add 50 μL of 100 mM hydrogen peroxide solution and 50 μL of 4.8 g / L 3,3′,5,5′-tetramethylbenzidine solution, respectively. Then add 350 μL of acetic acid-sodium acetate buffer and react at 35°C in the dark for 10 min. Measure the UV absorbance at 654 nm.

[0123] (3) Calculate the test results

[0124] (3.1) Observe the color of the reaction solution. When the reaction solution is colorless and transparent, the test result is negative; when the reaction solution is blue, the test result is positive. Substitute the fluorescence intensity value I of the reaction solution of the positive sample at 456 nm into the detection equation. The detection equation is: Y=0.14843X-0.06199, where X is the logarithm of the concentration of the Escherichia coli O157:H7 bacterial solution, Y is I, and I is the fluorescence intensity value at 456 nm when the present invention detects the test solution. Calculate the concentration of Escherichia coli O157:H7 in the test solution, and the test is completed. Take another 200 μL of the positive reaction solution, measure its ultraviolet absorption intensity value A at 654 nm, and substitute it into the standard curve 2 to calculate the concentration of Escherichia coli O157:H7 in the test solution, and calibrate it with the target bacteria concentration obtained by fluorescence.

[0125] The detection equation is obtained from Example 2;

[0126] (3.2) The reaction solution was transparent and the test result was negative, indicating that E. coli O157:H7 was not detected in the test object.

[0127] Example 6

[0128] Specific Detection Method for Escherichia coli O157:H7

[0129] (1) Sample processing

[0130] Take seven sterile centrifuge tubes and add 1 mL of sterile PBS buffer (control) and 10 6 cfu / mL of Escherichia coli O157:H7, Salmonella typhimurium, Pseudomonas aeruginosa, Cronobacter, Staphylococcus aureus, and Vibrio parahaemolyticus to obtain seven mixed solutions; the seven mixed solutions were centrifuged at 5000×g for 5 min, the supernatant was discarded, and the precipitate was resuspended in 1 mL of sterile PBS buffer with a concentration of 0.1 M and a pH value of 7.4 to obtain seven test solutions;

[0131] (2) Detection

[0132] (2.1) Take 200 μL of the graphene-loaded targeted copper-chloride co-doped carbon nanozyme aptamer complex and add 1 mL of the test solution. Incubate at 37°C for 40 min to obtain a complex solution.

[0133] The graphene-supported targeted copper-chloride co-doped carbon nanozyme aptamer complex is obtained from Example 1;

[0134] (2.2) Magnetic separation is performed on the composite solution. After standing on the magnetic rack for 2 minutes, the supernatant solution is collected to obtain the test solution.

[0135] (2.3) Add 50 μL of the test solution to 450 μL of 1× PBS solution and measure the fluorescence intensity at 456 nm under an excitation wavelength of 330 nm.

[0136] (2.4) Take another 50 μL of the test solution and add 50 μL of 100 mM hydrogen peroxide solution and 50 μL of 4.8 g / L 3,3′,5,5′-tetramethylbenzidine solution, respectively. Then add 350 μL of acetic acid-sodium acetate buffer and react at 35°C in the dark for 10 min. Measure the UV absorbance at 654 nm.

[0137] (3) Calculate the test results

[0138] (3.1) Observe the color of the reaction solution. When the reaction solution is colorless and transparent, the test result is negative; when the reaction solution is blue, the test result is positive. Substitute the fluorescence intensity value I of the reaction solution of the positive sample at 456 nm into the detection equation. The detection equation is: Y=0.14843X-0.06199, where X is the logarithm of the concentration of the Escherichia coli O157:H7 bacterial solution, Y is I, and I is the fluorescence intensity at 456 nm when the present invention detects the test solution. Calculate the concentration of Escherichia coli O157:H7 in the test solution, and the test is completed. Take another 200 μL of the positive reaction solution, measure its ultraviolet absorption intensity value A at 654 nm, and substitute it into the standard curve 2 to calculate the concentration of Escherichia coli O157:H7 in the test solution, and calibrate it with the target bacteria concentration obtained by fluorescence.

[0139] The detection equation is obtained from Example 2;

[0140] It was observed that the reaction solution with Escherichia coli O157:H7 turned blue, indicating a positive test result; the reaction solution with sterile PBS solution (control), Salmonella typhimurium, Listeria monocytogenes, Pseudomonas aeruginosa, Cronobacter, Staphylococcus aureus, and Vibrio parahaemolyticus turned colorless and transparent, indicating a negative test result; Figure 6 As shown in the figure, the fluorescence intensity values ​​of the seven reaction solutions at 456 nm were as follows: Escherichia coli O157:H7 was 0.9876; PBS solution was 0.20326; Salmonella typhimurium was 0.2790; Pseudomonas aeruginosa was 0.1987; Cronobacter 0.1979; Staphylococcus aureus was 0.2114; Vibrio parahaemolyticus was 0.2096; Figure 7As shown, the measured ultraviolet absorption intensity values ​​of the seven reaction solutions at 654 nm are: Escherichia coli O157:H7 is 1.1937; PBS solution is 0.1142; Salmonella typhimurium is 0.1275; Pseudomonas aeruginosa is 0.1047; Cronobacter is 0.0975; Staphylococcus aureus is 0.1142; and Vibrio parahaemolyticus is 0.1123. This shows that the present invention has good specificity for detecting Escherichia coli O157:H7.

Claims

1. A detection reagent for detecting Escherichia coli, characterized in that: The detection reagent is a graphene-loaded targeted copper-chloride co-doped carbon nanozyme aptamer complex, which is a light brown transparent liquid; The graphene-loaded targeted copper-chloride co-doped carbon nanozyme aptamer complex is prepared by reacting magnetic graphene, (3-dimethylaminopropyl)-3-ethylcarbodiimide, N-hydroxysuccinimide, copper-chloride co-doped carbon nanozyme and amino aptamer in a molar mass ratio of 15g:3mol:1.5mol:262.5g:0.3mol; The copper-chloride co-doped carbon nanozyme is prepared by mixing sodium lignin sulfonate and copper chloride in a mass ratio of 1:1; The copper-chloride co-doped carbon nanozyme reacts with the amino aptamer to prepare a targeted copper-chloride co-doped carbon nanozyme aptamer; The DNA sequence of the amino-terminated aptamer is shown in SEQ ID No:

1.

2. A method for preparing a detection reagent for detecting Escherichia coli, characterized in that: The steps are as follows: (1) Preparation of magnetic graphene (1.1) Graphene oxide and sodium hydroxide are mixed in ultrapure water and sonicated to obtain a graphene oxide solution; (1.2) Mixing ferric salt, ferrous salt, and hydrochloric acid to obtain a reaction solution; The mass ratio of the graphene oxide, the iron salt and the ferrous salt is 52:475:250; (1.3) The reaction solution was added dropwise to the graphene oxide solution to produce magnetic graphene with a mass volume concentration of 150 μg / mL. (2) Preparation of copper-chloride co-doped carbon nanozymes Sodium lignin sulfonate and copper chloride were ultrasonically dispersed in deionized water at a mass ratio of 1:1, incubated, cooled to room temperature, centrifuged, purified, dialyzed, concentrated, and freeze-dried to prepare a copper-chloride co-doped carbon nanozyme with a mass volume concentration of 525 μg / mL. (3) Preparation of targeted copper-chloride co-doped carbon nanozyme aptamers The (3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride solution, N-hydroxysuccinimide solution and copper-chloride co-doped carbon nanozyme were mixed at a molar mass ratio of 3 mol:1.5 mol:262.5 g:0.3 mol, and then added to the amino-modified aptamer solution and incubated to obtain a targeted copper-chloride co-doped carbon nanozyme aptamer; (4) Preparation of detection reagents for detecting Escherichia coli 1 mL of targeted copper-chloride co-doped carbon nanozyme aptamer was added to 100 μL of magnetic graphene with a mass volume concentration of 150 μg / mL and incubated to obtain a graphene-loaded targeted copper-chloride co-doped carbon nanozyme aptamer complex, which is a detection reagent for detecting Escherichia coli.

3. The method for preparing a detection reagent for detecting Escherichia coli according to claim 2, characterized in that: The specific operations in step (1) are as follows: (1.1) To 26 mL of 2 mg / mL graphene oxide (pH 8.23), add 1.4 g of sodium hydroxide pellets and 5.25 mL of ultrapure water. Mix by oscillation for 10 min and then sonicate for 30 min to obtain a graphene oxide solution. (1.2) Take 0.475 g of ferric chloride and 0.25 g of ferrous chloride, add 0.104 mL of 12 M hydrochloric acid and 3.021 mL of ultrapure water, respectively, and shake for 1 min to obtain 3.125 mL of reaction solution. (1.3) Slowly add 3.125 mL of the reaction solution to the graphene oxide solution and react at 80°C under nitrogen for 1 h to obtain magnetic graphene with a mass volume concentration of 150 μg / mL.

4. The method for preparing a detection reagent for detecting Escherichia coli according to claim 2, characterized in that: The specific operations in step (2) are as follows: (2.1) Add 100 mg of sodium lignin sulfonate and 100 mg of copper chloride to 39 mL of deionized water and sonicate for 10 min to obtain the sonicated product. (2.2) Incubate the sonicated product in a reaction vessel at 180°C for 8 h and allow to cool naturally to room temperature to obtain a suspension. (2.3) Centrifuging the suspension at 6500 rpm for 15 min, and filtering through a 0.22 µm membrane to obtain a filtrate; (2.4) The filtrate was dialyzed for 12 h using a 3500 Da dialysis bag, collected and concentrated, and then freeze-dried and dialyzed to obtain copper-chloride co-doped carbon nanozyme with a mass volume concentration of 525 μg / mL.

5. The method for preparing a detection reagent for detecting Escherichia coli according to claim 2, characterized in that: The specific operations in step (3) are as follows: (3.1) Add 300 μL of a 10 mM solution of (3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and a 5 mM solution of N-hydroxysuccinimide to 500 μL of a 525 μg / mL copper-chloride co-doped carbon nanozyme and shake at 150 rpm for 30 min. (3.2) Add 1 mL of 300 nM amino-modified aptamer solution and incubate at 200 rpm for 3 h to obtain the targeted copper-chloride co-doped carbon nanozyme aptamer.

6. The method for preparing a detection reagent for detecting Escherichia coli according to claim 2, characterized in that: In step (4), the incubation conditions are: incubation at 37°C for 10 min.

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