A method for detecting escherichia coli in food by Fe3O4@Mo-CDs nanozyme combined with aptamer

Abstract

The application discloses a method for detecting escherichia coli in food by Fe3O4@Mo-CDs nano-enzyme combined with aptamer, wherein the near-infrared light responsive molybdenum doped carbon dot modified Fe3O4@Mo-CDs nano-enzyme is synthesized by a hydrothermal method, the nano-enzyme has excellent NIR enhanced peroxidase-like activity and magnetism, oxidizes 3,3',5,5'-tetramethylbenzidine to generate blue oxidized TMB, after the surface of the nano-enzyme is functionalized by escherichia coli nucleic acid aptamer, the peroxidase-like activity of the Fe3O4@Mo-CDs nano-enzyme is inhibited, when escherichia coli exists, the nano-enzyme can be selectively combined with the escherichia coli E. coli , and further inhibits the peroxidase-like activity of the nano-enzyme, when the nano-enzyme is used for detecting escherichia coli in juice, cake and other samples E. coli , the detection limit is as low as 0.767 CFU / mL, the nano-enzyme has higher reliability and accuracy, and the application realizes magnetic target separation and visual detection of escherichia coli E. coli .

Description

Technical Field

[0001] This invention relates to the field of chemical analysis and detection technology, specifically to a method for detecting Escherichia coli in food using Fe3O4@Mo-CDs nanozyme-binding aptamers. Background Technology

[0002] Food safety is receiving increasing global attention. Despite significant success in treating pathogenic diseases, infections caused by pathogens remain a leading cause of morbidity and mortality worldwide. Escherichia coli (E. coli) Escherichia coli Escherichia coli (E. coli) is one of the most common foodborne pathogens that can cause serious illnesses, including inflammation, hemorrhagic colitis, and hemolytic uremic syndrome, and can even lead to death, especially in young people, the elderly, and immunocompromised individuals. Outbreaks of foodborne illnesses caused by E. coli are often linked to contaminated food and water. Therefore, in addition to effective treatment, rapid and reliable detection of foodborne pathogens is essential for food safety.

[0003] Fe3O4NPs, due to their excellent biocompatibility, superparamagnetism, and peroxidase-like activity, have been widely used in sensors, biomedicine, and microbial detection and treatment. However, Fe3O4NPs exhibit high chemical reactivity and are easily oxidized and lose their magnetism in air. Molybdenum-based nanomaterials possess excellent near-infrared light absorption and biocompatibility. Fe3O4NPs modified with molybdenum-based nanomaterials not only have enhanced peroxidase-like activity but also exhibit near-infrared light response properties. Nucleic acid aptamers (Apts) are concise single-stranded DNA or RNA molecules that can selectively bind to specific target molecules, including proteins, peptides, small molecules, and even whole cells. They are generated through the Systematic Evolution of Ligands by Exponential Enrichment (SELEX) method. Combining nanozymes with nucleic acid aptamers for rapid and specific detection of target substances has become a research hotspot in food safety control. Summary of the Invention

[0004] This invention provides a magnetic Fe3O4@Mo-CDs nanozyme-bound nucleic acid aptamer for the detection of Escherichia coli in food. E. coli The method described in this invention (O157:H7) first synthesizes a near-infrared (NIR, 808nm) light-responsive coral-like molybdenum-doped carbon-doped Fe3O4 composite material (Fe3O4@Mo-CDs nanozyme) via a hydrothermal method. The Fe3O4@Mo-CDs nanozyme exhibits excellent NIR-enhanced peroxidase (POD)-like activity and magnetism, oxidizing 3,3',5,5'-tetramethylbenzidine (TMB) to produce blue oxidized TMB. After functionalizing E. coli nucleic acid aptamers on its surface, the POD-like activity of the Fe3O4@Mo-CDs nanozyme is inhibited. E. coli In the presence of O157:H7, the nanozyme-aptamer complex can selectively bind. E. coli O157:H7 further inhibited the POD-like activity of Fe3O4@Mo-CDs nanozymes, thus constructing a method for detecting... E. coli The O157:H7 "capture-detection" colorimetric biosensor has a linear range of 10. 1 -10 7 CFU / mL.

[0005] The method of this invention can be used in food samples such as fruit juice and pastries. E. coli When detecting O157:H7, the superparamagnetism of Fe3O4@Mo-CDs nanozymes effectively eliminates matrix interference, exhibiting excellent detection performance with a detection limit as low as 0.767 CFU / mL. It also demonstrates high reliability and accuracy (recovery rates between 100.1% and 103.5%). This invention enables the detection of O157:H7. E. coli Magnetic targeting separation and visualization detection.

[0006] The method for detecting Escherichia coli O157:H7 in food using Fe3O4@Mo-CDs nanozyme-bound aptamers of the present invention is as follows: 1. Add 2.50-3.00g FeCl3·6H2O and 0.60-1.00g sodium citrate to 60-100mL ethylene glycol, stir and mix well, then add 4.50-5.00g sodium acetate, stir for 30-40min, transfer to a polytetrafluoroethylene container, place in a muffle furnace, heat at 180-210℃ for 12-14h, cool to room temperature, wash the solid product alternately with pure water and anhydrous ethanol 2-4 times, and vacuum dry to obtain magnetic nano Fe3O4; 2. Add 0.10-0.30g of nano Fe3O4, 0.30-0.90g of phosphomolybdic acid, 50-100μL of ethylenediamine, and 0.50-0.80g of citric acid to 30-50mL of deionized water, sonicate for 20-40min, react in a microwave at 170-200℃ for 1-2h, cool naturally to room temperature, separate the solid with a magnet, wash the solid 2-3 times each with anhydrous ethanol and deionized water, and vacuum dry to obtain Fe3O4@Mo-CDs nanozyme; 3. Mix the nucleic acid aptamer solution with the Fe3O4@Mo-CDs nanozyme solution and incubate at 37°C for 25-30 min to obtain the nanozyme-aptamer complex, wherein the nucleotide sequence of the nucleic acid aptamer is 5′-SH-CCGGACGCTTATGCCTTGCCATCTACAGAGGTGTGACGG-3′ (SEQ ID NO:1). The Fe3O4@Mo-CDs nanozyme solution concentration is 0.5 mg / mL, the nucleic acid aptamer solution concentration is 0.25 μmol / L, and the volume ratio of Fe3O4@Mo-CDs nanozyme solution to nucleic acid aptamer solution is 4-6:1. 4. Nanozyme and aptamer complex solution, Na+, etc., were added sequentially to Escherichia coli suspensions of different concentrations. + Solution, Mg 2+ Solution, 3,3',5,5'-tetramethylbenzidine solution, and H2O2 solution were diluted to volume with pH 4.0 acetate buffer solution, irradiated with near-infrared light at 808 nm for 10-20 min, separated by magnet, and the supernatant was taken and the absorbance was measured at a wavelength of 654 nm to determine the linear relationship between the concentration of Escherichia coli suspension and the absorbance value, and the regression equation was obtained. 5. The absorbance value of the sample solution to be tested is determined according to the method in step (4), and then substituted into the regression equation to obtain the concentration of Escherichia coli in the sample solution to be tested. The concentration of the Escherichia coli suspension was 10. 1 -10 7 CFU / mL; Na + The solution is a 50 mmol / L NaCl solution, and the addition volume is 50-100 μL; Mg 2+ The solutions were: 10 mmol / L MgCl2 solution, added in 50-100 μL; 50 mmol / L 3,3',5,5'-tetramethylbenzidine solution, added in 50-100 μL; 20 mmol / L H2O2 solution, added in 50-100 μL; and 0.1 mmol / L pH 4.0 acetate buffer solution.

[0007] The near-infrared light at 808nm has a power of 1.0-2.0 W / cm². 2 .

[0008] The advantages of this invention are: 1. The Fe3O4@Mo-CDs nanozyme prepared by this invention exhibits excellent peroxidase activity and paramagnetism. Near-infrared light irradiation further improves the enzyme-like activity of the Fe3O4@Mo-CDs nanozyme, and the rough surface is beneficial for… E. coli Adsorption of O157:H7 aptamer; Fe3O4@Mo-CDs nanozyme oxidizes 3,3',5,5'-tetramethylbenzidine (TMB) to produce blue oxidized TMB; after functionalization of nucleic acid aptamers on its surface, its POD-like activity is inhibited; when E. coli In the presence of O157:H7, the nanozyme-aptamer complex can selectively bind. E. coliO157:H7 further inhibited the POD-like activity of Fe3O4@Mo-CDs nanozymes, thus constructing a method for detecting... E. coli The O157:H7 "capture-detection" colorimetric biosensor has a linear range of 1-10. 7 The method, with CFU / mL, is characterized by its speed, high sensitivity, ease of operation, and high specificity.

[0009] 2. When the method of this invention is applied to the determination of food samples such as fruit juice and pastries, the superparamagnetism of Fe3O4@Mo-CDs nanozymes can effectively separate the matrix, eliminate interference, and simultaneously... E. coli O157:H7 was effectively enriched, exhibiting excellent detection performance with a detection limit as low as 0.767 CFU / mL. It also demonstrated high reliability and accuracy (recovery rates between 100.1% and 103.5%), achieving [the goal of] [the effective enrichment of O157:H7]. E. coli Magnetic targeting separation and visual detection of O157:H7. Attached Figure Description

[0010] Figure 1 This is a scanning electron microscope (TEM) image of the Fe3O4@Mo-CDs nanozyme in Example 1; Figure 2 The image shows the XRD pattern of the Fe3O4@Mo-CDs nanozyme synthesized in Example 1. Figure 3 This is a high-resolution XPS image of Fe3O4@Mo-CDs nanozyme synthesized in Example 1. Figure 4 shows the UV-Vis absorption spectra of Fe3O4@Mo-CDs nanozyme and Fe3O4@Mo-CDs nanozyme + NIR oxidation of TMB + H2O2 in Example 1; Figure 5 The Michaelis-Menten kinetics curve for the oxidation of TMB by Fe3O4@Mo-CDs nanoenzymes in Example 1 is shown. Figure 6 The Michaelis-Menten kinetics curve for the oxidation of H2O2 by Fe3O4@Mo-CDs nanoenzymes in Example 1 is shown. Figure 7 The Michaelis-Menten kinetics curve for the oxidation of TMB by Fe3O4@Mo-CDs+NIR in Example 1 is shown. Figure 8 The Michaelis-Menten kinetics curve for the oxidation of H2O2 by Fe3O4@Mo-CDs+NIR in Example 1 is shown. Figure 9 The diagrams show the ·OH groups detected by TA in Fe3O4@Mo-CDs and Fe3O4@Mo-CDs+NIR systems in Example 1. Figure 10This is a graph verifying the selective adsorption performance of Fe3O4@Mo-CDs. Figure 11 In Example 1 E. coli O157:H7 + UV-Vis absorption spectrum of Aptamer-Fe3O4@Mo-CDs inhibiting the oxidation of TMB by pseudoperoxidase after irradiation at 808 nm for 10 min (a) and linear equation (b). Figure 12 For the detection of 8 common pathogens E. coli The effects of O157:H7; Figure 13 For the determination of coexisting substances and metal ions E. coli Results of the influence of O157:H7. Detailed Implementation

[0011] The following examples further illustrate the content of the present invention, but these examples do not limit the scope of protection of the present invention. Unless otherwise specified, the methods in the examples are conventional methods, and unless otherwise specified, the reagents used are conventional commercial reagents or reagents prepared according to conventional methods. Example 1: In bread E. coli Determination of O157:H7 1. Preparation of Fe3O4 2.50 g FeCl3·6H2O and 0.80 g sodium citrate were added to 80 mL ethylene glycol and stirred until homogeneous. Then, 4.50 g sodium acetate was added and stirred for 30 min. The mixture was then transferred to a polytetrafluoroethylene container, placed in a muffle furnace, and heated at 200 °C for 12 h. After cooling to room temperature, the solid was collected by centrifugation at 4000 r / min for 15 min and washed three times alternately with pure water and anhydrous ethanol. The solid was then dried under vacuum at 60 °C to obtain magnetic nano-Fe3O4 material. 2. Preparation of Fe3O4@Mo-CDs nanozymes 0.20 g of nano-Fe3O4, 0.50 g of phosphomolybdic acid, 80 μL of ethylenediamine, and 0.60 g of citric acid were added to 40 mL of deionized water and sonicated for 25 min. The mixture was then reacted in a microwave at 190 °C for 2 h, allowed to cool naturally to room temperature, and separated using a magnet. The solid was washed three times each with anhydrous ethanol and deionized water, and then vacuum dried at 60 °C for 24 h to obtain Fe3O4@Mo-CDs nanozyme. The prepared Fe3O4@Mo-CDs nanozyme was analyzed by transmission electron microscopy (TEM). Figure 1 As shown, the synthesized Fe3O4@Mo-CDs nanozyme exhibits a coral-like rough surface structure. The X-ray diffraction (XRD) pattern analysis of the Fe3O4@Mo-CDs nanozyme is as follows... Figure 2 As shown, the main diffraction peak 2 θ=30.3°, 35.6°, 43.4°, 53.7°, 57.4° and 63.0°, corresponding to (220), (311), (400), (422), (511) and (440) cubic crystal planes of magnetite Fe3O4; X-ray photoelectron spectroscopy (XPS) analysis was used to determine the composition, valence state and binding energy of the elements present in Fe3O4@Mo-CDs nanozymes, and the O 1s spectrum of Fe3O4@Mo-CDs nanozymes ( Figure 3 Two peaks appeared at 530.7 eV and 532.2 eV, which belong to surface adsorbed oxygen (Os) and oxygen vacancy (Ov), respectively, indicating that there are oxygen vacancies on the surface of Fe3O4@Mo-CDs nanozymes, which is beneficial to promoting the enzyme-like activity of Fe3O4@Mo-CDs nanozymes.

[0012] 3. Preparation of nucleic acid aptamer-Fe3O4@Mo-CDs nanozyme complex 200 μL of a 0.5 mg / mL Fe3O4@Mo-CDs nanozyme solution was added to 50 μL of a 0.25 μmol / L (NaAc-HAcbuffer, pH 4.0) nucleic acid aptamer solution and incubated at 37 °C for 30 min to obtain the nanozyme-aptamer complex Aptamer-Fe3O4@Mo-CDs; the sequence of the nucleic acid aptamer was: 5′-SH-CCGGACGCTTATGCCTTGCCATCTACAGAGGTGTGACGG-3′.

[0013] 4. Evaluation of POD activity of Fe3O4@Mo-CDs nanozymes Using 3,3',5,5'-tetramethylbenzidine (TMB) as a substrate, 100 μL of 50 mmol / L TMB was added, along with 100 μL of 20 mmol / L H2O2, 50 μL of 2 mg / mL Fe3O4@Mo-CDs nanozyme, and 0.1 mmol / L pH 4.0 acetate buffer solution to a final volume of 3 mL. The mixture was shaken well and then analyzed using a power density of 1.0 W / cm². 2 Irradiate with near-infrared light at 808 nm for 10 min, or allow to stand for 10 min, and measure the absorbance at 654 nm. Figure 4 The results showed that the POD catalytic activity of Fe3O4@Mo-CDs nanozymes was significantly improved after irradiation at 808 nm.

[0014] This embodiment also included the determination of Michaelis catalytic kinetic parameters, the results of which are shown below. Figures 5-8 As shown in Table 1, regardless of whether TMB or H2O2 is used as the substrate, infrared light (NIR) significantly enhances the affinity of Fe3O4@Mo-CDs nanozymes for the substrate and the reaction rate. Table 1 Michaelis catalytic kinetic parameters ; 5. Adsorption performance test of nanozyme-aptamer-Fe3O4@Mo-CDs complex To investigate the effect of Aptamer-Fe3O4@Mo-CDs on E. coli The selective adsorption of O157:H7 will E. coli O157:H7 bacterial suspension was mixed with Aptamer-Fe3O4@Mo-CDs, and a blank control (Blank) without Aptamer-Fe3O4@Mo-CDs was also set up. Staphylococcus aureus was also compared. S.aureus ), the result is as follows Figure 9 , S.aureus There was very little adhesion to Aptamer-Fe3O4@Mo-CDs, and the turbidity of the supernatant remained unchanged after magnetic separation, while for E. coli O157:H7, after magnetic separation, a transparent supernatant was observed, indicating that... E. coli O157:H7 has been largely adsorbed onto Aptamer-Fe3O4@Mo-CDs.

[0015] 6. Free radical capture: To further investigate the ROS that play a major role in the catalytic process, ·OH and ·O2 were captured using isopropanol (IPA), 2,2,6,6-tetramethylpiperidine-1-oxo radical (TEMPOL), sodium oxalate (Na2C2O4), and silver nitrate (AgNO3), respectively. - h + and e - The result is as follows Figure 10 As shown, the absorbance values ​​of oxidized TMB exhibited different decreasing trends after the addition of IPA, TEMPOL, Na2C2O4, and AgNO3. The decrease was greatest after the addition of IPA, followed by TEMPOL, indicating that the order of influence is: ·OH > ·O2. - h + >e - These results indicate that, in catalytic reactions, ·OH and ·O2... - It is the main ROS.

[0016] 7. E. coli O157:H7 Working Curve Creation (1) E. coli O157:H7 culture: E. coliThe O157:H7 strain was cultured in LB medium at 37°C for 12 h, centrifuged at 3000 rpm for 5 min, washed twice with phosphate-buffered saline (PBS, 10 mM, pH 7.4), and resuspended in water to obtain a concentration of 10. 1 -10 7 CFU / mL E. coli bacterial suspension; (2) E. coli Preparation of O157:H7 working curve: Add 50µL of nanozyme and aptamer complex Aptamer-Fe3O4@Mo-CDs, 50µL of 50mmol / L NaCl solution, 50µL of 10mmol / L MgCl2 solution, and 10µL of MgCl2 solution to a 5mL stoppered colorimetric tube. 1 -10 7 CFU / mL E. coli 100 μL of O157:H7 bacterial suspension, 100 μL of 50 mmol / L TMB, and 100 μL of 20 mmol / L H2O2 were diluted to 3 mL with pH 4.0 acetate buffer, and the solution was shaken well. The solution was then passed through a power density of 1.0 W / cm³. 2 Irradiate with near-infrared light at 808 nm for 10 min, separate with a magnet, collect the supernatant and measure the absorbance at 654 nm. E. coli A standard curve was plotted with the concentration of the O157:H7 bacterial suspension on the x-axis and absorbance on the y-axis, yielding the regression equation. (See...) Figure 11 The regression equation, correlation coefficient, relative standard deviation, linear range, etc. are shown in Table 2. Table 2. Linear equation, correlation coefficient, relative standard deviation, and linear range .

[0017] 8. Method specificity investigation An investigation was conducted on eight common pathogens (Listeria). Listeria ),salmonella( Salmonella) Vibrio parahaemolyticus ( V. parahaemolyticus Staphylococcus aureus ( S. aureus ), Pseudomonas aeruginosa ( P. aeruginosa ), Citrobacter freundii ( C. freundii ), Shigella dysenteriae ( S. dysenteriae ) and Campylobacter jejuni ( C. jejuni The impact on the detection system, among which E. coli The concentration of O157:H7 was 10. 2 CFU / mL, the concentration of other pathogens is 10. 3 CFU / mL, results are shown in [link to results]. Figure 12 The results show that the detection system is only effective against... E. coliO157:H7 showed significant inhibitory effects, while other pathogens remained almost unchanged, demonstrating good selectivity specificity.

[0018] Simultaneously, coexisting substances (leucine, glycine, glutamic acid, vitamin A, vitamin C, vitamin B3, and vitamin B1) and metal ions (Cu) were examined. 2+ Na + Ca 2+ Zn 2+ Al 3+ K + Fe 2+ Fe 3+ The impact on the detection system, among which E. coli The concentration of O157:H7 was 10. 2 CFU / mL, other substances concentration was 1 mg / mL, results are shown in […]. Figure 13 The testing system only applies to E. coli O157:H7 has a significant inhibitory effect, while other substances do not affect the detection system.

[0019] 9. In the bread sample E. coli Determination of O157:H7 (1) Sample preparation: Take 25g of bread, cut it into pieces, add 250mL of PBS buffer solution, homogenize for 15min, centrifuge at 10000 r / min for 10min, and take out the supernatant to obtain the sample test solution; (2) Sample determination: In a 5 mL stoppered colorimetric tube, add 50 µL of the nanozyme-aptamer-Fe3O4@Mo-CDs complex, 50 μL of 50 mmol / L NaCl solution, 50 μL of 10 mmol / L MgCl2 solution, 100 μL of sample determination solution, 100 μL of 50 mmol / L TMB, and 100 μL of 20 mmol / L H2O2 sequentially. Dilute to 3 mL with pH 4.0 acetate buffer solution, shake well, and pass through a power density of 1.0 W / cm². 2 Irradiate with near-infrared light at 808 nm for 10 min, separate with a magnet, collect the supernatant and measure the absorbance at 654 nm. Substitute this value into the regression equation in step 7 to obtain the result of the bread sample. E. coli O157:H7 was not detected.

[0020] Example 2: Beverage sample E. coli Determination of O157:H7 1. Preparation of Fe3O4: 3.00g FeCl3·6H2O and 1.00g sodium citrate were added to 100mL ethylene glycol and stirred evenly. Then, 5.00g sodium acetate was added and stirred for 40min. The mixture was then transferred to a polytetrafluoroethylene container, placed in a muffle furnace, heated at 200℃ for 14h, cooled to room temperature, centrifuged at 5000r / min for 10min to collect the solid, and washed three times alternately with pure water and anhydrous ethanol. The solid was then dried under vacuum at 60℃ to obtain magnetic nano-Fe3O4 material. 2. Preparation of Fe3O4@Mo-CDs nanozyme: 0.30g of nano Fe3O4, 0.90g of phosphomolybdic acid, 60μL of ethylenediamine and 0.80g of citric acid were added to 50mL of deionized water and sonicated for 35min. The mixture was then reacted in a microwave at 200℃ for 1h. After cooling naturally to room temperature, the solid was separated by a magnet. The solid was washed twice each with anhydrous ethanol and deionized water and dried under vacuum at 60℃ for 24h to obtain Fe3O4@Mo-CDs nanozyme. 3. The preparation method of the nanozyme-aptamer-Fe3O4@Mo-CDs complex is the same as in Example 1; 4. E. coli The working curve for O157:H7 is created in the same way as in Example 1; 5. In fruit juice beverage samples E. coli Determination of O157:H7 (1) Sample preparation: Take 5 mL of fruit juice beverage sample, dilute it with distilled water at a ratio of 1:20, filter it through a 0.45 μm filter membrane, and obtain the sample test solution; (2) Sample determination: Same as in Example 1, no detectable sample. E. coli O157:H7; (3) Recovery and precision experiments: By analyzing the recovery rate and precision of milk samples... E. coli The feasibility of the constructed biosensor was verified by spiking O157:H7. Three different concentrations of O157:H7 were added to milk samples. E. coli O157:H7 was directly measured without any pretreatment except for dilution with buffer solution, and the results were also measured using the standard plate count method. The results are shown in Table 3. E. coli The spiked recoveries of O157:H7 ranged from 100.1% to 103.5%, with RSDs ranging from 2.5% to 3.4%. This method exhibits good accuracy and precision, comparable to the plate count method. Table 3. Spike recoveries and RSDs of samples (n = 3) .

Claims

1. A method for detecting Escherichia coli in food using Fe3O4@Mo-CDs nanozyme-bound aptamer, characterized in that, Includes the following steps: (1) Add 2.50-3.00g FeCl3·6H2O and 0.60-1.00g sodium citrate to 60-100mL ethylene glycol, stir and mix well, then add 4.50-5.00g sodium acetate, stir for 30-40min, transfer to a polytetrafluoroethylene container, place in a muffle furnace, heat at 180-210℃ for 12-14h, cool to room temperature, collect the solid product and wash it 2-4 times alternately with pure water and anhydrous ethanol, and vacuum dry to obtain magnetic nano Fe3O4; (2) Add 0.10-0.30g nano Fe3O4, 0.30-0.90g phosphomolybdic acid, 50-100μL ethylenediamine and 0.50-0.80g citric acid to 30-50mL deionized water, sonicate for 20-40min, react in microwave at 170-200℃ for 1-2h, cool naturally to room temperature, separate the solid with a magnet, wash the solid 2-3 times each with anhydrous ethanol and deionized water, and vacuum dry to obtain Fe3O4@Mo-CDs nanozyme; (3) Mix the nucleic acid aptamer solution with the Fe3O4@Mo-CDs nanozyme solution and incubate in a 37°C incubator for 25-30 min to obtain the nanozyme-aptamer complex, wherein the nucleotide sequence of the nucleic acid aptamer is 5′-SH-CCGGACGCTTATGCCTTGCCATCTACAGAGGTGTGACGG-3′. (4) Nanozyme and aptamer complex solution, Na+, etc. were added sequentially to Escherichia coli suspensions of different concentrations. + Solution, Mg 2+ Solution, 3,3',5,5'-tetramethylbenzidine solution, and H2O2 solution were diluted to volume with pH 4.0 acetate buffer solution, irradiated with near-infrared light at 808 nm for 10-20 min, separated by magnet, and the supernatant was taken and the absorbance was measured at a wavelength of 654 nm to determine the linear relationship between the concentration of Escherichia coli suspension and the absorbance value, and the regression equation was obtained. (5) The absorbance value of the sample solution to be tested is determined according to the method in step (4), and then substituted into the regression equation to obtain the concentration of Escherichia coli in the sample solution to be tested. Escherichia coli is E. coli O157:H7.

2. The method according to claim 1, characterized in that: In step (3), the concentration of Fe3O4@Mo-CDs nanozyme solution is 0.5 mg / mL, the concentration of nucleic acid aptamer solution is 0.25 μmol / L, and the volume ratio of Fe3O4@Mo-CDs nanozyme solution to nucleic acid aptamer solution is 4-6:

1.

3. The method according to claim 1, characterized in that: The concentration of the Escherichia coli suspension in step (4) is 10. 1 -10 7 CFU / mL; Na + The solution is a 50 mmol / L NaCl solution, and the addition volume is 50-100 μL; Mg 2+ The solutions were: 10 mmol / L MgCl2 solution, added in 50-100 μL; 50 mmol / L 3,3',5,5'-tetramethylbenzidine solution, added in 50-100 μL; 20 mmol / L H2O2 solution, added in 50-100 μL; and 0.1 mmol / L pH 4.0 acetate buffer solution.

4. The method according to claim 1, characterized in that: The power of near-infrared light at 808nm is 1.0-2.0W / cm². 2 .

Images