A method for detecting residual antibiotics in a water environment based on nano-enzyme

Abstract

The application discloses a method for detecting residual antibiotics in water environment based on nano-enzyme, and relates to the technical field of pollutant detection. The method comprises the following steps: uniformly mixing a water sample to be detected, TMB, nano-enzyme, H2O2 and a buffer to obtain a reaction system; after the reaction system is reacted, the absorbance at 652 nm is measured to obtain OD 652nm The OD 652nm is substituted into a standard curve to obtain the content of the antibiotics in the water sample to be detected. The method is simple in operation, strong in portability, short in detection period, capable of realizing qualitative and quantitative detection, optimized in detection steps, accelerated in reaction time, and expanded in the range of use scenarios, and has great potential in the field of on-site and real-time detection of antibiotics in water environment.

Description

Technical Field

[0001] This invention relates to the field of pollutant detection technology, and in particular to a method for detecting residual antibiotics in aquatic environments based on nanozymes. Background Technology

[0002] Nanozymes are a class of nanomaterials with biocatalytic functions. They represent a new generation of artificial enzyme mimics, capable of catalyzing substrates of natural enzymes based on specific nanostructures and serving as substitutes for natural enzymes. As a novel nanomaterial, nanozymes possess both the physicochemical properties of nanomaterials and catalytic functions similar to natural enzymes, combining the advantages of both natural and artificial enzymes. The nanostructure not only endows nanozymes with highly efficient catalytic function but also makes them more stable than natural enzymes, with easily tunable reactivity and easy large-scale production. With the continuous development and increasing variety of nanozymes, and the deepening research into their catalytic mechanisms, they are finding wide application in various fields such as catalytic medicine, sensing and detection, and environmental remediation.

[0003] Antibiotics are secondary metabolites or synthetic analogues produced by microorganisms or higher plants and animals during their life processes, possessing antipathogenic or other activities. Antibiotics are not fully absorbed by humans and animals, and most are excreted in their original form or as active metabolites, ending up in wastewater treatment plants or directly entering the environment. Residual antibiotics in the environment can re-enter the human body through various pathways. Long-term drug stimulation can cause some pathogenic bacteria to mutate and become drug-resistant strains. This resistance can be passed on to the next generation, potentially leading to the emergence of "superbugs." Currently, there are various methods for antibiotic detection, but high-performance liquid chromatography (HPLC) has stringent requirements, necessitating expensive equipment and cumbersome pretreatment processes. Enzyme-linked immunosorbent assay (ELISA) may produce false positives, and the results depend on the antibodies used, resulting in poor accuracy. Therefore, developing efficient, sensitive, and convenient detection methods is crucial.

[0004] Among various detection technologies, colorimetry is the most intuitive. Its principle involves an enzyme-catalyzed colorimetric reaction, allowing for direct visual observation of color changes for qualitative or semi-quantitative analysis. Rapid quantitative detection can then be achieved using instruments or smartphone image processing software. Colorimetric biosensor detection stands out due to its simplicity, speed, and efficiency. It has less dependence on analytical instruments, requires no complex operating procedures, and holds great potential for on-site analysis and immediate diagnosis. This invention aims to develop a method for detecting residual quinolone antibiotics in aquatic environments based on nanozymes, overcoming the problems of complex detection processes, high requirements for the expertise of testing personnel, long detection times, and high costs associated with existing detection technologies. Summary of the Invention

[0005] The purpose of this invention is to provide a method for detecting residual antibiotics in aquatic environments based on nanozymes, thereby addressing the problems existing in the prior art. This method is simple to operate, highly portable, and has a short detection cycle. It not only enables qualitative and quantitative detection but also optimizes the detection steps, accelerates the reaction time, and expands the range of applications, showing great potential for on-site, real-time detection of antibiotics in aquatic environments.

[0006] To achieve the above objectives, the present invention provides the following solution:

[0007] This invention provides a method for preparing nanozymes, comprising mixing Fe-MIL-100 and a soluble copper salt to obtain Fe-MIL-100@Cu. 2+ Then, the Fe-MIL-100@Cu 2+ The step involves calcining the nanozyme to obtain the nanozyme.

[0008] Furthermore, the soluble copper salt is CuCl2∙2H2O.

[0009] Furthermore, the mixing reaction is carried out at a temperature of 25°C for 2 hours.

[0010] Furthermore, the calcination treatment is carried out at a temperature of 750°C for 2 hours.

[0011] The present invention also provides a nanozyme prepared according to the above preparation method.

[0012] The present invention also provides the application of the above-described nanozyme in the detection of quinolone antibiotics.

[0013] Furthermore, the quinolone antibiotic is enrofloxacin, norfloxacin, or ciprofloxacin.

[0014] This invention also provides a method for detecting residual antibiotics in aquatic environments based on nanozymes, comprising mixing a water sample to be tested, TMB, the aforementioned nanozyme, H2O2, and a buffer solution to obtain a reaction system, reacting the reaction system, and measuring the absorbance at 652 nm to obtain the OD. 652 nm , to the OD 652 nm The step of substituting the standard curve to obtain the content of the antibiotic in the water sample to be tested;

[0015] The standard curve was obtained using different concentrations of antibiotics;

[0016] The antibiotic in question is a quinolone antibiotic.

[0017] Furthermore, the concentrations of the TMB, the nanozyme, and the H2O2 in the reaction system are 0.4 mg / mL, 0.16 mg / mL, and 0.25 mM, respectively.

[0018] Furthermore, the reaction is carried out at a pH of 3.5 and a temperature of 30°C.

[0019] The present invention discloses the following technical effects:

[0020] The metal-organic framework material Fe-MIL-100 possesses high specific surface area, thermal stability, and tunable porosity, exhibiting a high loading capacity for metal ions and bio-enzyme molecules. This invention utilizes Fe-MIL-100 as a support, leveraging its effective adsorption of large amounts of Cu. 2+ This allows it to be evenly distributed within the pores of Fe-MIL-100, resulting in Fe-MIL-100@Cu. 2+ The composite, after high-temperature calcination in a nitrogen atmosphere, forms a novel Fe-Cu nanozyme, thereby improving the performance of Fe-MIL-100 and Cu nanozymes used alone. 2+ and Fe-MIL-100@Cu 2+ Peroxidase activity of nanozymes prepared from composites as raw materials.

[0021] This invention develops a method for detecting residual antibiotics in aquatic environments based on nanozymes. The method uses Fe-Cu nanozymes as a reaction catalyst. The Fe-Cu nanozymes catalyze the conversion of H₂O₂ to H₂O, while simultaneously converting colorless 3,3',5,5'-tetramethylbenzidine (TMB) into blue oxidized TMB (oxTMB), which exhibits maximum characteristic absorbance at 652 nm. Quinolone antibiotics such as enrofloxacin (ENR), norfloxacin (NOR), and ciprofloxacin (CIP) can enhance the enzyme activity of Fe-Cu nanozymes and increase the superoxide anion (O₂) concentration. - The generation of oxTMB promotes the production of oxTMB, which can then be used to detect quinolone antibiotics based on changes in substrate color, i.e., changes in the absorbance of oxTMB at 652 nm.

[0022] Compared with existing detection methods, the method of the present invention is simple to operate, highly portable, and has a short detection cycle. It can not only achieve qualitative and quantitative detection, but also optimize the detection steps, speed up the reaction time, and expand the scope of application scenarios. It has great potential for on-site real-time detection of antibiotics in aquatic environments. Attached Figure Description

[0023] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0024] Figure 1 Fe-MIL-100, Fe-MIL-100@Cu 2+ XRD patterns of Fe-Cu nanozymes;

[0025] Figure 2 For Fe-MIL-100(A), Fe-MIL-100@Cu 2+ (B) and SEM images of Fe-Cu nanozymes (C);

[0026] Figure 3 A dotted line graph plotted using different concentrations of enrofloxacin;

[0027] Figure 4 A graph showing the fitted linear relationship using different concentrations of enrofloxacin;

[0028] Figure 5 A bar chart plotted using different concentrations of enrofloxacin;

[0029] Figure 6 A nonlinear relationship plotted using different concentrations of TMB;

[0030] Figure 7 The fitted linear relationship plots were created using different concentrations of TMB;

[0031] Figure 8 The nonlinear relationship plotted using different concentrations of H2O2;

[0032] Figure 9 The graph shows the fitted linear relationship plotted using different concentrations of H2O2;

[0033] Figure 10 Absorption spectra of the experiment investigating the mechanism by which quinolone antibiotics enhance the enzyme activity of Fe-Cu nanozymes;

[0034] Figure 11 OD of different Fe-Cu nanozyme dosages 652 nm Statistical chart;

[0035] Figure 12 OD for different TMB dosages 652 nm Statistical chart;

[0036] Figure 13 OD for different amounts of H2O2652 nm Statistical chart;

[0037] Figure 14 OD under different pH conditions 652 nm Statistical chart;

[0038] Figure 15 OD under different temperature conditions 652 nm Statistical chart;

[0039] Figure 16 For different concentrations of enrofloxacin and OD 652 nm Linear results graph;

[0040] Figure 17 Different concentrations of norfloxacin and OD 652 nm Linear results graph;

[0041] Figure 18 Different concentrations of ciprofloxacin and OD 652 nm Linear results graph;

[0042] Figure 19 OD for specific detection experiments 652 nm Statistical chart. Detailed Implementation

[0043] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0044] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0045] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0046] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be readily apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0047] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0048] Example 1: Preparation, characterization, and enzyme activity analysis of Fe-Cu nanozymes

[0049] I. Preparation of Fe-Cu Nanozymes

[0050] (1) Accurately weigh 1.676 g of terephthalic acid and dissolve it in 24 mL of 1 mol / L sodium hydroxide solution; accurately weigh 2.26 g of ferrous chloride tetrahydrate and dissolve it in 97.2 mL of ultrapure water. After both solutions are completely transparent, mix the two solutions and stir magnetically until homogeneous. Then, place them in a water bath and react at 25°C for 24 h. Centrifuge and wash three times each with ultrapure water and anhydrous ethanol (12000 rpm, 3 min) to obtain the precipitate. Dry in an oven at 55°C for 20 h to obtain Fe-MIL-100.

[0051] (2) Weigh 0.6 g of Fe-MIL-100 and dissolve it in 6 mL of ultrapure water. Add 6 mL of 1 mol / L CuCl2∙2H2O solution and place the mixture in a water bath at 25 °C for 2 h. After centrifugation, wash the precipitate twice with ultrapure water and twice with anhydrous ethanol (12000 rpm, 3 min each). Dry the precipitate in an oven at 55 °C for 20 h to obtain Fe-MIL-100@Cu 2+ .

[0052] (3) The obtained Fe-MIL-100@Cu 2+ The mixture was ground and then calcined at 750℃ for 2 h in a tube furnace under N2 conditions to obtain Fe-Cu nanozymes. The process involved dissolving 0.96 g of sodium hydroxide solid in 24 mL of ultrapure water in a 1 mol / L sodium hydroxide solution and dissolving 1.0229 g of CuCl2∙2H2O solid in 6 mL of ultrapure water in a 1 mol / L CuCl2∙2H2O solution.

[0053] II. Characterization of Fe-Cu nanozymes

[0054] To characterize Fe-MIL-100 and Fe-MIL-100@Cu 2+The crystal structure diffraction peaks of Fe-Cu nanozymes were obtained. The dry powder of each nanomaterial was placed in the sample chamber of an X-ray diffraction instrument for X-ray diffraction (XRD) scanning to characterize the crystal type and lattice parameters of the above three nanomaterials. λ=1.54056, scanning rate was 1° / min, and scanning range was 5~50°.

[0055] The results are as follows Figure 1 As shown, the results indicate that Fe-MIL-100 exhibits distinct X-ray diffraction peaks at positions 9°, 14°, and 16°, demonstrating that the Fe-MIL-100 nanoparticles synthesized in this invention possess a good crystal structure. When Cu is incorporated... 2+ Afterwards, its crystal structure did not change, indicating that Cu 2+ It can be uniformly distributed in Fe-MIL-100 without affecting the integrity of its crystal structure. When Fe-MIL-100@Cu 2+ The nanoparticles obtained after high-temperature calcination in a N2 atmosphere exhibit obvious diffraction peaks at 30°, 35°, and 45°, indicating the formation of new Fe-Cu bimetallic composite nanoparticles with a good crystal structure.

[0056] To observe Fe-MIL-100 and Fe-MIL-100@Cu 2+ To determine the morphology of Fe-Cu nanozymes, the nanomaterials were first placed in an EP tube containing anhydrous ethanol and sonicated for 10 min to ensure uniform dispersion. Then, 10 μL was dropped onto the matte side of aluminum foil (cut into a small piece, avoiding creases), dried in an oven, and stored in a clean EP tube. The surface structure and structural details of the nanomaterials were then observed under a scanning electron microscope.

[0057] like Figure 2 As shown in Figure A, Fe-MIL-100 exhibits uniform nanosheet-like structures; Cu doping... 2+ It has no effect on its appearance. Figure 2 (B); When Fe-MIL-100@Cu 2+ Uniform nanoflower particles were formed after high-temperature calcination in an N2 atmosphere. Figure 2 (C)

[0058] For accurate quantitative analysis of Fe-MIL-100@Cu 2+The Fe and Cu element content in Fe-Cu nanozymes obtained after high-temperature calcination was accurately determined. 50 mg of sample was dissolved in 5 mL of concentrated nitric acid, heated for digestion and decomposition, and then diluted to 25 mL. Finally, inductively coupled plasma optical emission spectrometry (ICP-OES) was used for analysis. The results showed that the Cu element content in Fe-MIL-100@Cu nanozymes was... 2+ There were no significant changes in the Fe-Cu nanozymes obtained after high-temperature calcination, which were 19.8 mg / g and 18.5 mg / g, respectively, indicating that the Fe-MIL-100 used in this invention to immobilize Cu showed no significant changes. 2+ It exhibits good stability, and high-temperature calcination treatment has no effect on the Cu content.

[0059] II. Enzyme Activity Analysis of Fe-Cu Nanozymes

[0060] (1) Effects of different concentrations of enrofloxacin on enzyme activity

[0061] Enrofloxacin standard stock solution was diluted from 5 mg / mL to 5, 10, 25, 50, 100, and 200 μg / mL. Then, 74 μL of acetate buffer (0.2 mM, pH 3.5), 10 μL of Fe-Cu nanozyme (0.2 mg / mL), 1 μL of TMB solution (20 mg / mL), 10 μL of different concentrations of enrofloxacin, and 5 μL of 30% H₂O₂ solution were added sequentially. After thorough mixing, the OD was measured using a multi-mode microplate reader. 652 nm Measured once every 10 seconds, for a total of 140 seconds.

[0062] Based on the measured OD 652 nm Plot a line graph with time on the x-axis and y-axis on the y-axis (see...). Figure 3 Take the OD measured in the first 60 seconds. 652 nm Plot a scatter plot with time on the x-axis and fit a linear relationship (see...). Figure 4 Using the measured OD 652 nm Substituting into the formula B = 100 / (39000 × 0.15) × ΔA, we obtain the enzyme activity B. Substituting into SA = B / m Fe-Cu The specific enzyme activity was determined, where m Fe-Cu ΔA represents the mass of Fe-Cu nanozymes, and ΔA represents OD. 652 nm A bar chart was created with enzyme activity on the ordinate and antibiotic concentration on the x-axis (see...). Figure 5 ).

[0063] (2) Enzyme activity analysis of Fe-Cu nanozymes was performed using TMB as a substrate. Groups 1-9 were treated with 1, 2, 4, 6, 8, 10, 15, 20, and 30 μL of TMB (4 mg / mL), respectively. Each group also contained 20 μL of Fe-Cu nanozyme (0.32 mg / mL) and 1 μL of 30% hydrogen peroxide. The mixture was balanced with acetate buffer (0.2 mM, pH=3.5) to a total volume of 200 μL. OD was measured every 10 s. 652 nm The measurement lasted for 300 seconds, and then the measured OD was used as the basis for the result. 652 nm Plot a scatter plot with time on the x-axis and the final TMB concentration on the x-axis, fit a linear relationship, and calculate the slope as the initial velocity V0. Use Origin to perform nonlinear fitting with the Enzyme Kinetics-Michaelismenten equation (see [link to Origin]). Figure 6 Vm and Km were calculated. A linear relationship was fitted by taking the reciprocals of the TMB concentration and the initial reaction rate (see...). Figure 7 ).

[0064] (3) Using H2O2 as a substrate, the enzyme activity of Fe-Cu nanozyme was analyzed. 1, 2, 4, 6, 8, 10, and 15 μL of 0.05% H2O2 solution were added to groups 1-7, respectively. 20 μL of Fe-Cu nanozyme (0.32 mg / mL) and 2 μL of TMB solution were added to each group, and the total volume was balanced with acetate buffer (0.2 mM, pH=3.5) to a total volume of 200 μL. OD was measured every 10 s. 652 nm The measurement lasted for 300 seconds, and then the measured OD was used as the basis for the result. 652 nm Plot a scatter plot with time on the x-axis and the final H₂O₂ concentration on the x-axis and the initial reaction velocity on the y-axis. Fit a linear relationship and calculate the slope as the initial velocity V₀. Use Origin for nonlinear fitting with the Enzyme Kinetics-Michaelismenten equation (see [link to Origin]). Figure 8 Vm and Km were calculated. A linear relationship was fitted by taking the reciprocals of the H2O2 concentration and the initial reaction rate (see...). Figure 9 ).

[0065] Example 2: Investigation into the mechanism by which quinolone antibiotics enhance the enzyme activity of Fe-Cu nanozymes

[0066] Nitrotetrazolium blue chloride (NBT) can be used as a detector for O2. - The probe utilizes O2 -Nitrogen blue tetrazolium can be reduced to form blue formazan, which has an absorption peak near 560 nm. The superoxide anion O2 is determined by observing the absorption peak at 560 nm in the reaction system and the control group, depending on whether enrofloxacin is added. - How much is produced.

[0067] Set up two experimental groups and one control group:

[0068] Experimental group 1: 20 μL nanozyme solution (0.32 mg / mL), 5 μL 30% H2O2 solution, 10 μL enrofloxacin solution (100 μg / mL), 65 μL NaOH solution (0.1 M) and 100 μL NBT solution (dissolved in 0.6 mM DMSO) were added.

[0069] Experimental group 2: Add 20 μL of nanozyme solution, 5 μL of 30% H2O2 solution, 75 μL of NaOH solution and 100 μL of NBT solution.

[0070] Blank group: 100 μL NaOH solution and 100 μL NBT solution.

[0071] The absorption spectra at wavelengths of 400-800 nm were measured, and the results are shown in [the table below]. Figure 10 .

[0072] Example 3: Optimization of Detection Reaction Conditions

[0073] (1) Dosage of Fe-Cu nanozyme

[0074] Fe-Cu nanozyme was used in groups of 4, 8, 16, 32, 64, and 128 μg, with three replicates per group. Each group consisted of 20 μL enrofloxacin solution (50 μg / mL), 20 μL Fe-Cu nanozyme solution, 10 μL 30% hydrogen peroxide solution, 2 μL TMB (20 mg / mL) solution, and 148 μL acetate buffer (pH 4, 0.2 M). The reaction mixture was incubated in a constant temperature shaker (37℃, 200 rpm) for 10 min, followed by centrifugation for 2 min (12000 rpm). The supernatant was diluted 4-fold and 100 μL was transferred to an ELISA plate. OD was measured using a multi-functional microplate reader. 652 nm .

[0075] The results are as follows Figure 11 As shown, when the amount of Fe-Cu nanozyme is between 4-32 μg, the OD increases with the increase of material dosage. 652 nm The OD continuously increases; when the dosage is between 32-128 μg, the OD increases with the increase of material dosage. 652 nmThe signal continuously decreased. This indicates that the signal was most pronounced at a nanozyme dosage of 32 μg, therefore, a nanozyme dosage of 32 μg is sufficient for the experimental requirements.

[0076] (2) Dosage of TMB

[0077] TMB dosages of 5, 10, 20, 40, 80, 160, and 320 μg were set up, with three replicates per group. Each group contained 20 μL of enrofloxacin solution (50 μg / mL), 10 μL of 30% hydrogen peroxide solution, 20 μL of Fe-Cu nanozyme solution (1.6 mg / mL), 20 μL of TMB solution, and 130 μL of acetate buffer (pH 4, 0.2 M). The reaction mixture was placed in a constant temperature shaker (37℃, 200 rpm) for 10 min, followed by centrifugation for 2 min (12000 rpm). The supernatant was diluted 4-fold and 100 μL was transferred to an ELISA plate. OD was measured using a multi-functional microplate reader. 652 nm .

[0078] The results are as follows Figure 12 As shown, when the amount of TMB is between 5-80 μg, the OD increases with the increase of TMB amount. 652 nm The OD level continuously increases; when the dosage is between 80-320 μg, the OD level increases with the increase of TMB dosage. 652 nm The signal continuously decreased. This indicates that the signal was most pronounced at a nanozyme dosage of 80 μg, therefore, a TMB dosage of 80 μg is sufficient for the experiment.

[0079] (3) Amount of H2O2

[0080] H₂O₂ concentrations of 0.05, 0.15, 0.25, 0.4, 0.5, 0.75, 1, and 1.5 mM were set up, with three replicates for each group. Each group contained 20 μL of enrofloxacin solution (50 μg / mL), 20 μL of TMB solution (4 mg / mL), different amounts of hydrogen peroxide solution, and 20 μL of Fe-Cu nanozyme solution (1.6 mg / mL). The reaction mixture was balanced to 200 μL with acetate buffer (pH 4, 0.2 M) and incubated in a constant temperature shaker (37℃, 200 rpm) for 10 min, followed by centrifugation for 2 min (12000 rpm). The supernatant was diluted 4-fold and 100 μL was transferred to an ELISA plate. OD was measured using a multi-mode microplate reader. 652 nm .

[0081] The results are as follows Figure 13 As shown, when the concentration of H2O2 is between 0.05 and 0.25 mM, the OD increases with increasing H2O2 concentration. 652 nmThe concentration continuously increases; when the concentration is between 0.25-1.5 mM, the OD increases with increasing H2O2 concentration. 652 nm The concentration of H2O2 continuously decreases. This indicates that the signal is most pronounced at a concentration of 0.25 mM. Therefore, an H2O2 concentration of 0.25 mM (i.e., 30% at 5 μL) is sufficient for the experiment.

[0082] (4) Optimization of reaction pH

[0083] Prepare NaAc-HAc buffer (0.2 M) with pH values ​​of 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, and 6. For each group, add 20 μL of enrofloxacin solution (50 μg / mL), 20 μL of TMB solution (4 mg / mL), 20 μL of Fe-Cu nanozyme solution (1.6 mg / mL), and 5 μL of 30% H2O2 solution. Add 135 μL of buffer solution at each pH to obtain the reaction system. Incubate the reaction system in a constant temperature shaker (37℃, 200 rpm) for 10 min, then centrifuge for 2 min (12000 rpm). Dilute the supernatant 4-fold, mix well, and transfer 100 μL to an ELISA plate. Measure the OD using a multi-functional microplate reader. 652 nm .

[0084] The results are as follows Figure 14 As shown, when the pH is between 2 and 3.5, OD increases with increasing pH. 652 nm OD continuously increases; when pH is between 3.5 and 6, OD increases with increasing pH. 652 nm The pH value continuously decreases. This indicates that the signal is most pronounced at a reaction pH of 3.5, therefore, pH 3.5 is the optimal pH for the reaction.

[0085] (5) Optimization of reaction temperature

[0086] The reaction temperatures were set at 15, 20, 25, 30, 37, and 45 °C. For each group, 20 μL of enrofloxacin solution (50 μg / mL), 20 μL of TMB solution (4 mg / mL), 20 μL of Fe-Cu nanozyme solution (1.6 mg / mL), 5 μL of 30% H₂O₂ solution, and 135 μL of acetate buffer (pH 3.5, 0.2 M) were added to obtain the reaction system. The reaction system was placed in a shaker at the above different temperatures (200 rpm) for 10 min, followed by centrifugation for 2 min (12000 rpm). The supernatant was diluted 4-fold and mixed thoroughly. 100 μL of the supernatant was then transferred to an ELISA plate, and the OD was measured using a multi-functional microplate reader. 652 nm .

[0087] The results are as follows Figure 15 As shown, when the temperature is between 15-30℃, OD increases with increasing temperature.652 nm The temperature continues to rise; when the temperature is between 30-45℃, the OD increases with increasing temperature. 652 nm The temperature continuously decreases. This indicates that the signal is most pronounced at a reaction temperature of 30℃, therefore, 30℃ is the optimal reaction temperature.

[0088] In summary, the optimal detection conditions determined in this embodiment are as follows: each group is prepared by adding 20 μL of the sample to be tested, 20 μL of TMB solution (4 mg / mL), 20 μL of Fe-Cu nanozyme solution (1.6 mg / mL), 5 μL of 30% H2O2 solution, and 135 μL of acetate buffer (pH 3.5, 0.2 M), and then reacting in a shaker (30℃, 200 rpm) for 10 min.

[0089] Example 4: Determination of standard curves for enrofloxacin, norfloxacin, and ciprofloxacin solutions and their detection in actual aquatic environments.

[0090] (1) Determination of standard curves for enrofloxacin, norfloxacin, and ciprofloxacin solutions

[0091] Using the optimal detection conditions obtained in Example 3, enrofloxacin standard solutions with concentrations of 0.1, 0.25, 0.5, 1, 2.5, 5, 10, 25, 50, 75, and 100 μg / mL; norfloxacin standard solutions with concentrations of 10, 25, 50, 75, 100, 125, and 150 μg / mL; and ciprofloxacin standard solutions with concentrations of 5, 10, 25, 50, 75, 100, 125, 150, 175, and 200 μg / mL were selected for detection. Figure 16-18 As shown, the B value decreases continuously with increasing concentration of the three antibiotics. The linear relationship between the concentration of different antibiotic solutions and the B value demonstrates that this biosensor detection system can effectively and sensitively detect the antibiotic content in solution, showing great potential in residual antibiotic detection applications. The linear equations, limits of detection, and R values ​​for the three antibiotics are also presented. 2 The values ​​are shown in Table 1.

[0092] Table 1. Linear equations, limits of detection, and R values ​​for various antibiotics. 2 value

[0093]

[0094] (2) Detection in actual aquatic environments

[0095] Sampling: One sample each of water from Longzihu Lake, Beihu Lake, and wastewater from the aquaculture farm.

[0096] Sample preparation: Filter using a 0.45 μm filter, centrifuge (12000 rpm, 2 min) and collect the supernatant.

[0097] Enrofloxacin standard solutions of 10, 25, and 50 μg / mL were added to the supernatant obtained above, respectively. Detection was performed using the optimal detection conditions obtained in Example 3. The B value was recorded, and the detected concentration was obtained by substituting it into the standard curve. The spiked recovery rate was calculated using the formula: Spiked recovery rate = (Detected concentration - Spiked concentration) / Spiked concentration × 100%. The recovery rate was between 93.0% and 115.3%, indicating that the constructed colorimetric biosensor system has good detection performance, and this detection method has greater advantages in on-site detection and emergency monitoring. The detection results of Longzihu Lake water, Beihu Lake water, and aquaculture farm wastewater are shown in Table 2.

[0098] Table 2. Test results of Longzihu Lake water, Beihu Lake water, and aquaculture farm wastewater.

[0099]

[0100] Example 5 Specificity Detection Experiment

[0101] Using the optimal detection conditions obtained in Example 3, enrofloxacin, norfloxacin, ciprofloxacin, florfenicol, erythromycin, polymyxin, streptomycin sulfate, tetracycline, kanamycin sulfate, and blank water samples were tested, and the OD values ​​were compared. 652 nm See the results of the differences Figure 19 The results showed that the detection method of the present invention is specific, with only enrofloxacin, norfloxacin, and ciprofloxacin showing significant differences from the blank water sample.

[0102] Example 6: Comparison of Detection Methods

[0103] The linear range and detection limit of the method of the present invention (using the optimal detection conditions obtained in Example 3) were compared with other detection methods, and the results are shown in Table 3.

[0104] Table 3 Comparison results of different detection methods

[0105]

[0106] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. The application of a nanozyme in the detection of quinolone antibiotics, characterized in that, The preparation method of the nanozyme includes mixing Fe-MIL-100 and a soluble copper salt to obtain Fe-MIL-100@Cu 2+ Then, the Fe-MIL-100@Cu 2+ The step of calcining to obtain the nanozyme; The calcination treatment was carried out at a temperature of 750°C for 2 hours. The mixing reaction was carried out at a temperature of 25°C for 2 hours.

2. The application according to claim 1, characterized in that, The soluble copper salt is CuCl2∙2H2O.

3. The application according to claim 1, characterized in that, The quinolone antibiotics mentioned are enrofloxacin, norfloxacin, or ciprofloxacin.

4. A method for detecting residual antibiotics in an aquatic environment based on nanozymes, characterized in that, The reaction system comprises uniformly mixing the water sample to be tested, TMB, the nanozyme described in any one of claims 1-2, H2O2, and buffer solution to obtain a reaction system. After reacting the reaction system, the absorbance at 652 nm is measured to obtain the OD. 652 nm , to the OD 652 nm The step of substituting the standard curve to obtain the content of the antibiotic in the water sample to be tested; The standard curve was obtained using different concentrations of antibiotics; The antibiotic in question is a quinolone antibiotic.

5. The method according to claim 4, characterized in that, The concentrations of TMB, nanozyme, and H2O2 in the reaction system are 0.4 mg / mL, 0.16 mg / mL, and 0.25 mM, respectively.

6. The method according to claim 4, characterized in that, The reaction was carried out at a pH of 3.5 and a temperature of 30°C.

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