Iron-based nanoszyme and application thereof in detection of quinolone antibiotics

Abstract

The application belongs to the technical field of pollutant detection, and relates to an iron-based nano-enzyme and application thereof in detection of quinolone antibiotics. Trichloride iron hexahydrate and cerium nitrate hexahydrate are added into water and uniformly mixed to obtain a mixed solution A, the mixed solution A is uniformly mixed with an organic polyacid ligand solution, and then a reflux reaction is carried out to prepare a Ce / Fe-MOF precursor. The Ce / Fe-MOF precursor and sodium hypophosphite are added into a tube furnace, and calcination is carried out under a nitrogen atmosphere to prepare Ce / Fe-P. The material synthesized by the application has a mesoporous structure of nanometer prisms, effectively increases the contact area with a substrate, and based on the principle that low-concentration NOR promotes the activity of Ce / Fe-P and high-concentration inhibits the activity of Ce / Fe-P, visual detection of NOR can be realized, and the application has high sensitivity, low detection limit, a wide linear response range, and good selectivity and stability.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of pollutant detection, and relates to an iron-based nanoscale enzyme and application thereof in quinolone antibiotic detection. BACKGROUND

[0002] As a class of drugs, antibiotics can kill bacteria, inhibit the growth and reproduction of bacteria, and treat infections caused by bacteria. Among them, norfloxacin (NOR) has high bacteriostatic activity on aerobic gram-negative bacteria. Due to its antibacterial properties, NOR plays an important role in the treatment of urinary tract infections, prostatitis, intestinal infections and infections caused by sensitive bacteria in the skin. However, long-term use of NOR can cause bacterial resistance, allergic reactions, and may pose a serious risk to human health. Therefore, it is of great significance to detect NOR residues in rivers, lakes and other water bodies. So far, scientists have developed various methods to determine NOR, including cyclic voltammetry, fluorescence spectroscopy, high-performance liquid chromatography and enzyme-linked immunosorbent assay. However, these methods usually require long incubation time, complex electrode preparation process or expensive equipment. SUMMARY

[0003] The application proposes an iron-based nanoscale enzyme and application thereof in quinolone antibiotic detection to solve the problems in traditional quinolone antibiotic detection.

[0004] To achieve the above purpose, the application is implemented by using the following technical solutions:

[0005] A preparation method of an iron-based nanoscale enzyme, comprising the following steps:

[0006] Step (1): add ferric chloride hexahydrate and cerium nitrate hexahydrate into water and mix uniformly to obtain a mixed solution A, then mix the mixed solution A with an organic polyacid ligand solution, and then perform a reflux reaction to prepare a Ce / Fe-MOF precursor.

[0007] Step (2): add the Ce / Fe-MOF precursor and sodium hypophosphite into a tube furnace, and calcine under a nitrogen atmosphere to prepare Ce / Fe-P.

[0008] Preferably, the organic polyacid in step (1) is fumaric acid, and the molar ratio of the ferric chloride hexahydrate to the fumaric acid is 1:1; the molar ratio of the cerium nitrate hexahydrate to the ferric chloride hexahydrate is 1:1-30, preferably 1:6.

[0009] Preferably, the reflux reaction temperature in step (1) is 90-110℃, and the reflux reaction time is 3-5h.

[0010] Preferably, the calcination temperature in step (2) is 330-360℃, and the holding time during calcination is 2-5h.

[0011] The application provides application of the iron-based nanoscale enzyme prepared by the method in detection of quinolone antibiotics, and the quinolone antibiotics are norfloxacin, enrofloxacin and ciprofloxacin.

[0012] The application method is as follows: the iron-based nanoscale enzyme, TMB, H2O2, quinolone antibiotics and NaAc-HAc buffer solution are uniformly mixed to obtain a reaction system, the reaction system is naturally and statically reacted for at least 5 min, then filtered, and the absorbance value at 652 nm is recorded by using an ultraviolet-visible spectrophotometer.

[0013] Preferably, the pH of the NaAc-HAc buffer solution is 3.0, the volume fraction of the NaAc-HAc buffer solution in the reaction system is 75-80%, the concentration of TMB is preferably 0.5 mmol / L, and the concentration of H2O2 is preferably 0.5 mmol / L during detection.

[0014] Compared with the prior art, the application has the advantages and positive effects that:

[0015] 1. The 0.5-Ce / Fe-P material synthesized by the application has a mesoporous structure of nanoprism, effectively increases the contact area with a substrate, and provides more active sites. The addition of Ce ions converts the alpha-Fe2O3 phase into the gamma-Fe2O3 phase, in addition, the introduction of appropriate Ce doping effectively improves the charge transfer rate, and is more conducive to the catalytic oxidation reaction of the nanoscale enzyme.

[0016] 2. The raw materials are cheap and rich in resources, the synthesis cost is low, the product performance is stable, and the product is suitable for large-scale synthesis.

[0017] 3. Based on the principle that low-concentration NOR promotes the activity of 0.5-Ce / Fe-P, and high-concentration NOR inhibits the activity of 0.5-Ce / Fe-P, the visual detection of NOR can be realized, and the method has high sensitivity, a low detection limit (0.78 nM), a wide linear response range (50 nM-2.5 μM and 200 μM-650 μM), and good selectivity and stability. BRIEF DESCRIPTION OF DRAWINGS

[0018] Figure 1 A is an X-ray diffraction pattern of Ce / Fe-MOF, Figure 1 B is an X-ray diffraction pattern of 0.5-Ce / Fe-P nanoscale enzyme.

[0019] Figure 2 A is an SEM image of Ce / Fe-MOF, Figure 2 B is a TEM image of Ce / Fe-MOF, Figure 2 C is an SEM image of 0.5-Ce / Fe-P, Figure 2D is a TEM image of 0.5-Ce / Fe-P.

[0020] Figure 3 D is a high-resolution transmission electron microscopy (HRTEM) image of 0.5-Ce / Fe-P.

[0021] Figure 4 D is a N2 adsorption desorption isotherm (inset: corresponding pore size distribution curve) of 0.5-Ce / Fe-P.

[0022] Figure 5 D is a comparison chart of POD-like activity of different nanoscale enzymes.

[0023] Figure 6 D is a specific detection chart of 0.5-Ce / Fe-P nanoscale enzyme.

[0024] Figure 7 D is a chart of the effect of pH value of buffer solution on the activity of 0.5-Ce / Fe-P nanoscale enzyme. DETAILED DESCRIPTION

[0025] In order to enable a more clear understanding of the above-mentioned objects, features and advantages of the present application, the present application will be further described below with specific examples. It should be noted that the examples of the present application and the features in the examples can be combined with each other without conflict.

[0026] In the following description, many specific details are set forth in order to provide a thorough understanding of the present application. However, the present application can be practiced in a variety of ways beyond the specific details set forth herein, and, accordingly, the present application is not limited to the specifics of the examples disclosed in the following description.

[0027] Example 1

[0028] 3 mmol FeCl3·6H2O and 0.5 mmol Ce(NO3)3·6H2O were added to 30 mL of deionized water, and 3 mmol of fumaric acid was dissolved in 30 mL of deionized water. The two solutions were mixed after being ultrasonicated for 5 min each, and stirred at room temperature for 30 min at a stirring rate of 200 rpm. The resulting mixture was subjected to a reflux reaction at 100°C for 4 h in an oil bath, and after the reaction was completed, the product was washed with ethanol and deionized water alternately by centrifugation 3 times at a centrifugation rate of 9000 rpm for 5 min each time. Then, the product was dried at 60°C for 12 h to obtain a yellow-brown solid (0.5-Ce / Fe-MOF precursor).

[0029] 200 mg of sodium hypophosphite was placed in the upstream of the tube furnace, and 50 mg of the 0.5-Ce / Fe-MOF precursor prepared above was placed in the downstream of the tube furnace. Nitrogen gas was introduced at a rate of 10 mL / min, and the temperature was raised to 300°C at a rate of 5°C / min. After the temperature reached 300°C, the nitrogen gas was replaced with hydrogen gas at a rate of 10 mL / min, and the reaction was allowed to proceed for 2 h. After the reaction was completed, the product was washed with deionized water by centrifugation 3 times at a centrifugation rate of 9000 rpm for 5 min each time, and then dried at 60°C for 12 h to obtain a yellow-brown solid (0.5-Ce / Fe-P). 3 / h, after 5 min of air into the tube furnace, the temperature of the tube furnace was raised to 350℃, the heating rate was 2℃ / min, and the temperature was kept for 5 h. During calcination, sodium hypophosphite decomposed and reacted with the precursor downstream under the action of nitrogen flow to form phosphating. After the temperature was kept, the sample was naturally cooled to room temperature to obtain a black solid, which was recorded as 0.5-Ce / Fe-P.

[0030] The prepared 0.5-Ce / Fe-MOF precursor and 0.5-Ce / Fe-P were characterized by XRD, and the results are shown in Figure 1 Figure 1 It can be seen from

[0031] The prepared 0.5-Ce / Fe-MOF precursor and 0.5-Ce / Fe-P were characterized by SEM and TEM, and the results are shown in Figure 2 Figure 2 It can be seen from Figure 2

[0032] The lattice state of 0.5-Ce / Fe-P is shown in Figure 3 Figure 3 The lattice spacing of 0.30 nm in the middle is the (210) face of Fe7(PO4)6, and the lattice fringes with spacings of 0.25 nm and 0.15 nm are the (112) and (4012) crystal faces of γ-Fe2O3, respectively.

[0033] The N2 adsorption-desorption curve of 0.5-Ce / Fe-P is shown in Figure 4 The curve has a significant hysteresis loop. According to the characterization results, 0.5-Ce / Fe-P has a high specific surface area of 23.7 m 2 / g and a mesoporous structure on the surface. The mesoporous property of nanoscale enzyme is beneficial to the contact between the substrate and the material, and the contact area between the two materials is large, which can provide more active sites, thereby enhancing the catalytic oxidation performance of the material.

[0034] Example 2

[0035] ​​​​The embodiment not specially explained is consistent with embodiment 1. 10 mmol of FeCl3·6H2O and 0.5 mmol of Ce(NO3)3·6H2O were added to 30 mL of deionized water, and 10 mmol of fumaric acid was dissolved in 30 mL of deionized water. After the two solutions were ultrasonically treated for 5 min, they were mixed, stirred at room temperature for 30 min, and the obtained mixture was refluxed in an oil bath at 95°C for 4.5 h. After the reaction was completed, the product was washed and centrifuged with ethanol and water alternately for 3 times, the centrifugal speed was 9000 rpm, and the centrifugal time was 5 min each time. Then, the product was dried at 60°C for 12 h to obtain a yellow-brown solid.

[0036] 200 mg of sodium hypophosphite was placed in the upstream of the tube furnace, 50 mg of the precursor prepared above was placed in the downstream, after the air was replaced by nitrogen, the temperature of the tube furnace was increased to 330°C at a rate of 2°C / min, and the temperature was kept for 4 h. After the calcination was completed, the product was naturally cooled to room temperature to obtain a black solid of iron-based nanoszyme.

[0037] Example 3

[0038] The embodiment not specially explained is consistent with embodiment 1. 10 mmol of FeCl3·6H2O and 0.5 mmol of Ce(NO3)3·6H2O were added to 30 mL of deionized water, and 10 mmol of fumaric acid was dissolved in 30 mL of deionized water. After the two solutions were ultrasonically treated for 5 min, they were mixed, stirred at room temperature for 30 min, and the obtained mixture was refluxed in an oil bath at 95°C for 4.5 h. After the reaction was completed, the product was washed and centrifuged with ethanol and water alternately for 3 times, the centrifugal speed was 9000 rpm, and the centrifugal time was 5 min each time. Then, the product was dried at 60°C for 12 h to obtain a yellow-brown solid.

[0039] 200 mg of sodium hypophosphite was placed in the upstream of the tube furnace, 50 mg of the precursor prepared above was placed in the downstream, after the air was replaced by nitrogen, the temperature of the tube furnace was increased to 330°C at a rate of 2°C / min, and the temperature was kept for 4 h. After the calcination was completed, the product was naturally cooled to room temperature to obtain a black solid of iron-based nanoszyme.

[0040] The iron-based nanoszyme prepared in embodiment 1 was used for performance test.

[0041] 1. POD-like activity determination of iron-based nanoszyme

[0042] The 0.5-Ce / Fe-P nanoscale enzyme prepared in Example 1 was added into ethanol to prepare a dispersion liquid with a concentration of 100 μg / mL, 800 μL of NaAc-HAc buffer solution (pH 3.0) was added with 50 μL of TMB (3,3',5,5'-tetramethylbenzidine disulfate, CAS number: 54827-18-8) and 50 μL of H2O2, then 100 μL of the dispersion liquid was added, and the reaction was allowed to proceed naturally at room temperature for 5 min, then filtered, and the filtrate was detected by a UV-visible spectrophotometer, and the UV-visible absorption spectrum at 652 nm was recorded. The results are shown in Figure 5 C, as shown in Figure 5 C, the 0.5-Ce / Fe-P nanoscale enzyme has excellent POD-like activity.

[0043] 2. Standard curve determination

[0044] By changing the concentration and volume of norfloxacin (NOR, purchased from Sigma-Aldrich), the detection was carried out respectively, and the volume of TMB and H2O2 added was kept at 50 μL, and the total volume of the system was made up to 1 ml by NaAc-HAc buffer solution with pH 3.0, and the UV-visible absorption spectrum at 652 nm was tested after reaction and filtration to test the linear range of the 0.5-Ce / Fe-P nanoscale enzyme. The detection limit was 0.78 nM. Among them, in the concentration range of 50 nM-2.5 μM, y=0.46x+1.57, R 2 =0.980; in the range of 200-650 μM, y=0.0020x+1.46, R 2 =0.984, x is the concentration of NOR, and y is the absorbance.

[0045] 3. Specificity test

[0046] Na + , Ni 2+ , Mn 2+ , K + , Ca 2+ , Cd 2+ , Cl - , glycine (Gly), sugars (glucose Glu, fructose Fru, maltose Mal), and other antibiotics (CIP, ENR) were detected for interference with the detection results. The interfering ions were provided by the following substances: ciprofloxacin (CIP), sodium chloride (Cl - , Na + ), manganese chloride (Mn 2+ ) purchased from China Macklin Biochemical Technology Co., Ltd. Potassium chloride (K +), nickel acetate tetrahydrate (Ni 2+ ), cadmium chloride (Cd 2+ ) were purchased from National Pharmaceutical Group Chemical Reagent Co., Ltd. D-fructose, maltose were purchased from China Huixing Biochemical Reagent Co., Ltd. Calcium chloride (Ca 2+ ) was purchased from Meril, glucose was purchased from Beijing Zhuangmeng International Biological Technology Co., Ltd., enrofloxacin (ENR) was purchased from Aladdin, glycine (Gly) was purchased from National Pharmaceutical Group Chemical Reagent Co., Ltd. The specific operation is as follows: the above-mentioned substances or sugar classes that provide interference ions were weighed and added into 10 ml of deionized water to prepare different interference solutions with the concentration of interference ions reaching 0.26 mol / L, and the concentration of antibiotics CIP and ENR reaching 13 mmol / L. The 0.5-Ce / Fe-P nanoscale enzyme prepared in Example 1 was added into ethanol to prepare a dispersion liquid with a concentration of 100 μg / mL, then 100 μL of the dispersion liquid was added into 750 μL of NaAc-HAc buffer solution (pH 3.0) containing 50 μL of TMB, 50 μL of H2O2 and 50 μL of the interference solution. After natural reaction at room temperature for 5 min, filtration was performed, the filtrate was detected by ultraviolet visible spectrophotometer, and the ultraviolet-visible absorption spectrum at 652 nm was recorded, and the results are shown in Figure 6 . Only after adding other antibiotics, the PO-like activity of 0.5-Ce / Fe-P has similar influence results as NOR.

[0047] 4. Real sample test

[0048] The standard addition method was used to detect the detection of 0.5-Ce / Fe-P on NOR in actual water samples, and the specific operation was as follows: Nanjing University of Technology water and Wahaha pure water were taken for real sample test, standard addition method was used to add different concentrations of NOR to each water sample, and 50 μL of water sample was taken for detection, and the actual content of NOR in each detection group was shown in Table 1. The 0.5-Ce / Fe-P nanoscale enzyme prepared in Example 1 was added into ethanol to prepare a dispersion liquid with a concentration of 100 μg / mL, then 100 μL of the dispersion liquid was added into 750 μL of NaAc-HAc buffer solution (pH 3.0) containing 50 μL of TMB, 50 μL of H2O2 and 50 μL of water sample, and natural reaction was performed at room temperature for 5 min, then filtration was performed, the filtrate was detected by ultraviolet visible spectrophotometer, and the ultraviolet-visible absorption spectrum at 652 nm was recorded, and the results were repeated three times. The working curve was brought in, and the detection amount was calculated. The results are shown in Table 1, and the relative standard deviation of the detection value is ≤3.75% (n = 3), which indicates that the colorimetric sensor has good repeatability in rapid detection of NOR content in water. In addition, the recovery rate is between 98.6-106.8%, which proves that the method has good accuracy and reliability for determination of NOR in actual water samples.

[0049] Table 1 Real sample detection results

[0050]

[0051] Comparative Example 1

[0052] The remaining preparation process was consistent with Example 1, except that no cerium nitrate hexahydrate was added, and the prepared nanoscale enzyme was denoted as Fe-P nanoscale enzyme. The Fe-P nanoscale enzyme was added to ethanol to prepare a dispersion liquid with a concentration of 100 μg / mL. Then, 100 μL of the dispersion liquid was added to 800 μL of NaAc-HAc buffer solution (pH 3.0) containing 50 μL of TMB and 50 μL of H2O2, and reacted naturally at room temperature for 5 min. Then, the solution was filtered, and the filtrate was detected by a UV-visible spectrophotometer, and the UV-visible absorption spectrum at 652 nm was recorded. The results are shown in Figure 5 B. The detection results after the same treatment process of 50 μL of TMB, 50 μL of H2O2, and 900 μL of NaAc-HAc buffer solution (pH 3.0) were compared, and the results are shown in Figure 5 A. and Figure 5 B, it can be seen that the POD activity of the Fe-P nanoscale enzyme prepared in this comparative example is lower than that of the 0.5-Ce / Fe-P nanoscale enzyme prepared in Example 1.

[0053] Comparative Example 2

[0054] The difference between this comparative example and Example 1 is that the pH of the buffer solution is different. The NaAc-HAc buffer solution with a pH value of 2, 4, 5, 6, 7, and 8 was sequentially replaced, and the remaining conditions were consistent with Example 1. After the sample was treated by the same procedure for the determination of the POD activity of the iron-based nanoscale enzyme, the UV-visible absorption spectrum at 652 nm was recorded, and the results are shown in Figure 7 A. and Figure 7 B. It can be seen that when the pH of the buffer solution is 3.0, the absorbance value is the highest.

[0055] The above is only a preferred embodiment of the present application, and is not intended to limit the other forms of the present application. Any person skilled in the art can use the disclosed technical content to make changes or modifications to equivalent embodiments applied to other fields, but any simple modification, equivalent change, and modification of the above embodiments made in accordance with the technical essence of the present application still falls within the protection scope of the technical solution of the present application.

Claims

1. A method for preparing an iron-based nanoszyme, characterized in that, The steps are as follows: Step (1): the ferric chloride hexahydrate and cerium nitrate hexahydrate are added into water and mixed uniformly to obtain a mixed solution A, the mixed solution A is mixed uniformly with an organic polybasic acid ligand solution, then reflux reaction is carried out, and a Ce / Fe-MOF precursor is prepared; Step (2): the Ce / Fe-MOF precursor and sodium hypophosphite are added into a tube furnace, and calcination is carried out under a nitrogen atmosphere to prepare a Ce / Fe-P.

2. The method of claim 1, wherein the iron-based nanoszyme is prepared by, The organic polybasic acid in step (1) is fumaric acid, the molar ratio of the ferric chloride hexahydrate to the fumaric acid is 1:1, and the molar ratio of the cerium nitrate hexahydrate to the ferric chloride hexahydrate is 1:1-30.

3. The method for preparing iron-based nanozymes according to claim 1, characterized in that, The reflux reaction temperature in step (1) is 90-110 DEG C, and the reflux reaction time is 3-5 h.

4. The method of claim 1, wherein the iron-based nanoscale enzyme is prepared by, The calcination temperature in step (2) is 330-360 DEG C, and the calcination time is 2-5 h.

5. The application of the iron-based nanoscale enzyme prepared by the method in any one of claims 1-4 in quinolone antibiotic detection.

6. Use according to claim 5, characterized in that, The application method is as follows: the iron-based nanoscale enzyme, TMB, H2O2, quinolone antibiotic and NaAc-HAc buffer solution are mixed uniformly to obtain a reaction system, the reaction is allowed to stand naturally for at least 5 min, then filtration is carried out, and then the absorbance value at 652 nm is recorded by using a UV-visible spectrophotometer.

7. Use according to claim 6, characterized in that, The pH of the NaAc-HAc buffer solution is 3.0, and the volume fraction of the NaAc-HAc buffer solution in the reaction system is 75-80%.

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