Cerium-based nano-enzyme, preparation method thereof and application of cerium-based nano-enzyme in organophosphorus pesticide detection

Abstract

The invention discloses a cerium-based nano-enzyme, a preparation method thereof and application of the cerium-based nano-enzyme in organophosphorus pesticide detection, and belongs to the technical field of pesticide residue detection. The cerium-based nano-enzyme has activity similar to that of organophosphorus hydrolase, and comprises a nitrogen-doped carbon nanosheet substrate and Ce2O2CN2 nano-particles loaded on the surface of the nitrogen-doped carbon nanosheet substrate. The nano-enzyme provided by the invention avoids the use of natural enzymes (such as organophosphorus hydrolase, acetylcholin esterase, alkaline phosphatase and the like) which are expensive in price, difficult to store and harsh in reaction condition, and realizes low-cost rapid detection of paraoxon. Compared with the reported Ce-based nano-enzyme, the nano-enzyme prepared by the invention is simple in preparation method, good in stability, small in usage amount, mild in reaction condition (37 DEG C), high in detection speed and wide in measurement range, a test result can be directly displayed by an ultraviolet-visible spectrophotometer, on-site real-time detection can be realized, and the pesticide paraoxon can be quickly and accurately detected.

Description

Technical Field

[0001] The present invention belongs to the technical field of pesticide residue detection, and specifically relates to a cerium-based nanozyme and a preparation method thereof, and application thereof in the detection of organophosphorus pesticides. Background Art

[0002] The information disclosed in this background technology section is only intended to enhance the understanding of the overall background of the invention, and should not necessarily be regarded as an admission or any form of suggestion that the information constitutes the prior art already known to a person skilled in the art.

[0003] At present, the detection methods of pesticide residues in agricultural products can be divided into two categories: one is based on traditional instrumental analysis, mainly including gas chromatography, high performance liquid chromatography, gas chromatography-mass spectrometry and liquid chromatography-mass spectrometry. These instrumental analysis methods have the advantages of high detection accuracy and good reproducibility, providing effective technical support for the detection of pesticide residues, but there are also some shortcomings, such as: the detection instrument is relatively expensive, the sample pretreatment steps are complicated, the operation is cumbersome, and the detection time is long. These shortcomings have caused certain troubles in realizing low-cost rapid detection of pesticide residue samples. The second category is based on the principle of biological detection technology for pesticide residue detection, mainly including immunoassay, enzyme inhibition detection, biosensor method, etc. The most widely used immunoassay is enzyme-linked immunosorbent assay, which is based on the specific binding between antigen and antibody to detect organophosphorus pesticide residues. Although this method has good specificity and sensitivity, the preparation of antibodies is too complicated and expensive, which also limits its application in pesticide residue detection.

[0004] Nanozymes are a type of nanomaterials with natural enzyme-like activity. They have both the properties of nanomaterials and the properties of enzymes. As a new generation of mimetic enzymes, their emergence has enriched the research on mimetic enzymes, expanding them from organic complexes to inorganic nanomaterials, and also broadening the scope of application of nanomaterials. Compared with traditional natural enzymes, nanozymes have the characteristics of low production cost, good stability, high catalytic efficiency and scalable preparation. They have been widely used in different fields such as materials science, medicine, chemistry, biology, and environment. So far, research on nanozymes has mainly focused on redox, and research on other types of nanozymes such as hydrolases is relatively rare. The development of nanozymes with organophosphorus hydrolase-like activity has become an important way to degrade and detect organophosphorus pesticides.

[0005] At present, the reported nanozymes with organophosphorus hydrolysis activity generally have problems such as narrow pH range and temperature range, large dosage, etc., such as the Ce-NC nanozyme disclosed in patent CN113418909A. Therefore, it is of great significance to design new nanozymes with high hydrolase activity to achieve rapid degradation of organophosphorus pesticides and detect their residual amount. Summary of the invention

[0006] In order to address the deficiencies of the prior art, the purpose of the present invention is to provide a cerium-based nanozyme and a preparation method thereof and application in the detection of organophosphorus pesticides. The cerium-based nanozyme provided by the present invention has the characteristics of simulating the activity of organophosphorus hydrolase (OPH) and can achieve low-cost rapid detection of paraoxon.

[0007] In order to achieve the above object, the technical solution of the present invention is:

[0008] The first aspect of the present invention provides a cerium-based nanozyme having organophosphorus hydrolase-like activity; the cerium-based nanozyme comprises: a nitrogen-doped carbon nanosheet substrate, and Ce2O2CN2 nanoparticles loaded on the surface of the nitrogen-doped carbon nanosheet substrate.

[0009] In some embodiments of the present invention, the loading amount of the Ce2O2CN2 nanoparticles is 1-20%, preferably 5-15%.

[0010] It should be noted that the term loading amount refers to the mass ratio of Ce2O2CN2 nanoparticles to the mass ratio of nitrogen-doped carbon nanosheet substrates. A loading amount of 1-20% means that the mass of Ce2O2CN2 nanoparticles is 1-20% of the mass of Ce2O2CN2 nanoparticles.

[0011] The second aspect of the present invention provides a method for preparing the cerium-based nanozyme, comprising:

[0012] Melting urea, then adding polyethylene glycol and cerium salt to obtain a mixed solution;

[0013] The mixed solution is pyrolyzed under a protective atmosphere to obtain a cerium-based nanozyme.

[0014] In some embodiments of the present invention, the mass ratio of urea, polyethylene glycol and cerium salt is 25:1:0.05-0.1, preferably 25:1:0.08.

[0015] In some embodiments of the present invention, the molecular weight of the polyethylene glycol is 3800-4200, preferably 4000.

[0016] In some embodiments of the present invention, the cerium salt includes one of cerium acetates.

[0017] In some embodiments of the present invention, the urea is melted at a temperature of 140-160°C.

[0018] In some embodiments of the present invention, the pyrolysis is carried out at a heating rate of 1-5°C / min, and the temperature is raised to 850-950°C and then kept at this temperature for 1-3 hours.

[0019] In some embodiments of the present invention, the protective atmosphere is an inert gas or nitrogen, preferably nitrogen.

[0020] The third aspect of the present invention provides a use of the cerium-based nanozyme as a hydrolase-like catalyst.

[0021] In some embodiments of the present invention, the application is: the cerium-based nanozyme according to claim 1 is used as an organophosphorus hydrolase-like catalyst for detecting organophosphorus pesticides.

[0022] A fourth aspect of the present invention provides a method for detecting an organophosphorus pesticide, comprising:

[0023] The cerium-based nanozyme or the cerium-based nanozyme prepared by the preparation method is added to the test solution. The cerium-based nanozyme catalyzes the hydrolysis of paraoxon into nitrophenol (p-NP). The organophosphorus pesticides are qualitatively detected by the color change of the test solution, and the organophosphorus pesticides are quantitatively detected by detecting the absorbance value of the supernatant within 300 to 500 nm.

[0024] In some embodiments of the present invention, the quantitative detection is: detecting the absorbance value of the supernatant at 400 nm, and inserting it into the standard working curve of the organophosphorus pesticide to obtain the concentration of the organophosphorus pesticide in the test solution.

[0025] The beneficial effects of the present invention are:

[0026] The present invention provides a novel cerium-based nanozyme Ce2O2CN2 / NC, and it is found that the Ce2O2CN2 / NC nanozyme has the characteristics of simulating the activity of organophosphorus hydrolase (OPH), and has excellent environmental adaptability, can be stored for a long time at different pH and / or different temperatures, is not inactivated, and has high stability. Compared with the traditional method for detecting paraoxon, the nanozyme provided by the present invention avoids the use of expensive, difficult to preserve, and harsh reaction conditions of natural enzymes (such as: organophosphorus hydrolase, acetylcholinesterase, alkaline phosphatase, etc.), and realizes low-cost rapid detection of paraoxon. Compared with the reported Ce-based nanozyme, the nanozyme prepared by the present invention has a simple preparation method, good stability, faster hydrolysis rate, less usage, mild reaction conditions (37°C), fast detection speed, wide measurement range, and the test results can be directly displayed by a UV-visible spectrophotometer, which can realize on-site real-time detection, which is conducive to rapid and accurate detection of the pesticide paraoxon. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] The accompanying drawings in the specification, which constitute a part of the present invention, are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute improper limitations on the present invention.

[0028] Figure 1This is the XRD pattern of the cerium-based nanozyme prepared in Example 1 of the present invention;

[0029] Figure 2 This is a transmission electron microscopy image of the cerium-based nanozyme prepared in Example 1 of the present invention;

[0030] Figure 3 This is a high-resolution TEM image of the cerium-based nanozyme prepared in Example 1 of the present invention;

[0031] Figure 4 High-angle annular dark-field scanning transmission electron microscopy (HAADF-STEM) and in-situ energy dispersive X-ray spectroscopy (EDS) elemental mapping diagrams of the cerium-based nanozyme prepared in Example 1 of the present invention;

[0032] Figure 5 This is a reaction principle diagram of the cerium-based nanozyme prepared in the present invention catalyzing the hydrolysis of paraoxon;

[0033] Figure 6 This is the simulation result of the organophosphorus hydrolase activity of the cerium-based nanozyme prepared in Example 1 of the present invention;

[0034] Figure 7 The paraoxon standard working curve prepared when the cerium-based nanozyme prepared in Example 1 of the present invention detects organophosphorus pesticides;

[0035] Figure 8 Comparison of the catalytic hydrolysis of paraoxon by the cerium-based nanozyme prepared in Example 1 of the present invention and commercial nano CeO2 at different concentrations;

[0036] Fig. 9 Comparison of the catalytic hydrolysis of paraoxon by the cerium-based nanozyme prepared in Example 1 of the present invention and natural organophosphorus hydrolase (OPH) at different pH values;

[0037] Fig.10 The catalytic hydrolysis of paraoxon by the cerium-based nanozyme prepared in Example 1 of the present invention and natural organophosphorus hydrolase (OPH) at different temperatures was compared. DETAILED DESCRIPTION

[0038] In order to enable those skilled in the art to more clearly understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below in conjunction with specific embodiments.

[0039] Example 1

[0040] A method for preparing a cerium-based nanozyme comprises the following steps:

[0041] Step 1: Heat 5g of urea solid at 150°C to melt it into liquid, then add 0.2g of polyethylene glycol (PEG, molecular weight 4000) and 0.016g of cerium acetate into the urea liquid, and stir for 5 minutes to form a uniform mixed solution.

[0042] Step 2: Pour the mixed solution into an alumina crucible, pyrolyze it at 900°C for 2 hours under nitrogen atmosphere protection, with a heating rate of 2°C / min, and finally obtain nitrogen-doped carbon-loaded Ce2O2CN2 composite material (abbreviated as Ce2O2CN2 / NC).

[0043] The crystal structure of Ce2O2CN2 / NC synthesized in this example was determined and analyzed by XRD. Figure 1 As shown, it has a good crystalline structure. The diffraction peaks at 10.561°, 26.041°, 28.181°, 32.081°, 33.841°, 41.081°, 45.971° and 47.311° correspond to the (001), (100), (101), (003), (102), (103), (110) and (111) crystal planes. These diffraction peaks are completely consistent with the peak positions of the standard tetragonal phase of Ce2O2CN2 / NC (ICSD PDF#49-1163), confirming the existence of Ce2O2CN2.

[0044] Transmission electron microscopy images show that the Ce2O2CN2 / NC material presents a sheet-like structure as a whole, and the surface is loaded with obvious nanoparticles ( Figure 2 ). The lattice spacing of the nanoparticles was measured by high-resolution TEM images to be 0.174 nm, which is attributed to the (110) crystal plane of Ce2O2CN2 ( Figure 3 ). High-angle annular dark-field scanning transmission electron microscopy (HAADF-STEM) and in situ energy dispersive X-ray spectroscopy (EDS) elemental mapping images show that Ce2O2CN2 nanoparticles are evenly distributed on the nitrogen-doped carbon nanosheet substrate ( Figure 4 ).

[0045] In summary, Ce2O2CN2 / NC was successfully prepared.

[0046] Example 2

[0047] A method for preparing a cerium-based nanozyme, the difference between the preparation method and Example 1 is that the amount of cerium acetate added is 0.01 g. The remaining steps are the same as those of Example 1.

[0048] Example 3

[0049] A method for preparing a cerium-based nanozyme, the difference between the preparation method and Example 1 is that the amount of cerium acetate added is 0.02 g. The remaining steps are the same as those of Example 1.

[0050] Experimental example: Simulating organophosphorus hydrolase activity

[0051] (1) Qualitative detection of paraoxon

[0052] Experimental system a: The catalytic reaction system contains 50 μg / mL paraoxon, 400 μg / mL Ce2O2CN2 / NC nanozyme (prepared in Example 1), and 10 mM CHES buffer with a pH of 9.0. The reaction is carried out at 37°C for 60 minutes, centrifuged, and the supernatant is taken and its absorbance value within 300-500 nm is detected by a UV-visible spectrophotometer.

[0053] Two other control experiments were conducted:

[0054] In the control experiment b, Ce2O2CN2 / NC nanozyme was not added to the catalytic reaction system, and the other conditions were the same as those of the experimental system a. The absorbance value was detected after the same reaction time.

[0055] In control experiment c, the catalytic reaction system did not contain paraoxon, and the other conditions were the same as those in experimental system a. The absorbance value was measured after the same reaction time.

[0056] Results: Figure 6 As shown, experimental system a showed an obvious ultraviolet absorption peak at around 400nm, and a yellow product p-nitrophenol (p-NP) was generated, indicating that Ce2O2CN2 / NC nanozyme has obvious organophosphorus hydrolase-like activity.

[0057] There is no obvious absorption peak near 400nm in control experiment b, indicating that without nanozyme as catalyst, paraoxon cannot be hydrolyzed; there is no obvious absorption peak near 400nm in control experiment c, indicating that the peak of experimental system a is not the response peak of Ce2O2CN2 / NC itself.

[0058] (2) Quantitative detection of paraoxon

[0059] Scheme: Different concentrations of paraoxon (0, 7.2 μmol / L, 18 μmol / L, 36 μmol / L, 72 μmol / L, 108 μmol / L and 144 μmol / L) were reacted with Ce2O2CN2 / NC nanozyme (400 μg / mL) respectively. After reacting at 37°C for 60 minutes, the mixture was centrifuged and the supernatant was taken.

[0060] Results: Observe the color of each supernatant. The color of the supernatant gradually changes from colorless to lighter bright yellow and then to rich bright yellow. That is, the color gradually deepens with the increase of paraoxon concentration. Take each of the above supernatants and use a UV-visible spectrophotometer or an enzyme marker to detect its absorbance within 300-500nm. Draw the paraoxon standard working curve after deducting the blank control from the absorbance at 400nm. Figure 7 As shown, the linear range is 7.2-144 μmol / L, y=0.0117x-0.0933 (R 2 =0.9998), and the limit of detection (LOD) was 0.135 μmol / L.

[0061] In summary, Ce2O2CN2 / NC has organophosphorus hydrolase-like activity and can catalyze the hydrolysis of organophosphorus pesticide paraoxon into p-nitrophenol (p-NP) at 37°C. p-Nitrophenol (p-NP) is bright yellow and has an obvious ultraviolet absorption peak at 400nm. The organophosphorus paraoxon can be qualitatively detected by the color of the supernatant and quantitatively detected by a UV-visible spectrophotometer or an enzyme reader.

[0062] (3) Advantages of Ce2O2CN2 / NC as an organophosphorus hydrolase

[0063] Compared with commercial nano-CeO2: The 400 μg / mL Ce2O2CN2 / NC in experimental system a was replaced with 400 μg / mL and 10 mg / mL commercial nano-CeO2, respectively, and the other conditions remained unchanged.

[0064] Results: Figure 8 As shown in the figure, at the same nanozyme concentration, nano-CeO2 has no obvious catalytic effect on the hydrolysis of paraoxon. When its concentration is increased to 10 mg / mL, nano-CeO2 shows a slight catalytic effect. It can be seen that the hydrolysis effect of Ce2O2CN2 / NC nanozyme is much better than that of nano-CeO2.

[0065] Comparison with natural organophosphorus hydrolase (OPH): To prove that Ce2O2CN2 / NC has higher stability and wider application range, Ce2O2CN2 / NC and OPH were first incubated at pH 2-10 or 10-90℃ for 2h, and then their activities were measured under standard conditions (37℃, pH 9) (the test conditions were the same as experimental system a).

[0066] Fig. 9 and 10It shows that the activity of OPH is significantly reduced after incubation at low pH or high temperature. In contrast, Ce2O2CN2 / NC still maintains high catalytic activity after incubation in a wide pH and temperature range. It can be seen that Ce2O2CN2 / NC has very excellent storage stability, making it suitable for various harsh storage environments and can be used more widely in biochemistry and industry, especially dephosphorylation reactions in high-temperature industries.

[0067] On the whole, the Ce2O2CN2 / NC nanozyme provided by the present invention has high organophosphorus hydrolase activity and strong stability.

[0068] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A cerium-based nanozyme, characterized in that: It has organophosphorus hydrolase-like activity; the cerium-based nanozyme comprises: a nitrogen-doped carbon nanosheet substrate, and Ce2O2CN2 nanoparticles loaded on the surface of the nitrogen-doped carbon nanosheet substrate.

2. The cerium-based nanozyme according to claim 1, characterized in that The loading amount of the Ce2O2CN2 nanoparticles is 1-20%, preferably 5-15%.

3. A method for preparing the cerium-based nanozyme according to claim 1 or 2, characterized in that: include: Melting urea, then adding polyethylene glycol and cerium salt to obtain a mixed solution; The mixed solution is pyrolyzed under a protective atmosphere to obtain a cerium-based nanozyme.

4. The preparation method according to claim 3, characterized in that: The mass ratio of urea, polyethylene glycol and cerium salt is 25:1:0.05-0.1, preferably 25:1:0.08; Preferably, the molecular weight of the polyethylene glycol is 3800-4200; Preferably, the cerium salt comprises one of cerium acetate; The urea is melted at a temperature of 140-160°C.

5. The preparation method according to claim 3, characterized in that: The pyrolysis is carried out at a heating rate of 1-5°C / min, and after heating to 850-950°C, the pyrolysis is maintained for 1-3 hours.

6. The preparation method according to claim 3, characterized in that: The protective atmosphere is an inert gas or nitrogen, preferably nitrogen.

7. Use of the cerium-based nanozyme according to claim 1 or 2 as a hydrolase-like catalyst.

8. The use according to claim 7, characterized in that The application is: the cerium-based nanozyme according to claim 1 is used as an organophosphorus hydrolase-like catalyst for detecting organophosphorus pesticides.

9. A method for detecting organophosphorus pesticides, characterized in that: include: The cerium-based nanozyme described in claim 1 or 2, or the cerium-based nanozyme prepared by the preparation method described in any one of claims 2 to 6, is added to the test solution. The cerium-based nanozyme catalyzes the hydrolysis of paraoxon into nitrophenol. The organophosphorus pesticides are qualitatively detected by the color change of the test solution, and the organophosphorus pesticides are quantitatively detected by detecting the absorbance value of the supernatant within 300 to 500 nm.

10. The detection method according to claim 9, characterized in that: The quantitative detection is as follows: detecting the absorbance value of the supernatant at 400 nm, and inserting it into the standard working curve of the organophosphorus pesticide to obtain the concentration of the organophosphorus pesticide in the solution to be tested.

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