A copper-chromium doped Prussian blue nanozyme and its preparation method and application in rapid detection of glyphosate

Abstract

The invention discloses a copper-chromium doped Prussian blue nanozyme and its preparation method and application in rapid detection of glyphosate, and is particularly related to the field of pesticide detection. The preparation method comprises the following steps: (1) adding citric acid and potassium ferrocyanide to water, stirring, and dissolving to obtain reaction solution A; (2) adding ferric nitrate and chromium chloride to water, stirring, and dissolving to obtain reaction solution B; (3) adding the reaction solution B obtained in step (2) dropwise to the reaction solution A obtained in step (1), stirring, and reacting; washing the product after the reaction with ethanol and deionized water and centrifuging three times, freeze-drying, and obtaining material PB-Cr; (4) adding copper sulfate and PB-Cr obtained in step (3) to a buffer solution, reacting, and obtaining copper-chromium doped Prussian blue nanozyme. Beneficial effect: The nanozyme prepared by the present invention has excellent catalase-like activity, can be combined with glyphosate, reduce its catalase-like activity, and indirectly realize the detection of glyphosate.

Description

Technical Field

[0001] The present invention relates to the field of pesticide detection, and in particular to a copper-chromium-doped Prussian blue nanozyme, a preparation method thereof, and an application thereof in rapid detection of glyphosate. Background Art

[0002] Glyphosate (GLY) is a commonly used non-selective broad-spectrum herbicide with high herbicidal activity and relatively low toxicity. It is widely used in tea gardens, farmlands, orchards, forestry, etc. With the large-scale planting of glyphosate-resistant genetically modified crops, the use of glyphosate has further increased. Glyphosate has now become the world's largest herbicide produced and used. Long-term and widespread use has led to a large amount of glyphosate residues in surface water, soil, and crops, which seriously threatens human health. Studies have shown that glyphosate has potential carcinogenic effects and was listed as a Class 2A carcinogen by the International Agency for Research on Cancer (IARC) in 2015. Glyphosate can also inhibit the activity of acetylcholinesterase (AChE) and cause myocardial, respiratory and neuromuscular dysfunction. Further studies have found that glyphosate can reduce the secretion of male steroid hormones, thereby leading to decreased fertility. At present, more than 30 countries or regions around the world have begun to ban or restrict the use of glyphosate and have strengthened the monitoring of glyphosate residues. my country's national standard "Maximum Residue Limits of Pesticides in Food GM2763-2019" limits the maximum residue of glyphosate in crops such as rice, wheat, corn, fruit, sugarcane and cottonseed oil; "Drinking Water Hygiene Standard GB5749-2006" limits the maximum concentration of glyphosate in drinking water to 0.7ppm. Therefore, it is urgent to develop a glyphosate detection method with high sensitivity, strong specificity, low cost, simple and effective.

[0003] At present, the detection methods of glyphosate are usually high performance liquid chromatography and gas chromatography combined with mass spectrometry. There is no chromophore or fluorophore in the structure of glyphosate, and it has the characteristics of strong polarity, high water solubility and low volatility. A derivatization procedure must be performed to improve the detection sensitivity of glyphosate. Other reported glyphosate detection methods include enzyme-linked immunosorbent assay, ion chromatography and inductively coupled plasma mass spectrometry. These methods require complex operating steps, expensive instruments and professional operators, which limit the application of such methods. For example, enzyme-linked antibodies are necessary for enzyme-linked immunosorbent assay, but antibodies are easily inactivated by high temperatures. Therefore, it has become a top priority to develop simple, low-cost, rapid and sensitive glyphosate detection methods.

[0004] In recent years, nanomaterials with bio-enzyme-like activity (referred to as nanozymes) have attracted widespread attention from researchers. The types of nanozymes mainly include metals, metal compounds, metal complexes and carbon-based materials. Metal compound or metal complex nanozymes usually exhibit peroxidase-like activity or catalase-like activity, while glyphosate contains phosphonate, carboxyl and amine groups, which can strongly coordinate with metal atoms and affect the catalytic activity of its nanozymes.

[0005] The Chinese patent application document with publication number CN115266702A discloses a method for preparing a nanomaterial with biomimetic enzyme activity and the application of the nanomaterial in glyphosate detection, wherein the nanomaterial with biomimetic enzyme activity is Fe3O4 nanoparticles modified with heptanoic acid and Prussian blue to obtain Fe3O4@C7 / PB with a core-shell structure, wherein the core is ferroferric oxide and the shell is compounded with heptanoic acid and Prussian blue. This patent synthesizes a nanozyme (Fe3O4@C7 / PB) with peroxidase-like activity by Fe3O4 nanoparticles modified with heptanoic acid and Prussian blue. The peroxidase-like activity is higher than that of Fe3O4@C7, and Fe3O4@C7 / PB can be kept stable by a monolayer PB to prevent agglomeration. However, this method is complicated to operate and has high detection costs, and needs to be further improved. Summary of the invention

[0006] The technical problem to be solved by the present invention is how to solve the problems of complicated operation and long detection time of the existing glyphosate detection method.

[0007] The present invention solves the above technical problems through the following technical means:

[0008] A method for preparing copper-chromium doped Prussian blue nanozyme, characterized in that it comprises the following steps:

[0009] (1) adding citric acid and potassium ferrocyanide into water, stirring, and dissolving to obtain a reaction solution A;

[0010] (2) adding ferric nitrate and chromium chloride into water, stirring, and dissolving to obtain reaction solution B;

[0011] (3) adding the reaction solution B obtained in step (2) dropwise into the reaction solution A obtained in step (1), stirring and reacting; washing the product after the reaction with deionized water, then adding alcohol and centrifuging, repeating the process three times, and freeze-drying to obtain a material named PB-Cr;

[0012] (4) Add copper sulfate and the PB-Cr obtained in step (3) to a buffer solution for reaction to obtain copper-chromium-doped Prussian blue nanozyme, named PB-Cr-Cu.

[0013] Beneficial effects: The present invention reacts chromium-doped Prussian blue with copper sulfate to obtain copper / chromium-doped Prussian blue, and the introduction of copper significantly improves the catalase-like activity of chromium-doped Prussian blue. At the same time, under weakly alkaline conditions, glyphosate coordinates with copper atoms, reducing the catalase-like activity of copper / chromium-doped Prussian blue nanozymes, thereby indirectly realizing the detection of glyphosate, which is simple to operate, short in time, and convenient and fast.

[0014] Preferably, the molar ratio of citric acid to potassium ferrocyanide in step (1) is (55-65):(1-3).

[0015] Preferably, the molar ratio of citric acid to potassium ferrocyanide in step (1) is 60:2.

[0016] Preferably, in step (2), the molar ratio of ferric nitrate to chromium chloride is 1:1.

[0017] Preferably, the solution dropping speed in step (3) is 0.3-0.8 mL / min.

[0018] Preferably, in step (3), the stirring speed is 350-450 r / min, and the stirring time is 2-4 h.

[0019] Preferably, in step (4), the mass ratio of copper sulfate to PB-Cr is (1-3): (4-6).

[0020] Preferably, in step (4), the mass ratio of copper sulfate to PB-Cr is 2:5.

[0021] Preferably, the buffer in step (4) is 0.1 M HEPES buffer.

[0022] Preferably, the pH of the buffer solution in step (4) is 8.0.

[0023] The present invention also provides a copper-chromium-doped Prussian blue nanozyme prepared by the above method.

[0024] The present invention also provides an application of the copper-chromium doped Prussian blue nanozyme prepared by the above method in the rapid detection of glyphosate.

[0025] The advantages of the present invention are:

[0026] (1) The present invention reacts chromium-doped Prussian blue with copper sulfate to obtain copper / chromium-doped Prussian blue, and the introduction of copper element significantly improves the catalase-like activity of chromium-doped Prussian blue. At the same time, under weak alkaline conditions, glyphosate coordinates with copper atoms, reducing the catalase-like activity of copper / chromium-doped Prussian blue nanozyme, thereby indirectly realizing the detection of glyphosate, which is simple to operate, short in time, and convenient and fast.

[0027] (2) The nanozyme prepared by the present invention has excellent catalase-like activity and can combine with glyphosate to reduce its catalase-like activity, thereby indirectly realizing the detection of glyphosate.

[0028] (3) Another beneficial effect of the present invention is that the preparation process of the nanozyme is simple and convenient, the preparation method is suitable for mass production, and the detection instrument is small in size and easy to carry, which is suitable for on-site detection.

[0029] (4) The present invention designs and prepares a simple, low-cost copper / chromium-doped Prussian blue nanozyme for rapid detection of glyphosate. Under weak alkaline conditions, the nanozyme can effectively catalyze the decomposition of H2O2 into O2, showing significant catalase-like activity. Glyphosate can inhibit its catalase-like activity, and glyphosate can be quantitatively detected by monitoring the changes in dissolved oxygen content in the system. The catalase-like activity of copper / chromium-doped Prussian blue nanozyme is used to detect glyphosate. The method is simple and rapid, and can be used for quantitative analysis of glyphosate.

[0030] (5) The nanozyme prepared in the present invention has a detection range of 1.6-13.3 μmol / L for glyphosate, and a detection limit of 0.53 μmol / L, which is lower than the national maximum concentration standard for glyphosate in drinking water. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 This is the XPS analysis chart of PB-Cr-Cu prepared in Example 1 of the present invention;

[0032] Figure 2 Comparison of catalase-like activity of PB-Cr nanozymes at different concentrations;

[0033] Figure 3 Comparison of catalase-like activity of PB-Cr nanozyme under different pH conditions;

[0034] Figure 4 This is the result diagram of the effect of copper ions on the activity of PB-Cr nanozyme catalase;

[0035] Figure 5 This is the result diagram of the effect of glyphosate on the activity of PB-Cr-Cu nanozyme catalase;

[0036] Figure 6 is the linear relationship diagram between glyphosate concentration and dissolved oxygen content;

[0037] Figure 7 This is a comparison chart of the inhibitory effects of different substances on the catalytic activity of PB-Cr-Cu type peroxidase. DETAILED DESCRIPTION

[0038] In order to make the purpose, technical solution and advantages of the embodiments of the present invention clearer, the technical solution in the embodiments of the present invention will be clearly and completely described in combination with the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0039] If no specific techniques or conditions are indicated in the examples of the present invention, the techniques or conditions described in the literature in the field or the product instructions are used. If the manufacturer of the reagents or instruments is not indicated, they are all conventional products that can be obtained through commercial purchase or other channels.

[0040] Embodiment 1:

[0041] A method for preparing chromium-doped Prussian blue nanozyme comprises the following steps:

[0042] (1) Add 3 g (15.6 mmol) of citric acid and 0.5 mmol of potassium ferrocyanide into 20 mL of deionized water, stir at room temperature, and dissolve to obtain reaction solution A;

[0043] (2) adding 0.25 mmol of chromium chloride and 0.25 mmol of ferric nitrate into 20 mL of deionized water, stirring at room temperature, and dissolving to obtain reaction solution B;

[0044] (3) adding the reaction solution B obtained in step (2) dropwise into the reaction solution A obtained in step (1) at a rate of 0.5 mL / min, stirring at 400 r / min for 3 hours at room temperature, washing the reaction product with deionized water, then adding alcohol and centrifuging, repeating the process three times, and freeze-drying to obtain the material PB-Cr;

[0045] (4) The PB-Cr obtained in step (3) was prepared into a 1 mg / mL PB-Cr solution, 200 μL was added to 750 μL, 0.1 M HEPES buffer (pH = 8.0), and then 50 μL, 10 mM copper sulfate solution was added. The reaction was carried out at room temperature for 10 minutes, centrifuged, and freeze-dried to obtain copper-chromium doped Prussian blue nanozyme (PB-Cr-Cu).

[0046] XPS analysis of the copper-chromium doped Prussian blue nanozyme PB-Cr-Cu prepared in this example showed that Figure 1 As shown in the figure, it can be seen that copper ions and chromium ions are successfully doped into Prussian blue.

[0047] Embodiment 2:

[0048] A method for preparing chromium-doped Prussian blue nanozyme comprises the following steps:

[0049] (1) Add 27.5 mmol of citric acid and 0.5 mmol of potassium ferrocyanide into 20 mL of deionized water, stir at room temperature, and dissolve to obtain reaction solution A;

[0050] (2) adding 0.25 mmol of chromium chloride and 0.25 mmol of ferric nitrate into 20 mL of deionized water, stirring at room temperature, and dissolving to obtain reaction solution B;

[0051] (3) adding the reaction solution B obtained in step (2) dropwise to the reaction solution A obtained in step (1) at a rate of 0.3 mL / min, stirring at 350 r / min for 4 hours at room temperature, washing the reaction product with deionized water, then adding alcohol and centrifuging, repeating three times, and freeze-drying to obtain the material PB-Cr;

[0052] (4) The PB-Cr obtained in step (3) was prepared into a 1 mg / mL PB-Cr solution, 320 μL was added to 750 μL, 0.1 M HEPES buffer (pH = 8.0), and then 50 μL, 10 mM copper sulfate solution was added. The reaction was carried out at room temperature for 10 minutes, centrifuged, and freeze-dried to obtain copper-chromium doped Prussian blue nanozyme (PB-Cr-Cu).

[0053] Embodiment 3:

[0054] A method for preparing chromium-doped Prussian blue nanozyme comprises the following steps:

[0055] (1) Add 21.7 mmol of citric acid and 1 mmol of potassium ferrocyanide into 20 mL of deionized water, stir at room temperature, and dissolve to obtain reaction solution A;

[0056] (2) adding 0.25 mmol of chromium chloride and 0.25 mmol of ferric nitrate into 20 mL of deionized water, stirring at room temperature, and dissolving to obtain reaction solution B;

[0057] (3) adding the reaction solution B obtained in step (2) dropwise to the reaction solution A obtained in step (1) at 0.8 mL / min, stirring at 450 r / min at room temperature for 2 hours, washing the reaction product with deionized water, then adding alcohol and centrifuging, repeating three times, and freeze-drying to obtain the material PB-Cr;

[0058] (4) The PB-Cr obtained in step (3) was prepared into a 1 mg / mL PB-Cr solution, 160 μL was added to 750 μL, 0.1 M HEPES buffer (pH = 8.0), and then 50 μL, 10 mM copper sulfate solution was added. The reaction was carried out at room temperature for 10 minutes, centrifuged, and freeze-dried to obtain copper-chromium doped Prussian blue nanozyme (PB-Cr-Cu).

[0059] Embodiment 4:

[0060] The catalase-like activity of PB-Cr and PB-Cr-Cu prepared in Example 1 was determined, specifically comprising the following steps:

[0061] (1) Prepare PB-Cr solutions with concentrations of 0.05, 0.1, 0.2, 0.5, and 0.8 mg / mL, take 1 mL of each solution and add it to 30 mL of 0.01 M HEPES buffer (pH = 8.0), then add 600 μL of 30% H2O2, and measure the O2 content in the solution using a dissolved oxygen meter for 6 min.

[0062] (2) Prepare a 0.5 mg / mL PB-Cr solution, take 1 mL and add it to 30 mL of 0.01 M HEPES buffer (pH = 6.0, 6.5, 7.0, 7.5, 8.0), then add 600 μL of 30% H2O2, and use a dissolved oxygen meter to measure the O2 content in the solution for 6 min.

[0063] (3) Prepare 0.2 mg / mL PB-Cr solution, take 1 mL and mix with 1 mL of 0.1, 0.2, 0.3, 0.4, 0.5 mM CuSO4 solution, and react at room temperature for 10 min. Then add to 30 mL of 0.01 M HEPES buffer (pH = 8.0), add 600 μL of 30% H2O2, and measure the O2 content in the solution using a dissolved oxygen meter for 6 min.

[0064] like Figure 2 As shown, the dissolved oxygen content showed a positive correlation with the PB-Cr concentration, reflecting the concentration dependence of PB-Cr-type catalase activity.

[0065] like Figure 3 As shown: the dissolved oxygen content increases with the increase of pH value, indicating that the activity of PB-Cr type catalase is pH-dependent and is more active under alkaline conditions.

[0066] like Figure 4 As shown: dissolved oxygen content increases with Cu 2+ The increase of doping amount indicates that the activity of PB-Cr-Cu catalase is related to Cu 2+ The doping amount changes in a positive correlation.

[0067] Embodiment 5:

[0068] The inhibitory effect of glyphosate on the catalytic activity of the PB-Cr-Cu type catalase prepared in Example 1 was evaluated, specifically comprising the following steps:

[0069] (1) Prepare 1 mL of 0.2 mg / mL PB-Cr solution, add 1 mL of 0.5 mM CuSO4, and react at room temperature for 10 min to obtain PB-Cr-Cu.

[0070] (2) Take 30 mL of 0.01 M HEPES buffer (pH = 8.0) and add glyphosate to make the glyphosate concentrations 1.6, 3.3, 6.6, 10, 13.3 and 16.7 μM.

[0071] (3) The PB-Cr-Cu obtained in step (1) was added to the glyphosate solution in step (2), stirred at room temperature for 10 min, and then 600 μL of 30% H2O2 was added. The O2 content in the solution was measured using a dissolved oxygen meter for 6 min.

[0072] like Figure 5 As shown: As the glyphosate concentration increases, the dissolved oxygen content decreases. When the glyphosate concentration is 13.3 μM, the dissolved oxygen content is almost reduced to the level of PB-Cr, indicating that the catalase-like activity of PB-Cr-Cu is inhibited by glyphosate. The quantitative analysis of glyphosate is achieved through the change of dissolved oxygen content.

[0073] like Figure 6 As shown: within the concentration range of 1.6-13.3 μM, there is a good linear relationship between dissolved oxygen and glyphosate concentration, and the working curve is y=23.4720-0.9958x, R2=0.9917.

[0074] Embodiment 6:

[0075] The inhibitory effect of other different substances on the catalytic activity of the PB-Cr-Cu type catalase prepared in Example 1 was evaluated, specifically comprising the following steps:

[0076] (1) Prepare 1 mL of 0.2 mg / mL PB-Cr solution, add 1 mL of 0.5 mM CuSO4, and react at room temperature for 10 min to obtain PB-Cr-Cu.

[0077] (2) Take 30 mL of 0.01 M HEPES buffer (pH = 8.0) and add glyphosate, glufosinate, promethazine, potassium phosphate, ferric nitrate, and calcium chloride to a concentration of 4 μM.

[0078] (3) Add the PB-Cr-Cu obtained in step (1) to the respective solutions in step (2), mix at room temperature for 10 min, then add 600 μL of 30% H2O2, and measure the O2 content in the solution using a dissolved oxygen meter for 6 min.

[0079] like Figure 7As shown: Among the tested substances, glyphosate has the greatest inhibitory effect on the activity of PB-Cr-Cu type peroxidase, followed by glufosinate ammonium and potassium phosphate, and the others have basically no inhibitory effect.

[0080] The above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit the same. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that the technical solutions described in the aforementioned embodiments may still be modified, or some of the technical features may be replaced by equivalents. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for preparing copper-chromium doped Prussian blue nanozyme, characterized in that: The following steps are involved: (1) adding citric acid and potassium ferrocyanide into water, stirring, and dissolving to obtain a reaction solution A; the molar ratio of citric acid to potassium ferrocyanide is (55-65):(1-3); (2) adding ferric nitrate and chromium chloride into water, stirring, and dissolving to obtain a reaction solution B; the molar ratio of the ferric nitrate to the chromium chloride is 1:1; (3) adding the reaction solution B obtained in step (2) dropwise into the reaction solution A obtained in step (1), stirring and reacting; washing the product after the reaction with deionized water, then adding alcohol and centrifuging, repeating the process three times, and freeze-drying to obtain the material PB-Cr; (4) Adding copper sulfate and the PB-Cr obtained in step (3) to a buffer solution, reacting to obtain copper-chromium doped Prussian blue nanozyme PB-Cr-Cu; the mass ratio of the copper sulfate to the PB-Cr is (1-3): (4-6); the buffer solution is 0.1 M HEPES buffer, and the pH of the buffer solution is 8.

0.

2. The method for preparing copper-chromium doped Prussian blue nanozyme according to claim 1, characterized in that: The molar ratio of citric acid to potassium ferrocyanide in step (1) is 55:

1.

3. The method for preparing the copper-chromium doped Prussian blue nanozyme according to claim 1, characterized in that: The molar ratio of citric acid to potassium ferrocyanide in step (1) is 60:

2.

4. The method for preparing the copper-chromium doped Prussian blue nanozyme according to claim 3, characterized in that: The solution dropping speed in step (3) is 0.3-0.8 mL / min.

5. The method for preparing copper-chromium doped Prussian blue nanozyme according to claim 4, characterized in that: The stirring speed in step (3) is 350-450 r / min, and the stirring time is 2-4 h.

6. The method for preparing copper-chromium doped Prussian blue nanozyme according to claim 1, characterized in that: In the step (4), the mass ratio of copper sulfate to PB-Cr is 2:

5.

7. The method for preparing copper-chromium doped Prussian blue nanozyme according to claim 1, characterized in that: In the step (4), the mass ratio of copper sulfate to PB-Cr is 1:

4.

8. The method for preparing copper-chromium doped Prussian blue nanozyme according to claim 5, characterized in that: The stirring speed in step (3) is 400 r / min and the stirring time is 3 h.

9. Copper-chromium-doped Prussian blue nanozyme prepared by the preparation method according to any one of claims 1 to 8.

10. Use of the copper-chromium doped Prussian blue nanozyme prepared by the preparation method according to any one of claims 1 to 8 in the rapid detection of glyphosate.

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