A Visualized and Rapid Detection Method and Application of Medium-Entropy Nanozymes for Thiocarbofuran

Abstract

This invention provides a rapid and visualized detection method for the pesticide thiocarbamate using medium-entropy nanozymes, and its application, belonging to the field of analytical chemistry. This invention is based on a metal-ligand crosslinking strategy, utilizing the chelating ability of dopamine phenolic hydroxyl groups and Fe... 3+ Ce 3+ and Cu 2+ The invention utilizes a multi-metal ion self-assembly synthesis of medium-entropy nanozymes (FeCeCu MEzymes), and designs a medium-entropy nanozyme (FeCeCu MEzymes) sensor combined with a smartphone to achieve rapid and visual detection of carbofuran. The rapid and visual detection method for carbofuran provided by this invention has the advantages of being fast, highly sensitive, selective, and providing intuitive detection results. It is also simple to operate and has a low cost.

Description

Technical Field

[0001] This invention belongs to the field of analytical chemistry technology, specifically relating to a visual rapid detection method and application of medium-entropy nanozymes for thiocarbamate pesticide. Background Technology

[0002] Thiophanate-carbofuran belongs to the carbamate pesticide class and has advantages such as high efficacy, easy decomposition, and short residual period, making it widely used for the control of crop pests. However, the overuse of pesticides can contaminate food and water sources, leading to biofouling in the food chain. Furthermore, thiophanate-carbofuran is an endocrine disruptor that can inhibit acetylcholinesterase activity in the central nervous system and may cause birth defects, mutations, and cancer. Therefore, accurate measurement of thiophanate-carbofuran pesticide residues is crucial for monitoring its environmental behavior and foodborne residue levels.

[0003] Currently, common detection methods for thiophanate-methyl pesticides include GC-MS, GC, HPLC, electrochemical methods, and fluorescence methods. However, these methods all suffer from drawbacks such as cumbersome operation, expensive equipment, and high costs. Therefore, there is still a need to develop a rapid, convenient, and inexpensive detection method. Among the many detection methods, nanozyme colorimetry, based on the relationship between the intensity of solution color and absorbance, has been widely applied in the detection field by combining nanozyme colorimetry with smartphones. Its characteristics include visualization, low cost, simple operation, and portable on-site detection, thus expanding its practical applications. Summary of the Invention

[0004] To address the shortcomings of the prior art, this invention aims to provide a rapid and visual detection method for the pesticide thiocarbofuran using medium-entropy nanozymes, and its application. The medium-entropy nanozymes FeCeCu Mezymes of this invention exhibit oxidase-like activity that allows dissolved oxygen to generate free radicals, which then catalyze the formation of a blue TMBox from colorless 3,3',5,5'-tetramethylbenzidine (TMB), resulting in a colorimetric reaction. When different concentrations of thiocarbofuran are added to the FeCeCu Mezymes sensing system, under pH conditions, thiocarbofuran hydrolyzes to generate -SH groups. These -SH groups can interact with the metals in the FeCeCu Mezymes, inhibiting TMBox formation and enabling rapid and visual detection of thiocarbofuran. The FeCeCu Mezymes of this invention are simple to synthesize, and their colorimetric detection method for thiocarbofuran is rapid, convenient, and provides clear and intuitive results.

[0005] To achieve the above objectives, the present invention provides the following technical solution:

[0006] This invention provides a method for rapid detection of carbofuran, specifically including the following steps:

[0007] S1: Mix ethylene glycol and water evenly, then add ammonia, dopamine hydrochloride, ferric nitrate nonahydrate, copper nitrate, and cerium nitrate hexahydrate in sequence, and stir evenly; after the reaction is complete, wash, centrifuge, dry, and calcine the obtained sample to obtain the medium-entropy nanozyme FeCeCu Mezymes;

[0008] S2: Add carbofuran and medium-entropy nanozyme FeCeCu Mezymes to HAc-NaAc acetate-sodium acetate buffer, incubate for 30 min, then add TMB, and perform visual detection of the solution after incubation.

[0009] Furthermore, in step S1, the volume ratio of ethylene glycol, water, and ammonia solution is 8:5:0.3, and the molar ratio of dopamine hydrochloride to ferric nitrate nonahydrate, copper nitrate, and cerium nitrate hexahydrate is 6:1:1:1. The mixture is stirred at 60°C for 11 h.

[0010] Furthermore, in step S2, the HAc-NaAc acetate-sodium acetate buffer solution was 0.1 M at pH 4 and incubated at 37°C.

[0011] Furthermore, in step S2, the visualization detection is performed using a UV spectrophotometer or by extracting and detecting the RGB grayscale values ​​from a smartphone.

[0012] Furthermore, the ultraviolet spectrophotometer detects and records the absorbance of the solution at 652 nm, and a standard curve and linear equation are derived based on the absorbance changes of different concentrations of carbofuran.

[0013] Furthermore, the RGB grayscale value extraction and detection includes the following steps: taking pictures of different concentrations of carbofuran added to the medium-entropy nanoenzyme FeCeCu Mezymes sensing system, extracting the RGB grayscale values ​​from each picture, the vertical axis of the linear equation is (G+B) / 2×R, and the horizontal axis is the concentration of carbofuran. The linear equation is obtained through the RGB values ​​and different concentrations of carbofuran.

[0014] On the other hand, the present invention also provides a method for visually detecting thiocarbamate pesticides, as described in the above examples, for rapid visual detection of pesticides.

[0015] Furthermore, carbofuran, dichlorvos, ethyl parathion, methamidophos, thiamethoxam, and glyphosate were added during the testing process for selective testing.

[0016] Furthermore, Na is added during the detection process. + K + Al 3+ Mg 2+ Mn 2+ Ca 2+ Zn 2+ Cu2+ Conduct anti-interference tests.

[0017] Furthermore, it can be applied to the rapid and visual detection of carbofuran pesticide in actual soil and water samples.

[0018] Compared with the prior art, the beneficial effects of the present invention are:

[0019] 1. The medium-entropy nanozymes FeCeCu Mezymes synthesized in this invention possess excellent oxidase-like activity. A colorimetric sensor was constructed using this nanozyme activity to detect the pesticide thiocarbofuran. This method, when applied to the detection of thiocarbofuran, offers advantages such as convenient and rapid operation, high detection sensitivity, and low cost.

[0020] 2. The medium-entropy nanozyme FeCeCu Mezymes synthesized in this invention exhibits a detection range of 0.15µM-50µM for carbofuran pesticide, with a detection limit of 0.13µM. Furthermore, it demonstrates selectivity for structurally similar pesticides such as carbofuran, dichlorvos, ethyl parathion, methamidophos, thiamethoxam, and glyphosate, and also shows selectivity for common Na+ ions in solution. + K + Al 3+ Mg 2+ Mn 2+ Ca 2 + Zn 2+ Cu 2+ It exhibits good anti-interference properties. When used for the detection of carbofuran in soil and water sources, combined with color changes, it enables real-time on-site detection of carbofuran.

[0021] 3. The preparation method of the present invention is simple and convenient, has high detection sensitivity, low cost, and good anti-interference performance. Attached Figure Description

[0022] Figure 1 The image shows a scanning electron microscope (SEM) image of the medium-entropy nanozyme FeCeCu Mezymes prepared under the conditions of Example 1.

[0023] Figure 2 This is a graph showing the oxidase-like activity of the entropy nanoenzyme FeCeCu Mezymes in different concentration TMB systems in the present invention, based on the UV-Vis absorption spectra.

[0024] Figure 3 The UV-Vis spectra of the TMB-FeCeCu MEzymes system with different concentrations of carbofuran pesticide added in this invention are shown.

[0025] Figure 4The graph shows the linear relationship between the concentration of thiocarbamate detected by the medium-entropy nanozyme FeCeCu Mezymes in this invention.

[0026] Figure 5 This invention uses a smartphone color analysis application to generate colorimetric photographs and standard curves for different concentrations of carbofuran. Specific implementation methods

[0027] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be clearly and completely described below in conjunction with embodiments. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all commercially available products. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. The terminology used in this specification is for the purpose of describing particular embodiments only and is not intended to limit the invention. The term "and / or" as used herein includes any and all combinations of one or more of the associated listed items.

[0028] Example 1: Preparation of medium-entropy nanozymes FeCeCu Mezymes

[0029] 1. Add 80 mL of ethylene glycol and 50 mL of water to a clean three-necked flask and mix well. Then add 3 mL of ammonia solution (28% by mass). Heat and stir to 60°C and stabilize for 10 min to obtain the ethylene glycol ammonia reaction system.

[0030] 2. First, add 10 mL of 50 mg / mL dopamine hydrochloride solution to the ethylene glycol ammonia reaction system, then slowly add 10 mL of 17.7 mg / mL ferric nitrate nonahydrate solution, 10.6 mg / mL copper nitrate, and 19.0 mg / mL cerium nitrate hexahydrate dropwise. The molar ratio of dopamine hydrochloride to ferric nitrate, copper nitrate, and cerium nitrate is 6:1:1:1. After reacting for 11 h, crude medium-entropy nanozyme FeCeCu Mezymes is obtained.

[0031] 3. The crude FeCeCu Mezymes nanozyme was collected by centrifugation, washed three times with a 1:1 volume ratio of ethanol and water, freeze-dried, and the resulting powder was calcined in a tube furnace at 900℃ for 7 h to obtain the FeCeCu Mezymes nanozyme sample.

[0032] Figure 1 The image shows a scanning electron microscope (SEM) image of the medium-entropy nanozyme FeCeCu Mezymes prepared under the conditions of Example 1. As can be seen from the image, the medium-entropy nanozyme FeCeCu Mezymes particles have a uniform morphology and self-assemble into flower-like shapes.

[0033] Example 2: Study on the oxidase-like activity of the medium-entropy nanozyme FeCeCu Mezymes

[0034] Prepare 50 mL of HAc-NaAc acetate-sodium acetate buffer (0.1 M ion concentration, pH 4) and 4 mL of 1 mM TMB; then, draw 1900 μL and 100 μL from each buffer and place them into cuvette #1. Next, draw 1900 μL of HAc-NaAc acetate-sodium acetate buffer and 100 μL of 1 mg / mL FeCeCu Mezymes nanozyme and place them into cuvette #2. Cuvette #3 is prepared by adding another 100 μL of 1 mg / mL FeCeCu Mezymes nanozyme to cuvette #1. After incubating for 10 min, measure the absorption spectra of all three cuvettes in the 300 nm–800 nm wavelength range.

[0035] Figure 2 The image shows the oxidase-like activity of the medium-entropy nanozyme FeCeCu Mezymes under the conditions of this embodiment. It can be seen that the medium-entropy nanozyme FeCeCu Mezymes has high oxidase-like activity, which oxidizes the chromogenic agent TMB into blue TMBox.

[0036] Example 3: Detection of the standard thiocarbamate solution

[0037] The TMB-FeCeCu MEzymes system and different concentrations of carbofuran were added to HAc-NaAc acetate-sodium acetate buffer and incubated at 37°C for 30 minutes. TMB was then added, and the mixture was incubated for another 10 minutes. Finally, the relationship between the absorbance at 652 nm and different carbofuran concentrations was investigated.

[0038] Figure 3 The image shows the UV-Vis spectrum of the TMB-FeCeCu MEzymes system with added carbofuran. As can be seen from the image, the absorbance at 652 nm gradually decreases with the increase of carbofuran concentration, indicating a decrease in the intensity of blue. Figure 4 The linear relationship between the concentration of thiocarbamate and the detection of thiocarbamate by the medium-entropy nanozyme FeCeCu Mezymes was plotted, and the linear relationship was obtained as: y = -0.00972x + 0.709(R) 2 =0.997), where x is the concentration of thiocarbofuran, y is the absorbance of the solution, the detection range is 0.15-50µM, and the limit of detection is 0.13µM.

[0039] Example 4: Detection of carbofuran in soil and water sources

[0040] Following standard spiking experiments, standard solutions of carbofuran (6µM, 30µM, 50µM) were added to extracts from soil and water sources. The test solution and the medium-entropy nanozyme FeCeCu Mezymes were then added to HAc-NaAc acetate-sodium acetate buffer solution and incubated at 37°C for 30 min. TMB was added, followed by another 10 min of incubation. The absorbance at 652 nm was then measured using a UV spectrophotometer. The experimental results are shown in Table 1.

[0041] Table 1

[0042]

[0043] As can be seen from the analysis of the test results in Table 1, the test results of this method on the content of spiked solutions in soil and water sources are similar to those of standard large-scale instruments, indicating that the entropy nanozyme FeCeCu Mezymes in this invention is feasible for the visual and rapid detection of carbofuran.

[0044] Example 5: Real-time detection of carbofuran using R, G, and B grayscale values ​​(RGB) color analysis program

[0045] The medium-entropy nanozymes FeCeCu Mezymes and thiocarbofuran were added to HAc-NaAc acetate-sodium acetate buffer and incubated at 37°C for 30 min. After adding TMB, the mixture was incubated for another 10 min. Photos were taken with a smartphone, and the average gray values ​​of the R, G, and B components were extracted from the photos using a color analysis program.

[0046] Figure 5 RGB colorimetric images and standard curves of different concentrations of carbofuran at a smartphone are provided. The linear relationship is as follows: y = -0.0385x + 2.941(R 2 =0.992), and the LOD is calculated to be 0.14µM.

[0047] The embodiments described above are merely preferred embodiments of the present invention, and while the descriptions are specific and detailed, they are not intended to limit the present invention. It should be noted that various changes and modifications can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the concept and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A visual detection method for thiocarbamate pesticide, characterized in that, Includes the following steps: S1: Mix ethylene glycol and water evenly, then add ammonia, dopamine hydrochloride, ferric nitrate nonahydrate, copper nitrate, and cerium nitrate hexahydrate in sequence, and stir evenly; after the reaction is complete, wash, centrifuge, dry, and calcine the obtained sample to obtain the medium-entropy nanozyme FeCeCuMEzymes; S2: Add the sample to be tested and the medium-entropy nanozyme FeCeCu Mezymes to HAc-NaAc acetate-sodium acetate buffer, incubate for 30 min, then add TMB, incubate for 10 min, and then perform visual detection of the solution.

2. The method according to claim 1, characterized in that, In step S1, the volume ratio of ethylene glycol, water, and ammonia solution is 8:5:0.3, and the molar ratio of dopamine hydrochloride to ferric nitrate nonahydrate, copper nitrate, and cerium nitrate hexahydrate is 6:1:1:

1. The mixture is stirred at 60°C for 11 h.

3. The method according to claim 1, characterized in that, In step S2, the HAc-NaAc acetate-sodium acetate buffer solution concentration is 0.1M, pH=4, and incubation is carried out at 37°C.

4. The method according to claim 1, characterized in that, In S2, visual detection is performed using a UV spectrophotometer or by extracting RGB grayscale values ​​from a smartphone.

5. The method according to claim 4, characterized in that, The ultraviolet spectrophotometer was used to detect and record the absorbance of the solution at 652 nm. A standard curve and linear equation were derived based on the absorbance changes of different concentrations of carbofuran.

6. The method according to claim 4, characterized in that, The extraction and detection of RGB grayscale values ​​in the smartphone includes the following steps: taking pictures of different concentrations of carbofuran added to the medium-entropy nanoenzyme FeCeCu Mezymes sensing system, extracting the RGB grayscale values ​​from each picture, the vertical axis of the linear equation is (G+B) / 2×R, and the horizontal axis is the concentration of carbofuran. The linear equation is obtained through the RGB values ​​and different concentrations of carbofuran.

7. The method according to claim 1, characterized in that, During the testing process, carbofuran, dichlorvos, ethyl parathion, methamidophos, thiamethoxam, and glyphosate were added for selective testing.

8. The method according to claim 7, characterized in that, Na is added during the detection process + K + Al 3+ Mg 2+ Mn 2+ Ca 2 + Zn 2+ Cu 2+ Conduct anti-interference tests.

9. The method according to claim 7, characterized in that, The sample to be tested is specifically soil or water.

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