Preparation of iron-based nano-enzyme hydrogel reactor and thiram monitoring application

Abstract

The invention discloses a colorimetric hydrogel reactor based on iron-based nano horseradish peroxidase, and provides a sensor capable of detecting thiram residues on site and a preparation method. The iron-based nano-enzyme with natural horse radish peroxidase activity is synthesized by taking ferrous chloride and glutathione as materials through a hydrothermal method. The iron-based nano-enzyme shows huge potential in the aspect of detection and application of pesticide thiram, and when thiram exists, active sites of the iron-based nano-enzyme are passivated, the catalytic activity is reduced, and the output color is changed. The construction of a hydrogel colorimetric platform based on a nano material simulating horseradish peroxidase can realize rapid visual and quantitative detection of thiram residues on site, and can promote the development of nano enzyme-based sensors and on-site monitoring application. The method has the advantages of being convenient to operate, economical and effective in mechanism, high in stability and the like, has excellent on-site practicability, and provides a new ideal platform for on-site monitoring of precision agriculture and food safety risk assessment.

Description

Technical Field

[0001] The present invention belongs to the technical field of biosensors, and particularly relates to a hydrogel colorimetric platform for on-site monitoring of thiram by applying an iron-based nanozyme that mimics the activity of natural horseradish peroxidase. Background Art

[0002] As a typical dithiocarbamate fungicide, thiram is widely used in the prevention and control of fungi, weeds and pests in fruits and vegetables to increase yields. However, thiram exposure can induce the biotoxicity of the endocrine and nervous systems and produce high cytotoxicity to fibroblasts by inactivating transcription factors NF-kB and hypoxia-inducible factor. Therefore, it is of great significance to establish a practical monitoring method with high specificity and sensitivity to ensure food safety and human health. Currently, the established monitoring methods such as chromatography, enzyme-linked analysis and surface-enhanced Raman scattering strategy require high-cost biorecognition elements, suffer from poor stability and high costs, and lack practical applicability. Nanozymes with intrinsic enzyme-like properties are regarded as ideal recognition elements for fabricating biosensors due to their excellent stability, sensitivity and selectivity. At present, nanozymes are usually combined with biological enzymes to form an enzyme-nanozyme cascade platform, react with the products mediated by biological enzymes and further regulate signal output, but the system still faces problems of instability and cost. Applying iron-based nanozymes to the sensing system can directly and specifically recognize thiram, not only improving cost-effectiveness, but also the unique stability of iron-based nanozymes making them have good application prospects in sensor manufacturing. In addition, encapsulating iron-based nanozymes in agarose hydrogels improves on-site practicability and provides a practical tool for developing a convenient and economical pesticide detection method. Summary of the Invention

[0003] The purpose of the present invention is to improve the selectivity, stability and economic benefits of thiram detection, and develop a hydrogel colorimetric platform for on-site monitoring of thiram based on an iron-based nanozyme that mimics the activity of natural horseradish peroxidase, which can simply and efficiently detect the content of thiram.

[0004] The hydrogel colorimetric platform for on-site monitoring of thiram based on an iron-based nanozyme that mimics the activity of natural horseradish peroxidase described in the present invention is established as follows:

[0005] (1) Provide a method for preparing an iron-based nanozyme. An aqueous solution of ferrous chloride and an aqueous solution of glutathione are added to an N,N-dimethylformamide solution (≥99.9%) and mixed in a ratio of 1:2:4. The mixture is transferred to a 20 mL Teflon-lined autoclave and heated in a constant-temperature oven at 140 °C for 4.5 h. After cooling to room temperature, the iron-based nanozyme is collected by centrifugation (15,000 rpm, 5 min) and washed three times with an N,N-dimethylformamide solution (≥99.9%) and deionized water to remove excess reactants. Finally, the precipitate is collected and freeze-dried to obtain a freeze-dried powder of the iron-based nanozyme ( Figure 1 ).

[0006] (2) Provide a method for on-site monitoring of thiram based on the activity of an iron-based nanozyme mimicking horseradish peroxidase. At 37 °C, different concentrations of thiram solutions are mixed with the iron-based nanozyme solution and reacted for 25 min, and then 3,3’,5,5’-tetramethylbenzidine solution and an aqueous hydrogen peroxide solution are mixed with a NaAC buffer solution (pH = 4.0) in a ratio of 1:1:1 and reacted at room temperature for 30 min. The absorbance curve generated by the color reaction of different concentrations of thiram and the iron-based nanozyme is measured by an ultraviolet spectrophotometer ( Figure 2 ).

[0007] (3) Provide a method for constructing a hydrogel colorimetric sensor for on-site monitoring of the pesticide thiram. The iron-based nanozyme is mixed with an agarose solution, and then quickly added to a 96-well microplate and cooled from 40 °C to room temperature. The hydrogel reactor is stored at 4 °C. Different concentrations of thiram solutions are added to the hydrogel reactor and reacted for 10 min. Then, a mixture of hydrogen peroxide, TMB, and a NaAC buffer solution (pH = 4.0) is used as the substrate system in a ratio of 2:1:1 and reacted at room temperature for 30 min. Observe the change in the color of the hydrogel, and use a smartphone to collect the color image of the hydrogel. It can be observed that as the concentration of the pesticide thiram increases, the color of the obtained picture changes from dark blue to light blue, and the red signal increases ( Figure 3 ).

[0008] The mechanism of the present invention is as follows:

[0009] Based on the prepared iron-based nanozyme material, in the presence of the pesticide thiram, thiram is anchored on the surface of the iron-based nanozyme through an S-Fe bond, passivating the active sites of the iron-based nanozyme, restricting the catalytic activity of the nanozyme, and causing a change in the colorimetric signal. The iron-based nanozyme can be used to construct a hydrogel reactor to achieve rapid visualization and qualitative detection of thiram. By taking pictures with a smartphone and converting the image parameters into data information using ImageJ software, quantitative detection of thiram is achieved.

[0010] The present invention has the following characteristics:

[0011] (1) The hydrogel-based thiram detection platform prepared by the present invention has the advantages of high sensitivity, high selectivity, and high stability;

[0012] (2) The construction process of the present invention has the advantages of high cost-effectiveness and simple operation.

[0013] The detection of thiram content based on hydrogel in the present invention shows great application potential in improving the selectivity of pesticide detection and constructing a stable portable detection device for point-of-care testing. Description of the Drawings

[0014] Figure 1 It is the scanning electron microscope image of the iron-based nanozyme material in Example 1;

[0015] Figure 2 It is the absorption spectrum of thiram at different concentrations tested by ultraviolet-visible spectrophotometer in Example 2;

[0016] Figure 3 It is the color image of the (R+G+B) value and thiram concentration analyzed by ImageJ in Example 3;

[0017] Figure 4 It is the linear relationship diagram of the (R+G+B) value and thiram concentration analyzed by ImageJ in Example 3. Detailed Embodiments

[0018] Example 1: Preparation of Iron-Based Nanozyme Material

[0019] Add an aqueous solution of ferrous chloride (0.24 mol L -1 , 2.5 mL) and an aqueous solution of glutathione (0.12 mol L -1 , 5 mL) into 10.0 mL of N,N-dimethylformamide solution (≥99.9%). After mixing evenly at room temperature for 5 min, transfer the mixed liquid to a 20 mL Teflon-lined autoclave and heat it at 140 °C in a constant-temperature oven for 4.5 h. After cooling, collect the iron-based nanozyme by centrifugation (15,000 rpm, 5 min), wash it three times with N,N-dimethylformamide solution (≥99.9%) and deionized water respectively, and finally obtain the iron-based nanozyme precipitate by freeze-drying. The scanning electron microscope image is as Figure 1 .

[0020] Example 2: Thiram Detection Method Based on Iron-Based Nanozyme with Simulated Horseradish Peroxidase Activity

[0021] At 37 °C, add different concentrations of thiram solution (10 μL) to the iron-based nanozyme solution (0.005 mg mL -1, 10 μL) were mixed and reacted for 25 min, and then 3,3',5,5'-tetramethylbenzidine solution (1 mg mL -1 , 20 μL) and aqueous hydrogen peroxide solution (0.1 mM, 20 μL) were mixed with NaAC buffer solution (30 mM, pH = 4.0, 20 μL), and reacted at room temperature for 30 min. The absorbance curves and linear relationships generated by the color reaction of thiram at different concentrations with the iron-based nanozyme were measured by an ultraviolet spectrophotometer ( Figure 2 ). A sensor for on-site monitoring of thiram was constructed using an iron-based nanozyme that mimics the activity of horseradish peroxidase.

[0022] Example 3: Construction of a hydrogel colorimetric sensor for on-site detection of the pesticide thiram

[0023] The iron-based nanozyme (1.0 mg mL -1 , 10 μL) was mixed with the agarose solution (10.0 mg mL -1 , 40 μL), then quickly added to a 96-well microplate and cooled from 40 °C to room temperature, and the hydrogel reactor was stored at 4 °C. 50 μL of thiram solutions at different concentrations (0.3, 0.7, 1.7, 3.3, 5, 6.7, 10 μg mL -1 ) were added to the hydrogel reactor and reacted for 10 min. Then, a mixture of hydrogen peroxide (0.1 mM, 100 μL), TMB (1.0 mg mL -1 , 50 μL) and NaAC buffer solution (30 mM, pH = 4.0, 50 μL) was used as the substrate system and reacted at room temperature for 30 min. The color change of the hydrogel was observed, and the color image of the hydrogel reactor was collected using the CMOS camera of a smartphone. It can be observed that as the concentration of the pesticide thiram increases, the color of the image changes from dark blue to light blue ( Figure 3 ). The ImageJ software was used to convert the image parameters into data information, and it was found that the red signal of the hydrogel reactor was positively correlated with the thiram concentration. The depth change of the hydrogel could be observed with the naked eye. According to several groups of (R + G + B) values and the corresponding pesticide concentration values, a relationship curve was fitted by regression analysis. Thus, the linear relationship between the (R + G + B) value of the hydrogel reactor and the thiram concentration was obtained, realizing the quantitative detection of thiram ( Figure 4 ).

[0024] Example 4: Detection of thiram in actual samples

[0025] To verify the practicality of the thiram detection strategy, environmental samples (lake water, tap water) and biological samples (pear juice, orange juice) were selected for testing. Different concentrations of thiram standard solutions (0.005, 0.05 and 0.5 μg L -1) to simulate the actual detection environment. During the analysis, to eliminate background interference, the lake water, tap water, pear juice, and orange juice samples were diluted with deionized water according to the sample characteristics, and the dilution factors were 1, 1, 200, and 200, respectively. As shown in Table 1, the spiked recoveries of thiram in the actual samples (the spiked recovery is the ratio of the result obtained after analysis using the established method to the added quantitative value when a certain amount of the analyte is added to the sample matrix) were 92.4 - 106.9%, and the relative standard deviation (RSD) was less than 7.60%. This data indicates that this thiram detection strategy has good applicability and accuracy in actual samples, providing reliable technical support for the monitoring of thiram in the actual environment.

[0026] Table 1: Detection of thiram in actual samples based on the ultraviolet-visible method

[0027]

Claims

1. A method for preparing an iron-based nanozyme based on mimicking the activity of horseradish peroxidase, which is constructed by the following method: The ferrous chloride aqueous solution (0.24 mol L -1 , 2.5 mL) and glutathione aqueous solution (0.12 mol L -1 , 5 mL) was added to 10.0 mL N, N-dimethylformamide solution (≥ 99.9%) and mixed at room temperature for 5 min. The mixed liquid was transferred to a 20 mL Teflon-lined autoclave and incubated in a constant temperature oven at 140 o C for 4.5 h. After cooling, the iron-based nanozymes were collected by centrifugation (15000 rpm, 5 min), washed three times with N, N-dimethylformamide solution (≥ 99.9%) and deionized water, and finally the iron-based nanozyme precipitate was obtained by freeze drying.

2. The method for preparing the iron-based nanozyme with horseradish peroxidase-like activity according to claim 1, characterized in that the synthesized iron-based nanozyme is centrifuged at 15,000 rpm for 5 min, and the precipitate is freeze-dried and collected using a condensation freeze dryer.

3. An application of iron-based nanozyme based on pesticide detection, characterized in that: Iron-based nanozymes are applied to enzyme-free sensors, and enzyme-free sensors are applied to the detection of pesticides.

4. The application of an iron-based nanozyme based on pesticide detection according to claim 3, characterized in that: The pesticide tested is Thiram.

5. The application of an iron-based nanozyme based on pesticide detection according to claim 3, characterized in that: The enzyme-free sensor for detecting thiram comprises the following steps: At 37 °C, different concentrations of thiram solution were mixed with iron-based nanozyme solution for reaction for 25 min, and then 3,3',5,5'-tetramethylbenzidine solution and aqueous hydrogen peroxide solution were added and mixed with NaAC buffer (pH = 4.0) in a ratio of 1:1:

1. The mixture was reacted at room temperature for 30 min, and the absorbance curve generated by the color development reaction of different concentrations of thiram and iron-based nanozyme was measured by UV spectrophotometer.

6. An application of an iron-based nanozyme based on the detection of the pesticide thiram, characterized in that: The enzyme-free hydrogel colorimetric sensor for detecting thiram comprises the following steps: The iron-based nanozyme was mixed with agarose solution, then quickly added to a 96-well microplate and cooled from 40°C to room temperature. The hydrogel reactor was stored at 4°C. Thiram solution of different concentrations was added to the hydrogel reactor for reaction for 10 min. Then, a mixture of hydrogen peroxide, TMB and NaAC buffer (pH = 4.0) was used as a substrate system with a ratio of 2:1:1 and reacted at room temperature for 30 min. The color change of the hydrogel was observed, and the color image of the hydrogel was collected using a smartphone. The image parameters were converted into data information using ImageJ software to obtain the corresponding (R+G+B) values ​​at different concentrations. According to several groups of (R+G+B) values ​​and corresponding pesticide concentration values, regression analysis was used to fit the relationship curve. Thus, the linear relationship between the (R+G+B) value of the hydrogel reactor and the concentration of Thiram was obtained, and the quantitative detection of Thiram was achieved.

Images