Organochlorine pesticide visual detection method based on metal organic framework immobilized enzyme composite material

Abstract

The invention discloses an organochlorine pesticide visual detection method based on a metal organic framework immobilized enzyme composite material, and belongs to the technical field of biosensors. The method is characterized in that a metal organic framework (MOFs) with a three-dimensional structure is used for encapsulating alkaline phosphatase (ALP) in natural enzyme, and the problems that in a traditional enzyme immobilization technology, bio-enzyme is prone to falling off, and the activity of immobilized enzyme is reduced or lost due to the fact that a solid-phase carrier is broken or dissolved are solved. The MOFs loading capacity is high, so that the target pesticide can effectively inhibit the activity of ALP, the ALP is influenced to hydrolyze p-nitrophenyl phosphonate to generate yellow p-nitrophenol, and an obvious colorimetric signal is generated. According to the present invention, based on the principle, the rapid detection of 2, 4-dichlorophenoxyacetic acid can be achieved, the operation is simple, the cost is low, the sensitivity is high, and the new approach is provided for the rapid detection of 2, 4-dichlorophenoxyacetic acid.

Description

Technical Field

[0001] The present invention belongs to the technical field of biosensors, and in particular relates to an innovative technology for a visual detection method of organochlorine pesticides based on a specific metal organic framework immobilized enzyme composite material (ALP@ZIF-8). Background Art

[0002] 2,4-Dichlorophenoxyacetic acid (2,4-D) is widely used in agricultural production as a herbicide and plant growth agent. Although it is highly effective, it also has high toxicity and potential carcinogenic risks, and is an organochlorine pesticide. Due to the improper use of 2,4-D in agricultural production, its residue problem in organisms has become increasingly serious, which not only poses a threat to the ecological environment, but also poses a huge challenge to human health, especially in the field of food safety. Therefore, in food safety supervision, the detection of pesticide residues, especially on-site rapid detection, is particularly important. At present, traditional detection methods such as chromatography, mass spectrometry and electrochemical analysis have been widely used in pesticide residue detection. They have certain advantages in cost-effectiveness, stability, sensitivity and detection efficiency. Chromatography can accurately separate and quantify a variety of compounds, mass spectrometry can provide high-precision molecular mass information, and electrochemical analysis is favored for its high sensitivity. Despite this, these traditional methods also have obvious limitations, such as cumbersome operation steps, high cost, complex detection process, and strict requirements on the experimental environment. These limitations make these methods difficult to apply to on-site real-time monitoring of pesticide residues in complex samples, and they cannot meet the actual needs of regulatory authorities, small and medium-sized enterprises and individual users for rapid and portable pesticide residue detection. Therefore, it is particularly important to research and develop new pesticide detection methods that are easy to operate, fast to detect and highly sensitive, which can not only improve detection efficiency, but also provide more efficient technical support for food safety supervision, which has far-reaching social and economic significance.

[0003] In recent years, the emergence of metal-organic framework (MOFs) materials with three-dimensional structures has brought new opportunities to the field of food safety. Due to its unique microporous structure and properties, MOFs materials provide new possibilities for the research and development of new methods for pesticide detection that are simple to operate, fast to detect and highly sensitive. As a type of porous functional coordination material, MOFs have significant advantages over traditional inorganic porous solid materials, such as micropore size adjustability, high specific surface area and adjustability of skeleton structure. These characteristics make MOFs widely used in biosensing, gas separation, catalysis, drug delivery and other fields. Recently, researchers have adopted an embedded synthesis strategy to use rigid MOFs as an immobilization carrier skeleton to protect biomacromolecules. This strategy has become an emerging frontier in MOFs research. In the implementation of this strategy, biomacromolecules of different sizes, such as enzymes, are encapsulated into the pores of MOFs during the synthesis process, and the size of these pores is significantly smaller than that of the biomacromolecules themselves, thereby achieving enzyme immobilization. The porous framework structure of MOFs can not only improve the stability of the encapsulated enzyme through the structural confinement effect, but also allow the guest to be selectively transported through its microporous network structure. This symbiotic strengthening effect between MOFs and enzymes further endows MOFs-immobilized enzymes with great application potential in the field of enzyme-based pesticide sensors, providing a new technical approach for the rapid and highly sensitive detection of pesticide residues.

[0004] Based on this, this project proposes an innovative technical solution, which is to use the porous framework structure of MOFs to achieve the encapsulation of alkaline phosphatase (ALP), and then prepare MOFs-immobilized enzyme composite materials. A colorimetric sensor based on the enzyme inhibition mechanism is constructed for the rapid and sensitive detection of 2,4-dichlorophenoxyacetic acid (2,4-D) in food matrices. This unique combination of technologies not only makes full use of the structural advantages of metal organic frameworks and the catalytic properties of enzymes, but also cleverly realizes the visualization of the detection process, effectively improving the convenience and accuracy of detection, and providing an effective reference for the development of rapid monitoring and screening technologies for food contaminants. Summary of the invention

[0005] The present invention aims to solve the problems existing in the prior art and provide a visual detection method for 2,4-dichlorophenoxyacetic acid (2,4-D) based on a metal organic framework immobilized enzyme composite material (ALP@ZIF-8), so as to achieve rapid, portable and sensitive detection of 2,4-D residues in test samples.

[0006] The mechanism of visual detection of pesticides based on ALP@ZIF-8 in the present invention is as follows: When the sample does not contain 2,4-D, the activity of alkaline phosphatase (ALP) in ALP@ZIF-8 is not inhibited. At this time, ALP can catalyze the decomposition reaction of the substrate p-nitrophenylphosphonate (P-NPP) to generate a yellow water-soluble product p-nitrophenol (P-NP). However, when 2,4-D is added to the sample, the activity of ALP encapsulated by ALP@ZIF-8 is inhibited, resulting in a significant reduction in the decomposition amount of P-NPP. This change in decomposition amount will cause a significant color change, thereby achieving visual detection. Specifically, this detection method utilizes the difference in catalytic activity of ALP under different conditions, and judges whether 2,4-D is present in the sample by observing the color change. In the absence of 2,4-D interference, ALP performs a normal catalytic effect to produce an observable yellow product. When 2,4-D is present, its inhibitory effect on ALP will change the reaction process and cause the color change to deviate from the normal state. The tester can quickly and intuitively judge the residual 2,4-D in the sample. This method has the advantages of simple operation, rapid detection and high sensitivity.

[0007] The technical solution of the present invention is as follows:

[0008] A method for visually detecting 2,4-dichlorophenoxyacetic acid based on a metal organic framework immobilized enzyme composite material, characterized in that it comprises the following steps:

[0009] (1) Preparation of ALP@ZIF-8: Zinc nitrate (0.1 mM) and 2-methylimidazole (0.1 mM) in a molar ratio of 1:1 were mixed in deionized water, and a certain amount of alkaline phosphatase was added. The mixture was allowed to stand at room temperature for 12 h, and ALP@ZIF-8 was obtained after washing, centrifugation, and drying.

[0010] (2) Construction of a colorimetric sensor and detection method for 2,4-D based on ALP@ZIF-8: ALP@ZIF-8, 2,4-D, and buffer were added to a centrifuge tube and mixed, incubated at an appropriate temperature for 30 min, and P-NPP was added, and then incubated at an appropriate temperature for another 20 min. The P-NPP hydrolysis product was used as a sensing probe to detect the change in its absorbance at 405 nm. At the same time, a control group (without 2,4-D, and other conditions were the same) was set up to compare the color difference to achieve visual detection.

[0011] Beneficial effects of the present invention

[0012] (1) Successfully synthesized high-quality ALP@ZIF-8 composite materials, effectively encapsulating ALP in ZIF-8. Compared with free natural enzymes, this composite material has stronger tolerance to environmental factors such as temperature and pH value, greatly improving the stability of the sensor and creating stable conditions for accurate detection.

[0013] (2) The detection method based on the enzyme inhibition principle adopted by the present invention is simple to operate, has a fast response speed and low cost. It is particularly important that alkaline phosphatase is used as an enzyme probe, and the enzyme inhibition principle induced by organochlorine pesticides is combined with optical technology to achieve sensor detection of organochlorine pesticides, effectively improving the reliability and accuracy of the method.

[0014] (3) A new colorimetric sensor was constructed using ALP@ZIF-8 composite material, and the detection method was visualized with the help of a simple catalytic color development reaction.

[0015] The 2,4-D visualization detection method of the present invention has the characteristics of convenience, high efficiency and sensitivity, can fully meet the needs of rapid on-site detection of pesticides, and has great application potential. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 Schematic diagram of the method for visual detection of 2,4-D based on metal organic framework immobilized enzyme composite material constructed in the present invention

[0017] Figure 2 Feasibility verification diagram of the method for visual detection of 2,4-D by the colorimetric sensor constructed in Example 2

[0018] Figure 3 The optimization diagram of Example 3 for the catalytic hydrolysis of P-NPP by ALP@ZIF-8 at different concentrations

[0019] Figure 4 The linear regression curve of ALP@ZIF-8 catalyzing 2,4-D (concentration of 0-0.5 μg / mL) in Example 4

[0020] Figure 5 The linear regression curve of ALP@ZIF-8 catalyzing 2,4-D (concentration of 1.0-20 μg / mL) in Example 4

[0021] Figure 6 The anti-interference diagram of the colorimetric sensor in Example 5 with multiple interfering substances DETAILED DESCRIPTION

[0022] Example 1: Preparation of ALP@ZIF-8

[0023] 0.0744g Zn(NO3)2·6H2O (0.1mM) and 0.0154g 2-MI (0.1mM) were dissolved in 10mL deionized water, respectively, named solution 1 and solution 2, and 0.1mg alkaline phosphatase (ALP) was weighed and dissolved in 1mL water to prepare 100mU / mL solution 3, then solution 3 was added to solution 1, and then solution 2 was added, the three solutions were fully mixed, and aged at room temperature for 12h. Then the suspension was centrifuged at a speed of 10000r / min for 5min, the supernatant was discarded, and the obtained product was washed 3 times with deionized water, and finally placed in a vacuum drying oven to dry to obtain ALP@ZIF-8.

[0024] Example 2: Construction of a colorimetric sensor for visual detection of 2,4-D

[0025] Mix 20μL ALP@ZIF-8 (375μg / mL) and 20μL 2,4-D (20μg / mL) evenly, add 140μL PBS buffer (0.01M, pH=9), incubate at 37°C for 30min, then add 20μL P-NPP (5mM), and continue incubating at 37°C for 20min. Set up a control group without adding target pesticides, use the hydrolysis product of P-NPP as the sensing probe, use an enzyme reader to record the change in the absorbance value of the reaction system at 405nm, and measure the absorption spectrum of 390nm-500nm to construct a colorimetric sensor. In addition, the absorption spectra of ALP and ZIF-8 at 390nm-500nm were measured to exclude the influence of other substances in the reaction system on the absorbance value. The results are as follows: Figure 2 shown.

[0026] Example 3: Catalytic hydrolysis of P-NPP by ALP@ZIF-8 at different concentrations

[0027] Mix 20 μL of different concentrations of ALP@ZIF-8 (0, 5, 25, 50, 125, 250, 375, 500 μg / mL) and 20 μL of 2,4-D (20 μg / mL), add 140 μL of PBS buffer (0.01M, pH=9), and incubate at 37°C for 30 min. Then add 20 μL of P-NPP (5 mM) and incubate at 37°C for 20 min. Use an enzyme reader to record the absorbance changes of the reaction system at 405 nm. At the same time, set the control group without adding the target pesticide, and repeat each experiment three times. Data processing is the same as the above steps. The absorbance value of the control group without adding the target pesticide is recorded as A0, the absorbance value of the experimental group with the target pesticide is recorded as A, and ΔA 405 =(A0-A) as the signal value of the reaction system for data analysis. The results are as follows Figure 3 shown.

[0028] Example 4: Linear regression curve of ALP@ZIF-8 detecting 2,4-D

[0029] 20 μL ALP@ZIF-8 (375 μg / mL) and 20 μL of different concentrations of 2,4-D (0, 0.01, 0.05, 0.1, 0.5, 1.0, 2.5, 5.0, 7.5, 20 μg / mL) were mixed evenly, and 140 μL PBS buffer (0.01 M, pH = 9) was added. The mixture was incubated at 37°C for 30 min, and then 20 μL P-NPP (5 mM) was added. The mixture was incubated at 37°C for 20 min. The absorbance of the reaction system at 405 nm was recorded using an ELISA reader. Each experiment was repeated three times. Figure 4 , 5 shown.

[0030] Example 5: Anti-interference experiment

[0031] 20 μL of ALP@ZIF-8 (375 μg / mL) and 20 μL of 2,4-D (20 μg / mL) were mixed evenly, and 20 μL of common biomolecules (glucose Glu, lysine Lys, glycine Gly, histidine His, serine Ser) and ions (Na + , K + Mg 2 + ), then add 120 μL PBS buffer (0.01 M, pH = 9) and incubate at 37°C for 30 min. Then add 20 μL P-NPP (5 mM) and incubate at 37°C for 20 min. Use an ELISA reader to record the absorbance changes of the reaction system at 405 nm. Each group of experiments was repeated three times. The results are shown in Figure 6 shown.

Claims

1. A visual detection method for organochlorine pesticides based on metal organic framework immobilized enzyme composite materials, characterized in that: The following steps are involved: (1) Preparation of metal-organic framework-immobilized enzyme composite materials; (2) construct a colorimetric sensor for visual detection of 2,4-dichlorophenoxyacetic acid; (3) Different concentrations of ALP@ZIF-8 catalyzed the hydrolysis of p-nitrophenyl phosphate; (4) The anti-interference ability of colorimetric sensors when coexisting with multiple interferents.

2. The method for visual detection of organochlorine pesticides based on a metal organic framework immobilized enzyme composite material according to claim 1, characterized in that: In step (1), the preparation of the metal organic framework-based immobilized enzyme composite material specifically includes: dissolving 0.0744g Zn(NO3)2·6H2O, 0.1mM and 0.0154g 2-MI, 0.1mM, respectively, in 10mL deionized water, respectively named solution 1 and solution 2, and at the same time weighing 0.1mg alkaline phosphatase and dissolving it in 1mL water to prepare 100mU / mL solution 3, then adding solution 3 to solution 1, and then adding solution 2, mixing the three solutions thoroughly, and aging at room temperature for 12h, then centrifuging the suspension at a speed of 10000r / min for 5min, discarding the supernatant, washing the resulting product with deionized water 3 times, and finally placing it in a vacuum drying oven to dry to obtain ALP@ZIF-8.

3. The method for visual detection of organochlorine pesticides based on a metal organic framework immobilized enzyme composite material according to claim 1, characterized in that: In step (2), the construction process of the colorimetric sensor is as follows: 20 μL ALP@ZIF-8, 375 μg / mL and 20 μL 2,4-D, 20 μg / mL are mixed evenly, 140 μL PBS buffer, 0.01 M, pH = 9 is added, and the mixture is incubated at 37°C for 30 min, followed by the addition of 20 μL P-NPP, 5 mM, and the mixture is incubated at 37°C for 20 min. A control group without adding target pesticide is set as the control group, and the hydrolyzate of P-NPP is used as the sensing probe. The absorbance change of the reaction system at 405 nm is recorded using an ELISA instrument, and the absorption spectrum of 390 nm-500 nm is measured at the same time to construct a colorimetric sensor. In addition, the absorption spectra of ALP and ZIF-8 at 390 nm-500 nm are measured to exclude the influence of other substances in the reaction system on the absorbance value.

4. The method for visual detection of organochlorine pesticides based on metal organic framework immobilized enzyme composite materials according to claim 1, characterized in that: In step (3), 20 μL of ALP@ZIF-8 of different concentrations and 20 μL of 2,4-D were mixed evenly, 140 μL of PBS buffer was added, and the mixture was incubated at 37°C for 30 min. Subsequently, 20 μL of P-NPP was added and the mixture was incubated at 37°C for 20 min. The absorbance change of the reaction system at 405 nm was recorded using an ELISA instrument. At the same time, a control group without the target pesticide was set. Each experiment was repeated three times, and the data processing was the same as the above steps. The absorbance value of the control group without the target pesticide was recorded as A0, the absorbance value of the experimental group with the target pesticide added was recorded as A, and ΔA was recorded as ΔA. 405 =A0-A was used as the signal value of the reaction system for data analysis.

5. The method for visual detection of organochlorine pesticides based on metal organic framework immobilized enzyme composite materials according to claim 1, characterized in that: In step (4), 20 μL of ALP@ZIF-8 and 20 μL of 2,4-D were mixed evenly, and 20 μL of common biomolecules such as glucose Glu, lysine Lys, glycine Gly, histidine His, serine Ser and ions such as Na + , K + Mg 2+ , then add 120 μL PBS buffer and incubate at 37°C for 30 min, then add 20 μL P-NPP and incubate at 37°C for 20 min, use a microplate reader to record the absorbance changes of the reaction system at 405 nm, and repeat each experiment three times.

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