Application of self-cascaded cerium-based MOF nano-enzyme in detection of profenofos

Abstract

The invention discloses an application of a self-cascade cerium-based MOF (Metal Organic Framework) nano-enzyme in detection of profenofos, the cerium-based MOF nano-enzyme is a cerium-based metal organic framework material HMUiO-66 (Ce) nano-enzyme, and the organophosphorus-like hydrolase activity and laccase-like activity of the HMUiO-66 (Ce) nano-enzyme can be sequentially exerted by adjusting the pH value. The method comprises the following steps: firstly, hydrolyzing profenofos by HMUiO-66 (Ce) nano enzyme under an alkaline condition to produce 4-bromo-2-chlorophenol; the method comprises the following steps: dissolving 4-bromine-2-chlorophenol and 4-aminoantipyrine in water to obtain a solution, regulating the solution to be acidic, performing laccase-like activity by HMUiO-66 (Ce) nano-enzyme under an acidic condition, complexing 4-bromine-2-chlorophenol and 4-aminoantipyrine into quinoneimine, detecting the ultraviolet absorbance of the quinoneimine at 508nm, and calculating the concentration of profenofos. The invention finds that the nano enzyme has good selectivity on profenofos, is extremely simple and convenient in detection operation and low in cost, and makes on-site rapid detection of profenofos possible.

Description

Technical Field

[0001] The invention belongs to the technical field of detecting organophosphorus pesticides, and particularly relates to the application of self-cascading cerium-based MOF nanozymes in detecting propylbromophos. Background Art

[0002] Organophosphorus pesticides (OPs) are a class of chemical substances that can irreversibly inhibit the activity of acetylcholinesterase. In agricultural practice, OPs are widely used to control insect pests to improve crop yield and quality. They play a positive role in preventing and controlling agricultural diseases and improving the quality and yield of agricultural products. However, due to the long-term abuse of OPs, serious pesticide residue problems have occurred, which not only pollutes the environment but also poses potential risks to human health. Profenofos (PFF) is a widely used OP pesticide that is mostly used to control pests and diseases of a variety of crops. Although it has significant effects in agriculture, it is moderately toxic and can significantly inhibit acetylcholinesterase in the human body, causing damage to the nervous system. With the banning of other highly toxic pesticides, the use of OPs has continued to increase, and its residue problem has become increasingly prominent.

[0003] Traditional organophosphorus pesticide detection methods such as high performance liquid chromatography and gas chromatography have the disadvantages of being time-consuming, costly and relying on professional techniques, while immunoassays are limited by the instability of enzymes or antibodies. As a simple, rapid, economical and visual detection method, colorimetric analysis has become a viable alternative for detecting bromophos residues and has received increasing attention in recent years. Yang et al. disclosed a method for preparing hierarchical macroporous-microporous metal-organic frameworks (HMMOFs) with thin macropore walls by realizing open channels with macropore sizes of up to 100 nm and macropore walls of 11-12 nm in cerium-based MOF (HMUiO-66 (Ce)). Specifically, a microemulsion-guided assembly strategy was adopted, Pluronic P123 and Pluronic F127 were used as stabilizers, and toluene was used as the oil phase to form a columnar microemulsion to template HMUiO-66 (Ce); the HMMOFs can be used as nuclease mimics to hydrolyze and cleave stable phosphodiester bonds in various DNAs (Yang, J.; Li, K.; Gu, J. Hierarchically Macro-Microporous Ce-Based MOFs for the Cleavage of DNA. ACS Mater. Lett. 2022, 4, 385-391.), but there is no information about this cerium-based MOF. Related reports on the application of HMUiO-66(Ce) nanozyme in the detection of bromophos. Summary of the invention

[0004] The purpose of the present invention is to overcome the above-mentioned defects and shortcomings in the prior art and provide an application of cerium-based metal organic framework material HMUiO-66 (Ce) nanozyme in detecting bromophos.

[0005] The second object of the present invention is to provide an application of cerium-based metal organic framework material HMUiO-66 (Ce) nanozyme in the preparation of a product for detecting bromophos.

[0006] The third object of the present invention is to provide a method for detecting bromophos using cerium-based metal organic framework material HMUiO-66 (Ce) nanozyme.

[0007] The above-mentioned object of the present invention is achieved through the following technical solutions:

[0008] The present invention synthesizes the cerium-based metal organic framework material HMUiO-66 (Ce) nanozyme according to the literature (Yang, J.; Li, K.; Gu, J. Hierarchically Macro-Microporous Ce-Based MOFs for the Cleavage of DNA. ACS Mater. Lett. 2022, 4, 385-391.). The present invention finds that under pH = 9, the HMUiO-66 (Ce) nanozyme can exert the activity of a hydrolase-like enzyme, hydrolyze bromophos (PFF) to generate colorless 4-bromo-2-chlorophenol (BCP), and after complete hydrolysis, add phosphate buffer to adjust the pH value of the solution to pH = 6, so that the HMUiO-66 (Ce) nanozyme exerts a laccase-like activity, catalyzing the complexation of 4-bromo-2-chlorophenol with the color developer 4-aminoantipyrine (4-AP) to generate red quinone imine (QI), and the red product exhibits strong ultraviolet absorption at a wavelength of 508nm. As the concentration of bromophos increases, the color of the quinoneimine solution gradually changes from colorless to pink. Therefore, when the concentration of bromophos is high, the concentration of bromophos can be quantitatively analyzed by UV-visible spectrophotometer or by RGB mode of smartphone according to the color of the solution.

[0009] Therefore, the present invention provides the use of cerium-based metal organic framework material HMUiO-66 (Ce) nanozyme in detecting bromophos.

[0010] The present invention also provides the use of cerium-based metal organic framework material HMUiO-66 (Ce) nanozyme in the preparation of a product for detecting bromophos.

[0011] Furthermore, the product includes but is not limited to a kit or a colorimetric sensor.

[0012] Furthermore, the preparation of the HMUiO-66(Ce) nanozyme comprises the following steps:

[0013] S1. Pluronic P123 and Pluronic F127 were added to water and stirred to form linear micelles;

[0014] S2. Add toluene, acetic acid, sodium perchlorate monohydrate, mix well to form a milky white microemulsion;

[0015] S2. Add terephthalic acid and ammonium cerium nitrate, stir, centrifuge, wash and dry.

[0016] In the preparation method of HMUiO-66(Ce) nanozyme of the present invention, Pluronic P123 and Pluronic F127 are used as costabilizers, and toluene is used as an oil phase to form a columnar microemulsion template to synthesize HMUiO-66(Ce). First, Pluronic P123 combines with a small portion of Pluronic F127 to form linear micelles in the aqueous phase; when toluene is added, the transparent micelle solution is rapidly converted into a milky white microemulsion, which can form a crown ether complex with a multivalent metal, so that the periphery of each microemulsion will be surrounded by Ce metal clusters to form a metal organic framework (MOF); secondly, the triblock polymer surfactant (such as Pluronic P123 and Pluronic F127) can be in ClO 4- With the help of Pluronic P123, the metal organic framework grows into an ordered mesoporous structure in aqueous solution, forming open, continuous, thin-walled macrochannels. Among them, the ratio of Pluronic P123 and Pluronic F127 can greatly affect the morphology and stability of the microemulsion, and then affect the structure of MOF, forming a self-cascading cerium-based MOF nanozyme HMUiO-66 (Ce).

[0017] Furthermore, in step S1, the mass ratio of Pluronic P123 to Pluronic F127 is 1.5-2.5:1.

[0018] Furthermore, in step S1, the mass ratio of Pluronic P123 to Pluronic F127 is 2:1.

[0019] Furthermore, in step S2, the mass volume ratio of toluene, acetic acid and sodium perchlorate monohydrate is 2-3 mL: 1 mL: 2-4 g.

[0020] Furthermore, in step S2, the mass volume ratio of toluene, acetic acid and sodium perchlorate monohydrate is 2.4 mL: 1 mL: 3 g.

[0021] Furthermore, in step S3, the mass ratio of phthalic acid to ammonium cerium nitrate is 10-15:53-57.

[0022] Furthermore, in step S3, the mass ratio of phthalic acid to ammonium cerium nitrate is 30:137.

[0023] Furthermore, the washing method in step S3 is washing with water for 1 to 3 times, washing with dimethylformamide for 1 to 2 times, and soaking in acidic dimethylformamide solution for 10 to 14 hours, and soaking in ethanol for 2 to 3 days.

[0024] Furthermore, the soaking temperature is 55-65°C.

[0025] Furthermore, the soaking temperature is 60°C.

[0026] Preferably, after adding terephthalic acid and ammonium cerium nitrate in step S3, the mixture is stirred at 400 rpm for 40 minutes at 40°C, and the solid obtained by centrifugation is washed twice with water and once with dimethylformamide. Then, the solid after centrifugation is soaked in 4 mL of dimethylformamide solution containing 0.1 wt% HCl for 12 hours at a soaking temperature of 60°C. Then, the sample is soaked in ethanol at 60°C for two days, and the ethanol is replaced every day. Finally, the product is dried overnight in a vacuum drying oven at 60°C.

[0027] The present invention also provides a method for detecting bromophos using a cerium-based metal organic framework material HMUiO-66 (Ce) nanozyme, comprising the following steps:

[0028] S1. Incubate the HMUiO-66(Ce) nanozyme with known different concentrations of bromofos solution under alkaline conditions, adjust the pH to acidic conditions, add 4-aminoantipyrine, incubate, centrifuge and separate the supernatant;

[0029] S2. Detect the absorbance value at 508nm, with the bromophos concentration as the abscissa and the ultraviolet absorbance as the ordinate, and construct a bromophos concentration-ultraviolet absorbance standard curve; or analyze the RGB value according to the color of the supernatant, with the bromophos concentration as the abscissa and the RGB value as the ordinate, and construct a bromophos concentration-RGB value standard curve;

[0030] S3. After treating the sample to be tested with the HMUiO-66(Ce) nanozyme according to the conditions of step S1, the absorbance value of the sample at 508nm is detected, and the concentration of bromophos is calculated according to the standard curve of bromophos concentration-ultraviolet absorbance; or the RGB value of the sample is detected, and the concentration of bromophos is calculated according to the standard curve of bromophos concentration-RGB value.

[0031] Furthermore, the alkaline condition in step S1 is pH=8.5-9.5;

[0032] Furthermore, the alkaline condition in step S1 is adjusted using a Tris-HCl buffer solution.

[0033] Furthermore, the acidic condition in step S1 is pH=5.5-6.5.

[0034] Furthermore, the acidic condition in step S1 is adjusted using a phosphate buffer.

[0035] Furthermore, the alkaline condition in step S1 is pH=9; and the acidic condition in step S2 is pH=6.

[0036] Furthermore, the method for detecting the absorbance value at 508 nm in step S2 is to perform quantitative analysis by ultraviolet-visible spectrophotometer.

[0037] Furthermore, the method for detecting the RGB value in step S2 is to perform quantitative analysis using the RGB mode of a smart phone.

[0038] Compared with the prior art, the present invention has the following beneficial effects:

[0039] The present invention discloses the application of self-cascading cerium-based MOF nanozyme in the detection of bromophos. The cerium-based MOF nanozyme is a cerium-based metal organic framework material HMUiO-66 (Ce) nanozyme. By adjusting the pH, the organophosphorus hydrolase and laccase activities of the HMUiO-66 (Ce) nanozyme can be exerted successively. First, under alkaline conditions, the HMUiO-66 (Ce) nanozyme hydrolyzes bromophos to produce 4-bromo-2-chlorophenol; then the solution is adjusted to acidic, and under acidic conditions, the HMUiO-66 (Ce) nanozyme exerts laccase-like activity, 4-bromo-2-chlorophenol and 4-aminoantipyrine are complexed to synthesize quinone imine, and its ultraviolet absorbance at 508nm is detected, and the concentration of bromophos is calculated. The present invention finds that the HMUiO-66 (Ce) nanozyme has good selectivity for bromophos. The present invention is extremely simple in detection operation, has a rapid detection process, is low in cost, and has low requirements on detection instruments. The RGB mode of a smart phone can perform quantitative analysis, making it possible to quickly detect bromophos on site, and is convenient for wide promotion and application. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Figure 1 This is a scanning electron micrograph of the HMUiO-66(Ce) nanozyme of the present invention.

[0041] Figure 2 This is a scanning electron micrograph of the HMUiO-66(Ce) nanozyme of the present invention.

[0042] Figure 3 Schematic diagram of EDS Mapping characterization of HMUiO-66(Ce) nanozyme in the present invention.

[0043] Figure 4 This is a schematic diagram of the HMUiO-66(Ce) nanozyme detection principle in the present invention.

[0044] Figure 5 The figure is a standard curve of bromophos-ultraviolet absorbance in the present invention, wherein the abscissa is the concentration (ppm) of bromophos and the ordinate is the ultraviolet absorbance (au).

[0045] Figure 6 It is a relationship diagram between the RGB value and the bromophos concentration in the present invention.

[0046] Figure 7 This is a graph showing the detection results of different organophosphorus pesticides in the present invention. DETAILED DESCRIPTION

[0047] The present invention is further described below in conjunction with the accompanying drawings and specific examples, but the examples do not limit the present invention in any form. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art.

[0048] Unless otherwise specified, the reagents and materials used in the following examples are commercially available.

[0049] The technical solution of the present invention will be described in detail and completely in conjunction with the accompanying drawings, in order to enable those skilled in the art to have a deeper understanding of the advantages and characteristics of the present invention, thereby providing a clearer definition of the protection scope of the present invention. It should be noted that the embodiments described in the present invention are only illustrative examples of the present invention, rather than exhaustive of all possible implementation methods. Based on the embodiments disclosed in the present invention, all other embodiments that can be derived by those of ordinary skill in the art without creative work are deemed to be within the protection scope of the present invention.

[0050] Example 1 Preparation of HMUiO-66 (Ce) Nanozyme

[0051] HMUiO-66(Ce) nanozyme was prepared according to the preparation method disclosed in (Yang, J.; Li, K.; Gu, J. Hierarchically Macro-Microporous Ce-Based MOFs for the Cleavage of DNA. ACS Mater. Lett. 2022, 4, 385-391.). The specific preparation method is as follows:

[0052] First, 100 mg Pluronic P123 (PEO 20 PPO 70 PEO 20 )、50mg Pluronic F127(PEO 106PPO 70 PEO 106 ) was dissolved in 6 mL of deionized water, and Pluronic P123 combined with Pluronic F127 to form linear micelles in the aqueous phase. Next, 240 μL of toluene was added to the solution. Next, 0.1 mL of acetic acid and 300 mg of sodium perchlorate monohydrate (NaClO4·H2O) were added, and the mixture was vigorously stirred for 15 minutes to form a white microemulsion. Subsequently, 120 mg of phthalic acid (H2BDC) and 548 mg of cerium ammonium nitrate ((NH4)2Ce(NO3)6) were added to the microemulsion, and the mixture was then stirred at 400 rpm for 40 minutes at 40°C. The PEO fragments on the Pluronic P123 and Pluronic F127 molecules can form crown ether complexes with multivalent metal species, so the periphery of each microemulsion will be surrounded by Ce metal clusters. Subsequently, the metal organic frameworks (MOFs) will crystallize along the anchored Ce metal clusters and form Ce metal clusters in the ClO 4- In the presence of , an ordered mesoporous structure is assembled. After the reaction is completed, the solid is separated by centrifugation, washed twice with water and once with dimethylformamide (DMF). The centrifuged solid is then soaked in 4 mL of a dimethylformamide solution containing 0.1 wt% HCl for 12 hours at 60°C to remove residual ligands. The sample is then soaked in ethanol at 60°C for two days, with the ethanol replaced every day. Finally, the product is dried overnight in a 60°C vacuum oven.

[0053] The scanning electron microscopy image of HMUiO-66(Ce) nanomaterials is shown in Figure 1 As shown, the HMUiO-66(Ce) particles have highly open macropores and uniform thin walls.

[0054] Transmission electron microscopy images of HMUiO-66(Ce) nanomaterials Figure 2 As shown, the images further confirm that uniform macroporous channels are distributed in each HMUiO-66(Ce) particle.

[0055] Schematic diagram of EDS Mapping characterization of HMUiO-66(Ce) nanomaterials Figure 3 As shown, C, O and Ce elements are uniformly distributed on the HMUiO-66(Ce) particles.

[0056] Example 2 Construction of Propylene Bromide Concentration-UV Absorbance Standard Curve

[0057] Profenofos stock solutions with concentrations of 0.002 mg / mL, 0.01 mg / mL, 0.04 mg / mL, 0.1 mg / mL, 0.2 mg / mL, 0.4 mg / mL, 0.6 mg / mL, 0.8 mg / mL and 1 mg / mL were prepared with methanol and stored in a refrigerator at 4°C. 200 μL of HMUiO-66(Ce) (1 mg / mL) was added to a 2 mL centrifuge tube, followed by 700 μL of deionized water and 50 μL of Tris-HCl buffer solution (1 M, pH = 9), and finally 50 μL of different concentrations of profenofos was added, and the mixture was stirred at 40°C for 20 minutes, wherein the concentrations of profenofos were 0.1 μg / mL, 0.5 μg / mL, 1 μg / mL, 2 μg / mL, 5 μg / mL, 10 μg / mL, 20 μg / mL, 30 μg / mL, 40 μg / mL and 50 μg / mL. Then, 50 μL of phosphate buffer (1M, pH=2) was added to adjust the pH value of the solution to pH=6, and then 20 μL of 4-aminoantipyrine solution (2 mg / mL) was added, and stirring was continued at 40°C for 10 minutes. The sample was then centrifuged at a speed of 10,000 rpm for 3 minutes, and 800 μL of the supernatant was taken after centrifugation. The absorbance value of the supernatant at 508 nm was measured by an ultraviolet spectrophotometer; the bromophos concentration was used as the horizontal coordinate and the ultraviolet absorbance was used as the vertical coordinate to construct a bromophos concentration-ultraviolet absorbance standard curve.

[0058] Schematic diagram of the principle of HMUiO-66(Ce) nanozyme detecting bromophos Figure 4 As shown, under alkaline conditions (pH = 9), HMUiO-66 (Ce) nanozyme hydrolyzes bromophos to produce 4-bromo-2-chlorophenol; secondly, under acidic conditions (pH = 6), HMUiO-66 (Ce) nanozyme exerts laccase-like activity, complexes 4-bromo-2-chlorophenol and 4-aminoantipyrine to synthesize quinoneimine, and then detects its ultraviolet absorbance at 508 nm, and calculates the concentration of bromophos.

[0059] The standard curve of bromophos concentration-ultraviolet absorbance is as follows Figure 5 As shown, the fitted linear equation is: y = 0.0154x + 0.0082, where x is the concentration of bromophos, in μg / mL, and y is the ultraviolet absorbance.

[0060] The relationship between RGB value and bromophos concentration is as follows Figure 6 As shown in the figure, the RGB mode of the smartphone can be used for quantitative analysis of bromophos concentration. The RGB value represents the brightness of the three colors red, green, and blue, ranging from 0 to 255. Different combinations of RGB values ​​can produce a variety of colors. By comparing the RGB values ​​of each color, the bromophos concentration can be obtained.

[0061] Example 3: Verification of the Selectivity of HMUiO-66(Ce) Hydrolysis / Detection of Profenofos

[0062] The following sample solutions were prepared with methanol: 5 mg / L concentrations of profenofos, phoxim, trichlorfon, fenthion, fenitrothion and chlorpyrifos. The method of Example 2 was used to incubate with the HMUiO-66(Ce) nanozyme to verify the selectivity of the HMUiO-66(Ce) nanozyme constructed in Example 1 of the present invention.

[0063] The test results are as follows Figure 7 As shown, the absorbance value of bromophos is much higher than that of other organophosphorus pesticides, indicating that the HMUiO-66(Ce) nanozyme of the present invention has a high selectivity for bromophos.

[0064] In summary, the HMUiO-66(Ce) nanozyme prepared by the present invention can recognize and hydrolyze bromophos, can be used for the concentration detection of bromophos, and has the advantages of simple operation, low detection cost, fast detection speed and low requirements for detection instruments.

Claims

1. Application of cerium-based metal-organic framework material HMUiO-66 (Ce) nanozyme in the detection of bromofos.

2. Application of cerium-based metal-organic framework material HMUiO-66 (Ce) nanozyme in the preparation of products for detecting bromophos.

3. A method for detecting bromophos using cerium-based metal organic framework material HMUiO-66 (Ce) nanozyme, characterized in that: The following steps are involved: S1. Incubate the HMUiO-66(Ce) nanozyme with known different concentrations of bromofos solution under alkaline conditions, adjust the pH to acidic conditions, add 4-aminoantipyrine, incubate, centrifuge and separate the supernatant; S2. Detect the absorbance value of the supernatant at 508nm, with the bromophos concentration as the abscissa and the ultraviolet absorbance as the ordinate, and construct a bromophos concentration-ultraviolet absorbance standard curve; or according to the RGB value of the supernatant color analysis, with the bromophos concentration as the abscissa and the RGB value as the ordinate, construct a bromophos concentration-RGB value standard curve; S3. After treating the sample to be tested with the HMUiO-66(Ce) nanozyme according to the conditions of step S1, the absorbance value of the sample at 508nm is detected, and the concentration of bromophos is calculated according to the standard curve of bromophos concentration-ultraviolet absorbance; or the RGB value of the sample is detected, and the concentration of bromophos is calculated according to the standard curve of bromophos concentration-RGB value.

4. The method according to claim 3, characterized in that: The alkaline condition in step S1 is pH=8.5-9.

5.

5. The method according to claim 3, characterized in that: The alkaline condition in step S1 is adjusted using a Tris-HCl buffer solution.

6. The method according to claim 3, characterized in that: The acidic condition in step S1 is pH=5.5-6.

5.

7. The method according to claim 3, characterized in that: The acidic condition in step S1 is adjusted using phosphate buffer.

8. The method according to claim 3, characterized in that: The method for detecting the absorbance value at 508 nm in step S2 is to perform quantitative analysis using an ultraviolet-visible spectrophotometer.

9. The method according to claim 3, characterized in that: The method for detecting the RGB value in step S2 is to perform quantitative analysis using the RGB mode of the smart phone.

10. The use according to claim 2, characterized in that: The product is a reagent kit or a colorimetric sensor.

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