An enzyme@COFs composite material, preparation method and application thereof in isocarbophos detection

Abstract

An enzyme@COFs composite material, a preparation method and its application in isocarbophos detection belong to the field of biosensor technology. The present invention prepares HRP@COFs-PB based on HRP encapsulated in COFs-PB, connects the synthesized HRP@COFs-PB to an isocarbophos secondary antibody to form an Ab2@HRP@COFs-PB composite material, and forms an immunosensor platform through antigen-antibody specific recognition and isocarbophos pesticide recognition of antigen. There is a competitive relationship between isocarbophos and antibodies. The introduction of isocarbophos can reduce the recognition of antigens by the Ab2@HRP@COFs-PB composite material, thereby inhibiting the occurrence of color development and making the blue output of the entire biosensor lighter, thereby achieving the conversion of pesticide residue concentration into color change, and realizing the basis for quantitative analysis of isocarbophos.

Description

Technical Field

[0001] The present invention belongs to the technical field of biosensors, and in particular relates to an enzyme@COFs composite material, a preparation method and application thereof in isocarbophos detection. Background Art

[0002] With the development of modern agriculture, pesticides are widely used. However, they can cause water and soil pollution, lead to a decline in biodiversity, and lead to environmental damage and potential toxicity to human health, which has attracted great attention. Isocarbophos is a broad-spectrum organophosphorus pesticide with systemic characteristics. It can be absorbed through plant roots and transported throughout the plant, thereby providing more comprehensive protection. It is mainly used in agriculture to control various pests, especially aphids and red spiders. Its mechanism of action is mainly through inhibiting cholinesterase activity in insects, which leads to nervous system dysfunction and ultimately the death of the pests, achieving the desired pest control effect. Because isocarbophos is a relatively toxic pesticide, practical methods with high selectivity and sensitivity are urgently needed to monitor isocarbophos to ensure human health and food safety.

[0003] With the development of biotechnology, enzyme-based sensors that utilize bioenzymes as sensing elements have garnered widespread attention. These methods achieve quantitative analysis by detecting the inhibitory or activating effects of pesticides on specific enzyme activities. In recent years, photoelectric sensors have also gained increasing attention. These sensors utilize light (fluorescence, colorimetry, Raman spectroscopy, etc.) and electricity (current, voltage, or resistance, etc.) as detection signals, enabling rapid and sensitive analysis. The advent of screen-printed electrodes has made electrochemical detection more convenient and economical, and researchers have begun utilizing modified electrodes to improve detection sensitivity and selectivity. Paper-based analytical methods are widely used due to their low cost, portability, and ease of operation. Researchers have developed various types of test strips for rapid on-site detection of pesticide residues. For example, fluorescent and electrochemical test strips can be used directly for on-site testing, meeting the demand for rapid testing in agricultural production and food safety. These advances provide more efficient and convenient solutions for pesticide monitoring.

[0004] Isocarbophos is highly toxic to humans and ecosystems. Detecting its residues not only ensures food safety but also protects the ecological environment. Therefore, developing a highly sensitive and selective detection method for on-site monitoring of the pesticide isocarbophos is crucial. Summary of the Invention

[0005] The purpose of the present invention is to overcome the problems of poor test stability, cumbersome test methods, inconvenient portability and low selectivity in existing pesticide detection technologies. An enzyme@COFs composite material, a preparation method and its application in the detection of isocarbophos are provided. The detection sensor can detect the content of isocarbophos simply and highly selectively.

[0006] The preparation method of the enzyme@COFs composite material of the present invention comprises the following steps:

[0007] (1) First, 1-20 mg of trimesaldehyde (BTC) was dissolved in 50-500 μL of dimethylformamide (DMF), then transferred to 5-25 mL of deionized water and ultrasonically treated for 1-60 min; then 1 mL of 0.05-5.0 M acetic acid aqueous solution, 100 μL of 1-20 mg / mL horseradish peroxidase (HRP) aqueous solution and 0.1-10 mL of 1-20 mg of p-phenylenediamine (PPD) aqueous solution were added; after reacting for 5-60 min, the pH of the reaction system was adjusted to 3.5-4.5 with 0.1-10 mL of 4 M sodium hydroxide aqueous solution. The resulting precipitate was washed 2-4 times with deionized water by centrifugation and then suspended in 1 mL of deionized water to obtain an HRP@COFs-PB aqueous solution. The HRP enzyme was encapsulated in the COFs-PB material to form nanoparticles. The morphology of the nanoparticles was rough spherical with a size of 300-500 nm.

[0008] (2) 50 μL of 0.2-10 mg / mL aqueous solution of EDC and 50 μL of 0.2-10 mg / mL aqueous solution of hydroxysuccinimide (NHS) were added to 20-500 μL of HRP@COFs-PB aqueous solution and reacted in a metal bath at 25°C and 500-1000 rpm for 2-60 min. The resulting precipitate was washed 2-4 times with deionized water by centrifugation and then dispersed in 50-5000 μL of PBS buffer with a pH value of 7.0-7.5. 50 μL of 0.1 A secondary antibody animal immunoglobulin (Ab2) aqueous solution with a concentration of 100 to 5.0 mg / mL is reacted in a metal bath at 25°C and 500 to 1000 rpm for 2 to 240 minutes; after being placed at 4°C for 10 to 15 hours, 10 to 500 μL of an ovalbumin aqueous solution with a mass percentage of 0.5 to 5% is added, and the solution is reacted in a metal bath at 37°C and 500 to 1000 rpm for 10 to 600 minutes. The resulting precipitate is washed 2 to 4 times by centrifugation with PBS buffer to obtain the enzyme@COFs composite material, which has a smooth spherical morphology of 150 to 300 nm.

[0009] The enzyme@COFs composite material described in the present invention is prepared by the above method.

[0010] The enzyme@COFs composite material of the present invention can be used in the detection of isocarbophos, specifically: the enzyme@COFs composite material is dispersed in 50-5000 μL of PBS buffer to obtain Ab2@HRP@COFs-PB solution; 10-100 μL of 60 ng / mL isocarbophos antigen aqueous solution (A-BSA) is dropped into a 96 microliter plate and incubated at 37°C for 2 hours; 50-300 μL of PBS solution containing 0.05% (volume percentage) Tween-20 is used to detect the enzyme@COFs composite material. The wells were washed 2-4 times with PBST solution, and then 50-300 μL of 20 mg / mL OVA aqueous solution was added and incubated at 37°C for 30-120 min; the wells were washed 2-4 times with 50-300 μL of PBST solution, and then 30-150 μL of 0.05-1000 ng / mL isocarbothion aqueous solution and 30-150 μL of 60 ng / mL isocarbothion primary antibody aqueous solution were added (the amount of both isocarbothion and primary antibody was 0 as a pure blank control, and the amount of isocarbothion was 0). As a single control experiment), incubate at 37°C for 30-120 min; wash the wells 2-4 times with 50-300 μL of PBST solution, then add 10-300 μL of Ab2@HRP@COFs-PB solution, incubate at 37°C for 30-120 min; wash the wells 2-4 times with 50-300 μL of PBST solution; then add 50-300 μL of 14 mg / mL sodium dihydrogen phosphate substrate buffer at pH 4.0-4.2, and 50-300 μL of 0.8 m A 0.1 M tetramethylbenzidine (TMB) aqueous solution and 5 to 30 μL of a 0.1 M H2O2 aqueous solution were incubated at 37°C for 30 to 120 minutes, and the absorbance was measured at 630 nm (the reaction appears blue at this point. Measuring absorbance at 630 nm is to determine whether the reaction has occurred and whether the reaction is sufficient. A sufficient reaction is defined as an absorbance of approximately 0.5 in a single control experiment. Stop solution was then added to stop the reaction, and the absorbance was measured at 450 nm, at which point the color appears yellow). The reaction was terminated by adding a 2 M H2SO4 solution, and the absorbance was again measured at 450 nm. An image of the 96-μL plate (i.e., the biosensor) was captured using a mobile phone. Color processing was performed using ImageJ software to obtain an RGB image containing color information and the values ​​of the three RGB channels of the biosensor. The G and B values ​​were then subjected to a G×B calculation to obtain the G×B value. Finally, regression analysis was used to fit a linear curve representing the "G×B value-logarithm of isocarbophos concentration," thereby enabling the detection of isocarbophos.

[0011] The mechanism of the present invention is as follows:

[0012] HRP encapsulated in the prepared COF material COFs-PB is fabricated as HRP@COFs-PB. HRP@COFs-PB oxidizes TMB using a TMB / H2O2 colorimetric system to form oxTMB, which displays a blue color in aqueous solution. The synthesized HRP@COFs-PB is then linked to a secondary antibody, isocarbophos, to form an Ab2@HRP@COFs-PB composite. This composite utilizes antigen-antibody specificity and isocarbophos's antigen recognition, creating an immunosensor platform. Since isocarbophos competes with antibodies, the introduction of isocarbophos reduces antigen recognition by the Ab2@HRP@COFs-PB composite, thereby suppressing color development and reducing the blue output of the biosensor. This converts pesticide residue concentration into a color change, providing the basis for quantitative analysis of isocarbophos.

[0013] ImageJ is further combined to collect and analyze the image information of the biosensor, and the image is processed according to the G×B algorithm of the pixel, so as to render the image in a pseudo-color, making it clearer and more identifiable.

[0014] The present invention has the following characteristics:

[0015] (1) Using the room temperature synthesis method of COFs-PB, a COF-encapsulated enzyme biosensor was prepared to achieve the binding of COFs-PB with Ab2.

[0016] (2) Utilizing the enzymatic activity of HRP@COFs-PB, an enzyme-linked immunosorbent assay (ELISA) platform was prepared to achieve the binding of HRP@COFs-PB with Ab2. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 : Transmission electron microscopy image of COFs-PB obtained in Example 1;

[0018] Figure 2 : Transmission electron microscopy image of HRP@COFs-PB obtained in Example 2;

[0019] Figure 3 : Transmission electron microscopy image of Ab2@HRP@COFs-PB obtained in Example 3;

[0020] Figure 4 : Schematic diagram of the biosensor color and pseudo color obtained in Example 4;

[0021] Figure 5 : Linear relationship diagram of pseudo color G×B value obtained in Example 5 and logarithm of isocarbophos concentration;

[0022] Figure 6 : IE value bar graph under different pesticides obtained in Example 6. DETAILED DESCRIPTION

[0023] Example 1: Preparation of COFs material COFs-PB (PB represents ligands PPD and BTC)

[0024] First, 5 mg of trimesaldehyde (BTC) was dissolved in 250 μL of DMF, then transferred to 10 mL of deionized water and ultrasonicated for 5 minutes. Subsequently, 1 mL of 1.75 M acetic acid aqueous solution and 1 mL of PPD aqueous solution (PPD mass was 10 mg) were added. After the mixture reacted for 20 minutes, the pH was adjusted to 4 with 1 mL of sodium hydroxide aqueous solution (4 M). The resulting precipitate was washed three times with deionized water to obtain nanoparticles (the product mass was 3 mg, the morphology was a rough sphere of 400 nm, and its transmission electron microscopy image was shown in FIG. Figure 1 ) was resuspended in 1 mL of deionized water to obtain a COFs-PB aqueous solution.

[0025] Example 2: Preparation of COFs material HRP@COFs-PB

[0026] First, 5 mg of trimesaldehyde (BTC) was dissolved in 250 μL of DMF, then transferred to 10 mL of deionized water and ultrasonicated for 5 minutes. Subsequently, 1 mL of 1.75 M acetic acid aqueous solution and 100 μL of 5 mg / mL HRP aqueous solution were added, and then 1 mL of PPD aqueous solution (the mass of PPD was 10 mg) was added. After the mixture reacted for 20 minutes, the pH was adjusted to 4 with 1 mL of sodium hydroxide aqueous solution (4 M). The precipitate was washed three times with deionized water to obtain nanoparticles (the product mass was 3 mg, the morphology was a smooth sphere of 200 nm, and its transmission electron microscopy image was shown in FIG. Figure 2 ) was suspended in 1 mL of deionized water to obtain an HRP@COFs-PB aqueous solution.

[0027] The nanoparticles in Example 1 were formed by reacting BTC and PPD. In Example 2, BTC and HRP were first reacted to form a precursor, which was then repaired and synthesized by PPD. The precursor restricted the outward growth of the nanoparticles, as well as the Gibbs free energy and Ostwald ripening processes, resulting in a smaller size. The smoothness is due to the presence of the precursor, which allows for more stable and orderly growth.

[0028] Example 3: Preparation of Ab2@HRP@COFs-PB composite material

[0029] 50 μL of 2 mg / mL EDC aqueous solution and 50 μL of 4 mg / mL NHS aqueous solution were added to 400 μL of HRP@COFs-PB aqueous solution and reacted in a metal bath at 25°C and 800 rpm for 30 min; the resulting precipitate was centrifuged and washed three times with deionized water, then dispersed in 450 μL of PBS buffer with a pH value of 7.2, and then 50 μL of 2.5 mg / mL Ab2 aqueous solution was added, and reacted in a metal bath at 25°C and 800 rpm for 60 min; after being placed in a refrigerator at 4°C for 12 h, 100 μL of 1% by mass ovalbumin aqueous solution was added, and the reaction was carried out in a metal bath at 37°C and 800 rpm for 60 min. The resulting precipitate was centrifuged and washed three times with PBS buffer to obtain the enzyme@COFs composite material, which had a smooth spherical morphology of 200 nm (see transmission electron microscopy image). Figure 3 ).

[0030] Example 4: Analysis of the color produced by the biosensor using ImageJ images

[0031] The enzyme@COFs composite material obtained in Example 3 was dispersed in 100 μL of PBS buffer to obtain an Ab2@HRP@COFs-PB solution.

[0032] 100 μL of aqueous solution of azocarb antigen (A-BSA, concentration of 60 ng / mL) was dropped into a 96 μL plate (i.e., biosensor) and incubated at 37°C for 2 hours; the wells were washed three times with 200 μL of PBST solution, and then 200 μL of OVA aqueous solution (20 mg / mL) was added and incubated at 37°C for 50 minutes; the wells were washed three times with 200 μL of PBST solution, and then 50 μL of aqueous solution of azocarb (concentrations of 1000, 333, 111, 37, 4, 0.4, 0.15, 0.05 ng / mL) and 50 μL of aqueous solution of primary antibody of azocarb (concentration of 60 ng / mL) were added and incubated at 37°C for 5 minutes. 60min; wash the wells three times with 200μL PBST solution, then add 100μL Ab2@HRP@COFs-PB solution and incubate at 37°C for 60min; wash the wells three times with 200μL PBST solution, then add 100μL sodium hydrogen phosphate substrate buffer with a pH of 4.0, 50μL TMB aqueous solution and 15μL H2O2 aqueous solution (the concentration of sodium hydrogen phosphate substrate buffer is 14mg / mL, the concentration of TMB aqueous solution is 0.8mg / mL, and the concentration of H2O2 aqueous solution is 0.1M), incubate at 37°C for 60min, and measure the absorbance at a wavelength of 630nm; finally, add 2M H2SO4 solution to terminate the reaction, measure the absorbance again at a wavelength of 450nm, and use a mobile phone to take an image of the 96-microliter plate (i.e., biosensor) (as shown in Figure 2). Figure 4Finally, the color processing was performed using ImageJ software to obtain the RGB image of the biosensor containing color information and the values ​​of the three RGB channels. The G value and B value were calculated by G×B to obtain the G×B value. The RGB image was then processed by the G×B algorithm to obtain a pseudo-color image (as shown in the figure). Figure 4 As shown in the second row of images); isocarbothion and the isocarbothion primary antibody will compete for binding to the isocarbothion antigen, and the Ab2@HRP@COFs-PB composite material will bind to the isocarbothion primary antibody. Therefore, isocarbothion and color development are negatively correlated, that is, as the concentration of isocarbothion decreases, the color becomes darker, as shown in Figure 4 shown.

[0033] Example 5: Detection of pesticide residue concentration using an immunosensor platform

[0034] According to the G×B value of the pseudo-color image obtained in Example 4 and the corresponding pesticide concentration, a linear relationship curve of "G×B value-logarithm of isocarbophos concentration" was obtained by regression analysis, thereby realizing the detection of isocarbophos (such as Figure 5 shown).

[0035] Example 6: By adding negative controls, the selectivity and anti-interference ability of isocarbophos recognition were studied.

[0036] The test method is to use 9 pesticides, including chlorpyrifos, paraoxon, cypermethrin, tebuconazole, imidacloprid, chlorthiophanate, thiacloprid, chlorfenapyr, and carbaryl. Figure 6 The 2nd to 10th horizontal axes correspond to the above 9 pesticides in order) are negative controls, that is, the isocarbophos in Example 4 is replaced by these 9 pesticides for experiments (0.1 ng / mL). The results show that the inhibition rate (IE = (maximum absorbance - current value) / (maximum absorbance - blank value)) does not change significantly ( Figure 6 The IE values ​​of the pesticides corresponding to the 2nd to 10th horizontal axes are shown in Figure 1), indicating that even if these pesticide compounds coexist, they will not interfere with the recognition of isocarbophos (e.g. Figure 6 shown).

[0037] The blank value refers to the absorbance value measured at a wavelength of 450 nm when the pesticide concentration and the isocarbophos primary antibody concentration are both 0; the current value refers to the absorbance measured at a wavelength of 450 nm when the isocarbophos primary antibody concentration is fixed and the pesticide concentration is 0.1 ng / mL; the maximum absorbance refers to the absorbance measured at a wavelength of 450 nm when the isocarbophos primary antibody concentration is fixed and the pesticide concentration is the lowest (0.005 ng / mL).

Claims

1. A method for preparing an enzyme@COFs composite material, comprising the following steps: (1) First, 1-20 mg of trimesaldehyde was dissolved in 50-500 μL of dimethylformamide, then transferred to 5-25 mL of deionized water and ultrasonically treated for 1-60 minutes; then, 1 mL of 0.05-5.0 M acetic acid aqueous solution, 100 μL of 1-20 mg / mL horseradish peroxidase aqueous solution, and 0.1-10 mL of 1-20 mg of p-phenylenediamine aqueous solution were added; after reacting for 5-60 minutes, the pH of the reaction system was adjusted to 3.5-4.5 with 0.1-10 mL of 4 M sodium hydroxide aqueous solution. The resulting precipitate was washed 2-4 times with deionized water by centrifugation and then suspended in 1 mL of deionized water to obtain an HRP@COFs-PB aqueous solution. The HRP enzyme was encapsulated in the COFs-PB material to form nanoparticles. The morphology of the nanoparticles was rough spherical with a size of 300-500 nm. (2) 50 μL of 0.2-10 mg / mL carbodiimide aqueous solution and 50 μL of 0.2-10 mg / mL hydroxysuccinimide aqueous solution were added to 20-500 μL of HRP@COFs-PB aqueous solution, and the mixture was reacted in a metal bath at 25°C and 500-1000 rpm for 2-60 min. The resulting precipitate was washed 2-4 times with deionized water by centrifugation, and then dispersed in 50-5000 μL of PBS buffer with a pH value of 7.0-7.5, and then 50 μL of PBS was added. A 0.1-5.0 mg / mL aqueous solution of secondary animal immunoglobulin was added to a metal bath at 25°C and 500-1000 rpm for 2-240 minutes; after being placed at 4°C for 10-15 hours, 10-500 μL of a 0.5-5% by mass ovalbumin aqueous solution was added, and the mixture was reacted in a metal bath at 37°C and 500-1000 rpm for 10-600 minutes. The resulting precipitate was centrifuged and washed 2-4 times with PBS buffer to obtain the enzyme@COFs composite material.

2. An enzyme@COFs composite material, characterized by: The method is prepared by the method according to claim 1.

3. Use of the enzyme@COFs composite material according to claim 2 in the detection of isocarbophos.

4. The use of an enzyme@COFs composite material in the detection of isocarbophos according to claim 3, characterized in that: The enzyme@COFs composite material was dispersed in 50-5000 μL of PBS buffer to obtain Ab2@HRP@COFs-PB solution; 10-100 μL of 60 ng / mL aqueous solution of isocarbophos antigen was dropped into a 96-μL plate and incubated at 37°C for 2 hours; the wells were washed 2-4 times with 50-300 μL of PBST solution, and then 50-300 μL of 20 mg / mL OVA aqueous solution was added and incubated at 37°C for 30-120 minutes; the wells were washed 2-4 times with 50-300 μL of PBST solution, and then 30-150 μL of 0.05-1000 ng / mL aqueous solution of isocarbophos and 30-150 μL of 60 ng / mL aqueous solution of isocarbophos were added. The cells were incubated at 37°C for 30-120 min with a solution of primary antibody containing thiophosphine; the wells were washed 2-4 times with 50-300 μL of PBST solution, and then 10-300 μL of Ab2@HRP@COFs-PB solution was added and incubated at 37°C for 30-120 min; the wells were washed 2-4 times with 50-300 μL of PBST solution; then 50-300 μL of 14 mg / mL sodium dihydrogen phosphate substrate buffer with a pH of 4.0-4.2, 50-300 μL of 0.8 mg / mL tetramethylbenzidine aqueous solution and 5-30 μL of 0.1 M H2O2 aqueous solution were added and incubated at 37°C for 30-120 min, and the absorbance was measured at a wavelength of 630 nm; 2 M The reaction was terminated with H2SO4 solution, and the absorbance was measured at a wavelength of 450nm. An image of the 96-microliter plate was captured using a mobile phone. ImageJ software was used for color processing to obtain an RGB image containing color information of the biosensor and the values ​​of the three RGB channels. The G×B value was then calculated by performing a G×B calculation on the G and B values. Finally, regression analysis was used to fit a linear curve representing the "G×B value-logarithm of isocarbophos concentration," thereby enabling the detection of isocarbophos.

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