Enzyme-coated COFs composite material, preparation method and application of enzyme-coated COFs composite material in isocarbophos detection

Abstract

The invention discloses an enzyme-coated COFs composite material, a preparation method and application of the enzyme-coated COFs composite material in isocarbophos detection, and belongs to the technical field of biosensors. According to the invention, HRP (at) COFs-PB is prepared based on COFs-PB packaged HRP, the synthesized HRP (at) COFs-PB is connected with an isocarbophos second antibody to form an Ab2 (at) HRP (at) COFs-PB composite material, and an immunosensing platform is formed through antigen-antibody specific recognition and recognition of isocarbophos pesticide on an antigen. The isocarbophos and the antibody have a competitive relationship, and the introduction of the isocarbophos can reduce the recognition of the Ab2-coated HRP-coated COFs-PB composite material on the antigen, so that the color development is inhibited, the blue color output by the whole biosensor becomes light, the pesticide residue concentration is converted into the color change, and the basis of quantitative analysis on the isocarbophos is realized.

Description

Technical Field

[0001] The 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 the detection of isocarbophos. Background Art

[0002] With the development of modern agriculture, pesticides are widely used, but they can cause water pollution, soil pollution, and biodiversity decline, leading 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 the roots of plants and transported to the whole plant body, thus providing more comprehensive protection. It is mainly used in agriculture to control a variety of pests, especially aphids, red spiders, etc. Its mechanism of action is mainly to inhibit the activity of cholinesterase in insects, thereby causing nervous system dysfunction and ultimately leading to the death of pests, achieving the effect of pest control. Since isocarbophos is a relatively toxic pesticide, there is an urgent need for practical methods with high selectivity and sensitivity to monitor isocarbophos to ensure human health and food safety.

[0003] With the development of biotechnology, enzyme-based sensors using biological enzymes as sensing elements have received 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 received increasing attention. These sensors use light (fluorescence, colorimetry, Raman, etc.) and electricity (current, voltage or resistance, etc.) as detection signals to achieve rapid and sensitive analysis. The emergence of screen-printed electrodes has made electrochemical detection more convenient and economical, and researchers have begun to use modified electrodes to improve the sensitivity and selectivity of detection. Paper-based analytical methods are widely used because of their low cost, lightness and ease of operation. Researchers have developed various types of test papers for rapid on-site detection of pesticide residues. For example, fluorescent test papers and electrochemical test papers can be directly used for on-site detection, meeting the needs of agricultural production and food safety for rapid detection. These advances provide more efficient and convenient solutions for pesticide monitoring.

[0004] Isocarbophos has significant toxicity to humans and ecosystems, and the detection of its residues can not only ensure food safety, but also protect the ecological environment. Therefore, it is very necessary to develop a highly sensitive and selective detection method to achieve on-site monitoring of isocarbophos pesticides. Summary of the invention

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

[0006] The preparation method of an enzyme@COFs composite material described in 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 minutes; 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 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 obtained precipitate was washed 2-4 times with deionized water by centrifugation, and then suspended in 1 mL of deionized water to obtain HRP@COFs-PB aqueous solution, and the HRP enzyme was encapsulated in the COFs-PB material to form nanoparticles, and 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 carbodiimide (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 the mixture was reacted in a metal bath at 25°C and 500-1000 rpm for 2-60 min. The obtained precipitate was washed 2-4 times by centrifugation with deionized water, and then dispersed in 50-5000 μL of PBS buffer with a pH value of 7.0-7.5, and then 50 μL of 0.1 A secondary antibody animal immunoglobulin (Ab2) aqueous solution with a concentration of 5.0 mg / mL is reacted in a metal bath at 25°C and 500-1000 rpm for 2-240 min; after being placed at 4°C for 10-15 h, 10-500 μL of an ovalbumin aqueous solution with a mass percentage of 0.5-5% is added, and the solution is reacted in a metal bath at 37°C and 500-1000 rpm for 10-600 min. The resulting precipitate is washed 2-4 times by centrifugation with PBS buffer to obtain the enzyme@COFs composite material, which has a smooth spherical morphology of 150-300 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 isothiocarb, 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 isothiocarb 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 50-300 μL of 20 mg / mL OVA aqueous solution 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 azothion aqueous solution and 30-150 μL of 60 ng / mL azothion primary antibody aqueous solution were added (the dosage of azothion and primary antibody was 0 as a pure blank control, and the dosage of azothion was 0 As a single control experiment), incubate at 37°C for 30-120 min; wash the wells with 50-300 μL of PBST solution 2-4 times, then add 10-300 μL of Ab2@HRP@COFs-PB solution, incubate at 37°C for 30-120 min; wash the wells with 50-300 μL of PBST solution 2-4 times; then add 50-300 μL of 14 mg / mL sodium dihydrogen phosphate substrate buffer with a pH of 4.0-4.2, and 50-300 μL of 0.8 m g / mL tetramethylbenzidine (TMB) aqueous solution and 5-30 μL 0.1M H2O2 aqueous solution are incubated at 37°C for 30-120 minutes, and the absorbance is measured at a wavelength of 630 nm (the reaction is blue at this time, and the absorbance is measured at a wavelength of 630 nm to see whether there is a reaction and whether the reaction is sufficient. Sufficient reaction means that the absorbance of a single control experiment is about 0.5, and then a stop solution is added to stop the reaction, and the absorbance is measured at a wavelength of 450 nm, and the color is yellow at this time); 2M H2SO4 solution is added to stop the reaction, and the absorbance is measured at a wavelength of 450 nm, and a mobile phone is used to take an image of a 96-microliter plate (i.e., a biosensor); ImageJ software is used for color processing to obtain an RGB image of the biosensor containing color information and the values ​​of the three RGB channels, and the G value and the B value are calculated by G×B to obtain the G×B value; finally, a regression analysis method is used to fit a linear relationship curve of "G×B value-logarithm of isocarbophos concentration", thereby realizing the detection of isocarbophos.

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

[0012] Based on the prepared COF material COFs-PB, HRP encapsulated with HRP is made into HRP@COFs-PB. HRP@COFs-PB can oxidize TMB through the TMB / H2O2 color development system to form oxTMB, which appears blue in aqueous solution. The synthesized HRP@COFs-PB is connected to the secondary antibody of isocarbamide to form Ab2@HRP@COFs-PB composite material, and an immunosensor platform is formed through antigen-antibody specific recognition and isocarbamide pesticide recognition of antigen. There is a competitive relationship between isocarbamide and antibodies. The introduction of isocarbamide can reduce the recognition of antigens by Ab2@HRP@COFs-PB composite materials, thereby inhibiting the appearance of color development and making the blue output of the entire biosensor lighter, thereby achieving the conversion of pesticide residue concentration into color changes, and realizing the basis for quantitative analysis of isocarbamide.

[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 combination of COFs-PB and Ab2.

[0016] (2) Utilizing the enzymatic activity of HRP@COFs-PB, an enzyme-linked immunosorbent sensing 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 color and pseudo color of the biosensor obtained in Example 4;

[0021] Figure 5 : A linear relationship diagram between the pseudo-color G×B value obtained in Example 5 and the logarithm of the concentration of isocarbophos;

[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 is 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 is 3 mg, the morphology is a rough sphere of 400 nm, and its transmission electron microscopy image is shown in 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 ultrasonically treated 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 Figure 2 ) was suspended in 1 mL of deionized water to obtain an HRP@COFs-PB aqueous solution.

[0027] The nanoparticles of Example 1 are formed by the reaction of BTC and PPD, and the nanoparticles of Example 2 are formed by the reaction of BTC and HRP to form a precursor, and then PPD is used for repair synthesis. The precursor limits the outward growth of the nanoparticles, as well as the Gibbs free energy and Ostwald ripening process, so that the size becomes smaller. The smoothness is because the presence of the precursor allows it to grow more stably and orderly.

[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 obtained precipitate was washed three times with deionized water by centrifugation, and 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% ovalbumin aqueous solution was added, and reacted in a metal bath at 37°C and 800 rpm for 60 min, and the obtained precipitate was washed three times with PBS buffer by centrifugation to obtain the enzyme@COFs composite material, which has 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 through 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 bisphosphonium 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 3 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 min; the wells were washed 3 times with 200 μL of PBST solution, and then 50 μL of aqueous solution of bisphosphonium (concentrations of 1000, 333, 111, 37, 4, 0.4, 0.15, 0.05 ng / mL) and 50 μL of aqueous solution of bisphosphonium primary antibody (concentration of 60 ng / mL) were added and incubated at 37°C for 50 min. 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 disodium hydrogen phosphate substrate buffer with a pH of 4.0, 50μL TMB aqueous solution and 15μL H2O2 aqueous solution (the concentration of disodium 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 4The first row of images shows the image); finally, the ImageJ software is used to perform color processing to obtain the RGB image of the biosensor containing color information and the values ​​of the three RGB channels, and the G value and the B value are calculated by G×B to obtain the G×B value; the RGB image is then processed by the G×B algorithm to obtain a pseudo-color image (as shown in Figure 4 As shown in the second row of images); isocarb and isocarb primary antibody will compete for binding to the isocarb antigen, and the Ab2@HRP@COFs-PB composite material will bind to the isocarb primary antibody. Therefore, isocarb and color development are negatively correlated, that is, the color will become darker as the concentration of isocarb decreases, such as 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: The selectivity and anti-interference ability of isocarbophos recognition were studied by adding negative controls.

[0036] The test method is to use 9 pesticides: chlorpyrifos, paraoxon, cypermethrin, tebuconazole, imidacloprid, chlorthiazolin, thiacloprid, chlorfenapyr, and carbaryl. Figure 6 The 2nd to 10th horizontal axes correspond to the above 9 pesticides in order) as negative controls, that is, the isocarbophos in Example 4 was replaced with these 9 pesticides for experiments (0.1 ng / mL), and the results showed that the inhibition rate (IE = (maximum absorbance - current value) / (maximum absorbance - blank value)) did not change significantly ( Figure 6 The IE values ​​of the pesticides corresponding to the 2nd to 10th horizontal axes of the graph 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 450nm when the pesticide concentration and the concentration of the isocarbophos primary antibody are both 0; the current value refers to the absorbance measured at a wavelength of 450nm when the concentration of the isocarbophos primary antibody is fixed and the pesticide concentration is 0.1ng / mL; the maximum absorbance refers to the absorbance measured at a wavelength of 450nm when the concentration of the isocarbophos primary antibody is fixed and the pesticide concentration is the lowest (0.005ng / mL).

Claims

1. A method for preparing an enzyme@COFs composite material, the steps of which are as follows: (1) First, 1-20 mg of trimesaldehyde is 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 are added; after reacting for 5-60 minutes, the pH of the reaction system is adjusted to 3.5-4.5 with 0.1-10 mL of 4 M sodium hydroxide aqueous solution, the obtained precipitate is 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, wherein the HRP enzyme is encapsulated in the COFs-PB material to form nanoparticles, and the morphology of the nanoparticles is a rough sphere 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 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 A 0.1-5.0 mg / mL aqueous solution of secondary animal immunoglobulin is reacted in a metal bath at 25°C and 500-1000 rpm for 2-240 min; after being placed at 4°C for 10-15 h, 10-500 μL of an ovalbumin aqueous solution with a mass percentage of 0.5-5% is added, and the solution is reacted in a metal bath at 37°C and 500-1000 rpm for 10-600 min. The resulting precipitate is centrifuged and washed 2-4 times with PBS buffer to obtain the enzyme@COFs composite material.

2. An enzyme@COFs composite material, characterized in that: The method is prepared by the method of 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 as claimed in 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 phosphamidon 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 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 aqueous solution of phosphamidon and 30-150 μL of 60 ng / mL aqueous solution of phosphamidon were added. The first antibody aqueous solution of thiophosphorus was incubated at 37°C for 30-120 min; 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 the wells were 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 the wells were 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. A mobile phone was used to capture an image of a 96-microliter plate. ImageJ software was used for color processing to obtain an RGB image of the biosensor containing color information and the values ​​of the three RGB channels. The G and B values ​​were then subjected to G×B calculation to obtain the G×B value. Finally, regression analysis was used to fit the linear relationship curve of "G×B value-logarithm of isocarbophos concentration", thereby realizing the detection of isocarbophos.

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