Light response type oxidase-like simulant with donor-receptor structure as well as preparation method and application of light response type oxidase-like simulant

Abstract

The invention belongs to the technical field of material chemistry, and provides a light response type oxidase-like simulant with a donor-receptor structure as well as a preparation method and application of the light response type oxidase-like simulant. The oxidase-like simulant is obtained through an ammonia-aldehyde condensation reaction of 1, 3, 6, 8-tetra (4-aminophenyl) pyrene or 2, 4, 6-tri (4-aminophenyl)-1, 3, 5-triazine and BDP (1, 3, 6, 8-tetra (4-aminophenyl) pyrene. The oxidase-like enzyme can form a sensor or a kit for detecting organophosphorus pesticides together with TMB, AChE and AChE substrates; qualitative determination can be realized through visual inspection of color change, and quantitative determination can also be realized through assistance of a colorimetric instrument. The light response type nano enzyme and AChE cascade reaction can realize visual rapid detection of various organophosphorus pesticides in traditional Chinese medicinal materials such as astragalus membranaceus and angelica sinensis, and has the advantages of being simple, rapid, high in sensitivity, good in practicability and the like.

Description

Technical Field

[0001] The present invention belongs to the technical field of material chemistry, and relates to a covalent organic polymer-based oxidase and its application in detecting pesticide residues. Background Art

[0002] Disclosing the information of this background art is intended to enhance the understanding of the overall background of the present invention, and it is not necessarily regarded as an admission or an implication in any form that this information constitutes the prior art already known to those of ordinary skill in the art.

[0003] Organophosphorus pesticides (OPs) are derivatives of phosphoric acid or phosphonic acid, and most contain a thiophosphoryl group (P=S) or a phosphoryl group (P=O). They are a type of insecticides and fungicides commonly used in agriculture. The main mechanism of action is to inhibit the activity of acetylcholinesterase (AChE) in the insect nervous system, thereby causing poisoning and death. Although organophosphorus pesticides have a highly effective killing effect on pests, the potential safety hazards of their residues in food have increasingly attracted attention. Long-term exposure to organophosphorus pesticides may lead to chronic poisoning, harming the nervous system, immune system, and endocrine system, and may even cause chronic diseases such as cancer in severe cases. Therefore, the residues of organophosphorus pesticides in traditional Chinese medicines such as Astragalus membranaceus and Angelica sinensis must be strictly monitored to ensure the health of consumers.

[0004] Currently, the methods for detecting OPs internationally mainly include instrumental analysis methods and immunoassay methods. Instrumental analysis methods include gas chromatography (GC), high-performance liquid chromatography (HPLC), gas chromatography-mass spectrometry (GC-MS), etc. Although these methods have strong specificity and high sensitivity, they also have disadvantages such as high cost, the need for detection in a laboratory environment, complex operation, and the need for skilled operators. Enzyme-linked immunosorbent assay is relatively commonly used in immunoassay methods, but it has disadvantages such as long detection time and low detection efficiency, resulting in the inability of these analysis methods to meet the requirements of on-site rapid detection. Therefore, the development of simple, rapid, and practical OPs detection methods with good analytical performance has attracted increasing attention. Currently, fluorescence (FL) and electrochemical (EC) sensing strategies using covalent organic frameworks (COFs) and metal-organic frameworks (MOFs) have attracted considerable attention in the detection of environmental pollutants. Although a relatively low detection limit can be achieved, it is easily interfered by the sample matrix and false positive results may occur. In addition, the stability of electrochemical detection needs to be improved, and it has high requirements for equipment maintenance and operators, and is not suitable for all laboratory or on-site detection scenarios.

[0005] Therefore, there is a need to develop a simple, green, rapid, and economical detection method for visual monitoring of OPs pesticide residues in foods. A covalent organic polymer (POP)-based oxidase-like enzyme involved in the present invention can not only achieve highly sensitive detection of various organophosphorus pesticides, but also increase the universality of the application scenarios with visible colorimetric effects. Summary of the Invention

[0006] Aiming at the problems of high detection limits for organophosphorus pesticide residues in foods, the need for pretreatment such as enrichment, and complex detection, the present invention provides a donor-acceptor-structured photo-responsive oxidase-like enzyme mimic, which uses 1,3,6,8-tetrakis(4-aminophenyl)pyrene or 2,4,6-tris(4-aminophenyl)-1,3,5-triazine as an electron donor and a substituted 5,5-difluoro-1,3,7,9-tetramethyl-10-phenyl-5H-4,5-dipyrrolo[1,2-c:2’,1’-f][1,3,2]diazaborinine-2,8-dicarbaldehyde as an electron acceptor, has the function of an oxidase-like enzyme, and has photo-responsive performance, and can form a reagent for detecting organophosphorus pesticide residues together with TMB, acetylcholinesterase, and its substrate.

[0007] Another object of the present invention is to provide a preparation method for the above-mentioned donor-acceptor-structured photo-responsive oxidase-like enzyme mimic.

[0008] Another object of the present invention is to provide an application of the above-mentioned donor-acceptor-structured photo-responsive oxidase-like enzyme mimic in detecting organophosphorus pesticide residues.

[0009] To achieve the above object, the present invention adopts the following technical solutions.

[0010] A preparation method for a porous organic polymer, comprising the following steps: performing an ammonia-aldehyde condensation reaction on a ligand of an electron donor and a ligand of an electron acceptor; The ligand of the electron donor is selected from 1,3,6,8-tetrakis(4-aminophenyl)pyrene (Py) or 2,4,6-tris(4-aminophenyl)-1,3,5-triazine (TAPT); the electron acceptor is 5,5-difluoro-1,3,7,9-tetramethyl-10-phenyl-5H-4,5-dipyrrolo[1,2-c:2’,1’-f][1,3,2]diazaborinine-2,8-dicarbaldehyde (BDP, CAS No. 2022166-57-8).

[0011] Preferably, the electron donor is 1,3,6,8-tetrakis(4-aminophenyl)pyrene (Py).

[0012] Preferably, the above preparation method comprises the following steps: Dispersing the ligand of the electron donor, the ligand of the electron acceptor, and a catalyst in a solvent to obtain a precursor mixture; The precursor mixture was repeatedly freeze-thawed and degassed, and reacted at 120 °C for 72 h under oxygen-free conditions. The reaction system was separated and purified to obtain a brownish-black powder, which was the porous organic polymer.

[0013] Preferably, in the above steps, the solvent is a mixture of n-butanol and benzyl alcohol; the catalyst is a 6 M acetic acid solution; the volume ratio of n-butanol, benzyl alcohol and acetic acid solution is 1:3:0.4.

[0014] In the above steps, the molar ratio of the electron donor to the electron acceptor is 1:2.

[0015] A porous organic polymer obtained by the above preparation method has an approximate spherical morphology and a particle size of 62-80 nm.

[0016] The above porous organic polymer can be used as a peroxidase-mimicking enzyme.

[0017] A sensor for detecting organophosphorus pesticides comprises the following components: The above peroxidase-mimicking enzyme, 3,3',5,5'-tetramethylbenzidine (TMB), acetylcholinesterase (AChE) and the substrate of acetylcholinesterase.

[0018] Preferably, the substrate of acetylcholinesterase is selected from acetylthiocholine (ATCh) or acetylcholine, and can also be selected from methylcholine, ethylcholine, butyrylcholine. The hydrolysis efficiency of these substrates is usually lower than that of acetylcholine or acetylthiocholine.

[0019] The present invention also provides the application of the above photo-responsive peroxidase-mimicking enzyme or the above sensor in detecting organophosphorus pesticides and a kit prepared therefrom.

[0020] Preferably, the kit can also contain a buffer solution and a standard of organophosphorus pesticide.

[0021] The organophosphorus pesticide is preferably a thiophosphate, phosphate, phosphoramide organophosphorus pesticide, such as parathion-methyl, chlorpyrifos, phoxim, triazophos, trichlorfon, malathion, dichlorvos. More preferably, the organophosphorus pesticide is selected from parathion-methyl, chlorpyrifos, phoxim or triazophos.

[0022] A method for detecting organophosphorus pesticide residues comprises the following steps: (1) The test solution was pre-incubated with acetylcholinesterase at 37 °C, and then the substrate of acetylcholinesterase was added and incubated continuously to obtain an incubation solution; (2) The incubation solution, the suspension of the above porous organic polymer and the TMB solution were mixed in a buffer solution with pH 4 and irradiated with a 635 nm excitation light source for reaction. The absorbance value at 652 nm was measured for the supernatant after centrifugation.

[0023] Preferably, the solution to be measured is obtained by extracting the sample to be measured with ethyl acetate and then removing the particulate matter from the extract.

[0024] In order to achieve quantitative detection, the method further includes the steps of detecting the absorbance values of a series of standard solutions of organophosphorus pesticides under the same conditions and preparing a standard curve of organophosphorus pesticide concentration - absorbance.

[0025] The mechanism of the present invention is as follows: The photo-responsive nanozyme with a donor-acceptor structure provided by the present invention is constructed from an electron donor and an electron acceptor. Under the irradiation of an excitation light source, it can promote electron transfer to generate reactive oxygen species (ROS) to oxidize 3,3’,5,5’-tetramethylbenzidine (TMB) into blue ox-TMB. At this time, introducing the natural enzyme acetylcholinesterase (AChE), the choline (TCH) generated by its reaction with acetylthiocholine (ATCh) can prevent the oxidation of TMB. At this time, the presence of organophosphorus pesticides (OPs) can inhibit the activity of AChE, thereby restoring the blue color of oxTMB. The darker the blue color, the larger the proportion of OPs. Qualitative determination can be achieved by visually observing the color change. Within a certain concentration range, the color change is correlated with the OPs concentration, and quantitative detection can be achieved with the assistance of a colorimetric instrument.

[0026] The present invention has the following advantages: The photo-responsive nanozyme of the present invention has a narrow energy band gap, can accelerate the electron transfer rate, and thus has good nanozyme-like catalytic activity. Under the irradiation of an excitation light source, it can oxidize TMB into blue ox-TMB, avoiding the use of H2O2 with poor stability and high toxicity; the photo-responsive nanozyme of the present invention solves the problem of complex and cumbersome analysis of the target by large-scale instruments, greatly improving the efficiency of analysis and detection. The cascade reaction of the photo-responsive nanozyme of the present invention with acetylcholinesterase (AChE) can realize the visual and rapid detection of various organophosphorus pesticides in Chinese medicinal materials and food materials such as Astragalus membranaceus and Angelica sinensis, with the advantages of simplicity, rapidity, high sensitivity, and good practicability, providing technical support for food safety in China and having important practical application significance. Description of the Drawings

[0027] Figure 1 are the Fourier transform infrared spectra of the Py-BDP material (Py-BDP), the TAPT-BDP material (TAPT-BDP), and their raw materials; Figure 2 are the scanning electron microscope image (A), particle size distribution diagram (B), XPS full spectrum (C), specific surface area analysis and pore size analysis diagram (D), and thermogravimetric analysis diagram (E) of the Py-BDP material; Figure 3are the solid UV-Vis diffuse reflectance spectra and Tauc plots of Py-BDP material (A) and TAPT-BDP material (B); Figure 4 are the fluorescence emission spectra of Py-BDP material (A) and TAPT-BDP material (B) (E x = 257 nm); Figure 5 are the Mott-Schottky plots of Py-BDP material (A) and TAPT-BDP material (B); Figure 6 are the energy band structure diagrams (A), electrochemical impedance diagrams (B) and transient photocurrent response diagrams (C) of Py-BDP material and TAPT-BDP material; Figure 7 are the UV absorption spectra of Py-BDP material, TAPT-BDP material and their ligands; Figure 8 are the optimization diagrams of the reaction conditions of Py-BDP-like oxidase. Among them, (A) is the optimization diagram of the pH value of the buffer solution, (B) is the optimization diagram of the Py-BDP concentration, (C) is the optimization diagram of the TMB concentration, (D) is the optimization diagram of the reaction time, and (E) is the optimization diagram of the reaction temperature; Figure 9 are the UV absorption spectra (A) and linear calibration curves (B) of Py-BDP-like oxidase for different concentrations of acetylcholinesterase; Figure 10 are the UV absorption spectra, colorimetric diagrams (A) and linear calibration curves (B) of Py-BDP-like oxidase for parathion-methyl; Figure 11 are the UV absorption spectra, colorimetric diagrams (A) and linear calibration curves (B) of Py-BDP-like oxidase for chlorpyrifos; Figure 12 are the UV absorption spectra, colorimetric diagrams (A) and linear calibration curves (B) of Py-BDP-like oxidase for phoxim; Figure 13 are the UV absorption spectra, colorimetric diagrams (A) and linear calibration curves (B) of Py-BDP-like oxidase for triazophos; Figure 14 are the UV-Vis spectra for the detection of OPs in Astragalus membranaceus and Angelica sinensis from different origins. Detailed implementation mode

[0028] The present invention will be further described below in conjunction with the embodiments and the drawings, but the present invention is not limited by the following embodiments.

[0029] Example 1 Preparation of a light-responsive porous organic polymer material 1. Py-BDP material Take a 10 mL Pyrex tube and add Py (11.3 mg, 0.02 mM), BDP (15.2 mg, 0.04 mM), 0.25 mL n-BuOH, 0.75 mL BnOH, and 0.1 mL of 6 M aqueous acetic acid solution. Stir the mixture for 15 minutes, degas it through three freeze-pump-thaw cycles, heat it at 120 °C for 72 h after vacuum sealing. Cool the reaction mixture to room temperature, filter the precipitate, wash it 5 times with THF and acetone, and finally dry it under vacuum at 60 °C for 12 h to obtain the Py-BDP material with a yield of 79.6%.

[0030] The Fourier transform infrared spectrum of the Py-BDP material is as Figure 1 shown. The amino stretching vibration peak (-NH2) of Py is clearly visible at 3345 cm -1 -1, the aldehyde stretching vibration peak (C=O) of BDP disappears at 1667 cm -1 -1, and the imine stretching vibration peak (C=N) of Py-BDP appears at approximately 1612 cm -1 -1, indicating that the Py-BDP has been successfully synthesized.

[0031] The surface morphology of the Py-BDP material was analyzed by scanning electron microscopy experiments. The results show that the surface of Py-BDP is rough and spherical in shape ( Figure 2 A), and the particle diameter is about 62 - 80 nm ( Figure 2 B). The full XPS spectrum ( Figure 2 C) shows that Py-BDP mainly contains C, N, and O elements, which is in line with the theory.

[0032] N2 adsorption and desorption were carried out on it at 77 K ( Figure 2 D), and the adsorption isotherm shows a type I; according to the BET model, the surface area of Py-BDP is 21.37 m 2 2 -1 / g; the main pore size calculated by non-local density functional theory (NLDFT) is 1.27 nm; and the pore size distribution of Py-BDP is uniform, including both micropores and mesopores.

[0033] The thermal stability of Py-BDP was analyzed using a thermogravimetric analyzer. The results show that from 30 °C to 280 °C, the mass loss of Py-BDP is only 8.05%, while the polymer structure mainly degrades between 280 °C and 535 °C, during which 23.45% of the mass is lost. When the temperature reaches 1000 °C, 60% of the volume of the Py-BDP substance remains, indicating its good thermal stability ( Figure 2E).

[0034] 2. TAPT-BDP Material Take a 10 mL Pyrex tube, add TAPT (10.6 mg, 0.03 mM), BDP (17.1 mg, 0.045 mM), 0.25 mL n-Bu OH, 0.75 mL Bn OH, and 0.1 mL of 6 M aqueous acetic acid solution. Stir the mixture for 15 minutes, degas it through three freeze-pump-thaw cycles, seal it under vacuum, and heat it at 120 °C for 72 h. Filter the resulting precipitate and wash it 5 times with THF and acetone, and finally dry it under vacuum at 60 °C for 12 h to obtain the TAPT-BDP material.

[0035] The Fourier transform infrared spectrum of the TAPT-BDP material is as Figure 1 shown. The amino stretching vibrations (-NH2) of TAPT at 3464 cm -1 , 3325 cm -1 , and 3214 cm -1 and the aldehyde stretching vibration (C=O) of BDP at 1667 cm -1 disappear, while an imine stretching vibration (C=N) appears at approximately 1604 cm -1 for TAPT-BDP, confirming that an aldol condensation reaction has occurred for TAPT-BDP.

[0036] 3. Comparison of the Optoelectronic Properties of Different Materials The optoelectronic properties of the Py-BDP material and the TAPT-BDP material were studied using solid-state UV-visible diffuse reflectance spectroscopy, fluorescence emission spectroscopy, and Mott-Schottky barriers to screen out the material with the optimal photocatalytic activity.

[0037] The results of the ultraviolet / visible light diffuse reflectance spectrum (UV / vis DRS) are as Figure 3 shown: Py-BDP exhibits effective absorption characteristics in the ultraviolet to infrared range, with an absorption edge located at approximately 1015 nm, which is slightly red-shifted by 105 nm compared to TAPT-BDP. This indicates that Py-BDP has a stronger ability to absorb visible light, which helps to improve the photocatalytic efficiency. Tauc plots were used to determine the band gaps (E g ) of Py-BDP and TAPT-BDP to be 1.16 eV and 1.39 eV, respectively, demonstrating that the energy band gap of Py-BDP is significantly lower than that of TAPT-BDP, which allows light to excite more electrons of Py-BDP into the conduction band and accelerate the electron transfer efficiency.

[0038] The results of the fluorescence emission spectrum are as Figure 4As shown: Py-BDP and TAPT-BDP can effectively induce photoinduced charge separation; compared with the monomers, the fluorescence intensities of Py-BDP and TAPT-BDP are significantly reduced, indicating that the generation of porous organic polymers (POPs) effectively prevents the recombination of photo-generated carriers.

[0039] Mott-Schottky plots of Py-BDP and TAPT-BDP ( Figure 5 ) show that both obtained materials are n-type semiconductors, mainly generating electron carriers. According to the M-S plots with x-axis intercepts, the conduction band potentials (E CB ) of Py-BDP and TAPT-BDP relative to the normal hydrogen electrode (NHE) are -0.55 eV and -0.77 eV, respectively, slightly higher than the O2 / O2 •− redox potential (-0.33 eV vs NHE), which enables them to convert O2 into O2 •− . Further calculations show that according to the formula E VB = E g + E CB , the valence band potentials (E VB ) of Py-BDP and TAPT-BDP are 0.75 eV and 0.89 eV, respectively ( Figure 6 A).

[0040] Electrochemical impedance spectroscopy (EIS) is shown in Figure 6 B: compared with TAPT-BDP, the arc radius of Py-BDP is significantly smaller. Accordingly, Py-BDP reduces the interfacial hindrance of charge transport, enabling carriers to move quickly and then participate in the reaction.

[0041] The transient photocurrent sensitivity of Py-BDP is significantly stronger than that of TAPT-BDP ( Figure 6 C), demonstrating that Py-BDP can effectively achieve photo-generated charge separation, and the generation of Py-BDP can also promote charge separation, accelerate the transport rate of photo-generated carriers, thereby improving the photocatalytic activity.

[0042] The above results indicate that the photocatalytic activity of Py-BDP is superior to that of TAPT-BDP.

[0043] For further verification, the visible-infrared absorption spectra of Py-BDP and TAPT-BDP were measured under the same conditions (40 mM TMB; pH 4.0; reaction time: 5 minutes; reaction temperature 25 °C). The absorbance of Py-BDP is higher than that of TAPT-BDP ( Figure 7 ), also confirming that the catalytic activity of Py-BDP nanozyme is higher than that of TAPT-BDP nanozyme.

[0044] Example 2 Application of Photo-responsive Porous Organic Polymer Material as a Peroxidase-like Enzyme and Condition Optimization 1. Optimization of pH In HAc-NaAc buffer solutions (3.9 mL, 0.1 M) with different pH values (3.5 - 5.5), add Py-BDP material (1 mL, 0.4 mg / mL) and TMB solution (0.1 mL, 40 mM) respectively. Then irradiate with an excitation light source (635 nm) at 25 °C for 5 minutes, then centrifuge at 6000 rpm for 5 minutes, and finally take the supernatant to measure the absorbance at 652 nm. The results are as Figure 8 shown in A: As the pH increases from 3.5 to 4.0, the absorbance of the supernatant increases. When the pH exceeds 4, the absorbance of the supernatant rapidly decreases; therefore, the pH of the reaction system is preferably 4 when the Py-BDP material acts as a peroxidase-like enzyme.

[0045] 2. Optimization of Py-BDP Concentration In HAc-NaAc buffer solution (pH 4, 3.9 mL, 0.1 M), add Py-BDP materials with different concentrations (0.1 - 2.5 mg / mL) (1 mL) and TMB solution (0.1 mL, 40 mM) respectively. Then irradiate with an excitation light source (635 nm) at 25 °C for 5 minutes, then centrifuge at 6000 rpm for 5 minutes, and finally take the supernatant to measure the absorbance at 652 nm. The results are as Figure 8 shown in B: As the concentration of Py-BDP increases in the range of 0.1 - 1.5 mg / mL, the absorbance value of the supernatant also increases rapidly. When the concentration exceeds 1.5 mg / mL, the absorbance of the supernatant basically no longer changes; therefore, the concentration of the Py-BDP material is preferably 1.5 mg / mL.

[0046] 3. Optimization of TMB Concentration In HAc-NaAc buffer solution (pH 4, 3.9 mL, 0.1 M), add Py-BDP material (1 mL, 1.5 mg / mL) and TMB solutions with different concentrations (10 - 60 mM) (0.1 mL) respectively. Then irradiate with an excitation light source (635 nm) at 25 °C for 5 minutes, then centrifuge at 6000 rpm for 5 minutes, and finally take the supernatant to measure the absorbance value at 652 nm; The results are as Figure 8 shown in C: As the concentration of TMB increases in the range of 10 - 40 mM, the absorbance value of the supernatant also increases rapidly. When the concentration exceeds 40 mM, the absorbance of the supernatant basically no longer increases; therefore, the concentration of the TMB material is preferably 40 mM.

[0047] Preferably, the concentration of TMB is 40 mM.

[0048] 4. Optimization of reaction time Add Py-BDP material (1 mL, 1.5 mg / mL) and TMB solution (0.1 mL, 40 mM) to the HAc-NaAc buffer solution (pH 4, 3.9 mL, 0.1 M) respectively, then irradiate with an excitation light source (635 nm) for different times (1 - 25 minutes) under the environmental condition of 25°C, then centrifuge at 6000 rpm for 5 minutes, and finally take the supernatant to measure the absorbance at 652 nm; the results are as Figure 8 shown in Figure D: As the reaction time increases, the absorbance value of the supernatant increases. When the reaction time is 15 minutes, the reaction is complete; therefore, the reaction time is 15 minutes.

[0049] 5. Optimization of reaction temperature Add Py-BDP material (1 mL, 1.5 mg / mL) and TMB solution (0.1 mL, 40 mM) to the HAc-NaAc buffer solution (pH 4, 3.9 mL, 0.1 M) respectively, then irradiate with an excitation light source (635 nm) for 5 minutes under the environmental conditions of different temperatures (25 - 65°C), then centrifuge at 6000 rpm for 5 minutes, and finally take the supernatant to measure the absorbance at 652 nm. The results are as Figure 8 shown in Figure E: As the reaction temperature increases, the absorbance value decreases slightly, indicating that Py-BDP as a nanozyme-like oxidase is insensitive to temperature and has a wide suitable temperature range.

[0050] According to the screening of single-factor conditions, the conditions for Py-BDP as a nanozyme-like oxidase to catalyze the oxidation of TMB are as follows: Add Py-BDP material (1 mL, 1.5 mg / mL) and TMB solution (0.1 mL, 40 mM) to the HAc-NaAc buffer solution (pH 4, 3.9 mL, 0.1 M) respectively, then irradiate with an excitation light source (635 nm) for 5 minutes under the environmental condition of 25°C.

[0051] Example 3 Construction of a sensor for the detection of organophosphorus pesticides Mix different concentrations (0.5 - 400 mU / mL, 700 μL) of acetylcholinesterase (AChE) solution with an excessive amount of acetylthiocholine (ATCh) solution (300 μL, 10 mM); incubate the resulting mixture at 37°C for 30 minutes to obtain an incubation solution; 1 mL of incubation solution, Py-BDP material (1 mL, 1.5 mg / mL), and TMB solution (0.1 mL, 40 mM) were added to 3.9 mL of HAc-NaAc buffer solution (pH 4, 0.1 M), and then irradiated with an excitation light source (635 nm) at 25 °C for 5 minutes to obtain a detection solution; The detection solution was centrifuged at 6000 rpm for 5 minutes, and finally the absorbance of the supernatant was scanned at 525 - 775 nm, and the absorbance at 652 nm was recorded to establish the relationship between AChE and the absorbance value.

[0052] The dose range of AChE was 0.5 to 400 mU / mL, and the color of the supernatant gradually became lighter, showing an inverse correlation with the absorption peak at 652 nm wavelength ( Figure 9 A). With the AChE concentration as the abscissa and the absorbance as the ordinate, the linear relationship between the absorbance and the AChE concentration was established, and the results of the linear relationship were as shown in Figure 9 B: The regression equation was y = -0.00493x + 2.02606, and the correlation coefficient (R 2 ) was 0.9999. According to 3σ / K, the detection limit (LOD) of AChE was 0.021 mU / mL (n = 10).

[0053] The above results indicate that the change in AChE activity can be reflected by the change in the color of the supernatant, and there is a linear relationship within a certain range. The Py-BDP nanozyme, AChE, its substrate, and TMB in Example 1 can be used to construct a sensor for detecting organophosphorus pesticides.

[0054] Application Example 1 Py-BDP-like oxidase for detecting organophosphorus pesticides 1. Methyl parathion (MP) 100 μL of methyl parathion (MP) solutions with different concentrations (0.4 - 200 μg / L) were pre-incubated with acetylcholinesterase (AChE) (350 μL, 400 mU / mL) at 37 °C for 30 minutes, and then acetylthiocholine (ATCh) (150 μL, 10 mM) was added and incubated for another 30 minutes to obtain an incubation solution; 1000 μL of the incubation solution was irradiated with an excitation light source (635 nm) at 25 °C for 15 minutes together with HAc-NaAc buffer solution (3.85 mL, 0.1 M, pH 4), the Py-BDP nanozyme suspension in Example 1 (500 μL, 1.5 mg / mL), and TMB solution (50 μL, 40 mM), and then centrifuged at 6000 rpm for 5 minutes, and the absorbance value of the supernatant at 652 nm was measured.

[0055] The results are as Figure 10 shown. As the concentration of MP increased, the absorption ability at 652 nm gradually rose. This confirmed that a higher concentration of MP had a stronger inhibitory effect on AChE, thus leading to a significant increase in the amount of TMB catalyzed by Py-BDP. In the concentration range of 0.4 - 200 μg / L, the correlation coefficient (R 2 ) between MP and absorbance was 0.999, and the LOD value was only 0.21 μg / L.

[0056] 2. Chlorpyrifos The solutions of chlorpyrifos at different concentrations (10 - 1600 μg / L) were detected according to the above method. The results are as Figure 11 shown. As the dose of chlorpyrifos increased, the absorption ability at 652 nm gradually rose. This confirmed that a higher concentration of chlorpyrifos had a stronger inhibitory effect on AChE, thus leading to a significant increase in the amount of TMB catalyzed by Py-BDP. In the concentration range of 10 to 1600 μg / L, the correlation coefficient (R 2 ) between chlorpyrifos and absorbance was 0.999, and the LOD value was only 2.48 μg / L.

[0057] 3. Phoxim The solutions of phoxim at different concentrations (5 - 200 μg / L) were detected according to the above method. The results are as Figure 11 shown. As the dose of phoxim increased, the absorption ability at 652 nm gradually rose. This confirmed that phoxim had a stronger inhibitory effect on AChE, thus leading to a significant increase in the amount of TMB catalyzed by Py-BDP. In the concentration range of 5 - 200 μg / L, the correlation coefficient (R 2 ) between phoxim and absorbance was 0.999, and the LOD value was only 0.38 μg / L.

[0058] Application Example 2: Py-BDP-based oxidase for detecting organophosphorus pesticide residues in Astragalus membranaceus and Angelica sinensis The OPs in Astragalus membranaceus and Angelica sinensis from different origins were identified using a Py-BDP sensor to evaluate the applicability of this method in actual samples. The specific steps are as follows: 1. Preparation of samples to be measured A total of 6 batches of samples were detected. Among them, 3 batches of Astragalus membranaceus came from Shanxi Province, Gansu Province and Jilin Province, and 3 batches of Angelica sinensis came from Min County, Longxi County and Zhang County in Gansu Province. First, Astragalus membranaceus or Angelica sinensis from different origins were ground into powders. 5 g of the powder was added to 1 g of sodium chloride and shaken immediately. Then, 50 mL of acetonitrile was added, and homogenized at a speed not less than 12000 rpm for 2 min, centrifuged at 4000 rpm, and the supernatant was separated. Another 50 mL of acetonitrile was added to the precipitate, homogenized for 1 min in the same way, centrifuged again, and the supernatants extracted twice were combined. Then, it was concentrated under reduced pressure to about 5 mL, and the mixture was cooled. Finally, it was diluted to 10.0 mL with acetonitrile. Pretreated extracts of Astragalus membranaceus and Angelica sinensis with known concentrations were used instead of OPs to determine the residue amounts of OPs in the actual samples. The solution prepared above was used as the sample to be tested.

[0059] 2. Colorimetric detection 1 mL of the sample to be tested was pre-incubated with acetylcholinesterase (AChE) (350 μL, 400 mU / mL) at 37 °C for 30 minutes, and then acetylthiocholine (ATCh) (150 μL, 10 mM) was added and incubated for another 30 minutes to obtain an incubation solution; 1000 μL of the incubation solution was irradiated with an excitation light source (635 nm) at 25 °C for 15 minutes together with HAc-NaAc buffer solution (3.85 mL, 0.1 M, pH 4), the Py-BDP nanozyme suspension in Example 1 (500 μL, 1.5 mg / mL) and TMB solution (50 μL, 40 mM), and then centrifuged at 6000 rpm for 5 minutes. The absorbance of the supernatant was scanned at 550 - 750 nm, and the absorbance at 652 nm was recorded.

[0060] Figure 14 The results showed that MP was found in Astragalus membranaceus from Shanxi, and the presence of MP could be inferred from the linear ranges of the four OPs. The detected value of this substance in Astragalus membranaceus from Shanxi was within the safe range and far lower than the maximum residue limit of 0.02 mg / kg specified in the Chinese Pharmacopoeia (2020 Edition).

[0061] 3. Spike recovery To further evaluate the accuracy of this method, Astragalus membranaceus from Shanxi was selected for the recovery study of 4 OPs.

[0062] Table 1 Determination of the recoveries of methyl parathion, chlorpyrifos, phoxim and triazophos in Astragalus membranaceus from Shanxi The data in Table 1 shows that the determined recovery rates of the 4 OPs are 87.73% - 100.31%, and the relative standard deviations (RSDs are all less than 5%). This indicates that even in complex situations, the accuracy and reliability of this analytical method are excellent.

[0063] The above are only examples of the present application and are not intended to limit the present application. For those skilled in the art, various changes and modifications can be made to the present application. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present application shall be included within the scope of the claims of the present application.

Claims

1. A method for preparing a porous organic polymer, characterized in that, It includes the following steps: Performing an ammonia aldol condensation reaction on the ligand of the electron donor and the ligand of the electron acceptor; The ligand of the electron donor is selected from 1,3,6,8-tetrakis(4-aminophenyl)pyrene or 2,4,6-tris(4-aminophenyl)-1,3,5-triazine; the electron acceptor is 5,5-difluoro-1,3,7,9-tetramethyl-10-phenyl-5H-4,5-dipyrrolo[1,2-c:2’,1’-f][1,3,2]diazaborininane-2,8-dicarboxaldehyde.

2. The preparation method according to claim 1, characterized in that, It includes the following steps: Dispersing the ligand of the electron donor, the ligand of the electron acceptor and a catalyst in a solvent to obtain a precursor mixture; The precursor mixture is repeatedly freeze-thawed and degassed, and reacted at 120 °C for 72 h under an oxygen-free condition. The reaction system is separated and purified to obtain a brownish-black powder, which is the porous organic polymer.

3. The preparation method according to claim 2, characterized in that, The solvent is a mixture of n-butanol and benzyl alcohol; the catalyst is an acetic acid solution with a concentration of 6 M; the volume ratio of n-butanol, benzyl alcohol and acetic acid solution is 1:3:0.4; The molar ratio of the electron donor to the electron acceptor is 1:

2.

4. The preparation method according to claim 1, wherein The electron donor is 1,3,6,8-tetrakis(4-aminophenyl)pyrene.

5. A porous organic polymer obtained by the preparation method according to any one of claims 1-4, characterized in that, The morphology is approximately spherical, and the particle size is 62 - 80 nm.

6. Application of a porous organic polymer as claimed in claim 5 as a peroxidase mimetic.

7. A sensor for detecting organophosphorus pesticides, characterized in that, It includes the following components: the porous organic polymer as claimed in claim 5, 3,3’,5,5’-tetramethylbenzidine, acetylcholinesterase and a substrate of acetylcholinesterase.

8. Application of a porous organic polymer as claimed in claim 5 and a sensor as claimed in claim 7 in detecting organophosphorus pesticides and a kit prepared therefrom.

9. The application according to claim 8, characterized in that, The organophosphorus pesticides are thiophosphate esters, phosphate esters, phosphoramide organophosphorus pesticides, such as methyl parathion, chlorpyrifos, phoxim, triazophos, trichlorfon, malathion, dichlorvos; the organophosphorus pesticides are preferably methyl parathion, chlorpyrifos, phoxim or triazophos.

10. A method for detecting organophosphorus pesticide residues, characterized in that, It includes the following steps: (1) Pre-incubating the test solution with acetylcholinesterase at 37 °C, and then adding a substrate of acetylcholinesterase and continuing the incubation to obtain an incubation solution; (2) Mixing the incubation solution, a suspension of the porous organic polymer as claimed in claim 5 or the sensor as claimed in claim 7 and TMB solution in a buffer solution with pH 4, irradiating the reaction with a 635 nm excitation light source, and measuring the absorbance value at 652 nm for the supernatant after centrifugation; The test solution is obtained by extracting the test sample with ethyl acetate and removing particulate matter from the extract; Preferably, the method further includes the step of detecting the absorbance values of a series of standard solutions of organophosphorus pesticides under the same conditions and making an organophosphorus pesticide concentration-absorbance standard curve.

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