A method for preparing a cerium-based covalent organic framework composite material with alkaline phosphatase-like activity and its application.

Abstract

This invention belongs to the field of pesticide residue detection technology, specifically relating to a method for preparing a cerium-based covalent organic framework composite material with alkaline phosphatase activity and its application. Using COF, L-valine, and Ce(NO3)3·6H2O as raw materials, a COF-OMe@Valine-CeO2 nanozyme was prepared via in-situ growth. This nanozyme exhibits alkaline phosphatase activity, and the constructed sensor demonstrates good selectivity, repeatability, and stability. The preparation method of the CeO2-Val@COF-OMe nanozyme is simple and can achieve rapid detection and hydrolysis of the organophosphorus pesticide methyl parathion.

Description

Technical Field

[0001] This invention belongs to the technical field of pesticide residue detection, and relates to a method for preparing a cerium-based covalent organic framework composite material with alkaline phosphatase activity and its application. More specifically, it relates to a method for preparing cerium oxide nanoparticles loaded with covalent organic framework nanozymes based on the in-situ complexation reaction of valine and cerium ions and its application. Background Technology

[0002] Organophosphorus pesticides (OPs) are a class of highly toxic phosphate compounds with the general structure O=P(OR)3, playing a crucial role in controlling agricultural diseases and improving the quality and yield of agricultural products. Methyl parathion (MP) is the most typical sulfur-containing OP, and due to its severe neurotoxicity and respiratory toxicity, it has attracted increasing attention from researchers in recent years. Therefore, developing a new and sensitive detection method for MP is essential for health protection and public safety.

[0003] Currently, methods for detecting pesticide residues in agricultural products can be divided into two main categories: The first category is based on traditional instrumental analysis methods, mainly including gas chromatography, high-performance liquid chromatography, gas chromatography-mass spectrometry, and liquid chromatography-mass spectrometry. These instrumental analysis methods have advantages such as high accuracy and good reproducibility, providing effective technical support for pesticide residue detection. However, they also have some drawbacks, such as the relatively high cost of the instruments, complex sample pretreatment steps, cumbersome operation, and long detection time. These shortcomings hinder the realization of low-cost and rapid detection of pesticide residues. The second category is based on the principles of biodetection technology for pesticide residue detection, mainly including immunoassay, enzyme inhibition assay, and biosensor methods. Among immunoassays, enzyme-linked immunosorbent assay (ELISA) is the most widely used. It detects organophosphorus pesticide residues based on the specific binding between antigens and antibodies. Although this method has good specificity and sensitivity, the preparation of antibodies is too complex and expensive, which limits its application in pesticide residue detection.

[0004] In recent years, inorganic nanomaterials have been developed for MP detection to improve the applicability of rapid detection methods and address the potential instability of biological enzymes. Nanozymes are a class of nanomaterials with enzyme-like activities. Due to their good stability, strong catalytic ability, and simple preparation, they are widely used to establish rapid detection methods. Among these nanozyme-based detection methods, electrochemical detection has received increasing attention in recent years due to its advantages such as short analysis time, high sensitivity, low cost, small sample size, and ease of operation. In particular, cerium (Ce)-based nanoparticles have shown promising potential due to their superior Ce content. 3+ and Ce 4+The mixed valence states and oxygen vacancies of CeO2, along with its catalase and peroxidase-like activities, endow it with the ability to function as nanozymes. Furthermore, due to the good affinity of CeO2 for phosphate groups, CeO2-based composites possess unique functions such as phosphopeptide recognition and enrichment. However, metal nanoparticles, due to their high surface energy, tend to aggregate into larger particles, leading to reduced catalytic activity. To address this issue, a supporting material is typically needed to prevent the aggregation of metal nanoparticles while maintaining their catalytic activity. Schiff-based covalent organic frameworks (COFs) are an emerging type of crystalline porous material with structural periodicity and inherent porosity. The ordered nanopores and tunable Schiff bonds provide favorable opportunities for enhancing reactant enrichment and introducing active sites via the Schiff reaction. Moreover, the well-defined structure of Schiff-based COFs facilitates the uniform dispersion of active sites, thereby shortening the catalytic pathway and resulting in higher catalytic activity. Therefore, this paper synthesizes a core-shell structured COF-based composite material supported on cerium dioxide (CeO2) and valine via an in-situ method and uses it as a rapid detection method for MP. Summary of the Invention

[0005] To address the shortcomings of existing technologies, the present invention aims to provide a method for preparing a cerium-based covalent organic framework composite material with alkaline phosphatase activity and its application. Using covalent organic framework material (COF), valine, and cerium nitrate hexahydrate Ce(NO3)3·6H2O as raw materials, a COF-OMe@Valine-CeO2 composite material was prepared via in-situ growth. The CeO2 component has the advantage of being a precursor to Ce... 3+ and Ce 4+ The mixed valence state and oxygen vacancies of the nanozyme can effectively catalyze the hydrolysis of MP to p-nitrophenol. The COF-OMe@Valine-CeO2 nanozyme prepared in this invention has good alkaline phosphatase catalytic activity and also has a large specific surface area.

[0006] To achieve the objectives of this invention, the following technical solution was adopted:

[0007] A method for preparing alkaline phosphatase-like cerium-based covalent organic framework composite material COF-OMe@Valine-CeO2 includes the following steps:

[0008] (1) Synthesis of COF-OMe@Valine: First, a certain amount of COF-OMe precursors 2,5-dimethoxybenzene-1,4-dicarboxaldehyde (DMTP) and 1,3,5-tris(4-aminophenyl)benzene (TAPB) were ultrasonically dissolved in a pre-designed 1,4-dioxane-n-butanol-methanol mixture (v / v / v, 4:4:1). A certain amount of L-valine and a certain amount of aqueous acetic acid were added to the COF-OMe synthesis precursors. The mixture was transferred to a stainless steel autoclave lined with polytetrafluoroethylene and heated at 50-100℃ for 24 h. After cooling to room temperature, the precipitate was collected by centrifugation and washed three times with tetrahydrofuran (THF). Finally, the prepared sample was dried overnight in a vacuum oven at 80℃ and labeled as COF-OMe@Valine.

[0009] (2) Synthesis of COF-OMe@Valine-CeO2: A certain amount of COF-OMe@Valine and a certain amount of Ce(NO3)3·6H2O were mixed and stirred continuously at room temperature. Then, the solution was placed in a stainless steel autoclave lined with polytetrafluoroethylene and heated at 150-200℃ for 12 h to form a COF-OMe@Valine-CeO2 sample. The obtained precipitate was washed several times with deionized water and then vacuum dried overnight at 80℃.

[0010] Further, in step (1), the molar ratio of DMTP, TAPB and L-Valine is 4.5:3:1-5; preferably, the molar ratio of DMTP, TAPB and L-Valine is 4.5:3:3-5.

[0011] Further, in step (2), the ratio of COF-OMe@Valine to Ce(NO3)3·6H2O is 20g:0.6-6mol; preferably, the ratio of COF-OMe@Valine to Ce(NO3)3·6H2O is 20g:3-6mol.

[0012] This invention also provides the application of the above-mentioned COF-OMe@Valine-CeO2 composite material, specifically, for constructing a sensor for detecting methyl paraoxon. The construction method is as follows: the COF-OMe@Valine-CeO2 composite material is uniformly dispersed in water to form a suspension; the suspension is drop-coated onto the surface of a glassy carbon electrode, dried at room temperature, and then 0.50% naphthol solution is added to its surface as a fixative to fix the composite material on the electrode. After drying, the COF-OMe@Valine-CeO2 / GCE modified electrode is obtained.

[0013] Furthermore, the concentration of the COF-OMe@Valine-CeO2 composite material in the suspension was 1 mg / mL; the drop volume of the suspension was 2.4 μL / cm. 2 .

[0014] The present invention also provides a detection method for the sensor for detecting methyl paraphosphine, specifically, using square wave voltammetry to detect the electrochemical response of the sensor to the sample to be tested, and calculating the concentration of methyl paraphosphine in the sample to be tested based on the standard curve regression equation.

[0015] The parameters for the square wave voltammetry method were set as follows: frequency 30Hz, potential -0.8 to 0V, and settling time 2s; the electrolyte was a PBS buffer solution with a pH of 7.0 and a concentration of 0.1mol / L.

[0016] Compared with existing technologies, this application achieves the following beneficial effects: COF-OMe@Valine-CeO2 nanocomposite materials with a core-shell structure are prepared using the complexation reaction of valine and cerium ions; furthermore, the constructed sensor exhibits good selectivity, repeatability, and stability. The process of this invention is simple, and the raw materials used are inexpensive and readily available, resulting in low cost and meeting environmentally friendly requirements. Since this method does not require pretreatment such as high temperature or calcination, it reduces energy consumption and reaction costs, facilitating mass production. Attached Figure Description

[0017] Figure 1 These are SEM images of COF-OMe@Valine-CeO2 at different magnifications in Embodiment 1 of the present invention;

[0018] Figure 2 The TEM spectrum of COF-OMe@Valine-CeO2 in Example 1 of this invention;

[0019] Figure 3 The XRD patterns of COF-OMe@Valine-CeO2 and COF-OMe in Embodiment 1 of the present invention;

[0020] Figure 4 The XPS spectrum of COF-OMe@Valine-CeO2 in Example 1 of this invention;

[0021] Figure 5 The figures show the current response of the composite materials prepared in Examples 1-8 of this invention to MP.

[0022] Figure 6 Electrochemical response diagrams of COF-OMe@Valine-CeO2 prepared in Example 1 of the present invention, COF-OMe prepared in Comparative Example 1, and CeO2 electrode and bare GCE electrode.

[0023] Figure 7 The response diagram (A) and standard curve (B) of the composite material COF-OMe@Valine-CeO2 modified electrode prepared in Example 1 of this invention to MP current are shown. Detailed Implementation

[0024] The invention will be further explained below with reference to specific implementation examples.

[0025] Example 1

[0026] The preparation method of COF-OMe@Valine-CeO2 nanozyme is as follows:

[0027] (1) Synthesis of COF-OMe@Valine: First, the COF-OMe precursors DMTP (87 mg, 0.045 mmol) and TAPB (105 mg, 0.03 mmol) were ultrasonically dissolved in a 45 mL solution of a pre-designed 1,4-dioxane-n-butanol-methanol mixture (v / v / v, 4:4:1). 0.03 mmol of Valine and 0.5 mL of acetic acid aqueous solution (12 mol / L) were added to the COF-OMe precursor, and the mixture was reacted at room temperature for 2 h. Then, 4.5 mL of acetic acid aqueous solution (12 mol / L) was added. The mixture was transferred to a PTFE-lined stainless steel autoclave and heated at 70 °C for 24 h. After cooling to room temperature, the precipitate was collected by centrifugation and washed three times with tetrahydrofuran (THF). Finally, the prepared sample was dried overnight in a vacuum oven at 80 °C and labeled as COF-OMe@Valine.

[0028] (2) Synthesis of COF-OMe@Valine-CeO2: 20.0 mg of COF-OMe@Valine and 20 mL of Ce(NO3)3·6H2O aqueous solution (0.24 mol / L) were continuously stirred until homogeneous at room temperature. Then, the solution was placed in a stainless steel autoclave lined with polytetrafluoroethylene and heated at 180 °C for 12 h. The resulting precipitate was washed several times with deionized water and then vacuum dried overnight at 80 °C to obtain the COF-OMe@Valine-CeO2 composite material.

[0029] Example 2

[0030] The difference between this embodiment and Example 1 is that the amount of Valine added is 0.01 mmol. All other steps are the same as in Example 1. The resulting product is labeled COF-OMe@Valine. 0.01 -CeO2 composite material.

[0031] Example 3

[0032] The difference between this embodiment and Example 1 is that the amount of Valine added is 0.02 mmol. All other steps are the same as in Example 1. The resulting product is labeled COF-OMe@Valine. 0.02 -CeO2 composite material.

[0033] Example 4

[0034] The difference between this embodiment and Example 1 is that the amount of Valine added is 0.04 mmol. All other steps are the same as in Example 1. The resulting product is labeled COF-OMe@Valine. 0.04 -CeO2 composite material.

[0035] Example 5

[0036] The difference between this embodiment and Example 1 is that the amount of Valine added is 0.05 mmol. All other steps are the same as in Example 1. The resulting product is labeled COF-OMe@Valine. 0.05 -CeO2 composite material.

[0037] Example 5

[0038] The difference between this embodiment and Example 1 is that the concentration of the Ce(NO3)3·6H2O aqueous solution is 0.03 mol / L. The other steps are the same as in Example 1. The resulting product is labeled COF-OMe@Valine-CeO 20.03 Composite materials.

[0039] Example 6

[0040] The difference between this embodiment and the previous one is that the concentration of the Ce(NO3)3·6H2O aqueous solution is 0.08 mol / L. The other steps are the same as in Example 1, and the resulting product is labeled COF-OMe@Valine-CeO. 20.08 Composite materials.

[0041] Example 7

[0042] The difference between this embodiment and the previous one is that the concentration of the Ce(NO3)3·6H2O aqueous solution is 0.15 mol / L. The other steps are the same as in Example 1, and the resulting product is labeled COF-OMe@Valine-CeO. 20.15 Composite materials.

[0043] Example 8

[0044] The difference between this embodiment and the previous one is that the concentration of the Ce(NO3)3·6H2O aqueous solution is 0.30 mol / L. The other steps are the same as in Example 1, and the resulting product is labeled COF-OMe@Valine-CeO. 20.30 Composite materials.

[0045] Comparative Example 1:

[0046] Synthesis of COF-OMe: First, the COF-OMe precursors DMTP (87 mg, 0.045 mmol) and TAPB (105 mg, 0.03 mmol) were sonicated and dissolved in 45 mL of a pre-designed 1,4-dioxane-n-butanol-methanol mixture (v / v / v, 4:4:1). Then, 0.5 mL of 12 mol / L aqueous acetic acid (solution A) was added, and the reaction was carried out at room temperature for 2 h. Then, 4.5 mL of 12 mol / L aqueous acetic acid was added. The mixture was transferred to a PTFE-lined stainless steel autoclave and heated at 70 °C for 24 h. After cooling to room temperature, the precipitate was collected by centrifugation and washed three times with THF. Finally, the prepared sample was dried overnight in a vacuum oven at 80 °C and labeled as COF-OMe.

[0047] Performance testing:

[0048] The composite material suspensions (1 mg / mL, 6 μL) prepared in Examples 1-8 were modified onto a glassy carbon electrode GCE (0.25 cm⁻¹). 2 The surface of the composite material is dried at room temperature, and then 3.0 μL of 0.50% naphthol solution is added to the surface as a fixative to fix the composite material onto the electrode, thus obtaining a modified electrode containing different modifiers.

[0049] Cyclic voltammetry (CV) was performed (parameter settings: potential -0.8-0.6 V, scan rate 100 mV / s, etc.) in a typical electrochemical quartz electrolytic cell containing 5 mL of PBS (0.1 M pH 7.0) solution, with all measurements performed at least three times. The current response to MP was measured by CV under the same conditions, such as... Figure 5 As shown, the COF-OMe@Valine nanocomposite containing 0.03 mol / L Valine and the COF-OMe@Valine-CeO2 nanocomposite containing 0.24 mol / L Ce(NO3)3·6H2O exhibited the best current response to MP. To further investigate the electrochemical performance of this sensor, a nanocomposite containing 15 μmol / L Valine was used. -1 In 5 mL of PBS (pH = 7.0) containing MP, the scan rate was 100 mV s. -1 CV measurements were performed on GCEs with different modifications. For example... Figure 6As shown, COF-OMe@Valine-CeO2 / GCE exhibits the best electrochemical response. The peak anolyte current of COF-OMe@Valine-CeO2 / GCE is approximately 5 times larger than that of bare GCE. Under the same conditions, CeO2 / GCE and COF-OMe@Valine-CeO2 / GCE were studied, and it is evident that COF-OMe and CeO2 exhibit a synergistic amplification effect in the catalytic process of MP. This is mainly reflected in three aspects: firstly, the porous crystalline material of COFs-OMe has a large specific surface area and an attractive two-dimensional structure, which can increase the effective electroactive surface area; secondly, CeO2 NPs are conductive, which can improve its oxidation process of MP; and thirdly, COFs are organic polymers that endow compounds with a large number of π-π bonds, and the benzene rings on MP can be adsorbed onto the modified material through π-π stacking interactions.

[0050] The successful preparation of the COF-OMe@Valine-CeO2 composite material of this invention was demonstrated by scanning electron microscopy (SEM), transmission electron microscopy (TEM), X-ray photoelectron spectroscopy (XPS), and X-ray diffraction (XRD). Figure 3 The strong diffraction peaks of the composite material at 28.55°, 33.08°, 47.48°, 56.33°, 59.09°, 69.40°, 76.70°, and 79.07° in the XRD pattern point to the (111), (200), (220), (311), (222), (400), (331), and (420) planes, respectively, matching the CeO2 crystal (JCPDS No. 34-0394). This demonstrates the successful synthesis of the composite material. Figure 1 SEM and Figure 2 TEM images confirmed the bonding of the composite material. The particle size of COF-OMe@Valine-CeO2 is approximately 500 nm. Figure 4 XPS spectra confirmed the presence of C, N, O, and Ce elements in the composite material, indicating the successful synthesis of the composite material.

[0051] The present invention also provides the above-mentioned composite material for MP detection in the field of pesticide residues.

[0052] Evaluation of MP testing:

[0053] (1) Electrode preparation: The bare GCE was polished to a mirror finish using a 0.030 μm alumina slurry and ultrasonically rinsed with ethanol and deionized water. Then, 1.0 mg COF-OMe@Valine-CeO2 was dispersed in 1.0 mL H2O and ultrasonicated to form a homogeneous solution. 6.0 μL of COF-OMe@Valine-CeO2 was dropped onto the surface of the glassy carbon electrode GCE (effective drop coating area 0.25 cm²). 2The composite material was dried at room temperature, and then 3.0 μL of 0.50% naphthol solution was added to its surface as a fixative to fix the composite material onto the electrode. After drying, the final electrode was labeled COF-OMe@Valine-CeO2 / GCE.

[0054] (2) Using square wave voltammetry (SWV) to measure the saturation of different standard solutions (0.034-76 μmol) -1 The electrochemical response of MP was studied.

[0055] Square wave voltammetry (SWV) with parameters set as follows (frequency 30 Hz, potential -0.8 to 0 V, settling time 2 sec, etc.) was performed in a typical electrochemical quartz electrolytic cell containing 5 mL of PBS (0.1 M pH 7.0) solution for parameter optimization and quantitative analysis. All measurements were performed at least three times. Figure 7 As shown in Figure A, the electrochemical response at different MP concentrations was evaluated using SWV. The results showed that the peak current increased with increasing MP concentration, and remained constant in the range of 0.034–76 μmol. -1 Within the concentration range, the peak current showed a clear linear relationship with the MP concentration. Figure 7 B). The linear regression equation is y = 0.4433x + 0.8816, with a correlation coefficient of 0.998. The detection limit is 0.011 μmol. -1 / L.

[0056] The embodiments described above are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments. Any obvious improvements, substitutions or modifications that can be made by those skilled in the art without departing from the essence of the present invention shall fall within the protection scope of the present invention.

Claims

1. A method for preparing a cerium-based covalent organic framework composite material with alkaline phosphatase-like activity, characterized in that, The specific steps include the following: (1) Synthesis of COF-OMe@Valine: 2,5-dimethoxybenzene-1,4-dicarboxaldehyde and 1,3,5-tris(4-aminophenyl)benzene were ultrasonically dissolved in a mixture of 1,4-dioxane-n-butanol-methanol to obtain the COF-OMe synthesis precursor; L-valine and acetic acid aqueous solution were added to the COF-OMe synthesis precursor to obtain a mixed solution; the mixed solution was heated at 50-100℃ for 24h, cooled to room temperature, centrifuged to collect the precipitate, washed with tetrahydrofuran, and dried to obtain COF-OMe@Valine; wherein, the mixture of 1,4-dioxane-n-butanol-methanol was composed of 1,4-dioxane, n-butanol and methanol in a volume ratio of 4:4:1; (2) Synthesis of COF-OMe@Valine-CeO2: COF-OMe@Valine and Ce(NO3)3·6H2O were mixed and stirred evenly at room temperature. The mixture was then heated at 150-200℃ for 12 hours. The precipitate was washed with deionized water and dried to obtain the COF-OMe@Valine-CeO2 composite material.

2. The method for preparing the alkaline phosphatase-like cerium-based covalent organic framework composite material according to claim 1, characterized in that, In step (1), the molar ratio of 2,5-dimethoxybenzene-1,4-dicarboxaldehyde, 1,3,5-tris(4-aminophenyl)benzene and L-valine is 4.5:3:1-5.

3. The method for preparing the alkaline phosphatase-like cerium-based covalent organic framework composite material according to claim 1, characterized in that, In step (2), the ratio of COF-OMe@Valine to Ce(NO3)3·6H2O is 20g:0.6-6mol.

4. A method for constructing a sensor for detecting methyl paraoxon, characterized in that, The alkaline phosphatase-like cerium-based covalent organic framework composite material prepared by the method of any one of claims 1-3 is uniformly dispersed in water to form a suspension; the suspension is drop-coated onto the surface of a glassy carbon electrode, dried at room temperature, and then 0.50% naphthol solution is added to its surface as a fixative to fix the composite material onto the electrode. After drying, the COF-OMe@Valine-CeO2 / GCE modified electrode is obtained.

5. The method for constructing a sensor for detecting methyl paraoxon according to claim 4, characterized in that, The concentration of alkaline phosphatase-like cerium-based covalent organic framework composite material in the suspension was 1 mg / mL; the drop volume of the suspension was 2.4 μL / cm. 2 .

6. An application of a sensor for detecting methyl paraoxon, constructed by the method described in claim 4, characterized in that, The electrochemical response of the sensor to the test sample was detected using square wave voltammetry, and the concentration of methyl para-oxygen phosphorus in the test sample was calculated based on the standard curve regression equation.

7. The application of the sensor for detecting methyl paraphosphine according to claim 6, characterized in that, Square wave voltammetry parameters: frequency 30 Hz, potential -0.8 to 0 V, settling time 2 s; electrolyte is PBS buffer solution with pH 7.0 and concentration of 0.1 mol / L.

Images