A copper-based nanoszyme, products containing the same, preparation method and application

Abstract

The application discloses a copper-based nano-enzyme, a product containing the same, a preparation method and application, and belongs to the technical field of pesticide detection, and a high-activity and high-stability peroxidase-like material is obtained by self-assembly of a copper source, H3BTC and PVP at room temperature. 2+ / Cu + Redox cycle, significantly improving the catalytic activity of TMB-H2O2 system. The method is simple to operate, does not require complex instruments, can realize high sensitivity, high selectivity and rapid detection of nicosulfuron, and is suitable for on-site screening of pesticide residues in water and agricultural products, overcoming the defects of the prior art, such as strong dependence on equipment, high cost and poor portability.

Description

Technical Field

[0001] This invention belongs to the field of pesticide detection technology, and particularly relates to a copper-based nanozyme, products containing the same, preparation methods, and applications. Background Technology

[0002] Currently, there are various analytical methods available both domestically and internationally for the detection of nicosulfuron pesticides, mainly including the following categories: 1. Chromatography and Chromatography-Mass Spectrometry (GC-MS) techniques Gas chromatography (GC), high-performance liquid chromatography (HPLC), gas chromatography-mass spectrometry (GC-MS), and ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS / MS) are all methods used in this field. These methods typically involve sample pretreatment (such as extraction, purification, and concentration), followed by separation of the target analyte using a chromatographic column, and then qualitative and quantitative analysis using detectors (such as ECD, UV, and MS). However, these methods have the following drawbacks and reasons: (1) The equipment has a complex structure and large size: it relies on large precision instruments, requires a fixed laboratory environment, and cannot achieve on-site testing; (2) Cumbersome pretreatment: It usually requires multiple steps such as solid phase extraction and liquid-liquid extraction, which is time-consuming and requires a large amount of reagents. (3) High level of professional operation requirements: It requires professionally trained technicians to operate, and its popularity is poor. (4) Long detection cycle: It usually takes several hours or even longer from sample preparation to result output.

[0003] 2. Immunoassay Methods Enzyme-linked immunosorbent assay (ELISA), fluorescence polarization immunoassay (FPIA), etc. These methods are based on antigen-antibody specific recognition, combined with enzyme labeling or fluorescent labeling to achieve signal amplification and detection. They have the following drawbacks and reasons: (1) Poor antibody stability: Biological antibodies are easily affected by environmental conditions (temperature, pH), and the storage and transportation conditions are harsh; (2) Cross-reaction interference: The selectivity for structural analogs is limited, and false positive or false negative results are likely to occur; (3) High reagent cost: Antibody preparation is complex and the detection cost is high; (4) Limited throughput: It is usually a single target detection, making it difficult to achieve high throughput or multi-residue analysis.

[0004] 3. Traditional Molecularly Imprinted Polymer (MIP) Technology Molecularly imprinted materials based on monolithic polymerization are used for solid-phase extraction or sensor construction. These materials, which specifically recognize holes, are formed through the polymerization of template molecules and functional monomers under the action of a cross-linking agent. However, the following drawbacks exist and are explained below: (1) Recognition site embedding depth: Template molecules are difficult to completely wash out, recognition site accessibility is poor, resulting in low binding efficiency; (2) Slow mass transfer rate: The materials are mostly in block or particulate form, which is not conducive to the rapid diffusion of the target to the recognition site; (3) Difficult to integrate with miniaturized devices: The material form and size are not suitable for miniaturized platforms such as microfluidic chips.

[0005] 4. Sensors based on metal-based nanomaterials Sensors based on carbon quantum dots, other metallic materials, etc., utilize changes in the optical properties of nanomaterials to induce a signal response through the interaction between the target object and the material. However, they have the following drawbacks and reasons: (1) Insufficient selectivity: Most materials lack the ability to specifically identify the target and are easily affected by the sample matrix; (2) Poor signal stability: Optical signals are easily affected by factors such as ambient light and temperature, resulting in poor repeatability; (3) Extremely expensive: Platinum, gold and palladium are all rare precious metals with high raw material costs, which severely limits their large-scale production and practical application; (4) Limited catalytic mechanism and efficiency: Some noble metals (such as Au) have low intrinsic catalytic activity and need to be improved by reducing size and constructing special crystal faces; (5) Poor biocompatibility and metabolism: It is difficult to degrade and metabolize in the body and may remain in the reticuloendothelial system such as the liver and spleen for a long time, raising long-term concerns about biosafety.

[0006] To address the numerous problems existing in current methods for detecting nicosulfuron pesticides, this invention aims to provide a low-cost, portable, simple-to-operate, rapid, highly sensitive, and highly selective colorimetric sensor based on copper-based nanozymes, suitable for detecting nicosulfuron pesticide residues in food. Summary of the Invention

[0007] In view of the many shortcomings of the existing technologies, such as strong equipment dependence, complex operation, long detection cycle, high cost, poor portability, insufficient selectivity and difficulty in achieving rapid on-site screening, the present invention aims to provide a copper-based nanozyme, products containing the same, preparation method and application.

[0008] To achieve the above objectives, the present invention provides the following technical solution: A method for preparing a copper-based nanozyme includes the following steps: The copper source, organic ligand H3BTC, and nitrogen-containing structure directing agent were dissolved in a solvent and reacted by stirring at room temperature. After the reaction was completed, the copper-based nanozyme was obtained by centrifugation, washing, and drying.

[0009] Beneficial effects: This invention systematically studies the effects from three aspects: the structural design of core materials, the regulation of catalytic active centers, and the precision of detection mechanisms. The details are as follows: Research on catalytic and detection mechanisms: The catalytic activity of the copper-based nanozyme disclosed in this invention is highly dependent on the Cu on the nanozyme surface. + Cu with the solution to be tested / interface 2+ The dynamic redox pair formed between them (Cu) 2+ / Cu + Cooperative cycling. The lone pair electrons of the sulfonylurea group interact with Cu. 2+ Coordination occurs, transferring Cu 2+ Transformation into Cu + Nitrogen (urea group) and oxygen (sulfonyl group) can react with Cu + Formation of stable complexes, inhibiting Cu + The re-oxidation of Cu + Increased content. Complexes can alter Cu content. 2+ The redox potential of H2O2 increased Cu + The efficiency of formation. Therefore, the presence of pesticides (nicosulfuron) promotes the formation of redox duplexes (Cu). 2+ / Cu + The synergistic effect between them enhances the catalytic activity of copper-based nanozymes.

[0010] This specific chelating effect on the copper active site acts like a "molecular key," precisely unlocking the core of the catalytic cycle and linking the generation chain of •OH radicals. Therefore, other pesticides, common ions, and organic acids that do not possess strong metal chelating groups can hardly produce a similar enhancing effect, thus enabling the sensor to exhibit excellent selectivity for nicosulfuron pesticides.

[0011] Optionally, the copper source is Cu(NO3)2·3H2O; The nitrogen-containing structure directing agent is polyvinylpyrrolidone (PVP). The solvent is methanol.

[0012] Beneficial Effects: This invention specifies polyvinylpyrrolidone (PVP) as the nitrogen-containing structure-directing agent because, compared to 2-methylimidazole, PVP is suitable for constructing various MOF systems. It stabilizes the MOF surface through physical coating and weak interactions, avoiding violent reactions that damage the framework structure, thus achieving a balance between morphology control and stability. Compared to triethylamine, PVP is less toxic and environmentally friendly, and the modified MOF exhibits stable morphology, less intermolecular aggregation, and better dispersibility in solution. Therefore, PVP is not only a nitrogen source but also an excellent morphology-directing agent and dispersant, effectively controlling the size and shape of MOF crystals, which is difficult to achieve with other small-molecule nitrogen sources. Therefore, this invention ultimately limits PVP to being used as the raw material.

[0013] Furthermore, the mass ratio of the copper source, H3BTC, and polyvinylpyrrolidone is 0.875:0.42:0.4.

[0014] Beneficial effects: This invention limits the mass ratio of copper source, H3BTC, and polyvinylpyrrolidone. If the amount of polyvinylpyrrolidone is too small, it is insufficient to regulate the morphology of the copper-based nanozyme; if the amount is too large, the nanozyme particle size begins to increase. The nanozyme particles with the optimal size and dispersion are obtained at this specific ratio.

[0015] Optionally, the room temperature reaction time is 20-28 hours.

[0016] Optionally, the drying is vacuum drying, with a drying temperature of 55-65℃ and a drying time of 10-24 hours.

[0017] A copper-based nanozyme was prepared by the above-described method.

[0018] Optionally, the copper-based nanozyme is a nitrogen-doped copper-metal-organic framework material, whose crystal particles have a regular octahedral morphology and are uniformly dispersed.

[0019] The above-mentioned copper-based nanozymes are used in the detection of nicosulfuron pesticides or in the preparation of colorimetric sensors for the detection of nicosulfuron pesticides.

[0020] A colorimetric sensor for detecting nicosulfuron includes: Core material: copper-based nanozymes; Oxidizing agent H2O2; The developing substrate is TMB.

[0021] Optionally, the colorimetric sensor may also include a fluorescent probe, terephthalic acid (PTA).

[0022] A method for detecting nicosulfuron using the above-mentioned colorimetric sensor includes the following steps: Mix the copper-based nanozyme dispersion with the sample to be tested; Add H2O2 and the chromogenic substrate TMB; Incubate at 20-30℃ for 20-30 minutes, and measure the change in absorbance using a UV-Vis spectrophotometer. The concentration of nicosulfuron was quantitatively analyzed based on changes in absorbance.

[0023] Construction of the colorimetric sensor: This invention integrates the aforementioned copper-based nanozyme material with a well-defined mechanism into a homogeneous reaction system in the form of a 96-well microplate or an equivalent test strip / detection tube. The operation steps can be simplified to three steps: ① Add sample and copper-based nanozyme reagent, ② Add TMB / H2O2 substrate for color development, ③ Incubate at ~25℃ for ~25 minutes. The entire detection process can be completed within 30 minutes, and the main reaction time (25 minutes) is incubation, requiring no manual intervention. The color change is clearly visible to the naked eye and can be read using a portable microplate reader or smartphone, completely eliminating the dependence on large instruments, making it fast, convenient, and truly enabling on-site detection. Furthermore, the core material disclosed in this invention uses relatively inexpensive copper, requiring no precious metals or complex preparation processes, resulting in low cost and ease of promotion.

[0024] Compared with the prior art, the present invention has the following advantages and technical effects: (1) This invention provides a copper-based nanozyme with high activity and high stability: N-doped copper-based nanozymes with well-defined and uniformly distributed octahedral shapes were synthesized by introducing PVP. The introduction of highly negatively charged N species can change the electronic properties of the material, thereby effectively improving the relevant performance of the nanocatalyst. Therefore, this invention provides a core sensing material that combines high catalytic activity similar to peroxidase with excellent physicochemical stability. This structure aims to fundamentally solve the problems of low catalytic activity and easy aggregation and deactivation of ordinary copper nanozymes and traditional inorganic nanozymes.

[0025] (2) This invention constructs a highly sensitive and selective pesticide detection method: based on the prepared copper-based nanozyme, an "enhanced" colorimetric sensing platform is constructed. This invention aims to enhance the copper redox pair (Cu) on the nanozyme surface through a unique enhancement mechanism—namely, the specificity of nicosulfuron pesticides in enhancing this redox pair. 2+ / Cu + The synergistic effect of nicosulfuron is used to achieve detection. This mechanism aims to achieve a highly selective response to nicosulfuron pesticides, significantly reduce interference from other common pesticides and coexisting substances, and utilize its high catalytic activity to achieve highly sensitive detection of nicosulfuron pesticides, obtaining a lower limit of detection.

[0026] (3) This invention discloses a rapid, simple, and reliable practical detection scheme: integrating a copper-based nanozyme-based colorimetric sensor and detection method into a complete operating procedure. This method aims to eliminate the need for complex and expensive instruments, allowing for semi-quantitative or quantitative analysis by visually observing color changes or using simple portable spectrometers. Therefore, it is suitable for rapid screening and monitoring of nicosulfuron pesticide residues in complex matrices such as water bodies and agricultural products at the field and grassroots levels, addressing the shortcomings of existing instrumental methods being unsuitable for field application and the poor reliability of bio-enzyme methods.

[0027] (4) Optimize and integrate the overall performance of the sensor to meet the needs of rapid on-site detection: This invention simplifies the operation process, employing a few steps of "mixing-incubation-colorimetry" for detection, eliminating the need for complex pretreatment and precision instruments. Furthermore, this invention enhances signal output, based on the vivid color change (colorless-blue-deep blue) generated by high catalytic activity, ensuring reliable semi-quantitative judgment by the naked eye, while also being compatible with portable photometers for precise quantification. Ensuring stability, the copper-based nanozyme-based colorimetric sensor prepared by this invention maintains stable performance across different temperature, pH, and ionic strength ranges, overcoming the weakness of natural enzyme sensors' high environmental requirements, making it truly suitable for non-laboratory environments such as fields, markets, and ports. Attached Figure Description

[0028] The accompanying drawings, which form part of this invention, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an undue limitation of the invention. In the drawings: Figure 1 In Figure 1, a and b are SEM images of Cu-MOF synthesized with the participation of PVP in Example 1 of the present invention; c and d are SEM images of Cu-MOF synthesized without the participation of PVP in Comparative Example 1. Figure 2 The XPS spectrum of the Cu-MOF synthesized in Example 1 of this invention; Figure 3 Typical UV-Vis absorption spectra of different reaction systems; Figure 4 (a) shows the effect of nicosulfuron on the activity of Cu-MOF peroxidases; (b) shows the effect of Cu-MOF on the formation of hydroxyl radicals; (c) shows the effect of nicosulfuron on the formation of hydroxyl radicals; and (d) shows the XPS spectrum of Cu-MOF after the addition of nicosulfuron. Figure 5 In the diagram, a is the Michaelis constant plot of TMB; b is the Michaelis constant plot of H2O2; c is the double reciprocal plot of TMB; and d is the double reciprocal plot of H2O2. Figure 6In Figure a, the UV-Vis absorption spectra of the Cu-MOF synthesized in Example 1 of this invention catalyzing the reaction of the TMB-H2O2 system in the presence of different concentrations of nicosulfuron are shown; in Figure b, the relationship between different concentrations of nicosulfuron and the absorbance at 652 nm is shown. Figure 7 This invention provides the specificity of the rapid detection method for nicosulfuron pesticides using a colorimetric sensor based on copper-based nanozymes. Figure 8 (a) shows the change in ultraviolet absorbance intensity on different monitoring days; (b) shows the relative standard deviation of Cu-MOF between the same batch and different batches. Figure 9 (a) shows the UV-Vis absorption spectra of the TMB-H2O2 system catalyzed by Cu-MOF in the presence of maize samples with different concentrations of nicosulfuron; (b) shows the relationship between maize samples with different concentrations of nicosulfuron and the absorbance at 652 nm. Figure 10 Electron micrograph of the copper-based nanozyme of 2-methylimidazole prepared in Comparative Example 2; Figure 11 Electron micrograph of the copper-based nanozyme with triethylamine prepared in Comparative Example 3. Detailed Implementation

[0029] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0030] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Every smaller range between any stated value or intermediate value within a stated range, and any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0031] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0032] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0033] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0034] Unless otherwise specified, "room temperature" in this invention refers to 20-30℃.

[0035] All raw materials used in this invention were purchased from the market.

[0036] The technical solution of the present invention will be further illustrated by the following embodiments.

[0037] Example 1 A method for preparing a colorimetric sensor based on copper-based nanozymes includes the following steps: 0.875 g Cu(NO3)2·3H2O and 0.4 g PVP (polyvinylpyrrolidone) were dissolved in 50 mL of methanol solution using the room temperature method.

[0038] Subsequently, 50 mL of a methanol solution containing 0.42 g H3BTC (1,3,5-benzenetricarboxylic acid) was poured into the above solution, and the mixture was stirred continuously at room temperature for 24 hours.

[0039] The blue precipitate was collected by centrifugation, washed three times with fresh methanol solution, and dried under vacuum at 60°C overnight. The blue solid was collected and ground into powder, and Cu-MOF (based on copper-based nanozymes) was prepared.

[0040] Comparative Example 1 The difference from Example 1 is that PVP is not added during the preparation process. Other preparation processes and conditions are the same as in Example 1.

[0041] Comparative Example 2 The difference from Example 1 is that an equal mass of 2-methylimidazole was used to replace polyvinylpyrrolidone in the preparation process. The final product was a copper-based nanozyme based on 2-methylimidazole.

[0042] Figure 10 The electron microscope image of the copper-based nanozyme of 2-methylimidazole prepared in Comparative Example 2 shows that the morphology of the nanozyme is blurred, the crystal edges are indistinct, and severe aggregation leads to structural obscuration.

[0043] Comparative Example 3 The difference from Example 1 is that, in the preparation process, triethylamine was used instead of polyvinylpyrrolidone by an equal mass. The final product was a copper-based nanozyme containing triethylamine.

[0044] Figure 11 The image shows an electron micrograph of the copper-based triethylamine nanozyme prepared in Comparative Example 3. It can be seen that the nanozyme has an irregular and disordered morphology, poor synthesis control, unclear pore structure, and severe aggregation.

[0045] By comparison, the copper-based nanozyme prepared using polyvinylpyrrolidone as the raw material has clear crystal particle boundaries, uniform dispersion, and a regular octahedral shape. The particle size is not significantly different, which is more conducive to carrying out subsequent experiments.

[0046] Effect verification: (1) Peroxidase-like properties of Cu-MOF Under H2O2 conditions, the peroxidase-like activity of the chromogenic substrate TMB (3,3',5,5'-tetramethylbenzidine) was investigated by Cu-MOF catalytic oxidation. The specific test procedure was as follows: 100 μL of 1.0 mmol / L TMB (solvent: anhydrous ethanol), 200 μL of 30% H2O2, and 200 μL of 0.5 mmol / L Cu-MOF dispersion (solvent: anhydrous ethanol) were added to 1500.0 μL of HAC-NaAC buffer (pH=4.8). This mixture was designated as the TMB-H2O2 system. After incubation for 25 minutes, the mixture was transferred to a quartz cuvette and placed in a UV-Vis spectrophotometer to record the UV-Vis absorption spectrum.

[0047] (2) Determination and mechanism study of hydroxyl radicals (·OH) The 2 mL reaction system included Cu-MOF (0.5 mmol / L), H₂O₂ (60 mmol / L), PTA (terephthalic acid, 0.5 mmol / L), and HAC-NaAC buffer (pH=4.0). The reaction was carried out at room temperature for 5 min, and the fluorescence spectra of different systems were measured using a fluorescence spectrophotometer at an excitation wavelength of 300 nm. Furthermore, Cu-MOF (0.5 mmol / L) was mixed with different concentrations of nicosulfuron (0, 5, and 10 μg / mL), and the reaction was repeated at room temperature for 5 min.

[0048] (3) Steady-state dynamics test of Cu-MOF To further investigate the peroxidase-like activity of Cu-MOF, steady-state kinetics were tested. The absorbance of Cu-MOF at 652 nm was monitored within the TMB and H2O2 concentration range, and the kinetic process was measured over time. The system was incubated under optimal experimental conditions, and kinetic data were collected.

[0049] (4) Quantitative detection of nicosulfuron and preparation of standard curve First, add 1300 μL of HAC-NaAC buffer and 200 μL of Cu-MOF dispersion (0.5 mmol / L) to centrifuge tubes. Next, add 200 μL of nicosulfuron at different concentrations (0.2, 2, 4, 6, 8, 10 μg / mL) and incubate at 25 °C for 1 min. Finally, add 200 μL of H₂O₂ and 100 μL of TMB, and incubate the mixture at 25 °C for 24 min. Record the UV-Vis absorption spectra of the solution under optimal experimental conditions.

[0050] (5) Specificity, stability and accuracy tests of Cu-MOF To investigate the specificity of this detection method, other common pesticides such as glyphosate, methyl parathion, chlorpyrifos, and carbaryl were studied, with the concentration of nicosulfuron and other pesticides all at 10 μg / mL. In addition, the effects of metal ions such as Co were also investigated. 2+ Na + K + The influence of potential interfering ions or substances, such as antibiotics like chloramphenicol and xylose, on the detection system was assessed. The concentration of interfering ions or substances was 20 μg / mL.

[0051] To ensure the reliability of subsequent experimental results, the changes in absorbance intensity of Cu-MOF were recorded after 30 days of storage. Absorbance was measured on days 1, 6, 12, 18, 24, and 30, and the absorbance value at 652 nm was recorded. All experiments were repeated three times in parallel to examine the stability of Cu-MOF.

[0052] To evaluate the accuracy of this method, Cu-MOFs from the same batch and different batches were synthesized using the same preparation method. The absorbance changes of nicosulfuron were then measured. All experiments were repeated in triplicate to assess the accuracy of Cu-MOF synthesis.

[0053] (6) Actual sample testing To further verify the feasibility of this detection method in actual sample testing, purchased corn was crushed and ground, and passed through an 80-mesh sieve. 5g of corn flour was weighed and added to a centrifuge tube. 200μL of nicosulfuron standard solution of different concentrations (0.2, 2, 4, 6, 8, 10μg / mL) was added to the corn flour, and an appropriate amount of methanol was added to moisten the corn flour, ensuring uniform distribution of nicosulfuron. Then, the mixture was carefully stirred and placed in a fume hood to allow the methanol to evaporate completely. Next, 1g of NaCl and 25 mL of 80% (w / v) methanol extraction solution were added to the centrifuge tube, and the mixture was thoroughly mixed. The mixture was homogenized for 3 min on a high-speed homogenizer. The homogenized sample was then extracted by shaking at 25℃ and 180 rp / min for 30 min. The sample was filtered using Whatman 4 qualitative filter paper, and the filtrate was passed through a 0.22µm filter membrane. The collected filtrate was the sample to be tested, and the recovery rate was calculated.

[0054] The specific steps and conditions for testing using copper-based nanozymes are as follows: Add 1300 μL of HAC-NaAC buffer and 200 μL of Cu-MOF dispersion (0.5 mmol / L) to centrifuge tubes. Next, add corn flour extract containing different concentrations of nicosulfuron (0.2, 2, 4, 6, 8, 10 μg / mL, 200 μL) and incubate at 25 °C for 1 min. Finally, add 200 μL of H2O2 and 100 μL of TMB, and incubate the mixture at 25 °C for 24 min. Record the UV-Vis absorption spectra of the solutions under optimal experimental conditions.

[0055] All experiments were repeated 3 times.

[0056] Figure 1 In Figures a and b, SEM images of Cu-MOF synthesized with PVP in Example 1 of this invention are shown; in Figures c and d, SEM images of Cu-MOF synthesized without PVP in Comparative Example 1 are shown. It can be seen from the figures that the copper-based nanozyme particles synthesized without the introduction of N-source PVP (…) Figure 1 The sizes of c and d are significantly different, and the grain boundaries are blurred. Copper-based nanoenzyme particles synthesized with the aid of N-source PVP (… Figure 1 In (a) and (b), the crystal particles have clear boundaries, are uniformly dispersed, and have a regular octahedral shape with little difference in size. This phenomenon indicates that the addition of PVP plays a crucial role in the morphology and structure of copper-based nanozyme particles.

[0057] To further investigate the elemental composition and morphology of Cu-MOF, XPS was used to test Cu-MOF.

[0058] Figure 2This is the XPS spectrum of the Cu-MOF synthesized in Example 1 of this invention; it can be seen from the figure that: 2p 3 / 2 The peak is located at 934.3 eV, 2p. 1 / 2 The peak is located at 953.8 eV, and the binding energy with Cu is... 2+ Standard value (2p) 3 / 2 933-935 eV, 2p 1 / 2 The values ​​(953-955 eV) are consistent with the satellite peaks, further confirming the presence of Cu. 2+ The presence of Cu. Cu-MOF is mainly composed of Cu element. 2+ The existence of the valence state further proves the successful synthesis of Cu-MOF.

[0059] Figure 3 The figures show typical UV-Vis absorption spectra of different reaction systems. It can be seen from the figures that the TMB+Cu-MOF system has no absorption peak at 652 nm; the TMB+H2O2 system has an absorption peak at 652 nm, but the absorbance is low; the TMB+Cu-MOF+H2O2 system shows an increased absorption peak at 652 nm and a higher absorbance, confirming that Cu-MOF has peroxidase-like activity. The absorbance of the TMB+Cu-MOF+H2O2+nicosulfuron system significantly increased after the addition of nicosulfuron. Therefore, the peroxidase-like activity of Cu-MOF can be used for the quantitative detection of nicosulfuron.

[0060] Figure 4 (a) Effect of nicosulfuron on the activity of Cu-MOF peroxidases; (b) Effect of Cu-MOF on the formation of hydroxyl radicals; (c) Effect of nicosulfuron on the formation of hydroxyl radicals; (d) XPS spectrum of Cu-MOF after the addition of nicosulfuron; Figure 4 It can be seen from this: like Figure 4 As shown in (a), the Cu-MOF-TMB-H2O2 system exhibits an absorption signal at 652 nm, and the solution is blue. Upon addition of nicosulfuron, the Cu-MOF-TMB-H2O2 system shows a strong absorption signal at 652 nm, and the blue color of the solution deepens, indicating enhanced catalytic activity of Cu-MOF. Therefore, the enhanced peroxidase activity based on Cu-MOF can be used for the quantitative detection of nicosulfuron.

[0061] The changes in hydroxyl radicals (•OH) were tracked using fluorescence experiments, and their detection mechanism was explored. The interaction between terephthalic acid and •OH can produce fluorescent products, such as... Figure 4In Figure (b), the fluorescence intensity of the PTA+Cu-MOF system with PTA is relatively weak; the fluorescence intensity of the PTA+H2O2 system is significantly increased, indicating the presence of •OH in the catalytic process. The fluorescence intensity of the PTA+Cu-MOF+H2O2 system is greater than that of the PTA+H2O2 system. The fluorescence intensity increases with the presence of Cu-MOF; therefore, the peroxidase-like activity of Cu-MOF mainly comes from the decomposition of H2O2 by Cu to generate •OH.

[0062] like Figure 4 As shown in (c), when different concentrations of nicosulfuron were added to the PTA+Cu-MOF+H2O2 system, the fluorescence intensity increased with increasing nicosulfuron concentration. The results indicate that nicosulfuron enhances the peroxidase-like activity of Cu-MOF by promoting the conversion of H2O2 to •OH.

[0063] like Figure 4 As shown in (d), the lone pair electrons of the sulfonylurea group in nicosulfuron react with Cu 2+ Coordination occurs, transferring Cu 2+ Transformation into Cu + Nitrogen (urea group) and oxygen (sulfonyl group) can react with Cu + Formation of stable complexes, inhibiting Cu + The re-oxidation of Cu + Increased content. Cu + -Nicosulfuron complexes can alter Cu 2+ The redox potential of H2O2 increased Cu + The efficiency of formation. Therefore, the presence of nicosulfuron promotes the redox duplex (Cu) formation. 2+ / Cu + The synergistic effect between Cu and MOF enhances their catalytic activity.

[0064] Figure 5 In the graph, a is the Michaelis constant plot of TMB; b is the Michaelis constant plot of H2O2; c is the double reciprocal plot of TMB; and d is the double reciprocal plot of H2O2. From the graphs, it can be seen that there is a good linear relationship between 1 / v and 1 / c (R0). 2 TMB=0.981, R 2 H2O2 = 0.991). Compared with reported horseradish peroxidase (HRP) and other forms of Cu nanozymes, Cu-MOFs using TMB or H2O2 as substrates have smaller Km values ​​(TMB = 0.991). Km =0.38, H2O 2 Km =1.80), indicating that Cu-MOF has a high affinity for TMB and H2O2, which proves that Cu-MOF has highly efficient peroxidase-like activity.

[0065] Figure 6 In Figure a, the UV-Vis absorption spectra of the Cu-MOF synthesized in Example 1 of this invention catalyzing the reaction of the TMB-H2O2 system in the presence of different concentrations of nicosulfuron are shown; in Figure b, the relationship between different concentrations of nicosulfuron and the absorbance at 652 nm is shown. It can be seen from the figure that: Figure 6 In Figure 'a', the UV-Vis absorption spectra of the TMB colorimetric reaction catalyzed by different concentrations of nicosulfuron mixed with Cu-MOF are shown (each line from bottom to top represents 0.2, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 μg / mL, respectively). Figure 6 As shown in Figure b, the absorbance of the system at 652 nm increased significantly with increasing nicosulfuron concentration. The ratio of nicosulfuron concentration to absorbance showed a linear relationship in the range of 0.2–10 μg / ml, and the linear regression equation was A = 0.05718x + 0.02265 (R²). 2 =0.9913).

[0066] Figure 7 To enhance the specificity of the rapid detection method for nicosulfuron pesticide using a colorimetric sensor based on copper-based nanozymes, this invention investigated the response of Cu-MOF to several other interfering substances. The results showed that the Cu-MOF exhibited relatively high catalytic activity after the addition of nicosulfuron, and only nicosulfuron could induce ΔA. 652 The value increased significantly, and in the presence of other pesticides and interfering substances, compared with the absorbance value of the blank sample, ΔA 652 The values ​​did not change significantly or even decreased. The results indicate that the Cu-MOF-based colorimetric method has high specificity for nicosulfuron.

[0067] Figure 8 (a) shows the change in UV absorbance intensity on different monitoring days; (b) shows the relative standard deviation of Cu-MOF from the same batch to different batches (Cu-MOF prepared according to the same steps and parameter conditions as in Example 1); it can be seen from the figure that: Compared to the absorbance on day 1, the absorbance of Cu-MOF decreased by only 9.7% after 30 days of storage. The absorbance showed no significant change over 30 days, indicating the stability of Cu-MOF. Furthermore, the precision of this method was evaluated. The catalytic activity of Cu-MOF from the same batch and different batches tested for nicosulfuron was relatively similar, with relative standard deviations (RSD) all <3.0%, indicating good precision of the method.

[0068] Figure 9(a) shows the UV-Vis absorption spectra of the TMB-H2O2 system catalyzed by Cu-MOF in the presence of maize samples with different concentrations of nicosulfuron; (b) shows the relationship between maize samples with different concentrations of nicosulfuron and the absorbance at 652 nm. From the figures, it can be seen that: The prepared Cu-MOF was used to detect six concentrations of nicosulfuron in maize samples from Heilongjiang Province. Figure (a) shows the UV-Vis absorption spectra of maize samples containing different concentrations of nicosulfuron catalyzed by the TMB colorimetric reaction after mixing with Cu-MOF. The concentrations, from bottom to top, are 0.2, 2, 4, 6, 8, and 10 μg / mL. Figure 9 As shown in (b), the absorbance of the system at 652 nm increased significantly with the increase of nicosulfuron concentration in the maize sample. The ratio of nicosulfuron concentration to absorbance in the maize sample showed a linear relationship in the range of 0.2–10 μg / ml, with a linear regression equation of A = 0.0512x + 0.2354 (R² = 0.993). The limit of detection was 0.02 mg / kg (the limit of detection is 3σ / K, where σ is the standard deviation of the blank group and K is the slope). The results are shown in Table 1. The recoveries of all samples ranged from 90.88% to 117.33%, with RSDs of 0.69%–3.17%. The results indicate that this colorimetric detection method has high accuracy and can be used for the rapid detection of nicosulfuron in real samples.

[0069] Table 1. Detection of nicosulfuron in maize from Heilongjiang Province In addition, to evaluate the applicability of the sensor, maize samples from six different origins (Shandong, Jiangsu, Jilin, Gansu, Guizhou, and Guangxi) were analyzed. The nicosulfuron spiking concentration for maize samples from different origins was 4 μg / ml. The recoveries of all samples ranged from 98.96% to 106.98%, with RSDs of 2.52% to 12.21%. Unspecified nicosulfuron analysis was performed on maize samples from the six different origins. Compared with the national standard maximum residue limit of 0.1 mg / kg for nicosulfuron, the nicosulfuron residues in maize samples from all six origins met the national standard, with relative standard deviations ranging from 1.97% to 6.73%. All these results indicate that Cu-MOF has certain applicability in detecting nicosulfuron in real-world samples.

[0070] The above are merely preferred embodiments of the present invention, but the scope of protection of the present invention is not limited thereto. Any variations or substitutions that can be easily conceived by those skilled in the art within the scope of the technology disclosed in the present invention should be included within the scope of protection of the present invention.

Claims

1. A method for preparing a copper-based nanozyme, characterized in that, Includes the following steps: The copper source, organic ligand H3BTC, and nitrogen-containing structure directing agent were dissolved in a solvent and reacted by stirring at room temperature. After the reaction was completed, the copper-based nanozyme was obtained by centrifugation, washing, and drying.

2. The method for preparing a copper-based nanozyme according to claim 1, characterized in that, The copper source is Cu(NO3)2·3H2O; The nitrogen-containing structure directing agent is polyvinylpyrrolidone; The solvent is methanol.

3. The method for preparing a copper-based nanozyme according to claim 1, characterized in that, The mass ratio of the copper source, the organic ligand H3BTC, and polyvinylpyrrolidone is 0.875:0.42:0.

4.

4. The method for preparing a copper-based nanozyme according to claim 1, characterized in that, The stirring reaction time is 20-28 hours; The drying process is vacuum drying, with a drying temperature of 55-65℃ and a drying time of 10-24 hours.

5. A copper-based nanozyme, characterized in that, It is prepared by the preparation method according to any one of claims 1-4.

6. The copper-based nanozyme according to claim 5, characterized in that, The copper-based nanozyme has a regular octahedral morphology in its crystal particles.

7. The application of the copper-based nanozyme as described in claim 5 or 6 in the detection of nicosulfuron pesticide or in the preparation of a colorimetric sensor for the detection of nicosulfuron pesticide.

8. A colorimetric sensor for detecting nicosulfuron, characterized in that, include: The core material is the copper-based nanozyme as described in claim 5 or 6; Oxidizing agent H2O2; The developing substrate is TMB.

9. A colorimetric sensor for detecting nicosulfuron according to claim 8, characterized in that, The colorimetric sensor also includes a fluorescent probe, terephthalic acid.

10. A method for detecting nicosulfuron using the colorimetric sensor of claim 8, characterized in that, Includes the following steps: Mix the copper-based nanozyme dispersion with the sample to be tested; Add the oxidant H2O2 and the chromogenic substrate TMB; Incubate at 20-30℃ for 20-30 minutes, and measure the change in absorbance using a UV-Vis spectrophotometer. The concentration of nicosulfuron was quantitatively analyzed based on changes in absorbance.

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