A supported bactericide coordination polymer, a preparation method and application thereof

Abstract

The application discloses a kind of immobilized bactericide coordination polymers and preparation method and application, belong to metal coordination polymer technical field, with seven hydrated zinc sulfate, naphthalene dicarboxylic acid and HEX are placed in mixed solvent, after dissolving solvent hot reaction is carried out, after reaction is ended, natural cooling to room temperature, the obtained reaction mixture is placed and volatilizes crystal, filtration, washing, drying, i.e. the immobilized bactericide coordination polymer of described preparation. BUAP8 as efficient, environmentally friendly bactericide delivery carrier, can simultaneously improve the bacteriostatic activity, stability and long-acting of reagent, while reducing ecological risk, has great application potential in the field of agricultural targeted disease prevention and control. The coordination polymer of this kind relies on unique self-assembly characteristics and non-covalent interaction, provides a new idea for the development of new bactericide delivery system, and also provides an innovative strategy for green crop protection.

Description

Technical Field

[0001] This invention belongs to the field of metal coordination polymer technology, and in particular relates to a coordination polymer for immobilized bactericides, its preparation method, and its application. Background Technology

[0002] Rice blast ( Rice Blast ) is caused by rice blast fungus ( Magnaporthe oryzae Rice blast, a devastating fungal disease affecting rice worldwide, is one of the most damaging diseases in rice cultivation globally, posing a serious threat to food security and economic stability. Widely distributed in rice-growing regions worldwide, rice blast poses a significant threat to rice yield, quality, and global food security, and is often referred to as "rice cancer" due to its devastating impact.

[0003] Traditional pesticides suffer from low utilization rates, easy loss, and high environmental residues, urgently requiring novel carrier materials to improve their bioavailability, prolong efficacy, and reduce impacts on the environment and non-target organisms. Controlled-release formulations can deliver encapsulated pesticides directly to the site of action at a controlled dose, minimizing direct contact between pesticide users and the environment, while significantly reducing pesticide losses due to various dissipation pathways. Porous CP materials can be used as encapsulation carriers to achieve effective pesticide loading. Yingjian Ma et al. developed a pH-responsive Fe-Zn dual MOF-MOF core-shell nanocarrier (MIL-101@ZIF-8), which combines fungicide delivery with micronutrient supplementation—an innovative approach in agricultural nanotechnology. Meanwhile, Saijie Song et al. reported a pH-responsive PDS using a carboxymethyl cellulose (CMC)-modified Zr-based metal-organic framework (UiO-66-NH2) as a nanocarrier for acetamiprid (ATP). In other studies, CP materials have served as the core framework of core-shell structures and participated in the formation of composite material systems. Furthermore, Vinayak Hegde et al. developed a dual-stimulus responsive pesticide delivery carrier using an iron-copper bimetallic-organic structure (Fe-Cu MOF), supported on diatomaceous earth (DE) and coated with lauric acid (LA). Yushan Hou et al. introduced a method of loading cyproconazole (MYC) into an Ag-based MOF (Ag-TCPP) and modifying it with a metallophenol network (MPN) via in-situ polymerization to construct a pH-responsive nanodelivery system (Ag-TCPP@MYC@MPN). Ag-TCPP@MYC@MPN exhibits a disordered nanoflower structure and numerous active sites, possessing the potential to enhance loading activity. This work provides new potential for developing improved responsive stimulus nanocarriers to achieve sustainable and controlled release of MYC. Covalent framework (CP)-based carriers, with their unique structural advantages, have shown great potential in constructing responsive pesticide delivery systems and achieving precise pest and disease control, and related research is increasingly attracting attention. However, existing systems generally have many problems that need to be solved, such as high raw material and synthesis costs, complex preparation routes, poor material stability in complex environments, low drug loading rate, short release cycle, and often accompanied by uncontrollable burst release behavior, which makes it difficult to meet the needs of actual field applications. Summary of the Invention

[0004] To address the above problems, this invention provides a polymer for immobilized bactericide coordination, its preparation method, and its application.

[0005] To achieve the above-mentioned objectives, the present invention adopts the following technical solution: In a first aspect, the present invention provides a method for preparing a solidified bactericide coordination polymer, comprising the following steps: zinc sulfate heptahydrate, H2NDC (naphthalenedicarboxylic acid), and HEX ((RS)-2-(2,4-dichlorophenyl)-1-(1H-1,2,4-triazol-1-yl)-hex-2-ol) are placed in a mixed solvent, dissolved, and subjected to a solvothermal reaction. After the reaction is completed, the mixture is naturally cooled to room temperature. The resulting reaction mixture is allowed to stand and evaporate to precipitate crystals. The mixture is then filtered, washed, and dried to obtain the solidified bactericide coordination polymer.

[0006] Furthermore, the molar ratio of zinc sulfate heptahydrate, naphthalenedicarboxylic acid, and (RS)-2-(2,4-dichlorophenyl)-1-(1H-1,2,4-triazol-1-yl)-hex-2-ol is 1:1:1.

[0007] Furthermore, the mixed solvent has a volume ratio of 1:1. N , N - A mixed solvent of dimethylacetamide (DMA) and water.

[0008] Furthermore, the solvothermal reaction is carried out at a temperature of 120 °C for a duration of 96 h.

[0009] Furthermore, the time for the crystals to be allowed to stand and evaporate is 7 days.

[0010] Furthermore, the washing process involves alternating between anhydrous ethanol and deionized water three times.

[0011] Secondly, the present invention provides a solidified bactericide coordination polymer prepared by the preparation method described above.

[0012] Thirdly, the present invention provides the application of immobilized fungicide coordination polymer in the control of rice blast fungus.

[0013] Compared with the prior art, the present invention has the following advantages and technical effects: The BUAP8 (immobilized fungicide coordination polymer) prepared in this invention exhibits acid-responsive slow-release properties in an aqueous environment. This immobilized fungicide coordination polymer achieves a HEX loading rate of 67.87%, extending the HEX release time to 5 days under normal temperature conditions, with a release amount of approximately 40.63%. In pathogen resistance experiments, BUAP8 significantly inhibited the growth of rice blast fungus, with an EC50 value of 0.13 μg / mL. Furthermore, compared to HEX alone, BUAP8 demonstrated significant advantages in biosafety tests on wheat seeds and zebrafish, showcasing good biocompatibility and environmental safety.

[0014] BUAP8, as a highly efficient and environmentally friendly fungicide delivery carrier, can simultaneously enhance the antifungal activity, stability, and long-lasting effect of fungicides while reducing ecological risks, demonstrating enormous application potential in the field of targeted disease control in agriculture. This type of coordination polymer, with its unique self-assembly properties and non-covalent interactions, provides a novel approach for the development of new fungicide delivery systems and offers innovative strategies for green crop protection. Attached Figure Description

[0015] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0016] Figure 1 [Zn(NDC)(HEX)2] n Synthesis roadmap for BUAP8; Figure 2 (a) shows the coordination environment of BUAP8, and (b) shows the one-dimensional chain structure. Figure 3 (a) shows the simulated and experimental P-XRD spectra; (b) shows the thermogravimetric analysis of BUAP8; (c) shows the Fourier transform infrared spectrum of BUAP8. Figure 4 The middle image (a) is a scanning electron microscope (SEM) image of BUAP8, and the other sub-images are EDS elemental spectra of C, N, Cl, O and Zn, respectively. Figure 5 (a) shows the pH cumulative release rate curve of BUAP8, (b) shows the zero-order, first-order, Higuchi, and Ritger-Peppas release kinetic models of HEX from BUAP8 suspension at different pH values, (c) shows the temperature cumulative release rate curve of BUAP8, and (d) shows the zero-order, first-order, Higuchi, and Ritger-Peppas release kinetic models of HEX from BUAP8 suspension at different temperatures. Figure 6 The standard curve for HEX standard solutions; Figure 7 (a) shows the inhibitory effects of HEX solution, BUAP8 supernatant, BUAP8 suspension and control group on rice blast fungus and the corresponding inhibition rates (b); Figure 8 (a) is a digital photograph of the growth of rice blast fungus; (b) shows the inhibition rates of different concentrations of H2NDC and ZnSO4. Figure 9(a) shows the infection of strawberry leaves with rice blast fungus by the blank control (sterile water treatment group), HEX solution and BUAP8 suspension, and digital photos of the strawberry leaves after treatment; (b) shows the area of ​​rice blast fungus lesions; (c) shows the inhibition rate of HEX solution and BUAP8 suspension against rice blast fungus. Figure 10 (a) shows the contact angle images of the control group, HEX solution, and BUAP8 suspension on strawberry leaves; (b) shows the evaporation of the control group, HEX solution, and BUAP8 suspension on filter paper over time; and (c) shows the liquid holding capacity of the control group, HEX solution, and BUAP8 suspension on strawberry leaves. Figure 11 (a) shows the UV resistance of HEX solution, HEX emulsion and BUAP8 suspension; (b) shows the storage stability of HEX solution and BUAP8 at 4, 27 and 54 °C; (c) shows the P-XRD pattern of BUAP8 after 2 weeks of water treatment. Figure 12 (a) is a digital photograph of wheat seed germination, (b) is the height of seedlings, and (c) is the result of a biosafety experiment on zebrafish to BUAP8. Detailed Implementation

[0017] 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.

[0018] 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.

[0019] 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.

[0020] 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.

[0021] 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.

[0022] The room temperature in this invention refers to 25±2℃.

[0023] Example 1: A method for preparing a coordinating polymer for immobilized bactericide. Weigh out ZnSO4·7H2O (14.4 mg, 0.05 mmol), naphthalenedicarboxylic acid (H2NDC, 10.81 mg, 0.05 mmol), and HEX (15.71 mg, 0.05 mmol). Add these solid reagents sequentially to a 1:1 mixture of DMA and deionized water, with a total solvent volume of 10 mL. Stir thoroughly until the solid reagents are completely dissolved to obtain a homogeneous precursor mixture. Transfer the precursor mixture to a 25 mL polytetrafluoroethylene-lined high-pressure reactor, seal it, and place it in a constant-temperature drying oven at 120 °C for 96 h. After the reaction is complete, allow the reactor to cool naturally to room temperature. Then, transfer the reaction mixture to a room-temperature environment and allow it to slowly evaporate for 7 days until a colorless, transparent single-crystal product spontaneously precipitates within the system. The target crystals were separated and collected using a vacuum filtration device. The product was then washed three times alternately with anhydrous ethanol and deionized water to thoroughly remove unreacted raw materials and residual organic solvents adsorbed on the crystal surface. The washed crystals were transferred to a vacuum drying oven and dried under vacuum at 60°C for 24 h, finally yielding a pale yellow crystalline product immobilized with a bactericide coordination polymer, denoted as [Zn(NDC)(HEX)2]. n BUAP8, yield 74.61% (22.15 mg; calculated based on H2NDC), Figure 1 [Zn(NDC)(HEX)2] n The synthetic route for BUAP8, and the formula for calculating the drug loading rate are shown below: ; 1. [Zn(NDC)(HEX)2] prepared in Example 1 n Structural characterization of BUAP8: 1.1. SC-XRD analysis of BUAP8: Figure 2(a) shows the coordination environment of BUAP8, and (b) shows the one-dimensional chain structure; Table 1 shows the crystal data of BUAP8. SC-XRD data shows that the fungicide content reaches 67.87%. Zn(II) adopts a six-atom tetrahedral coordination, in which the two imidazole N atoms come from two independent HEX ligands, and the four carboxylic acid O atoms come from two NDCs. 2- ligands ( Figure 2 (a)). NDC 2- Connect the two Zn(Ⅱ) pairs to form a one-dimensional chain along vector b. Figure 2 In (b), HEX is suspended on both sides of the chain as a terminal ligand.

[0024] Table 1 shows the crystal data for BUAP8. 1.2. P-XRD, TG and FT-IR analysis of BUAP8 Figure 3 (a) shows the simulated and experimental P-XRD patterns; (b) shows the thermogravimetric analysis of BUAP8; (c) shows the Fourier transform infrared spectrum of BUAP8. Figure 3 As shown in (a): The theoretical P-XRD simulation spectrum obtained by fitting the refined structure of single-crystal X-ray diffraction (SC-XRD) is compared and analyzed with the measured P-XRD spectrum. The peak positions, peak shapes and relative intensities of the characteristic diffraction peaks of the two are completely consistent, with no impurity peaks or peak position shifts. This fully confirms that the BUAP8 sample has excellent phase purity and a complete and regular crystal structure, which is consistent with the crystal configuration obtained by single-crystal analysis. Figure 3 As shown in (b): BUAP8 exhibits no significant mass loss and maintains structural stability in the temperature range below 300℃, demonstrating good mid-temperature thermal stability; however, when the temperature rises to the range of 310~400℃, the sample shows a sharp mass loss. Figure 3 As shown in (c): 1487 cm -1 With 1535 cm -1 The characteristic absorption peak at this location is attributed to the C=C stretching vibration of the aromatic ring in the ligand, and is consistent with HEX and NDC. 2- The aromatic ring structure of the ligands is highly consistent; 1394 cm -1 and 1446 cm -1 The absorption peaks at these locations correspond to the symmetric stretching vibrations of the carboxyl group, respectively. ν (COO - The presence of ligand functional groups is further corroborated by the CH bending vibration. Additionally, the values ​​at 700, 849, 913, and 935 cm⁻¹ further support the existence of these ligand functional groups. -1 The characteristic absorption peaks at these locations are attributed to the out-of-plane bending vibrations of the aromatic CH bonds and the stretching vibrations of the Zn-O / Zn-N coordination bonds, respectively. This result directly confirms that Zn 2+With NDC 2- Coordination bonds were successfully formed between HEX ligands, constructing a stable coordination polymer structure that is completely consistent with the one-dimensional chain coordination polymer configuration obtained by analysis.

[0025] 1.3. SEM and EDS spectral analysis of BUAP8 Figure 4 The middle image (a) is a scanning electron microscope (SEM) image of BUAP8. The other sub-images are EDS elemental spectra of C, N, Cl, O, and Zn, respectively. Figure 4 As shown in (a), BUAP8 exhibits a regular, plate-like microstructure with good crystallinity, uniform morphology, and no obvious agglomeration or morphological defects. EDS analysis further revealed the presence of five characteristic elements: Zn, N, C, Cl, and O. These elements were uniformly distributed on the sample surface, without local enrichment or deficiency, confirming the overall uniform composition of BUAP8. The SEM morphology characterization and EDS elemental analysis results corroborate each other, indicating that BUAP8 possesses a uniform plate-like structure and a stable, homogeneous elemental composition, providing crucial structural support for the stable performance of its sustained-release and antibacterial properties.

[0026] 2. Analysis of the sustained-release properties of immobilized bactericide coordination polymers 2.6 g of HEPES (hydroxyethylpiperazine ethanesulfonate sodium) was dissolved in 500 mL of ultrapure water. The pH of the system was adjusted to 5, 7, and 9 using HCl or NaOH solution, respectively, to prepare a series of HEPES buffer solutions with a concentration of 20 mmol / L. Different masses of HEX samples (0.625, 1.25, 2.50, 5.00, 10.00, and 20.00 mg) were accurately weighed and placed in 100 mL volumetric flasks. Chromatographic grade methanol was added to the mark, and the solutions were thoroughly mixed and allowed to stand at room temperature for 0.5 h to equilibrate, thus preparing a series of gradient standard solutions. The sample concentrations were determined using an Agilent 1200 series high-performance liquid chromatography system equipped with a G1314B UV detector and a ZORBAX Eclipse XDB-C18 column (4.6 × 150 mm). 2 The mobile phase was a mixture of methanol and ultrapure water at a volume ratio of 4:1, the flow rate was set to 1.0 mL / min, the column temperature was controlled at 30℃, and the detection wavelength was 272 nm. The corresponding chromatographic peak areas were recorded, and then a standard calibration curve was plotted.

[0027] BUAP8 (20 mg) was uniformly stirred in 100 mL of three HEPES buffer solutions at pH 5, 7, or 9. At regular intervals, 2 mL samples were taken, centrifuged, and the supernatant was used to determine the HEX concentration. The precipitate and 2 mL of buffer solution were then added to the release system. The cumulative release rate of HEX in the solution was calculated according to Formula 1.Q %)

[0028] in, Q This represents the cumulative release rate of HEX. V e The volume (2 mL) of each sample taken within a given time interval; V 0 This is the initial volume of the solution (200 mL); C i This represents the concentration in the supernatant during continuous sampling; C n The HEX concentration (μg / mL) at the nth sampling point; m HEX The total amount of HEX loaded in the material (mg).

[0029] In addition, the release kinetics of HEX in BUAP8 were fitted using various models: Zero-order (Equation 2), First-order (Equation 3), Higuchi (Equation 4), and Korsmeyer-Peppas (Equation 5). in, k It is a dynamic constant; n It is an indicator of model release: when n <0.43, 0.43< n <0.85 or 0.89 ≤ n When, represents Fick diffusion, non-Fick (or anomalous) diffusion, or zero-order migration model, respectively.

[0030] Figure 6 The standard curve for the HEX standard solution shows a linear quantification range of 0.625–10 μg / mL. Figure 5 (a) shows the pH cumulative release rate curve of BUAP8; (b) shows the zero-order, first-order, Higuchi, and Ritger-Peppas release kinetic models of HEX from BUAP8 suspension at different pH values; (c) shows the temperature cumulative release rate curve of BUAP8; and (d) shows the zero-order, first-order, Higuchi, and Ritger-Peppas release kinetic models of HEX from BUAP8 suspension at different temperatures. Figure 5As shown in (a), the HEX release trend is consistent across different pH environments: the first 13 hours are a rapid release phase with a rapid increase in the cumulative release rate, followed by a gradual slowdown in the release rate after 13 hours, with an overall sustained release duration of up to 120 hours, demonstrating good sustained-release characteristics. Significant differences in release rates were observed at different pH levels, with acidic conditions being more conducive to HEX release: at 13 hours, the cumulative release rates at pH 5, 7, and 9 were 47.1%, 33.4%, and 24.6%, respectively; by 120 hours, the cumulative release rate at pH 5 was close to 70%, at pH 7 approximately 47%, and at pH 9 nearly 38%. In summary, BUAP8 exhibits the best sustained-release and release efficiency of HEX under acidic conditions, demonstrating a clear acidic pH response characteristic.

[0031] Regarding the pH-responsive release characteristics of HEX in BUAP8, the core mechanism for its improved release efficiency under acidic conditions can be summarized in three aspects: First, in acidic media, H... + Will with NDC 2- Competition for Zn(II) coordination sites directly disrupts Zn-O / Zn-N coordination bonds, triggering rapid acid etching and disintegration of the coordination polymer backbone; secondly, NDC 2- Under acidic conditions, protonation occurs, significantly weakening the coordination ability and further accelerating skeletal dissociation, disrupting the original coordination equilibrium between Zn(II) and HEX. Thirdly, HEX is a weakly basic bactericide, and its solubility is significantly enhanced in acidic media, forming a dual driving force with skeletal disintegration, jointly promoting rapid and large-volume drug release. Furthermore, based on SC-XRD refinement results, the Zn-O bond length (1.983-2.017 Å) in BUAP8 is shorter than that of Zn-N (2.018-2.047 Å), but NDC... 2- The carboxylate group of HEX exhibits a bidentate coordination mode, resulting in significant steric hindrance; while HEX uses a terminal undentate coordination mode, offering greater flexibility. Under acidic conditions, H... + Preferred protonation NDC 2- The carboxylate ions break the Zn-O bond, while NDC... 2- The removal of bridging ligands and the collapse of the framework accelerate the release of HEX. This unique acid-responsive property enables it to target and release the active ingredient in the slightly acidic environment created by plant pathogen infection, making it easier and more efficient to exert antibacterial activity and possessing excellent potential for targeted field applications.

[0032] This invention employs four models—zero-order, first-order, Higuchi, and Ritger-Peppas—to dynamically fit the release curve. Figure 5 In (b), the fitting parameters and determination coefficients R of each model are combined. 2Analysis (Table 2) shows that the release process of HEX optimally conforms to the first-order kinetic model, and the release rate is directly proportional to the amount of unreleased drug in the system, which fully meets the typical sustained-release characteristics of drug gradual diffusion and dissolution from the polymer carrier. The cumulative release curves of HEX at different temperatures are shown in Table 2. Figure 5 As shown in (c), the results indicate that the HEX release rate is significantly positively correlated with ambient temperature; the release rate increases synchronously with increasing temperature. In each group of experiments, the HEX release concentration reached equilibrium after 72 h, with equilibrium concentrations of 30.42 mg / L at 15℃, 40.63 mg / L at 25℃, and 50.00 mg / L at 35℃, respectively. Significant differences in release were observed among the different temperature groups.

[0033] From the perspective of molecular interactions and coordination equilibrium mechanisms, increased temperature can intensify the thermal vibration amplitude of the Zn-O / Zn-N coordination bonds within the BUAP8 framework, directly driving bond breakage and initiating the gradual dissolution of the polymer framework, thus providing a structural basis for the release of the active ingredient. High temperatures can simultaneously increase the solubility of HEX in aqueous media, increase the drug molecule diffusion coefficient, and effectively reduce mass transfer resistance across the framework, further accelerating the release of the active ingredient. Furthermore, since coordination bond formation is a typical exothermic process, under increasing temperature, the coordination equilibrium of the system spontaneously shifts towards ligand dissociation, further intensifying framework disintegration and synergistically promoting the continuous release of HEX. This unique temperature-responsive release characteristic enables BUAP8 to rapidly release its active ingredient in high-temperature field environments, thereby exerting a highly effective antibacterial effect, providing solid theoretical support for the design and development of novel controllable-release agricultural antibacterial coordination polymers.

[0034] Table 2. Parameters of BUAP8 release kinetic model at different pH values. Table 3. Release kinetic model parameters of BUAP8 at different temperatures. 4. Determination of the bioactivity of immobilized bactericide coordination polymers 4.1 Antifungal Activity of Single Raw Materials: The antifungal activity of the target samples was systematically evaluated using the potato dextrose agar (PDA) plate culture method. A series of PDA mediums containing HEX, H2NDC, and ZnSO4 were prepared at concentrations of 1.00, 0.50, 0.25, 0.125, and 0.0625 μg / mL, respectively. A blank control group without any added agents was also included. 10 mL of each concentration of the above-mentioned medium was accurately measured and evenly poured into sterile 70 mm diameter petri dishes. The medium was allowed to solidify completely before use. Three replicates were set up for each concentration to ensure the statistical reliability of the experimental results. Rice blast fungus (… M. oryzaeAs the test fungus, 5 mm diameter mycelial discs were cut from the edge of the activated fungal colonies using a sterile punch and inoculated into the center of the solidified PDA medium in each group. The inoculated petri dishes were placed in a constant temperature incubator at 28℃, with a light-dark cycle of 16.0 h / 8.0 h. After 7 days of continuous culture, the diameter of the fungal colonies in each group was measured using the cross-sectional method. The mycelial growth inhibition rate under different agents and concentrations was calculated according to Formula 6 to quantitatively evaluate the antifungal activity of the samples.

[0035] in, N 0 and N t The mycelial diameters of the control group and the treatment group are shown respectively after 7 days of cultivation.

[0036] 4.2. Antifungal activity of immobilized bactericide coordination polymers: Preparation and antifungal test: 500 mL of BUAP8 (based on HEX active ingredient, 50 μg / mL) aqueous suspension was stirred for 0.5 h, then 200 mL of the mixture was centrifuged, and the HEX concentration in the supernatant was determined by HPLC. Bactericidal activity of the supernatant: PDA media (10 mL each) were prepared using the supernatant of the BUAP8 suspension instead of the HEX bactericide, following the procedure outlined in 4.1, to ensure HEX concentrations in the soluble mixtures were 1.00, 0.50, 0.25, 0.125, and 0.0625 μg / mL, respectively. Bactericidal activity of the suspension: PDA media solutions (10 mL each) were prepared using the BUAP8 suspension, following the procedure outlined in 4.1, to ensure HEX soluble concentrations in the mixtures were 1.00, 0.50, 0.25, 0.125, and 0.0625 μg / mL (based on HEX concentration in the supernatant).

[0037] Figure 7 Table 4 shows the inhibitory effects of HEX solution, BUAP8 supernatant, BUAP8 suspension, and control group on rice blast fungus and their corresponding inhibition rates (b), with concentrations of 0.0625, 0.125, 0.25, 0.5, and 1 μg / mL, respectively; Table 4 shows the inhibitory effects of HEX solution, BUAP8 supernatant, and BUAP8 suspension on rice blast fungus. M. oryzae Its antifungal activity.

[0038] Table 4 from Figure 7As shown in (a) and (b), when the concentration of the active ingredient reached 1 μg / mL, the growth inhibition rate of all three treatments against *Strombus oryzae* was higher than 78%, exhibiting excellent antibacterial activity. The supernatant of BUAP8 maintained a high inhibition level throughout the entire test concentration range, and the antibacterial effect gradually increased with increasing treatment concentration. At the same concentration, the inhibition rate of BUAP8 suspension against *Strombus oryzae* was close to 100%, demonstrating particularly outstanding antibacterial performance. In contrast, the antifungal activity of the single raw materials ZnSO4 and H2NDC was weaker. Under the same test concentration gradient, the inhibition rates of both against the tested strains were 7.71%–32.76% (e.g., ...). Figure 8 As shown in the figure, this fully demonstrates that the coordination polymer carrier can effectively promote the antibacterial effect of the active ingredients.

[0039] Quantitative activity data showed that pure HEX solution exhibited high activity against EC50 of rice blast fungus. 50 The concentration was 0.19 μg / mL, indicating some antibacterial activity, but its antibacterial effect was significantly weaker than that of the BUAP8 system. The BUAP8 suspension EC... 50 The value was 0.11 μg / mL, and the supernatant EC was... 50 The concentration was 0.13 μg / mL, indicating that the antibacterial activity of the drug-loaded system was significantly better than that of a single agent. The improved antibacterial performance of BUAP8 is attributed to the synergistic effect of multiple factors. The active components released by the system not only include HEX but also ZnSO4 and H2NDC fragment molecules. The synergistic effect of multiple components can significantly enhance the inhibitory effect on pathogens. At the same time, as a controllable release coordination polymer, BUAP8 can precisely regulate the release rate of HEX, maintain an effective antibacterial concentration in the environment for a long time, and achieve long-lasting antibacterial effect. In addition, the porous structure and high specific surface area of ​​BUAP8 can effectively improve the bioavailability of antibacterial components, further enhancing the overall antibacterial efficacy.

[0040] 5. Potted plant experiment A solution / suspension containing HEX and BUAP8 (based on the active ingredient HEX, 50 μg / mL) was prepared. Strawberry leaves of uniform growth were selected, and three evenly distributed artificial wounds were created on the midrib using a sterile needle-pricking method. All wounds were confined to a circular area with a diameter of 5 mm to simulate natural infection damage. Subsequently, 5 mm diameter fungal mycelium was precisely inoculated onto the injured leaf veins. Each treatment was performed in triplicate to ensure the reliability and reproducibility of the experimental results. After inoculation, the leaves were placed in an artificial climate chamber for cultivation, maintaining room temperature and a relative humidity of 85% to create suitable temperature and humidity conditions for fungal infection. After 24 hours of fungal infection, the corresponding fungicide solution or suspension was sprayed evenly on the test leaves every 24 hours. Subsequent treatments were carried out according to the experimental design, and the disease incidence and control efficacy of the fungicides were continuously observed. Figure 9(a) shows the infection of strawberry leaves with rice blast fungus by the blank control (sterile water treatment group), HEX solution and BUAP8 suspension, and digital photographs of the strawberry leaves after treatment; (b) shows the area of ​​rice blast fungus lesions; (c) shows the inhibition rate of HEX solution and BUAP8 suspension against rice blast fungus.

[0041] This invention uses a sterile water treatment group as a blank control, and sets up treatment groups with HEX solution and BUAP8 suspension, both with a concentration of 50 μg / mL, and a treatment period of 7 days. The results showed that after 7 days of incubation, the diseased leaf area in the blank control group reached 1.85 cm². 2 The diseased leaf area in the pure HEX treatment group decreased to 1.09 cm². 2 The BUAP8 suspension treatment group showed a further significant reduction in diseased area, to only 0.17 cm². 2 The diseased area in both treatment groups was significantly lower than that in the control group. Figure 9 (b) Calculations showed that the pure HEX treatment group had an inhibition rate of 40.84% ​​against rice blast fungus, while the BUAP8 treatment group had an inhibition rate as high as 90.94%, demonstrating significantly better antibacterial effects than single agents. Figure 9 (c)

[0042] 6. Determination of Leaf Retention of Immobilized Fungicide Coordination Polymer 6.1 Wetting and Adhesion Tests: Fresh strawberry leaves were used as the substrate for BUAP8. The contact angle of the corresponding suspension on the leaf surface was measured to evaluate the wettability of the material on plant leaves. Before the test, fresh strawberry leaves with uniform thickness, shape, and growth were selected and fixed flat on the surface of a glass slide to eliminate the interference of leaf wrinkles and thickness differences on the test results. Under room temperature conditions, 5 μL of suspension was accurately drawn using a microsyringe. BUAP8 suspension (the concentration of the effective component in the suspension was controlled at 100 μg / mL) and sterile water (blank control group) were transferred and evenly coated onto the leaf surface. Each group of samples was tested in parallel five times at different sites on the leaf to ensure the statistical validity of the experimental data. After coating, the samples were allowed to stand for 10 seconds until the droplet state stabilized to eliminate the interference of interface fluctuations caused by dynamic coating. Then, the corresponding contact angle values ​​for each group were collected and recorded.

[0043] 6.2. Liquid Holdup Test The leaf holding capacity (LHC) of strawberry leaves was quantitatively analyzed using an immersion method. Fresh, uniformly grown strawberry leaves were selected and cut into uniform 2×2cm pieces. 2Small leaf samples were prepared, with three parallel leaf samples per treatment group to ensure the repeatability and reliability of experimental data. The prepared leaf samples were immersed in BUAP8 suspension, with HEX as the active ingredient at a concentration of 50 μg / mL, for a uniform immersion time of 15 s. The LHC value was then calculated using Formula 7 based on the leaf weight.

[0044] in, m 1 and m 2 represents the weight of the leaf before and after immersion, respectively. S This represents the surface area of ​​the blade.

[0045] 6.3 Volatility Performance Test The specific procedure for determining the volatile content of a sample using the filter paper method is as follows: Before the experiment, weigh the initial mass of the quantitative filter paper and record it as follows: m 0; Subsequently, using a uniform spraying method, 1.0 mL of BUAP8 suspension or sterile water (blank control group) was added dropwise to the filter paper. The concentration of HEX active ingredient in the BUAP8 suspension was 50 μg / mL. After spraying, the wet weight of the filter paper was quickly measured and recorded as follows. m 1. Place the moistened filter paper in a temperature-controlled environment, maintaining room temperature. Weigh the filter paper periodically at preset time intervals and record the mass as follows. m n The evaporation rate of the sample was calculated based on the mass change at different time points. Finally, the evaporation rate was calculated using Equation 8.

[0046] Figure 10 (a) shows the contact angle images of the control group, HEX solution, and BUAP8 suspension on strawberry leaves; (b) shows the evaporation of the control group, HEX solution, and BUAP8 suspension on filter paper over time; and (c) shows the liquid holding capacity of the control group, HEX solution, and BUAP8 suspension on strawberry leaves. Figure 10 As shown in (a), BUAP8 has a contact angle of 56.16° on the strawberry leaf surface, indicating that it possesses moderate and suitable leaf wettability, ensuring effective spread and uniform adhesion of the pesticide on the plant surface, thus laying the foundation for improving pesticide utilization in the field. Leaf liquid retention tests showed that BUAP8 achieved a liquid retention capacity of 11.78 mg / cm² per unit area. 2 ( Figure 10 (c) This study confirmed its excellent leaf retention capacity, which significantly improved the adhesion and retention stability of the agent on the leaf surface, prolonged the action time of the fungicide on the target surface, and further enhanced the long-lasting antibacterial effect. Figure 10As shown in Figure (b), the pure HEX component exhibits a rapid volatilization rate, reaching 60.37% after 30 minutes at room temperature and surging to 91.82% after 60 minutes, indicating significant loss of active ingredients. In contrast, the BUAP8 system shows a significantly slower volatilization rate. Under the same test conditions, the volatilization rate is only 47.02% after 30 minutes and 78.74% after 60 minutes, far lower than that of the pure HEX component. The slow-vaporization characteristic of BUAP8 effectively reduces the volatilization loss of the active ingredient HEX into the atmosphere, allowing more fungicide to remain stably on the target surface of strawberry leaves, achieving slow release and sustained action of the active ingredient, and further enhancing the field antibacterial efficacy and persistence of the agent.

[0047] 7. Stability analysis of coordination polymers of immobilized bactericides 7.1. Ultraviolet Degradation Test: The UV resistance of the target samples was systematically investigated using the ultraviolet light degradation method. The specific experimental steps are as follows: HEX solution, HEX emulsion, and BUAP8 suspension were prepared separately. In the BUAP8 system, HEX was used as the active ingredient, with an active substance concentration controlled at 50 μg / mL. Each system was transferred to a 50 mL quartz bottle for later use. Under room temperature conditions, the quartz bottles containing various samples were fixed in an ultraviolet irradiation device, and a 340 W ultraviolet light source with a wavelength of 253 nm was used for continuous irradiation. The distance between the light source and the sample liquid surface was controlled at 20 cm to construct a standardized ultraviolet degradation system.

[0048] Following a preset time gradient (0, 0.5, 1.0, 2.0, 3.0, 4.0, 5.0, 6.0 h), 1 mL of sample was precisely drawn from each quartz bottle and aliquoted. Each aliquot was then thoroughly mixed with 1 mL of dimethyl sulfoxide / methanol mixed solvent to dissolve any precipitated solid components and prevent solute precipitation from affecting the quantitative results. Subsequently, the HEX concentration in the samples at each time point was determined using the aforementioned high-performance liquid chromatography (HPLC) method. The UV resistance of the material was evaluated based on the concentration change pattern.

[0049] 7.2. Water stability test The water stability of BUAP8 was systematically characterized as follows: 100 μg of BUAP8 containing the HEX active ingredient was weighed and immersed in 100 mL of ultrapure water for 14 days to construct an aqueous aging system. The system was periodically sampled and filtered to separate solid samples, which were then thoroughly dried in an oven preheated to 45°C to remove surface adsorbed moisture. Powder X-ray diffraction was used to characterize the structure of the solid samples before and after water immersion treatment. The evolution of the crystal structure after long-term immersion in the aqueous environment was compared and analyzed to verify the material's aqueous stability.

[0050] 7.3. Storage stability test Pure HEX and BUAP8 (both containing 50 μg of HEX active ingredient) were placed in brown, light-proof glass bottles and stored in sealed containers at three temperature gradients (4, 27, and 54 °C) for 7 days to systematically investigate the storage stability of the samples under different temperature conditions. After the storage period, the samples were dissolved in suitable solvents (methanol was used for BUAP8). The remaining HEX content in each sample was then accurately determined using the previously established high-performance liquid chromatography (HPLC) method to quantitatively assess the differences in storage stability of the samples under different temperature conditions.

[0051] Figure 11 (a) shows the UV resistance of HEX solution, HEX emulsion and BUAP8 suspension; (b) shows the storage stability of HEX solution and BUAP8 at 4, 27 and 54 °C; and (c) shows the P-XRD pattern of BUAP8 after 2 weeks of water treatment.

[0052] like Figure 11 As shown in (a), after 6 hours of continuous UV irradiation, the degradation rates of both commercially available HEX and pure HEX components exceeded 91%, indicating significant loss of active ingredients. In contrast, the degradation rate of HEX-loaded components in the BUAP8 system was 88%, lower than that of the single HEX component. This result demonstrates that loading HEX into the BUAP8 coordination polymer backbone can appropriately improve its resistance to UV degradation, enhance photostability, and thus prolong the duration of BUAP8's activity in complex field environments, ensuring long-term antibacterial effects and stable disease control. Figure 11 As shown in (c): After two weeks of water immersion treatment, the P-XRD pattern of BUAP8 was almost identical to the original pattern, with no characteristic peak shift, intensity reduction, or impurity peak generation. This demonstrates that BUAP8 possesses excellent structural stability in aqueous solution, maintaining its crystal framework intact without significant dissolution or collapse, and exhibits outstanding resistance to water environment stress. Long-term storage stability test (… Figure 11 (b) Three temperature gradients of 4, 27, and 54°C were selected, with a storage period of two weeks. The results showed that the content of HEX active ingredients in BUAP8 remained stable after storage under different temperature conditions, without significant reduction or fluctuation, fully demonstrating its excellent storage stability. This characteristic gives BUAP8 a longer shelf life in practical applications and makes its storage requirements less stringent.

[0053] 8. Biosafety assessment of immobilized bactericide coordination polymers 8.1 Seed Germination: Using wheat seeds as a model organism, the biosafety of BUAP8 and HEX was systematically evaluated by measuring seed germination rate and seedling height. Before the experiment, wheat seeds underwent sterilization pretreatment: the test seeds were soaked in a 10% sodium hypochlorite solution for 10 min, followed by repeated rinsing three times with ultrapure water to thoroughly remove residual sodium hypochlorite from the seed surface and avoid interference with seed germination. The sterilized wheat seeds were then soaked in HEX aqueous solution, BUAP8 suspension, and a blank control group (water) for 2.0 h, respectively. The concentration of the active ingredient in HEX was controlled at 50 μg / mL, and the concentration parameters were kept consistent across all treatment groups. Five mL of each treatment solution was measured and evenly sprayed into petri dishes lined with qualitative filter paper. Seven wheat seeds were evenly placed in each dish, and the dishes were sealed and incubated in a constant temperature and dark environment at 25 ± 2℃. The culture period lasted for 7 days. After the culture was completed, the germination rate of wheat seeds in each group was counted, and the seedling height was measured to quantitatively assess the biosafety of the test samples. All experimental groups were set up in triplicate.

[0054] 8.2. Zebrafish Toxicity: Zebrafish were selected as the model organism, and acute toxicity tests were conducted according to the standard testing procedures established by the US Environmental Protection Agency (EPA). Before the test, tap water was exposed to natural light for 72.0 h to complete dechlorination pretreatment before being used in the preparation of the test system to eliminate the toxic interference of chlorine substances in tap water on zebrafish. After adaptive culture, zebrafish were randomly divided into groups of 7 fish per test unit and exposed to a series of concentration gradients of test solutions. The concentrations for each group were as follows: HEX solution concentrations were 3.0, 2.5, 2.0, 1.5, and 1.0 μg / mL; BUAP8 suspension with HEX as the active ingredient was set at concentration gradients of 5.0, 4.5, 4.0, 3.5, and 3.0 μg / mL. All treatment groups were replicated three times. A blank control group was also set up. The zebrafish in the control group were raised in pretreated water without any test drugs, and all other culture conditions were exactly the same as those of the treatment groups. Zebrafish were continuously exposed and cultured in the corresponding experimental system for 96.0 h. During the experiment, the mortality rate of zebrafish in each group was observed and recorded at regular intervals. The mortality rate was calculated, and a toxicity regression equation was fitted based on the mortality rate data to calculate the median lethal concentration (LC50) of each test sample for zebrafish. 50 ), to quantitatively assess its aquatic organism toxicity and biosafety.

[0055] Figure 12 (a) is a digital photograph of wheat seed germination, (b) is the height of seedlings, and (c) is the result of a biosafety experiment on zebrafish to BUAP8.

[0056] The results of a 7-day hydroponic germination experiment are as follows Figure 12As shown in Figure (b), the average height of wheat seedlings in the blank control group was 7.47 cm, the average height of seedlings in the pure HEX treatment group was 6.00 cm, and the average height of seedlings in the BUAP8 treatment group was 7.05 cm. The seedling height in both treatment groups was slightly lower than that in the control group, indicating a mild growth inhibition effect. However, the seedling height in the BUAP8 treatment group was significantly higher than that in the pure HEX treatment group, indicating a significantly weaker inhibitory effect. This result is mainly attributed to the loading and sustained-release effect of the BUAP8 coordination polymer on HEX, which effectively reduced the instantaneous concentration of free HEX in the solution, thereby weakening the toxic stress of the active ingredient on plant seedlings. These results fully demonstrate that BUAP8, as a drug carrier, can reduce the potential negative impact on plant growth by precisely controlling the release behavior of the active ingredient HEX, significantly improving the overall biocompatibility of the material and making it more suitable for safe application in agricultural fields.

[0057] This invention uses zebrafish as a standard aquatic model organism to complete the aquatic organism safety verification of BUAP8. The results of the 96-hour acute exposure test are as follows: Figure 12 As shown in (c), the survival rate of zebrafish decreased in a gradient with increasing concentration of BUAP8 in the test solution, exhibiting typical concentration-dependent toxicity characteristics. Based on data fitting calculations, the median lethal concentration (LC50) of BUAP8 for zebrafish during the 96-hour exposure period was determined. 50 The LC50 concentration was 4.13 μg / mL, indicating that BUAP8 exhibits low acute toxicity to zebrafish within the tested concentration range. Compared to pure HEX, BUAP8 showed a lower LC50 concentration. 50 A higher value means that, under the same concentration conditions, its toxicity to zebrafish is significantly lower than that of pure HEX, resulting in superior safety for aquatic organisms. This advantage stems from BUAP8's unique coordination framework structure, which enables the slow and controlled release of the active ingredient, avoiding excessively high instantaneous drug concentrations in the water. This effectively reduces the acute toxic impact on aquatic organisms and improves overall environmental compatibility.

[0058] 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 supported biocide complex polymer, characterized by, Includes the following steps: Zinc sulfate heptahydrate, naphthalenedicarboxylic acid, and (RS)-2-(2,4-dichlorophenyl)-1-(1H-1,2,4-triazol-1-yl)-hex-2-ol were placed in a mixed solvent, dissolved, and subjected to a solvothermal reaction. After the reaction was completed, the mixture was naturally cooled to room temperature. The resulting reaction mixture was allowed to stand and evaporate to precipitate crystals. After filtration, washing, and drying, the solidified bactericide coordination polymer was obtained.

2. The method for preparing a solid supported bactericidal agent complex polymer according to claim 1, characterized in that, The molar ratio of zinc sulfate heptahydrate, naphthalenedicarboxylic acid, and (RS)-2-(2,4-dichlorophenyl)-1-(1H-1,2,4-triazol-1-yl)-hex-2-ol is 1:1:

1.

3. The method for preparing the immobilized bactericide coordination polymer according to claim 1, characterized in that, The mixed solvent is a mixed solvent of dimethylacetamide and water at a volume ratio of 1:1 N , N - dimethylacetamide and water.

4. The method for preparing the immobilized bactericide coordination polymer according to claim 1, characterized in that, The solvothermal reaction was carried out at a temperature of 120 °C for 96 h.

5. The method for preparing the immobilized bactericide coordination polymer according to claim 1, characterized in that, The time for the crystals to be volatilized and precipitated by static evaporation is 7 days.

6. The method of preparing a supported biocide ligating polymer according to claim 1, wherein, The washing process involves alternating between anhydrous ethanol and deionized water three times.

7. A coordinating polymer for immobilized bactericide prepared by the preparation method according to any one of claims 1-6.

8. The application of the immobilized fungicide coordination polymer of claim 7 in the control of rice blast fungus.

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