Functional bio-based nanoszyme, preparation method thereof and synergistic application in agrochemicals

Abstract

The present application relates to a kind of functional biological-based nanometer enzyme and its preparation method and agricultural chemical synergistic application, the functional biological-based nanometer enzyme includes the porous structure Prussian blue analogue, and the complex attached to the surface of the Prussian blue analogue, the complex is formed by the metal ion in Prussian blue analogue and the antioxidant biomass molecule complex, the antioxidant biomass molecule is selected from at least one of biomass polyphenol, biomass polysaccharide or amphoteric quaternary ammonium salt alkaloid, the porosity of the functional biological-based nanometer enzyme is 35%~70%. When the functional biological-based nanometer enzyme of the present application is used as agricultural chemical synergist, it can actively regulate and effectively delay the resistance of pests, significantly improve the sensitivity of pests to insecticide;At the same time, the functional biological-based nanometer enzyme has good biocompatibility, can reduce the potential harm to non-target organisms, has higher ecological safety.

Description

Technical Field

[0001] This invention relates to the field of agricultural technology, and in particular to a functional bio-based nanozyme, its preparation method, and its synergistic effect on agricultural chemicals. Background Technology

[0002] Insecticides are currently the primary means of controlling pests; however, their frequent use leads to the development of resistance in pests. This resistance not only severely reduces the control efficacy of insecticides but also significantly shortens their effective lifespan. To delay the development of pest resistance, chemical synergists such as polyphenylene ether (PBO) and triphenyl phosphate (TPP) are often added for synergistic control. However, these synergists mainly work by passively inhibiting the active sites of detoxification enzymes, lacking the ability to inhibit the synthesis of detoxification enzymes in pests at the source. They generally suffer from insufficient target specificity and are prone to inducing cross-resistance, making it difficult to fundamentally solve the problem of insecticide resistance in pests. Summary of the Invention

[0003] Therefore, it is necessary to address the above problems by providing a functional bio-based nanozyme, its preparation method, and its synergistic effect on agricultural chemicals. When used as an synergist for agricultural chemicals, the functional bio-based nanozyme can regulate the resistance of pests and significantly improve the sensitivity of pests to insecticides.

[0004] A functional bio-based nanozyme, comprising a porous Prussian blue analogue and a complex attached to the surface of the Prussian blue analogue, wherein the complexue is formed by the complexation of metal ions in the Prussian blue analogue with antioxidant biomass molecules, wherein the antioxidant biomass molecules are selected from at least one of biomass polyphenols, biomass polysaccharides, or amphoteric quaternary ammonium alkaloids.

[0005] In one embodiment, the porosity of the functional bio-based nanozyme is 35% to 70%.

[0006] And / or, the particle size of the functional bio-based nanozyme is 50nm~200nm;

[0007] And / or, the thickness of the complex on the surface of the Prussian blue analogue is 2 nm to 15 nm;

[0008] And / or, when the antioxidant biomass molecule is biomass polyphenol and biomass polysaccharide, the mass ratio of the biomass polyphenol to the biomass polysaccharide is 1:5 to 5:1.

[0009] In one embodiment, the biomass polyphenols are selected from at least one of dopamine, tannic acid, quercetin, chlorogenic acid, phytic acid, tea polyphenols, or gallic acid;

[0010] And / or, the biomass polysaccharide is selected from at least one of fucoidan, chitosan oligosaccharide, chitosan or sodium alginate;

[0011] And / or, the amphoteric quaternary ammonium alkaloid is selected from at least one of betaine or berberine.

[0012] In one embodiment, the Prussian blue analogue is selected from at least one of manganese-based Prussian blue, cobalt-based Prussian blue, magnesium-based Prussian blue, or molybdenum-based Prussian blue.

[0013] A method for preparing the above-described functional bio-based nanozyme includes the following steps:

[0014] Ferric cyanide, biomass end-capping agent and biomass acid source are mixed and dissolved, then metal salt is added, and then a hydrothermal reaction is carried out to obtain Prussian blue analogues.

[0015] Antioxidant biomass molecules, the Prussian blue analogue, and a solvent are mixed and subjected to a complexation reaction to obtain a functional bio-based nanozyme.

[0016] The antioxidant biomass molecule is selected from at least one of biomass polyphenols, biomass polysaccharides, or amphoteric quaternary ammonium alkaloids.

[0017] In one embodiment, the mass ratio of the biomass acid source to the ferricyanide is 1:0.1 to 1:0.2;

[0018] And / or, the mass ratio of the antioxidant biomass molecule to the Prussian blue analogue is 5:1 to 1:5;

[0019] And / or, the mass ratio of the metal salt to the ferricyanide is 1:5 to 1:10;

[0020] And / or, the metal salt is selected from at least one of manganese salt, cobalt salt, magnesium salt or molybdenum salt;

[0021] And / or, the biomass acid source in the functional bio-based nanoenzyme is selected from at least one of citric acid, fulvic acid or tannic acid;

[0022] And / or, the biomass capping agent in the functional bio-based nanozyme is selected from at least one of tea seed cake or rhamnolipid.

[0023] In one embodiment, the hydrothermal reaction temperature is 70°C to 90°C, and the reaction time is 10h to 24h.

[0024] And / or, the temperature of the complexation reaction is 20℃~30℃, and the reaction time is 5h~12h.

[0025] A type of functional bio-based nanozyme, as described above, is used as an synergist for agricultural chemicals.

[0026] Application of a functional bio-based nanozyme as described above in the control of reed aphids.

[0027] In one embodiment, the functional bio-based nanozyme is used in combination with a neonicotinoid insecticide.

[0028] In this invention, Prussian blue analogs with porous structures provide excellent electron transport capabilities and abundant catalytic active sites, providing a structural basis for the catalysis of multiple enzyme activities. A functional interface layer is formed on the surface of the Prussian blue analog by a complex of metal ions and antioxidant biomass molecules. This functional interface layer, on the one hand, constructs an efficient electron transport channel, promotes redox cycles, and enhances the antioxidant enzyme activity of the functional bio-based nanozyme; on the other hand, this interface layer works synergistically with the metal ions in the Prussian blue analog framework, endowing the functional bio-based nanozyme with excellent multi-enzyme catalytic activity, thereby achieving efficient and synergistic scavenging of endogenous reactive oxygen species (ROS).

[0029] Furthermore, the functional bio-based nanozymes provided by this invention have good biocompatibility, can effectively reduce the environmental risks caused by traditional chemical synergists, and reduce potential harm to non-target organisms, thus exhibiting high ecological safety. Attached Figure Description

[0030] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments recorded in the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0031] Figure 1 Transmission electron microscope images of the functional bio-based nanozymes prepared in Examples 1-5, 7 and 9-10 of this invention;

[0032] Figure 2 A statistical graph showing the mortality rate of different concentrations of imidacloprid against the reed aphid;

[0033] Figure 3 A statistical chart showing the mortality rate of *Imidaclopridus spp.* when imidacloprid is used in combination with the functional bio-based nanoenzyme prepared in Example 1 of this invention against *Imidaclopridus spp.*

[0034] Figure 4 A statistical graph showing the effect of imidacloprid combined with the functional bio-based nanozyme prepared in Example 1 of this invention on the specific activities of catalase (CAT), superoxide dismutase (SOD), and peroxidase (POD) in the body of the grass aphid.

[0035] Figure 5 A statistical chart showing the effect of the functional bio-based nanozyme prepared in Example 1 of this invention on the survival rate of the reed aphid.

[0036] Figure 6 A statistical chart showing the effect of applying the functional bio-based nanozyme prepared in Example 1 of this invention on the survival rate of earthworms;

[0037] Figure 7 This is a photograph of wheat growth when the functional bio-based nanozyme prepared in Example 1 of this invention is applied.

[0038] Figure 8 A statistical chart showing the plant height, root length, chlorophyll SPAD value, and fresh weight of wheat when the functional bio-based nanozyme prepared in Example 1 of this invention is applied. Detailed Implementation

[0039] To facilitate understanding of the present invention, it will be described in more detail below. However, it should be understood that the present invention can be implemented in many different forms and is not limited to the embodiments or examples described herein. Rather, these embodiments or examples are provided to make the disclosure of the present invention more thorough and complete.

[0040] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The terminology used herein in the specification of this invention is for the purpose of describing particular embodiments or examples only and is not intended to limit the invention. The optional scope of the term "and / or" as used herein includes any one of two or more of the related listed items, as well as any and all combinations of the related listed items, including any two related listed items, any more related listed items, or a combination of all related listed items.

[0041] In this invention, numerical ranges are involved. Unless otherwise specified, the numerical ranges are considered continuous and include the minimum and maximum values ​​of the range, as well as every value between the minimum and maximum values. Furthermore, when the range refers to integers, it includes every integer between the minimum and maximum values ​​of the range. Additionally, when multiple ranges are provided to describe features or characteristics, the ranges may be merged. In other words, unless otherwise specified, all ranges disclosed herein should be understood to include any and all subranges to which they are included.

[0042] Studies have shown that when pests are stimulated by pesticides, their ROS levels increase significantly, especially in resistant pest populations. Excessive ROS does not cause lethal damage to pests; instead, it acts as a signaling molecule to activate the ROS / CncC signaling pathway, thereby inducing the upregulation of the expression of detoxification enzyme genes such as cytochrome P450 and enhancing the activity of detoxification enzymes. This accelerates the metabolic degradation of pesticides, thus reducing the control effect of pesticides.

[0043] To address the problem of pest resistance at its source and improve the sensitivity of pests to pesticides, this invention provides a functional bio-based nanozyme. The functional bio-based nanozyme comprises a porous Prussian blue analogue and a complex attached to the surface of the Prussian blue analogue. The complexue is formed by the complexation of metal ions in the Prussian blue analogue with antioxidant biomass molecules. The antioxidant biomass molecules are selected from at least one of biomass polyphenols, biomass polysaccharides, or amphoteric quaternary ammonium alkaloids.

[0044] The Prussian blue derivative described in this invention forms a highly ordered three-dimensional porous framework structure by bridging metal ions with cyano groups. This structure possesses excellent electron transport capabilities and abundant accessible catalytic active sites, mimicking the activities of various antioxidant enzymes such as SOD and CAT, thereby achieving the catalytic degradation of ROS. Based on this structure, antioxidant functional molecules such as biomass polyphenols, polysaccharides, and alkaloids are introduced, and through complexation and coordination, they couple with the metal active centers in the framework to form a stable biomass functionalized interface layer. The phenolic hydroxyl, alcoholic hydroxyl, and cationic groups in this interface layer not only act as electron donors in redox reactions, promoting efficient electron transfer between biomass components and metal centers, but also accelerate the catalytic cycle, significantly enhancing the material's simulated antioxidant enzyme activity. Simultaneously, a synergistic catalytic system is formed between the biomass functional layer and the metal active centers in the Prussian blue derivative framework. Through a dual mechanism of "the porous framework providing active sites - the biomass interface layer enhancing electron transport and free radical scavenging," efficient ROS removal is achieved.

[0045] Optionally, the porosity of the functional bio-based nanozyme is 35%–70%, for example, any value or range of 42.5%, 48.2%, 55.6%, 62.4%, or 68.7%. Within this porosity range, the material exhibits a moderately open porous structure, which is beneficial for increasing the spatial exposure of active sites, allowing the multi-metal centers inside the Prussian blue analogue to fully participate in the catalytic reaction. Compared to a dense structure with low porosity, a porosity of 35%–70% significantly increases the specific surface area and the number of interfacial active sites, providing more accessible reaction sites for substrates such as ROS. Furthermore, the surface-modified polyphenol layer is not only distributed on the outer surface of the particles but also extends to some of the internal interfaces of the pores, forming an "internal-external synergistic" interface regulation structure. This structure helps to construct continuous electron transport channels, promoting efficient electron migration between the polyphenol layer and the metal active centers, thereby enhancing the redox cycle process.

[0046] Optionally, the particle size of the functional bio-based nanozyme is preferably 50nm~200nm, and can be selected as any value or a range between 50nm, 80nm, 110nm, 140nm, 170nm or 200nm. This control can, on the one hand, increase the specific surface area of ​​the functional bio-based nanozyme and increase the exposure area of ​​the complex, and on the other hand, improve the uptake efficiency of the functional bio-based nanozyme by pests, thereby enhancing the ability of the nanozyme to remove ROS in the pest's body.

[0047] Optionally, in order to construct an effective electron transport channel, promote the redox cycle process, and thereby enhance the antioxidant enzyme activity of the functional bio-based nanozyme, the thickness of the complex on the surface of the Prussian blue analogue is 2 nm to 15 nm, and can be selected as any value among 3 nm, 5 nm, 7 nm, 9 nm, 11 nm, 13 nm or 15 nm or any range between two.

[0048] Optionally, the Prussian blue analogue can be a bimetallic center, a trimetallic center, a tetrametallic center, etc., preferably a bimetallic center. For example, the Prussian blue analogue is at least one of manganese-based Prussian blue (Mn-PB), cobalt-based Prussian blue (Co-PB), magnesium-based Prussian blue (Mg-PB), or molybdenum-based Prussian blue (Mo-PB), preferably Mn-PB. This allows the complex to synergize more effectively with the multivalent Prussian blue analogue, endowing the functional bio-based nanozyme with excellent multi-enzyme catalytic activity.

[0049] Optionally, the antioxidant biomass molecule coordinated with the metal ion can be any one of biomass polyphenols, biomass polysaccharides, or amphoteric quaternary ammonium salt alkaloids, or any combination of two or three. When the antioxidant biomass molecule is biomass polyphenols and biomass polysaccharides, the mass ratio of biomass polyphenols to biomass polysaccharides is 1:5 to 5:1, for example, any ratio of 1:5, 2:4, 3:3, 4:2, or 5:1.

[0050] Optionally, the biomass polyphenols may be selected from at least one of dopamine, tannic acid, quercetin, chlorogenic acid, phytic acid, tea polyphenols, or gallic acid; the biomass polysaccharides may be selected from at least one of fucoidan, chitosan oligosaccharide, chitosan, or sodium alginate; and the amphoteric quaternary ammonium salt alkaloids may be selected from at least one of betaine or berberine.

[0051] The functional bio-based nanozyme provided by this invention has good biocompatibility, can effectively reduce the environmental risks caused by traditional chemical synergists, and reduce potential harm to non-target organisms, thus exhibiting high ecological safety.

[0052] This invention also provides a method for preparing the aforementioned functional bio-based nanozyme, comprising the following steps:

[0053] Ferric cyanide, biomass capping agent, and biomass acid source are mixed and dissolved, then a metal salt is added, followed by a hydrothermal reaction to obtain a Prussian blue analog. Antioxidant biomass molecules, the Prussian blue analog, and a solvent are mixed and subjected to a complexation reaction to obtain a functional bio-based nanozyme. The antioxidant biomass molecules are selected from at least one of biomass polyphenols, biomass polysaccharides, or amphoteric quaternary ammonium alkaloids.

[0054] In this invention, by adding a bio-capping agent, a porous structure can be introduced into the Prussian blue analogue, thereby increasing the porosity of the functional bio-based nanozyme and exposing more metal ion active sites, thus accelerating the removal of ROS. Furthermore, the Prussian blue analogue is a multi-metallic center; the iron ions within it have a high ligand field stabilization energy and are not easily released, while non-ferrous metal ions have unstable coordination structures and are easily released. Therefore, when the Prussian blue analogue reacts with antioxidant biomass molecules, a large number of free non-ferrous metal ions and a small number of free iron ions graft onto the antioxidant biomass molecules to form a complex. This complex not only does not shield the active sites but also constructs a highly efficient electron transport channel, promoting the redox cycle and enhancing the antioxidant enzyme activity of the functional bio-based nanozyme, thus removing ROS more efficiently. Moreover, the synergistic effect of this complex with the metal ions in the Prussian blue analogue framework gives the functional bio-based nanozyme excellent multi-enzyme catalytic activity, thereby achieving highly efficient synergistic removal of endogenous ROS.

[0055] Furthermore, these biomass polyphenols, biomass polysaccharides, biomass acid sources, and biomass end-capping agents are rich in active groups such as hydroxyl groups, which not only possess excellent antioxidant and reducing capabilities but also coordinate with metal ions in Prussian blue analogues, thereby effectively enhancing the dispersibility and stability of functional bio-based nanozymes. In addition, the biomass components endow functional bio-based nanozymes with excellent interfacial adhesion properties, significantly enhancing the wetting, spreading, and deposition efficiency of pesticide solutions on leaf surfaces and insect bodies, reducing pesticide loss, and prolonging the duration of effect, thus overcoming the problems of easy loss and low utilization rate of traditional synergists.

[0056] Furthermore, the preparation method of the present invention uses natural biomass as raw material, and the preparation process is mild with no toxic solvents involved, thus possessing the potential for low cost and large-scale production.

[0057] Optionally, the mass ratio of the biomass acid source to the ferricyanide is preferably 1:0.1 to 1:0.2, and can be selected as any ratio among 1:0.145, 1:0.175, 1:0.185 or 1:0.195. Within this range, a uniform interface layer can be constructed on the surface of the Prussian blue analogue, which significantly improves the exposure of metal ions, thereby facilitating the subsequent formation of complexes with antioxidant biomass molecules.

[0058] Optionally, the mass ratio of the antioxidant biomass molecule to the Prussian blue analog is 5:1 to 1:5, and can be selected as any ratio of 5:1, 4:2, 3:3, 2:4 or 1:5. Within this ratio range, a complex can be formed better on the surface of the Prussian blue analog.

[0059] To provide sufficient non-ferrous metal ions to promote the formation of complexes by grafting onto the surface of Prussian blue analogues and to inhibit the formation of a coating layer by physical encapsulation of antioxidant biomass molecules, the mass ratio of the metal salt to the ferricyanide salt is preferably 1:5 to 1:10, for example, any ratio of 1:5, 1:6, 1:7, 1:8, 1:9 or 1:10; the metal salt may be selected from at least one of manganese salt, cobalt salt, magnesium salt or molybdenum salt, preferably manganese salt, and further preferably manganese chloride.

[0060] Optionally, the biomass acid source in the functional bio-based nanozyme can be selected from at least one of citric acid, fulvic acid, or tannic acid; the biomass capping agent in the functional bio-based nanozyme can be selected from at least one of tea seed cake or rhamnolipid.

[0061] Optionally, the temperature of the hydrothermal reaction is preferably 70℃~90℃, and can be any value of 70℃, 75℃, 80℃, 85℃ or 90℃ or any range between two; the reaction time of the hydrothermal reaction is preferably 10h~24h, and can be any value of 10h, 15h, 20h or 24h or any range between two.

[0062] Optionally, the temperature of the complexation reaction is preferably 20℃~30℃, and can be any value of 20℃, 24℃, 25℃, or 30℃, or any range between two values; the reaction time of the complexation reaction is preferably 5h~12h, and can be any value of 5h, 7h, 9h, 11h, or 12h, or any range between two values. Under the above-mentioned temperature and time synergistic control conditions, the complexation reaction can proceed stably in a mild environment. On the one hand, the low temperature system of 20℃~30℃ can effectively reduce the activation energy of the interfacial complexation reaction, allowing biomass polyphenols, polysaccharides, and alkaloid molecules to undergo ordered interfacial coupling on the surface of manganese-based Prussian blue analogs at a moderate rate, thereby achieving the controllable construction of the functional modification layer. On the other hand, the reaction time of 5h~12h provides a sufficient kinetic window for interfacial assembly, allowing the complex layer to grow uniformly and remain stable within a reasonable thickness range of 2nm~15nm, thereby avoiding incomplete surface coverage due to insufficient reaction, or excessive coating layer thickness and limited mass transfer due to excessive reaction. In addition, mild reaction conditions help to maintain the structural integrity and functional activity of biomass molecules to the greatest extent, especially key antioxidant functional groups such as phenolic hydroxyl and alcoholic hydroxyl groups, which can effectively prevent polyphenols from undergoing oxidative polymerization or structural deactivation, thereby ensuring that the interface modification layer has good chemical stability and biological activity.

[0063] This invention also provides a functional bio-based nanoenzyme for use as an synergist in agricultural chemicals, such as an insecticide synergist or a plant growth regulator. When used as a plant growth regulator, the functional bio-based nanoenzyme can promote plant growth and development, exhibiting positive regulatory effects on plant height, root development, and chlorophyll content.

[0064] The present invention also provides an application of the aforementioned functional bio-based nanoenzyme in the control of reed aphids.

[0065] Optionally, functional bio-based nanozymes can be used in combination with neonicotinoid insecticides, especially when functional bio-based nanozymes are used in combination with imidacloprid to control reed aphids.

[0066] When functional bio-based nanozymes are used in combination with imidacloprid, the functional bio-based nanozymes, after entering the body of the reed aphid, can continuously scavenge ROS induced by insecticide stress, inhibit the activation of the ROS-mediated CncC / P450 signaling pathway from the source, thereby downregulating the expression of toxic enzymes and the activity of related enzymes, and inhibiting the metabolism and decomposition of insecticides by the reed aphid.

[0067] Therefore, the functional bio-based nanozyme of the present invention can actively regulate and effectively delay the resistance development of *Aphidius chinensis* by precisely intervening in the "CncC / P450" resistance pathway, significantly improving the pest's sensitivity to insecticides. It has the advantages of "reduced dosage, strong control effect, and reversible resistance", which helps to achieve pesticide reduction and efficiency improvement and resistance management. Specifically, when the functional bio-based nanozyme is used in combination with imidacloprid, in a *Aphidius chinensis* population with a resistance multiple of 21.09 times, the toxicity index of imidacloprid increases by 1.98 times after adding 200 mg / L of functional bio-based nanozyme.

[0068] The technical solution of the present invention will be further described below through specific embodiments. However, those skilled in the art will understand that the following embodiments are only for illustrating the present invention and should not be regarded as limiting the scope of the present invention. Unless otherwise specified, specific conditions in the embodiments are performed under conventional conditions or conditions recommended by the manufacturer. Reagents or instruments used without specified manufacturers are all commercially available conventional products.

[0069] Example 1

[0070] 1.95 g of potassium ferricyanide and 6 g of tea seed cake were mixed evenly, then 10 g of fulvic acid was added, and the mixture was stirred continuously for 10 min to form a homogeneous solution. Then, 250 mg of manganese source was added, and the mixture was stirred continuously at room temperature until a uniform dispersion was formed. The dispersion was then transferred to an oven and subjected to a hydrothermal reaction at 80 °C for 12 h. After the reaction, porous Mn-PB was obtained through separation and purification.

[0071] 40 mg of dopamine (DA) was dissolved in 20 mL of pure water and sonicated until completely dissolved. 20 mg of the porous Mn-PB prepared above was added, and the mixture was stirred continuously at 25 °C and 700 rpm in the dark for 7 h. The mixture was then centrifuged at 11000 rpm for 8 min, the precipitate was collected, and dried at 60 °C to obtain DA@Mn-PB functional bio-based nanozyme.

[0072] Example 2

[0073] The difference between Example 2 and Example 1 is only that: 60 mg of tannic acid (TA) was dissolved in 20 mL of deionized water, and 20 mg of the porous Mn-PB prepared above was added. The mixture was stirred continuously at 25 °C and 600 rpm in the dark for 7 h, and then centrifuged at 11000 rpm for 8 min. The precipitate was collected and dried at 60 °C to obtain TA@Mn-PB functional bio-based nanozyme.

[0074] Example 3

[0075] The difference between Example 3 and Example 1 is only that: 80 mg of tea polyphenols (TP) was dissolved in 20 mL of deionized water, 20 mg of the porous Mn-PB prepared above was added, and the mixture was stirred continuously in the dark at 25 °C and 600 rpm for 7 h, then centrifuged at 11000 rpm for 8 min, the precipitate was collected and dried at 60 °C to obtain TP@Mn-PB functional bio-based nanozyme.

[0076] Example 4

[0077] The only difference between Example 4 and Example 1 is that 40 mg of gallic acid (GA) was dissolved in 20 mL of deionized water, and 20 mg of the porous Mn-PB prepared above was added. The mixture was stirred continuously at 25 °C and 600 rpm in the dark for 7 h, and then centrifuged at 11000 rpm for 8 min. The precipitate was collected and dried at 60 °C to obtain GA@Mn-PB functional bio-based nanozyme.

[0078] Example 5

[0079] The difference between Example 5 and Example 1 is only that: 40 mg of betaine (BE) was dissolved in 20 mL of deionized water, and 20 mg of the porous Mn-PB prepared above was added. The mixture was stirred continuously in the dark at 25 °C and 600 rpm for 7 h, and then centrifuged at 11000 rpm for 8 min. The precipitate was collected and dried at 60 °C to obtain BE@Mn-PB functional bio-based nanozyme.

[0080] Example 6

[0081] The difference between Example 6 and Example 1 is only that: 40 mg of fucoidan (FU) was dissolved in 20 mL of deionized water, and 20 mg of the porous Mn-PB prepared above was added. The mixture was stirred continuously in the dark at 25 °C and 600 rpm for 7 h, and then centrifuged at 11000 rpm for 8 min. The precipitate was collected and dried at 60 °C to obtain FU@Mn-PB functional bio-based nanozyme.

[0082] Example 7

[0083] The only difference between Example 7 and Example 1 is that 40 mg of dopamine is replaced with 20 mg of dopamine.

[0084] Example 8

[0085] The only difference between Example 8 and Example 1 is that 40 mg of dopamine is replaced with 60 mg of dopamine.

[0086] Example 9

[0087] The only difference between Example 9 and Example 1 is that 40 mg of dopamine is replaced with 80 mg of dopamine.

[0088] Example 10

[0089] The only difference between Example 10 and Example 1 is that 40 mg of dopamine is replaced with 100 mg of dopamine.

[0090] Example 11

[0091] The only difference between Example 11 and Example 1 is that 40 mg of dopamine was replaced with 20 mg of tannic acid and 20 mg of betaine to prepare TA+BE@Mn-PB functional bio-based nanozyme.

[0092] Comparative Example 1

[0093] 1.95g of potassium ferricyanide and 6g of tea seed cake were mixed evenly, and then 10g of humic acid was added. The mixture was stirred continuously at room temperature until a uniform dispersion was formed. The dispersion was then transferred to an oven and subjected to a hydrothermal reaction at 80℃ for 12 hours. After the reaction was completed, Prussian blue (PB) was obtained by separation and purification.

[0094] 40 mg of dopamine (DA) was dissolved in 20 mL of pure water and sonicated until completely dissolved. 20 mg of the PB prepared above was added, and the mixture was stirred continuously at 25 °C and 700 rpm in the dark for 7 h. The mixture was then centrifuged at 11000 rpm for 8 min, the precipitate was collected, and dried at 60 °C to obtain DA@PB functional bio-based nanozyme.

[0095] Comparative Example 2

[0096] 1.95 g of potassium ferricyanide and 6 g of tea seed cake were mixed evenly, then 10 g of nitric acid was added, and the mixture was stirred continuously for 10 min to form a homogeneous solution. Then, 250 mg of manganese source was added, and the mixture was stirred continuously at room temperature until a uniform dispersion was formed. The dispersion was then transferred to an oven and subjected to a hydrothermal reaction at 80 °C for 12 h. After the reaction was completed, Mn-PB was obtained through separation and purification.

[0097] 60 mg of dopamine (DA) was dissolved in 20 mL of pure water and sonicated until completely dissolved. 20 mg of the Mn-PB prepared above was added, and the mixture was stirred continuously at 25 °C and 700 rpm in the dark for 7 h. The mixture was then centrifuged at 11000 rpm for 8 min, the precipitate was collected, and dried at 60 °C to obtain DA@Mn-PB functional bio-based nanozyme.

[0098] Comparative Example 3

[0099] The only difference between Comparative Example 3 and Comparative Example 2 was that 10g of tannic acid was added to 1.95g of potassium ferricyanide, and the mixture was stirred continuously for 10 minutes to form a homogeneous solution. Then, 250mg of manganese source was added, and the mixture was stirred continuously at room temperature until a uniform dispersion was formed. The dispersion was then transferred to an oven and subjected to a hydrothermal reaction at 80℃ for 12 hours. After the reaction was completed, Mn-PB was obtained by separation and purification.

[0100] Comparative Example 4

[0101] The only difference between Comparative Example 4 and Example 1 is that dopamine is replaced with cysteine.

[0102] The functional bio-based nanozymes prepared in Examples 1-5, 7, and 9-10 were characterized by transmission electron microscopy, and the results are as follows: Figure 1 As shown, most embodiments yielded nanoparticles with regular cubic shapes, exhibiting good monodispersity and a particle size distribution ranging from approximately 50 nm to 200 nm. The complex layer on the surface of the nanoparticles can be clearly observed in the high-magnification electron microscope images.

[0103] The particle size and complex thickness of the functional bio-based nanozymes prepared in the above examples and comparative examples were detected using transmission electron microscopy. The porosity of the functional bio-based nanozymes was detected using a fully automated specific surface area and porosity analyzer. The results are shown in Table 1.

[0104] Table 1

[0105]

[0106] The activities of CAT and SOD in the functional bio-based nanozymes prepared in the above examples and comparative examples were determined. The CAT activity was quantitatively determined by colorimetric method using a catalase kit (provided by Suzhou Keming Biotechnology Co., Ltd.), and the SOD activity was determined and calculated by standard micro-method using a matching SOD assay kit (provided by Suzhou Keming Biotechnology Co., Ltd.).

[0107] Table 2

[0108]

[0109] As can be seen from Example 1 and Comparative Example 1, the CAT and SOD activities of the resulting functional bio-based nanozymes were lower after replacing the Prussian blue analog with Prussian blue. This is because the degree of iron ion freeness in Prussian blue is low, and the antioxidant biomass molecules on its surface mainly exist in the form of physical coating rather than forming complexes. In contrast, Example 1 forms a stable complex layer on the surface of the Prussian blue analog. This complex is embedded in the metal center coordination environment reconstruction on the surface of the Prussian blue analog in the form of coordination bonds. This interfacial coordination structure can regulate the electronic structure of the metal center, reduce the activation energy of ROS-related redox reactions, thereby significantly improving the enzyme-like catalytic efficiency and achieving efficient removal of ROS.

[0110] As can be seen from Example 1 and Comparative Examples 2-3, the porosity of the functional bio-based nanozyme has a significant impact on the CAT and SOD activities. As can be seen from Example 1 and Comparative Example 4, when cysteine ​​is used as the antioxidant biomass molecule, the prepared functional bio-based nanozyme exhibits low CAT and SOD activities. This is because cysteine ​​does not contain hydroxyl groups or anions, and therefore cannot form complexes with Prussian blue analogues.

[0111] The following specific application examples, using the resistant reed aphid as the target and combined with the field-recommended insecticide imidacloprid, further verify in detail the toxicity-enhancing effect of the functional bio-based nanoenzyme of the present invention in the control of agricultural pests and diseases, the in vivo resistance target regulation mechanism, and the safety to non-target ecological environments.

[0112] Application Example 1: The toxicity-enhancing effect of nanozymes in actual agricultural pest and disease control

[0113] 1. Experimental Procedure

[0114] 1) Test insects: The imidacloprid-sensitive *Symplocos edulis* strain (SS) used in this invention was provided by the Insect Toxicology Laboratory of China Agricultural University and was raised for more than 5 years without exposure to any insecticides. The imidacloprid-resistant *Symplocos edulis* strain was collected from a wheat field in Tengzhou City, Shandong Province (35.09°N, 117.17°E).

[0115] 2) The wheat aphids are raised using hydroponics: (1) Soaking seeds: Soak wheat seeds in a sterile container with water covering the seeds by about 2cm for 12 hours to improve the germination rate of wheat seeds. (2) Seedling raising: Place a 9cm diameter piece of filter paper on the bottom of a petri dish with holes, spread a layer of wheat seeds on top, and place it in a constant temperature incubator for cultivation (temperature 25±1℃, relative humidity 60%~70%, photoperiod L17:D7). When the seedlings grow to 3cm~5cm, they are used for subsequent insect inoculation. (3) Inoculation and propagation: Place the wheat seedlings of the appropriate period into a breeding cage containing the reed aphids. The cage is 40cm long, 40cm wide, and 40cm high, and the cage is surrounded by 100-mesh nylon mesh. The reed aphids will crawl onto the new seedlings to reproduce. Add 20mL of clean water every 24 hours to maintain the breeding conditions of the reed aphids (temperature 25±1℃, relative humidity 60%~70%, photoperiod L17:D7).

[0116] 3) Bioassay Method - Leaf Immersion Method: First, dissolve the imidacloprid technical grade in 5 mL of acetone to prepare a 20000 mg / L imidacloprid stock solution, then refrigerate and protect from light for later use. Next, dilute the imidacloprid stock solution with 0.05% Triton X-100 aqueous solution to five different concentration gradients of imidacloprid solutions. Take 4-day-old hydroponic wheat seedlings (whole seedlings, approximately 5 cm in length from seed base to leaf tip) and immerse them in different concentrations of imidacloprid solutions for 15 seconds. After removal, allow them to air dry on a clean surface. Place the seedlings in petri dishes containing moistened filter paper, with 5 seedlings per dish. Set up 3 biological replicates for each concentration gradient. Wheat seedlings immersed in 0.05% Triton X-100 aqueous solution served as a blank control group. Take 15 healthy and vigorous wingless adult aphids and place them in each petri dish. Cover the petri dish and fix it with sealing film. Then place it in the insect rearing cage. After 24 hours, check and calculate the mortality rate of the test insects in each petri dish. Gently stimulate the test insects with a fine brush. If only one leg of the aphid moves or the whole insect does not react at all, it is judged as dead.

[0117] 2. Resistance test of imidacloprid to the reed aphid

[0118] The toxicity of imidacloprid to sensitive and resistant strains of *Aphidius simonii* was determined using the leaf-dip method. Imidacloprid technical was prepared in acetone. For the assay, the stock solution was diluted to a series of concentration gradients using an aqueous solution containing 0.05% Triton X-100. The concentrations for imidacloprid-sensitive *Aphidius simonii* strains were 0 mg / L, 2.5 mg / L, 5 mg / L, 10 mg / L, 25 mg / L, 50 mg / L, and 100 mg / L; the concentrations for imidacloprid-resistant *Aphidius simonii* strains were 0 mg / L, 10 mg / L, 50 mg / L, 100 mg / L, 200 mg / L, 400 mg / L, 800 mg / L, and 1600 mg / L. Mortality was assessed 24 hours after treatment, and corrected mortality was calculated. The results are shown below. Figure 2 As shown in the figure, a and b represent the insecticidal ability of different imidacloprid concentrations on imidacloprid-sensitive and imidacloprid-resistant *Aphidius chinensis* strains, respectively. It can be seen that the insecticidal effect on *Aphidius chinensis* is better as the imidacloprid concentration increases.

[0119] The concentration of imidacloprid (LC50) at which the mortality rate of the reed aphid reaches 50% was calculated using SPSS statistical software. 50 ), 95% confidence interval and toxicity regression equation, wherein the formula for calculating the resistance multiple is: The results are shown in Table 3.

[0120] Table 3

[0121]

[0122] As shown in Table 3, compared with the imidacloprid-sensitive *Aphidius chinensis* strain, the imidacloprid-resistant *Aphidius chinensis* strain showed a significant decrease in sensitivity to imidacloprid. Calculations using the formula showed a resistance multiple of 21.09, indicating that this imidacloprid-resistant *Aphidius chinensis* strain has reached a moderate level of resistance.

[0123] 3. Determination of the combined toxicity of functional bio-based nanozymes and imidacloprid on *Aphidius chinensis*

[0124] The toxicity of imidacloprid was determined by leaf dipping method and using the DA@Mn-PB functional bio-based nanozyme prepared in Example 1 as an insecticidal synergist: immobilized imidacloprid on LC 30 The concentrations were combined with five concentrations of DA@Mn-PB functional bio-based nanozymes (50 mg / L, 100 mg / L, 200 mg / L, 400 mg / L, and 800 mg / L) to form mixed treatment groups, and a single imidacloprid LC-LC-T was also set up. 30Concentration treatments served as controls, with each treatment having three replicates. Mortality rates of the *Gnaphalium affine* were assessed 24 hours after treatment. Duncan's new multiple range test (labeled with significance letters such as a, b, ab, etc.) was used to analyze the statistical differences between the treatment groups and the blank control group (0 mg / LDA@Mn-PB functional bio-based nanozyme). Results are as follows: Figure 3 As can be seen from 'a' in the figure, compared with the use of imidacloprid alone, when the concentration of DA@Mn-PB functional bio-based nanozyme is in the range of 50 mg / L to 800 mg / L, the mortality rate of the reed aphid increases by 1.06 times to 2.19 times.

[0125] Subsequently, toxicity was determined using the leaf-dip method: Imidacloprid stock solution was diluted with 0.1% Triton X-100 aqueous solution to create a series of concentration gradients of 50 mg / L, 100 mg / L, 200 mg / L, 400 mg / L, 800 mg / L, and 1600 mg / L, serving as the single-agent imidacloprid treatment group (Imi). This series of imidacloprid solutions was then combined with 200 mg / L DA@Mn-PB functional bio-based nanozymes to form the combined synergistic treatment group (Imi+DA@Mn-PB). A distilled aqueous solution containing 0.1% Triton X-100 served as a blank control. The above treatment solutions were used to treat imidacloprid-resistant *Reticulitermes rubrum* strains to further verify the synergistic effect of the functional bio-based nanozymes. Figure 3 As can be seen from b, the combination of DA@Mn-PB functional bio-based nanozyme and imidacloprid significantly enhances the toxicity of imidacloprid to the imidacloprid-resistant reed aphid strain.

[0126] The concentration of imidacloprid (LC50) at which the mortality rate of the reed aphid reaches 50% was calculated using SPSS statistical software. 50 The 95% confidence interval and toxicity regression equation are given, where the synergistic effect ratio is calculated using the following formula: The results are shown in Table 4.

[0127] Table 4

[0128]

[0129] As shown in Table 4, at the optimal application concentration of 200 mg / L, the DA@Mn-PB functional bio-based nanoenzyme provided by this invention, when used as an insecticide synergist in combination with an insecticide, significantly increased the toxicity index against moderately resistant *Aphidius simonii* by 1.98 times. Therefore, the functional bio-based nanoenzyme of this invention can overcome the detoxification and metabolic defense of resistant *Aphidius simonii*, greatly reducing the lethal concentration of the insecticide, and achieving the technical effects of "reduced dosage and increased efficiency" and "targeted management of resistance" in pesticides.

[0130] Application Example 2: Validation of the mechanism by which nanozymes regulate the activity of antioxidant stress kinases in target pests

[0131] Imidacloprid-resistant *Aphidius oryzae* strains were selected, and four treatment groups were set up in parallel: (1) Control group: treated with sterile water containing an equal amount of solvent; (2) Imidacloprid single-agent treatment group: treated with LC-4. 30 (3) Single-agent functional bio-based nanozyme treatment group (DA@Mn-PB): treated with only 200 mg / L of the DA@Mn-PB functional bio-based nanozyme dispersion prepared in Example 1 of the present invention; (4) Combined synergistic treatment group (Imi+DA@Mn-PB), with simultaneous application of LC 30 A concentration of imidacloprid and 200 mg / L of DA@Mn-PB functional bio-based nanozyme prepared in Example 1 of this invention were used. After treating imidacloprid-resistant *Gnaphalium affine* with the pesticide, surviving *Gnaphalium affine* aphids from each group were collected, and total protein was extracted from the insect tissues using liquid nitrogen flash freezing and grinding. The specific activities of SOD, CAT, and POD in *Gnaphalium affine* aphids from each treatment group were determined and recorded using a high-sensitivity biochemical reagent kit. The results are as follows: Figure 4 As shown, compared with the control group, the introduction of functional bio-based nanozymes significantly increased the activities of three antioxidant enzymes in the imidacloprid-resistant *Aphidius chinensis*, indicating a significant regulation of its redox homeostasis. Notably, this increased enzyme activity was not solely due to the pest's own stress response, but primarily attributed to the exogenous catalytic effect of the introduced nanozymes. These nanozymes possess highly efficient ROS scavenging capabilities, effectively reducing intracellular oxidative stress levels. With the decrease in ROS levels, the expression of detoxification-related genes such as P450 is inhibited, leading to a reduction in detoxification enzyme activity. This weakens the detoxification ability of the resistant pest, increasing its sensitivity to imidacloprid, thus achieving a significant synergistic effect between the nanozymes and the insecticide.

[0132] Application Example 3: Evaluation of the safety of non-target organisms and the growth-promoting effect on target crops

[0133] 1. Non-toxicity test against target pests

[0134] Two parallel treatment groups were set up: (1) Control group: treated with sterile water containing an equal amount of solvent; (2) Single-agent functional bio-based nanozyme treatment group (DA@Mn-PB): treated with 200 mg / L DA@Mn-PB functional bio-based nanozyme dispersion prepared in Example 1 of this invention. Healthy and consistent imidacloprid-resistant *Aphidius oryzae* strains were selected, and the above two treatment group dispersions were applied by slide immersion method. The survival status of *Aphidius oryzae* was observed and recorded, and the process was repeated three times. The results are as follows: Figure 5As shown, even when functional bio-based nanozymes are applied alone at a concentration as high as 200 mg / L, the survival rate of the imidacloprid-resistant *Aphidius chinensis* strain remains at 100%. This indicates that the functional bio-based nanozymes themselves do not possess contact toxicity or insecticidal activity; their effect of reducing pesticide dosage and enhancing efficacy stems from the regulatory mechanism of the ROS signaling pathway within the pest, rather than a simple additive effect of toxicity.

[0135] 2. Acute toxicity test of non-target environmental organisms (earthworms)

[0136] Healthy adult Eisenia fetidae earthworms were selected, and experiments were conducted using the OECD standard artificial soil method: parallel treatment groups with different concentration gradients of DA@Mn-PB functional bio-based nanoenzymes were set up (0 mg / kg, 25 mg / kg, 50 mg / kg, 100 mg / kg, 200 mg / kg, 400 mg / kg dry weight of potted soil), with each concentration replicated three times. The results are as follows: Figure 6 As shown in the figure, A and B represent the survival rates of earthworms in each group on the 7th and 14th days after application of the drug, respectively. It can be seen that the survival rate of earthworms in all treatment groups is relatively high. Although the earthworm survival rate decreased slightly after 14 days of treatment with high concentration (400 mg / kg), statistical analysis showed that there was no significant difference compared with the control group, proving that the functional bio-based nanozyme is highly safe for soil environmental indicator organisms.

[0137] 3. Safety and growth-promoting effects of target crops (wheat)

[0138] A pot experiment was conducted using wheat seeds. Parallel treatment groups with different concentration gradients of DA@Mn-PB functional bio-based nanozymes (0 mg / L, 25 mg / L, 50 mg / L, 100 mg / L, 200 mg / L, and 400 mg / L aqueous solutions) were established, and wheat seedlings were treated using a combination of root irrigation and foliar spraying. Seven days after treatment, plant height, root length, and aboveground fresh weight of wheat in each group were measured, and the relative chlorophyll content of leaves was determined using a SPAD meter. The statistical differences between the treatment groups and the blank control group (0 mg / L) were analyzed using Duncan's new multiple range method. Figure 7 The wheat growth chart shows the growth of wheat seedlings 7 days after treatment. It can be seen that the higher the concentration of functional bio-based nanozymes added, the better the growth of wheat seedlings. Figure 8A, B, C, and D in the figure represent the changes in wheat plant height, root length, chlorophyll SPAD value, and fresh weight, respectively. It can be seen that plant height, root length, chlorophyll SPAD value, and fresh weight all show an increasing trend with the increase of functional bio-based nanozyme concentration. Furthermore, under the high concentration treatment of 400 mg / L, the above four indicators are significantly higher than those of the blank control group, proving that the functional bio-based nanozyme of the present invention can significantly promote wheat growth and increase chlorophyll level at high concentrations, and can be used as a plant growth regulator.

[0139] The technical features of the above embodiments can be combined in any way. For the sake of brevity, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0140] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the protection scope of the present invention. Therefore, the protection scope of this invention patent should be determined by the appended claims.

Claims

1. A functional bio-based nanozyme, characterized in that, The functional bio-based nanozyme includes a porous Prussian blue analogue and a complex attached to the surface of the Prussian blue analogue. The complexue is formed by the complexation of metal ions in the Prussian blue analogue with antioxidant biomass molecules. The antioxidant biomass molecules are selected from at least one of biomass polyphenols, biomass polysaccharides, or amphoteric quaternary ammonium alkaloids.

2. The functional bio-based nanozyme according to claim 1, characterized in that, The porosity of the functional bio-based nanozyme is 35%~70%; And / or, the particle size of the functional bio-based nanozyme is 50nm~200nm; And / or, the thickness of the complex on the surface of the Prussian blue analogue is 2 nm to 15 nm; And / or, when the antioxidant biomass molecule is biomass polyphenol and biomass polysaccharide, the mass ratio of the biomass polyphenol to the biomass polysaccharide is 1:5 to 5:

1.

3. The functional bio-based nanozyme according to claim 1 or 2, characterized in that, The biomass polyphenols are selected from at least one of dopamine, tannic acid, tea polyphenols, quercetin, chlorogenic acid, phytic acid, or gallic acid; And / or, the biomass polysaccharide is selected from at least one of fucoidan, chitosan oligosaccharide, chitosan or sodium alginate; And / or, the amphoteric quaternary ammonium alkaloid is selected from at least one of betaine or berberine.

4. The functional bio-based nanozyme according to claim 1, characterized in that, The Prussian blue analogue is selected from at least one of manganese-based Prussian blue, cobalt-based Prussian blue, magnesium-based Prussian blue, or molybdenum-based Prussian blue.

5. A method for preparing a functional bio-based nanozyme as described in any one of claims 1 to 4, characterized in that, Includes the following steps: Ferric cyanide, biomass end-capping agent and biomass acid source are mixed and dissolved, then metal salt is added, and then a hydrothermal reaction is carried out to obtain Prussian blue analogues. Antioxidant biomass molecules, the Prussian blue analogue, and a solvent are mixed and subjected to a complexation reaction to obtain a functional bio-based nanozyme. The antioxidant biomass molecule is selected from at least one of biomass polyphenols, biomass polysaccharides, or amphoteric quaternary ammonium alkaloids.

6. The method for preparing the functional bio-based nanozyme according to claim 5, characterized in that, The mass ratio of the biomass acid source to the ferricyanide is 1:0.1 to 1:0.2; And / or, the mass ratio of the antioxidant biomass molecule to the Prussian blue analogue is 5:1 to 1:5; And / or, the mass ratio of the metal salt to the ferricyanide is 1:5 to 1:10; And / or, the metal salt is selected from at least one of manganese salt, cobalt salt, magnesium salt or molybdenum salt; And / or, the biomass acid source in the functional bio-based nanoenzyme is selected from at least one of citric acid, fulvic acid or tannic acid; And / or, the biomass capping agent in the functional bio-based nanozyme is selected from at least one of tea seed cake or rhamnolipid.

7. The method for preparing the functional bio-based nanozyme according to claim 5 or 6, characterized in that, The hydrothermal reaction temperature is 70℃~90℃, and the reaction time is 10h~24h; And / or, the temperature of the complexation reaction is 20℃~30℃, and the reaction time is 5h~12h.

8. A functional bio-based nanozyme as described in any one of claims 1 to 4, used as an synergist for agrochemicals.

9. The application of a functional bio-based nanozyme as described in any one of claims 1 to 4 in the control of aphids.

10. The application of the functional bio-based nanozyme according to claim 9 in the control of aphids, characterized in that, The functional bio-based nanozyme is used in combination with neonicotinoid insecticides.

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