Preparation method of Fe-CDs nanozyme, Fe-CDs nanozyme and its application in improving plant resistance to stress

Abstract

The present invention relates to a preparation method of Fe-CDs nanozyme, comprising the following steps: S1: mixing: mixing anhydrous citric acid, ferric chloride hexahydrate, o-phenylenediamine and a small molecule mixture with deionized water to obtain a mixture; S2: stirring and dissolving: magnetic stirring until completely dissolved to obtain a dissolved mixture solution; S3: hydrothermal reaction: transferring the dissolved mixture solution to a Teflon-lined stainless steel hydrothermal kettle for hydrothermal reaction to obtain a reacted solution; S4: post-treatment: filtering, centrifuging and dialyzing the reacted solution to obtain a negatively charged Fe-CDs nanozyme with multi-enzyme activity. Fe-CDs nanozyme has high-intensity fluorescence and high enzyme activity, which can enhance the antioxidant properties of the material itself. At the same time, a method for applying Fe-CDs nanozyme to improve plant stress resistance is proposed. Based on the multi-enzyme activity of Fe-CDs nanozyme, crops are helped to alleviate the oxidative stress caused by abiotic stress, activate the body's antioxidant defense system, and thus improve the stress resistance of crops.

Description

Technical Field

[0001] The present invention relates to the technical field of nanomaterials, and in particular to a preparation method of Fe-CDs nanozyme, Fe-CDs nanozyme and application thereof in improving plant resistance to stress. Background Art

[0002] In recent years, the rapid development of industry and agriculture in my country has been accompanied by increasing emissions of industrial wastewater, waste residue, and exhaust gas, sewage irrigation, and the application of chemical fertilizers and pesticides. This has led to varying degrees of heavy metal contamination in soils across the country. Excessive accumulation of trace metals in plants can disrupt various physiological processes, including oxidative stress and genotoxicity, adversely affecting plant growth and development. Due to increased human activity, trace metal soil contamination is a growing problem worldwide. Trace metal contamination can cause plant toxicity and growth inhibition, as well as accumulation in edible parts, reaching or exceeding levels that threaten food safety and human health. As a staple food, wheat-derived products are a major source of cadmium exposure in humans, potentially leading to renal dysfunction, cancer, and hypertension. The development of plant-based nanomodulators with resistance to heavy metal stress is crucial for improving plant growth and food safety in contaminated soils.

[0003] The application of nanotechnology in plant science holds enormous potential. Through the application of nanomaterials, nanobiosensors, nanogene delivery systems, and drug delivery systems, it can effectively improve plant growth, increase yields, and enhance disease resistance and tolerance to adverse conditions. These innovative applications are of great significance in the agricultural sector and will contribute to the realization of sustainable agricultural development.

[0004] Nanozymes are a class of artificially synthesized catalysts with enzyme properties. They possess advantages such as high catalytic activity, ease of preparation, low cost, good biocompatibility, and stable properties, making them extremely versatile. Iron plays a crucial role in many physiological processes in plants, including the biosynthesis of photosynthetic pigments, photosynthesis, and respiration. However, research on the application of iron-based nanozymes in plants is relatively rare, and further research on their application in nano-agrochemicals warrants exploration. Summary of the Invention

[0005] In order to solve the above-mentioned problems of the prior art, the present invention provides a method for preparing Fe-CDs (iron-doped carbon dots) nanozymes. The prepared Fe-CDs nanozymes have multiple enzyme activities, including peroxidase activity (POD), superoxide dismutase (SOD), and catalase (CAT), so that the material has high-intensity fluorescence and high enzyme activity at the same time, achieving the effect of improving the antioxidant properties of the material itself. At the same time, an application of Fe-CDs nanozymes in improving plant resistance to stress is proposed, that is, based on the multi-enzyme activity of Fe-CDs nanozymes, it helps crops alleviate the oxidative stress caused by abiotic stress, activates the body's antioxidant defense system, thereby improving the stress resistance of crops, and also accelerating the growth rate of plants.

[0006] In order to achieve the above objectives, the main technical solutions adopted by the present invention include:

[0007] According to the first aspect of the present invention: a method for preparing Fe-CDs nanozyme, comprising the following steps:

[0008] S1: mixing: mixing anhydrous citric acid, ferric chloride hexahydrate, o-phenylenediamine and the small molecule mixture with deionized water to obtain a mixture;

[0009] S2: stirring and dissolving: stirring magnetically until completely dissolved to obtain a dissolved mixture solution;

[0010] S3: hydrothermal reaction: transferring the dissolved mixture solution to a Teflon-lined stainless steel hydrothermal reactor for hydrothermal reaction to obtain a reacted solution;

[0011] S4: Post-treatment: The reaction solution is filtered, centrifuged, and dialyzed to obtain negatively charged Fe-CDs nanozymes with multi-enzyme activity;

[0012] in:

[0013] The molar ratio of anhydrous citric acid, ferric chloride hexahydrate and o-phenylenediamine is 1:1:2;

[0014] The mass ratio of anhydrous citric acid to the small molecule mixture is 320:1;

[0015] The small molecule mixture is cysteine ​​and resveratrol, and the mass ratio of cysteine ​​to resveratrol is 1:1.

[0016] Preferably, in step S3:

[0017] The hydrothermal reaction temperature is 180°C and the reaction time is 10-12h;

[0018] The heating rate and cooling rate are both 180℃ / h.

[0019] Preferably, in step S4:

[0020] In the filtration process, the filter membrane is a water filter membrane with a pore size of 0.22um and a diameter of 50mm;

[0021] During the centrifugation process, the centrifugal speed is 8000-10000 rpm / min, the centrifugal time is 5-6 minutes, and the number of centrifugation times is 3-5 times.

[0022] Preferably, in step S4:

[0023] During the dialysis treatment, the dialysis bag specification is 1000Da, and the dialyzed solution is freeze-dried to a powder to obtain negatively charged Fe-CDs nanozymes with multi-enzyme activity.

[0024] Preferably, S1: mixing: mixing 5 mmol of anhydrous citric acid, 5 mmol of ferric chloride hexahydrate, 10 mmol of o-phenylenediamine and 3 mg of the small molecule mixture with 40 mL of deionized water to obtain a mixture;

[0025] S2: stirring and dissolving: stirring magnetically until completely dissolved to obtain a dissolved mixture solution;

[0026] S3: Hydrothermal reaction: The dissolved mixture solution was transferred to a 100 ml Teflon-lined stainless steel hydrothermal autoclave and reacted at 180°C for 10 hours. After the reaction was completed, the autoclave was cooled to room temperature to obtain a reacted solution;

[0027] S4: Post-treatment: The reaction solution was filtered using a water filter membrane with a pore size of 0.22um and a diameter of 50mm, and then centrifuged at a centrifugal speed of 10000rpm / min for 5min, repeated three times, and finally, dialyzed using a dialysis bag with a specification of 1000Da and freeze-dried to a powder to obtain negatively charged Fe-CDs nanozyme with multi-enzyme activity.

[0028] Preferably, the Fe-CDs have a spherical structure, and the particle size d of the Fe-CDs is ≤3 nm.

[0029] According to the second aspect of the present invention, a Fe-CDs nanozyme is used to improve plant resistance to stress. The Fe-CDs nanozyme prepared above is dispersed in water to obtain a Fe-CDs nanozyme dispersion, and the Fe-CDs nanozyme dispersion is applied to the roots of plants.

[0030] Preferably, the concentration of the Fe-CDs nanozyme dispersion is 5-10 mg / mL.

[0031] Preferably, the plant is the monocotyledonous plant wheat.

[0032] Preferably, the stress-resistant species is resistant to heavy metal cadmium.

[0033] The beneficial effects of the present invention are:

[0034] (1) The preparation method of the Fe-CDs nanozyme of the present invention does not require complex instruments and equipment and is low in cost. The prepared Fe-CDs nanozyme is highly stable, negatively charged, and has multiple enzyme activities, including peroxidase activity (POD), superoxide dismutase (SOD), and catalase (CAT). The material has both high-intensity fluorescence and high enzyme activity, thereby achieving the effect of improving the material's own antioxidant properties.

[0035] (2) Fe-CDs nanozymes were prepared into water-soluble nanoregulators. Under cadmium stress, the applied Fe-CDs nanozymes were able to interact with wheat, making the wheat show better cadmium tolerance, and could significantly promote the elongation of wheat roots and accelerate its growth rate.

[0036] (3) The application of a lower concentration of Fe-CDs nanozyme can achieve a significant effect in promoting wheat root elongation, which can minimize soil pollution and is environmentally friendly. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 This is the XRD spectrum of Fe-CDs nanozyme.

[0038] Figure 2 Transmission electron microscopy (TEM) image of Fe-CDs.

[0039] Figure 3 Zeta potential diagram of Fe-CDs.

[0040] Figure 4 is the fluorescence spectrum of Fe-CDs: Ex is the optimal fluorescence excitation spectrum, Em is the optimal fluorescence emission spectrum, (A) is a photo of the Fe-CDs nanozyme dispersion taken under natural light, and (B) is a photo of the Fe-CDs nanozyme dispersion taken under ultraviolet light.

[0041] Figure 5 These are the multi-enzyme activity test diagrams of Fe-CDs: (A) is the peroxidase activity test diagram of Fe-CDs; (B) is the superoxide dismutase activity test diagram of Fe-CDs.

[0042] Figure 6 The promoting effect of applying different concentrations of Fe-CDs on wheat growth.

[0043] Figure 7Figure 3. Effect of Fe-CDs on alleviating cadmium stress in wheat. (A) is the wheat root system diagram, (B) is the wheat plant diagram, (C) is the root length statistical comparison diagram, (D) is the aboveground fresh weight statistical comparison diagram, (E) is the aboveground dry weight statistical comparison diagram, (F) is the underground fresh weight statistical comparison diagram, and (G) is the underground dry weight statistical comparison diagram.

[0044] Figure 8 Statistical comparison chart of the reduction of cadmium content in wheat by the application of Fe-CDs, where: (A) is the statistical comparison chart of cadmium content in the aboveground part and (B) is the statistical comparison chart of cadmium content in the underground part.

[0045] Figure 9 The figures show the effect of Fe-CDs on promoting plant photosynthesis, including: (A) a statistical comparison diagram of chlorophyll a changes, (B) a statistical comparison diagram of chlorophyll b changes, (C) a statistical comparison diagram of carotenoid changes, (D) a statistical comparison diagram of total chlorophyll changes, (E) a statistical comparison diagram of net photosynthetic rate changes, (F) a statistical comparison diagram of transpiration rate changes, (G) a statistical comparison diagram of stomatal conductance changes, and (H) a statistical comparison diagram of intercellular CO2 concentration changes. DETAILED DESCRIPTION

[0046] In order to better explain the present invention and facilitate understanding, the present invention is described in detail below through specific implementation methods in conjunction with the accompanying drawings.

[0047] The technical solution of the present invention is summarized as follows: The present invention provides a method for preparing Fe-CDs (iron-doped carbon dots) nanozymes. The prepared Fe-CDs nanozymes have multiple enzyme activities, including peroxidase (POD), superoxide dismutase (SOD), and catalase (CAT), so that the material has high-intensity fluorescence and high enzyme activity, achieving the effect of improving the material's own antioxidant properties. At the same time, a method for using Fe-CDs nanozymes in improving plant resistance to stress is proposed. That is, based on the multi-enzyme activity of Fe-CDs nanozymes, it helps crops alleviate oxidative stress caused by abiotic stress, activates the body's antioxidant defense system, thereby improving the stress resistance of crops and accelerating plant growth.

[0048] In order to illustrate the solution and technical advancement of the present invention, the technical solution and technical application are as follows:

[0049] A method for preparing Fe-CDs nanozyme, comprising the following steps:

[0050] S1: mixing: mixing anhydrous citric acid, ferric chloride hexahydrate, o-phenylenediamine and the small molecule mixture with deionized water to obtain a mixture;

[0051] S2: stirring and dissolving: stirring magnetically until completely dissolved to obtain a dissolved mixture solution;

[0052] S3: hydrothermal reaction: transferring the dissolved mixture solution to a Teflon-lined stainless steel hydrothermal reactor for hydrothermal reaction to obtain a reacted solution;

[0053] S4: Post-treatment: The reaction solution is filtered, centrifuged, and dialyzed to obtain negatively charged Fe-CDs nanozymes with multi-enzyme activity;

[0054] in:

[0055] The molar ratio of anhydrous citric acid, ferric chloride hexahydrate and o-phenylenediamine is 1:1:2;

[0056] The mass ratio of anhydrous citric acid to the small molecule mixture is 320:1;

[0057] The small molecule mixture is cysteine ​​and resveratrol, and the mass ratio of cysteine ​​to resveratrol is 1:1.

[0058] In step S3: the hydrothermal reaction temperature is 180°C and the reaction time is 10-12 hours;

[0059] The heating rate and cooling rate are both 180℃ / h.

[0060] In step S4: during the filtration process, the filter membrane is a water filter membrane with a pore size of 0.22 μm and a diameter of 50 mm;

[0061] During the centrifugation process, the centrifugal speed is 8000-10000 rpm / min, the centrifugal time is 5-6 minutes, and the number of centrifugation times is 3-5 times.

[0062] In step S4: during the dialysis treatment, the dialysis bag specification is 1000Da, and the dialyzed solution is freeze-dried to a powder to obtain negatively charged Fe-CDs nanozymes with multi-enzyme activity.

[0063] A more preferred solution is: S1: mixing: 5 mmol of anhydrous citric acid, 5 mmol of ferric chloride hexahydrate, 10 mmol of o-phenylenediamine, and 3 mg of the small molecule mixture are mixed with 40 mL of deionized water to obtain a mixture;

[0064] S2: stirring and dissolving: stirring magnetically until completely dissolved to obtain a dissolved mixture solution;

[0065] S3: Hydrothermal reaction: The dissolved mixture solution was transferred to a 100 ml Teflon-lined stainless steel hydrothermal autoclave and reacted at 180°C for 10 hours. After the reaction was completed, the autoclave was cooled to room temperature to obtain a reacted solution;

[0066] S4: Post-treatment: The reaction solution was filtered through a 0.22 μm pore size, 50 mm diameter water filter membrane. The solution was then centrifuged at 10,000 rpm for 5 minutes, repeated three times. Finally, the solution was dialyzed through a 1,000 Da dialysis bag and freeze-dried to a powder, yielding negatively charged Fe-CD nanozymes with multienzyme activity. The Fe-CDs were spherical in structure, with a particle size d ≤ 3 nm.

[0067] A Fe-CDs nanozyme is used to enhance plant stress resistance. The Fe-CDs nanozyme prepared above is dispersed in water to obtain a Fe-CDs nanozyme dispersion, which is then applied to plant roots. The concentration of the Fe-CDs nanozyme dispersion is 5-10 mg / mL. The plant is a monocotyledonous wheat plant. The stress resistance is resistant to the heavy metal cadmium.

[0068] It should be noted that in the design of the preparation method and the selection of materials used in this invention, ① the enzymatic activity of unmodified or undoped CDs is generally low, making them difficult to use directly. By utilizing methods such as surface modification and passivation, as well as element doping, the optical and physical properties of carbon dots can be modified to meet experimental requirements. Therefore, in this invention, iron is incorporated into CDs to dope the carbon dots, producing highly active nanozymes with peroxidase- and superoxide dismutase-like activities and other desirable physicochemical properties. ② Citric acid has excellent heavy metal ion chelating properties and can also serve as an organic acid in plants, providing a carbon source and regulating their physiological metabolism. Furthermore, citric acid is biodegradable and environmentally friendly. Therefore, citric acid was selected as the carbon source for the synthesis of carbon dot materials. ③ In this invention, a small molecule modification method was specifically employed to functionally modify the Fe-CDs nanozyme. By introducing cysteine ​​and resveratrol, the multi-enzyme activity of the material was enhanced, resulting in the material exhibiting both high-intensity fluorescence and high enzymatic activity, thereby enhancing its inherent antioxidant properties. ④ By designing reaction conditions such as reaction temperature and reaction time, the best-performing Fe-CDs nanozyme is obtained. ⑤ The Fe-CDs nanozyme of the present invention is not limited to being used in wheat to alleviate cadmium stress, but can also be used to alleviate stress induced by other heavy metals or non-metals on plants and to alleviate the aforementioned stresses in other plant species.

[0069] The Fe-CDs nanozyme obtained by the preparation method of the present invention has the following characteristics or functions: ① It has multiple enzyme activities, such as peroxidase activity (POD), superoxide dismutase (SOD), and catalase (CAT), which can help crops alleviate the oxidative stress caused by abiotic stress and activate the body's antioxidant defense system, thereby improving the stress resistance of crops; ② It has a small particle size and can enter plant cells to act, and can be easily transported to various parts of the plant body, which is beneficial to promoting the growth and development of plants; ③ It has good solubility. After the material is fully dissolved in water, it can be left standing for 24 hours without sedimentation or agglomeration; ④ It has high stability. The aqueous solution of the material can be placed for a long time without deterioration; ⑤ It has good biocompatibility. After the material enters the plant, it has no toxicity to the plant and can promote the growth of the plant; ⑥ It has low cost. The reagents used in the preparation process are cheap and easily available, and the preparation conditions are relatively simple; ⑦ The effective application amount is low and there is no pollution to the environment and soil.

[0070] Example:

[0071] (1) Preparation example of Fe-CDs nanozyme

[0072] S1: Mixed

[0073] 5mmol anhydrous citric acid (C6H8O7), 5mmol ferric chloride hexahydrate (FeCl3·6H2O), 10mmol o-phenylenediamine (C6H8N2) and 3mg small molecule mixture (by mass, cysteine ​​C3H7NO2S: resveratrol C 14 H 12 O3 = 1:1) was mixed with 40 mL of deionized water to obtain a mixture;

[0074] S2: stirring and dissolving: stirring magnetically until completely dissolved to obtain a dissolved mixture solution;

[0075] S3: Hydrothermal reaction: Transfer the dissolved mixture to a 100ml Teflon-lined stainless steel hydrothermal autoclave and react at 180°C for 10-12 hours. After the reaction is complete, cool the autoclave to room temperature to obtain a reacted solution. The heating and cooling rates are both 180°C / h.

[0076] S4: Post-treatment: The reaction solution was filtered through a 0.22 μm pore size, 50 mm diameter water filter membrane. The solution was then centrifuged at 10,000 rpm for 5 minutes, repeated three times. Finally, the solution was dialyzed through a 1,000 Da dialysis bag and freeze-dried to a powder, yielding negatively charged Fe-CD nanozymes with multienzyme activity. The Fe-CDs were spherical in structure, with a particle size d ≤ 3 nm.

[0077] like Figures 1 to 4As shown, the Fe-CDs nanozyme was prepared using the above 4-step method, wherein: Figure 1 The diffraction peak corresponding to position C (carbon) appears in the Figure 2 It can be seen that Fe-CDs are spherical structures with a particle size of d≤3nm. Figure 3 It can be seen that Fe-CDs are negatively charged. Figure 4 It can be seen that this nanozyme has a strong emission peak near 440nm ( Figure 4 Em fluorescence best emission spectrum), and has blue fluorescence ( Figure 4 B) Demonstrates the successful preparation of Fe-CDs nanozymes in the present invention.

[0078] The Fe-CDs nanozyme prepared by the present invention has multi-enzyme activity. Figure 5 From the multi-enzyme activity test diagram of Fe-CDs shown in the figure, Figure 5 A is the test diagram of Fe-CDs having peroxidase activity, Figure 5 B is a test diagram of the superoxide dismutase activity of Fe-CDs. Figure 5 In A, TMB is 3,3',5,5'-tetramethylbenzidine, which is used as a colorimetric agent for peroxidase activity determination. TMB+H2O2 is hydrogen peroxide + colorimetric agent (substrate), TMB+H2O2+CDs is substrate + hydrogen peroxide + undoped iron material, and TMB+H2O2+Fe-CDs is substrate + hydrogen peroxide + doped iron material. Figure 5 As shown in Figure A, the TMB + H₂O₂ + Fe-CDs curve exhibits the largest absorbance peak compared to the other curves. It is well known that nanomaterials with peroxidase activity can catalyze the oxidation of H₂O₂, and the resulting color product is blue. The darker the color, the higher the enzyme activity and the higher the absorbance. Therefore, Fe-CDs demonstrate peroxidase activity (POD). Figure 5 In B, the reaction substrates used were riboflavin, disodium EDTA, methionine, and nitroblue tetrazolium, and the designed comparative experimental conditions were: light control, dark control, CDs, and Fe-CDs. Figure 5As shown in Figure B, the absorbance of the curves obtained with Fe-CDs is lower than that obtained with the light control and CDs. It is well known that the chromogenic agent nitroblue tetrazolium (NBT) undergoes photoreduction in the presence of riboflavin upon illumination, producing a chromogenic substance with a maximum absorption peak at 560 nm. Nanomaterials with superoxide dismutase (SOD) activity can inhibit the photoreduction of NBT. The degree of inhibition is proportional to enzyme activity. Therefore, Fe-CDs demonstrate SOD activity. Furthermore, catalytic experiments with H₂O₂ using blank controls, undoped iron materials (CDs), and iron-doped materials (Fe-CDs) revealed that the Fe-CDs assay produced the most bubbles. It is well known that nanomaterials with catalase activity can catalyze the oxidation of H₂O₂ to produce oxygen. Under the same experimental conditions, the greater the number of bubbles, the higher the enzyme activity. Therefore, Fe-CDs demonstrate catalase (CAT) activity.

[0079] In summary, through the method of the present invention, Fe-CDs nanozymes with negative charge, high-intensity fluorescence and multiple enzyme activities were prepared.

[0080] (2) Application examples of Fe-CDs nanozymes

[0081] Relevant test conditions:

[0082] (1) In the present invention, the wheat variety selected for testing was Shumai 2203, which was cultivated by the Wheat Research Institute of Sichuan Agricultural University. The hydroponic test of wheat was conducted in a light culture room with a room temperature of 25°C, a relative humidity of 80% ± 5%, and a photoperiod of 14 h light / 10 h dark.

[0083] (2) Dispersing the Fe-CDs nanozyme in water to obtain a Fe-CDs nanozyme dispersion, and preparing 2.5 mg / mL, 5 mg / mL, 10 mg / mL, 20 mg / mL, 40 mg / mL, and 80 mg / mL water-soluble nanoregulators for later use.

[0084] Test method:

[0085] (1) Experiment on the optimal application concentration of Fe-CDs nanozymes on wheat ( Figure 6 )

[0086] Dried wheat seeds were placed in an oven at 32-35°C for 48 hours, then disinfected with 5% sodium hypochlorite for 20 minutes, rinsed three times with deionized water, and stored in a refrigerator at 4°C for 48 hours before being transferred to petri dishes and placed in a greenhouse for further germination. When the seeds germinated to one week old, different concentrations of Fe-CDs were applied to the roots (referred to as root application) at a gradient of 0 mg / mL, 2.5 mg / mL, 5 mg / mL, 10 mg / mL, 20 mg / mL, 40 mg / mL, and 80 mg / mL. Treatment continued for 14 days, with the hydroponic nutrient solution (1 / 2 Hoagland's) replaced every three days. At the end of the experiment, all plants were harvested, photographed for comparative morphological characteristics, and their fresh and dry biomass and root length were measured. The photosynthetic pigment content, malondialdehyde content, and antioxidant enzyme activity of the plants were also measured. A comprehensive comparison of their growth and development was conducted to identify the optimal application concentration of the nanomaterial.

[0087] The optimal concentration of Fe-CDs nanozymes applied to wheat was tested. Figure 6 The results of the 14-day greenhouse hydroponic experiment shown in the figure show that the applied concentration of 5-10 mg / mL has a better effect on promoting wheat root elongation.

[0088] (2) Wheat stress resistance test ( Figure 7-9 )

[0089] One-week-old wheat seedlings of uniform growth were transplanted and divided into four groups: a blank control (CK), a cadmium stress group (Cd), a cadmium stress + material group (abbreviated as: Cd+NMs), and a material group (abbreviated as: NMs). The material was Fe-CDs, the cadmium stress concentration was set at 30 μmol / L, and the Fe-CDs application rate was set at 10 mg / mL. The root application experiment lasted for 14 days, and the hydroponic nutrient solution (1 / 2 concentration Hoagland's) was changed every three days. After the experiment, all plants were harvested, and their morphological characteristics were observed and photographed. Various physiological indicators were measured to assess the level of the plant's antioxidant defense system and comprehensively analyze the ability of Fe-CDs to mitigate plant oxidative damage.

[0090] Through the wheat stress resistance test:

[0091] like Figure 7 As shown, after 14 days of greenhouse hydroculture, the NMs group alone grew better and had longer roots than the blank control CK group ( Figure 7 A), the plant is slightly sturdy ( Figure 7 A and Figure 7 B); The effects of Cd stress alone and Cd+NMs experimental group were the same as above. Figure 7 C root length statistical comparison chart, Figure 7 D. Statistical comparison of fresh weight of above-ground parts, Figure 7 E. Statistical comparison of dry weight of aboveground parts, Figure 7 F underground part fresh weight statistical comparison chart and Figure 7 G The results of the statistical comparison of underground dry weight: the root system of the group with NMs alone was longer than that of the blank control CK group ( Figure 7 C), the dry and fresh weights of both above-ground and underground parts of the plant were heavier ( Figure 7 D- Figure 7 G); the effects of Cd stress alone and Cd+NMs experimental groups were compared as above. This further verified the growth-promoting effect of Fe-CDs on wheat and its resistance to heavy metal cadmium stress.

[0092] like Figure 8 As shown in the figure, after 14 days of greenhouse hydroculture, the cadmium content of the four experimental groups was quantitatively analyzed using ICP-MS technology. The experimental results showed that after applying low concentrations of Fe-CDs, both wheat leaves ( Figure 8 A, the Cd content of the Cd stress group alone was about 76.3 mg / Kg, and the Cd content of the Cd+NMs group was about 69 mg / Kg) or the root system ( Figure 8 B, The Cd content in the Cd stress group alone was about 1000 mg / Kg, and the Cd content in the Cd+NMs group was about 905 mg / Kg), and the cadmium content was significantly reduced.

[0093] like Figure 9 As shown, after 14 days of greenhouse hydroculture, the chlorophyll a ( Figure 9 A) Chlorophyll b Figure 9 B), carotenoids ( Figure 9 C) and total chlorophyll ( Figure 9 The content of photosynthetic pigments in the group with single application of NMs was higher than that in the blank control CK; the effects of single application of Cd stress and Cd+NMs experimental group were the same as above, that is, the content of photosynthetic pigments in each control group with low concentration of Fe-CDs was higher than that in the group without application; and the corresponding photosynthetic parameters of each treatment group, such as net photosynthetic rate ( Figure 9 E), transpiration rate ( Figure 9 F), stomatal conductance ( Figure 9 G) and intercellular CO2 concentration ( Figure 9 H) also showed an obvious improvement effect, indicating that the application of Fe-CDs can significantly promote wheat photosynthesis.

[0094] In summary, the continuous treatment and application of Fe-CDs prepared in the present invention on seedlings after germination demonstrated a good correlation and promotion between the long-term effect of the material on seedlings and the relief of stress. Analysis suggests the following reasons:

[0095] ① Fe-CDs, a material with multi-enzyme activity, can affect and enhance the plant's antioxidant defense system after entering the plant body. Application of the material can increase the activity of several related antioxidant enzymes (at the enzyme level) and the content of several antioxidant substances (at the non-enzyme level), while reducing the content of harmful substances produced by the plant due to oxidative stress.

[0096] ② Induce the upregulation of some genes related to heavy metal ion transport in plants, enhance the heavy metal chelation and excretion capabilities of plants, reduce the toxicity of cadmium to plants, and effectively resist the oxidative damage caused by heavy metal stress.

[0097] ③ Induce the upregulation of some genes related to photosynthesis in plants, enhance the light capture ability of chloroplasts and improve the efficiency of photosynthetic electron transfer, effectively improve the photosynthesis efficiency of plants, promote the accumulation of plant biomass, make plants stronger, and promote plant growth.

[0098] ④The application of Fe-CDs materials will stimulate plants to secrete a variety of secondary metabolites, among which the flavonoids with the highest content have anti-inflammatory, bactericidal and antioxidant effects, and may have a certain inhibitory effect on certain root-harmful fungi, thereby promoting the elongation of plant roots and allowing plants to develop and grow healthily.

[0099] Finally, it should be noted that the embodiments described above are only used to illustrate the technical solutions of the present invention, rather than to limit them. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some or all of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for preparing Fe-CDs nanozyme, characterized by: The steps include: S1: mixing: mixing anhydrous citric acid, ferric chloride hexahydrate, o-phenylenediamine and the small molecule mixture with deionized water to obtain a mixture; S2: stirring and dissolving: stirring magnetically until completely dissolved to obtain a dissolved mixture solution; S3: hydrothermal reaction: transferring the dissolved mixture solution to a Teflon-lined stainless steel hydrothermal reactor for hydrothermal reaction to obtain a reacted solution; S4: Post-treatment: The reaction solution is filtered, centrifuged, and dialyzed to obtain negatively charged Fe-CDs nanozymes with multi-enzyme activity; in: The molar ratio of anhydrous citric acid, ferric chloride hexahydrate and o-phenylenediamine is 1:1:2; The mass ratio of anhydrous citric acid to the small molecule mixture is 320:1; The small molecule mixture consists of cysteine ​​and resveratrol, with a mass ratio of cysteine ​​to resveratrol of 1:1; In step S3: the hydrothermal reaction temperature is 180°C and the reaction time is 10-12 hours; The heating rate and cooling rate are both 180℃ / h.

2. The method for preparing the Fe-CDs nanozyme according to claim 1, wherein: In step S4: In the filtration process, the filter membrane is a water filter membrane with a pore size of 0.22um and a diameter of 50mm; During the centrifugation process, the centrifugal speed is 8000-10000 rpm / min, the centrifugal time is 5-6 minutes, and the number of centrifugation times is 3-5 times.

3. The method for preparing the Fe-CDs nanozyme according to claim 2, wherein: In step S4: During the dialysis treatment, the dialysis bag specification is 1000Da, and the dialyzed solution is freeze-dried to a powder to obtain negatively charged Fe-CDs nanozymes with multi-enzyme activity.

4. The method for preparing the Fe-CDs nanozyme according to claim 3, wherein: S1: Mixing: 5 mmol of anhydrous citric acid, 5 mmol of ferric chloride hexahydrate, 10 mmol of o-phenylenediamine, and 3 mg of the small molecule mixture with 40 mL of deionized water to obtain a mixture; S2: stirring and dissolving: stirring magnetically until completely dissolved to obtain a dissolved mixture solution; S3: Hydrothermal reaction: The dissolved mixture solution was transferred to a 100 ml Teflon-lined stainless steel hydrothermal autoclave and reacted at 180°C for 10 hours. After the reaction was completed, the autoclave was cooled to room temperature to obtain a reacted solution; S4: Post-treatment: The reaction solution was filtered using a water filter membrane with a pore size of 0.22um and a diameter of 50mm, and then centrifuged at a centrifugal speed of 10000rpm / min for 5min, repeated three times, and finally, dialyzed using a dialysis bag with a specification of 1000Da and freeze-dried to a powder to obtain negatively charged Fe-CDs nanozyme with multi-enzyme activity.

5. The method for preparing the Fe-CDs nanozyme according to claim 4, wherein: Fe-CDs have a spherical structure and the particle size of Fe-CDs is d≤3nm.

6. An application of Fe-CDs nanozyme in improving plant resistance to stress, characterized by: The Fe-CDs nanozyme prepared according to any one of claims 1 to 5 is dispersed in water to obtain a Fe-CDs nanozyme dispersion, and the Fe-CDs nanozyme dispersion is applied to the roots of plants.

7. The use of the Fe-CDs nanozyme according to claim 6 in improving plant resistance to stress, characterized in that: The concentration of Fe-CDs nanozyme dispersion is 5-10 mg / mL.

8. The use of the Fe-CDs nanozyme in improving plant resistance to stress according to claim 6, characterized in that: The plant is the monocotyledonous plant wheat.

9. The use of the Fe-CDs nanozyme in improving plant resistance to stress according to claim 6, characterized in that: The stress-resistant type is resistant to heavy metal cadmium.

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