Nitrogen-doped carbon dots capable of reducing tomato pesticide residues as well as preparation method and application of nitrogen-doped carbon dots

Abstract

The invention discloses nitrogen-doped carbon dots for reducing tomato pesticide residues and a preparation method and application of the nitrogen-doped carbon dots, and belongs to the technical field of carbon materials. The preparation method comprises the following steps: uniformly mixing citric acid, urea and water, then carrying out a hydrothermal reaction, centrifuging after the reaction is completed, taking supernate, dialyzing, and filtering to obtain the N-doped carbon dots (N-CDs). According to the invention, the carbon dots (CDs) are used for promoting the metabolism of plants, so that the pesticide residue of tomatoes is reduced. CDs can enhance photosynthesis and play a role of a biological stimulant, so that tomato growth is promoted.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of carbon materials, and particularly relates to nitrogen-doped carbon dots for reducing pesticide residues of tomatoes as well as a preparation method and application thereof. BACKGROUND

[0002] Tomato is an annual or perennial herbaceous plant of the Solanaceae family. Tomato is one of the important export-oriented agricultural products in China due to its long planting time and large planting area. Tomato is rich in nutrients and has good taste, and thus is favored by consumers, resulting in a large market demand. However, diseases and insect pests are prone to occur in the process of tomato planting, which seriously affects the yield and economic benefits of tomatoes. At present, the prevention and control of diseases and insect pests in tomato production mainly relies on chemical control. As a common non-systemic pesticide, chlorothalonil (CHT) has the advantages of low toxicity, small particle size, good adhesion, long residual period, and is widely used in the prevention and control of fungal leaf diseases of vegetables, field crops and other crops. Due to the large increase in the amount of application every year, chlorothalonil has gradually become a major pesticide in the family of fungicides. However, due to the pursuit of high prevention and control effect on diseases and insect pests, excessive application of chlorothalonil often occurs in the production process. Excessive pesticide residues can cause damage to tomato crops, resulting in the overall wilting of tomato plants and poor growth. Excessive pesticide residues on tomatoes can pollute the environment and harm human health. At present, pesticide residues have become the most prominent problem in the safety of tomatoes in China, and are also an important factor restricting the quality and yield of tomatoes.

[0003] At present, the problem of pesticide residues in tomato crops is mainly solved by improving the application method of pesticides and removing pesticides during harvesting and processing, but the removal effect often fails to meet the expected effect. Tomato has a perfect detoxification system that can convert and decompose toxic substances that are not conducive to its growth and development. Antioxidant enzymes (SOD, POD, CAT, APX) and glutathione play an important role in the process of reducing pesticide residues in plants. The fundamental way to solve the problem of pesticide residues is to promote the metabolism of tomatoes and thus reduce pesticide residues.

[0004] Carbon dots (CDs) are a new type of fluorescent nanomaterial, which has many excellent properties in stability, biological safety and other aspects, and is widely used in agriculture, biology and other fields. In plants, some carbon dots act as biological stimulants, which can enhance photosynthesis and thus promote plant growth and improve stress resistance. However, there is no report on the research and application of nitrogen-doped carbon dots (N-CDs) in relieving the toxicity of pesticides to tomatoes, reducing pesticide residues and promoting the growth of tomatoes. SUMMARY

[0005] In view of this, the purpose of this invention is to provide a nitrogen-doped carbon dot for reducing pesticide residues in tomatoes, its preparation method, and its application. This invention utilizes carbon dots (CDs) to promote plant metabolism, thereby reducing pesticide residues in tomatoes. CDs can enhance photosynthesis, acting as biostimulants and thus promoting tomato growth.

[0006] To achieve the above objectives, the present invention provides the following technical solution:

[0007] In a first aspect, the present invention provides a method for preparing nitrogen-doped carbon dots that reduce pesticide residues in tomatoes, comprising the following steps:

[0008] Citric acid, urea, and water are mixed evenly and then subjected to a hydrothermal reaction. After the reaction is completed, the mixture is centrifuged, and the supernatant is dialyzed and filtered to obtain nitrogen-doped carbon dots.

[0009] Preferably, the mass ratio of citric acid, urea and water is (5-15):(1-5):(15-25).

[0010] Preferably, the hydrothermal reaction is carried out at a temperature of 120–200°C for 4–10 hours; more preferably, the hydrothermal reaction is carried out at a temperature of 160°C for 6 hours.

[0011] Preferably, the centrifugation rate is 8,000 to 15,000 rpm, more preferably 10,000 rpm, and the time is 10 to 30 min, more preferably 15 min.

[0012] Preferably, the dialysis membrane has a molecular weight cutoff of 500-5000 Da, more preferably 1000 Da, a dialysis time of 4-24 h, more preferably 12 h, and a dialysate volume of 200-1200 mL, more preferably 500 mL.

[0013] Preferably, the filtration uses a regenerated cellulose filter membrane with a pore size of 0.22–0.45 μm, more preferably 0.22 μm.

[0014] Secondly, the present invention provides a nitrogen-doped carbon dot for reducing pesticide residues in tomatoes, which is prepared by the above-described preparation method.

[0015] Preferably, the particle size of the nitrogen-doped carbon dots (N-CDs) is 1–4.5 nm.

[0016] Thirdly, the present invention provides the application of the above-mentioned nitrogen-doped carbon dots in reducing pesticide residues in tomatoes.

[0017] Fourthly, the present invention provides the application of the above-mentioned nitrogen-doped carbon dots in promoting tomato growth.

[0018] Preferably, the concentration of the nitrogen-doped carbon dots is 100–200 mg·L⁻¹. -1 .

[0019] Preferably, the application method is to uniformly spray a nitrogen-doped carbon dot aqueous solution onto the surface of tomato leaves.

[0020] It contains at least the following beneficial technical effects:

[0021] This invention synthesizes N-CDs using citric acid and urea as raw materials via a hydrothermal method. The preparation process is simple and easy to operate, with low production cost, environmentally friendly, and suitable for large-scale production applications. N-CDs have a particle size of approximately 2.42 nm and possess abundant hydrophilic groups. N-CDs exhibit strong biocompatibility, blue fluorescence, and good biosafety. It is evident that the raw materials are common chemical products, the preparation steps are simple, equipment requirements are low, and it is easy to scale up applications. N-CDs have good biocompatibility and no secondary pollution, overcoming the problems of toxic side effects or residues associated with traditional chemical regulators. At 150 mg·L⁻¹... -1 At certain concentrations, N-CDs significantly activate the tomato's antioxidant system, accelerate the degradation of chlorothalonil, and reduce leaf residue by 66%, demonstrating remarkable effects. In addition to reducing residues, N-CDs also enhance photosynthetic efficiency, promote plant growth, and improve the overall productivity of tomatoes. Attached Figure Description

[0022] Figure 1 The morphological characteristics, fluorescence properties, and safety of N-CD prepared in Example 1 are shown in (a) transmission electron microscopy (TEM) results, (b) particle size distribution of N-CD, (c) three-dimensional fluorescence spectrum of N-CD, and (d) cytotoxicity test.

[0023] Figure 2 The elemental groups of N-CD are characterized as follows: (a) absorption in the visible region and fluorescence emission spectrum of N-CD, (b) Fourier transform infrared (FT-IR) spectrum, (c) X-ray photoelectron spectroscopy (XPS) spectrum of N-CD, (d) deconvolution map of C1s spectrum of N-CD, (e) deconvolution map of N-1s spectrum of N-CD, and (f) deconvolution map of O-1s spectrum of N-CD.

[0024] Figure 3 The effect of different concentrations of N-CD on chlorothalonil (CHT) residues in tomatoes.

[0025] Figure 4 The effect of N-CD treatment on reducing the toxicity of chlorothalonil to tomatoes was studied. Among them, (a) showed the phenotypes of tomato plants treated with control and N-CD spraying under control or CHT treatment, and the plant height (b), leaf area (c), dry weight (d), fresh weight (e), MDA content (f), and CHT residue (g).

[0026] Figure 5 The N-CD treatment alleviates the inhibition of tomato photosynthesis induced by chlorothalonil, where (ad) represents total chlorophyll, net photosynthetic rate (Pn), maximum photochemical quantum yield of photosystem II (Fv / Fm), and photochemical efficiency of photosystem II (Fv' / Fm'), respectively.

[0027] Figure 6 To investigate the effects of N-CD treatment on the antioxidant activity of tomatoes, the following parameters were measured: (a) superoxide dismutase (SOD) activity, (b) peroxidase (POD) activity, (c) catalase (CAT) activity, (d) ascorbate peroxidase (APX) activity, H2O2 content, and (e) O2 content. ·- content.

[0028] Figure 7 N-CD treatment altered the synthesis and metabolism of glutathione in tomatoes, including (a) glutathione S-transferase activity, (b) glutathione (GSH) content, (c) oxidized glutathione (GSSG) content, (d) total glutathione content (GSH+GSSG), and (e) GSH / GSSG ratio. Detailed Implementation

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

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

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

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

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

[0034] Unless otherwise specified, "room temperature" and "normal temperature" in this invention refer to 25±2℃.

[0035] Unless otherwise specified, all raw materials or instruments used in the following embodiments of the present invention are commercially available.

[0036] The measurement method used in this invention is as follows:

[0037] 1. Determination of net synergistic rate (Pn) of plants

[0038] Pn in tomato leaves was measured using a CIRAS-3 photosynthesis system manufactured by PPSYSTEMS, USA. During the measurement, the instrument was set to a light intensity of 300 μmol / m. -2 s -1 The CO2 concentration was 400 ppm, and the relative humidity was set to 60%–70%.

[0039] 2. Determination of chlorophyll content in plants

[0040] The chlorophyll content determination method was based on Li Hesheng's method with modifications. Weigh 0.05g of fresh tomato leaf sample, carefully avoiding the veins. Grind thoroughly in a mortar under ice, transfer to 2mL centrifuge tubes, add 1.5mL of 80% acetone to each tube, and place on a shaker at 28℃ and 400rpm for 4 hours, completely covering the shaker with aluminum foil. After 4 hours, centrifuge at 8,000rpm for 8 minutes, and transfer the supernatant to a new 1.5mL tube.

[0041] The Varioskan LUX multi-functional microplate reader measured the absorbance at wavelengths of 470 nm, 647 nm, and 663 nm and calculated the chlorophyll content.

[0042] 3. Determination of chlorophyll fluorescence parameters in plants

[0043] The chlorophyll fluorescence parameters of tomato leaves were determined using PlantExplorerPRO+ manufactured by Huinuo Ruide Co., Ltd. in China. The tomato leaves were subjected to a 3-minute dark treatment before the parameters such as the maximum photochemical quantum yield of photosystem II (Fv / Fm) and the photochemical efficiency of photosystem II (Fq' / Fm') were measured.

[0044] 4. Determination of the activities of superoxide dismutase (SOD), peroxidase (POD), catalase (CAT), ascorbate peroxidase (APX), and malondialdehyde (MDA) in plants.

[0045] Methods for extracting supernatant from plant leaves:

[0046] Weigh 0.1 g of fresh tomato leaves and add 600 μL of 0.05 mol·L⁻¹ solution. -1 Phosphate buffer solution with pH 7.8 was vortexed thoroughly on ice and centrifuged at 10,500 rpm for 20 min at 4°C. The supernatant was collected after centrifugation and stored at 4°C in the dark for subsequent determination of SOD, POD, CAT, APX activities, and MDA content. Preparation of phosphate buffer solution: 0.2 mol·L⁻¹ - 1 Na₂HPO₄ solution: 71.64 g Na₂HPO₄⁻¹²H₂O + sterile water to a final volume of 1 L, 0.2 mol·L⁻¹ -1 Na₂HPO₄ solution: 7.6g Na₂HPO₄-H₂O₂ + sterile water to a final volume of 1L, 0.1mol·L⁻¹ -1 Preparation of buffer solution: 0.2 mol·L⁻¹ -1 Na₂HPO₄ solution + 0.2 mol·L⁻¹ -1 Na₂HPO₄ solution diluted with sterile water to 200 mL, 0.05 mol·L⁻¹ -1 Preparation of buffer solution: 0.2 mol·L⁻¹ -1 Na₂HPO₄ solution + 0.2 mol·L⁻¹ -1 Na2HPO4 solution diluted with sterile water to 400mL.

[0047] 4.1 Determination of superoxide dismutase (SOD) activity in plants

[0048] The SOD activity of plants was determined using the nitroblue tetrazolium method:

[0049] 0.05 mol·L -1 Preparation of phosphate buffer solution with pH = 7.8: 91.5 mL 0.2 mol·L⁻¹ -1 Na₂HPO₄ solution + 8.5 mL 0.2 mol·L⁻¹ -1 Dilute Na₂HPO₄ solution with sterile water to 400 mL. Prepare Met solution: 0.388 g Met + 20 mL 0.05 mol·L⁻¹ - 1 Phosphate buffer (pH 7.8), NBT preparation: 0.012 g NBT + 20 mL 0.05 mol / L -1Preparation of EDTA-2Na phosphate buffer (pH 7.8): 0.0074 g EDTA-2Na + 20 mL 0.05 mol·L⁻¹ -1 Phosphate buffer (pH 7.8) for riboflavin preparation: 0.015 g riboflavin + 200 mL sterile water. Volume ratio of reaction solution components: water : 0.05 mol / L. -1 pH 7.8 Phosphate buffer: Met: NBT: EDTA-2Na: Riboflavin; 5:30:6:6:6:6. Add 25 μL of supernatant to centrifuge tubes, and add 25 μL of 0.05 mol·L⁻¹ to two control tubes. -1 Add 1.5 mL of reaction solution to phosphate buffer (pH 7.8) and mix well. Place one control tube in the dark and the rest in an incubator for light reaction for 20 min. The control tube not placed in the dark serves as a blank. Measure the absorbance at 560 nm and calculate the SOD activity.

[0050] 4.2 Determination of plant peroxidase (POD) activity

[0051] The guaiacol method was used to determine the plant's POD activity.

[0052] 0.1 mol·L -1 Preparation of pH 6.0 phosphate buffer: 12.3 mL 0.2 mol·L⁻¹ -1 Na₂HPO₄ solution + 87.7 mL 0.2 mol·L⁻¹ -1 Na₂HPO₄ solution diluted with sterile water to 200 mL; reaction solution preparation: 0.1 mol·L⁻¹ -1 pH 6.0 phosphate buffer 50 mL + guaiacol 28 μL + 30% H2O2 19 μL. Add 10 μL of the supernatant to a 2.2 mL well of 96, and add 20 μL of 0.1 mol·L⁻¹ for the control. -1 Add 1.5 mL of phosphate buffer (pH 6.0) to a 96-well microplate using a pipette, and immediately read the absorbance at 470 nm. Read the absorbance every 1 minute until the 15th minute, and calculate the POD activity.

[0053] 4.3 Determination of plant catalase (CAT) activity

[0054] The method for determining CAT activity in plants is as follows:

[0055] 0.1 mol·L -1 Preparation of pH 7.0 phosphate buffer: 61 mL 0.2 mol·L⁻¹ -1 Na₂HPO₄ solution + 39 mL 0.2 mol·L⁻¹ -1 Na₂HPO₄ solution diluted with sterile water to 200 mL, 0.1 mol·L⁻¹-1 Preparation of H2O2: Add 2.84 mL of 30% H2O2 and sterile water to a final volume of 500 mL. Preparation of reaction solution: 0.1 mol·L⁻¹ -1 pH 7.0 phosphate buffer 20 mL + 5 mL 0.1 mol·L⁻¹ - 1 H2O2. For the assay, use a quartz microplate. Add 5 μL of the supernatant to each of the 96 wells. For the control, add 5 μL of 0.1 mol·L⁻¹. -1 Use a phosphate buffer solution of pH 7.0 to pipette 150 μL of the reaction solution into a 96-well microplate. After mixing, immediately read the absorbance at 240 nm and start timing. Read the absorbance every 1 minute until the 5th minute, then calculate the CAT activity.

[0056] 4.4 Determination of ascorbate peroxidase (APX) activity in plants

[0057] The APX activity of the plant was determined as follows:

[0058] Preparation of 20mM H2O2: 102μL 30% H2O2 + 50mL sterile water, 5mmol·L⁻¹ -1 Preparation of reduced ascorbic acid (ASA): 44g ASA + 50mL sterile water, 0.1mol·L⁻¹ -1 Preparation of pH 7.0 phosphate buffer: 61 mL 0.2 mol·L⁻¹ - 1 Na₂HPO₄ solution + 39 mL 0.2 mol·L⁻¹ -1 Na₂HPO₄ solution diluted with sterile water to 200 mL; reaction solution preparation: 17 mL 0.1 mol·L⁻¹ -1 pH 7.0 phosphate buffer + 100 μL 5 mmol·L -1 ASA + 100 μL 20 mmol·L -1 For H2O2 assay, a quartz microplate was used. 10 μL of the supernatant was placed into the quartz microplate, and 190 μL of the reaction solution was drawn up with a pipette. After mixing, the absorbance value at 290 nm was read immediately. The absorbance was read once every 1 min to calculate the APX activity.

[0059] 4.5 Determination of malondialdehyde (MDA) content in plants

[0060] The TBA method was used to determine the MDA content in plants.

[0061] Preparation of 0.67% TBA: 0.67g TBA + a small amount of 1mol·L⁻¹ -1 TBA dissolved in NaOH solution; preparation of 10% trichloroacetic acid: 10 mg / mL -1Trichloroacetic acid (TCA) was added to 90 mL of sterile water. 300 μL of the supernatant was collected, and 300 μL of water was added to the control. 1 mL / 600 μL of 67% TBA was added, and the mixture was dissolved in water for 15 min. The mixture was then rapidly cooled on ice. Centrifuged at 4,000 rpm for 20 min. The supernatant was then analyzed at 600 nm, 532 nm, and 450 nm to calculate the MDA content.

[0062] 5. Plant-derived oxidized glutathione (GSSG), reduced glutathione (GSH), and superoxide anion (O2) ·- Determination of hydrogen peroxide (H2O2) content and glutathione S-transferase (GST) activity

[0063] 5.1 Determination of oxidized glutathione (GSSG) content in plants

[0064] The kit (BC1185-100T / 96S) used for the determination of oxidized glutathione (GSSG) content was purchased from Solarbio (Beijing). The microplate reader was preheated for 30 minutes. The absorbance was measured at 412 nm for 30 seconds and 150 seconds. The GSSG content was calculated, expressed in μg / g. The experimental procedure was performed according to the instruction manual.

[0065] 5.2 Determination of reduced glutathione (GSH) content in plants

[0066] The kit (BC1175-100T / 96S) used for the determination of reduced glutathione (GSH) content was purchased from Solarbio (Beijing). The microplate reader was preheated for 30 minutes. The absorbance was measured at 412 nm using the microplate reader, and the GSH content was calculated in μg / g. The experimental procedure was performed according to the instruction manual.

[0067] 5.3 Plant superoxide anion (O2) ·- Determination of content

[0068] Superoxide anion (O2) ·- The reagent kit (BC1295-100T / 96S) used for the content determination was purchased from Solarbio (Beijing). The microplate reader was preheated for 30 minutes, and the absorbance was measured at 530 nm. O2 was then calculated. ·- Content is expressed in μmol / g. Please refer to the instruction manual for experimental procedures.

[0069] 5.4 Determination of hydrogen peroxide (H2O2) content in plants

[0070] The reagent kit (BC3595-100T / 96S) used for the determination of hydrogen peroxide (H2O2) content was purchased from Solarbio (Beijing). The microplate reader was preheated for 30 minutes beforehand. The absorbance was measured at 415 nm using the microplate reader, and the H2O2 content was calculated. The unit is expressed as μmol / gmol. The experimental procedure was performed according to the instruction manual.

[0071] 5.5 Detection of glutathione S-transferase (GST) activity in plants

[0072] The kit (BC0355-100T / 96S) used for the glutathione S-transferase (GST) activity assay was purchased from Solarbio (Beijing). The microplate reader was preheated for 30 minutes. The absorbance was measured at 340 nm using the microplate reader, and the GST activity was calculated. The unit is expressed as U / g. The experimental procedure was performed according to the instruction manual.

[0073] 6. Detection of pesticide residues in plants

[0074] High-performance liquid chromatography (HPLC) was used to analyze CHT residues in tomato leaves. Standard solutions were prepared as follows: chlorothalonil standards were accurately weighed, dissolved in acetonitrile, and prepared into stock solutions with a concentration of 1.0 mg / mL. These stock solutions were stored at -18℃ for later use. The solutions were serially diluted with the mobile phase to concentrations of 0.05, 0.1, 0.5, 1.0, 2.0, 5.0, 10.0, 25.0, 50.0, 100.0, and 200.0 mg / L. -1 A mixed standard working solution.

[0075] Sample preparation and extraction: Take an appropriate amount of tomato leaf sample, place the sample in aluminum foil, and crush it in liquid nitrogen. Place the crushed tomato leaf sample in a centrifuge tube and store it at -80℃ for later use. Accurately weigh 0.2g of the crushed tomato leaf sample into a 10mL centrifuge tube, add 2.0mL of acetonitrile and 0.2g of NaCl, and vortex for 30s to ensure homogeneity. Let the mixture stand at room temperature for 30min.

[0076] Sample purification: Transfer the supernatant from the centrifuge tube to a 10 mL centrifuge tube, place the centrifuge tube in a multi-channel constant-temperature nitrogen evaporator, and evaporate to near dryness in a 60°C water bath with nitrogen. Add 2 mL of n-hexane and vortex for 30 s to mix thoroughly, then proceed with purification. Place the Alumina-N column (1 g / 6 mL) on a solid-phase extraction apparatus and activate it sequentially with 2 mL of n-hexane / acetone (90:10, V:V) and 2 mL of n-hexane. When the solvent level reaches the surface of the column adsorption layer, pour in the solution to be purified and collect the eluent. Wash the sample tube with 1 mL of n-hexane / acetone (90:10, V:V) and rinse the Alumina-N column, repeating once. Place the sample tube containing the collected eluent in a multi-channel constant-temperature nitrogen evaporator and evaporate to near dryness in a 50°C water bath with nitrogen. Add 2.0 mL of mobile phase, vortex to mix thoroughly, filter through a 0.22 μm organic microporous membrane, and perform analysis using liquid chromatography.

[0077] Liquid chromatography conditions: Column: ZORBAX Eclipse XDB-C18 (250 mm × 4.6 mm, 5 μm); Mobile phase: Acetonitrile: Water = 55:45 (V:V); Flow rate: 0.6 mL / min; Detection wavelength: 232 nm; Injection volume: 10 μL; Column temperature: 30 ℃.

[0078] Example 1

[0079] Preparation of nitrogen-doped carbon dots:

[0080] (1) Accurately weigh 1.0248g of citric acid powder and 0.3762g of urea powder, add them to 20mL of ultrapure water, stir and mix evenly to form a transparent solution;

[0081] (2) Transfer the mixed solution into a 50 mL polytetrafluoroethylene liner, place it in a high-pressure reactor, and carry out a hydrothermal reaction at 160 °C for 6 h.

[0082] (3) After the reaction is completed, the reaction vessel is cooled to room temperature, and the resulting reaction solution is centrifuged at 14,000 r / min for 15 min. The precipitate is discarded and the supernatant is collected.

[0083] (4) Place the collected supernatant in a dialysis bag with a molecular weight cutoff of 1000 Da and dialyze it in ultrapure water for 12 hours until the color of the dialysate no longer changes.

[0084] (5) After dialysis, the solution is filtered through a 0.22 μm filter membrane to obtain the prepared nitrogen-doped carbon dots (N-CDs), which are stored at 4 °C under light-protected conditions for later use.

[0085] Example 2

[0086] Preparation of nitrogen-doped carbon dots:

[0087] (1) Accurately weigh 1.0248g of citric acid powder and 0.0752g of urea powder, add them to 15mL of ultrapure water, stir and mix evenly to form a transparent solution;

[0088] (2) Transfer the mixed solution into a 50 mL polytetrafluoroethylene liner, place it in a high-pressure reactor, and carry out a hydrothermal reaction at 120 °C for 4 h.

[0089] (3) After the reaction is completed, the reaction vessel is cooled to room temperature, and the resulting reaction solution is centrifuged at 8,000 r / min for 10 min. The precipitate is discarded and the supernatant is collected.

[0090] (4) Place the collected supernatant in a dialysis bag with a molecular weight cutoff of 500 Da and dialyze it in ultrapure water for 4 hours until the color of the dialysate no longer changes.

[0091] (5) After dialysis, the solution is filtered through a 0.22 μm filter membrane to obtain the prepared nitrogen-doped carbon dots (N-CDs), which are stored at 4 °C under light-protected conditions for later use.

[0092] Example 3

[0093] Preparation of nitrogen-doped carbon dots:

[0094] (1) Accurately weigh 1.0248g of citric acid powder and 1.0248g of urea powder, add them to 25mL of ultrapure water, stir and mix evenly to form a transparent solution;

[0095] (2) Transfer the mixed solution into a 50 mL polytetrafluoroethylene inner liner, place it in a high-pressure reactor, and carry out a hydrothermal reaction at 200°C for 10 h.

[0096] (3) After the reaction is completed, the reaction vessel is cooled to room temperature, and the resulting reaction solution is centrifuged at 15,000 r / min for 30 min. The precipitate is discarded and the supernatant is collected.

[0097] (4) Place the collected supernatant in a dialysis bag with a molecular weight cutoff of 5000 Da and dialyze it in ultrapure water for 24 hours until the color of the dialysate no longer changes.

[0098] (5) After dialysis, the solution is filtered through a 0.45 μm filter membrane to obtain the prepared nitrogen-doped carbon dots (N-CDs), which are stored at 4 °C under light-protected conditions for later use.

[0099] Experimental Example 1

[0100] Characterization methods:

[0101] (1) Transmission electron microscopy (TEM): Images are acquired using a transmission electron microscope to observe the morphological characteristics, size and dispersion state of carbon dots in order to confirm whether the particle size range meets the requirements of nanoscale.

[0102] (2) Fluorescence 3D scanning spectrum: Measured by a fluorescence spectrophotometer, used to analyze the emission characteristics of carbon dots under different excitation wavelengths, thereby determining their optical properties;

[0103] (3) Ultraviolet-visible absorption spectrum (UV-Vis): Measured by a UV-visible spectrophotometer, it is used to obtain electronic structure information of carbon dots and can reflect information such as size, structure and surface modification.

[0104] (4) Fourier transform infrared spectroscopy (FTIR): Fourier transform infrared spectroscopy is used to determine the chemical structure and functional group information by detecting the absorption of infrared light by molecular vibration or rotation and combining the analysis of spectral peak position and intensity.

[0105] (5) X-ray photoelectron spectroscopy (XPS): X-ray photoelectron spectroscopy is used to determine the elemental composition and chemical valence state by exciting the inner-shell electrons of the atoms on the sample surface with high-energy X-rays and measuring the photoelectron energy.

[0106] 1. Characterization and biosafety testing of N-CDs

[0107] (1) In Example 1, N-doped citric acid precursors N-CDs were prepared using the amino group in urea as a nitrogen source. The morphology of the prepared material was studied by TEM. Figure 1 a). The prepared N-CDs exhibited uniform size and high monodispersity. Furthermore, the particle size range of the N-CDs was 1–4.5 nm, with an average particle size of (2.42 ± 0.24) nm. Figure 1 b). Furthermore, fluorescence three-dimensional scanning spectroscopy showed that N-CDs in the water emitted strong blue fluorescence under excitation light of 315–500 nm. Figure 1 c).

[0108] (2) Cell viability assay: The CCK-8 assay was used, and the results were detected using a UV-Vis absorption spectrometer to evaluate the biocompatibility and safety of the cell. The steps of the CCK-8 assay are as follows:

[0109] Human embryonic kidney cells 293T in good growth condition were seeded at a density of approximately 1 × 10⁶ cells per well. 4Cells were transferred at a density of 100 μL to 96-well plates, with three replicates per sample. The plates were incubated for 24 hours. Cells were then gently washed with phosphate-buffered saline (PBS), followed by the addition of 200 μL of N-CDs culture medium dilution to each well. A water-treated group served as a control, and the plates were incubated for another 24 hours. Next, 10 μL of CCK-8 solution (from a CCK-8 kit) was added to each well, and the plates were gently agitated to ensure even distribution. The plates were returned to the incubator for another 4 hours. Finally, the absorbance of each well was measured at 450 nm using a microplate reader, and cell viability was calculated. The experiment was performed in triplicate.

[0110] like Figure 1 As shown in Figure d, the histogram corresponds to the absorbance of N-CDs and the blank control at 450 nm within 0–4 days after injection, and the line graph corresponds to the relative survival rate of cells in the N-CDs wells relative to the blank control within 0–4 days. After 4 days, although the cell survival rate treated with N-CDs decreased to 93.14%, there was no significant difference between the water-treated cells and the N-CDs-treated cells. These results indicate that the experimentally prepared N-CDs have high biosafety and are expected to be applied in actual production.

[0111] 2. Elemental group analysis of N-CDs

[0112] The ultraviolet spectra of the prepared N-CDs aqueous solution were further investigated. Figure 2 a). The orange lines of N-CDs show strong absorption bands at 235 nm and 340 nm. The first absorption peak at 235 nm is due to the π-π* transition in the sp2 domain. The absorption peak at 340 nm corresponds to the n-π* transition of the C=O or C=N bond. The optimal excitation wavelength is 378 nm; under this wavelength, N-CDs exhibit strong absorption at 488 nm. Figure 2 The strongest blue fluorescence is emitted at the location indicated by the blue line. FT-IR spectroscopy characterizes the distribution of functional groups on the N-CD surface. Figure 2 As shown in b, it is at 3200cm -1 The nearby peaks belong to the absorption peaks of the NH stretching vibration. 3417 cm⁻¹ -1 The strong absorption peak at 2932 cm⁻¹ is caused by the stretching vibration of the intermolecular hydrogen bond OH. -1 The absorption peak at 1443 cm⁻¹ corresponds to the stretching vibration of CH₄. -1 This is CH's fingerprint area. 1700cm -1 The characteristic absorption peak at 1611 cm⁻¹ is attributed to the stretching vibrations of C=O and C=C. -1 The absorption peak at 1585 cm⁻¹ is attributed to the bending vibration of primary amine (NH₃). -1The nearby absorption peak is attributed to the bending vibration of NH, 1409 cm⁻¹ -1 The absorption peaks at these locations are attributed to the stretching vibration absorption of CN. These peaks indicate that the surface and edges of N-CDs are rich in carboxyl, ester, hydroxyl, and carbonyl groups. Furthermore, XPS measurements show that the main components of N-CNs are carbon, nitrogen, and oxygen in a ratio of 62:15:23. Figure 2 c). Further analysis of the valence states and composition of the N-CD surface elements was conducted using refined XPS spectra. For example... Figure 2 As shown in d to f, C1s fitted peaks of CC / C=C and C=O / C=N at 284.8 eV and 288.12 eV, N1s fitted only one peak of NH at 399.06 eV, and O1s fitted two peaks of C=O at 530.63 eV. Combined with FT-IR spectra, it is proved that N-CDs have abundant hydrophilic functional groups.

[0113] Experiment Example 2

[0114] Different concentrations of N-CDs reduced pesticide residues in tomato leaves.

[0115] Two-week-old tomato seedlings of uniform growth were selected, and N-CDs aqueous solutions of different concentrations were sprayed evenly on each seedling, including N-CDs1 (50 mg·L⁻¹). -1 ), N-CDs2 (100 mg·L) -1 ), N-CDs3 (150 mg·L) -1 ), N-CDs4 (200 mg·L) -1 ) and N-CDs5 (300 mg·L -1 Two days after treatment, a uniform 37.6 mM chlorothalonil (CHT) aqueous solution was sprayed. Seven days after CHT treatment, the fifth leaf was taken, and the CHT residue was determined by high performance liquid chromatography (HPLC). Each treatment was performed in at least three biological replicates.

[0116] The results are as follows Figure 3 As shown, all N-CDs treatments significantly reduced CHT residues in tomato leaves, with 150 mg·L⁻¹ being the most effective. -1 The treatment with (N-CDs3) showed the most significant effect, reducing the residue to 34%. Therefore, 150 mg·L⁻¹ -1 N-CDs are the optimal concentration for reducing pesticide residues in tomatoes.

[0117] Experimental Example 3

[0118] Application of N-CDs as plant growth regulators in promoting tomato growth and reducing pesticide residues

[0119] The experiment consisted of a control group and a CHT group, each subdivided into water treatment and N-CDs treatment. Each group contained 10 tomato plants: 5 in the water treatment group and 5 in the N-CDs treatment group. First, 2-week-old tomato seedlings of uniform growth in both the water and N-CDs treatment groups were uniformly sprayed with equal amounts of sterilized water and a solution at a concentration of 150 mg·L⁻¹. -1 Two days later, equal volumes of sterile water and a 37.6 mM CHT aqueous solution were sprayed evenly onto the control group and the CHT group, respectively. All solutions were prepared and used immediately. Seven days after CHT treatment, plant phenotypes were photographed and the fifth leaf was taken to detect relevant indicators. At least three biological replicates were performed.

[0120] 1. N-CDs promote tomato growth and alleviate CHT toxicity.

[0121] like Figure 4 As shown in figure a, N-CDs-treated plants exhibited growth-promoting effects in both the control and CHT groups. Under CHT treatment, the growth status of N-CDs-treated plants was similar to that of the water-treated control plants. Further results showed that ( Figure 4 (b~e) N-CDs treatment significantly increased plant height, leaf area, fresh weight (FW), and dry weight (DW), which returned to control levels under CHT treatment. Simultaneously, in the CHT group, the malondialdehyde (MDA) content in N-CDs-treated plants was significantly lower than that in water-treated plants, indicating that N-CDs alleviated the oxidative damage caused by CHT. Furthermore, N-CDs significantly reduced the residual amount of CHT in leaves (b~e). Figure 4 f).

[0122] 2. N-CDs alleviate CHT-induced photosynthetic inhibition

[0123] like Figure 5 As shown in a–d, N-CDs treatment significantly increased chlorophyll content (Chl), net photosynthetic rate (Pn), maximum photochemical quantum yield of photosystem II (Fv / Fm), and photochemical efficiency of photosystem II (Fv′ / Fm′). Under CHT treatment, photosynthetic parameters generally decreased, while the Chl content and Pn of N-CDs-treated plants basically recovered to the control group level, indicating that N-CDs alleviated the inhibition of photosynthesis by CHT by promoting chlorophyll synthesis.

[0124] 3. N-CDs inhibit the accumulation of reactive oxygen species induced by CHT and activate the activity of antioxidant enzymes.

[0125] In the control group, the levels of superoxide anions (O2) in tomato leaves treated with water and N-CDs were significantly higher. ·- There was no significant difference in the content of ) and hydrogen peroxide (H2O2). Figure 4 (a~b). In the CHT group, the O2 content in the leaves of all tomato plants was... ·-Both O2 and H2O2 content increased significantly. However, compared with water-treated tomato plants, N-CDs-treated tomato plants had significantly higher O2 content in their leaves. ·- The accumulation of H2O2 is relatively low. Figure 4 (a~b).

[0126] In the control group, compared with water-treated tomato plants, the activities of superoxide dismutase (SOD), catalase (CAT), and ascorbate peroxidase (APX) were significantly increased in N-CDs-treated tomato plants. Importantly, in the CHT group, the activities of SOD, POD, CAT, and APX were further activated in N-CDs-treated tomato plants compared with water-treated plants. Figure 4 c~f) indicates that N-CDs enhance the antioxidant defense capacity of tomatoes.

[0127] 4. N-CDs enhance tomatoes' ability to detoxify and metabolize CHT.

[0128] The activities of glutathione S-transferase (GST), glutathione (GSH) content, oxidized glutathione (GSSG) content, total glutathione content (GSH+GSSG), and the GSH / GSSG ratio were analyzed. Compared with the control group, the CHT group significantly increased the contents of GSSG and GSH+GSSG, but significantly decreased the GSH / GSSG ratio. Figure 7 c~e). In the CHT group, N-CDs treatment significantly increased the contents of GST and GSH, respectively, compared to water-treated tomatoes ( Figure 7 a-c) increased by 50% and 75%, respectively, while decreasing GSSG content by 14%. Notably, although N-CDs treatment did not affect GSH content in the control group, it increased GST content, leading to a higher GSH / GSSG ratio. Figure 7 The ratios of a and b) increased. Changes in GSH and GSSG led to a significant decrease in the GSH / GSSG ratio under CHT treatment; however, in the CHT group, N-CDs treatment significantly increased the GSH / GSSG ratio in N-CDs-treated tomato plants compared to water-treated plants. Figure 7 e). These results indicate that N-CDs enhance the detoxification and metabolism of CHT in tomatoes by regulating glutathione metabolism.

[0129] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for preparing nitrogen-doped carbon dots that reduce pesticide residues in tomatoes, characterized in that, Includes the following steps: Citric acid, urea, and water are mixed evenly and subjected to a hydrothermal reaction. After the reaction is completed, the mixture is centrifuged, and the supernatant is dialyzed and filtered to obtain nitrogen-doped carbon dots.

2. The preparation method according to claim 1, characterized in that, The mass ratio of citric acid, urea and water is (5-15):(1-5):(15-25).

3. The preparation method according to claim 1, characterized in that, The hydrothermal reaction is carried out at a temperature of 120–200°C for 4–10 hours.

4. A nitrogen-doped carbon dot for reducing pesticide residues in tomatoes, characterized in that, It is prepared by the preparation method according to any one of claims 1-3.

5. The nitrogen-doped carbon dot according to claim 4, characterized in that, The particle size of the nitrogen-doped carbon dots is 1–4.5 nm.

6. The application of the nitrogen-doped carbon dots as described in claim 4 in reducing pesticide residues in tomatoes.

7. The application of the nitrogen-doped carbon dots as described in claim 4 in promoting tomato growth.

8. The application according to claim 6 or 7, characterized in that, The concentration of the nitrogen-doped carbon dots used is 100–200 mg·L. -1 .

9. The application according to claim 8, characterized in that, The application method involves uniformly spraying a nitrogen-doped carbon dot aqueous solution onto the surface of tomato leaves.

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