Application of rhodamine fluorescent probe in detection of herbicide benzfendizone

Abstract

The application discloses application of a fluorochloridone specific fluorescent probe based on a rhodamine skeleton, and the fluorochloridone specific fluorescent probe can be used for detecting fluorochloridone in agricultural products, environmental samples and biological samples. Specifically, test paper is immersed in a 40 muM probe 4 solution for 1 hour, and then is dried at room temperature; the test paper is cut into multiple hearts, and is placed on a glass slide; fluorochloridone with different concentrations is dropped on the test paper strip as an experimental group, and water treatment is used as a control group; the droplets completely cover the surface of the test paper; then the test paper is dried at room temperature; and the fluorescent image of the test paper strip is photographed under natural light and 365 nm ultraviolet light irradiation in a dark environment; wherein the fluorescent probe 4 shows good performance; the fluorescent probe 4 is integrated into the test paper and a hydrogel; and the combination of the fluorescent probe 4 and a smart phone based platform facilitates portable and instantaneous detection in actual samples; and the method is a simple and accurate method for detecting the content of fluorochloridone.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of environment, rapid detection of agricultural products and biological imaging, and particularly relates to a rhodamine fluorescent probe and a preparation method and application thereof. BACKGROUND

[0002] With the continuous increase of global population, the demand for food supply is also increasing. In order to improve the yield of food, pesticides have become an important tool for disease prevention, weed control and insect control in modern agriculture. As the first major class of pesticides, herbicides account for about 30% of the total production of Chinese pesticides. Among them, benzfosulfuron-methyl is widely used in the control of broadleaf weeds in farmland due to its high selectivity and excellent herbicidal effect. Even at a low application rate of 280-430 g / hm 2 , benzfosulfuron-methyl also has strong biological activity and has become one of the most widely used herbicides in the world. However, its slow degradation and long-term presence in soil pose a major challenge. Long-term and intensive application can seriously affect the succession of sensitive crops, increasing the risk of crop damage in subsequent planting seasons. In addition, it destroys soil microbial communities and enzyme activity, leading to reduced crop yields. The widespread use of benzfosulfuron-methyl, even sometimes improper use, also leads to the accumulation of residues on the surface of food products. Ingestion of food or water contaminated with excessive benzfosulfuron-methyl residues can cause serious health risks, including neurotoxicity, immune dysfunction, and liver damage. Considering its harmful effects on the environment, ecosystems, and human health, there is an urgent need to develop effective benzfosulfuron-methyl detection systems. These systems are crucial for ensuring the safety and quality of agricultural products and protecting public health.

[0003] To date, various methods for detecting benzfosulfuron-methyl have been developed, including HPLC-PDA, LC-MS / MS, nanoscale enzyme, immunoassay, etc. However, these techniques are often limited by factors such as complex procedures, time-consuming, high cost, dependence on specialized equipment, and the need for skilled personnel, which limit their application in real-time detection and on-site application. Currently, fluorescent probes have the advantages of simple operation, rapid response, visualization, and suitability for real-time analysis, and play a prominent role in environmental pollution monitoring, hazardous substance detection, food safety, and other applications. Among them, rhodamine has excellent photophysical properties, high biocompatibility, superior fluorescence quantum yield, and switchable spirolactone "on-off" structure, making it a particularly powerful fluorescent probe skeleton for labeling and tracking biological species. Despite these advantages, no small-molecule fluorescent probe has been developed for the detection of benzfosulfuron-methyl, mainly due to the low chemical reactivity of benzfosulfuron-methyl. Therefore, designing a fast-response fluorescent probe for detecting benzfosulfuron-methyl is a major challenge. SUMMARY

[0004] The main content of the application is to develop a series of rhodamine-based fluorescent probes to realize the detection of benfluralin in environmental samples and agricultural products, and to be applied to biological imaging. Among them, probe 4 shows good performance, with high sensitivity, selectivity and fast response time of less than 60 seconds, which can be used for the detection of benfluralin in environment and agricultural environment. In addition, probe 4 is integrated into a multifunctional detection platform which combines test paper and image analysis software. This method creates an intelligent visualization system with the ability to detect benfluralin residues in agricultural products. The probe further realizes the real-time monitoring of the distribution of benfluralin in Arabidopsis thaliana root tips and zebrafish, providing valuable visual data for exploring the application of benfluralin imaging in biological bodies.

[0005] One of the technical solutions of the application is a qualitative detection method for soil surface analysis experiment, comprising the following steps:

[0006] (1) Collect soil from local farmland without any pretreatment.

[0007] (2) Put 1g of soil sample in a culture dish and divide it into two groups: control group and experimental group. The control group is sprayed with 10uM fluorescent probe solution for imaging; the experimental group is sprayed with 50uM, 100uM, 200uM and 300uM different concentrations of benfluralin, and then 10uM fluorescent probe solution is sprayed on the surface of the soil, and finally the photo under 365nm ultraviolet lamp is captured.

[0008] To achieve the above purpose, the application provides the following scheme:

[0009] The second technical solution of the application is a method for detecting vegetable surface analysis experiment, comprising the following steps:

[0010] (1) Several types of vegetable samples including wood ear, spinach and baby vegetables are purchased from local supermarkets.

[0011] (2) The experimental group of vegetables: take wood ear, spinach and baby vegetables as examples, respectively spray 10uM, 50uM and 100uM of benfluralin solution, while the control group is pretreated with water. Then, the vegetables are dried to simulate actual benfluralin residues. Subsequently, the fluorescent probe solution 10uM is sprayed on the surface of the vegetables.

[0012] (3) The experimental group of vegetables: take sweet potato and potato as examples, respectively spray 400uM, 800uM, 1mM and 2.5mM of benfluralin solution, while the control group is pretreated with water. The vegetables are dried, and the test paper is soaked in 40uM fluorescent probe solution, then the test paper is attached to the surface of the dried vegetables.

[0013] (4)Finally, the photos of these sprays and test papers were captured by a smartphone under natural light and dark environment with 365nm ultraviolet light irradiation.

[0014] Based on the above technical scheme, the present application has the following technical effects: six rhodamine B-based fluorochloridone fluorescent probes are designed and synthesized. Probe 4 has extremely high sensitivity and selectivity to fluorochloridone, and has a clear fluorescence "off-on" mode. After adding fluorochloridone, the color of the solution can be observed to change from colorless to red with the naked eye. In order to prove the practicability of the probe, soil surface analysis experiments are carried out to evaluate the quantitative detection of fluorochloridone in actual samples, and the accuracy of the method is verified.

[0015] Compared with the prior art, the present application has the following beneficial effects:

[0016] (1) A series of fluorochloridone fluorescent probes 1-6 are designed and synthesized, among which probe 4 has strong selectivity and high sensitivity to fluorochloridone.

[0017] (2) By combining the portability of test paper with the RGB value analysis function of a smartphone, a portable, simple, accurate and qualitative and quantitative detection sensor platform for detecting the content of fluorochloridone is established.

[0018] (3) Quantitative and qualitative detection of fluorochloridone is realized through soil surface analysis experiments.

[0019] (4) Visualization research on Arabidopsis thaliana root tips and zebrafish is realized through confocal laser scanning microscopy, and quantitative detection of fluorochloridone by probe 4 is realized. BRIEF DESCRIPTION OF DRAWINGS

[0020] Figure 1 : Fluorescence spectra and enhancement multiples of probes 1 to 6;

[0021] Figure 2 : Soil treated with fluorochloridone (0, 50, 100, 200, 300 μM) was placed in a culture dish containing a solution of probe 4 (10.0 μM), and imaged under 365nm ultraviolet light;

[0022] Figure 3 : Photos of wood ear, spinach and baby bok choy containing different concentrations of fluorochloridone under ultraviolet light (365nm) irradiation;

[0023] Figure 4 : Application diagram of test paper sensor on the surface of vegetables: sweet potato and potato. (a) Schematic diagram of fluorochloridone detection in test paper. (b) Photos of test paper for detecting different concentrations of fluorochloridone. (c) Photos of test paper used in experiments on the surface of sweet potato and potato: testing different concentrations of fluorochloridone.

[0024] Figure 5 Fluorescence imaging of Arabidopsis roots with fentrazamide (20 μΜ, 50 μΜ, 100 μΜ), where the scale bar: 20 μιη.

[0025] Figure 6 Zebrafish imaging experiment. (a) Schematic diagram of probe 4 for zebrafish fentrazamide (20 μΜ, 50 μΜ, 80 μΜ) detection. (b) Schematic diagram of zebrafish embryo digestive system simulation (1 : gall bladder; 2: intestine). (c) Fluorescence microscope images of zebrafish fentrazamide detection with probe 4, where the scale bar: 200 μιη. DETAILED DESCRIPTION

[0026] The detailed description set forth below is intended as a description of various example embodiments of the application and is not intended to represent the only embodiments in which the application can be practiced. The detailed description is intended to be read with the understanding that both the comprehensive and careful presentation of the details as well as the careful presentation of the completely detailed description are not intended to limit the scope of the application but merely to illustrate certain aspects of the application.

[0027] It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the application. Additionally, the use of the term "about" in relation to a value or a range of values is intended to include each individual intermediate value and each smaller range that falls within the range of values. The upper and lower limits of these smaller ranges can be independently included or excluded from the range.

[0028] Unless defined otherwise, 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 application belongs. Although preferred methods and materials are described, any methods and materials similar or equivalent to those described herein can be used in the practice of the present application. All documents mentioned herein are incorporated by reference to disclose and describe the methods and / or materials in connection with which the documents are cited. In case of conflict between the present specification and any document incorporated by reference, the present specification will control.

[0029] Various modifications and changes can be made to the specific embodiments of the application described herein without departing from the scope or spirit of the application. Other embodiments of the application will be apparent to those of ordinary skill in the art from the description and examples presented herein. The specification and examples given should be considered exemplary only, with the true scope of the application being indicated by the following claims.

[0030] As used herein, the terms "comprises", "comprising", "includes", "including", "has", "having", "contains", "containing", or variations thereof, are intended to be open-ended terms that mean including, but not limited to.

[0031] The technical solutions of the present application are conventional solutions in the art if not specifically stated, and the reagents or raw materials used are purchased from commercial channels or are publicly disclosed if not specifically stated.

[0032] Example 1

[0033] The synthesis method of probes 1-6 includes the following steps:

[0034] (1) Probes 1-5: Rhodamine B (200 mg, 450.89 μmol), EDCI (103.72 mg, 541.07 μmol) and HOBT (54.18 mg, 450.89 μmol) were dissolved in acetonitrile (8 mL), stirred at room temperature for 30 min, and then 2-amino oxazole, 2-amino thiazole, 2-amino benzoxazole, 2-amino benzothiazole, 5-methyl-2-amino benzoxazole, 5-nitro-2-amino benzoxazole (73.11 mg, 450.89 μmol) were slowly added dropwise into the above solution, and the reaction mixture was stirred at room temperature for 4 h. After the reaction was completed, the excess solvent was removed by pressure. The obtained residue was purified by silica gel column chromatography (eluent: PE:EA = 2:1, v:v) to obtain probes 1-5 (yield: 85%, 51%, 78%, 70%, 45%, respectively).

[0035] (2) Probe 6: 0.5 mL of phosphorus oxychloride was added to a stirred solution of Rhodamine B (0.3 g, 0.6 mol) and 1,2-dichloroethane (7 mL). The solution was refluxed for 4 h and evaporated to concentrate. The obtained crude chloric acid was dissolved in acetonitrile (2 ml). 5-nitro-2-benzoxazole amine (0.1340 g, 0.001 mol) and triethylamine (0.5 mL) were added to acetonitrile (7 mL), and the obtained crude chloric acid solution was added dropwise into it within 30 min. After refluxing for 4 h, the solvent was removed under reduced pressure, and probe 6 was obtained as a light yellow crystal (0.05 g, yield: 12%) after purification by silica gel column chromatography (eluent: PE:EA = 2:1, v:v).

[0036]

[0037] The physicochemical properties, nuclear magnetic resonance and high resolution mass spectrometry of the synthesized probes 1 to 6 are shown in Table 1.

[0038] Table 1. Physicochemical properties, nuclear magnetic resonance and high resolution mass spectrometry data of the target probes

[0039]

[0040]

[0041] Performance test of probes 1 to 6 on SA spectrum:

[0042] We test evaluated the fluorescence spectrum and enhancement factor of probe 1 to probe 6, as shown in Figure 1 Under the same conditions, the fluorescence intensity of the 6 probes (10 μM) after adding 10 μM benzenesulfonamide herbicide was compared (a) and the enhancement factor (b), as shown in the fluorescence intensity of probe 4 was significantly enhanced by 7.68221 times, and the fluorescence intensity of probe 1, probe 2, probe 3, probe 5 and probe 6 was enhanced by 2.78194 times, 2.85285 times, 3.40891 times, 3.79072 and 3.94263 times, respectively. In summary, the experiment clearly shows that probe 4 has the optimal sensitivity enhancement factor for benzenesulfonamide herbicide. Therefore, in the follow-up study, we prefer to choose probe 4 as the probe for detecting benzenesulfonamide herbicide.

[0043] Example 2

[0044] In order to further study the feasibility of probe 1 in detecting benzenesulfonamide herbicide in the environment, we selected soil samples collected from local fields for detection and qualitative analysis experiments.

[0045] Experimental method:

[0046] (1) Collect soil from local farmland without any pretreatment.

[0047] (2) Put 1 g of soil sample in a culture dish and divide it into two groups: control group and experimental group. The control group is sprayed with 10 μM probe 4 solution for imaging; the experimental group is sprayed with 1 mM, 5 mM and 10 mM benzenesulfonamide herbicide, then 10 μM probe 4 solution is sprayed on the surface of the soil, and finally the photo under 365 nm ultraviolet light is taken.

[0048] The results show that in the initial soil surface imaging experiment, there is no obvious difference in fluorescence between the uncontaminated soil and the benzenesulfonamide herbicide contaminated soil. However, when probe 4 solution is sprayed on the surface of the soil contaminated by different concentrations of benzenesulfonamide herbicide, obvious red fluorescence emission is observed under ultraviolet light, as shown in Figure 2 The results show that fluorescence probe 4 can effectively detect the contamination of benzenesulfonamide herbicide in soil, and the fluorescence signal is clear and easy to identify. These results show that fluorescence probe 4 can be effectively used for on-site, sensitive and selective detection of benzenesulfonamide herbicide in actual soil samples.

[0049] Example 3

[0050] Application of fluorescence probe 4 to detect benzenesulfonamide herbicide in wood ear, spinach and baby bok choy. As a widely used herbicide, it is inevitable that some residues will remain on the surface of vegetables during the spraying of benzenesulfonamide herbicide. Therefore, it is urgent to develop an effective detection system for benzenesulfonamide herbicide to ensure the safety and quality of agricultural products and protect public health.

[0051] 1. Experimental method:

[0052] (1) Several types of vegetable samples were purchased from local supermarkets including: wood ear, spinach, and baby corn.

[0053] (2) The experimental group of vegetables: wood ear, spinach, and baby corn were sprayed with 10 μΜ, 50 μΜ, and 100 μΜ of fomesafen solution, respectively, while the control group was pretreated with water. Then, the vegetables were air-dried to simulate the actual fomesafen residue. Subsequently, 40 μΜ of fluorescent probe 4 solution was sprayed onto the surface of the vegetables.

[0054] (3) Finally, the photos of these sprays and strips were captured by a smartphone under the illumination of a 365 nm ultraviolet lamp in a dark environment.

[0055] The results show that probe 4 can be used to detect fomesafen in various actual samples. As shown in Figure 3 , the preparation and imaging process is revealed. The vegetables were sprayed with different concentrations of fomesafen solution, and then 40 μΜ of fluorescent probe 4 solution was sprayed on the surface of the vegetables. The colorless turned red can be clearly observed with the naked eye under natural light. This study shows that probe 4 can be used as a direct and effective tool for detecting fomesafen, thereby ensuring environmental, agricultural product safety, and human health.

[0056] Example 4

[0057] To evaluate the practical application of fluorescent probe 4-loaded test strips, fomesafen detection was performed on actual agricultural products. As shown in Figure 4 , the test strips were placed on the surface of vegetables with different concentrations of fomesafen, and the fluorescence change was obvious.

[0058] Experimental method:

[0059] (1) The experimental group of sweet potatoes and potatoes were sprayed with fomesafen solution: 400 μΜ, 800 μΜ, and 1 mM pretreatment, respectively, while the control group of sweet potatoes and potatoes were treated with water. Subsequently, these vegetables were air-dried. After the test strips were soaked in 40 μΜ of fluorescent probe 4 solution, the test strips were attached to the surface of the vegetables for detection.

[0060] (2) Finally, the photos of these sprays and strips were captured by a smartphone under the illumination of a 365 nm ultraviolet lamp in a dark environment.

[0061] The results show that different concentrations of fomesafen solution were sprayed on the surface of the vegetables to simulate the presence of fomesafen residues, and then the prepared 40 μΜ of fluorescent probe 4 was loaded on the test strips and attached to the surface of the fruits. As shown in Figure 3The photos were taken under natural light and 365 nm ultraviolet light. Under natural light, as the concentration of benzenesulfonamide herbicide increased, the test paper gradually turned pink. In addition, the color of the test paper also transitioned from cyan fluorescence to pink fluorescence and gradually deepened under ultraviolet irradiation. Probe 4 can reliably and quantitatively detect benzenesulfonamide herbicide residues in actual agricultural products

[0062] Example 5

[0063] To further verify the precision and accuracy of the fluorescence probe 4 in detecting the content of benzenesulfonamide herbicide in food samples, the recovery rate was 92.9% to 106.3%, as shown in Table 2. The preparation and detection procedures are as follows Figure 3

[0064] Table 2. Recovery rate determination of benzenesulfonamide herbicide in various actual agricultural and sideline products.

[0065]

[0066]

[0067] Experimental method:

[0068] In the quantitative experiment of agricultural products, the sample was cut into small pieces, each piece weighing 1 g, soaked in 5 mL of deionized distilled water or a certain amount of benzenesulfonamide herbicide solution, the mixture was ultrasonically treated for 5 minutes, and the supernatant extracted from each sample was used as the test solution. Different concentrations of benzenesulfonamide herbicide were added to the solution with probe 4 for spectral testing, and the spectral signal was recorded at a wavelength of 588 nm, with each experiment being performed in triplicate.

[0069] Example 6

[0070] Fluorescence imaging technology is an important technology in chemical biology for observing and monitoring various species to understand their pathogenicity or function. The present application utilizes laser confocal fluorescence microscopy for imaging applications of benzenesulfonamide herbicide in Arabidopsis thaliana root tips.

[0071] 1. Experimental method:

[0072] (1) Arabidopsis thaliana samples were placed in culture dishes and divided into two groups: a control group and an experimental group;

[0073] (2) The control group was soaked in 10 μM of fluorescence probe 4 solution for 0.5 h; the experimental group was soaked in 0.5 h of Arabidopsis thaliana root tips, washed three times with deionized distilled water, and then soaked in 20 μM, 50 μM, and 100 μM of benzenesulfonamide herbicide for 0.5 h;

[0074] (3) Finally, the photos were taken under a laser scanning confocal microscope.

[0075] ​Results show that no fluorescence signal was detected in the root tips of Arabidopsis thaliana before and after treatment with fluorescent probe 4. However, when the root system of Arabidopsis thaliana was treated with probe 4 for 30 minutes and then incubated with different concentrations of benfluralin, the fluorescence intensity increased significantly in a concentration-dependent manner. These results indicate that fluorescent probe 4 can effectively penetrate the cell wall of the root tip tissue and monitor and quantitatively detect benfluralin, as shown in Figure 5 .

[0076] Example 7

[0077] Benfluralin inevitably enters the aquatic ecosystem and poses a threat to aquatic organisms. Therefore, we used zebrafish as a model to perform fluorescence imaging experiments on the enrichment of benfluralin.

[0078] 1. Experimental method

[0079] (1) Zebrafish samples were placed in culture dishes and divided into two groups: a control group and an experimental group;

[0080] (2) The control group was incubated with 10 μM fluorescent probe 4 solution for 10 min; the experimental group was incubated with 20 μM, 50 μM, and 80 μM benfluralin for 5 min after being washed three times with deionized distilled water;

[0081] (3) Finally, photographs were taken under a laser scanning confocal microscope;

[0082] The results show that no obvious fluorescence signal was detected before and after treatment with fluorescent probe 4, indicating that the probe can image without interference. In contrast, the fluorescence intensity in the eyes, gallbladder, and intestines of zebrafish incubated with benfluralin and fluorescent probe 4 increased significantly. At the same time, as the concentration of benfluralin increased, the fluorescence intensity increased, as shown in Figure 6 , which further verifies that fluorescent probe 4 has good biocompatibility and high detection performance.

Claims

1. Application of rhodamine fluorescent probe in detection of herbicide benzfendizone, characterized in that: The structure is shown as formula (I) below: using rhodamine fluorescent probe 1-6 can quickly and real-time detect fluorodifen in environment and agricultural products and be applied to biological imaging; 2. The application of rhodamine fluorescent probe in detecting herbicide benzfendizine according to claim 1, characterized in that: The synthesis method of the designed fluorescent probe is as follows:

3. The application of rhodamine fluorescent probe in detecting herbicide benzfendizone according to claim 1, characterized in that: The above rhodamine-based fluorescent probe 4 can be used to quickly and real-time detect fluorodifen in environment and agricultural products and be applied to biological imaging. ​ 4.The application of rhodamine fluorescent probe in the detection of benzenesulfonamide herbicide according to claim 1, characterized in that: A method for detecting the content of fluorodifen is designed and synthesized, specifically, the test paper is immersed in 40 muM fluorescent probe solution for 1 hour, then dried at room temperature, cut into multiple hearts, placed on a glass slide, different concentrations of fluorodifen are dropped on the test paper as the experimental group, and water treatment as the control group, let the droplets completely cover the surface of the test paper, then dry at room temperature, and take the fluorescence image of the paper under natural light and 365nm ultraviolet lamp irradiation in the dark environment.

5. The application of rhodamine fluorescent probe in detecting herbicide benzfendizone according to claim 4, characterized in that: The specific method for detecting fluorodifen in soil using rhodamine-based fluorescent probe is as follows: 1g of soil sample is placed in a culture dish and divided into two groups: control group and experimental group, the soil surface of the experimental group is respectively sprayed with 50 muM, 100 muM, 200 muM, 300 muM fluorodifen solution for pretreatment, and the soil surface of the control group is treated with water, the soil is naturally dried, 10-40 muM fluorescent probe solution is sprayed on the soil surface, and finally the photo is taken by smart phone under 365nm ultraviolet lamp irradiation in the dark environment.

6. The application of rhodamine fluorescent probe in detecting herbicide benzfendizone according to claim 4, characterized in that: The specific method for detecting fluorodifen in agricultural products using rhodamine-based fluorescent probe is as follows: (1) The experimental group of vegetables: agaric, spinach and baby vegetables, respectively spray 10 muM, 50 muM and 100 muM fluorodifen solution, while the control group is pretreated with water, the surface solution of the vegetables is naturally dried to simulate the actual fluorodifen residue, 10 muM fluorescent probe solution is sprayed on the surface of the vegetables, and finally the photo is taken by smart phone under 365nm ultraviolet lamp irradiation in the dark environment; (2) The experimental group of vegetables: sweet potato, potato, cut into small pieces, each piece 1g, soaked in 5mL deionized distilled water or a certain amount of fluorodifen solution, the mixture is ultrasonically treated for 5 minutes, and the supernatant extracted from each sample is used as the test solution, 400 muM, 800 muM, 1mM and 2.5mM fluorodifen solution is respectively sprayed in the solution with fluorescent probe, while the control group is pretreated with water, the surface solution is naturally dried, the test paper is immersed in 40 muM fluorescent probe solution, then the test paper is pasted on the surface of the vegetables, and finally the photo is taken by smart phone or the spectral signal is recorded at 588nm wavelength under natural light and 365nm ultraviolet lamp irradiation in the dark environment, each experiment is repeated three times; (3) The Arabidopsis samples are divided into two groups: control group and experimental group, the control group is soaked in 10 muM fluorescent probe solution for 0.5h; the experimental group of Arabidopsis is incubated in 10 muM fluorescent probe solution for 0.5h, then washed with deionized water for three times, and then incubated in 20 muM, 50 muM and 100 muM fluorodifen solution for 0.5h, finally the photo is taken under laser confocal microscope.

7. The application of rhodamine fluorescent probe in detecting herbicide benzfendizone according to claim 5, characterized in that: the herbicide is benzfendizone. The specific method for detecting flumetralin in animal samples using rhodamine-based fluorescent probes is as follows: zebrafish samples are divided into two groups: a control group and an experimental group. The zebrafish in the experimental group are incubated in a 10 μM fluorescent probe solution for 10 minutes, then washed three times with deionized water, and then placed in 20 μM, 50 μM and 80 μM flumetralin solutions for 5 minutes. Finally, photographs are taken under a laser scanning confocal microscope.

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