Application of rhodamine-based fluorescent probe in detection of herbicide fomesafen

Abstract

The invention discloses application of a fomesafen specific fluorescent probe based on rhodamine skeleton fluorene. The fomesafen specific fluorescent probe can be used for detecting fomesafen in agricultural products, environmental samples and biological samples. The method specifically comprises the following steps: immersing test paper into a 40 [mu] M probe 4 solution for 1 h, airing at room temperature, cutting the test paper into a plurality of love hearts, placing the love hearts on a glass slide, dripping fomesafen with different concentrations on the test paper to form an experimental group, performing water treatment to form a control group, enabling liquid drops to completely cover the surface of the test paper, airing at room temperature, and determining the fomesafen content of the test paper. A fluorescent probe 4 is arranged on a paper strip, a fluorescent image of the paper strip is shot under irradiation of a 365 nm ultraviolet lamp in a natural light and dark environment, the fluorescent probe 4 shows excellent performance, the fluorescent probe 4 is integrated into test paper and hydrogel, and portable and instantaneous detection in an actual sample is facilitated in combination with a platform based on a smart phone. The method is a portable, simple and accurate method for detecting the fomesafen content in real time.

Description

Technical Field

[0001] The invention relates to the field of rapid detection technology of environment and agricultural products and biological imaging, and in particular to a rhodamine-based fluorescent probe and a preparation method and application thereof. Background Art

[0002] As the global population continues to increase, the demand for food supply is also increasing. In order to increase food production, pesticides have become an important tool for disease prevention, weed control, and pest control in modern agriculture. Herbicides, as the largest category of pesticides, account for about 30% of China's pesticides in total output. Among them, fomesafen is widely used in the control of broadleaf weeds in farmland due to its high selectivity and excellent weed control effect. Even at 280-430g / hm 2 Fomesulfuron has strong biological activity even at low application rates and has become one of the most widely used herbicides worldwide. However, its slow degradation and long-term presence in soil pose major challenges, and long-term and intensive application can seriously affect the succession of sensitive crops and increase the risk of crop damage in subsequent planting seasons. In addition, it destroys soil microbial communities and enzyme activities, leading to reduced crop yields. The widespread use of fomesulfuron, and sometimes even improper use, has also led to the accumulation of residues on the surface and inside of food products. Ingestion of food or water contaminated with excessive fomesulfuron residues can cause serious health risks, including neurotoxicity, immune dysfunction, and liver damage. Given its harmful effects on the environment, ecosystems, and human health, there is an urgent need to develop effective fomesulfuron detection systems. These systems are essential to ensure the safety and quality of agricultural products and protect public health.

[0003] To date, various methods have been developed for the detection of fomesafen, including HPLC-PDA, LC-MS / MS, nanozymes, immunoassays, etc. However, these techniques are often limited by complex procedures, time-consuming, high cost, dependence on specialized equipment, and the need for skilled operators, which restrict their real-time detection and application in the field. Currently, fluorescent probes have the advantages of simple operation, rapid response, visualization, and suitability for real-time analysis, and they occupy a prominent position in applications such as environmental pollution monitoring, hazardous substance detection, and food safety. Among them, rhodamine has excellent photophysical properties, high biocompatibility, superior fluorescence quantum yield, and switchable spironolactone "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 fomesafen, mainly because of the low chemical reactivity of fomesafen. Therefore, it is a major challenge to design fast-response fluorescent probes for the detection of fomesafen. Summary of the invention

[0004] The main content of the present invention is that a series of new rhodamine-based fluorescent probes have been developed for the first time to achieve specific, rapid and real-time detection of fomesulfuron in environmental samples and agricultural products, and applied to biological imaging. Among them, probe 4 shows excellent performance, with high sensitivity, selectivity and a fast response time of less than 60 seconds, and can be used for fomesulfuron detection in environmental and agricultural environments. In addition, probe 4 is integrated into a multifunctional detection platform that combines test paper and image analysis software. This innovative method creates an intelligent visualization system with a powerful ability to detect fomesulfuron residues in agricultural products. The probe further realizes real-time monitoring of the distribution of fomesulfuron in Arabidopsis root tips and zebrafish, providing valuable visual data for exploring the application of fomesulfuron imaging in organisms.

[0005] One of the technical solutions of the present invention is a method for qualitatively detecting soil surface analysis experiments, comprising the following steps:

[0006] (1) Soil was collected from local farmland without any pretreatment.

[0007] (2) 1g soil sample was placed in a petri dish and divided into two groups: a control group and an experimental group. The control group was sprayed with 10μM fluorescent probe solution for imaging; the experimental group was sprayed with 50μM, 100μM, 200μM, and 300μM of different concentrations of fomesafen, and then 10μM fluorescent probe solution was sprayed on the soil surface, and finally photos were taken under 365nm ultraviolet light.

[0008] To achieve the above object, the present invention provides the following solutions:

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

[0010] (1) Several vegetable samples including wood ear vegetable, spinach and baby cabbage were purchased from a local supermarket.

[0011] (2) Vegetables in the experimental group: Taking Auricularia auricula, spinach and baby cabbage as examples, they were sprayed with 10μM, 50μM and 100μM fomesafen solutions respectively, while the control group was pretreated with water. Then, the vegetables were air-dried to simulate the actual fomesafen residue. Subsequently, 10μM of the fluorescent probe solution was sprayed on the surface of the vegetables.

[0012] (3) Vegetables in the experimental group: Taking sweet potatoes and potatoes as examples, they were sprayed with 400 μM, 800 μM, 1 mM and 2.5 mM fomesafen solutions, respectively. The control group was pretreated with water. The vegetables were air-dried, and the test paper was soaked in a 40 μM fluorescent probe solution and then attached to the surface of the dry vegetables.

[0013] (4) Finally, a smartphone was used to capture photos of the spray and test strips under natural light and 365 nm UV light in a dark environment.

[0014] Based on the above technical scheme, the present invention has the following technical effects: The present invention designs and synthesizes six flufenacet fluorescent probes based on rhodamine B. Probe 4 has extremely high sensitivity and selectivity to flufenacet and has an obvious fluorescence "off-on" mode. After adding flufenacet, the color of the solution can be observed to change from colorless to red with the naked eye. In order to prove the practicality of the probe, we conducted a soil surface analysis experiment to evaluate the quantitative detection of flufenacet in actual samples and verify the accuracy of its method.

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

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

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

[0018] (3) Quantitative and qualitative detection of fomesafen was achieved through soil surface analysis experiments.

[0019] (4) Using confocal laser scanning microscopy, we visualized the root tips of Arabidopsis thaliana and zebrafish and achieved quantitative detection of fomesafen by probe 4. BRIEF DESCRIPTION OF THE DRAWINGS

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

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

[0022] Figure 3 : Photos of Auricularia auricula, spinach and baby cabbage containing different concentrations of fomesafen under ultraviolet light (365nm);

[0023] Figure 4 : Application diagram of test paper sensors on vegetable surfaces: sweet potatoes and potatoes. (a) Schematic diagram of fomesulfuron detection in test paper. (b) Photo of test paper used to detect different concentrations of fomesulfuron. (c) Photo of test paper used in sweet potato and potato skin experiments: testing different concentrations of fomesulfuron.

[0024] Figure 5 : Fluorescence imaging of fomesafen (20μM, 50μM, 100μM) in Arabidopsis roots, where scale bar: 20μm.

[0025] Figure 6 : Zebrafish imaging experiment. (a) Schematic diagram of probe 4 used for zebrafish fomesafen (20μM, 50μM, 80μM) detection. (b) Schematic diagram of zebrafish embryo digestive system simulation (1: gallbladder; 2: intestine). (c) Fluorescence microscope image of zebrafish fomesafen detection probe 4, where the scale bar is 200μm. DETAILED DESCRIPTION

[0026] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0027] It should be understood that the terms described in the present invention are only for describing special embodiments and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. Each smaller range between the intermediate value in any stated value or stated range and any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0028] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0029] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present application description and examples are exemplary only.

[0030] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0031] The technical solutions described in the present invention, unless otherwise specified, are all conventional solutions in the art, and the reagents or raw materials used, unless otherwise specified, are purchased from commercial channels or have been disclosed.

[0032] Example 1

[0033] The synthesis method of probes 1 to 6 comprises 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). After stirring at room temperature for 30 minutes, 2-aminooxazole, 2-aminothiazole, 2-aminobenzoxazole, 2-aminobenzothiazole, 5-methyl-2-aminobenzoxazole, and 5-nitro-2-aminobenzoxazole dissolved in acetonitrile (73.11 mg, 450.89 μmol) were slowly added dropwise to the above solution. The reaction mixture was stirred at room temperature for 4 hours. After the reaction was completed, the excess solvent was removed under pressure. The residue was purified by silica gel column chromatography (eluent: PE:EA = 2:1, v:v) to obtain probes 1-5 (yields: 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 hours and concentrated by evaporation. The obtained crude chloric acid was dissolved in acetonitrile (2 ml). 5-Nitro-2-benzoxazolamine (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 thereto within 30 min. After reflux for 4 h, the solvent was removed under reduced pressure and purified by silica gel column chromatography (eluent: PE:EA=2:1, v:v). Probe 6 was light yellow crystals (0.05 g, yield: 12%).

[0036]

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

[0038] Table 1. Physicochemical properties, NMR and high-resolution mass spectrometry data of target probes

[0039]

[0040]

[0041] Probe 1 to probe 6 for SA spectrum performance test:

[0042] We tested and evaluated the fluorescence spectra and enhancement factors of probes 1 to 6, such as Figure 1 As shown in the figure, under the same conditions, the fluorescence intensity of the six probes (10 μM) after adding 10 μM fomesafen (a) and the enhancement factor (b), 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 times and 3.94263 times, respectively. In summary, the experiment clearly shows that probe 4 has the best sensitivity enhancement factor for fomesafen. Therefore, in subsequent studies, we prefer probe 4 as the probe for fomesafen detection.

[0043] Example 2

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

[0045] Experimental methods:

[0046] (1) Soil was collected from local farmland without any pretreatment.

[0047] (2) 1g soil sample was placed in a petri dish and divided into two groups: a control group and an experimental group. The control group was sprayed with 10μM probe 4 solution for imaging; the experimental group was sprayed with different concentrations of 1mM, 5mM and 10mM fomesafen, and then 10μM probe 4 solution was sprayed on the soil surface, and finally photos were taken under 365nm ultraviolet light.

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

[0049] Example 3

[0050] Fluorescent probe 4 was used to detect fomesulfuron in Auricularia auricula, spinach and baby cabbage. As a widely used herbicide, it is inevitable that some fomesulfuron will remain on the surface of vegetables during the spraying process. Therefore, it is urgent to develop an effective fomesulfuron detection system to ensure the safety and quality of agricultural products and protect public health.

[0051] 1. Experimental methods:

[0052] (1) Several vegetable samples including wood ear vegetable, spinach and baby cabbage were purchased from a local supermarket.

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

[0054] (3) Finally, a smartphone was used to capture photos of the spray and paper strips under 365nm UV light in a dark environment.

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

[0056] Example 4

[0057] In order to evaluate the practical application of the fluorescent probe 4 loaded test strips, fomesafen was detected on actual agricultural products. Figure 4 As shown, when the test strips were placed on the surface of vegetables with different fomesafen concentrations, the fluorescence changes were obvious.

[0058] Experimental methods:

[0059] (1) The surfaces of sweet potatoes and potatoes in the experimental group were sprayed with 400 μM, 800 μM, and 1 mM fomesafen solutions, respectively, and the surfaces of sweet potatoes and potatoes in the control group were treated with water. Subsequently, the vegetables were air-dried. After the test paper was soaked in a 40 μM solution of fluorescent probe 4, it was attached to the surface of the vegetables for detection.

[0060] (2) Finally, photos of the spray and paper strips were captured by a smartphone under natural light and dark conditions using a 365nm UV light.

[0061] The results showed that different concentrations of fomesafen solutions were sprayed on the surface of vegetables to simulate the presence of fomesafen residues, and then the prepared 40 μM fluorescent probe 4 was loaded on the test paper and attached to the surface of these fruits. Figure 3As shown, the photos were taken under natural light and 365nm ultraviolet light. Under natural light, as the concentration of fomesafen increases, the colorless test paper gradually turns pink. In addition, the color of the test paper also transitions from cyan fluorescence to pink fluorescence and gradually deepens under ultraviolet light. Probe 4 can reliably and quantitatively detect fomesafen residues in actual agricultural products.

[0062] Example 5

[0063] The precision and accuracy of fluorescent probe 4 in detecting the content of fomesafen in food samples were further verified, and the recovery rate was 92.9% to 106.3%, as shown in Table 2. Preparation and detection procedures are as follows Figure 3 shown.

[0064] Table 2. Determination of the recovery of fomesafen in various actual agricultural and sideline products.

[0065]

[0066]

[0067] Experimental methods:

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

[0069] Example 6

[0070] Fluorescence imaging is an important technique in chemical biology for observing and monitoring various species to understand their pathogenicity or function. The present invention uses laser confocal fluorescence microscopy to image fomesafen in Arabidopsis root tips.

[0071] 1. Experimental methods:

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

[0073] (2) The control group soaked the Arabidopsis root tips with 10 μM fluorescent probe 4 solution for 0.5 h; the experimental group took out the Arabidopsis rhizomes that had been soaked for 0.5 h, washed them three times with deionized distilled water, and then soaked them with 20 μM, 50 μM, and 100 μM fomesafen at different concentrations for 0.5 h;

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

[0075] The results showed that no fluorescence signal was emitted from the root tips of Arabidopsis thaliana, whether untreated or treated with fluorescent probe 4. However, when the roots of Arabidopsis thaliana were treated with probe 4 for 30 minutes and then incubated with different concentrations of fomesafen, the fluorescence intensity increased significantly in a concentration-dependent manner. These results indicate that fluorescent probe 4 can effectively penetrate the cell wall of root tip tissue and monitor and quantitatively detect fomesafen. Figure 5 shown.

[0076] Example 7

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

[0078] 1. Experimental methods

[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 incubated zebrafish with 10 μM fluorescent probe 4 solution for 10 min; the experimental group took out the zebrafish after incubation for 10 min, washed them three times with deionized distilled water, and then soaked them in different concentrations of 20 μM, 50 μM, and 80 μM fomesafen for 5 min;

[0081] (3) Finally, take photos under a laser scanning confocal microscope;

[0082] The results showed that no obvious fluorescence signal was found before and after the treatment with fluorescent probe 4, indicating that the probe can be imaged without interference. In contrast, the fluorescence intensity in the eyes, gallbladder and intestine of zebrafish incubated with fomesafen and fluorescent probe 4 increased significantly. At the same time, as the concentration of fomesafen increased, the fluorescence intensity increased. Figure 6 As shown, the experimental results further verified that fluorescent probe 4 has good biocompatibility and efficient detection performance.

Claims

1. An application of a rhodamine fluorescent probe in detecting the herbicide fomesafen, characterized in that: Its structure is shown in the following formula (I): The use of rhodamine-based fluorescent probes 1-6 can be used to quickly detect fomesafen in the environment and agricultural products in real time and applied to biological imaging; 2. The use of a rhodamine fluorescent probe in detecting the herbicide fomesafen according to claim 1, characterized in that: The synthesis method of the designed fluorescent probe is as follows:

3. The use of a rhodamine fluorescent probe in detecting the herbicide fomesafen according to claim 1, characterized in that: The above-mentioned rhodamine-based fluorescent probe 4 can be selected for rapid and real-time detection of fomesafen in the environment and agricultural products and applied to biological imaging.

4. The use of a rhodamine fluorescent probe in detecting fomesafen according to claim 1, characterized in that: A portable, simple, accurate and real-time method for detecting the content of fomesafen was designed and synthesized. Specifically, the test paper was immersed in a 40 μM fluorescent probe solution for 1 hour and then dried at room temperature. The test paper was cut into multiple hearts and placed on a glass slide. Different concentrations of fomesafen were dropped on the test paper as the experimental group, and water treatment was used as the control group. The droplets were allowed to completely cover the surface of the test paper, and then dried at room temperature. The fluorescent image of the paper was taken under natural light and 365 nm ultraviolet light in a dark environment.

5. The use of a rhodamine fluorescent probe in detecting the herbicide fomesafen according to claim 4, characterized in that: The specific method for detecting fomesafen in soil using rhodamine-based fluorescent probe is as follows: 1 g of soil sample was placed in a culture dish and divided into two groups: a control group and an experimental group. The soil surface of the experimental group was pretreated by spraying 50 μM, 100 μM, 200 μM, and 300 μM fomesafen solutions, respectively. The soil surface of the control group was treated with water. The soil was dried naturally, and 10-40 μM fluorescent probe solution was sprayed on the soil surface. Finally, photos were taken with a smartphone under 365 nm ultraviolet light in a dark environment.

6. The use of a rhodamine fluorescent probe in detecting the herbicide fomesafen according to claim 4, characterized in that: The specific method for detecting fomesulfuron in agricultural products using rhodamine-based fluorescent probe is as follows: (1) The vegetables in the experimental group, including Auricularia auricula, spinach and baby cabbage, were sprayed with 10 μM, 50 μM and 100 μM fomesafen solutions, respectively, while the control group was pretreated with water. The solution on the surface of the vegetables was naturally dried to simulate the actual fomesafen residue, and a 10 μM fluorescent probe solution was sprayed on the surface of the vegetables. Finally, photos were taken with a smartphone under 365 nm ultraviolet light in a dark environment; (2) Vegetables in the experimental group: sweet potato and potato. Cut the samples into small pieces, 1 g each, and soak them in 5 mL of deionized distilled water or a certain amount of fomesafen solution. The mixture was ultrasonically treated for 5 minutes, and the supernatant extracted from each sample was used as the test solution. 400 μM, 800 μM, 1 mM and 2.5 mM fomesafen solutions were sprayed in the solution with the fluorescent probe, respectively. The control group was pretreated with water. The surface solution was naturally dried. After the test paper was soaked in the 40 μM fluorescent probe solution, it was attached to the surface of the vegetables. Finally, under natural light and dark environment, 365 nm ultraviolet light was used to irradiate, and photos were taken with a smartphone or the spectral signal was recorded at a wavelength of 588 nm. Each experiment was performed in triplicate. (3) The Arabidopsis samples were divided into two groups: a control group and an experimental group. The Arabidopsis root tips in the control group were immersed in a 10 μM fluorescent probe solution for 0.5 h. The Arabidopsis in the experimental group were incubated in a 10 μM fluorescent probe solution for 0.5 h, then taken out and washed three times with deionized water, and then incubated in 20 μM, 50 μM, and 100 μM fluazifop solutions for 0.5 h, respectively. Finally, photos were taken under a laser confocal microscope.

7. The use of a rhodamine-based fluorescent probe in detecting the herbicide fomesafen according to claim 5, characterized in that: The specific method for detecting fomesafen in animal samples using a rhodamine-based fluorescent probe is as follows: the 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 taken out and washed three times with deionized water, and then incubated in 20 μM, 50 μM, and 80 μM fomesafen solutions for 5 minutes. Finally, photos are taken under a laser scanning confocal microscope.

Images