A Cu for the specific detection of glyphosate 2+ Preparation and application of coordination fluorescent compounds

Abstract

This invention discloses a Cu for the specific detection of glyphosate. 2+ Preparation and application of coordination fluorescent compounds, specifically (E)-N'-((1-hydroxynaphth-2-yl)methylene)-3-oxo-3H-benzo(f)chromene-2-formylhydrazide copper, which can specifically detect glyphosate in vivo; this Cu 2+ Coordination fluorescent compounds are simple to prepare, use readily available raw materials, exhibit good anti-interference ability and strong stability, and possess good cell membrane permeability and low cytotoxicity. They can enter living cells and animal tissues and react with exogenous glyphosate to produce a strong, visually recognizable green fluorescence. Ultraviolet absorption and fluorescence spectroscopy analysis revealed that this Cu... 2+ Coordination fluorescent compounds exhibit superior selectivity for glyphosate under various interfering conditions and strong resistance to interference from the same type of organophosphorus pesticides. They can not only selectively recognize exogenous glyphosate, but also quantitatively detect glyphosate with high sensitivity under various living cell growth environments, and have been successfully applied to live cell and zebrafish imaging.

Description

Technical Field

[0001] This invention relates to the field of organic small molecule fluorescent probe technology, specifically a Cu for the specific detection of glyphosate. 2+ Preparation and application of coordination fluorescent compounds. Background Technology

[0002] Inorganic pollutants in the environment have a wide range of sources and are complex in type. Most of them will enter the human body. When the concentration of these pollutants in the environment exceeds the safety threshold, they may pose a threat to the environment and human health. Glyphosate, as a widely used broad-spectrum non-selective herbicide in agriculture, can lead to large amounts of residues in soil, food and water if used improperly, causing direct or indirect damage to organisms and affecting heredity, development and reproduction. In addition to its widespread use in agriculture, glyphosate is also often used in non-agricultural scenarios, such as weed control in public green spaces, lawns and roadside slopes. In these scenarios, glyphosate may enter the environment through multiple pathways, including residues after spraying, soil infiltration and runoff. With the increase in the amount of glyphosate used, its residues in the environment also increase accordingly, bringing a series of problems to the environment and ecosystem. Therefore, it is urgent to establish a reliable, rapid, convenient and efficient analytical method to detect glyphosate in the environment.

[0003] Currently, there are many methods for detecting glyphosate. Besides high-performance liquid chromatography (HPLC) and gas chromatography (GC), there are other traditional methods such as mass spectrometry (MS) and spectrophotometry. These methods can achieve accurate detection of glyphosate to a certain extent, but they also have some shortcomings. While mass spectrometry has high sensitivity and accuracy, it requires expensive equipment and complex sample pretreatment steps, making it difficult to operate and unsuitable for rapid detection of large quantities of samples. Spectrophotometry mainly includes ultraviolet-visible spectroscopy and infrared spectroscopy. Although it is rapid and simple, its detection sensitivity at low concentrations is low, and it is easily affected by sample matrix interference. In contrast, fluorescent probes have attracted much research attention due to their high specificity and sensitivity. In addition to attracting the interest of researchers, the ease of operation of fluorescent probes is also one of their advantages. Compared with traditional chromatography and mass spectrometry, fluorescent probe methods are simple and fast to operate, requiring no complex instruments or professional skills. They can be performed in the laboratory or on-site, making fluorescent probe technology more operable and practical, suitable for glyphosate monitoring needs in different scenarios. Therefore, due to its excellent characteristics, fluorescent probe technology has broad application prospects in environmental monitoring, agricultural production, life science research, and disease diagnosis. With increasing attention to glyphosate residue issues, fluorescent probe technology will undoubtedly play an increasingly important role in the field of glyphosate detection, providing reliable technical support for protecting the environment and human health.

[0004] Glyphosate molecules contain functional groups such as amino, phosphate, and carboxyl groups, which can react with Cu. 2+ With strong coordination, the fluorescence detection of glyphosate can be based on the substitution of Cu by glyphosate in the fluorescent probe. 2+ The fluorescence change caused by the fluorescence is used to achieve the fluorescence detection of glyphosate. Furthermore, regarding the use of fluorescent Cu... 2+ There are few reports on the recognition of glyphosate by complexes. Based on this, a fluorescent probe (E)-N'-((1-hydroxynaphth-2-yl)methylene)-3-oxo-3H-benzo[f]chromene-2-formylhydrazide was developed, which, along with Cu... 2+ After coordination, it can specifically recognize glyphosate, as demonstrated by NMR and HRMS for Cu. 2+ The coordination fluorescent compound was characterized, and the Cu was verified by Job curves, HRMS, and DFT calculations. 2+ The reaction mechanism of coordination fluorescent compounds with glyphosate, Cu 2+ Coordination fluorescent compounds exhibit high selectivity and strong anti-interference ability for glyphosate, and can effectively identify trace amounts of glyphosate. Furthermore, Cu... 2+ Coordination fluorescent compounds have been successfully applied to imaging exogenous glyphosate in HeLa cells and zebrafish. Therefore, it is necessary to provide a Cu for the specific detection of glyphosate. 2+ The preparation and application of coordination fluorescent compounds solve the above-mentioned technical problems. Summary of the Invention

[0005] The first objective of this invention is to develop a Cu-based method for selectively detecting the environmental pollutant glyphosate. 2+ Coordination fluorescent compound, this Cu 2+ Coordination fluorescent compounds can distinguish the presence of glyphosate even in the presence of interference from other analytes.

[0006] The second objective of this invention is to provide a Cu that can specifically detect the environmental pollutant glyphosate. 2+ Preparation method of coordination fluorescent compounds.

[0007] The third objective of this invention is to provide a method for detecting exogenous glyphosate in solutions, biological tissues, and cells.

[0008] To achieve the above objectives, the present invention provides the following technical solution: a Cu for the specific detection of glyphosate. 2+ Coordination fluorescent compound, wherein Cu 2+ The coordination fluorescent compound is (E)-N'-((1-hydroxynaphth-2-yl)methylene)-3-oxo-3H-benzo[f]chromene-2-carboxylhydrazide copper, with the molecular formula C 25 H 15 CuN2O4, the chemical structural formula is shown in formula (I): ;

[0009] Formula (I).

[0010] A Cu for the specific detection of glyphosate 2+ A method for preparing coordination fluorescent compounds, comprising the following steps:

[0011] (1) First, add 30 mmol of 2-hydroxy-1-naphthaldehyde, 33 mmol of diethyl malonate, 0.5 mL of pyrrolidine and 120 mL of ethanol to a 250 mL three-necked flask and reflux the reaction. Monitor the reaction progress by TLC. After the reaction is completed, slowly pour the reaction solution into 240 mL of water. A solid precipitates out. Filter, wash with water, and dry to obtain a white solid ethyl 3-oxo-3H-benzo[f]chromene-2-carboxylate.

[0012] (2) Next, 10 mmol of ethyl 3-oxo-3H-benzo[f]chromene-2-carboxylate, 80% hydrazine hydrate (50 mmol) and 100 mL of tetrahydrofuran were added to a 250 mL three-necked flask and refluxed for 6 h. After the reaction was complete, the mixture was concentrated to dryness under reduced pressure and recrystallized from ethanol to obtain a yellow solid 3-oxo-3H-benzo[f]chromene-2-carboxyhydrazide.

[0013] (3) Then, 5 mmol of 3-oxo-3H-benzo[f]chromene-2-carboxylhydrazine, 5.5 mmol of 2-hydroxy-1-naphthaldehyde, 0.5 mL of glacial acetic acid and 100 mL of anhydrous ethanol were added to a 250 mL three-necked flask and refluxed for 15 h. After cooling to room temperature, the mixture was filtered and dried, and recrystallized using N,N-dimethylformamide to obtain the fluorescent probe (E)-N'-((1-hydroxynaphth-2-yl)methylene)-3-oxo-3H-benzo[f]chromene-2-carboxylhydrazine (yellow solid, yield 68%).

[0014] (4) Then, 10 mmol of (E)-N'-((1-hydroxynaphth-2-yl)methylene)-3-oxo-3H-benzo[f]chromene-2-carboxylhydrazine and 10 mmol of copper sulfate were dissolved in a molar ratio of 1:1 in a mixed solution of N,N-dimethylformamide (DMF) and 4-hydroxyethylpiperazine ethanesulfonic acid (HEPES) (DMSO:HEPES=7:3) to obtain Cu 2+ Coordination fluorescent compounds.

[0015] Applications of Cu2+ coordination fluorescent compounds for the specific detection of glyphosate.

[0016] Includes the following steps:

[0017] (1) First, the Cu 2+The coordination fluorescent compound was prepared into a 10 μM working solution using a buffer solution, which was prepared by N,N-dimethylformamide and 4-hydroxyethylpiperazine ethanesulfonic acid in a volume ratio of 7:3, and the pH of the buffer solution was 7.4.

[0018] (2) Next, take 27 portions of 3 mL of 10 μM Cu. 2+ A solution of a coordination fluorescent compound was prepared by adding 100 μL of a 3 × 10⁻⁶ solution to each compound. -3 The analytes were glyphosate, profenofos, pyridaben, trichlorfon, diazinon, propargite, malathion, quinalphos, and glyphosate. Each analyte was composed of three parallel units, and 27 reactants were obtained. The final concentration of the analytes in the reactants was 100 μM. After the reaction was complete, the fluorescence intensity of the 27 reactants was measured. (3) The results showed that glyphosate could increase Cu 2+ Fluorescence intensity of the working solution of the coordination fluorescent compound; the Cu 2+ The coordination fluorescent compound can only undergo a fluorescence-enhancing reaction with glyphosate, i.e., the Cu... 2+ Coordination fluorescent compounds enable specific recognition of glyphosate.

[0019] The Cu 2+ The procedure for using coordination fluorescent compounds in the cyclic detection of glyphosate is as follows:

[0020] (1) Preparation of the Cu 2+ The coordination fluorescent compound detection reagent uses dimethylformamide and 4-hydroxyethylpiperazine ethanesulfonic acid in a buffer solution with a volume ratio of 7:3 to react the Cu... 2+ The coordination fluorescent compound was prepared into a working solution with a concentration of 10 μM.

[0021] (2) To 10 μM Cu 2+ Alternating addition of 100 μM glyphosate and Cu to a solution of a coordination fluorescent compound 2+ The reaction was carried out for 1 minute, and the fluorescence intensity at 514 nm was measured.

[0022] (3) The results show that in Cu 2+ During the alternating addition of Cu and glyphosate, 2+ The coordination fluorescent compound can stably detect glyphosate more than 5 times in cycles, indicating that the fluorescence intensity of this system has high stability, which verifies the Cu... 2+ The practical application potential of coordination fluorescent compounds.

[0023] The Cu 2+ The procedure for detecting glyphosate in HeLa cells using coordination fluorescent compounds is as follows:

[0024] (1) The Cu2+ Preparation of working solutions for coordination fluorescent compounds;

[0025] The Cu was prepared with a buffer solution. 2+ The coordination fluorescent compound was prepared into a working solution with a concentration of 10 μM; the buffer solution was prepared by N,N-dimethylformamide and 4-hydroxyethylpiperazine ethanesulfonic acid in a volume ratio of 7:3, and the pH value of the buffer solution was 7.4.

[0026] (2) Take 6 experimental groups: A, B, C, D, E, and F;

[0027] Group A was the blank control group: HeLa cells that had not undergone any treatment were the test items in Group A;

[0028] Group B was the glyphosate treatment control group: 100 μL of 50 μM glyphosate was used in a solution containing 5 × 10⁻⁶ glyphosate. 4 The sample was incubated with HeLa cells in a well plate containing 1000 HeLa cells for 30 minutes to obtain the analyte B for detection.

[0029] Group C is Cu 2+ Treatment control group: 100 μL of 50 μM Cu 2+ In containing 5×10 4 The sample was incubated with HeLa cells in a well plate containing 1000 HeLa cells for 30 minutes to obtain the analyte C for detection.

[0030] Group D is Cu 2+ Control group treated with coordination fluorescent compound: 100 μL of 20 μM Cu 2+ Coordination fluorescent compounds containing 5×10 4 The sample was incubated with HeLa cells in a well plate containing 1000 HeLa cells for 30 minutes to obtain the D group of test samples for detection.

[0031] Group E consists of uncoordinated fluorescent compounds and Cu. 2+ Treatment group: 100 μL of 20 μM Cu was used as a pretreatment agent. 2+ Pretreatment of coordinated fluorescent compounds containing 5 × 10 4 A well plate containing HeLa cells was prepared for 30 minutes, followed by 100 μL of 50 μM Cu. 2+ Incubate together for 30 minutes to obtain group E of test samples for testing;

[0032] Group F is Cu 2+ Coordination fluorescent compounds and glyphosate treatment group: 100 μL of 20 μM Cu 2+ Pretreatment with coordination fluorescent compounds containing 5×10 4 The cells were incubated in a well plate with 100 μL of 50 μM glyphosate for 30 minutes to obtain the F group of test samples for detection.

[0033] (3) The above 6 groups of test substances were placed under a fluorescence microscope with an excitation wavelength of 375 nm for observation;

[0034] (4) Fluorescence imaging results showed that no fluorescence was detected in group A, group B, and group C; while group D showed obvious green fluorescence, which increased as the fluorescent compound reacted with Cu. 2+ After coordination, the fluorescence of the analytes in group E exhibited a quenching effect; the analytes in group F, after further treatment with glyphosate, showed a quenching effect due to competition from glyphosate for Cu. 2+ Cu on coordination fluorescent compounds 2+ Fluorescence recovery is achieved by releasing a fluorescent compound; fluorescence imaging results indicate that the Cu... 2+ The coordination fluorescent compound can enter HeLa cells and react with exogenous glyphosate to produce strong green fluorescence, enabling the specific detection of exogenous glyphosate in HeLa cells.

[0035] The Cu 2+ A method for detecting exogenous glyphosate in zebrafish using coordination fluorescent compounds includes the following steps:

[0036] (1) The Cu 2+ Preparation of working solutions of coordination fluorescent compounds

[0037] The Cu was prepared with a buffer solution. 2+ The coordination fluorescent compound was prepared into a working solution with a concentration of 20 μM. The buffer solution was prepared by N,N-dimethylformamide and 4-hydroxyethylpiperazine ethanesulfonic acid in a volume ratio of 7:3, and the pH value of the buffer solution was 7.4.

[0038] (2) Take 6 experimental groups: A, B, C, D, E, and F.

[0039] Group A, the blank control group: 3-day-old zebrafish that were not treated, and the test samples were obtained from Group A.

[0040] Group B, the glyphosate treatment control group: 3-day-old zebrafish with normal development were incubated with 10 mL of 50 μM glyphosate for 30 minutes to obtain the test sample for Group B.

[0041] Group C, Cu 2+ Treatment control group: 10 mL of 50 μM Cu 2+ Incubate with 3-day-old zebrafish with normal development for 30 minutes to obtain the test sample C for testing;

[0042] Group D, which is not associated with Cu 2+ Control group treated with coordinated fluorescent compound: 3-day-old zebrafish were treated with 10 mL of 20 μM of the aforementioned uncoordinated fluorescent compound.2+ Treatment with the coordinated fluorescent compound for 30 minutes yielded the analytes in group D for detection.

[0043] Group E consists of uncoordinated fluorescent compounds and Cu. 2+ Treatment group: 3-day-old zebrafish were treated with 10 mL of 20 μM Cu-containing solution. 2+ The coordinated fluorescent compound was treated for 30 minutes, followed by treatment with 10 mL of 50 μM Cu. 2+ Incubate together for 30 minutes to obtain group E of test samples for testing;

[0044] Group F, for Cu 2+ Coordination fluorescent compound and glyphosate treatment group: 10 mL of 20 μM Cu 2+ Three-day-old zebrafish were pretreated with a coordination fluorescent compound for 30 minutes, and then incubated with 10 mL of 50 μM glyphosate for 30 minutes to obtain the F group of analytes for detection.

[0045] The six groups of test samples were observed under a fluorescence microscope with an excitation wavelength of 375 nm. The results showed that no fluorescence was detected in groups A, B, and C; while group D showed obvious green fluorescence in zebrafish, with the fluorescent compound reacting with Cu. 2+ After coordination, the fluorescence in group E zebrafish was quenched; and the fluorescence in group F zebrafish treated with glyphosate was further quenched due to competition from glyphosate for Cu. 2+ Cu on coordination fluorescent compounds 2+ Fluorescence recovery is achieved by releasing a fluorescent compound; fluorescence imaging results indicate that the Cu... 2+ The coordination fluorescent compound can enter the zebrafish and react with exogenous glyphosate to produce strong red fluorescence, enabling the specific detection of exogenous glyphosate in zebrafish.

[0046] Compared with the prior art, the beneficial effects of the present invention are:

[0047] 1. The Cu described in this invention 2+ Coordination fluorescent compounds are easy to prepare, use readily available raw materials, are easy to synthesize, and require mild reaction conditions.

[0048] 2. The Cu described in this invention 2+ The amino, phosphate, and carboxyl functional groups in the glyphosate molecule, a coordination fluorescent compound, can interact with Cu. 2+ A strong coordination reaction occurs; therefore, glyphosate can be utilized to interact with Cu. 2+ Cu in coordination fluorescent compounds 2+ A strong 1:1 coordination reaction releases a fluorescent compound, causing a change in fluorescence intensity, thus enabling the fluorescence detection of glyphosate (ESI MS [C]).25 H 15 N2O4 + H] + m / z: 409.1825; ESI MS [glyphosate-Cu + H] + (m / z: 232.1075), produced recognizable fluorescence ( Figure 1 -2), thus eliminating interference from common analytes in solutions and organisms, and with a detection limit as low as 0.12 μM, compared to most similar Cu... 2+ Coordination fluorescent compounds have significant advantages; Figure 3 The results show that the Cu 2+ Coordination fluorescent compounds exhibit good selectivity for glyphosate. Figure 4 As shown, Cu 2+ The fluorescence intensity of the coordination fluorescent compound increased with increasing glyphosate content, and exhibited a good linear relationship (R0). 2 =0.9811), that is, the Cu 2+ Coordination fluorescent compounds can be used to quantitatively detect trace amounts of glyphosate in the aquatic environment;

[0049] 3. The present invention, wherein the Cu 2+ Coordination fluorescent compounds have potential application value, and this Cu 2+ Coordination fluorescent compounds can be used for multiple-cycle detection of glyphosate, in 10 μM Cu 2+ Alternating addition of 100 μM glyphosate and Cu to a solution of a coordination fluorescent compound 2+ After the reaction is complete, the fluorescence intensity at 514 nm is measured in Cu. 2+ During alternating detection with glyphosate, Cu 2+ The coordination fluorescent compound can be stably cycled more than 5 times, and the Cu 2+ The good cell membrane permeability and low cytotoxicity of coordination fluorescent compounds have enabled their successful application in imaging glyphosate in live cells and zebrafish, demonstrating good potential for biological applications.

[0050] (1) The Cu 2+ Coordination fluorescent compounds have potential applications, see Figure 5 Contains Cu 2+ The detection solution of the coordination fluorescent compound can stably detect glyphosate more than 5 times in cycles.

[0051] (2) The Cu 2+ The coordination fluorescent compound has a logP value of 3.839. It is a lipophilic compound that can easily enter cells and has good cell membrane permeability.

[0052] (3) The Cu 2+ Coordination fluorescent compounds exhibit low cytotoxicity, such as Figure 6 As shown in a, at a concentration of 50 μM Cu 2 + Even in the presence of the coordination fluorescent compound, the survival rate of HeLa cells remained above 80%, indicating that the Cu... 2+ Coordination fluorescent compounds exhibit low cytotoxicity;

[0053] (4) The Cu 2+ Coordination fluorescent compounds have good potential for biological applications, such as Figure 6 As shown in result b, untreated HeLa cells were treated with Cu 2+ The coordination fluorescent compound and glyphosate showed significant green fluorescence after incubation for 30 minutes; for example... Figure 7 As shown, when zebrafish and Cu 2+ After incubation for 30 minutes, green fluorescence was observed under a fluorescence microscope after coordinating with fluorescent compounds (20 μM) and glyphosate (50 μM). Attached Figure Description

[0054] Figure 1 High-resolution mass spectrum of the product of the reaction between (E)-N'-((1-hydroxynaphth-2-yl)methylene)-3-oxo-3H-benzo[f]chromene-2-carbamoylhydrazide copper and glyphosate;

[0055] Figure 2 shows the Job curve of the reaction product of (E)-N'-((1-hydroxynaphth-2-yl)methylene)-3-oxo-3H-benzo[f]chromene-2-carboxylhydrazide copper with glyphosate;

[0056] Figure 3 The fluorescence emission and UV spectra of the reaction of different analytes and other compounds in the working solution of (E)-N'-((1-hydroxynaphth-2-yl)methylene)-3-oxo-3H-benzo[f]chromene-2-carbamoylhydrazide copper were obtained.

[0057] Figure 4 The fluorescence emission spectrum of (E)-N'-((1-hydroxynaphth-2-yl)methylene)-3-oxo-3H-benzo[f]chromene-2-carboxylhydrazide copper with glyphosate;

[0058] Figure 5 The graph shows the cyclic detection of glyphosate by copper (E)-N'-((1-hydroxynaphth-2-yl)methylene)-3-oxo-3H-benzo[f]chromene-2-carboxylhydrazide.

[0059] Figure 6 Fluorescence imaging of (E)-N'-((1-hydroxynaphth-2-yl)methylene)-3-oxo-3H-benzo[f]chromene-2-carbamoylhydrazide copper in HeLa cells;

[0060] Figure 7 Fluorescence imaging of (E)-N'-((1-hydroxynaphth-2-yl)methylene)-3-oxo-3H-benzo[f]chromene-2-carboxylhydrazide copper incubated in 3-day-old zebrafish. Detailed Implementation

[0061] This invention discloses a Cu that can specifically detect glyphosate. 2+ Coordination fluorescent compounds and their preparation methods. The Cu... 2+ Coordination fluorescent compounds are characterized by their two-part composition, including Cu 2+ And (E)-N'-((1-hydroxynaphth-2-yl)methylene)-3-oxo-3H-benzo[f]chromene-2-carboxylhydrazine. The reported glyphosate is able to react with Cu... 2+ Cu on coordination fluorescent compounds 2+ Strong coordination occurs, releasing (E)-N'-((1-hydroxynaphth-2-yl)methylene)-3-oxo-3H-benzo[f]chromene-2-carboxylhydrazine, which produces fluorescence enhancement, thereby enabling specific detection of glyphosate.

[0062] The present invention will be further described below with reference to embodiments.

[0063] Example 1: Cu for glyphosate detection 2+ Preparation of Coordination Fluorescent Compounds

[0064] The Cu2+ coordination fluorescent compound of this invention is (E)-N'-((1-hydroxynaphth-2-yl)methylene)-3-oxo-3H-benzo[f]chromene-2-formylhydrazide copper (HYBC), and the specific preparation process is as follows: ;

[0065] (1) 30 mmol of 2-hydroxy-1-naphthaldehyde, 33 mmol of diethyl malonate, 0.5 mL of pyrrolidine and 120 mL of ethanol were added to a 250 mL three-necked flask and refluxed. The reaction was monitored by TLC. After the reaction was completed, the reaction solution was slowly poured into 240 mL of water. A solid precipitated out. The solid was filtered, washed with water and dried to obtain a white solid ethyl 3-oxo-3H-benzo[f]chromene-2-carboxylate.

[0066] (2) 10 mmol of ethyl 3-oxo-3H-benzo[f]chromene-2-carboxylate, 80% hydrazine hydrate (50 mmol) and 100 mL of tetrahydrofuran were added to a 250 mL three-necked flask and refluxed for 6 h. After the reaction was complete, the mixture was concentrated to dryness under reduced pressure and recrystallized from ethanol to obtain a yellow solid 3-oxo-3H-benzo[f]chromene-2-carboxyhydrazide.

[0067] (3) Add 5 mmol of 3-oxo-3H-benzo[f]chromene-2-carboxylhydrazine, 5.5 mmol of 2-hydroxy-1-naphthaldehyde, 0.5 mL of glacial acetic acid, and 100 mL of anhydrous ethanol to a 250 mL three-necked flask, and reflux for 15 h; after cooling to room temperature, filter and dry, and recrystallize using N,N-dimethylformamide (DMF) to obtain the fluorescent probe (E)-N'-((1-hydroxynaphth-2-yl)methylene)-3-oxo-3H-benzo[f]chromene-2-carboxylhydrazine (yellow solid, yield 68%). The characterization results of (E)-N'-((1-hydroxynaphth-2-yl)methylene)-3-oxo-3H-benzo[f]chromene-2-carboxylhydrazine are as follows: 1H NMR spectroscopy determination: 13 CNMR(151 MHz, DMSO-d6): 168.7, 161.6, 159.4, 150.5, 146, 144.2, 137.0, 130.3,130.1, 128.8, 128.5, 127.9, 126.8, 126.6, 126.3, 125.8, 123.6, 123.0, 122.3,121.1, 117.7, 115.5, 114.1, 111.4. 1 HNMR (400 MHz, DMSO-d6): δ 12.91 (s, 1H), 11.56 (s, 1H), 8.74 (s, 1H), 8.46 (d, J = 7.0, 1.8 Hz, 2H), 8.11 (t, J = 7.5, 2.2 Hz, 2H), 7.93 (m, J = 7.0, 2.0 Hz, 2H), 7.69 (m, J = 7.0, 1.8 Hz, 2H), 7.49 (m, J = 7.0, 2.0 Hz, 2H), 7.20–7.26 (m, J = 7.0, 2.0 Hz, 3H); High-resolution mass spectrometry: HRMS (ESI, m / z) calculated for [C 25 H 16 N2O4+H] + : 409.11883, found: 409.18277; (4) Dissolve (E)-N'-((1-hydroxynaphth-2-yl)methylene)-3-oxo-3H-benzo[f]chromene-2-carboxylhydrazine (10 mmol) and copper sulfate (10 mmol) in a molar ratio of 1:1 in a mixed solution of N,N-dimethylformamide (DMF) and 4-hydroxyethylpiperazine ethanesulfonic acid (HEPES) (DMSO:HEPES=7:3) to obtain Cu 2+ Coordination fluorescent compounds;

[0068] The Cu 2+ The coordination fluorescent compound is (E)-N'-((1-hydroxynaphth-2-yl)methylene)-3-oxo-3H-benzo[f]chromene-2-carboxylhydrazide copper, and its chemical structural formula is shown in formula (I): ;

[0069] Formula (I).

[0070] The detection mechanism of the Cu2+ coordination fluorescent compound (E)-N'-((1-hydroxynaphth-2-yl)methylene)-3-oxo-3H-benzo[f]chromene-2-carboxylhydrazide copper described in this invention is as follows: ;

[0071] (1) The Cu described in this invention 2+ Coordination fluorescent compound, (E)-N'-((1-hydroxynaphth-2-yl)methylene)-3-oxo-3H-benzo[f]chromene-2-carboxylhydrazide copper, reacts with glyphosate. The amino, phosphate, and carboxyl functional groups in the glyphosate molecule can react with Cu. 2+ A strong coordination reaction occurs. Therefore, glyphosate can be utilized to interact with Cu. 2+ Cu in coordination fluorescent compounds 2 + A strong 1:1 coordination reaction releases a fluorescent compound, causing a change in fluorescence intensity, thus enabling the fluorescence detection of glyphosate (ESI MS [C]). 25 H 15 N2O4 + H] + m / z: 409.1825; ESI MS [glyphosate-Cu + H] + (m / z: 232.1075) Figure 1 -2 shows the Cu 2+ Job curves and high-resolution mass spectra of the reaction products of coordination fluorescent compounds and glyphosate;

[0072] (3) The results eliminated the interference of common analytes in solutions and organisms, and the detection limit was as low as 0.12 μM, which is a great advantage compared with most similar fluorescent compounds.

[0073] Example 2: Selective detection of glyphosate in solution systems

[0074] (1) The Cu prepared in Example 1 2+ The coordination fluorescent compound was prepared into a 10 μM Cu solution using a buffer solution. 2+A solution of a coordination fluorescent compound, wherein the buffer solution is prepared by N,N-dimethylformamide (DMF) and 4-hydroxyethylpiperazine ethanesulfonic acid (HEPES) in a volume ratio of 7:3, and the pH of the buffer solution is 7.4;

[0075] (2) Using 10 μM Cu 2+ The coordination fluorescent compound solution selectively detects glyphosate in solution;

[0076] Take 27 portions of 3 mL of 10 μM Cu 2+ A solution of a coordination fluorescent compound was prepared by adding 100 μL of a 3 × 10⁻⁶ solution to each compound. -3 Nine analytes were tested at a concentration of mol / L. Each analyte was tested in groups of three parallel units, resulting in a total of 27 reactants. The nine analytes were 100 μM glyphosate, 100 μM glyphosate, 100 μM profenofos, 100 μM pyridaben, 100 μM trichlorfon, 100 μM diazinon, 100 μM propargite, 100 μM malathion, and 100 μM quinalphos.

[0077] (3) The reaction was complete, and 27 reactants were obtained. The fluorescence intensity of each of the 27 reactants was measured.

[0078] (4) Figure 3 a and 3b represent the results of fluorescence intensity and ultraviolet absorption intensity, respectively. The results show that only glyphosate showed significant changes in fluorescence intensity and ultraviolet absorption intensity, indicating that (E)-N'-((1-hydroxynaphth-2-yl)methylene)-3-oxo-3H-benzo[f]chromene-2-formylhydrazide has good selectivity for glyphosate; moreover, Figure 3 c shows the Cu 2+ The results of the interference resistance of coordination fluorescent compounds to glyphosate indicate that other interfering analytes do not affect Cu. 2+ The response of the coordination fluorescent compound to glyphosate; in summary, the Cu 2+ Coordination fluorescent compounds can specifically recognize glyphosate and have strong anti-interference capabilities.

[0079] Example 3: Cu in solution system 2+ The detection sensitivity of coordination fluorescent compounds to glyphosate (1) The Cu prepared in Example 1 2+ The coordination fluorescent compound was prepared into a 10 μM Cu solution using a buffer solution. 2+ A solution of a coordination fluorescent compound, wherein the buffer solution is prepared by N,N-dimethylformamide (DMF) and 4-hydroxyethylpiperazine ethanesulfonic acid (HEPES) in a volume ratio of 7:3, and the pH of the buffer solution is 7.4;

[0080] (2) Using 10 μM Cu2+ Fluorescence detection of glyphosate at different concentrations (0µM-20µM) using coordination fluorescent compound solutions;

[0081] (3) Add 3 mL of 10 μM Cu to 63 portions of the prepared solution. 2+ The solutions of coordination fluorescent compounds were respectively added with different concentrations (0µM-20µM) of glyphosate, with three parallel units for each concentration;

[0082] (4) The reaction was complete, and 63 reactants were obtained. The fluorescence intensity of each of the 63 reactants was measured.

[0083] (5) By Figure 4 It is evident that glyphosate can increase Cu 2+ The fluorescence intensity of the coordination fluorescent compound solution increased with increasing glyphosate concentration, Cu 2+ The fluorescence intensity of the solution of the coordination fluorescent compound increases accordingly; Figure 4 b shows Cu 2+ The fluorescence intensity of the coordination fluorescent compound solution showed a good linear relationship with the concentration of glyphosate (Rf). 2 =0.9811), the calculated detection limit of Cu2+ coordinated fluorescent compound for glyphosate is as low as 0.12 μM, that is, the Cu2+ coordinated fluorescent compound has a detection limit as low as 0.9811. 2+ Coordination fluorescent compounds can be used to quantitatively detect trace amounts of glyphosate in solution.

[0084] Example 4: Application in the cyclic detection of glyphosate

[0085] Cu 2+ A method for cyclic detection of glyphosate using coordination fluorescent compounds, characterized by the following steps: (1) preparing the Cu 2+ The coordination fluorescent compound detection reagent, using a buffer solution (DMF:HEPES=7:3) to react the Cu... 2+ (2) The coordination fluorescent compound was prepared into a working solution with a concentration of 10 μM; 2+ Alternating addition of 100 μM glyphosate Cu to the solution of the coordination fluorescent compound 2+ The reaction was carried out for 1 minute, and the fluorescence intensity at 514 nm was measured.

[0086] (3) The results show that in Cu 2+ During the alternating addition of Cu and glyphosate, 2+ It can quench the fluorescence of fluorescent compounds, while glyphosate will... 2+ Cu on coordination fluorescent compounds 2+ The competition for fluorescent compounds leads to enhanced fluorescence.

[0087] Cu 2+The coordination fluorescent compound can stably detect glyphosate more than 5 times in cycles, indicating that the fluorescence intensity of this system has high stability, which verifies the Cu... 2+ The practical application potential of coordination fluorescent compounds ( Figure 5 ).

[0088] Example 5: Intracellular fluorescence imaging of HeLa cells

[0089] (1) When used for glyphosate detection in HeLa cells, the Cu was prepared with a buffer solution. 2+ The coordination fluorescent compound was prepared into a working solution with a concentration of 20 μM; the buffer solution was prepared by N,N-dimethylformamide (DMF) and 4-hydroxyethylpiperazine ethanesulfonic acid (HEPES) in a volume ratio of 7:3, and the pH value of the buffer solution was 7.4.

[0090] (2) Take 6 experimental groups: A, B, C, D, E, and F;

[0091] Group A was the blank control group: HeLa cells that had not undergone any treatment were the test items in Group A;

[0092] Group B was the glyphosate treatment control group: 100 μL of 50 μM glyphosate was used in a solution containing 5 × 10⁻⁶ glyphosate. 4 The sample was incubated with HeLa cells in a well plate containing 1000 HeLa cells for 30 minutes to obtain the analyte B for detection.

[0093] Group C is Cu 2+ Treatment control group: 100 μL of 50 μM Cu 2+ In containing 5×10 4 The sample was incubated with HeLa cells in a well plate containing 1000 HeLa cells for 30 minutes to obtain the analyte C for detection.

[0094] Group D is Cu 2+ Control group treated with coordination fluorescent compound: 100 μL of 20 μM Cu 2+ Coordination fluorescent compounds containing 5×10 4 The sample was incubated with HeLa cells in a well plate containing 1000 HeLa cells for 30 minutes to obtain the D group of test samples for detection.

[0095] Group E consists of uncoordinated fluorescent compounds and Cu. 2+ Treatment group: 100 μL of 20 μM Cu was used as a pretreatment agent. 2+ Pretreatment of coordinated fluorescent compounds containing 5 × 10 4 A well plate containing HeLa cells was prepared for 30 minutes, followed by 100 μL of 50 μM Cu. 2+ Incubate together for 30 minutes to obtain group E of test samples for testing;

[0096] Group F is Cu2+ Coordination fluorescent compounds and glyphosate treatment group: 100 μL of 20 μM Cu 2+ Pretreatment with coordination fluorescent compounds containing 5×10 4 The cells were incubated in a well plate with 100 μL of 50 μM glyphosate for 30 minutes to obtain the F group of test samples for detection.

[0097] (3) The above 6 groups of test substances were placed under a fluorescence microscope with an excitation wavelength of 375 nm for observation;

[0098] (4) Fluorescence imaging results showed that no fluorescence was detected in group A, group B, and group C; while group D showed obvious green fluorescence, with (E)-N'-((1-hydroxynaphth-2-yl)methylene)-3-oxo-3H-benzo[f]chromene-2-formylhydrazine reacting with Cu 2+ After coordination, the fluorescence of the analytes in group E exhibited a quenching effect; the analytes in group F, after further treatment with glyphosate, showed a quenching effect due to competition from glyphosate for Cu. 2+ Cu on coordination fluorescent compounds 2+ The release of fluorescent compounds achieves fluorescence recovery; fluorescence imaging results show that the (E)-N'-((1-hydroxynaphth-2-yl)methylene)-3-oxo-3H-benzo[f]chromene-2-carboxylhydrazide copper can enter HeLa cells and react with exogenous glyphosate, producing strong green fluorescence, thus achieving specific detection of exogenous glyphosate in HeLa cells. Figure 6 ).

[0099] Example 6: Fluorescence Imaging of Zebrafish

[0100] (1) When used for the detection of exogenous glyphosate in zebrafish, the Cu is dissolved in a buffer solution. 2+ The coordination fluorescent compound was prepared into a working solution with a concentration of 20 μM. The buffer solution was prepared by N,N-dimethylformamide (DMF) and 4-hydroxyethylpiperazine ethanesulfonic acid (HEPES) in a volume ratio of 7:3, and the pH value of the buffer solution was 7.4.

[0101] (2) Take 6 experimental groups: A, B, C, D, E, and F.

[0102] Group A was the blank control group: 3-day-old zebrafish that had not been treated, and the test samples were obtained from Group A.

[0103] Group B was the glyphosate treatment control group: 3-day-old zebrafish with normal development were incubated with 10 mL of 50 μM glyphosate for 30 minutes to obtain the test sample from Group B.

[0104] Group C is Cu 2+Treatment control group: 10 mL of 50 μM Cu 2+ Incubate with 3-day-old zebrafish with normal development for 30 minutes to obtain the test sample C for testing;

[0105] Group D was not associated with Cu. 2+ Control group treated with coordinated fluorescent compound: 3-day-old zebrafish were treated with 10 mL of 20 μM of the aforementioned uncoordinated fluorescent compound. 2+ Treatment with coordinated (E)-N'-((1-hydroxynaphth-2-yl)methylene)-3-oxo-3H-benzo[f]chromene-2-formylhydrazide for 30 minutes yielded the analyte in group D for detection.

[0106] Group E consists of uncoordinated (E)-N'-((1-hydroxynaphth-2-yl)methylene)-3-oxo-3H-benzo[f]chromene-2-carboxylhydrazine and Cu 2+ Treatment group: 3-day-old zebrafish were treated with 10 mL of 20 μM Cu-containing solution. 2+ The coordinated (E)-N'-((1-hydroxynaphth-2-yl)methylene)-3-oxo-3H-benzo[f]chromene-2-carboxylhydrazine was treated for 30 minutes, followed by treatment with 10 mL of 50 μM Cu. 2+ Incubate together for 30 minutes to obtain group E of test samples for testing;

[0107] Group F is Cu 2+ Coordination fluorescent compound and glyphosate treatment group: 10 mL of 20 μM Cu 2+ Three-day-old zebrafish were pretreated with coordination (E)-N'-((1-hydroxynaphth-2-yl)methylene)-3-oxo-3H-benzo[f]chromene-2-carboxylhydrazine for 30 minutes, and then incubated with 10 mL of 50 μM glyphosate for 30 minutes to obtain the F group of analytes for detection.

[0108] (3) The above 6 groups of test substances were placed under a fluorescence microscope with an excitation wavelength of 375 nm for observation;

[0109] The results showed that no fluorescence was detected in group A, group B, and group C; however, group D showed obvious green fluorescence in zebrafish, with (E)-N'-((1-hydroxynaphth-2-yl)methylene)-3-oxo-3H-benzo[f]chromene-2-carboxylhydrazine reacting with Cu 2+ After coordination, the fluorescence in group E zebrafish was quenched; and the fluorescence in group F zebrafish treated with glyphosate was further quenched due to competition from glyphosate for Cu. 2+ Cu on coordination (E)-N'-((1-hydroxynaphth-2-yl)methylene)-3-oxo-3H-benzo[f]chromene-2-formylhydrazine 2+The release of fluorescent compounds enables fluorescence recovery; fluorescence imaging results show that the (E)-N'-((1-hydroxynaphth-2-yl)methylene)-3-oxo-3H-benzo[f]chromene-2-carboxylhydrazide copper can enter zebrafish and react with exogenous glyphosate, producing strong green fluorescence, thus achieving specific detection of exogenous glyphosate in zebrafish. Figure 7 ).

[0110] Contents not described in detail in this specification are prior art known to those skilled in the art. In the description of this invention, unless otherwise stated, "a plurality of" means two or more. Terms such as "upper," "lower," "left," "right," "inner," "outer," "front end," "rear end," "head," and "tail" indicate orientations or positional relationships based on the accompanying drawings, and are used only for ease of description and simplification, not to indicate or imply that the device or element referred to must have a specific orientation, or be constructed and operated in a specific orientation, and therefore should not be construed as limiting the invention. Furthermore, terms such as "first," "second," and "third" are used for descriptive purposes only and should not be construed as indicating or implying relative importance. In the description of this invention, it should be noted that unless otherwise explicitly specified and limited, the terms "connected" and "linked" should be interpreted broadly. For example, they can refer to a fixed connection, a detachable connection, or an integral connection; they can refer to a mechanical connection or an electrical connection; they can refer to a direct connection or an indirect connection through an intermediate medium. Those skilled in the art can understand the specific meaning of the above terms in this invention based on the specific circumstances.

[0111] It will be apparent to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above, and that the invention can be implemented in other specific forms without departing from its spirit or essential characteristics. Therefore, the embodiments should be considered in all respects as exemplary and non-limiting, and the scope of the invention is defined by the appended claims rather than the foregoing description. Thus, all variations falling within the meaning and scope of equivalents of the claims are intended to be included within the present invention. No reference numerals in the claims should be construed as limiting the scope of the claims.

Claims

1. A Cu-based method for the specific detection of glyphosate 2+ Coordination fluorescent compounds, characterized in that: The Cu 2+ The coordination fluorescent compound is (E)-N'-((1-hydroxynaphth-2-yl)methylene)-3-oxo-3H-benzo[f]chromene-2-carboxylhydrazide copper, with the molecular formula C 25 H 15 CuN2O4, the chemical structural formula is shown in formula (I): ； Formula (I).

2. The Cu method for specific detection of glyphosate according to claim 1 2+ A method for preparing coordination fluorescent compounds, characterized in that, Includes the following steps: (1) First, add 30 mmol of 2-hydroxy-1-naphthaldehyde, 33 mmol of diethyl malonate, 0.5 mL of pyrrolidine and 120 mL of ethanol to a 250 mL three-necked flask and reflux the reaction. Monitor the reaction progress by TLC. After the reaction is completed, slowly pour the reaction solution into 240 mL of water. A solid precipitates out. Filter, wash with water, and dry to obtain a white solid ethyl 3-oxo-3H-benzo[f]chromene-2-carboxylate. (2) Next, 10 mmol of ethyl 3-oxo-3H-benzo[f]chromene-2-carboxylate, 50 mmol of hydrazine hydrate and 100 mL of tetrahydrofuran were added to a 250 mL three-necked flask and refluxed for 6 h. After the reaction was complete, the mixture was concentrated to dryness under reduced pressure and recrystallized from ethanol to obtain a yellow solid 3-oxo-3H-benzo[f]chromene-2-carboxyhydrazide. (3) Then, 5 mmol of 3-oxo-3H-benzo[f]chromene-2-carboxylhydrazide, 5.5 mmol of 2-hydroxy-1-naphthaldehyde, 0.5 mL of glacial acetic acid and 100 mL of anhydrous ethanol were added to a 250 mL three-necked flask and refluxed for 15 h. After cooling to room temperature, the mixture was filtered and dried, and recrystallized using N,N-dimethylformamide to obtain the fluorescent probe (E)-N'-((1-hydroxynaphth-2-yl)methylene)-3-oxo-3H-benzo[f]chromene-2-carboxylhydrazide, which was a yellow solid with a yield of 68%. (4) Then, 10 mmol of (E)-N'-((1-hydroxynaphth-2-yl)methylene)-3-oxo-3H-benzo[f]chromene-2-carboxylhydrazine and 10 mmol of copper sulfate were dissolved in a mixed solution of N,N-dimethylformamide and 4-hydroxyethylpiperazine ethanesulfonic acid in a molar ratio of 1:1, with N,N-dimethylformamide:4-hydroxyethylpiperazine ethanesulfonic acid = 7:3, and finally Cu was obtained. 2+ Coordination fluorescent compounds.

3. The Cu according to claim 1 2+ The use of coordination fluorescent compounds, characterized in that, For the specific detection of glyphosate.

4. The Cu according to claim 3 2+ The use of coordination fluorescent compounds, characterized in that, Includes the following steps: (1) First, the Cu 2+ The coordination fluorescent compound was prepared into a 10 μM working solution using a buffer solution, which was prepared by N,N-dimethylformamide and 4-hydroxyethylpiperazine ethanesulfonic acid in a volume ratio of 7:3, and the pH of the buffer solution was 7.

4. (2) Next, take 27 portions of 3 mL of Cu with a concentration of 10 μM. 2+ A solution of a coordination fluorescent compound was prepared by adding 100 μL of a 3 × 10⁻⁶ solution to each compound. -3 The analytes were glyphosate, profenofos, pyridaben, trichlorfon, diazinon, propargite, malathion, quinalphos, and glyphosate. Each analyte was composed of three parallel units, and 27 reactants were obtained. The final concentration of the analyte in the reactants was 100 μM. After the reaction was complete, the fluorescence intensity of the 27 reactants was measured. (3) The results showed that glyphosate could increase Cu 2+ Fluorescence intensity of the working solution of the coordination fluorescent compound; the Cu 2+ Coordination fluorescent compounds can only undergo fluorescence enhancement reactions with glyphosate.

5. The Cu according to claim 1 2+ A method for cyclic detection of glyphosate using coordination fluorescent compounds, characterized in that... The operation steps are as follows: (1) Making the Cu as described in claim 1 2+ The coordination fluorescent compound detection reagent uses dimethylformamide and 4-hydroxyethylpiperazine ethanesulfonic acid in a buffer solution with a volume ratio of 7:3 to react the Cu... 2+ The coordination fluorescent compound was prepared into a working solution with a concentration of 10 μM. (2) To 10 μM Cu 2+ Alternating addition of 100 μM glyphosate and Cu to the solution of the coordination fluorescent compound 2+ The reaction was carried out for 1 minute, and the fluorescence intensity at 514 nm was measured. (3) The results show that in Cu 2+ During the alternating addition of Cu and glyphosate, 2+ Coordination fluorescent compounds can stably detect glyphosate more than 5 times in cycles.

6. The Cu according to claim 1 2+ A method for detecting glyphosate in HeLa cells using coordination fluorescent compounds, characterized in that... The operation steps are as follows: (1) The Cu as described in claim 1 2+ Preparation of working solutions of coordination fluorescent compounds The Cu of claim 1 is prepared with a buffer solution. 2+ The coordination fluorescent compound was prepared into a working solution with a concentration of 10 μM; the buffer solution was prepared by N,N-dimethylformamide and 4-hydroxyethylpiperazine ethanesulfonic acid in a volume ratio of 7:3, and the pH value of the buffer solution was 7.

4. (2) Take 6 experimental groups: A, B, C, D, E, and F; Group A was the blank control group: HeLa cells that had not undergone any treatment were the test items in Group A; Group B was the glyphosate treatment control group: 100 μL of 50 μM glyphosate was used in a solution containing 5 × 10⁻⁶ glyphosate. 4 The sample was incubated with HeLa cells in a well plate containing 1000 HeLa cells for 30 minutes to obtain the analyte B for detection. Group C is Cu 2+ Treatment control group: 100 μL of 50 μM Cu 2+ In containing 5×10 4 The sample was incubated with HeLa cells in a well plate containing 1000 HeLa cells for 30 minutes to obtain the analyte C for detection. Group D is Cu 2+ Control group treated with coordination fluorescent compound: 100 μL of 20 μM Cu 2+ Coordination fluorescent compounds containing 5×10 4 The sample was incubated with HeLa cells in a well plate containing 1000 HeLa cells for 30 minutes to obtain the D group of test samples for detection. Group E consists of uncoordinated fluorescent compounds and Cu. 2+ Treatment group: 100 μL of 20 μM Cu was used as a pretreatment agent. 2+ Pretreatment of coordinated fluorescent compounds containing 5 × 10 4 A well plate containing HeLa cells was prepared for 30 minutes, followed by 100 μL of 50 μM Cu. 2+ Incubate together for 30 minutes to obtain group E of test samples for testing; Group F is Cu 2+ Coordination fluorescent compounds and glyphosate treatment group: 100 μL of 20 μM Cu 2+ Pretreatment with coordination fluorescent compounds containing 5×10 4 The cells were incubated in a well plate with 100 μL of 50 μM glyphosate for 30 minutes to obtain the F group of test samples for detection. (3) The above 6 groups of test substances were placed under a fluorescence microscope with an excitation wavelength of 375 nm for observation; (4) Fluorescence imaging results showed that no fluorescence was detected in group A, group B, and group C; while group D showed obvious green fluorescence, which increased as the fluorescent compound reacted with Cu. 2+ After coordination, the fluorescence of the analytes in group E exhibited a quenching effect; the analytes in group F, after further treatment with glyphosate, showed a quenching effect due to competition from glyphosate for Cu. 2+ Cu on coordination fluorescent compounds 2+ Fluorescence recovery is achieved by releasing a fluorescent compound; fluorescence imaging results show that the Cu described in claim 1 2+ The coordination fluorescent compound can enter HeLa cells and react with exogenous glyphosate to produce strong green fluorescence, enabling the specific detection of exogenous glyphosate in HeLa cells.

7. The Cu according to claim 1 2+ A method for detecting exogenous glyphosate in zebrafish using coordination fluorescent compounds, characterized in that, Includes the following steps: (1) The Cu as described in claim 1 2+ Preparation of working solutions of coordination fluorescent compounds The Cu of claim 1 is prepared with a buffer solution. 2+ The coordination fluorescent compound was prepared into a working solution with a concentration of 20 μM. The buffer solution was prepared by N,N-dimethylformamide and 4-hydroxyethylpiperazine ethanesulfonic acid in a volume ratio of 7:3, and the pH value of the buffer solution was 7.

4. (2) Take 6 experimental groups: A, B, C, D, E, and F. Group A was the blank control group: 3-day-old zebrafish that had not been treated, and the test samples were obtained from Group A. Group B was the glyphosate treatment control group: 3-day-old zebrafish with normal development were incubated with 10 mL of 50 μM glyphosate for 30 minutes to obtain the test sample from Group B. Group C is Cu 2+ Treatment control group: 10 mL of 50 μM Cu 2+ Incubate with 3-day-old zebrafish with normal development for 30 minutes to obtain the test sample C for testing; Group D was not associated with Cu. 2+ Control group treated with coordinated fluorescent compound: 3-day-old zebrafish treated with 10 mL of 20 μM fluorescent compound as described in claim 1, without Cu. 2+ Treatment with the coordinated fluorescent compound for 30 minutes yielded the analytes in group D for detection. Group E consists of uncoordinated fluorescent compounds and Cu. 2+ Treatment group: 3-day-old zebrafish treated with 10 mL of 20 μM Cu as described in claim 1, without Cu. 2+ The coordinated fluorescent compound was treated for 30 minutes, followed by treatment with 10 mL of 50 μM Cu. 2+ Incubate together for 30 minutes to obtain group E of test samples for testing; Group F is Cu 2+ Coordination fluorescent compound and glyphosate treatment group: 10 mL of 20 μM Cu 2+ Three-day-old zebrafish were pretreated with a coordination fluorescent compound for 30 minutes, and then incubated with 10 mL of 50 μM glyphosate for 30 minutes to obtain the F group of analytes for detection. The six groups of test samples were observed under a fluorescence microscope with an excitation wavelength of 375 nm. The results showed that no fluorescence was detected in groups A, B, and C; while group D showed obvious green fluorescence in zebrafish, with the fluorescent compound reacting with Cu. 2+ After coordination, the fluorescence in group E zebrafish was quenched; and the fluorescence in group F zebrafish treated with glyphosate was further quenched due to competition from glyphosate for Cu. 2+ Cu on coordination fluorescent compounds 2+ Fluorescence recovery is achieved by releasing a fluorescent compound; fluorescence imaging results show that the Cu described in claim 1 2+ The coordination fluorescent compound can enter the zebrafish and react with exogenous glyphosate to produce strong red fluorescence, enabling the specific detection of exogenous glyphosate in zebrafish.

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