A novel method for the continuous and specific detection of Cu based on acylhydrazone derivatives 2+ and glyphosate fluorescent chemical sensors

Abstract

The present invention discloses a method for continuous specific detection of Cu based on acylhydrazone derivatives. 2+ The fluorescent chemical sensor for Cu(III)-glyphosate and (E)-N'-(4-(diethylamino)-2-hydroxybenzylidene)-3-oxo-3H-benzo[f]chromene-2-carboxylic acid hydrazide is disclosed. The fluorescent chemical sensor has the advantages of easy availability of raw materials, low cost, ideal yield, mild reaction conditions and strong stability. It can specifically and continuously identify Cu(III)-glyphosate in organisms. 2+ and glyphosate; the continuous dual recognition design significantly improves the application potential of the fluorescent probe; the fluorescent chemical sensor can detect Cu in the presence of various interferents 2+ It has excellent selectivity for the recognition of glyphosate and has strong anti-interference ability for common biological molecules; the fluorescent chemical sensor can enter HeLa cells and zebrafish and interact with Cu 2+ It reacts rapidly with glyphosate, producing fluorescence quenching and fluorescence enhancement phenomena that can be distinguished by the naked eye, enabling clear observation of Cu 2+ and the distribution of glyphosate in active cells.

Description

Technical Field

[0001] The present invention relates to the technical field of organic small molecule fluorescent probes, in particular to a method for continuous and specific detection of Cu based on acylhydrazone derivatives. 2+ and fluorescent chemical sensors for glyphosate. Background Art

[0002] Copper ions play a vital role in the human body. They are an essential trace element and a key component of proteins and enzymes. They can be found inside and outside red blood cells, supporting iron transfer proteins and catalyzing hemoglobin synthesis. If copper ion levels in the body exceed the normal range, they can burden the body and lead to copper poisoning, which is harmful to the body. These can include jaundice, liver necrosis, and gastric ulcers. Accidentally ingesting large amounts of metal objects containing copper ions can easily lead to acute copper poisoning, which can cause nausea, vomiting, abdominal pain, hematemesis, and hematuria. In severe cases, complications such as hepatitis, hypotension, and renal failure can occur, and can even lead to death. Copper ions are highly toxic to aquatic organisms. For example, copper contamination can cause oyster meat to turn green, negatively impacting the growth, development, and physiological metabolism of aquatic animals. High concentrations of copper ions can also affect genetic expression in aquatic animals, even causing poisoning and death. When copper-containing wastewater is used to irrigate farmland, it can lead to copper accumulation in the soil and crops, affecting crop growth, potentially causing poor growth, and contaminating grains. In addition, elevated copper levels in the soil also pose challenges to crop cultivation and ecological environmental protection. Copper ions inhibit plant photosynthesis, reduce chloroplast function, affect leaf pigmentation, and reduce chlorophyll content. The impact of this metal stress on plants is related to the degree of heavy metal stress.

[0003] Glyphosate is an organophosphorus herbicide. Although it has low toxicity, it is consumed in large quantities and has a wide range of application. It may affect people through the food chain, production and life, and has great harm to the human body, mainly manifested in hepatotoxicity, immunotoxicity, endocrine toxicity, and reproductive toxicity. Glyphosate has obvious damaging effects on liver cells, mainly because glyphosate can cause liver cell oxidation, mitochondrial collapse, etc. High concentrations of glyphosate can have a slight inhibitory effect on human immune function, mainly because high concentrations of glyphosate can suppress the body's immune function by inhibiting the release of multiple cytokines. Glyphosate not only reduces the activity of cytochrome P450 aromatase, but also inhibits related reductases. It is a potential environmental endocrine disruptor that can have adverse effects on the human endocrine system. Glyphosate can inhibit the activity of mitochondrial succinate dehydrogenase and release adenylate kinase in the cytoplasm to cause cell membrane rupture, thereby inducing reproductive toxicity. Not only that Small doses of glyphosate can also irritate the oral mucosa and throat, causing nausea and vomiting. People who are exposed to high concentrations of glyphosate for a long time will also experience eye and skin irritation. Therefore, when using glyphosate, standard operations should be followed to avoid causing harm to the body. As a relatively powerful herbicide, glyphosate's main function is to inhibit plant growth. Long-term and large-scale use will lead to poor plant growth in the soil, resulting in a lack of nutrients in the soil, making the soil barren. The use of glyphosate will affect various microorganisms in the soil, causing changes in the types and numbers of microorganisms. The impact on the diversity and number of soil microorganisms is an effect that only long-term use of glyphosate will have. The use of glyphosate will also pollute the water environment. Glyphosate is usually sprayed directly on farmland or lawns. When rainwater or irrigation water flows through, glyphosate may flow into the water body, thereby polluting the water body and may cause damage to the ecological environment.

[0004] With researchers' increasing concern about heavy metal ion and pesticide pollution, a variety of detection methods have been developed, including atomic absorption spectrometry (AAS), inductively coupled plasma mass spectrometry (ICP-MS), enzyme-linked immunosorbent assay (ELISA), gas chromatography (GC), high-performance liquid chromatography (HPLC), mass spectrometry (MS), surface-enhanced Raman scattering (SERMS), and electrochemical analysis. Although these methods can achieve the detection goals to a certain extent, their shortcomings, such as complex sample pretreatment, time-consuming, high cost, and the need for professional operators, limit their widespread use in reagent applications. Therefore, the continuous development of new, more efficient, simple, and sensitive detection methods is a current research focus. In recent years, fluorescent probe detection technology has been favored by researchers due to its high sensitivity, strong specificity, short response time, diversity, ease of operation, non-destructiveness, real-time detection, and visualization capabilities. In particular, fluorescent probes can achieve non-invasive real-time visualization and analysis and can be applied to fluorescence imaging of living cells and even whole organisms. To date, fluorescent probes have become the preferred method for detecting certain environmental pollutants and endogenous biomacromolecules.

[0005] In summary, a novel acylhydrazone derivative-based method for the continuous and specific detection of Cu 2+ The fluorescent chemical sensor (E)-N'-(4-(diethylamino)-2-hydroxybenzylidene)-3-oxo-3H-benzo[f]chromene-2-carboxylic acid hydrazide is a fluorescent chemical sensor for glyphosate. The fluorescent chemical sensor utilizes the imino and hydroxyl groups on its structure to react with Cu 2+ Coordination produces fluorescence quenching effect to identify Cu 2+ , while glyphosate molecules contain amino, phosphate and carboxyl functional groups, which can react with Cu 2+ Stronger coordination, competing for Cu on fluorescent chemical sensors 2+ The fluorescent chemical sensor was released to generate fluorescence enhancement to detect glyphosate. The fluorescent chemical sensor was characterized by NMR and HRMS. The Job curve, HRMS and DFT calculations verified the fluorescence chemical sensor and Cu 2+ and glyphosate reaction mechanism, fluorescent chemical sensor for Cu 2+ and glyphosate have high selectivity and strong anti-interference ability, and can effectively identify trace Cu 2+ In addition, the fluorescent chemical sensor was also successfully applied to HeLa cells and zebrafish for exogenous Cu 2+ and imaging of glyphosate;

[0006] Therefore, it is necessary to provide a fluorescent chemical sensor based on acylhydrazone derivatives for continuous and specific detection of Cu2+ and glyphosate to solve the above technical problems. Summary of the Invention

[0007] The first technical problem to be solved by the present invention is to develop a method for continuously and specifically recognizing Cu 2+ and glyphosate, which can distinguish Cu 2+ / Glyphosate with other analytes.

[0008] The second technical problem to be solved by the present invention is to provide a method for continuously and specifically recognizing Cu 2+ The invention discloses a preparation method of an acylhydrazone-based fluorescent chemical sensor for glyphosate.

[0009] To achieve the above object, the present invention provides the following technical solution: a method for continuous specific detection of Cu based on acylhydrazone derivatives 2+ The fluorescent chemical sensor for glyphosate and oxaline is characterized by a fluorescent probe (E)-N'-(4-(diethylamino)-2-hydroxybenzylidene)-3-oxo-3H-benzo[f]chromene-2-carboxylic acid hydrazide, the chemical structure of which is shown in formula (I):

[0010]

[0011] In a further embodiment, a method for preparing a fluorescent chemical sensor comprises the following steps:

[0012] (1) 2-hydroxy-1-naphthaldehyde, diethyl malonate and pyrrolidine were reacted in ethanol to prepare ethyl 3-oxo-3H-benzo[f]chromene-2-carboxylate as a white solid;

[0013] (2) 3-Oxo-3H-benzo[f]chromene-2-carboxylic acid ethyl ester was modified with hydrazine hydrate in tetrahydrofuran solution to obtain yellow solid 3-Oxo-3H-benzo[f]chromene-2-carboxylic acid hydrazide;

[0014] (3) (E)-N'-(4-(diethylamino)-2-hydroxybenzylidene)-3-oxo-3H-benzo[f]chromene-2-carboxylic acid hydrazide was prepared by reacting 3-oxo-3H-benzo[f]chromene-2-carboxylic acid hydrazide, 2-hydroxy-4-diethylamino salicylaldehyde and glacial acetic acid in anhydrous ethanol.

[0015] In a further embodiment, step (1) includes the following steps:

[0016] 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 into a 250 mL three-necked flask, mix well, and reflux. Then, monitor the reaction process using a TLC plate.

[0017] ② After the reaction is completed, the reaction solution is slowly poured into 240 mL of water until solid is precipitated, which is then filtered and washed with water, and finally dried to obtain white solid 3-oxo-3H-benzo[f]chromene-2-carboxylic acid ethyl ester.

[0018] In a further embodiment, step (2) includes the following steps:

[0019] ①Using 100 mL of tetrahydrofuran as solvent, add 10 mmol of ethyl 3-oxo-3H-benzo[f]chromene-2-carboxylate and 80% hydrazine hydrate (50 mmol) into a 250 mL three-necked flask and dissolve and mix evenly;

[0020] ② Reflux for 6 hours. After the reaction is complete, concentrate to dryness under reduced pressure and recrystallize from ethanol to obtain a yellow solid 3-oxo-3H-benzo[f]chromene-2-carboxylic acid hydrazide.

[0021] In a further embodiment, step (3) includes the following steps:

[0022] ① First, add 5 mmol of 3-oxo-3H-benzo[f]chromene-2-carboxylic acid hydrazide, 5.5 mmol of 2-hydroxy-4-diethylamino salicylaldehyde, 0.5 mL of glacial acetic acid, and 100 mL of anhydrous ethanol into a 250 mL three-necked flask, and then reflux for 15 hours;

[0023] ② After cooling to room temperature, the mixture is filtered, dried, and recrystallized in DMF to obtain a yellow solid fluorescent probe (E)-N'-(4-(diethylamino)-2-hydroxybenzylidene)-3-oxo-3H-benzo[f]chromene-2-carboxylic acid hydrazide.

[0024] In a further embodiment, the application of the fluorescent compound of formula (I) is used for the fluorescent chemical sensor for Cu in a solution system. 2+ The screening of factors affecting the identification of glyphosate includes the following steps:

[0025] (3) The fluorescent chemical sensor was prepared into a working solution with a concentration of 10 μM using different solvents, wherein the solvents were dimethyl sulfoxide, N,N-dimethylformamide, acetonitrile, methanol, ethanol, acetone and ethyl acetate; 100 μM Cu was added to the 10 μM fluorescent chemical sensor solution prepared with different solvents. 2+ and glyphosate, three parallels were set for each solution; the reaction was complete to obtain 21 reactants, and the fluorescence intensity of the 21 reactants was measured respectively. Dimethyl sulfoxide made the fluorescent chemical sensor 2+ It has better fluorescence quenching effect and fluorescent chemical sensor-Cu 2+The system exhibited good fluorescence enhancement in the presence of glyphosate, so dimethyl sulfoxide was selected as the standard solvent for subsequent experiments;

[0026] (4) The fluorescent chemical sensor was prepared into a 10 μM fluorescent chemical sensor solution using different ratios of dimethyl sulfoxide and 4-hydroxyethylpiperazineethanesulfonic acid, wherein the ratios of dimethyl sulfoxide and 4-hydroxyethylpiperazineethanesulfonic acid were 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, and 90%, respectively; 100 μM Cu was added to the 10 μM fluorescent chemical sensor solution prepared using different ratios of dimethyl sulfoxide, respectively. 2+ and glyphosate, with three replicates for each solution. The reaction was complete, yielding 27 reactants. Fluorescence intensity measurements were performed on each of the 27 reactants. The results showed that a dimethyl sulfoxide:4-hydroxyethylpiperazineethanesulfonic acid ratio of 7:3 maximized the fluorescence performance of the fluorescent chemical sensor. Therefore, a dimethyl sulfoxide / 4-hydroxyethylpiperazineethanesulfonic acid ratio of 7:3 was selected as the buffer solution for subsequent experiments.

[0027] The fluorescent chemical sensor solution was prepared with a buffer solution having a concentration of 10 μM. 2+ The working solution of the glyphosate detection system was prepared by mixing methyl sulfoxide and 4-hydroxyethylpiperazineethanesulfonic acid in a volume ratio of 7:3; the pH of the buffer solution was set to 2, 3, 4, 5, 6, 6.5, 7, 7.4, 8, 9, 10, 11, and 12; 100 μM glyphosate and Cu were added to the fluorescent chemical sensor solution with a concentration of 10 μM at different pH values. 2+ , three parallels were set for each solution; the reaction was complete, and 78 reactants were obtained. The fluorescence intensity of 78 reactants was measured respectively. The results showed that Cu 2+ The optimal pH of the detection system of dapoxetine and glyphosate is around 7.4. Considering that the subsequent imaging experiment will be carried out in vivo, the physiological pH of 7.4 is selected as the pH of the subsequent experimental solution.

[0028] In a further embodiment, the application of the fluorescent compound of formula (I) is used as a fluorescent probe for continuous specific detection of Cu 2+ and glyphosate, cyclic detection of Cu 2+ and glyphosate, comprising the steps of:

[0029] (1) First, the fluorescent chemical sensor detection reagent is prepared, and then the fluorescent chemical sensor is prepared into a working solution with a concentration of 10 μM using a buffer solution prepared by N,N-dimethylformamide:4-hydroxyethylpiperazineethanesulfonic acid = 7:3;

[0030] (2) Then, 100 μM Cu was added alternately to the 10 μM fluorescent chemical sensor solution. 2+ and glyphosate, and then react for 1 min, and measure the fluorescence intensity at 517 nm;

[0031] (3) Finally, the results show that in Cu 2+ During the alternating addition of glyphosate, Cu 2+ The coordinated fluorescent compound can stably detect glyphosate more than 6 times in a cycle, that is, the fluorescence intensity of the system has a high stability, which verifies the Cu 2+ Practical application potential of coordination fluorescent compounds.

[0032] In a further embodiment, the application of the fluorescent compound of formula (I) is used for Cu 2+ The invention relates to a fluorescent chemical sensor for the specific detection of Cu and glyphosate. The fluorescent chemical sensor is prepared into a 10 μM 4-hydroxyethylpiperazineethanesulfonic acid and dimethyl sulfoxide buffer solution. The volume ratio of 4-hydroxyethylpiperazineethanesulfonic acid to dimethyl sulfoxide in the buffer solution is 3:7, and the pH value of the buffer solution is 7.4. 100 μM analyte solutions are added to the prepared 10 μM fluorescent chemical sensor solution, including various metal ions: barium chloride, mercuric sulfate, cobalt chloride, manganese chloride, nickel chloride, zinc chloride, potassium hydroxide, ferric chloride, sodium chloride, calcium chloride, magnesium chloride, ferrous chloride tetrahydrate, and copper sulfate pentahydrate; and various commercial organophosphorus pesticides: pyridabenzyl, glyphosate, profenofos, trichlorfon, diazinon, propargite, malathion, quinalphos, and glyphosate. After the reaction is complete, the fluorescence intensity is measured. The change in fluorescence intensity shows that the fluorescent probe can react with Cu and glyphosate. 2+ The fluorescent chemical sensor can continuously and specifically detect Cu 2+ and glyphosate.

[0033] In a further embodiment, the application of the fluorescent compound of formula (I) is used to detect Cu in HeLa cells of human cervical cancer tissue. 2+ When distributing glyphosate, the fluorescent chemical sensor was prepared into a buffer solution with a concentration of 20 μM and divided into 6 groups: a, b, c, d, e, and f:

[0034] Group a, blank control: HeLa cells without any treatment, used as the test object of group a;

[0035] Group b, glyphosate control: HeLa cells were incubated with 50 μM glyphosate for 30 min as the test substance for group b;

[0036] Group c, Cu 2+ Control: 50 μM Cu 2+HeLa cells were incubated for 30 min and used as test objects in group c;

[0037] Group d, fluorescent compound control: HeLa cells were incubated with 20 μM (E)-N'-(4-(diethylamino)-2-hydroxybenzylidene)-3-oxo-3H-benzo[f]chromene-2-carboxylic acid hydrazide for 30 min, which was used as the detection compound for group d;

[0038] Group e, fluorescent chemical sensors and Cu 2+ Experimental group 1: HeLa cells were incubated with 20 μM (E)-N'-(4-(diethylamino)-2-hydroxybenzylidene)-3-oxo-3H-benzo[f]chromene-2-carboxylic acid hydrazide for 30 minutes and then with 50 μM Cu 2+ Treated for 30 minutes, used as test objects in group e;

[0039] Group f, fluorescent chemical sensor-Cu 2+ -Glyphosate experimental group 2: HeLa cells were treated with 20 μM (E)-N'-(4-(diethylamino)-2-hydroxybenzylidene)-3-oxo-3H-benzo[f]chromene-2-carboxylic acid hydrazide and 50 μM Cu 2+ HeLa cells were treated for 30 min and then incubated with 50 μM glyphosate for 30 min as the test substance in group f;

[0040] The fluorescence imaging results of groups d, e, and f showed that (E)-N'-(4-(diethylamino)-2-hydroxybenzylidene)-3-oxo-3H-benzo[f]chromene-2-carboxylic acid hydrazide could enter the cells and interact with Cu 2+ Reacting with glyphosate, cell imaging can clearly show Cu 2+ and the distribution of glyphosate in HeLa cells.

[0041] In a further embodiment, the application of the fluorescent compound of formula (I) is used for Cu 2+ For the continuous specific detection of glyphosate, the fluorescent chemical sensor was prepared into a working solution with a concentration of 20 μM and divided into 6 groups: a, b, c, d, e, and f:

[0042] Group a, blank control: three-day-old zebrafish without any treatment, used as test objects in group a;

[0043] Group b, glyphosate control: three-day-old zebrafish were incubated with 50 μM glyphosate for 30 minutes as the test substance for group b;

[0044] Group c, Cu 2+ Control: 50 μM Cu 2+ Three-day-old zebrafish were incubated for 30 minutes and used as test objects in group C;

[0045] Group d, fluorescent compound control: three-day-old zebrafish were incubated with 20 μM (E)-N'-(4-(diethylamino)-2-hydroxybenzylidene)-3-oxo-3H-benzo[f]chromene-2-carboxylic acid hydrazide for 30 minutes as the test substance for group d;

[0046] Group e, fluorescent chemical sensors and Cu 2+ Experimental group 1: Three-day-old zebrafish were incubated with 20 μM (E)-N'-(4-(diethylamino)-2-hydroxybenzylidene)-3-oxo-3H-benzo[f]chromene-2-carboxylic acid hydrazide for 30 minutes and then with 50 μM Cu 2+ Treated for 30 minutes, used as the detection object in group e; group f, fluorescent chemical sensor-Cu 2+ -Glyphosate experimental group 2: Three-day-old zebrafish were treated with 20 μM (E)-N'-(4-(diethylamino)-2-hydroxybenzylidene)-3-oxo-3H-benzo[f]chromene-2-carboxylic acid hydrazide and 50 μM Cu 2+ Three-day-old zebrafish were treated for 30 min, and then incubated with 50 μM glyphosate for 30 min as the test object in group f;

[0047] The results showed that zebrafish treated with (E)-N'-(4-(diethylamino)-2-hydroxybenzylidene)-3-oxo-3H-benzo[f]chromene-2-carboxylic acid hydrazide at 28°C showed obvious fluorescence, while zebrafish treated with (E)-N'-(4-(diethylamino)-2-hydroxybenzylidene)-3-oxo-3H-benzo[f]chromene-2-carboxylic acid hydrazide and Cu 2+ The treated zebrafish showed a certain fluorescence quenching phenomenon, and the fluorescence in the zebrafish recovered in the presence of glyphosate; the fluorescence imaging results showed that (E)-N'-(4-(diethylamino)-2-hydroxybenzylidene)-3-oxo-3H-benzo[f]chromene-2-carboxylic acid hydrazide could enter the zebrafish and interact with Cu 2+ Continuously reacting with glyphosate, it produces fluorescence quenching and recovery phenomena, thereby achieving the detection effect.

[0048] Compared with the prior art, the present invention has the following beneficial effects:

[0049] 1. The fluorescent chemical sensor of the present invention has the advantages of readily available raw materials, low cost, ideal yield, mild reaction conditions and strong stability;

[0050] 2. Continuous recognition of Cu in this fluorescent chemical sensor 2+ and glyphosate significantly improve the practical application value of the fluorescent probe;

[0051] 3. This fluorescent chemical sensor does not react with other common analytes in the body and can effectively and continuously identify Cu under the interference of common analytes. 2+ and glyphosate, that is, the fluorescent chemical sensor has strong anti-interference ability. Moreover, the fluorescent chemical sensor can effectively detect exogenous Cu in various living cell growth environments. 2+ and glyphosate;

[0052] 4. This fluorescent chemical sensor has the ability to detect Cu 2+ and the potential of glyphosate;

[0053] 5. The logP value of the fluorescent chemical sensor is 5.35, indicating that the fluorescent chemical sensor is a lipophilic compound with good cell membrane permeability. Cytotoxicity tests at different concentrations show that the fluorescent chemical sensor has low cytotoxicity.

[0054] 6. This fluorescent chemical sensor can effectively and specifically identify Cu in the body 2+ and glyphosate, and has been successfully applied to HeLa cells and zebrafish imaging. BRIEF DESCRIPTION OF THE DRAWINGS

[0055] Figure 1 (E)-N'-(4-(diethylamino)-2-hydroxybenzylidene)-3-oxo-3H-benzo[f]chromene-2-carboxylic acid hydrazide and Cu 2+ High-resolution mass spectra of the reaction products with glyphosate;

[0056] Figure 2 (E)-N'-(4-(diethylamino)-2-hydroxybenzylidene)-3-oxo-3H-benzo[f]chromene-2-carboxylic acid hydrazide and Cu 2+ Screening of solvents and solvent ratios for reactions with glyphosate;

[0057] Figure 3 (E)-N'-(4-(diethylamino)-2-hydroxybenzylidene)-3-oxo-3H-benzo[f]chromene-2-carboxylic acid hydrazide and Cu 2+ Effect of pH of the reaction solution with glyphosate;

[0058] Figure 4 Recognition of Cu by (E)-N'-(4-(diethylamino)-2-hydroxybenzylidene)-3-oxo-3H-benzo[f]chromene-2-carboxylic acid hydrazide 2+ Specificity and anti-interference study;

[0059] Figure 5 (E)-N'-(4-(diethylamino)-2-hydroxybenzylidene)-3-oxo-3H-benzo[f]chromene-2-carboxylic acid hydrazide-Cu 2+Study on the specificity and anti-interference ability of the system in identifying glyphosate;

[0060] Figure 6 Recognition of Cu by (E)-N'-(4-(diethylamino)-2-hydroxybenzylidene)-3-oxo-3H-benzo[f]chromene-2-carboxylic acid hydrazide 2+ Sensitivity studies;

[0061] Figure 7 (E)-N'-(4-(diethylamino)-2-hydroxybenzylidene)-3-oxo-3H-benzo[f]chromene-2-carboxylic acid hydrazide-Cu 2+ Study on the sensitivity of the system to recognize glyphosate;

[0062] Figure 8 Recognition of Cu by (E)-N'-(4-(diethylamino)-2-hydroxybenzylidene)-3-oxo-3H-benzo[f]chromene-2-carboxylic acid hydrazide 2+ and a cyclic detection diagram of glyphosate;

[0063] Figure 9 The bar graph shows the effect of different concentrations of (E)-N'-(4-(diethylamino)-2-hydroxybenzylidene)-3-oxo-3H-benzo[f]chromene-2-carboxylic acid hydrazide on cell survival rate.

[0064] Figure 10 This is a fluorescence microscopic image of (E)-N'-(4-(diethylamino)-2-hydroxybenzylidene)-3-oxo-3H-benzo[f]chromene-2-carboxylic acid hydrazide in HeLa cells;

[0065] Figure 11 Fluorescence microscopic imaging of (E)-N'-(4-(diethylamino)-2-hydroxybenzylidene)-3-oxo-3H-benzo[f]chromene-2-carboxylic acid hydrazide incubated in 3-day-old zebrafish. DETAILED DESCRIPTION

[0066] The present invention discloses a method for continuous specific detection of Cu 2+ The fluorescent chemical sensor is characterized in that it can continuously identify Cu 2+ and glyphosate, using the imino and hydroxyl groups in their structures to react with Cu 2+ Coordination produces fluorescence quenching effect to identify Cu 2+ , while glyphosate molecules contain amino, phosphate and carboxyl functional groups, which can react with Cu 2+ Stronger coordination, competing for Cu on fluorescent chemical sensors 2 +The fluorescent chemical sensor is released to generate fluorescence enhancement to detect glyphosate, thereby achieving Cu 2+ and continuous specific detection of glyphosate.

[0067] The present invention is further described below with reference to the embodiments

[0068] Example 1 is used for continuous specific detection of Cu 2+ Preparation of fluorescent chemical sensors for chloramphenicol and glyphosate

[0069] The fluorescent chemical sensor of the present invention is (E)-N'-(4-(diethylamino)-2-hydroxybenzylidene)-3-oxo-3H-benzo[f]chromene-2-carboxylic acid hydrazide, and the specific preparation process is as follows:

[0070]

[0071] (1) 2-Hydroxy-1-naphthaldehyde (30 mmol), diethyl malonate (33 mmol), pyrrolidine (0.5 mL), and ethanol (120 mL) were added to a 250 mL three-necked flask and refluxed. The reaction progress was monitored by TLC. After the reaction was completed, the reaction solution was slowly poured into 240 mL of water. Solid precipitated, which was filtered, washed with water, and dried to obtain ethyl 3-oxo-3H-benzo[f]chromene-2-carboxylate as a white solid.

[0072] (2) Ethyl 3-oxo-3H-benzo[f]chromene-2-carboxylate (10 mmol), 80% hydrazine hydrate (50 mmol) and tetrahydrofuran (100 mL) 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 3-oxo-3H-benzo[f]chromene-2-carboxylic acid hydrazide as a yellow solid.

[0073] (3) 3-Oxo-3H-benzo[f]chromene-2-carboxylic acid hydrazide (5 mmol), 2-hydroxy-4-diethylamino salicylaldehyde (5.5 mmol), 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 from DMF to obtain a yellow solid fluorescent probe (E)-N'-(4-(diethylamino)-2-hydroxybenzylidene)-3-oxo-3H-benzo[f]chromene-2-carboxylic acid hydrazide (DHBC). 13CNMR(151MHz,DMSO-d6):168.7,162.0,159.4,153.3,150.5,146.0,144.2,132.8,130.3,130 .1,128.8,128.5,126.8,123.6,122.3,117.7,115.5,114.1,108.0,104.5,99.1,47.1,12.9. 1 HNMR (400MHz, DMSO-d6): δ12.35(s,1H),10.70(s,1H),8.82(s,1H),8.62(s,1H),8.15–8.25(d,J=7.5,2.5Hz,2H),7.5 6–7.62(d,J=7.0,2.2Hz,2H),7.32–7.39(d,J=7.5,2.5Hz,2H),7.25(m,1H),6.31(s,1H),3.61–3.35(dd,J=7.5,3.4Hz 4H),1.12(s,6H).HRMS(ESI,m / z)calculated for[C 25 H 23 N3O4+H] + : 430.17668, found: 430.19290. The fluorescent chemical sensor of the present invention is (E)-N'-(4-(diethylamino)-2-hydroxybenzylidene)-3-oxo-3H-benzo[f]chromene-2-carboxylic acid hydrazide. The detection principle is as follows:

[0074]

[0075] The fluorescent chemical sensor of the present invention, (E)-N'-(4-(diethylamino)-2-hydroxybenzylidene)-3-oxo-3H-benzo[f]chromene-2-carboxylic acid hydrazide, can continuously and specifically detect Cu 2+ The mechanism of glyphosate is as follows:

[0076] Continuous and specific detection of Cu with (E)-N'-(4-(diethylamino)-2-hydroxybenzylidene)-3-oxo-3H-benzo[f]chromene-2-carboxylic acid hydrazide 2+ and glyphosate mechanism: The fluorescent chemical sensor uses the imino and hydroxyl groups on its structure to react with Cu 2+ Coordination produces fluorescence quenching effect to identify Cu 2+ , while glyphosate molecules contain amino, phosphate and carboxyl functional groups, which can react with Cu 2+ Forming a stronger coordination, competing for Cu on the fluorescent chemical sensor 2+ The fluorescent chemical sensor is released to generate fluorescence enhancement to detect glyphosate, thereby achieving Cu2+ Furthermore, high resolution mass spectrometry (HRMS) revealed that the fluorescent chemical sensor was compatible with Cu 2+ The molecular ion peak after coordination is m / z 492.8832 (the theoretical value of m / z is 492.0974). 2+ The molecular ion peak m / z after coordination was 231.1351 (the theoretical value of m / z was 231.9425), and the results confirmed that the fluorescent chemical sensor-Cu 2+ and glyphosate-Cu 2+ The complexes were generated in a 1:1 ratio ( Figure 1 ).

[0077] Example 2 Fluorescent chemical sensor for Cu in solution system 2+ Screening of factors affecting the continuous specific recognition of glyphosate

[0078] (1) The fluorescent chemical sensor was prepared into a working solution with a concentration of 10 μM using different solvents, wherein the solvents were dimethyl sulfoxide (DMSO), N,N-dimethylformamide (DMF), acetonitrile (Acetonitrile), methanol (Methanol), ethanol (EtOH), acetone (Acetone) and ethyl acetate (EA); 100 μM Cu was added to the 10 μM fluorescent chemical sensor solutions prepared in different solvents respectively. 2+ and glyphosate, with three parallels for each solution; the reaction was complete, and 42 reactants were obtained. The fluorescence intensity of the 42 reactants was measured ( Figure 2 a). The results showed that the fluorescent chemical sensor detected Cu 2+ The best results were obtained with DMSO in the glyphosate process, and DMSO was selected as the solvent for the detection of Cu 2+ and glyphosate.

[0079] (2) The fluorescent chemical sensor was prepared into a 10 μM fluorescent chemical sensor solution using different ratios of dimethyl sulfoxide (DMSO) and 4-hydroxyethylpiperazineethanesulfonic acid (HEPES), wherein the DMSO ratios were 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, and 90%; 100 μM Cu was added to the 10 μM fluorescent chemical sensor solution prepared using different ratios of DMSO. 2+ and glyphosate, with three parallels for each solution; after the reaction was complete, 60 reactants were obtained, and the fluorescence intensity of each of the 60 reactants was measured ( Figure 2 b) The results showed that the fluorescent chemical sensor detected Cu 2+ The optimal ratio of 4-hydroxyethylpiperazineethanesulfonic acid (HEPES) to DMSO in the glyphosate system is 3:7.

[0080] (3) The fluorescent chemical sensor solution was prepared with a buffer solution having a concentration of 10 μM. 2+ The working solution of the glyphosate detection system was prepared by mixing 4-hydroxyethylpiperazineethanesulfonic acid (HEPES) and DMSO in a volume ratio of 3:7. The pH of the buffer solution was set to 2, 3, 4, 5, 6, 6.5, 7, 7.4, 8, 9, 10, 11, and 12. 100 μM Cu was added to the near-infrared fluorescent chemical sensor solution with different pH concentrations of 10 μM. 2+ and glyphosate, with three parallels for each solution; the reaction was complete, yielding 78 reactants, which were each subjected to fluorescence intensity measurements ( Figure 3 ). The results showed that Cu 2+ The optimal pH of the detection system of dapoxetine and glyphosate is around 7.4. Considering that the subsequent imaging experiment will be carried out in vivo, the physiological pH of 7.4 is selected as the pH of the subsequent experimental solution.

[0081] Example 3: Cu in solution system 2+ Exploration of the effectiveness of continuous identification of glyphosate and

[0082] The fluorescent chemical sensor prepared in Example 1 was prepared into a fluorescent chemical sensor detection solution with a concentration of 10 μM using 4-hydroxyethylpiperazineethanesulfonic acid (HEPES) / dimethyl sulfoxide (DMSO) buffer to selectively detect Cu in the solution. 2+ The specific operation process is as follows:

[0083] 100 μM Cu was added to the prepared 10 μM fluorescent chemical sensor solution. 2+ and glyphosate, Figure 4 and 5 The results showed that the fluorescence of the fluorescent chemical sensor itself was very strong, and when Cu 2+ The fluorescence quenching phenomenon occurs after the addition of glyphosate, and the fluorescence intensity increases with the addition of glyphosate. The results show that the fluorescent chemical sensor can effectively and continuously identify Cu in the solution system. 2+ and glyphosate.

[0084] Example 4: Cu in environmental solution medium 2+ and glyphosate-specific detection

[0085] The fluorescent chemical sensor is prepared into a 10 μM 4-hydroxyethylpiperazineethanesulfonic acid (HEPES) and dimethyl sulfoxide (DMSO) buffer solution; the Cu 2+The working solution of the glyphosate detection system was prepared from 4-hydroxyethylpiperazineethanesulfonic acid (HEPES) and DMSO in a volume ratio of 3:7. 100 μM analyte solutions were added to the prepared 10 μM fluorescent probe solution, including various metal ions in the organism: barium chloride, mercuric sulfate, cobalt chloride, manganese chloride, nickel chloride, zinc chloride, potassium hydroxide, ferric chloride, sodium chloride, calcium chloride, magnesium chloride, ferrous chloride tetrahydrate, and copper sulfate pentahydrate; and various commercial organophosphorus pesticides: pyridabenzyl, glyphosate, profenofos, trichlorfon, diazinon, propargite, malathion, quinalphos, and glyphosate. Three replicates were set for each solution. The reaction was complete, obtaining 132 reactants, and the fluorescence intensity of each of the 132 reactants was measured ( Figure 4 and 5 ); The study of the change in fluorescence intensity showed that the fluorescent probe can 2+ The fluorescent chemical sensor can specifically and continuously recognize Cu 2+ To simulate the complex environment in cells, we also conducted competition experiments to determine the anti-interference ability of the fluorescent chemical sensor by adding potential interferents. The results showed that the fluorescent chemical sensor has strong anti-interference ability.

[0086] Example 5 Fluorescent Chemical Sensor for Cu 2+ Study on the sensitivity of continuous identification of glyphosate

[0087] The fluorescent chemical sensor was prepared into a 10 μM 4-hydroxyethylpiperazineethanesulfonic acid (HEPES) and dimethyl sulfoxide (DMSO) buffer solution; Cu 2+ at a concentration of 0-15 μM was gradually added to the prepared 10 μM fluorescent probe solution. 2+ and glyphosate, three replicates per group, a total of 90 reactants were obtained, and the fluorescence intensity of each of the 90 reactants was measured. The results showed that the fluorescence intensity was consistent with that of Cu 2+ Inversely proportional to the concentration ( Figure 6 ), which is proportional to glyphosate ( Figure 7 ), has a good linear relationship, that is, the fluorescent chemical sensor can quantitatively detect Cu in liquid medium 2+ and glyphosate.

[0088] Example 6 Fluorescent chemical sensor for cyclic detection of Cu 2+ and glyphosate

[0089] (1) preparing the fluorescent chemical sensor detection reagent, using a buffer solution (DMF:HEPES=7:3) to prepare a working solution of the fluorescent chemical sensor with a concentration of 10 μM;

[0090] (2) Alternately add 100 μM Cu to the 10 μM fluorescent chemical sensor solution. 2+ and glyphosate, and reacted for 1 min, and the fluorescence intensity at 517 nm was measured;

[0091] (3) The results show that in Cu 2+ During the alternating addition of glyphosate, Cu 2+ The coordinated fluorescent compound can stably detect glyphosate more than 6 times in a cycle, that is, the fluorescence intensity of the system has a high stability, which verifies the Cu 2+ Practical application potential of coordination fluorescent compounds ( Figure 8 ).

[0092] Example 7: Investigation of the cytotoxicity of fluorescent chemical sensors to HeLa cells

[0093] A series of probe solution concentration gradients (10, 20, 30, 40 and 50 μM) were added to the wells containing HeLa cells (5 × 10 4 Cells) were cultured in a 96-well culture plate, and untreated HeLa cells were used as a control. Then, after 24 hours of incubation, the cell viability of HeLa cells was verified by CCK-8 assay ( Figure 9 The results showed that when the concentration of the fluorescent chemical sensor was 50 μM, the HeLa survival rate was above 80%, indicating that the fluorescent chemical sensor has low cytotoxicity and can be used for the next step of in vivo imaging applications.

[0094] Example 8 Cu in HeLa cells 2+ and glyphosate fluorescence imaging

[0095] Cu in human cervical cancer HeLa cells 2+ When detecting glyphosate, a working solution with a concentration of 20 μM was prepared using 4-hydroxyethylpiperazineethanesulfonic acid (HEPES) / dimethyl sulfoxide (DMSO) buffer and the fluorescent chemical sensor of the present invention;

[0096] The specific operation process is as follows: divided into 6 groups: a, b, c, d, e, and f:

[0097] Group a (blank control): HeLa cells without any treatment, used as the test object of group a;

[0098] Group b (glyphosate control): HeLa cells were incubated with glyphosate (50 μM) for 30 min as the test substance for group b;

[0099] c group (Cu 2+ Control): Cu 2+ (50 μM) was incubated in HeLa cells for 30 min as the test substance in group c;

[0100] Group d (fluorescent compound control): HeLa cells were incubated with (E)-N'-(4-(diethylamino)-2-hydroxybenzylidene)-3-oxo-3H-benzo[f]chromene-2-carboxylic acid hydrazide (20 μM) for 30 min as the detection compound for group d;

[0101] e group (fluorescent chemical sensors and Cu 2+ Experimental group 1): HeLa cells were incubated with (E)-N'-(4-(diethylamino)-2-hydroxybenzylidene)-3-oxo-3H-benzo[f]chromene-2-carboxylic acid hydrazide (20 μM) for 30 minutes and then treated with Cu 2+ (50 μM) for 30 min as the test substance in group e;

[0102] f Group (fluorescent chemical sensor-Cu 2+ -Glyphosate experimental group 2): HeLa cells were treated with (E)-N'-(4-(diethylamino)-2-hydroxybenzylidene)-3-oxo-3H-benzo[f]chromene-2-carboxylic acid hydrazide (20 μM) and Cu 2+ (50 μM) for 30 min, and then HeLa cells were incubated with glyphosate (50 μM) for 30 min as the test substance in group f;

[0103] The fluorescence imaging results of groups d, e, and f showed that (E)-N'-(4-(diethylamino)-2-hydroxybenzylidene)-3-oxo-3H-benzo[f]chromene-2-carboxylic acid hydrazide could enter the cells and interact with Cu 2+ Reacting with glyphosate, cell imaging can clearly show Cu 2+ and the distribution of glyphosate in HeLa cells ( Figure 10 ).

[0104] Example 9 Cu in zebrafish 2+ and glyphosate fluorescence imaging

[0105] Cu in zebrafish 2+ When imaging glyphosate, a 4-hydroxyethylpiperazineethanesulfonic acid (HEPES) / dimethyl sulfoxide (DMSO) buffer solution is used to prepare a working solution with a concentration of 20 μM with the fluorescent chemical sensor of the present invention;

[0106] The specific operation process is as follows:

[0107] Divided into 6 groups: a, b, c, d, e, f:

[0108] Group a (blank control): three-day-old zebrafish without any treatment, used as the test object of group a;

[0109] Group b (glyphosate control): Three-day-old zebrafish were incubated with glyphosate (50 μM) for 30 minutes as the test substance for group b;

[0110] c group (Cu 2+ Control): Cu 2+ (50 μM) was incubated in three-day-old zebrafish for 30 minutes as the test substance in group c;

[0111] Group d (fluorescent compound control): Three-day-old zebrafish were incubated with (E)-N'-(4-(diethylamino)-2-hydroxybenzylidene)-3-oxo-3H-benzo[f]chromene-2-carboxylic acid hydrazide (20 μM) for 30 minutes as the test substance for group d;

[0112] e group (fluorescent chemical sensors and Cu 2+ Experimental group 1): Three-day-old zebrafish were incubated with (E)-N'-(4-(diethylamino)-2-hydroxybenzylidene)-3-oxo-3H-benzo[f]chromene-2-carboxylic acid hydrazide (20 μM) for 30 minutes and then treated with Cu 2+ (50 μM) for 30 min as the test substance in group e;

[0113] f Group (fluorescent chemical sensor-Cu 2+ -Glyphosate experimental group 2): Three-day-old zebrafish were treated with (E)-N'-(4-(diethylamino)-2-hydroxybenzylidene)-3-oxo-3H-benzo[f]chromene-2-carboxylic acid hydrazide (20 μM) and Cu 2+ (50 μM) for 30 min, and then three-day-old zebrafish were incubated with glyphosate (50 μM) for 30 min as the test substance in group f;

[0114] The results showed that zebrafish treated with (E)-N'-(4-(diethylamino)-2-hydroxybenzylidene)-3-oxo-3H-benzo[f]chromene-2-carboxylic acid hydrazide at 28°C showed obvious fluorescence, while zebrafish treated with (E)-N'-(4-(diethylamino)-2-hydroxybenzylidene)-3-oxo-3H-benzo[f]chromene-2-carboxylic acid hydrazide and Cu 2+ The treated zebrafish showed a certain fluorescence quenching phenomenon, and the fluorescence in the zebrafish recovered in the presence of glyphosate; the fluorescence imaging results showed that (E)-N'-(4-(diethylamino)-2-hydroxybenzylidene)-3-oxo-3H-benzo[f]chromene-2-carboxylic acid hydrazide could enter the zebrafish and interact with Cu 2+ Continuous reaction with glyphosate produces fluorescence quenching and recovery, thus achieving the detection effect ( Figure 11 ).

Claims

1. A method for the continuous and specific detection of Cu based on acylhydrazone derivatives 2+ A fluorescent chemical sensor for glyphosate and oxaline, characterized by: The fluorescent probe is (E)-N'-(4-(diethylamino)-2-hydroxybenzylidene)-3-oxo-3H-benzo[f]chromene-2-carboxylic acid hydrazide, and its chemical structure is shown in formula (I): ； Formula (I).

2. The method for preparing a fluorescent chemical sensor according to claim 1, wherein: The following steps are involved: (1) 2-Hydroxy-1-naphthaldehyde, diethyl malonate and pyrrolidine were reacted in ethanol to obtain ethyl 3-oxo-3H-benzo[f]chromene-2-carboxylate as a white solid; (2) 3-Oxo-3H-benzo[f]chromene-2-carboxylic acid ethyl ester was modified with hydrazine hydrate in tetrahydrofuran solution to obtain yellow solid 3-Oxo-3H-benzo[f]chromene-2-carboxylic acid hydrazide; (3) (E)-N'-(4-(diethylamino)-2-hydroxybenzylidene)-3-oxo-3H-benzo[f]chromene-2-carboxylic acid hydrazide was prepared by reacting 3-oxo-3H-benzo[f]chromene-2-carboxylic acid hydrazide, 2-hydroxy-4-diethylamino salicylaldehyde and glacial acetic acid in anhydrous ethanol.

3. The method for preparing a fluorescent chemical sensor according to claim 2, wherein: Step (1) 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 into a 250 mL three-necked flask, mix well, and reflux. Then, monitor the reaction process using a TLC plate. ② After the reaction is completed, the reaction solution is slowly poured into 240 mL of water until solid is precipitated, which is then filtered and washed with water, and finally dried to obtain white solid 3-oxo-3H-benzo[f]chromene-2-carboxylic acid ethyl ester.

4. The method for preparing a fluorescent chemical sensor according to claim 2, wherein: Step (2) includes the following steps: ①Use 100 mL of tetrahydrofuran as solvent to add 10 mmol of ethyl 3-oxo-3H-benzo[f]chromene-2-carboxylate and 50 mmol of hydrazine hydrate into a 250 mL three-necked flask and dissolve and mix evenly; ② Reflux for 6 hours. After the reaction is complete, concentrate to dryness under reduced pressure and recrystallize from ethanol to obtain a yellow solid 3-oxo-3H-benzo[f]chromene-2-carboxylic acid hydrazide.

5. The method for preparing a fluorescent chemical sensor according to claim 2, wherein: Step (3) includes the following steps: ① First, add 5 mmol of 3-oxo-3H-benzo[f]chromene-2-carboxylic acid hydrazide, 5.5 mmol of 2-hydroxy-4-diethylamino salicylaldehyde, 0.5 mL of glacial acetic acid, and 100 mL of anhydrous ethanol into a 250 mL three-necked flask, and then reflux for 15 hours; ② After cooling to room temperature, the mixture is filtered, dried, and recrystallized in DMF to obtain a yellow solid fluorescent probe (E)-N'-(4-(diethylamino)-2-hydroxybenzylidene)-3-oxo-3H-benzo[f]chromene-2-carboxylic acid hydrazide.

6. The use of the fluorescent chemical sensor according to claim 1, characterized in that: Fluorescent chemical sensors for Cu in solution systems 2+ The screening of factors affecting the identification of glyphosate includes the following steps: (1) The fluorescent chemical sensor according to claim 1 was prepared into a working solution with a concentration of 10 μM using different solvents, wherein the solvents were dimethyl sulfoxide, N, N-dimethylformamide, acetonitrile, methanol, ethanol, acetone and ethyl acetate; 100 μM Cu was added to the 10 μM fluorescent chemical sensor solutions prepared in different solvents. 2+ and glyphosate, three parallels were set for each solution; the reaction was complete to obtain 21 reactants, and the fluorescence intensity of the 21 reactants was measured respectively. Dimethyl sulfoxide made the fluorescent chemical sensor 2+ It has better fluorescence quenching effect and fluorescent chemical sensor-Cu 2+ The system exhibited good fluorescence enhancement in the presence of glyphosate, so dimethyl sulfoxide was selected as the standard solvent for subsequent experiments; (2) The fluorescent chemical sensor according to claim 1 is prepared into a fluorescent chemical sensor solution with a concentration of 10 μM using different ratios of dimethyl sulfoxide and 4-hydroxyethylpiperazineethanesulfonic acid, wherein the ratios of dimethyl sulfoxide to 4-hydroxyethylpiperazineethanesulfonic acid are 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, and 90%, respectively; 100 μM Cu is added to the 10 μM fluorescent chemical sensor solution prepared using different ratios of dimethyl sulfoxide, respectively. 2+ and glyphosate, with three replicates for each solution. The reaction was complete, yielding 27 reactants. Fluorescence intensity measurements were performed on each of the 27 reactants. The results showed that a dimethyl sulfoxide:4-hydroxyethylpiperazineethanesulfonic acid ratio of 7:3 maximized the fluorescence performance of the fluorescent chemical sensor. Therefore, a dimethyl sulfoxide / 4-hydroxyethylpiperazineethanesulfonic acid ratio of 7:3 was selected as the buffer solution for subsequent experiments. The fluorescent chemical sensor buffer according to claim 1 is prepared into a fluorescent chemical sensor solution with a concentration of 10 μM, wherein the Cu 2+ The working solution of the glyphosate detection system was prepared by mixing methyl sulfoxide and 4-hydroxyethylpiperazineethanesulfonic acid in a volume ratio of 7:3; the pH of the buffer solution was set to 2, 3, 4, 5, 6, 6.5, 7, 7.4, 8, 9, 10, 11, and 12; 100 μM glyphosate and Cu were added to the fluorescent chemical sensor solution with a concentration of 10 μM at different pH values. 2+ , three parallels were set for each solution; the reaction was complete, and 78 reactants were obtained. The fluorescence intensity of 78 reactants was measured respectively. The results showed that Cu 2+ The optimal pH of the detection system for glyphosate and ethanol is 7.

4. Considering that the subsequent imaging experiment will be carried out in vivo, the physiological pH of 7.4 is selected as the pH of the subsequent experimental solution.

7. The use of the fluorescent chemical sensor according to claim 1, characterized in that: The fluorescent compound of formula (I) is used as a fluorescent probe for continuous and specific detection of Cu 2+ and glyphosate, cyclic detection of Cu 2+ and glyphosate, comprising the steps of: (1) First, prepare the fluorescent chemical sensor detection reagent according to claim 1, and then use a buffer solution prepared with N, N-dimethylformamide:4-hydroxyethylpiperazineethanesulfonic acid = 7:3 to prepare the fluorescent chemical sensor to a working solution with a concentration of 10 μM; (2) Then 100 μM Cu was added alternately to the 10 μM fluorescent chemical sensor solution. 2+ and glyphosate, and the reaction was allowed to proceed for another 1 min, and the fluorescence intensity at 517 nm was measured; (3) Finally, the results show that in Cu 2+ During the alternating addition of glyphosate, Cu 2+ The fluorescent compound of coordination formula (I) can stably detect glyphosate for more than 6 times, that is, the fluorescence intensity of the system has high stability, which verifies the Cu 2+ Practical application potential of fluorescent compounds with coordination formula (I).

8. The use of the fluorescent chemical sensor according to claim 1, characterized in that: For use in environmental solution media Cu 2 + The invention relates to a method for the specific detection of Cu and glyphosate, wherein the fluorescent chemical sensor according to claim 1 is prepared into a 10 μM 4-hydroxyethylpiperazineethanesulfonic acid and dimethyl sulfoxide buffer solution; the volume ratio of 4-hydroxyethylpiperazineethanesulfonic acid to dimethyl sulfoxide in the buffer solution is 3:7, and the pH value of the buffer solution is 7.4; 100 μM analyte solutions are added to the prepared 10 μM fluorescent chemical sensor solution, including various metal ions: barium chloride, mercuric sulfate, cobalt chloride, manganese chloride, nickel chloride, zinc chloride, potassium hydroxide, ferric chloride, sodium chloride, calcium chloride, magnesium chloride, ferrous chloride tetrahydrate, copper sulfate pentahydrate; and various commercial organophosphorus pesticides: pyridabenzyl, glyphosate, profenofos, trichlorfon, diazinon, propargite, malathion, quinalphos and glyphosate; after the reaction is complete, the fluorescence intensity is measured; and the change in fluorescence intensity shows that the fluorescent probe can react with Cu and glyphosate. 2+ The fluorescent chemical sensor can continuously and specifically detect Cu 2+ and glyphosate.

9. The use of the fluorescent chemical sensor according to claim 1, characterized in that: For the detection of Cu in HeLa cells of human cervical cancer tissue 2+ When distributing glyphosate, the fluorescent chemical sensor according to claim 1 is prepared into a buffer solution with a concentration of 20 μM; and divided into 6 groups: a, b, c, d, e, and f: Group a, blank control: HeLa cells without any treatment, used as the test object of group a; Group b, glyphosate control: HeLa cells were incubated with 50 μM glyphosate for 30 min as the test substance for group b; Group c, Cu 2+ Control: 50 μM Cu 2+ HeLa cells were incubated for 30 min and used as test objects in group c; Group d, fluorescent compound control: HeLa cells were incubated with 20 μM (E)-N'-(4-(diethylamino)-2-hydroxybenzylidene)-3-oxo-3H-benzo[f]chromene-2-carboxylic acid hydrazide for 30 min, which was used as the detection compound for group d; Group e, fluorescent chemical sensors and Cu 2+ Experimental group 1: HeLa cells were incubated with 20 μM (E)-N'-(4-(diethylamino)-2-hydroxybenzylidene)-3-oxo-3H-benzo[f]chromene-2-carboxylic acid hydrazide for 30 minutes and then with 50 μM Cu 2+ Treated for 30 minutes, used as test objects in group e; Group f, fluorescent chemical sensor-Cu 2+ -Glyphosate experimental group 2: HeLa cells were treated with 20 μM (E)-N'-(4-(diethylamino)-2-hydroxybenzylidene)-3-oxo-3H-benzo[f]chromene-2-carboxylic acid hydrazide and 50 μM Cu 2+ HeLa cells were treated for 30 min and then incubated with 50 μM glyphosate for 30 min as the test substance in group f; The fluorescence imaging results of groups d, e, and f showed that (E)-N'-(4-(diethylamino)-2-hydroxybenzylidene)-3-oxo-3H-benzo[f]chromene-2-carboxylic acid hydrazide could enter the cells and interact with Cu 2+ Reacting with glyphosate, cell imaging can clearly show Cu 2+ and the distribution of glyphosate in HeLa cells. The above methods are not used for diagnosis and treatment of diseases.

10. The use of the fluorescent chemical sensor according to claim 1, characterized in that: For Cu in zebrafish 2+ When continuously and specifically detecting glyphosate, the fluorescent chemical sensor according to claim 1 is prepared into a working solution with a concentration of 20 μM; and divided into 6 groups a, b, c, d, e, and f: Group a, blank control: three-day-old zebrafish without any treatment, used as test objects in group a; Group b, glyphosate control: three-day-old zebrafish were incubated with 50 μM glyphosate for 30 minutes as the test substance for group b; Group c, Cu 2+ Control: 50 μM Cu 2+ Three-day-old zebrafish were incubated for 30 minutes and used as test objects in group C; Group d, fluorescent compound control: three-day-old zebrafish were incubated with 20 μM (E)-N'-(4-(diethylamino)-2-hydroxybenzylidene)-3-oxo-3H-benzo[f]chromene-2-carboxylic acid hydrazide for 30 minutes as the test substance for group d; Group e, fluorescent chemical sensors and Cu 2+ Experimental group 1: Three-day-old zebrafish were incubated with 20 μM (E)-N'-(4-(diethylamino)-2-hydroxybenzylidene)-3-oxo-3H-benzo[f]chromene-2-carboxylic acid hydrazide for 30 minutes and then with 50 μM Cu 2+ Treated for 30 minutes, used as test objects in group e; Group f, fluorescent chemical sensor-Cu 2+ -Glyphosate experimental group 2: Three-day-old zebrafish were treated with 20 μM (E)-N'-(4-(diethylamino)-2-hydroxybenzylidene)-3-oxo-3H-benzo[f]chromene-2-carboxylic acid hydrazide and 50 μM Cu 2+ Three-day-old zebrafish were treated for 30 min, and then incubated with 50 μM glyphosate for 30 min as the test object in group f; The results showed that zebrafish treated with (E)-N'-(4-(diethylamino)-2-hydroxybenzylidene)-3-oxo-3H-benzo[f]chromene-2-carboxylic acid hydrazide at 28°C showed obvious fluorescence, while zebrafish treated with (E)-N'-(4-(diethylamino)-2-hydroxybenzylidene)-3-oxo-3H-benzo[f]chromene-2-carboxylic acid hydrazide and Cu 2+ The treated zebrafish showed a certain fluorescence quenching phenomenon, and the fluorescence in the zebrafish recovered in the presence of glyphosate; the fluorescence imaging results showed that (E)-N'-(4-(diethylamino)-2-hydroxybenzylidene)-3-oxo-3H-benzo[f]chromene-2-carboxylic acid hydrazide could enter the zebrafish and interact with Cu 2+ Continuous reaction with glyphosate produces fluorescence quenching and recovery phenomena, thereby achieving the detection effect. The above method is not used for diagnosis and treatment of diseases.

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