A fluorescent probe and its use in fluorescent detection of glyphosate

Abstract

The present invention relates to the fields of chemistry, agriculture, and environmental protection, and discloses a fluorescent probe and its use in fluorescent detection of glyphosate, specifically a fluorescent probe technology for rapid detection of glyphosate in water and agricultural products. The colorimetric fluorescent probe, which combines a 2-chloroaniline sensing unit with the fluorophore dansyl chloride, induces fluorescence enhancement through a substitution reaction with glyphosate. Specifically, the probe can detect glyphosate in the range of 0 to 70 μmol·L. ‑1 Fluorescence titration experiments were conducted with glyphosate at varying concentrations within a specific range. With the gradual addition of glyphosate, the probe's fluorescence spectrum rapidly increased, establishing a satisfactory relationship between fluorescence intensity and glyphosate concentration. Common anions and metal ions found in water did not interfere with the reaction, demonstrating the detection method's excellent anti-interference properties. Furthermore, this probe was used to detect glyphosate in tap water, Xiangxi River Reservoir, soil, soybeans, and corn, with recoveries ranging from 94.7% to 109.9%. This open-type fluorescent probe can specifically detect glyphosate with the naked eye under ultraviolet light.

Description

Technical Field

[0001] The present invention relates to a technology for rapidly detecting glyphosate in water and agricultural products, and more particularly to the preparation of a fluorescent probe for rapidly detecting glyphosate in water and agricultural products. Background Art

[0002] Glyphosate is a highly effective, broad-spectrum organophosphine herbicide launched by Monsanto in 1974. It is the fastest-growing, most widely sold, and most widely used herbicide worldwide. Glyphosate is widely used in orchards, rice, corn, soybeans, wheat, tea plantations, and other cash crops. Due to its frequent use, glyphosate has been found in surface water and streams from agricultural, urban, and forestry sectors, posing potential risks to human health. It has been classified by the International Agency for Research on Cancer (IARC) as genotoxic and potentially carcinogenic to humans, sparking intense controversy among the public and regulators.

[0003] Glyphosate's highly polar molecular structure and lack of chromophores or fluorophores make its detection difficult. Conventional analytical methods for its detection include gas chromatography (GC), gas chromatography-mass spectrometry (GC-MS), high-performance liquid chromatography (HPLC), enzyme-linked immunosorbent assay (ELISA), capillary electrophoresis (CE), ion chromatography (IC), and inductively coupled plasma mass spectrometry (ICP-MS). While these methods are generally suitable for laboratory analysis with sufficient sensitivity and accuracy, they still have some inevitable disadvantages, such as time-consuming derivatization procedures (4-16 hours) and the requirement for highly trained personnel, which limits their practical application.

[0004] In recent years, fluorescent probes have attracted extensive attention due to their low cost and ease of operation. Although a variety of fluorescent probes have been reported for the detection of glyphosate, the general design strategy is a chemical sensor based on competitive coordination, Cu 2+ However, compared with the “signal on-off-on” type fluorescent probes, the on-type fluorescent probes usually have a higher signal-to-noise ratio under dark background and can effectively avoid false positive signals, which means they are more suitable for practical applications. Summary of the Invention

[0005] Designing a fluorescent probe that directly recognizes glyphosate is challenging. This colorimetric fluorescent probe, consisting of a 2-chloroaniline sensing unit coupled to the fluorophore dansyl chloride, undergoes a substitution reaction with glyphosate to induce fluorescence enhancement. This probe exhibits a weak fluorescence signal, but upon addition of glyphosate, the PET fluorescence intensity increases by more than tenfold through a photoinduced electron transfer process. This on-type fluorescent probe achieves a stable fluorescence signal within two minutes and can specifically detect glyphosate with the naked eye under ultraviolet light, showing great promise for on-site determination.

[0006] The present invention is achieved through the following technical solutions:

[0007] Fluorescent probes are used to rapidly detect glyphosate in water and agricultural products. Its molecular structure is as follows:

[0008]

[0009] The synthesis method of the fluorescent probe described above:

[0010]

[0011] 2-Chloroaniline and dansyl chloride are dissolved in chloroform, a small amount of triethylamine is added, and the reaction is heated to reflux. The reaction product is evaporated and purified by silica gel column chromatography to obtain a fluorescent probe product. The reflux reaction temperature is 60-80°C and the reaction time is 10-20 hours.

[0012] On the one hand, triethylamine mainly acts as a base catalyst and participates in acid-base neutralization reactions; on the other hand, triethylamine can act as a proton intermediary and participate in proton transfer reactions, promoting proton transfer and accelerating the reaction rate.

[0013] The technical solution of the present invention also provides a fluorescent probe for detecting glyphosate, wherein the fluorescent probe is the fluorescent probe described above or the fluorescent probe prepared by the method described above.

[0014] The reaction principle of the present invention is that dansyl chloride linked to 2-chloroaniline acts as a fluorescent probe, which undergoes a substitution reaction with glyphosate to induce fluorescence enhancement. The possible response mechanism is shown in the following formula:

[0015]

[0016] The fluorescent probe for detecting glyphosate is used for detecting glyphosate in a non-disease diagnosis and treatment method during the glyphosate detection process.

[0017] The fluorescent probe detects glyphosate in a water environment, a solvent environment, a soil environment, and a plant environment.

[0018] The water environment includes any one of tap water, river water, and reservoir water;

[0019] The solvent environment includes N,N-dimethylformamide, tetrahydrofuran, dimethyl sulfoxide, ethyl acetate, and acetone;

[0020] The soil environment includes a soil environment to which glyphosate has been applied;

[0021] The plant environment includes any one of soybean, corn, rapeseed, wheat, tea, citrus, and cotton;

[0022] The present invention also provides a new use of the fluorescent probe for detecting glyphosate in selectively detecting glyphosate.

[0023] The fluorescent probe for detecting glyphosate is used for detecting glyphosate in a non-disease diagnosis and treatment method during the process of selectively detecting glyphosate.

[0024] The fluorescent probe for detecting glyphosate has a new application in selectively detecting glyphosate in a cationic environment; the cation includes Na + , K + 、Cu 2+ Mg 2+ 、Zn 2+ 、Co 2+ 、Al 3+ 、Fe 3+ and Cr 3+ Any one or more combinations of .

[0025] The fluorescent probe for detecting glyphosate has a new application in selectively detecting glyphosate in an anion environment; the anions include SO2-4, HCO-3, H2PO-4, CH3COO - 、Cl - , NO- 3, or any one or more combinations thereof.

[0026] The fluorescent probe for detecting glyphosate has a fluorescence emission peak intensity of approximately 530 nm during the process of selectively detecting glyphosate;

[0027] The minimum detection limit was lower than 4.0 nmol·L -1 The minimum detection limit is preferably less than 3 nmol·L -1 The minimum detection limit is preferably less than 2 nmol·L -1 ;

[0028] The minimum detection time is less than 8 minutes, more preferably the minimum detection time is less than 5 minutes, and even more preferably the minimum detection time is less than 3 minutes.

[0029] Advantages of the present invention:

[0030] The fluorescent probe prepared by the invention has a simple synthesis process and is easy to prepare.

[0031] The fluorescent probe prepared by the present invention is sensitive to 0-70 μmol·L -1 The linear relationship between glyphosate and the concentration of glyphosate was good within the range, and the minimum detection limit was 2.0 nmol·L -1It can meet the detection requirements of my country's drinking water hygiene standards (GB5749-2006), has high sensitivity, and realizes trace detection of glyphosate.

[0032] The fluorescent probe prepared by the present invention has good anti-interference ability to glyphosate and is not interfered by other metal ions and common anions.

[0033] The fluorescent probe prepared by the present invention can detect glyphosate in tap water, Xiangxi River Reservoir, soil, soybeans and corn, with a recovery rate of 94.7% to 109.9%.

[0034] The fluorescent probe prepared by the present invention can identify and detect glyphosate with the naked eye and has good application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 The diagram shows the mechanism of substitution reaction between glyphosate and fluorescent probe.

[0036] Figure 2 For fluorescent probes 1 H NMR spectrum.

[0037] Figure 3 For fluorescent probes 13 C HMR spectrum.

[0038] Figure 4 The fluorescence spectra of the probe in response to different concentrations of glyphosate.

[0039] Figure 5 Create a graph for the glyphosate working curve.

[0040] Figure 6 is the response time of the fluorescent probe to glyphosate.

[0041] Figure 7 This is a diagram showing the effect of coexisting metal ions on glyphosate detection.

[0042] Figure 8 Graph showing the effect of coexisting anions on glyphosate degradation. DETAILED DESCRIPTION

[0043] The present invention will be further described below with reference to specific examples, but the present invention is not limited to the following examples.

[0044] Example 1 is a method for synthesizing a fluorescent probe:

[0045] 2-Chloroaniline (1.27 g, 10 mmol) and dansyl chloride (2.967 g, 11 mmol) were dissolved in chloroform (150 mL), and triethylamine (1.6 mL) was added dropwise. The mixture was refluxed at 65°C for 12 h. After cooling, the reaction solution was concentrated by rotary evaporation, extracted with dichloromethane, and dried over anhydrous sodium sulfate. After rotary evaporation again, the mixture was purified by silica gel column chromatography using petroleum ether / ethyl acetate as the eluent (1:20, v / v) to obtain a light green solid with a yield of 85%. 1 H NMR, 13 C HMR spectroscopy (e.g. Figure 2 and 3 ) and high-resolution mass spectrometry analysis fully demonstrated the synthesis of the probe. 1 H NMR (400 MHz, DMSO- d 6) δ 10.26 (s, 1H), 8.47(d, J = 8.0 Hz, 1H), 8.37 (d, J = 8.0 Hz, 1H), 8.03 (dd, J1= 2.0 Hz, J2= 8.0Hz, 1H), 7.59-7.54 (m, 2H), 7.32 (dd, J1= 2.0 Hz, J2= 8.0 Hz, 1H), 7.26-7.13(m, 4H), 2.82 (s, 6H). 13 C NMR (100 MHz, DMSO- d 6) δ 151.73, 136.36. 134.1,130.56, 130.32, 129.62, 129.47, 129.42, 128.32, 128.16, 127.91, 127.54,123.50, 119.66, 115.64. HR-MS (ESI): [M+H] + calcd for C 18 H 17 ClN2O2S, 361.8573; found, 361.8570.

[0046] Example 2 is the fluorescence probe for different concentrations of glyphosate (C0 = 0 ~70 μmol·L -1 ) Fluorescence response

[0047] The fluorescent probe prepared in Example 1 was prepared into a 5 μmol / L stock solution using N,N-dimethylformamide (DMF) solution. 3 mL of the fluorescent probe stock solution was taken into a cuvette and then 0-70 μmol·L -1The glyphosate standard solution was placed at room temperature for 5 minutes, and the fluorescence emission spectrum of the probe was measured under 340 nm excitation light. The results are as follows: Figure 4 As shown. Record the peak fluorescence emission intensity at 530 nm (as shown Figure 5 ), the linear relationship between fluorescence intensity and PMG concentration was obtained as y = 800.8x + 4334.6 (R 2 =0.995).

[0048] Example 3 is the response time of the fluorescent probe and glyphosate

[0049] This example uses the fluorescent probe stock solution prepared in Example 2 to detect its response time to glyphosate. Take 3 mL of the fluorescent probe stock solution in a cuvette and add 30 μmol·L -1 After waiting for 0, 2, 5, and 10 minutes respectively, the fluorescence emission spectrum of the probe was measured under 340nm excitation light. The results are as follows Figure 6 As shown, the fluorescence intensity hardly changed significantly after 2 minutes, indicating that the response time of the fluorescent probe was fast and stable after interacting with glyphosate.

[0050] Example 4 is the selectivity of the fluorescent probe for glyphosate in the presence of metal ions

[0051] This example uses the fluorescent probe stock solution prepared in Example 2 to test its selectivity for glyphosate. Take 3 mL of the fluorescent probe stock solution in a cuvette and add 30 μmol·L -1 glyphosate solution, and then add 10 μmol·L -1 Common metal ions (Na + 、Cu 2+ Mg 2+ 、Zn 2+ 、Co 2+ 、Al 3+ 、Fe 3+ and Cr 3+ In the present invention, only these common cations are used as experimental cases. The technical solutions of this application are not limited to these metal cations. The technical solutions for detecting glyphosate in the above cations are all within the protection scope of this case). Under the action of 340 nm excitation light, the fluorescence emission spectrum of the probe is measured, and the results are as follows. Figure 7 As shown in the figure, the fluorescence of glyphosate hardly changed significantly, indicating that the presence of metal ions did not interfere with the detection of glyphosate.

[0052] Example 5 is the selectivity of the fluorescent probe for glyphosate in the presence of anions

[0053] This example uses the fluorescent probe stock solution prepared in Example 2 to test its selectivity for glyphosate. Take 3 mL of the fluorescent probe stock solution in a cuvette and add 30 μmol·L -1 glyphosate solution, and then add 10 μmol·L -1 Common anions (SO2- 4, HCO- 3, H2PO- 4, CH3COO - 、Cl - , NO- 3, the present invention only uses these common anions as experimental cases, and the technical solutions of this application are not limited to these anions. The technical solutions for detecting glyphosate in the above anions are all within the protection scope of this case). Under the action of 340 nm excitation light, the fluorescence emission spectrum of the probe is measured, and the results are as follows Figure 8 As shown in the figure, the fluorescence of PMG hardly changes significantly. The results indicate that the presence of anions will not interfere with the detection of PMG. Therefore, the probe has good anti-interference ability for the detection of PMG.

[0054] Example 6: Detection of glyphosate in actual water samples using a fluorescent probe

[0055] To investigate the potential application of the probe in a practical environment, two water samples, tap water and Xiangxi River Reservoir (Yichang, Hubei), were selected and pretreated: the water samples were centrifuged at 10,000 rpm for 10 min and filtered through a 0.45 μm filter membrane to prepare the following concentrations: 10 μmol·L -1 , 30 μmol·L -1 , 50 μmol·L -1 of glyphosate solution.

[0056] Using the fluorescent probe stock solution prepared in Example 2, the glyphosate solutions of varying concentrations were added to the fluorescent probe. Under 340 nm excitation light, the peak fluorescence emission intensity of the fluorescent probe at 530 nm was measured. This value was then substituted into the equation in Example 2 to calculate the concentration of the glyphosate solution. The test results are shown in Table 1.

[0057] Example 7: Detection of glyphosate in agricultural products using fluorescent probes

[0058] To investigate the potential application of the probe in a real-world setting, campus soil, soybeans, and corn (purchased from a local supermarket) were tested using the standard addition method. Pretreatment involved weighing a certain amount of soybean or corn sample, grinding it, mixing it with 100 mL of water, ultrasonicating it for 15 minutes, and centrifuging it at 10,000 rpm for 10 minutes.

[0059] Using the fluorescent probe stock solution prepared in Example 2, the glyphosate solutions of varying concentrations were added to the fluorescent probe. Under 340 nm excitation light, the peak fluorescence emission intensity of the fluorescent probe at 530 nm was measured. This value was then substituted into the equation in Example 2 to calculate the concentration of the glyphosate solution. The test results are shown in Table 1.

[0060] As shown in Table 1, the recovery rates of glyphosate in actual water samples are 94.7-109.9%. These results indicate that the fluorescent probe prepared by the present invention has high accuracy in detecting glyphosate in actual water samples and agricultural products.

[0061] Table 1 Fluorescent probes for glyphosate detection in actual water samples and agricultural products

[0062]

Claims

1. A fluorescent probe for detecting glyphosate for non-disease diagnosis and treatment purposes, characterized in that: The structural formula of the fluorescent probe is .

2. The use according to claim 1, characterized in that The fluorescent probe detects glyphosate in a water environment, a solvent environment, a soil environment, and a plant environment.

3. The use according to claim 2, characterized in that The water environment includes any one of tap water, river water, and reservoir water; The solvent environment includes N,N-dimethylformamide, tetrahydrofuran, dimethyl sulfoxide, ethyl acetate, and acetone; The soil environment includes a soil environment to which glyphosate has been applied; The plant environment includes any one of soybean, corn, rapeseed, wheat, tea, citrus and cotton.

4. The use according to claim 3, characterized in that The fluorescent probe is used for selectively detecting glyphosate.

5. The use according to claim 4, characterized in that The fluorescent probe is used to selectively detect glyphosate in a cationic environment; the cations include Na + , K + 、Cu 2+ Mg 2+ 、Zn 2+ 、Co 2+ 、Al 3+ 、Fe 3+ and Cr 3+ Any one or more combinations of .

6. The use according to claim 5, characterized in that The fluorescent probe is used to selectively detect glyphosate in an anionic environment; the anions include SO4 2- 、HCO3 - 、H2PO4 - 、CH3COO - 、Cl - 、NO3 - Any one or more combinations of .

7. The use according to claim 6, characterized in that The fluorescent probe for detecting glyphosate has a fluorescence emission peak intensity of approximately 530 nm during the process of selectively detecting glyphosate; The minimum detection limit was lower than 4.0 nmol·L -1 ; The minimum detection time is less than 8 minutes.

8. The use according to claim 7, characterized in that The fluorescent probe for detecting glyphosate has a fluorescence emission peak intensity of approximately 530 nm during the process of selectively detecting glyphosate; The minimum detection limit is less than 3 nmol·L -1 ; The minimum detection time is less than 5 minutes.

9. The use according to claim 8, characterized in that The fluorescent probe for detecting glyphosate has a fluorescence emission peak intensity of approximately 530 nm during the process of selectively detecting glyphosate; The minimum detection limit is less than 2 nmol·L -1 ; The minimum detection time is less than 3 minutes.

10. The use according to any one of claims 1 to 9, characterized in that The preparation method of the fluorescent probe is as follows: 2-chloroaniline and dansyl chloride are dissolved in chloroform, triethylamine is added, and the reaction is carried out under reflux at elevated temperature. The reaction product is evaporated and purified by silica gel column chromatography to obtain the fluorescent probe product; the reaction formula is as follows: The reflux reaction temperature is 60-80°C and the reaction time is 10-20 h.

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