Fluorescent probe for detecting glyphosate as well as preparation method and application of fluorescent probe

Abstract

The invention discloses a fluorescent probe for detecting glyphosate as well as a preparation method and application of the fluorescent probe. The fluorescent probe is composed of a complex formed by 2-(2-hydroxyphenyl) benzoxazole and Cu < 2 + > according to the stoichiometric ratio of 2: 1. The complexing ability of the glyphosate and Cu < 2 + > is stronger than that of the 2-(2-hydroxyphenyl) benzoxazole and Cu < 2 + >, so that when the glyphosate is added into the 2-(2-hydroxyphenyl) benzoxazole-Cu < 2 + > complex, Cu < 2 + > is replaced by the glyphosate, a probe is decomplexed, and a remarkable fluorescence enhancement phenomenon is generated. The fluorescent probe not only can efficiently detect glyphosate, but also is not interfered by other types of pesticides, has the advantages of extremely simple and convenient preparation method, high selectivity, high sensitivity and quick response, and realizes trace detection of glyphosate. In addition, test paper prepared from the fluorescent probe can visually detect glyphosate under ultraviolet irradiation.

Description

Technical Field

[0001] The present invention belongs to the field of glyphosate analysis and detection, and particularly relates to a fluorescent probe for detecting glyphosate, a preparation method thereof, and an application thereof. Background Art

[0002] Glyphosate (GLY), a water-soluble, broad-spectrum, and highly effective herbicide, has been widely used for weed control in agricultural production since 1974. The long-term and widespread application of glyphosate has resulted in its persistent accumulation in crops, soil, and aquatic environments. As a Class 2A carcinogen, glyphosate poses significant threats to human health, including disruption of neural signaling, DNA damage, and various cancers such as melanoma of the skin, non-Hodgkin's lymphoma, breast cancer, liver cancer, kidney cancer, and thyroid cancer. Glyphosate residues have become a key concern in food safety and environmental monitoring, and accurate monitoring of residue levels is crucial for protecting health and the environment. The World Health Organization has set a maximum residue limit (MRL) in drinking water at 0.9 mg / L, and my country's national standard GB5479-2022 stipulates that the maximum residue limit for glyphosate in drinking water shall not exceed 0.7 μg / mL. Traditional methods for detecting glyphosate include spectrophotometry, gas chromatography-mass spectrometry (GC-MS), liquid chromatography-mass spectrometry (LC-MS), and ion chromatography. While these traditional glyphosate detection methods offer advantages such as high selectivity, excellent sensitivity, and low limits of detection and quantification, they still face numerous challenges in practical application. For example, they often require complex sampling techniques, derivatization steps, and cumbersome operational procedures, and rely on expensive precision instruments and specialized technical support. These issues limit the widespread application of these traditional detection methods in routine monitoring scenarios.

[0003] Compared with traditional detection methods, fluorescent probe detection of glyphosate has the advantages of being simple and rapid, with intuitive and visible results, requiring less sample, and not requiring complex instruments and professional knowledge. These characteristics make it more versatile and practical, and suitable for rapid detection of glyphosate in a variety of scenarios. Currently, some fluorescent probes for glyphosate detection have been developed. For example, Chinese patent CN118294420 A "Fluorescein for the detection of lead ions and glyphosate and detection method" uses acetone or alcohol reagents and buffer solution in a volume ratio of 99:1-50:50 to prepare a fluorescein solution, and uses the principle that fluorescein molecules form orange or orange-red substances with lead ions in a specific solvent system, and when glyphosate is present in the solvent system, no orange or orange-red substances can be formed, thereby achieving rapid detection of glyphosate. However, lead ions are introduced during the detection process, which may cause certain harm to the environment and humans, and quantitative detection is impossible, which to a certain extent limits its application. Chinese patent CN 115494033 A "A method for detecting glyphosate" is based on Cu2+ Can quench carbon dot fluorescence, glyphosate can specifically bind to Cu 2+ Based on the principle of carbon dot fluorescence recovery, a "turn-off-on" fluorescent carbon dot sensor was constructed to detect glyphosate. Although the detection limit of the fluorescent carbon dot sensor is low and it can specifically detect glyphosate, it has problems such as poor visualization and long response time. Chinese patent CN 119390611 A "A dual-recognition fluorescent molecular probe, its preparation method and application in the detection of lead ions and / or glyphosate" prepared a Schiff base fluorescent probe with dicyanoisophorone and benzoylhydrazide as fluorescent groups. After complexing with lead ions, it can accurately detect glyphosate with a detection limit as low as 10 -8 However, this fluorescent probe requires multiple synthesis steps, a complex reaction process, and high preparation cost. Therefore, it is of great significance to develop a simple, cost-effective, visual, and fast-response fluorescent probe for glyphosate detection. Summary of the Invention

[0004] In order to solve the above-mentioned shortcomings and deficiencies of the prior art, the present invention aims to provide a 2-(2-hydroxyphenyl)benzoxazole-Cu 2+ Complex fluorescent probe. This probe is easy to prepare and low-cost, enabling efficient, visual, sensitive, and rapid glyphosate detection. Furthermore, test strips made with this probe can directly identify and detect glyphosate with the naked eye under ultraviolet light.

[0005] The object of the present invention is achieved by at least one of the following technical solutions: The present invention provides a fluorescent probe for detecting glyphosate, wherein the fluorescent probe is 2-(2-hydroxyphenyl)benzoxazole-Cu 2+ Fluorescent probe (abbreviated as HBO-Cu 2+ ), the specific structure is as follows: .

[0006] The present invention provides a 2-(2-hydroxyphenyl)benzoxazole-Cu for detecting glyphosate 2+ The preparation method of the complex fluorescent probe and the synthetic route are as follows: .

[0007] The present invention provides a 2-(2-hydroxyphenyl)benzoxazole-Cu for detecting glyphosate 2+ The preparation method of the complex fluorescent probe specifically comprises the following steps: 2-(2-hydroxyphenyl)benzoxazole was added to ethanol and stirred until completely dissolved, and copper salt was added and stirred at room temperature to obtain 2-(2-hydroxyphenyl)benzoxazole-Cu2+ After the reaction, the precipitate was washed with anhydrous ethanol and water and vacuum dried to obtain a brown powdery solid, which is 2-(2-hydroxyphenyl)benzoxazole-Cu 2+ Complex fluorescent probe (HBO-Cu 2+ ).

[0008] Preferably, the copper salt is any one of copper sulfate, copper chloride and copper nitrate.

[0009] Preferably, the molar ratio of the 2-(2-hydroxyphenyl)benzoxazole to the copper salt is 2:(1-2).

[0010] Preferably, the 2-(2-hydroxyphenyl)benzoxazole-Cu 2+ The molar volume ratio to ethanol is 1.8mmol / 29mL~3mmol / 29mL.

[0011] Preferably, the stirring reaction time at room temperature is 1-2 hours.

[0012] Preferably, the vacuum drying temperature is 20-30° C. and the time is 12-24 hours.

[0013] The present invention also provides a HBO-Cu 2+ The test paper made of complex fluorescent probe can be used to identify and detect glyphosate with naked eyes under ultraviolet light. 2+ The complex is prepared by naturally drying in ethanol solution.

[0014] Compared with the prior art, the outstanding advantages of the fluorescent probe provided by the present invention include: (1) The fluorescent probe of the present invention is prepared by a one-step method, which is simple, efficient and low-cost, and can be applied to large-scale industrial production.

[0015] (2) HBO-Cu prepared by the present invention 2+ The complex fluorescent probe has good selectivity for glyphosate, is not interfered by other pesticides, and has a fast response speed. 2+ The detection limit of the complex fluorescent probe for glyphosate is 8.8×10 -7 mol / L (i.e. 0.149 mg / L), which can meet the detection requirements of my country's drinking water hygiene standards (GB5749-2022), has high sensitivity, and can realize trace detection of glyphosate.

[0016] (3) HBO-Cu prepared by the present invention 2+The complex fluorescent probe can be made into test strips, which can visually identify and detect glyphosate under ultraviolet light. It is highly practical and has good application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 It is a fluorescent probe HBO-Cu 2+ Fourier transform infrared spectrum.

[0018] Figure 2 It's HBO and Cu 2+ Complexation ratio determination diagram.

[0019] Figure 3 It is HBO-Cu 2+ The relationship between the fluorescence intensity of the complex probe and the concentration of glyphosate.

[0020] Figure 4 It is HBO-Cu 2+ Linear relationship between the fluorescence intensity of the complex fluorescent probe at 440 nm and the glyphosate concentration.

[0021] Figure 5 It is HBO-Cu 2+ Experimental diagram of the selectivity of the complex fluorescent probe for glyphosate detection.

[0022] Figure 6 It is HBO-Cu 2+ Response time diagram of the complex fluorescent probe to glyphosate.

[0023] Figure 7 Is the use of HBO-Cu 2+ The fluorescence of the test strip made of the complex fluorescent probe changes with the glyphosate concentration under ultraviolet light irradiation.

[0024] Figure 8 Is the use of HBO-Cu 2+ Grayscale graph showing the fluorescence of a test strip made of a complex fluorescent probe changing with glyphosate concentration under ultraviolet light. DETAILED DESCRIPTION

[0025] In order to further understand the present invention, the specific implementation of the present invention is further described below in conjunction with the accompanying drawings and examples, but the implementation and protection of the present invention are not limited thereto. It should be noted that if there are any processes that are not particularly described in detail below, they can be implemented or understood by those skilled in the art with reference to the prior art. If the manufacturer of the reagents or instruments used is not indicated, they are deemed to be conventional products that can be purchased commercially.

[0026] Example 1 Dissolve 63.37 mg (10 mmol / L) of 2-(2-hydroxyphenyl)benzoxazole in a mixed solution of 29 mL of ethanol and 1 mL of water, add 36 mg (7.5 mmol / L) of copper sulfate, and stir at room temperature for 2 h to obtain HBO-Cu 2+ After the reaction, the ethanol and water were removed by rotary evaporation, and the precipitate was washed with anhydrous ethanol and water several times, and vacuum dried at 30 ° C for 12 hours to obtain HBO-Cu 2+ The complex fluorescent probe was 72.6 mg (yield 83.16%).

[0027] Figure 1 For HBO and HBO-Cu 2+ The infrared spectrum data of the complex is shown in Figure 2. It was characterized by infrared spectroscopy at 580 cm -1 The characteristic peak of Cu-O exists at 1633 cm-1, indicating the formation of metal complex. -1 、1587cm -1 、1544cm -1 and 742cm -1 Due to the increased conjugation effect caused by the formation of the complex, the characteristic peaks of the benzene ring were red-shifted to 1610 cm -1 、1560cm -1 、1540cm -1 and 730cm -1 The characteristic peak of C=N bond in HBO is due to Cu 2+ Coordination with N causes its -1 Redshift to 1473 cm -1 HBO and Cu 2+ The complexation ratio curve ( Figure 2 ) shows that HBO and Cu 2+ The complexation ratio is 2:1. Through the analysis of the above infrared spectrum and complexation ratio, it can be determined that the synthesized product is the target fluorescent probe Example 2 50.7 mg (8 mmol / L) of 2-(2-hydroxyphenyl)benzoxazole was dissolved in a mixed solution of 29 mL of ethanol and 1 mL of water, and 19.2 mg (4 mmol / L) of copper sulfate was added. The mixture was stirred at room temperature for 1.5 h to obtain HBO-Cu 2+ After the reaction, the ethanol and water were removed by rotary evaporation, and the precipitate was washed with anhydrous ethanol and water several times, and vacuum dried at 25 ° C for 18 hours to obtain HBO-Cu 2+ The complex fluorescent probe was 60.25 mg (yield 86.27%).

[0028] The characterization results of the fluorescent probe obtained in this example are the same as those in Example 1.

[0029] Example 3 38.02 mg (6 mmol / L) of 2-(2-hydroxyphenyl)benzoxazole was dissolved in a mixed solution of 29 mL of ethanol and 1 mL of water, and 28.8 mg (6 mmol / L) of copper sulfate was added. The mixture was stirred at room temperature for 1 h to obtain HBO-Cu 2+ After the reaction, the ethanol and water were removed by rotary evaporation, and the precipitate was washed with anhydrous ethanol and water several times, and vacuum dried at 20 ° C for 24 h to obtain HBO-Cu 2+ Complex fluorescent probe 42.79 mg (yield 81.69%).

[0030] The characterization results of the fluorescent probe obtained in this example are the same as those in Example 1.

[0031] Example 4 Effect of glyphosate concentration on HBO-Cu 2+ Influence of the fluorescence emission spectrum of the complex probe: This example investigates the effect of glyphosate concentration on HBO-Cu 2+ The influence of the complex probe fluorescence emission spectrum. Take the HBO-Cu prepared in Example 1 2+ The complex fluorescent probe is dissolved in a mixed solution of ethanol and water (V(EtOH) / V(H2O)=1:2) and prepared into a test stock solution with a probe concentration of 1mmo1 / L. Each time, 60μL of the probe stock solution is taken into a centrifuge tube, diluted with ethanol and water, and then different volumes of glyphosate are added to make the concentrations of glyphosate 0, 1.0, 2.0, 3.0, 4.0, 5.0..., 10.0, 12.0, 16.0, 18.0, 20.0μmo1 / L, respectively. The volume ratio of the mixed solvent is fixed at V(EtOH) / V(H2O)=1:2, and the volume of the test solution is ensured to be 3mL. Under the action of 320nm excitation light, the fluorescence emission spectrum of the system is measured. The results are as follows Figure 3 As shown in Figure 2, as the concentration of glyphosate increases, the fluorescence intensity of the probe gradually increases. A linear fit is performed with the glyphosate concentration as the horizontal axis and the fluorescence intensity at 440 nm as the vertical axis. The results are shown in Figure 2. Figure 4 As shown in Figure 2, within the glyphosate concentration range of 0 to 10 μmo1 / L, the fluorescence intensity has a good linear relationship with the glyphosate concentration change. The standard curve equation obtained by fitting is: Y = 263 + 21.99X (X is the glyphosate concentration, Y is the fluorescence emission peak intensity value), and the linear correlation coefficient R 2 =0.99. According to the calculation formula of detection limit: detection limit = 3σ / k, HBO-Cu 2+ The detection limit of the complex fluorescent probe for glyphosate is 8.8×10-7 mo1 / L (i.e. 0.149 mg / L), which can meet the testing requirements of my country's drinking water hygiene standards (GB5749-2022). 2+ The complex fluorescent probe has high sensitivity in detecting glyphosate and can achieve quantitative detection of glyphosate.

[0032] Example 5 HBO-Cu 2+ Selective testing of complex probes for glyphosate: This example uses the HBO-Cu prepared in Example 1 2+ The HBO-Cu complex fluorescent probe prepared in Example 1 was used to determine the selectivity of the complex fluorescent probe to glyphosate. 2+ The complex fluorescent probe was dissolved in a 1:2 ethanol:water mixture to prepare a test solution with a probe concentration of 1 mmol / L. 60 μL of the probe solution was placed in a centrifuge tube and diluted with ethanol and water. Then, 60 μL of 1 mmol / L glyphosate, oxyfluorfen, mesotrione, thiophanate-methyl, trichlorfon, nicosulfuron, and imidacloprid were added in equal amounts. The volume ratio of the mixed solvent was fixed at V(EtOH) / V(H2O) = 1:2, while ensuring that the test solution volume was 3 mL. Fluorescence emission spectra were measured under 320 nm excitation light.

[0033] Example 6 HBO-Cu 2+ Time response test of complex probe to glyphosate: This example uses the HBO-Cu prepared in Example 1 2+ The time response of the complex fluorescent probe to glyphosate was measured. 2+ The complex fluorescent probe is dissolved in a mixed solution of ethanol and water (V(EtOH) / V(H2O)=1:2) to prepare a test solution with a probe concentration of 1mmo1 / L. Each time, 60μL of the probe solution is taken into a centrifuge tube, diluted with ethanol and water, and then 60μL of 1mmo1 / L glyphosate is added. The volume ratio of the mixed solvent is fixed at V(EtOH) / V(H2O)=1:2, and the volume of the test solution is ensured to be 3mL. The change of fluorescence intensity at 440nm with time is measured, and the excitation wavelength is 320nm. Figure 6 As shown in the figure, as the reaction time with glyphosate increases, the fluorescence intensity gradually increases and reaches a plateau at 80s. 2+ The complex fluorescent probe reacts quickly to glyphosate and can achieve rapid detection of glyphosate within 2 minutes.

[0034] Example 7 HBO-Cu 2+Detection of glyphosate in actual water samples using complex probe: Two water samples, laboratory tap water and lake water, were selected and pre-treated: the water samples were filtered twice using a filter membrane with a pore size of 0.22 μm, and a glyphosate solution with a concentration of 1 mmol / L was prepared using the filtered water samples. 2+ The complex fluorescent probe was dissolved in a mixture of ethanol and water (V(EtOH) / V(H2O) = 1:2) to prepare a test solution with a probe concentration of 1 mmol / L. 60 μL of the probe solution was then diluted with ethanol and water, and then added to the glyphosate solution to achieve glyphosate concentrations of 3.0 μmol / L, 6.0 μmol / L, and 9.0 μmol / L, respectively. The volume ratio of the mixed solvent was fixed at V(EtOH) / V(H2O) = 1:2, and the test solution volume was maintained at 3 mL. Under 320 nm excitation light, the peak fluorescence emission intensity of the fluorescent probe at 440 nm was measured and substituted into the following standard curve equation: Y = 263 + 21.99X (X is the glyphosate concentration, Y is the peak fluorescence emission intensity) to calculate the concentration of the glyphosate solution. The test results are shown in Table 1.

[0035] Table 1 HBO-Cu 2+ Detection of glyphosate in real water samples using a complex fluorescent probe

[0036] The data in Table 1 show that the recovery of glyphosate in actual water samples is 97.67%~101.56%, and the relative standard deviation is 1.01%~2.01%. These results show that HBO-Cu 2+ The complex fluorescent probe has high accuracy and practical performance in detecting glyphosate in actual water samples. Example 8 HBO-Cu 2+ Complex probe for detection of glyphosate in soil: The soil on campus was pretreated by weighing 1 g of soil and placing it in a centrifuge tube. 30 mL of water was added and ultrasonicated for 20 minutes. The soil was filtered with filter paper and then filtered twice with a 0.22 μm pore size membrane. The filtered water was used to prepare a 1 mmol / L glyphosate solution. 2+The complex fluorescent probe was dissolved in a mixture of ethanol and water (V(EtOH) / V(H2O) = 1:2) to prepare a test solution with a probe concentration of 1 mmol / L. 60 μL of the probe solution was then diluted with ethanol and water, and then added to the glyphosate solution, achieving glyphosate concentrations of 3.0 μmol / L, 6.0 μmol / L, and 9.0 μmol / L, respectively. The ratio of the mixed solvent was fixed at V(EtOH) / V(H2O) = 1:2, and the test solution volume was maintained at 3 mL. Under 320 nm excitation light, the peak fluorescence emission intensity of the fluorescent probe at 440 nm was measured and substituted into the following calibration curve equation: Y = 263 + 21.99X (X is the glyphosate concentration, Y is the peak fluorescence emission intensity) to calculate the concentration of the glyphosate solution. The test results are shown in Table 2.

[0037] Table 2 HBO-Cu 2+ Detection of glyphosate in soil samples using a complex fluorescent probe

[0038] The data in Table 2 show that the recovery rate of glyphosate in soil samples is 102.22%~105%, and the relative standard deviation is 0.4%~1.59%. These results show that HBO-Cu 2+ Complex fluorescent probe for detecting glyphosate in soil has high accuracy and practical performance Example 9 HBO-Cu 2+ Detection of glyphosate using a test strip made of a complex fluorescent probe The blank test strip was immersed in the HBO-Cu prepared in Example 1. 2+ The complex was incubated in a mixed solution of ethanol and water (V(EtOH) / V(H2O)=1:2) (at a concentration of 1 mmol / L) for 12 hours and then dried naturally to prepare glyphosate test strips. Glyphosate solutions at concentrations of 2.0 mmol / L, 4.0 mmol / L, 6.0 mmol / L, and 10.0 mmol / L were added to the cellulose test paper, respectively. After the test paper dried, it was illuminated with a 254 nm UV lamp and the color and brightness of the test paper were observed. Figure 7 and Figure 8 As shown, the color of the test paper without glyphosate (0mmol / L) is blue-purple. As the concentration of glyphosate increases, the color gradually changes to blue-green and the fluorescence brightness increases. This shows that the HBO-Cu 2+ Test strips made of complex fluorescent probes can achieve convenient and visual detection of glyphosate.

[0039] The above examples are preferred embodiments of the present invention, but the embodiments of the invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be considered as equivalent replacement methods and are included in the scope of protection of the present invention.

Claims

1. A 2-(2-hydroxyphenyl)benzoxazole-Cu-2-phosphate for the detection of glyphosate 2+ The complex fluorescent probe is characterized by: It has the following structural formula: 。 2. A 2-(2-hydroxyphenyl)benzoxazole-Cu-2-nitropropene for detecting glyphosate according to claim 1 2+ The method for preparing a complex fluorescent probe is characterized in that: The synthetic route is as follows: 。 3. The preparation method according to claim 2, characterized in that The following steps are involved: 2-(2-hydroxyphenyl)benzoxazole was added to ethanol and stirred until completely dissolved, and copper salt was added and stirred at room temperature to obtain 2-(2-hydroxyphenyl)benzoxazole-Cu 2+ After the reaction, the precipitate was washed with anhydrous ethanol and water and vacuum dried to obtain a brown powdery solid, which is 2-(2-hydroxyphenyl)benzoxazole-Cu 2+ Complex fluorescent probe.

4. The preparation method according to claim 3, characterized in that The copper salt is any one of copper sulfate, copper chloride and copper nitrate.

5. The preparation method according to claim 3, characterized in that The molar ratio of the 2-(2-hydroxyphenyl)benzoxazole to the copper salt is 2:(1-2).

6. The preparation method according to claim 3, characterized in that The molar volume ratio of the 2-(2-hydroxyphenyl)benzoxazole to ethanol is 1.8 mmol / 29 mL to 3 mmol / 29 mL.

7. The preparation method according to claim 3, characterized in that The time of the reaction under stirring at room temperature is 1-2 hours.

8. The preparation method according to claim 3, characterized in that The vacuum drying temperature is 20-30° C. and the time is 12-24 hours.

9. The 2-(2-hydroxyphenyl)benzoxazole-Cu according to claim 1 2+ Application of fluorescent probes in the detection of glyphosate.

10. A fluorescent probe test paper, characterized in that A blank filter paper strip is impregnated with 2-(2-hydroxyphenyl)benzoxazole and Cu prepared by the method of claim 3. 2+ The complex is removed from the ethanol solution and naturally dried, and glyphosate is visually identified and detected under ultraviolet light.

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