Method for detecting glyphosate based on Fe < 2 + >-mediated fluorescence sensor
The fluorescent sensor combined with the fluorescent probe PEI-PDA and Fe2+ solves the complexity and cost of glyphosate detection, and realizes the rapid, simple and sensitive detection of glyphosate, with good selectivity and anti-interference ability.
Patent Information
- Application Number
- CN202510721417.5
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-05-30
- Publication Date
- 2025-09-02
AI Technical Summary
In the prior art, glyphosate detection methods are complex, time-consuming and expensive, and there is a lack of a fluorescence sensor detection method based on Fe2+ mediated.
The fluorescent probe PEI-PDA is used to combine with Fe2+, and glyphosate is detected through the principle of fluorescence quenching, simplifying the operation process, and judging the concentration of glyphosate by using the fluorescence intensity changes.
It realizes fast, simple and sensitive detection of glyphosate, has good selectivity and anti-interference ability, and is suitable for accurate detection of actual samples.
Smart Images
Figure CN120577273A_ABST
Abstract
Description
Technical Field
[0001] The present invention belongs to the field of pesticide detection technology, and more specifically, relates to a method for detecting pesticides based on Fe 2+ A method for detecting glyphosate using a mediated fluorescence sensor. Background Art
[0002] The chemical name of glyphosate (GLY) is N-(phosphomethyl)glycine. It is a systemic organophosphorus herbicide. Due to its advantages of high efficiency, broad spectrum, non-selectivity and low cost, it is widely used in agriculture, forestry and other fields. It has now become the most used organophosphorus pesticide in the world. However, the high water solubility of GLY makes it easy for it to remain in soil, water or crops during long-term use. It will not only pollute the natural environment, but also accumulate in the human body through the food chain, causing adverse effects on the human endocrine system, reproductive system, nervous system, etc., causing liver damage, DNA damage, etc. The "Standard for Drinking Water Quality" sets the limit value of GLY in drinking water at 0.7μg / mL, and the National Food Safety Standard (GB2763-2021) stipulates that the maximum residue limit of GLY in most crops and tea is 0.1 and 1mg / kg. Therefore, the development of a rapid, simple and accurate GLY detection method is of great significance to food and environmental safety.
[0003] Currently, the main methods for detecting GLY include high-performance liquid chromatography (HPLC), gas chromatography (GC), colorimetry, electrochemical analysis, and capillary electrophoresis (CE). Although these methods have advantages in terms of accuracy and stability, disadvantages such as time-consuming and complex sample pretreatment, expensive equipment, and the requirement for skilled personnel have limited their widespread application in practical sample detection.
[0004] Compared with other detection methods, fluorescence analysis is widely used in pesticide trace detection due to its simple operation, fast response and high sensitivity. 2+ The method for detecting glyphosate based on the fluorescence “turn-on” principle is mediated by Fe 2+ A method for detecting glyphosate using the fluorescence "turn-off" principle has not yet been reported. Summary of the Invention
[0005] In view of the above problems existing in the prior art, the technical problem to be solved by the present invention is to provide a simple, rapid, highly sensitive and specific Fe-based 2+ A mediated fluorescence sensor was used to detect glyphosate.
[0006] In order to solve the above technical problems, the technical solutions adopted by the present invention are as follows:
[0007] A Fe-based 2+ The method of detecting glyphosate by using a mediated fluorescence sensor is as follows: the fluorescent probe PEI-PDA is dissolved in a buffer solution to form an initial solution. Under an excitation wavelength of 365 nm, the initial solution has a characteristic fluorescence intensity at 530 nm. Fe 2+ After the solution is added, the green fluorescence intensity emitted at 530 nm decreases slightly, forming a detection system. The test solution is added to the detection system. If the green fluorescence intensity emitted at 530 nm further decreases, the test solution contains glyphosate.
[0008] Preferably, the linear concentration range of glyphosate is 0-500 μmol / L, and the detection limit is 0.644 μmol / L.
[0009] Preferably, the preparation process of the fluorescent probe PEI-PDA is as follows: DA·HCl solution, PEI solution and Tris-HCl buffer solution are mixed, stirred in the dark at room temperature for 12 hours and then filtered to obtain a PEI-PDA solution.
[0010] Preferably, the buffer solution is a Tris-HCl buffer solution with a pH of 7.
[0011] Preferably, the volume concentration of the fluorescent probe PEI-PDA in the detection system is 3-8%, and more preferably, the volume concentration of the fluorescent probe PEI-PDA in the detection system is 7%.
[0012] Preferably, the Fe 2+ The concentration is 0.1 to 0.8 mmol / L. More preferably, the Fe 2+ The concentration is 0.4mmol / L.
[0013] A Fe-based 2+ The method for detecting glyphosate using a mediated fluorescence sensor comprises the following steps:
[0014] 1) Drawing of glyphosate standard concentration gradient curve;
[0015] PEI-PDA, Fe 2+ Then, glyphosate solution of different concentrations was added, and after the reaction in the dark, the fluorescence intensity of the system was measured at 530 nm using a fluorescence spectrometer; the fluorescence intensity quenching difference ΔF 530 (F0-F) draw a linear relationship curve;
[0016] 2) Detection of the system to be tested;
[0017] The same detection method as step 1) is used to determine the ΔF of the test system. 530The concentration of glyphosate in the test system is calculated using the linear relationship curve in step 1).
[0018] Preferably, in step 2), the linear regression equation of glyphosate is y=0.2108x+1.8352, and the detection limit is 0.644 μmol / L.
[0019] Preferably, in step 2), the volume concentration of the fluorescent probe PEI-PDA in the detection system is 7%, Fe 2+ The concentration in the detection system is 0.4 mmol / L, the concentration of the Tris-HCl buffer solution is 20 mmol / L, and the pH is 7.
[0020] Preferably, the reaction temperature is room temperature and the reaction time is 5 minutes.
[0021] Compared with the prior art, the present invention has the following beneficial effects:
[0022] 1) The present invention is the first to use Fe 2+ The mediated fluorescence sensor is used for GLY detection, which does not require complicated pre-treatment processes and achieves rapid, simple, and sensitive detection of GLY by simplifying the operation;
[0023] 2) The PEI-PDA used in the present invention has the advantages of simple preparation, low toxicity, low cost and good biocompatibility;
[0024] 3) The fluorescent sensor constructed in the present invention has good selectivity for GLY and is not affected by other organophosphorus pesticides and other interfering substances such as common metal ions, anions, and amino acids in water;
[0025] 4) The present invention has good spiked recovery rate in the detection of actual water samples, soil and tea, high accuracy and reliable detection results. BRIEF DESCRIPTION OF THE DRAWINGS
[0026] Figure 1 Characterization images of PEI-PDA prepared in Example 1; (A) is a fluorescence excitation (Ex.) and emission (Em.) spectrum and a UV-Vis absorption (Abs.) spectrum; the inset is a fluorescence image of PEI-PDA under fluorescent light and 365nm UV light; (B) is a TEM image;
[0027] Figure 2 The feasibility analysis diagram is shown in FIG. 1 , wherein the buffer solution is 20 mmol / L Tris-HCl buffer solution with a pH of 7, PEI-PDA accounts for 7% (v / v) of the detection system, and Fe 2+ The concentration of Fe was 0.2mmol / L, and the concentration of GLY was 0 and 500μmol / L; (A) is PEI-PDA and its Fe2+ / Fe 2+ -GLY fluorescence spectrum, (B) is the fluorescence intensity analysis diagram at 530 nm corresponding to each system in A;
[0028] Figure 3 For analysis, (A) is the fluorescence intensity difference ΔF of different buffer solutions for detecting GLY 530 The analysis diagram of each buffer solution is 20mmol / L; (B) is the fluorescence intensity difference ΔF of GLY detected by the system at different pH values 530 Analysis chart of
[0029] Figure 4 This is an analysis diagram of the effect of different PEI-PDA concentrations on the fluorescence intensity of GLY detection in this system;
[0030] Figure 5 For different Fe 2+ Analysis of the effect of concentration on the fluorescence intensity of GLY detected by this system;
[0031] Figure 6 This is an analysis diagram of the effect of different temperatures on the fluorescence intensity of GLY detected by this system;
[0032] Figure 7 This is an analysis diagram of the effect of different time on the fluorescence intensity of GLY detected by this system;
[0033] Figure 8 The fluorescence spectra of the system affected by different concentrations of GLY and the difference ΔF between GLY concentration and system fluorescence intensity 530 Linear relationship diagram; (A) is the fluorescence spectrum of the effect of different concentrations of GLY on this system; (B) is the difference ΔF between the GLY concentration at 530nm and the fluorescence intensity of the system 530 Linear relationship diagram;
[0034] Figure 9 This is the specificity analysis diagram of this detection method. DETAILED DESCRIPTION
[0035] In order to make the purpose, technical solutions and advantages of the present invention clearer, the present invention is further described below in conjunction with specific embodiments. Unless otherwise specified in the following embodiments, the technical means used are conventional means well known to those skilled in the art.
[0036] Example 1
[0037] 1. Preparation of PEI-PDA
[0038] 1) Preparation of PEI-PDA using a one-pot method
[0039] Mix 1 mL of 1% PEI (Mw = 600) solution, 1 mL of 1% DA·HCl solution, and 6 mL of 50 mmol / L pH 8.5 Tris-HCl buffer solution, then add ultrapure water to a total volume of 10 mL. Stir in the dark at room temperature for 12 h, then filter with a 0.22 μm microporous filter membrane to obtain a brown PEI-PDA solution, which was sealed and stored in the dark at 4°C.
[0040] 2) Characterization of prepared PEI-PDA
[0041] Depend on Figure 1 Figure A shows the fluorescence excitation (Ex.) and emission (Em.) spectra and UV-Vis absorption (Abs.) spectrum of the prepared PEI-PDA. The prepared PEI-PDA exhibits maximum fluorescence excitation and emission peaks at 365 nm and 530 nm, respectively. The UV-Vis absorption spectrum shows an absorption shoulder peak at 380 nm. Figure B shows the color of the prepared PEI-PDA solution. Under fluorescent light and 365 nm UV light, PEI-PDA appears light brown and green, respectively. Figure C shows a TEM image of the prepared PEI-PDA, showing that the prepared PEI-PDA is approximately spherical and uniform in size. These results demonstrate the successful preparation of PEI-PDA.
[0042] 2. PEI-PDA for the detection of glyphosate (GLY)
[0043] Combine PEI-PDA, Fe 2+ , the test system containing different concentrations of GLY was added into Tris-HCl buffer solution (20mmol / L, pH=7) in turn, and PEI-PDA and Fe 2+ The concentrations in the detection system were 7% (v / v) and 0.4 mmol / L, respectively. After reacting in the dark at room temperature for 5 minutes, the fluorescence intensity of the system was measured using a fluorescence spectrometer.
[0044] Figure 2 This is the feasibility analysis diagram of this application, Figure 2 It can be seen that PEI-PDA, Fe 2+ and GLY solution to make PEI-PDA and Fe 2+ The concentrations of the test system were 7% (v / v) and 0.2 mmol / L, and the concentrations of GLY were 0 and 500 μmol / L. After thorough mixing, the reaction was carried out in the dark at room temperature for 5 minutes, and the fluorescence intensity of the system was measured using a fluorescence spectrometer. Under 365 nm excitation, PEI-PDA had a characteristic fluorescence peak at 530 nm. When Fe 2+The fluorescence of PEI-PDA was slightly reduced after adding GLY, and the fluorescence was further quenched. 2+ It is feasible to use PEI-PDA as a fluorescent sensor to detect GLY.
[0045] 3. Determination of the buffer system and pH of the detection system
[0046] Combine PEI-PDA, Fe 2+ They were added to different buffer solutions (BR, MOPS, Tris-HCl, PB, PBS) at pH = 8, 20 mmol / L, to make PEI-PDA and Fe 2+ The concentrations in the detection system were 5% (v / v) and 0.2mmol / L, respectively. A control group and an experimental group (0 and 500mmol / L GLY) were set up. The fluorescence intensity of the detection system was as follows. Figure 3 shown.
[0047] Depend on Figure 3 It can be seen that when Tris-HCl buffer solution is used, ΔF 530 The value is the largest, so Tris-HCl buffer solution is selected for pH optimization ( Figure 3 (A)). When pH = 7, ΔF 530 The value is the largest, so the Tris-HCl buffer solution with pH = 7 is selected ( Figure 3 (as shown in (B)).
[0048] 4. Determination of PEI-PDA concentration in the detection system
[0049] Different concentrations of PEI-PDA, Fe 2+ were added to Tris-HCl buffer solution (pH=7, 20mmol / L) to make the concentration of PEI-PDA in the detection system 3%, 4%, 5%, 6%, 7%, 8% (v / v), respectively. 2+ The concentration was 0.2mmol / L, and a control group and an experimental group (0 and 500mmol / L GLY) were set up. The fluorescence intensity difference (ΔF 530 ), the result is as follows Figure 4 shown.
[0050] Depend on Figure 4 It can be seen that when the concentration of PEI-PDA in the detection system is 7%, ΔF 530 The value is the largest, so 7% PEI-PDA is selected to construct the detection system.
[0051] 5. Fe in the detection system 2+ Determination of concentration
[0052] PEI-PDA, different concentrations of Fe 2+ Add to Tris-HCl buffer solution (pH=7, 20mmol / L) to make the concentration of PEI-PDA in the detection system 7% (v / v), Fe 2+ The concentrations were 0.1, 0.2, 0.4, 0.6, and 0.8 mmol / L, and the control group and experimental group (0 and 50 mmol / L GLY) were set up. The fluorescence intensity difference (ΔF 530 ), the result is as follows Figure 5 shown.
[0053] Depend on Figure 5 It can be seen that when Fe 2+ When the concentration is 0.4mmol / L, ΔF 530 The maximum value, Fe 2+ When the concentration is too high, it is not conducive to the detection of GLY, so 0.4mmol / L Fe 2+ Build a detection system.
[0054] 6. Temperature and time optimization of the detection system
[0055] PEI-PDA, Fe 2+ , making PEI-PDA and Fe 2+ The concentrations in the test system were 7% (v / v) and 0.4 mmol / L, respectively. A control group and an experimental group (0 and 500 mmol / L GLY) were set up. The reaction temperatures were set at 15°C, 20°C, 25°C, 30°C, and 35°C, respectively. After the reaction time was the same, the fluorescence intensity difference (ΔF 530 ); and set the reaction time to 5min, 10min, 15min, 20min, 30min, 45min, and 60min, respectively. After the reaction at room temperature and in the dark, the fluorescence intensity difference (ΔF 530 ).like Figure 6 and Figure 7 As shown in the figure, temperature and time have little effect on the detection system, so the optimal reaction temperature is room temperature (25°C) and the reaction time is 5 minutes.
[0056] Example 2
[0057] 1. Drawing of GLY standard concentration gradient curve
[0058] PEI-PDA, Fe 2+ , making PEI-PDA and Fe 2+The concentrations in the test system were 7% (v / v) and 0.4 mmol / L, respectively. GLY solutions of different concentrations were then added to make the GLY concentrations in the test system 0.5, 1, 5, 10, 20, 30, 50, 60, 80, 100, 200, 500, 1000, and 2000 μmol / L, respectively. After reacting in the dark at room temperature for 5 minutes, the fluorescence intensity of the test system was measured using a fluorescence spectrometer. The results are as follows: Figure 8 shown.
[0059] Depend on Figure 8 It can be seen that with the increase of GLY concentration, the fluorescence intensity of the system at 530nm gradually decreased. 530 ) draws a linear relationship curve, which shows that GLY is in the range of 0~500μmol / L, ΔF 530 It showed a good linear relationship with the concentration of GLY, and the linear equation was y=0.2108x+1.8352, R 2 =0.970 (S / N=3), and the minimum detection limit LOD (S / N=3) was 0.644 μmol / L.
[0060] Example 3
[0061] 1. Specificity test of common organophosphorus pesticides and other interfering substances
[0062] PEI-PDA, Fe 2+ , making PEI-PDA and Fe 2+ The concentrations in the test system were 7% (v / v) and 0.4 mmol / L, and five organophosphorus pesticides (glyphosate GLY, glufosinate GLF, dimethoate DIM, acephate ACE, trichlorfon TRI), eight common ions in water (Cl - 、 K + 、Na + , Ca 2+ Mg 2+ ), amino acids (glycine Gly, glutamic acid Glu, histidine His, lysine Lys) and humic acid (fulvic acid FA, humic acid HA), wherein the concentration of organophosphorus pesticides in the detection system is 20mmol / L, and the concentration of other interfering substances in the detection system is 100mmol / L. After reacting in the dark for 5 minutes at room temperature, the fluorescence intensity difference (ΔF 530 ), the result is as follows Figure 9 shown.
[0063] Depend on Figure 9It can be seen that the interference has almost no effect on the detection system, indicating that the constructed fluorescence system has good selectivity for GLY and strong anti-interference ability.
[0064] 2. Spiking experiment of actual sample GLY
[0065] In order to verify the practical application capability of the sensor in this application, spike recovery experiments were conducted on pre-treated laboratory tap water, Nongfu Spring mineral water, lake water (Xuanwu Lake, Hongze Lake), school soil, and tea from the school supermarket. The GLY concentrations measured after the final spike of each sample were set at 5 μmol / L and 10 μmol / L, and the fluorescence intensity of each spiked sample was measured using a fluorescence spectrometer. Three sets of experiments were repeated to calculate ΔF. 530 The recovery rate was analyzed with ultrapure water as the control group. The results are shown in Table 1.
[0066] Table 1 Recovery of GLY in actual samples
[0067]
[0068]
[0069] As shown in Table 1, the spiked recoveries of GLY in two actual water samples, mineral water and lake water, as well as in soil and tea, ranged from 88.25% to 105.44%, and the relative standard deviations (RSDs) ranged from 1.84% to 8.82%. This indicates that the system is accurate and reliable for the detection of GLY in actual samples and is suitable for the qualitative and quantitative detection of GLY in various environments.
[0070] The descriptions and practices disclosed in this invention are easy to understand and comprehend for those skilled in the art, and modifications and refinements may be made without departing from the principles of the invention. Therefore, modifications and improvements made without departing from the spirit of the invention should also be considered within the scope of protection of this invention.
Claims
1. A Fe-based 2+ A method for detecting glyphosate using a fluorescence sensor, characterized in that: The fluorescent probe PEI-PDA was dissolved in a buffer solution to form an initial solution. Under an excitation wavelength of 365 nm, the initial solution had a characteristic fluorescence intensity at 530 nm. Fe 2+ After the solution is added, the green fluorescence intensity emitted at 530 nm decreases slightly, forming a detection system. The test solution is added to the detection system. If the green fluorescence intensity emitted at 530 nm further decreases, the test solution contains glyphosate.
2. Fe-based according to claim 1 2+ A method for detecting glyphosate using a fluorescence sensor, characterized in that: The linear concentration range of glyphosate is 0-500 μmol / L, and the detection limit is 0.644 μmol / L.
3. Fe-based according to claim 1 2+ A method for detecting glyphosate using a fluorescence sensor, characterized in that: The preparation process of the fluorescent probe PEI-PDA is as follows: DA·HCl solution, PEI solution and Tris-HCl buffer solution are mixed, stirred in the dark at room temperature for 12 hours and then filtered to obtain a PEI-PDA solution.
4. Fe-based according to claim 1 2+ A method for detecting glyphosate using a fluorescence sensor, characterized in that: The buffer solution is a Tris-HCl buffer solution with a pH of 7.
5. Fe-based according to claim 1 2+ A method for detecting glyphosate using a fluorescence sensor, characterized in that: The volume concentration of the fluorescent probe PEI-PDA in the detection system is 3-8%.
6. Fe-based according to claim 1 2+ A method for detecting glyphosate using a fluorescence sensor, characterized in that: The Fe 2+ The concentration is 0.1~0.8mmol / L.
7. A Fe-based 2+ A method for detecting glyphosate using a fluorescence sensor, characterized in that: The following steps are involved: 1) Drawing of glyphosate standard concentration gradient curve; PEI-PDA, Fe 2+ Then, glyphosate solutions of different concentrations were added, and after the reaction was protected from light, the fluorescence intensity of the system at 530 nm was measured using a fluorescence spectrometer; Using the fluorescence intensity quenching difference ΔF 530 (F0-F) draw a linear relationship curve; 2) Detection of the system to be tested; The same detection method as step 1) is used to determine the ΔF of the test system. 530 The concentration of glyphosate in the test system is calculated using the linear relationship curve in step 1).
8. Fe-based according to claim 7 2+ A method for detecting glyphosate using a fluorescence sensor, characterized in that: In step 2), the linear regression equation of glyphosate is y=0.2108x+1.8352, and the detection limit is 0.644 μmol / L.
9. Fe-based according to claim 7 2+ A method for detecting glyphosate using a fluorescence sensor, characterized in that: In the step 2), the volume concentration of the fluorescent probe PEI-PDA in the detection system is 7%, Fe 2+ The concentration in the detection system is 0.4 mmol / L, the concentration of the Tris-HCl buffer solution is 20 mmol / L, and the pH is 7.
10. Fe-based according to claim 9 2+ A method for detecting glyphosate using a fluorescence sensor, characterized in that: The reaction temperature is room temperature and the reaction time is 5 minutes.