A method for detecting glyphosate by in situ fluorescence reaction triggered by biological enzymes

Abstract

The present invention relates to the technical field of food safety detection, and in particular to a method for detecting glyphosate by an in-situ fluorescence reaction triggered by a biological enzyme. The present invention utilizes alkaline phosphatase (ALP) to catalyze the substrate L-ascorbic acid-2-phosphate trisodium salt to form L-ascorbic acid, which reacts with 3-aminopropyltrimethoxysilane at room temperature to generate fluorescent product silicon nanoparticles (SiNPs). When glyphosate is present, glyphosate inhibits ALP activity, weakens its catalytic ability, and then causes L-ascorbic acid and SiNPs to produce less, and the fluorescence intensity weakens. As the concentration of glyphosate increases, the fluorescence intensity of the system continuously weakens, thereby achieving quantitative detection of glyphosate. The present invention does not need to prepare nanomaterials, simplifies the detection method, reduces detection costs, and simultaneously the method has good selectivity and high sensitivity in the detection of glyphosate, and has a broader application prospect in glyphosate residue detection.

Description

Technical Field

[0001] The present invention relates to the technical field of food safety detection, and in particular to a method for detecting glyphosate through an in-situ fluorescence reaction triggered by a biological enzyme. Background Art

[0002] Glyphosate is a conductive, lethal organophosphorus herbicide. It is currently the most effective and widely used herbicide in agricultural production. It can effectively control weeds and significantly increase crop yields. However, the improper use of glyphosate in the ecosystem will directly lead to soil, water and air pollution, posing a serious threat to the ecological environment and human health. Currently, more than 30 countries and regions around the world have begun to ban or restrict the use of glyphosate and strengthen the detection of glyphosate residues in the environment and food. For example, the U.S. Environmental Protection Agency (EPA) stipulates that the maximum content of glyphosate in daily drinking water is 0.7μg / mL. -1 (4.14 μM); in China, the maximum limit for glyphosate in rice and fruit is 0.1 mg / kg and 0.5 mg / kg, respectively. Therefore, the development of a method that can detect glyphosate quickly, efficiently, sensitively, and at low cost is of great significance.

[0003] Currently, many sensors based on enzyme inhibition strategies and signal transduction technologies have been developed for the effective detection of glyphosate, such as enzyme-linked immunosorbent assay (ELISA), fluorescence sensing, colorimetry, electrochemical analysis, and surface-enhanced Raman spectroscopy. Among these, fluorescence sensing has become a preferred choice for glyphosate detection due to its high sensitivity, rapid response, ease of operation, and the lack of large-scale instrumentation, allowing for real-time detection and suitability for on-site analysis. Fluorescent probes are key components in fluorescent sensor design, and various types of fluorescent probes, such as MOFs, carbon nanomaterials, and metal oxides, have been used to detect glyphosate. However, the preparation of these fluorescent probes is complex and time-consuming, and some are highly toxic, limiting their widespread application. Silicon nanoparticles (SiNPs) are nanomaterials with high fluorescence quantum yields that are easy to synthesize, non-toxic, and have good biocompatibility.

[0004] In light of this, the present invention has designed an analytical method for detecting glyphosate using enzyme-triggered in situ generation of fluorescent SiNPs. Glyphosate's inhibitory effect on the enzyme regulates the amount of SiNPs produced, thereby generating a variable fluorescent signal and enabling detection of the target. This method, which requires no additional probe preparation and is simple and cost-effective, offers advantages such as high sensitivity, good selectivity, and a wide detection range for glyphosate detection, and holds great promise for future applications in glyphosate residue detection. Summary of the Invention

[0005] The purpose of the present invention is to provide a method for detecting glyphosate by an in situ fluorescence reaction triggered by an enzyme, so as to solve the problems in existing detection methods, such as the complex and time-consuming preparation of fluorescent probes, and the high toxicity and poor water solubility of some fluorescent probes, which limit their wide application.

[0006] To achieve the above objectives, the present invention adopts the following technical solution: a method for detecting glyphosate by an in situ fluorescence reaction triggered by an enzyme, comprising the following steps:

[0007] S1. Evenly mixing glyphosate solutions of different concentrations with alkaline phosphatase solutions to obtain a mixed solution;

[0008] S2, adding L-ascorbic acid-2-phosphate trisodium salt solution, magnesium chloride solution and Tris-HCl buffer to each mixed solution in S1, and adding 3-aminopropyltrimethoxysilane solution after the reaction is completed, and continuing the reaction;

[0009] S3. After the reaction is completed, the fluorescence intensity F of each mixed solution is measured, and the fluorescence intensity measured when no pesticide is added is recorded as F0;

[0010] S4. Based on the linear relationship between the F0 / F value and the glyphosate concentration, the glyphosate concentration can be quantitatively detected.

[0011] Furthermore, in the S1, the reaction time after uniform mixing is 25-40 minutes, and the reaction temperature is 37°C.

[0012] Furthermore, in the S2, the reaction time after adding L-ascorbic acid-2-phosphate trisodium salt solution, magnesium chloride solution and Tris-HCl buffer is 65-80 minutes, and the reaction temperature is 37°C; the reaction time after adding 3-aminopropyltrimethoxysilane solution is 25-35 minutes, and the reaction temperature is room temperature.

[0013] Furthermore, the concentration of the alkaline phosphatase solution is 200-300 U / L; the concentration of the L-ascorbic acid-2-phosphate trisodium salt solution is 40-60 mM; the concentration of the magnesium chloride solution is 10-20 mM; the concentration of the Tris-HCl buffer is 18-22 mM, and the pH is 8.5-9.5.

[0014] Furthermore, the volume of the glyphosate solution is 30-50 μL; the volume of the alkaline phosphatase solution, the L-ascorbic acid-2-phosphate trisodium salt solution, and the magnesium chloride solution is 80-120 μL; the volume of the Tris-HCl buffer is 400-600 μL; and the volume of the 3-aminopropyltrimethoxysilane solution is 150-200 μL.

[0015] Furthermore, in the S3, the fluorescence intensity is measured under the conditions of an excitation wavelength of 420 nm and an emission wavelength of 516 nm.

[0016] Furthermore, it is characterized in that: in the S4, when the glyphosate concentration range is 0.01 to 500 μM, the linear relationship between the F0 / F value and the glyphosate concentration is F0 / F=0.02168C glyphosate +1.055, R 2 =0.9989, and the detection limit was 0.785 nM.

[0017] Furthermore, the method has good selectivity for glyphosate in the selective detection of pesticides.

[0018] Furthermore, the pesticide is a non-organophosphine pesticide, including but not limited to spirotetramat, permethrin, tebuconazole, deltamethrin, carbaryl and thiamethoxam.

[0019] Beneficial effects of the present invention:

[0020] 1. This invention is the first to use SiNPs, a fluorescent product generated by an in-situ reaction triggered by ALP, for the detection of glyphosate, achieving trace and quantitative detection of glyphosate, and has broader application prospects in glyphosate residue detection;

[0021] 2. The method of the present invention is simple to operate, low in cost, does not require large-scale instruments and equipment, and does not require complicated sample pretreatment. It also has good selectivity for glyphosate, high detection sensitivity, and fast response speed, and can achieve rapid and routine detection of glyphosate.

[0022] 3. When the glyphosate concentration ranges from 0.01 to 500 μM, the emission peak intensity has a good linear relationship with the glyphosate concentration, and the detection line is as low as 0.785×10 -9 mol / L, which can realize trace and quantitative detection of glyphosate. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 It is a diagram showing the detection principle of the method of the present invention;

[0024] Figure 2 is the fluorescence excitation and emission spectra of SiNPs of the present invention;

[0025] Figure 3 The fluorescence intensity (A) and linear relationship diagram (B) of SiNPs after adding different concentrations of glyphosate in the present invention;

[0026] Figure 4 It is a histogram of the fluorescence intensity of SiNPs generated by reacting different types of pesticides with ALP in the reaction system of the present invention. DETAILED DESCRIPTION

[0027] The technical solution of the present invention will be clearly and completely described below in conjunction with the accompanying drawings in the embodiments of the present invention.

[0028] The principle of the present invention is:

[0029] like Figure 1 As shown, alkaline phosphatase (ALP) catalyzes the substrate L-ascorbic acid-2-phosphate trisodium salt (AA2P) to form L-ascorbic acid (AA), and AA reacts with 3-aminopropyltrimethoxysilane (APTMS) at room temperature to generate fluorescent product silicon nanoparticles (SiNPs), which emit a fluorescence emission peak at 516 nm.

[0030] When glyphosate is present, glyphosate inhibits ALP activity, weakening its catalytic ability, resulting in reduced production of AA and SiNPs and weakened fluorescence intensity, thereby achieving glyphosate detection.

[0031] The specific steps are as follows:

[0032] S1. Preparation of fluorescent SiNPs by in situ reaction triggered by biological enzymes

[0033] Alkaline phosphatase (ALP), L-ascorbic acid-2-phosphate trisodium salt (AA2P), magnesium chloride (MgCl2) and Tris-HCl (pH = 8.5-9.5) solution are mixed and reacted for 65-80 minutes. Subsequently, 3-aminopropyltrimethoxysilane (APTMS) solution is added to the above reaction solution, mixed evenly, and allowed to react at room temperature for 25-35 minutes to obtain silicon nanoparticles SiNPs with green fluorescence.

[0034] S2. Enzyme-triggered in situ synthesis of fluorescent SiNPs for glyphosate detection

[0035] Glyphosate at varying concentrations was mixed with ALP and reacted at 37°C for 25-40 minutes. AA2P solution, Tris-HCl solution, and MgCl2 solution were added to the mixture and reacted at 37°C for 65-80 minutes. APTMS solution was then added to the reaction system and reacted at room temperature for 25-35 minutes. Under an excitation wavelength of 420 nm, the peak fluorescence emission intensity of each mixed solution at 516 nm was recorded as F, where the fluorescence intensity measured at zero glyphosate concentration was F0. Based on the linear relationship between the F0 / F value and glyphosate concentration, glyphosate can be quantitatively detected.

[0036] Example 1

[0037] Preparation of SiNPs by in situ reaction triggered by biological enzymes.

[0038] Take 40 μL of water, 100 μL of 220 U / L ALP, 100 μL of 50 mM AA2P, 100 μL of 15 mM MgCl2 and 500 μL of 20 mM Tris-HCl (pH = 8.9) solution, mix them evenly, and react at 37 ° C for 70 minutes. Then add 160 μL APTMS solution to the above reaction solution, mix evenly, and let it stand at room temperature for 30 minutes to obtain silicon nanoparticles SiNPs with green fluorescence.

[0039] like Figure 2 As shown, the optimal excitation and emission wavelengths of SiNPs are 420 nm and 516 nm.

[0040] Example 2

[0041] Glyphosate was tested at different concentrations.

[0042] 40 μL of glyphosate solutions of different concentrations and 100 μL of 220 U / L ALP were reacted at 37°C for 30 min. Then, 100 μL of 50 mM AA2P, 100 μL of 15 mM MgCl2, and 500 μL of 20 mM Tris-HCl (pH = 8.9) solution were added to the reaction solution, mixed evenly, and the reaction was continued for 70 min. Finally, 160 μL of APTMS solution was added. After reaction at room temperature for 30 min, the fluorescence emission peak intensity of the glyphosate solutions of different concentrations at 516 nm was recorded under an excitation wavelength of 420 nm as F, where the fluorescence intensity measured when the glyphosate concentration was 0 was F0.

[0043] like Figure 3 As shown in the figure, it can be seen that with the increase of glyphosate concentration, the fluorescence of the reaction system continues to weaken. When the glyphosate concentration is in the range of 0.01 to 500 μM, the SiNPs fluorescence intensity ratio (F0 / F) shows a good linear relationship with the glyphosate concentration, and the linear equation is F0 / F = 0.02168C glyphosate +1.055, R 2 =0.9989.

[0044] Wherein, F0 and F are the fluorescence intensities of SiNPs in the absence and presence of glyphosate, respectively. According to the detection limit calculation formula: detection limit = 3σ / K, the detection limit of this analytical method for glyphosate is calculated to be as low as 0.785 nM. The results show that the analytical method of the present invention has a wide linear range and a low detection limit for the detection of glyphosate, and can achieve trace qualitative and quantitative measurement of glyphosate.

[0045] Example 3

[0046] Selective detection of pesticides.

[0047] Alkaline phosphatase was reacted with glyphosate, spirotetramat, permethrin, tebuconazole, deltamethrin, carbaryl, and thiamethoxam at 37°C for 30 minutes. Then, 100 μL of 50 mM AA2P, 100 μL of 15 mM MgCl2, and 500 μL of 20 mM Tris-HCl (pH = 8.9) solution were added and mixed well. The reaction was continued at 37°C for 70 minutes. Finally, 160 μL of APTMS solution was added. After reacting at room temperature for 30 minutes, the fluorescence intensity of the different pesticide reaction systems was tested at an excitation wavelength of 420 nm.

[0048] like Figure 4 As shown in Figure 2, where F represents the fluorescence intensity measured with different pesticides added to the reaction system, and F0 represents the fluorescence intensity measured without pesticides. It can be seen that only glyphosate has a significant inhibitory effect on ALP, while no significant change in the F0 / F ratio was observed in the presence of other pesticides. This demonstrates that the present invention has good selectivity for glyphosate detection.

[0049] Example 4

[0050] Determination of glyphosate concentration in actual samples.

[0051] Apples and cabbages were purchased from a local supermarket, and actual samples of water were taken from a lake.

[0052] First, 10 g of cabbage and apple peel samples were added to 50 mL of distilled water and ultrasonically treated for 60 min.

[0053] Subsequently, the mixed solution and lake water were centrifuged at 10,000 rpm for 8 minutes to remove insoluble matter; the supernatant was filtered through a 0.22 μm membrane filter to obtain the corresponding solution to be tested.

[0054] Then, known concentrations of glyphosate were added to the actual samples to prepare glyphosate solutions at concentrations of 25 μM, 50 μM, and 100 μM, respectively. 100 μL of 220 U / L ALP was mixed with 40 μL of apple, cabbage, and lake water samples containing varying concentrations of glyphosate. The mixtures were then reacted at 37°C for 30 minutes. Then, 100 μL of 50 mM AA2P, 100 μL of 15 mM MgCl2, and 500 μL of 20 mM Tris-HCl (pH 8.9) were added to the mixtures. The reaction was continued at 37°C for 70 minutes. Finally, 160 μL of APTMS solution was added. After 30 minutes of reaction at room temperature, fluorescence spectra were recorded at an excitation wavelength of 420 nm.

[0055] According to the equation obtained in Example 2, the determination results, recovery rate and relative standard deviation were calculated. The results are shown in Table 1.

[0056] Table 1 Glyphosate concentration determination in actual samples

[0057]

[0058] As shown in Table 1, the recoveries of glyphosate in actual samples were 99.1% to 106.9%, and the relative standard deviations (RSDs) were 1.20% to 3.75%. These results indicate that the present invention has good accuracy in detecting glyphosate in fruits, vegetables, and water samples.

[0059] The present invention is not limited to the above-mentioned optimal implementation mode. Anyone can derive other forms of products under the inspiration of the present invention. However, no matter what changes are made in the shape or structure, any technical solution that is the same or similar to that of the present application falls within the scope of protection of the present invention.

Claims

1. A method for detecting glyphosate by in situ fluorescence reaction triggered by a biological enzyme, characterized in that: The following steps are involved: S1. Evenly mixing glyphosate solutions of different concentrations with alkaline phosphatase solutions to obtain a mixed solution; S2, adding L-ascorbic acid-2-phosphate trisodium salt solution, magnesium chloride solution and Tris-HCl buffer to each mixed solution in S1, and adding 3-aminopropyltrimethoxysilane solution after the reaction is completed, and continuing the reaction; S3. After the reaction is completed, the fluorescence intensity F of each mixed solution is measured, and the fluorescence intensity measured when no pesticide is added is recorded as F0; S4. Based on the linear relationship between the F0 / F value and the glyphosate concentration, the glyphosate concentration can be quantitatively detected.

2. The method for detecting glyphosate by in situ fluorescence reaction triggered by an enzyme according to claim 1, characterized in that: In the above-mentioned S1, the reaction time after uniform mixing is 25-40 minutes, and the reaction temperature is 37°C.

3. The method for detecting glyphosate by in situ fluorescence reaction triggered by an enzyme according to claim 2, characterized in that: In the S2, the reaction time after adding the L-ascorbic acid-2-phosphate trisodium salt solution, magnesium chloride solution and Tris-HCl buffer is 65-80 minutes, and the reaction temperature is 37° C.; the reaction time after adding the 3-aminopropyltrimethoxysilane solution is 25-35 minutes, and the reaction temperature is room temperature.

4. The method for detecting glyphosate by in situ fluorescence reaction triggered by an enzyme according to claim 3, characterized in that: The concentration of the alkaline phosphatase solution is 200-300 U / L; the concentration of the L-ascorbic acid-2-phosphate trisodium salt solution is 40-60 mM; the concentration of the magnesium chloride solution is 10-20 mM; the concentration of the Tris-HCl buffer solution is 18-22 mM, and the pH is 8.5-9.

5.

5. The method for detecting glyphosate by in situ fluorescence reaction triggered by an enzyme according to claim 4, characterized in that: The volume of the glyphosate solution is 30-50 μL; the volumes of the alkaline phosphatase solution, L-ascorbic acid-2-phosphate trisodium salt solution, and magnesium chloride solution are 80-120 μL; the volume of the Tris-HCl buffer solution is 400-600 μL; and the volume of the 3-aminopropyltrimethoxysilane solution is 150-200 μL.

6. The method for detecting glyphosate by in situ fluorescence reaction triggered by an enzyme according to claim 1, characterized in that: In the above-mentioned S3, the fluorescence intensity was measured at an excitation wavelength of 420 nm and an emission wavelength of 516 nm.

7. The method for detecting glyphosate by in situ fluorescence reaction triggered by an enzyme according to any one of claims 1 to 6, characterized in that: In the above S4, when the glyphosate concentration range is 0.01 to 500 μM, the linear relationship between the F0 / F value and the glyphosate concentration is F0 / F=0.02168C glyphosate +1.055, R 2 =0.9989, and the detection limit was 0.785 nM.

8. The method for detecting glyphosate by in situ fluorescence reaction triggered by an enzyme according to claim 1, characterized in that: The method has good selectivity for glyphosate in the selective detection of pesticides.

9. The method for detecting glyphosate by in situ fluorescence reaction triggered by an enzyme according to claim 8, characterized in that: The pesticides are non-organophosphine pesticides, including but not limited to spirotetramat, permethrin, tebuconazole, deltamethrin, carbaryl and thiamethoxam.

Images