Method for detecting glyphosate through in-situ fluorescence reaction triggered by biological enzyme

Abstract

The invention relates to the technical field of food safety detection, in particular to a method for detecting glyphosate through in-situ fluorescence reaction triggered by biological enzyme. According to the method, alkaline phosphatase (ALP) is utilized to catalyze a substrate L-ascorbic acid-2-trisodium phosphate to form L-ascorbic acid, the L-ascorbic acid reacts with 3-aminopropyltrimethoxysilane at room temperature, and the fluorescent product silicon nanoparticles (SiNPs) are generated. When the glyphosate exists, the glyphosate can inhibit the activity of ALP, so that the catalytic ability of the glyphosate is weakened, the generation of L-ascorbic acid and SiNPs is reduced, the fluorescence intensity is weakened, and the fluorescence intensity of the system is continuously weakened along with the increase of the concentration of the glyphosate, so that the quantitative detection of the glyphosate is realized. According to the method, no nano material needs to be prepared, the detection method is simplified, the detection cost is reduced, and meanwhile, the method is good in selectivity and high in sensitivity in glyphosate detection and has a wider 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 by in-situ fluorescence reaction triggered by a biological enzyme. Background Art

[0002] Glyphosate is a conductive, lethal organophosphorus herbicide. It is the most effective and widely used herbicide in agricultural production. It can effectively control weeds and significantly increase crop yields. However, improper use of glyphosate in ecosystems 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 (4.14μM); in China, the maximum limit of glyphosate in rice and fruit is 0.1mg / kg and 0.5mg / kg, respectively. Therefore, it is of great significance to develop a method that can detect glyphosate quickly, efficiently, sensitively and at low cost.

[0003] Nowadays, 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, fluorescence sensing detection, colorimetric analysis, electrochemical analysis and surface enhanced Raman spectroscopy. Among them, fluorescence sensing detection has become a better choice for glyphosate detection due to its high sensitivity, fast response speed, convenient operation, no need for large-scale instruments and equipment, and can be used for real-time detection and suitable for on-site analysis. Fluorescent probes are key components in the design of fluorescent sensors. Various types of fluorescent probes, such as MOF, 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 fluorescent probes are highly toxic, which limits their widespread application. Silicon nanoparticles (SiNPs) are a type of nanomaterial with high fluorescence quantum yield, which are easy to synthesize, non-toxic and have good biocompatibility.

[0004] In view of this, the present invention designs an analytical method for detecting glyphosate by in situ generation of fluorescent SiNPs triggered by biological enzymes. The inhibitory effect of glyphosate on biological enzymes can regulate the amount of SiNPs produced, thereby generating a changing fluorescent signal to achieve detection of the target. The method designed by the present invention does not require additional preparation of probes, is easy to operate, has low cost, and has the advantages of high sensitivity, good selectivity, and a wide detection range in the detection of glyphosate, and has a broader application prospect in the detection of glyphosate residues. Summary of the invention

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

[0006] To achieve the above object, the present invention adopts the following technical solution: a method for detecting glyphosate by in situ fluorescence reaction triggered by a biological 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 after the reaction is completed, adding 3-aminopropyltrimethoxysilane solution to continue 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 volumes of the alkaline phosphatase solution, the L-ascorbic acid-2-phosphate trisodium salt solution, and the 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.

[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 ranges from 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, detection limit is 0.785nM.

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

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

[0019] Beneficial effects of the present invention:

[0020] 1. The present invention uses SiNPs, a fluorescent product generated by an in-situ reaction triggered by ALP, for the first time to detect glyphosate, thus achieving trace and quantitative detection of glyphosate, and has a broader application prospect 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 complex sample pretreatment, and 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 is in the range of 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 the detection principle diagram 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) with a fluorescence emission peak at 516 nm.

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

[0031] The specific steps are as follows:

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

[0033] Take alkaline phosphatase (ALP), L-ascorbic acid-2-phosphate trisodium salt (AA2P), magnesium chloride (MgCl2) and Tris-HCl (pH = 8.5-9.5) solution, mix well and react for 65-80 minutes, then add 3-aminopropyltrimethoxysilane (APTMS) solution to the above reaction solution, mix well, and let it stand at room temperature for 25-35 minutes to obtain silicon nanoparticles SiNPs with green fluorescence.

[0034] S2. In situ synthesis of fluorescent SiNPs triggered by bio-enzymes for glyphosate detection

[0035] Glyphosate of different concentrations was mixed with ALP respectively, and reacted at 37°C for 25-40 minutes; AA2P solution, Tris-HCl solution and MgCl2 solution were added to the above mixture, and reacted at 37°C for 65-80 minutes; then APTMS solution was added to the above reaction system, and reacted at room temperature for 25-35 minutes, and then the fluorescence emission peak intensity of each mixed solution at 516nm was recorded as F at an excitation wavelength of 420nm, wherein the fluorescence intensity measured when the glyphosate concentration was 0 was F0. According to the linear relationship between the F0 / F value and the 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 ALP with a concentration of 220 U / L, 100 μL of AA2P with a concentration of 50 mM, 100 μL of MgCl2 with a concentration of 15 mM, and 500 μL of Tris-HCl (pH = 8.9) solution with a concentration of 20 mM, mix them evenly, and react at 37°C for 70 minutes. Then, add 160 μL of APTMS solution to the above reaction solution, mix them evenly, and let them 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 testing at different concentrations.

[0042] Take 40 μL of glyphosate solutions of different concentrations and 100 μL of ALP with a concentration of 220 U / L, react at 37°C for 30 minutes, then add 100 μL of 50 mM AA2P, 100 μL of 15 mM MgCl2 and 500 μL of 20 mM Tris-HCl (pH=8.9) solution to the above reaction solution, mix well and continue to react for 70 minutes, finally add 160 μL of APTMS solution, react at room temperature for 30 minutes, and record the fluorescence emission peak intensity of glyphosate solutions of different concentrations at 516 nm under an excitation wavelength of 420 nm as F, where the fluorescence intensity measured when the glyphosate concentration is 0 is 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] Among them, 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, it is calculated that the detection limit of glyphosate by this analytical method is as low as 0.785nM. 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 realize 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 min, and 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. After mixing evenly, the mixture was reacted at 37°C for 70 min. Finally, 160 μL of APTMS solution was added. After reacting at room temperature for 30 min, the fluorescence intensity of different pesticide reaction systems was tested at an excitation wavelength of 420 nm.

[0048] like Figure 4 As shown, F is the fluorescence intensity measured when different types of pesticides are added to the reaction system, and F0 is the fluorescence intensity measured when no pesticide is added to the reaction system. It can be seen that only glyphosate has a significant inhibitory effect on ALP, and in the presence of other types of pesticides, no significant change in F0 / F is observed. The results show that the present invention has good selectivity for the detection of glyphosate.

[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 for testing.

[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 min 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, glyphosate of known concentrations was added to the actual samples to prepare glyphosate solutions with concentrations of 25μM, 50μM and 100μM, respectively. 100μL of 220U / L ALP was mixed with 40μL of apple, cabbage and lake water samples containing different concentrations of glyphosate and placed at 37℃ for 30min, then 100μL of 50mM AA2P, 100μL of 15mM MgCl2 and 500μL of 20mM Tris-HCl (pH=8.9) solution were added to the above mixtures, and the reaction was continued at 37℃ for 70min. Finally, 160μL of APTMS solution was added, and the fluorescence spectrum was recorded at an excitation wavelength of 420nm after reacting at room temperature for 30min.

[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 Determination of glyphosate concentration in actual samples

[0057]

[0058] As shown in Table 1, the recovery rate of glyphosate in actual samples is 99.1% to 106.9%, and the relative standard deviation (RSD) is 1.20 to 3.75%. These results show 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 various forms of products under the inspiration of the present invention. However, no matter what changes are made in the shape or structure, all technical solutions that are the same or similar to those of the present application fall within the protection scope of the present invention.

Claims

1. A method for detecting glyphosate by in situ fluorescence reaction triggered by biological enzymes, 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 after the reaction is completed, adding 3-aminopropyltrimethoxysilane solution to continue 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 a biological enzyme according to claim 1, characterized in that: In the S1, after uniform mixing, the reaction time is 25-40 minutes, and the reaction temperature is 37°C.

3. The method for detecting glyphosate by in situ fluorescence reaction triggered by a biological enzyme according to claim 2, characterized in that: 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.

4. The method for detecting glyphosate by in situ fluorescence reaction triggered by a biological 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 biological 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, the L-ascorbic acid-2-phosphate trisodium salt solution, and the 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 biological enzyme according to claim 1, characterized in that: 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.

7. A method for detecting glyphosate by in situ fluorescence reaction triggered by a biological enzyme according to any one of claims 1 to 6, characterized in that: In S4, when the glyphosate concentration ranges from 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, detection limit is 0.785nM.

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

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