Application of silver nanoclusters in detection of glyphosate, method for detecting glyphosate in plants and dynamic monitoring method

Abstract

The application belongs to the technical field of organic phosphorus pesticide detection and analysis, and provides application of silver nanoclusters in glyphosate detection, a glyphosate detection method in plants and a dynamic monitoring method. The application uses silver nanoclusters for glyphosate detection, does not need to add a detection reagent, is simple to operate, has good selectivity for glyphosate, fast response and high sensitivity, avoids false positives in detection, and improves the accuracy of glyphosate detection results. The detection method provided in the application uses an alcohol solvent to alcohol extract a plant to be detected to obtain an alcohol extract; silver nanoclusters are added to the alcohol extract to obtain a sample to be detected; fluorescence detection is performed on the sample to be detected to obtain a fluorescence value of the plant to be detected; and the fluorescence value of the plant to be detected is substituted into a predetermined standard curve to obtain the concentration of glyphosate in the plant to be detected. The detection method provided in the application does not need to add a detection reagent, is simple to operate, avoids false positives in detection, and improves the accuracy of detection.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of detection and analysis of organophosphorus pesticides, and particularly relates to application of silver nanoclusters in glyphosate detection, a glyphosate detection method in plants and a dynamic monitoring method. BACKGROUND

[0002] In recent years, with the widespread use of pesticides in modern agriculture, the influence of pesticide residues and pesticides on environmental pollution, food safety and human health has attracted more and more attention. Glyphosate is a broad-spectrum, non-selective, organophosphorus herbicide developed by the American Monsanto Company in 1971, and is one of the most widely used and largest herbicides in the world. Glyphosate can effectively inhibit the growth of annual weeds, perennial plants and herbaceous plants in a non-selective manner, and has the properties of low cost and low toxicity to mammals. However, the excessive and long-term use of glyphosate can cause its continuous enrichment in the environment and organisms, and enter the human body through the food chain, posing a potential threat to human health. Recent studies have shown that glyphosate can cause a variety of diseases, including lung cancer, lymphadenoma and pancreatic cancer, and has been listed as a 2A carcinogen by the World Health Organization (WHO). More than 30 countries or regions around the world have begun to prohibit or restrict the use of glyphosate, and have included the residual limit of glyphosate in relevant regulations and standards. Therefore, it is of great significance to analyze and detect glyphosate in the environment and biological samples.

[0003] The detection methods for glyphosate pesticide residues mainly include gas chromatography, high performance liquid chromatography, chromatography-mass spectrometry, electrochemical analysis and enzyme-linked immunoassay. These traditional methods have achieved good results in the detection of glyphosate pesticide residues, but usually require high professional requirements for experimental equipment and operating personnel, need large and expensive equipment, have long detection time, require complex sample pretreatment, and are difficult to perform high-throughput analysis.

[0004] Compared with the above method, the fluorescence sensing detection method has the advantages of simple operation, easy-to-obtain equipment, high sensitivity, strong specificity, short response time, real-time high-throughput identification and detection, etc., so the application of the fluorescence sensing technology to the detection of glyphosate residues has broad application prospects. At present, some reported fluorescence probes have been used for the detection and sensing research of organophosphorus pesticides and glyphosate. For example, Jiang Xingyu et al. (Liu, D.; Chen, W.; Wei, J.; Li, X.; Wang, Z.; Jiang, X. Anal. Chem. 2012, 84, 4185-4191.) of the National Center for Nanoscience reported a composite probe based on gold nanoparticles and rhodamine B dye, which realized the detection of organophosphorus pesticides through the mechanism of inhibiting acetylcholinesterase (AChE) activity. Ai Shiyun et al. (Hou, J.; Dong, J.; Zhu, H.; Teng, X.; Ai, S.; Mang, M. Biosens. Bioelectron. 2015, 15, 20-26.) of Shandong Agricultural University developed a carbon dot functionalized with L-tyrosine, which realized the selective detection of methyl parathion pesticide through the mechanism of inhibiting tyrosinase activity. Ding Lan et al. (Wang, L.; Bi, Y.; Gao, J.; Li, Y.; Ding, H.; Ding, L. RSC Adv. 2016, 6, 85820-85828.) of Jilin University realized the indirect detection of glyphosate by using a carbon dot-copper ion composite fluorescence probe. Guan Jie et al. (Guan, J.; Yang, J.; Zhang, Y.; Zhang, X.; Deng, H.; Xu, J.; Wang, J.; Yuan, M. S. Talanta 2021, 224, 121834-121839.) of Northwest A&F University also realized the indirect detection of glyphosate by using a rhodamine derivative-copper ion composite fluorescence probe. These probes have been used for the detection of organophosphorus and glyphosate pesticides, but still have obvious limitations, such as: the reported probes need to add two or more than two detection reagents, and this indirect detection mode will have the problem of false positive, resulting in inaccurate determination results. SUMMARY

[0005] Therefore, the purpose of the present application is to provide an application of silver nanoclusters in glyphosate detection, a glyphosate detection method in plants and a dynamic monitoring method.

[0006] In order to achieve the above-mentioned purpose of the application, the present application provides the following technical solutions:

[0007] The present application provides an application of silver nanoclusters in glyphosate detection.

[0008] Preferably, the silver nanoclusters are used in the form of a silver nanocluster aqueous solution; the concentration of the silver nanocluster aqueous solution is 10-30 g / L.

[0009] Preferably, the method for preparing the silver nanoclusters comprises the following steps:

[0010] The polymer, the soluble silver salt and water are mixed, and incubation and ultraviolet lamp irradiation are sequentially performed to obtain a precursor;

[0011] The precursor is sequentially subjected to dialysis and solvent removal to obtain the silver nanoclusters.

[0012] The polymer comprises polymethylacrylic acid and / or polyacrylic acid.

[0013] Preferably, the use amount ratio of the polymer to the soluble silver salt is 0.6 g: 0.001-0.003 mol.

[0014] Preferably, the incubation is performed under the condition of a constant-temperature water bath, the temperature of the constant-temperature water bath is 25-30 ℃, and the time is 10-20 min; the ultraviolet lamp wavelength of the ultraviolet lamp irradiation is 365 nm; the ultraviolet lamp irradiation is performed in the mode of stopping for 20-30 min every 1 h of irradiation; and the total time of the ultraviolet lamp irradiation is 3-4.5 h.

[0015] Preferably, the molecular weight cut-off of the dialysis bag for dialysis is 8000-14000 Da.

[0016] The application further provides a method for detecting glyphosate in plants, comprising the following steps:

[0017] An alcohol solvent is used to alcohol extract the plant to be detected to obtain an alcohol extract;

[0018] The silver nanoclusters are added into the alcohol extract to obtain a sample to be detected;

[0019] The sample to be detected is subjected to fluorescence detection to obtain the fluorescence value of the plant to be detected.

[0020] The fluorescence value of the plant to be detected is substituted into a predetermined standard curve to obtain the concentration of glyphosate in the plant to be detected.

[0021] The ordinate of the predetermined standard curve is F0-F / F0, and the abscissa is the concentration of glyphosate.

[0022] Preferably, the alcohol solvent comprises methanol; the use amount ratio of the alcohol solvent to the plant to be detected is 10-25 mL: 5.0 g; and the alcohol extraction is performed in the mode of ultrasonic extraction, and the ultrasonic extraction is performed twice, and each time of ultrasonic extraction is performed for 20 min.

[0023] Preferably, the excitation light wavelength of the fluorescence detection is 515 nm; and the fluorescence value of the plant to be detected is the fluorescence value at 605 nm.

[0024] The application further provides a method for dynamically monitoring glyphosate in plants, comprising the following steps:

[0025] The living plant is sequentially subjected to first incubation in a silver nanocluster aqueous solution and second incubation in a glyphosate aqueous solution, to obtain an incubated plant.

[0026] The incubated plant is subjected to dynamic monitoring.

[0027] The application provides application of silver nanoclusters in glyphosate detection.

[0028] The application provides a method for detecting glyphosate in plants, comprising the following steps: alcohol extraction is performed on a plant to be detected by using an alcohol solvent to obtain an alcohol extract; silver nanoclusters are added into the alcohol extract to obtain a sample to be detected; fluorescence detection is performed on the sample to be detected to obtain a fluorescence value of the plant to be detected; and the fluorescence value of the plant to be detected is substituted into a predetermined standard curve to obtain a concentration of glyphosate in the plant to be detected; the ordinate of the predetermined standard curve is F0-F / F0, and the abscissa is the concentration of glyphosate.

[0029] The application further provides a method for dynamically monitoring glyphosate in plants, comprising the following steps: a living plant is sequentially subjected to first incubation in a silver nanocluster aqueous solution and second incubation in a glyphosate aqueous solution, to obtain an incubated plant; and the incubated plant is subjected to dynamic monitoring. BRIEF DESCRIPTION OF DRAWINGS

[0030] Figure 1 UV absorption diagrams of silver nanoclusters before and after interaction with glyphosate;

[0031] Figure 2 Fluorescence emission diagrams of silver nanoclusters before and after interaction with glyphosate;

[0032] Figure 3 A diagram showing changes in fluorescence emission intensity of a silver nanocluster aqueous solution after addition of different concentrations of glyphosate (0-3 μM);

[0033] Figure 4A linear standard curve graph for fitting fluorescence intensity-glyphosate concentration;

[0034] Figure 5 A fluorescence intensity-time change graph for the silver nanocluster-glyphosate interaction system;

[0035] Figure 6 A graph of the change in fluorescence emission intensity at 605 nm after adding 10 μM of different interferents (potassium ions, sodium ions, calcium ions, silver ions, sulfate ions, nitrate ions and ascorbic acid), and organophosphorus pesticides (glyphosate, malathion, glufosinate, parathion, methamidophos, dichlorvos, chlorpyrifos and atrazine) to the silver nanocluster aqueous solution;

[0036] Figure 7 A fluorescence staining graph for the silver nanocluster for glyphosate detection in lettuce seedling root tip cells; wherein, A and E are imaging graphs of the lettuce seedlings after adding 1.2 mg / mL of the probe and incubating for 30 min; B and F are local enlarged graphs of the lettuce seedling root tip tissue after silver nanocluster staining under a high-power lens; C and G are imaging graphs of the lettuce seedling root tip tissue incubated with the silver nanocluster and then adding 3 μM of glyphosate and incubating for 30 min; D and H are imaging graphs of the lettuce seedling root tip tissue incubated with the silver nanocluster and then adding 5 μM of glyphosate and incubating for 30 min; wherein, E, F, G and H are imaging graphs of the red channel and the corresponding bright field under a confocal laser microscope. DETAILED DESCRIPTION

[0037] The application provides application of silver nanoclusters in glyphosate detection.

[0038] In the application, the silver nanoclusters are used in the form of a silver nanocluster aqueous solution; the concentration of the silver nanocluster aqueous solution is preferably 10-30 g / L, and is further preferably 24.64 g / L.

[0039] In the application, the preparation method of the silver nanoclusters comprises the following steps:

[0040] The polymer, the soluble silver salt and water are mixed, and then incubation and ultraviolet lamp irradiation are sequentially performed to obtain a precursor;

[0041] The precursor is sequentially subjected to dialysis and solvent removal to obtain the silver nanoclusters;

[0042] The polymer comprises polymethylacrylic acid and / or polyacrylic acid.

[0043] The present application mixes a polymer, a soluble silver salt and water, and then carries out incubation and ultraviolet lamp irradiation in sequence to obtain a precursor.In the present application, the polymer includes polymethacrylic acid and / or polyacrylic acid, and preferably is polymethacrylic acid (PMAA).In the present application, the molecular weight of the polymer is preferably 4000-6000, and further preferably 5000.In the present application, the soluble silver salt preferably includes silver nitrate.In the present application, the soluble silver salt is preferably used in the form of an aqueous solution of the soluble silver salt, and the concentration of the aqueous solution of the soluble silver salt is preferably 0.05-0.15 mol / L.In the present application, the ratio of the amount of the polymer to the amount of the soluble silver salt is preferably 0.6 g: 0.001-0.003 mol, and further preferably 0.6 g: 0.001 mol.

[0044] In the present application, the incubation is preferably carried out under the condition of a constant-temperature water bath, the temperature of the constant-temperature water bath is preferably 25-30℃, and the time is preferably 10-20 min; the incubation is preferably carried out under the protection of nitrogen.

[0045] In the present application, the wavelength of the ultraviolet lamp for the ultraviolet lamp irradiation is preferably 365 nm; the mode of the ultraviolet lamp irradiation is preferably 20-30 min of stopping for each 1 h of irradiation; and the total time of the ultraviolet lamp irradiation is preferably 3-4.5 h.

[0046] In the present application, the main functions of the ultraviolet lamp irradiation are: first photo-reduction, reducing silver ions in the positive one valence to silver atoms in zero valence; and second, polymerization of a plurality of silver atoms to form silver nanoclusters.

[0047] After obtaining the precursor, the present application carries out dialysis and solvent removal in sequence for the precursor to obtain the silver nanoclusters.In the present application, the cut-off molecular weight of the dialysis bag for the dialysis is preferably 8000-14000 Da, and further preferably 12000 Da.In the present application, the time for the dialysis is preferably 24-48 h.In the present application, the solvent for the dialysis preferably includes distilled water.In the present application, the dialysis can remove unreacted silver ions.

[0048] In the present application, the mode of the solvent removal is preferably rotary evaporation; and the present application does not make a specific limitation on the parameters of the rotary evaporation, as long as the solvent in the dialysis liquid obtained through the dialysis can be removed.

[0049] After the solvent removal, the present application preferably further includes dissolving the viscous substance obtained through the solvent removal, and the reagent for the dissolving preferably includes distilled water.

[0050] The preparation method of the silver nanoclusters of the present application is simple in operation.

[0051] The present application further provides a method for detecting glyphosate in plants, including the following steps:

[0052] The alcohol solvent is used to alcohol extract the plant to be tested to obtain an alcohol extract;

[0053] Silver nanoclusters are added to the alcohol extract to obtain a sample to be detected;

[0054] The sample to be detected is subjected to fluorescence detection to obtain the fluorescence value of the plant to be tested;

[0055] The fluorescence value of the plant to be tested is substituted into a predetermined standard curve to obtain the concentration of glyphosate in the plant to be tested.

[0056] The ordinate of the predetermined standard curve is F0-F / F0, and the abscissa is the concentration of glyphosate.

[0057] The alcohol solvent is used to alcohol extract the plant to be tested to obtain an alcohol extract. In the present application, the plant to be tested preferably includes vegetables, and the vegetables preferably include lettuce. In the present application, the alcohol solvent preferably includes methanol; the ratio of the amount of the alcohol solvent to the plant to be tested is preferably 10-25 mL:5.0 g. In the present application, the alcohol extraction is preferably ultrasonic extraction, the number of ultrasonic extractions is preferably 2, and the time of each ultrasonic extraction is preferably 20 min. In the present application, the power of the ultrasonic is preferably 80-200 W. Further preferably, the power is 100 W.

[0058] After the alcohol extraction, the present application further includes centrifuging the obtained extract, and filtering the obtained supernatant. In the present application, the speed of centrifugation is preferably 8000-10000 rpm, and the time is preferably 10-30 min. In the present application, the pore size of the filter membrane used for filtration is preferably 0.22 μm.

[0059] After obtaining the alcohol extract, the present application adds silver nanoclusters to the alcohol extract to obtain a sample to be detected. In the present application, the silver nanoclusters are preferably used in the form of a silver nanocluster aqueous solution; the concentration of the silver nanocluster aqueous solution is preferably 10-30 g / L. In the present application, the concentration of silver nanoclusters in the sample to be detected is preferably 0.5-1.5 g / L, and further preferably 1.0-1.2 g / L.

[0060] After obtaining the sample to be detected, the present application subjects the sample to be detected to fluorescence detection to obtain the fluorescence value of the plant to be tested. In the present application, the excitation light wavelength of the fluorescence detection is preferably 515 nm; and the fluorescence value of the plant to be tested is preferably the fluorescence value at 605 nm.

[0061] After obtaining the fluorescence value of the plant to be tested, the present application substitutes the fluorescence value of the plant to be tested into a predetermined standard curve to obtain the concentration of glyphosate in the plant to be tested.

[0062] In the present application, the ordinate of the predetermined standard curve is F0-F / F0, and the abscissa is the concentration of glyphosate.

[0063] In the present application, the predetermined standard curve is preferably obtained by the following steps:

[0064] Different concentrations of glyphosate are added to the quantitative silver nanocluster aqueous solution to obtain a series of on-machine samples;

[0065] The fluorescence of the series of on-machine samples is detected to obtain the fluorescence values of the series of on-machine samples;

[0066] The F0-F / F0 is taken as the ordinate, and the concentration of glyphosate is taken as the abscissa to linearly fit to obtain the predetermined standard curve;

[0067] F0 is the fluorescence value of the on-machine sample obtained when the concentration of glyphosate is 0;

[0068] F is the fluorescence value of the on-machine sample obtained when the concentration of glyphosate is not 0.

[0069] In the present application, the concentration of silver nanoclusters in the series of on-machine samples is preferably 0.5-15 g / L, and further preferably 1.2-10 g / L.

[0070] After obtaining the series of on-machine samples, the fluorescence of the series of on-machine samples is detected to obtain the fluorescence values of the series of on-machine samples. In the present application, the parameters of the fluorescence detection are preferably consistent with the above technical solution, and will not be repeated here.

[0071] After obtaining the fluorescence values of the series of on-machine samples, the F0-F / F0 is taken as the ordinate, and the concentration of glyphosate is taken as the abscissa to linearly fit to obtain the predetermined standard curve. The operation of the linear fitting in the present application is not specifically limited, and the operation well known to those skilled in the art can be used.

[0072] In the present application, F0 is the fluorescence value of the on-machine sample obtained when the concentration of glyphosate is 0; and F is the fluorescence value of the on-machine sample obtained when the concentration of glyphosate is not 0.

[0073] The present application also provides a method for dynamically monitoring glyphosate in plants, comprising the following steps:

[0074] The living plants are sequentially subjected to first incubation in silver nanocluster aqueous solution and second incubation in glyphosate aqueous solution to obtain incubated plants;

[0075] The incubated plants are dynamically monitored.

[0076] The living plant is sequentially subjected to first incubation in silver nanocluster aqueous solution and second incubation in glyphosate aqueous solution to obtain the incubated plant. In the present application, the living plant preferably comprises vegetables, and the vegetables preferably comprise lettuce. In the present application, the concentration of the silver nanocluster aqueous solution is preferably 0.5-15 g / L, and further preferably 1.2 g / L. In the present application, the concentration of the glyphosate aqueous solution is preferably 0-30 μM. In the present application, the temperature of the first incubation is preferably room temperature, i.e. neither additional heating nor additional cooling is needed; and the time of the first incubation is preferably 30 min. In the present application, the temperature of the second incubation is preferably room temperature, i.e. neither additional heating nor additional cooling is needed; and the time of the second incubation is preferably 30 min.

[0077] In the present application, the dynamic monitoring preferably comprises confocal laser scanning microscope imaging analysis. The present application does not make specific limitation to the parameters of the confocal laser scanning microscope imaging analysis, and the operation well known to those skilled in the art can be adopted.

[0078] The application of silver nanoclusters in glyphosate detection, the method for detecting glyphosate in plants and the dynamic monitoring method provided by the present application will be described in detail below in conjunction with examples, but they should not be understood as limiting the scope of protection of the present application.

[0079] Preparation of silver nanoclusters for detecting glyphosate in Example 1

[0080] comprising the following steps:

[0081] (1) Under magnetic stirring, 0.6 g of polymethacrylic acid (PMAA, M = 5000) was introduced into a freshly prepared 20 mL 0.05 mol / L silver nitrate solution.

[0082] (2) After incubation at 27℃ for 10 min in water bath under nitrogen protection in the dark, irradiation was performed under a 365 nm wavelength ultraviolet lamp, and the irradiation reaction was performed at intervals for 4.5 h, with 30 min of stop after each 1 h of irradiation to obtain a precursor.

[0083] (3) The precursor was dialyzed (the molecular weight cut-off of the dialysis bag was 12000 Da) in distilled water for 24 h to remove unreacted silver ions, and the final silver nanocluster solution was stored at room temperature in the dark.

[0084] (4) The solution after dialysis was placed in a round-bottom flask and rotary evaporated to be viscous, and then vacuum pumped to dryness to obtain 0.4926 g; and then dissolved with 20 mL of distilled water to obtain a silver nanocluster aqueous solution with a concentration of 24.64 mg / mL.

[0085] Application of silver nanoclusters for detecting glyphosate pesticide in Example 2

[0086] Take 3 mL of silver nanoclusters aqueous solution with a concentration of 1.2 mg / mL as the detection main solution, the UV absorption peak of silver nanoclusters is at 515 nm, and the corresponding solution color is reddish brown; after the silver nanoclusters and 3 μM glyphosate pesticide are allowed to react at room temperature for 1 min, the absorption peak at 515 nm decreases and disappears, and the solution color becomes light yellow, as shown in the results. Figure 1 Therefore, the present application can realize the detection of glyphosate by colorimetric method and naked eye observation method, and the detection method is simple and easy to operate, and has high sensitivity.

[0087] Figure 2 The fluorescence emission diagram of silver nanoclusters before and after reacting with glyphosate can be seen from Figure 2 It can be seen that the fluorescence emission peak of 3 mL of silver nanoclusters aqueous solution with a concentration of 1.2 mg / mL is at 605 nm before reacting with glyphosate, and under the irradiation of a 365 nm ultraviolet lamp, a deep red fluorescence is obviously presented; after reacting with 3 μM glyphosate, the fluorescence intensity at 605 nm is significantly weakened and quenched.

[0088] Take 3 mL of silver nanoclusters aqueous solution with a concentration of 1.2 mg / mL, and add glyphosate with a concentration of 0-3 μM respectively to form a series of samples, and then perform fluorescence detection on the obtained series of samples, and the obtained fluorescence spectrum is shown in Figure 3 It can be seen from Figure 3 that the linear range of silver nanoclusters for the detection of glyphosate is wide (0-3 μM), and the detection limit is as low as 21 nM.

[0089] Take F0-F / F0 as the vertical coordinate, and take the concentration of glyphosate in the series of samples as the horizontal coordinate, perform linear fitting, and obtain a standard curve, specifically Y=0.334X-0.013; wherein X is the concentration of glyphosate, and Y is F0-F / F0, and the results are shown in Figure 4 It can be seen from Figure 4 that silver nanoclusters have high sensitivity for the detection of glyphosate, and are suitable for the qualitative and quantitative detection of trace glyphosate.

[0090] Example 3 Time response and selectivity of silver nanoclusters for the detection of glyphosate

[0091] Take 1.2 mg / mL silver nanoclusters aqueous solution in a cuvette, add 3 μM glyphosate, and observe the fluorescence intensity of the silver nanoclusters and glyphosate reaction system changing with time, and the results are shown in Figure 5 It can be seen from Figure 5 that the reaction time of silver nanoclusters for the detection of glyphosate is short, and the reaction reaches equilibrium within 30 s, which is conducive to realizing the rapid and ultra-sensitive detection of glyphosate in a complex sample system.

[0092] A 1.2 mg / mL aqueous solution of silver nanoclusters was placed in a cuvette, and 10 μM of common biological interfering agents (potassium ions, sodium ions, calcium ions, silver ions, sulfate ions, nitrate ions, and ascorbic acid) and various organophosphorus pesticides (glyphosate, malathion, glufosinate, parathion, methamidophos, dichlorvos, chlorpyrifos, and atrazine) were added sequentially. The mixture was reacted at room temperature for 1 min to obtain the interfering solution. The fluorescence emission spectrum of the interfering solution was measured under 515 nm excitation light, and the results are as follows. Figure 6 As shown. From Figure 6 It can be seen that the fluorescence of silver nanoclusters was significantly weakened after the addition of glyphosate to the aqueous solution, while the addition of other interfering substances and organophosphorus pesticides did not cause significant fluorescence changes, indicating that silver nanoclusters have excellent selective recognition ability for glyphosate.

[0093] Example 4: Detection and Imaging Application of Glyphosate in Lettuce Root Tip Tissue

[0094] Place qualitative filter paper in a 9cm diameter petri dish, add 2mL of distilled water to soak it, transfer lettuce seeds into the petri dish, and incubate at 25℃ in an incubator. After 48 hours, take lettuce seedlings of similar size for the next experiment.

[0095] In a petri dish containing lettuce seedlings, 1 mL of a 1.2 g / L aqueous solution of silver nanoclusters was added and incubated at room temperature for 30 min. The lettuce seedlings were then washed with PBS buffer to remove any remaining silver nanoclusters from their surface. Next, 0–5 μM glyphosate solutions were added and incubated for 30 min each time. The lettuce seedlings were then observed under a confocal laser scanning microscope (Olympus Fluoview 1000). When the lettuce seedlings were treated solely with the silver nanocluster solution, a distinct bright red fluorescence was observed at the roots. Figure 7 (A and E in the diagram). Observe lettuce seedling root tip tissue cells under a high-power objective lens (A and E in the diagram). Figure 7 In the B and F groups, root tip cells, including the nuclei, were uniformly stained by silver nanoclusters, indicating that the silver nanoclusters were first absorbed by the root tip cells of lettuce seedlings via endocytosis and then further transported to other parts of the lettuce seedling root. With increasing glyphosate concentrations (0, 3 μM, 5 μM), the fluorescence of the lettuce seedling root tip tissue gradually decreased. Figure 7 (B, C, and D in the text). This indicates that silver nanoclusters entering the plant can interact with glyphosate to generate non-fluorescent substances. Silver nanoclusters can achieve real-time, in-situ, and dynamic detection and imaging of glyphosate residues at the cellular level in living plant tissues. Simultaneously, as... Figure 7 As shown in E, F, G, and H, the bright-field cell assays demonstrated that the plant cells maintained good cell morphology throughout the probe incubation and glyphosate detection processes, indicating that the silver nanoclusters possess good biocompatibility and low cytotoxicity.

[0096] Quantitative detection of glyphosate in lettuce seedlings

[0097] In order to investigate the potential application of silver nanoclusters in the detection of pesticide residues in actual vegetable samples, the laboratory-cultivated lettuce seedling (soilless cultivation for 48 h) sample was pretreated: 5.0 g of the cultivated lettuce seedling sample was added into 25 mL of methanol, and the lettuce seedling was ultrasonically extracted twice (the power of ultrasonic was 100 W) for 20 min each time. The extraction solutions were combined and centrifuged at 10000 rpm for 10 min, and the supernatant was collected. Subsequently, the supernatant was filtered with a 0.22 μm membrane to remove impurities, and then the filtrate was rotary evaporated to dryness, and was dissolved with 10 mL of a glyphosate solution to prepare glyphosate solutions with concentrations of 0.5 μM, 1.0 μM, 1.5 μM, 2.0 μM and 3.0 μM, respectively.

[0098] The silver nanocluster aqueous solution prepared in Example 1 was used, and the above glyphosate solutions with different concentrations were added into 1.2 g / L of the silver nanocluster aqueous solution. Under the action of excitation light at 515 nm, the fluorescence emission peak intensity value of the detection system at 605 nm was measured, and was brought into the following equation:

[0099] Y = 0.334X - 0.013;

[0100] Wherein: X is the concentration of glyphosate, and Y is F0-F / F0.

[0101] The concentration of the glyphosate solution to be detected was calculated, and the results are shown in Table 1.

[0102] Table 1: Detection of glyphosate in actual lettuce seedling samples by silver nanoclusters

[0103]

[0104]

[0105] As can be seen from Table 1, the recovery rate of glyphosate in the actual lettuce seedling sample detected by standard addition was 90.1% to 103.8%, and the relative standard deviation was 0.56% to 3.93%. The measured glyphosate concentration had a very small error with the corresponding standard addition concentration. This result shows that the silver nanoclusters provided by the present application have good accuracy in detecting glyphosate in actual vegetable samples, and can quantitatively detect glyphosate in the range of 0 to 3 μM, and have good practical performance.

[0106] The above only describes the preferred embodiments of the present application, and it should be noted that those skilled in the art can make several improvements and refinements without departing from the principles of the present application, and these improvements and refinements should also be considered as the protection scope of the present application.

Claims

1. A method for detecting glyphosate in a plant, characterized by, The method comprises the following steps: alcohol extraction is performed on the plant to be tested by using an alcohol solvent to obtain an alcohol extract; silver nanoclusters are added into the alcohol extract to obtain a sample to be tested; fluorescence detection is performed on the sample to be tested to obtain a fluorescence value of the plant to be tested; the fluorescence value of the plant to be tested is substituted into a predetermined standard curve to obtain a concentration of glyphosate in the plant to be tested; the ordinate of the predetermined standard curve is F0-F / F0, and the abscissa is the concentration of glyphosate; F0 is a fluorescence value of the sample obtained when the concentration of glyphosate is 0; F is a fluorescence value of the sample obtained when the concentration of glyphosate is not 0; the preparation method of the silver nanoclusters comprises the following steps: a precursor is obtained by mixing a polymer, a soluble silver salt and water, and then performing incubation and ultraviolet lamp irradiation in sequence; the precursor is subjected to dialysis and solvent removal in sequence to obtain the silver nanoclusters; the polymer is polymethacrylic acid.

2. The detection method according to claim 1, characterized in that, The silver nanoclusters are used in the form of a silver nanocluster aqueous solution; the concentration of the silver nanocluster aqueous solution is 10-30 g / L.

3. The method of claim 1, wherein The amount ratio of the polymer to the soluble silver salt is 0.6 g:0.001-0.003 mol.

4. The method of claim 1, wherein The incubation is performed under the condition of a constant-temperature water bath, the temperature of the constant-temperature water bath is 25-30 ℃, and the time is 10-20 min; the wavelength of the ultraviolet lamp for the ultraviolet lamp irradiation is 365 nm; the ultraviolet lamp irradiation is performed in the mode of stopping for 20-30 min every 1 h of irradiation; and the total time of the ultraviolet lamp irradiation is 3-4.5 h.

5. The method of claim 1, wherein The cut-off molecular weight of the dialysis bag for the dialysis is 8000-14000 Da.

6. The method of claim 1, wherein The alcohol solvent comprises methanol; the amount ratio of the alcohol solvent to the plant to be tested is 10-25 mL:5.0 g; and the alcohol extraction is performed in the mode of ultrasonic extraction, and the ultrasonic extraction is performed twice, and the time of each ultrasonic extraction is 20 min.

7. The method of claim 1, wherein, The excitation light wavelength of the fluorescence detection is 515 nm. The fluorescence value of the plant to be tested is a fluorescence value at 605 nm.

8. A method for dynamic monitoring of glyphosate in a plant, characterized in that, The method comprises the following steps: a plant is subjected to first incubation and second incubation in a silver nanocluster aqueous solution and a glyphosate aqueous solution in sequence to obtain an incubated plant; dynamic monitoring is performed on the incubated plant; the preparation method of the silver nanoclusters comprises the following steps: a precursor is obtained by mixing a polymer, a soluble silver salt and water, and then performing incubation and ultraviolet lamp irradiation in sequence; the precursor is subjected to dialysis and solvent removal in sequence to obtain the silver nanoclusters; the polymer is polymethacrylic acid.

Images