A fluorescent biopolysaccharide for in-situ fluorescence detection of nano-pesticides and its detection method

Abstract

The present invention discloses a fluorescent biopolysaccharide for in-situ fluorescence detection of nano-pesticides and its detection method. The fluorescent biopolysaccharide for in-situ fluorescence detection of nano-pesticides uses sodium alginate as a raw material, fluorescamine as a fluorescent agent, and tetrahydrofuran as a solvent to synthesize fluorescamine-labeled oxidized sodium alginate (FLA@OSA). Chitosan is used as a raw material, fluorescein isothiocyanate as a fluorescent agent, and methanol as a solvent to synthesize fluorescein isothiocyanate-labeled chitosan (FITC@Ch). By utilizing the luminescent properties of the materials and using them as fluorescent detection agents, in-situ detection of pesticide residues in the environment is realized, with advantages such as simple synthesis process, mild conditions, low cost, green environmental protection, and rapid and efficient in-situ detection.

Description

Technical Field

[0001] The present invention relates to a method for detecting nano-pesticides, specifically a method for in-situ fluorescence detection of nano-pesticides using fluorescein-labeled biological polysaccharides. Background Art

[0002] In the process of the development of Chinese planting industry, due to the convenience, relatively low cost and good control effect of chemical pesticides, which can meet the needs of farmers for pest control and increasing production and income, their use scope is relatively wide. Especially in large-scale grain production operations, the dependence on pesticides is even higher. However, when pesticides are used in large quantities for a long time, a part of them will enter the soil, water and atmosphere, destroying the ecological environment and causing air pollution and water pollution. Pesticide residues are the most common pollutants in the environment, which can cause water, soil and air pollution, and can also accumulate in organisms such as humans and animals.

[0003] Since pesticide residues have seriously affected the environment and human health, the detection of pesticide residues is an important guarantee to ensure the safety of agricultural products. Conventional pesticide detection methods include chromatographic analysis, enzyme-linked immunosorbent assay, capillary electrophoresis, molecular imprinting and electrochemical methods, etc. However, chromatographic methods have defects such as expensive equipment, high requirements for technical personnel, complicated experimental processes, and inconvenience for on-site detection. Moreover, the thermal instability of some pesticides or the non-volatile properties of some pesticides make it impossible to directly use this method, which limits the application of chromatographic methods in pesticide residue detection. Although enzyme-linked immunosorbent assay has advantages such as simple operation, fast response, high detection sensitivity, good selectivity and no need for complicated sample treatment processes, the key is that some biological factors such as the properties of antibodies are prone to change, and some antibodies are not suitable for labeling, are greatly affected by reaction conditions, have poor tolerance, and may also have cross-reactions. In addition, it is also limited by the lack of current agricultural residue antibody types and the difficulty in preparation, and its application is greatly restricted. Although electrochemical methods have many advantages, due to the lack of electrochemical activity of many pesticides, the application of ordinary electrochemical sensors is hindered. Molecular imprinting technology has advantages such as high stability, simplicity and low cost. However, there are also some defects. Generally, there are problems with uncontrollable sizes of nano-materials. Membrane electrodes often fall off, and there are generally problems with stability. Therefore, the nano-molecular sizes and molecular imprinting membranes in molecular imprinting technology also have this problem. Conventional pesticide detection methods cannot achieve specific detection and cannot be detected online.

[0004] Therefore, the current methods for the identification and quantitative analysis of pesticide residues are still very limited. In order to reduce the food safety and ecological environment problems caused by excessive pesticide residues and to meet the needs of the high-quality development of China's agricultural economy, there is an urgent need for a more mature and perfect pesticide detection system. Therefore, it is of great significance to establish an effective method for detecting pesticide residues.

[0005] Therefore, it is of great practical significance and application prospects to create a more selective, efficient, sensitive and reliable pesticide residue detection method. Summary of the Invention

[0006] Aiming at the above deficiencies, the first object of the present invention is to provide a nano-pesticide in-situ fluorescence detection method that is efficient, rapid, easy to operate, and environmentally friendly.

[0007] The second object of the present invention is to provide two kinds of fluorescent biopolysaccharides with rich substrate resources, low cost, easy availability and environmental friendliness for nano-pesticide in-situ fluorescence detection.

[0008] In order to achieve the above object, the first technical solution adopted by the present invention is:

[0009] A nano-pesticide in-situ fluorescence detection method, successively including the following steps:

[0010] 1) Dissolve the fluorescent biopolysaccharide for nano-pesticide in-situ fluorescence detection in a dispersant to obtain a blank sample suspension;

[0011] 2) Take the blank sample suspension and place it in a four-way fluorescence cuvette. Detect its emission spectrum under the conditions of excitation wavelength 300 - 550 nm, emission wavelength 480 - 600 nm, slit width 1 - 4 nm, and fluorescence peak wavelength 300 - 600 nm to obtain the fluorescence intensity of the blank sample;

[0012] 3) Then add the sample to be tested to the four-way fluorescence cuvette containing the blank sample suspension in step 2). After ultrasonic treatment, test its emission spectrum under the same conditions as in step 2) to obtain the fluorescence intensity after adding the sample to be tested;

[0013] 4) Calculate the fluorescence quenching rate

[0014] Where: I0 and I are the fluorescence intensity of the blank sample and the fluorescence intensity after adding nano-pesticide respectively.

[0015] Furthermore, in the above-mentioned nano-pesticide in-situ fluorescence detection method, the dispersant in step 1) is one of water, ethanol, methanol, dichloromethane, acetonitrile, and ethyl acetate.

[0016] Furthermore, in the above-mentioned nano-pesticide in-situ fluorescence detection method, the test conditions of the fluorescence spectrometer in step 2) are: excitation wavelength 450 - 550 nm, emission wavelength 480 - 600 nm, slit width 1 - 4 nm, and fluorescence peak wavelength 300 - 600 nm.

[0017] Further, in the above-mentioned method for in-situ fluorescence detection of nano-pesticides, the volume ratio of the blank sample suspension to the mass of the sample to be measured is 1:5 to 5:1 (mg / mL).

[0018] Further, in the above-mentioned method for in-situ fluorescence detection of nano-pesticides, the concentration of the blank sample suspension is 0.2 to 1 mg / mL.

[0019] Further, in the above-mentioned method for in-situ fluorescence detection of nano-pesticides, the biological polysaccharide used for in-situ fluorescence detection of nano-pesticides is prepared by the following method:

[0020] 1) Dissolve sodium alginate in distilled water, then add sodium periodate and stir for 4 to 8 h at room temperature in the dark;

[0021] 2) After the reaction is completed, add ethylene glycol to terminate the reaction and continue stirring for 0.5 to 1.5 h; finally, add ethanol to obtain a white precipitate, filter and freeze-dry to obtain white powder of oxidized sodium alginate;

[0022] 3) Dissolve the oxidized sodium alginate obtained in step 1) in distilled water, then dissolve fluorescamine in tetrahydrofuran and mix it with the oxidized sodium alginate solution, and stir the mixture at room temperature for 10 to 14 h;

[0023] 4) Remove the tetrahydrofuran solvent by rotary evaporation, and freeze-dry the fluorescamine-labeled sodium alginate to obtain a red powder, which is fluorescamine-labeled oxidized sodium alginate.

[0024] Further, in the above-mentioned method for in-situ fluorescence detection of nano-pesticides, the biological polysaccharide used for in-situ fluorescence detection of nano-pesticides is prepared by the following method:

[0025] 1) Add 5 to 15 mL of anhydrous methanol to 15 to 25 mL of 0.1 mol / L acetic acid aqueous solution containing 400 to 600 mg of chitosan and stir to dissolve;

[0026] 2) Add 4 to 6 mL of 1.5 to 2.5 mg / mL fluorescein isothiocyanate methanol solution and stir and react at room temperature in the dark for 2 to 6 h to make the amino group on chitosan react with the isothiocyanate group on FITC to form a thiourea bond;

[0027] 3) Add 50 to 150 mL of 0.15 to 0.25 mol / L NaOH solution to precipitate the labeled product, centrifuge the mixture for 12 to 18 min, wash the obtained precipitate with deionized water solution for several times until no fluorescence is detected in the supernatant, and collect the precipitate to obtain fluorescein isothiocyanate-labeled chitosan.

[0028] The second technical solution provided by the present invention is two fluorescent biopolysaccharides for in-situ fluorescence detection of nano-pesticides:

[0029] The first one uses sodium alginate as the raw material, fluorescamine as the fluorescent agent, and tetrahydrofuran as the solvent to synthesize fluorescamine-labeled oxidized sodium alginate (FLA@OSA);

[0030] It is specifically prepared by the following method:

[0031] 1) Dissolve sodium alginate in distilled water, then add sodium periodate and stir for 4 - 8 h at room temperature in the dark;

[0032] 2) After the reaction is completed, add ethylene glycol to terminate the reaction and continue stirring for 0.5 - 1.5 h; finally, add ethanol to obtain a white precipitate, filter and freeze-dry to obtain white powder oxidized sodium alginate;

[0033] 3) Dissolve the oxidized sodium alginate obtained in step 1) in distilled water, then dissolve fluorescamine in tetrahydrofuran and mix it with the oxidized sodium alginate solution, and stir the mixture at room temperature for 10 - 14 h;

[0034] 4) Remove the tetrahydrofuran solvent by rotary evaporation, and freeze-dry the fluorescamine-labeled sodium alginate to obtain a red powder, which is fluorescamine-labeled oxidized sodium alginate.

[0035] Its synthesis route is as follows:

[0036]

[0037] The second one uses chitosan as the raw material, fluorescein isothiocyanate as the fluorescent agent, and methanol as the solvent to synthesize fluorescein isothiocyanate-labeled chitosan (FITC@Ch).

[0038] It is specifically prepared by the following steps:

[0039] 1) In 15 - 25 mL of 0.1 mol / L acetic acid aqueous solution containing 400 - 600 mg of chitosan, add 5 - 15 mL of anhydrous methanol and stir to dissolve;

[0040] 2) Add 4 - 6 mL of 1.5 - 2.5 mg / mL fluorescein isothiocyanate methanol solution and stir and react at room temperature in the dark for 2 - 6 h to make the amino group on chitosan react with the isothiocyanate group on FITC to form a thiourea bond;

[0041] 3) Add 50 - 150 mL of a 0.15 - 0.25 mol / L NaOH solution to precipitate the labeled product. Centrifuge the mixture for 12 - 18 min. Wash the obtained precipitate with deionized water several times until no fluorescence is detected in the supernatant. Collect the precipitate to obtain fluorescein isothiocyanate - labeled chitosan.

[0042] The synthesis route is as follows:

[0043]

[0044] Compared with the prior art, the present invention has the following beneficial effects:

[0045] 1. By introducing fluorescamine into the biopolysaccharide sodium alginate, the present invention endows it with luminescent properties, and the synthesis is simple and the conditions are mild.

[0046] 2. The present invention uses the biopolysaccharides sodium alginate and chitosan as raw materials, which are rich in resources, inexpensive, easily available and environmentally friendly.

[0047] 3. Detecting the presence of pesticides by the change in fluorescence intensity is efficient, convenient and has good application prospects. Description of the Drawings

[0048] Figure 1 are the fluorescence emission spectrum and excitation spectrum of the FLA@OSA suspension provided in Example 1;

[0049] Figure 2 are the fluorescence emission spectrum and excitation spectrum of the FITC@Ch suspension provided in Example 2;

[0050] Figure 3 are the fluorescence emission spectra of the FLA@OSA and FLA@OSA + TH aqueous suspensions;

[0051] Figure 4 are the fluorescence emission spectra of the FLA@OSA and FLA@OSA + TH ethanol suspensions provided in Example 4;

[0052] Figure 5 are the fluorescence emission spectra of the FLA@OSA and FLA@OSA + TH methanol suspensions provided in Example 5;

[0053] Figure 6 are the fluorescence emission spectra of the FLA@OSA and FLA@OSA + TH dichloromethane suspensions provided in Example 6;

[0054] Figure 7 are the fluorescence emission spectra of the FLA@OSA and FLA@OSA + TH acetonitrile suspensions provided in Example 7;

[0055] Figure 8It is the fluorescence emission spectra of the FLA@OSA and FLA@OSA+TH ethyl acetate suspensions provided in Example 8;

[0056] Figure 9 It is the standard curve of the FLA@OSA dichloromethane suspension provided in Example 9 within the concentration range of 0.025 - 0.055 mM;

[0057] Figure 10 It is the standard curve of the FLA@OSA dichloromethane suspension provided in Example 10 within the concentration range of 0.06 - 0.10 mM;

[0058] Figure 11 It is the fluorescence emission spectra of the FITC@Ch and FITC@Ch+EB water suspensions provided in Example 12;

[0059] Figure 12 It is the fluorescence emission spectra of the FITC@Ch and FITC@Ch+EB ethanol suspensions provided in Example 13;

[0060] Figure 13 It is the fluorescence emission spectra of the FITC@Ch and FITC@Ch+EB methanol suspensions provided in Example 14;

[0061] Figure 14 It is the fluorescence emission spectra of the FITC@Ch and FITC@Ch+EB dichloromethane suspensions provided in Example 15;

[0062] Figure 15 It is the fluorescence emission spectra of the FITC@Ch and FITC@Ch+EB acetonitrile suspensions provided in Example 16. Detailed implementation manners

[0063] To make the objectives, technical solutions and advantages of the present invention clearer and more understandable, the following preferred embodiments are given to further elaborate on the present invention in detail. However, the protection scope of the present invention is not limited to the scope represented by the embodiments.

[0064] Example 1

[0065] A fluorescent biopolysaccharide for in-situ fluorescence detection of nano-pesticides, which is specifically prepared by the following method:

[0066] 1) Dissolve 2.0 g of sodium alginate in 200 mL of distilled water, then add 2.5 g of sodium periodate, and stir for 6 h at room temperature under lightless conditions;

[0067] 2) After the reaction ended, 3 mL of ethylene glycol was added to terminate the reaction, and stirring continued for 1 h; finally, 300 mL of ethanol was added to obtain a white precipitate. After passing through a 300-mesh filter screen, it was freeze-dried at -58 °C for 24 h to obtain white powdered oxidized sodium alginate;

[0068] 3) Dissolve 600 mg of the oxidized sodium alginate obtained in step 1) in 10 mL of distilled water. Then dissolve 200 mg of fluorescamine in 10 mL of tetrahydrofuran, and mix it with the oxidized sodium alginate solution. The mixture was stirred at room temperature for 12 h;

[0069] 4) Remove the tetrahydrofuran solvent by rotary evaporation (at a temperature of 50 °C). The fluorescamine-labeled sodium alginate was freeze-dried at -58 °C for 24 h to obtain a red powder, denoted as fluorescamine-labeled oxidized sodium alginate (FLA@OSA);

[0070] The fluorescence emission spectrum and excitation spectrum of the FLA@OSA suspension are referred to Figure 1 。

[0071] Its synthesis route is as follows:

[0072]

[0073] Example 2

[0074] Another fluorescent biopolysaccharide for in-situ fluorescence detection of nano-pesticides is specifically prepared by the following method:

[0075] In 20 mL of a 0.1 mol / L acetic acid solution containing 500 mg of chitosan, 10 mL of anhydrous methanol was added and stirred to dissolve. Then 5 mL of a 2 mg / mL fluorescein isothiocyanate (FITC) methanol solution was added. Stir the reaction in the dark at room temperature for 4 h to allow the amino group on chitosan to react with the isothiocyanate group on FITC to form a thiourea bond. Then 100 mL of a 0.2 mol / L NaOH solution was added to precipitate the labeled product (FITC@Ch). The mixture was centrifuged at 8000 r / min for 15 min. The obtained precipitate was washed with deionized water solution, and the washing and centrifugation operations were repeated several times until no fluorescence was detected in the supernatant. The fluorescence emission spectrum and excitation spectrum of the FITC@Ch suspension are referred to Figure 2 。

[0076] Its synthesis route is as follows:

[0077]

[0078] Example 3

[0079] Take 5 mg of the FLA@OSA prepared in Example 1 and add it to 10 mL of water to prepare a 0.5 mg / mL aqueous solution of FLA@OSA. Take 2 mL and place it in a four-way fluorescence cuvette, and use a fluorescence spectrometer to measure its emission spectrum (measurement conditions: λ ex = 350 nm, λ em = 525 nm, slit width = 3.10 nm), to obtain the fluorescence intensity of the blank sample. Then add 1 mg of thiamethoxam (TH), after ultrasonic treatment at 40 KHz for 30 min and then measure its emission spectrum under the same conditions (measurement conditions: λ ex = 350 nm, λ em = 525 nm, slit width = 3.10 nm), to obtain the fluorescence intensity after adding thiamethoxam. By the change in the fluorescence intensity of FLA@OSA at 525 nm before and after adding thiamethoxam, according to the fluorescence quenching rate I0 and I are the fluorescence intensities of the blank sample and the fluorescence intensity after adding thiamethoxam respectively, it can be calculated that the fluorescence quenching rate is 6.620%, indicating that the detection effect of FLA@OSA on thiamethoxam in water is not obvious. The fluorescence emission spectra of FLA@OSA and FLA@OSA+TH aqueous suspensions are referred to Figure 3 .

[0080] Example 4

[0081] Take 5 mg of the FLA@OSA prepared in Example 1 and add it to 10 mL of ethanol to prepare a 0.5 mg / mL ethanol suspension of FLA@OSA. Take 2 mL and place it in a four-way fluorescence cuvette, and use a fluorescence spectrometer to measure its emission spectrum (measurement conditions: λ ex = 350 nm, λ em = 525 nm, slit width = 3.10 nm), to obtain the fluorescence intensity of the blank sample. Then add 1 mg of thiamethoxam (TH), after ultrasonic treatment at 40 KHz for 30 min and then measure its emission spectrum under the same conditions, to obtain the fluorescence intensity after adding thiamethoxam. By the change in the fluorescence intensity of FLA@OSA at 433 nm before and after adding thiamethoxam, according to the fluorescence quenching rate I0 and I are the fluorescence intensities of the blank sample and the fluorescence intensity after adding thiamethoxam respectively, it can be calculated that the fluorescence quenching rate is 35.65%, indicating that the detection effect of FLA@OSA on thiamethoxam in ethanol is relatively obvious. The fluorescence emission spectra of FLA@OSA and FLA@OSA+TH ethanol suspensions are referred to Figure 4 .

[0082] Example 5

[0083] Take 5 mg of FLA@OSA prepared in Example 1 and add it to 10 mL of methanol to prepare a 0.5 mg / mL FLA@OSA methanol suspension. Take 2 mL and place it in a four-way fluorescence cuvette, and use a fluorescence spectrometer to measure its emission spectrum (measurement conditions: λ ex = 350 nm, λ em = 525 nm, slit width = 3.10 nm), and obtain the fluorescence intensity of the blank sample. Then add 1 mg of thiamethoxam (TH), after ultrasonic treatment at 40 KHz for 30 min, measure its emission spectrum under the same conditions again, and obtain the fluorescence intensity after adding thiamethoxam. By the change in the fluorescence intensity of FLA@OSA at 525 nm before and after adding thiamethoxam, according to the fluorescence quenching rate where I0 and I are the fluorescence intensities of the blank sample and the fluorescence intensity after adding thiamethoxam respectively, it can be calculated that the fluorescence quenching rate is 35.81%, indicating that the detection effect of FLA@OSA on thiamethoxam in methanol is relatively obvious. The fluorescence emission spectra of FLA@OSA and FLA@OSA+TH methanol suspensions are shown in Figure 5 .

[0084] Example 6

[0085] Take 5 mg of FLA@OSA prepared in Example 1 and add it to 10 mL of dichloromethane to prepare a 0.5 mg / mL FLA@OSA dichloromethane suspension. Take 2 mL and place it in a four-way fluorescence cuvette, and use a fluorescence spectrometer to measure its emission spectrum (measurement conditions: λ ex = 350 nm, λ em = 525 nm, slit width = 3.10 nm), and obtain the fluorescence intensity of the blank sample. Then add 1 mg of thiamethoxam (TH), after ultrasonic treatment at 40 KHz for 30 min, measure its emission spectrum under the same conditions again, and obtain the fluorescence intensity after adding thiamethoxam. By the change in the fluorescence intensity of FLA@OSA at 408 nm before and after adding thiamethoxam, it can be calculated that the fluorescence quenching rate is 97.01%, indicating that FLA@OSA has a good detection effect on thiamethoxam in dichloromethane. The fluorescence emission spectra of FLA@OSA and FLA@OSA+TH dichloromethane suspensions are shown in Figure 7 .

[0086] Example 7

[0087] Take 5 mg of FLA@OSA prepared in Example 1 and add it to 10 mL of acetonitrile to prepare a 0.5 mg / mL FLA@OSA acetonitrile suspension. Take 2 mL and place it in a four-way fluorescence cuvette, and use a fluorescence spectrometer to measure its emission spectrum (measurement conditions: λ ex = 350 nm, λ em= 525 nm, slit width = 3.10 nm), the fluorescence intensity of the blank sample was obtained. Then 1 mg of thiamethoxam (TH) was added, and after ultrasonic treatment at 40 KHz for 30 min, the emission spectrum was measured under the same conditions to obtain the fluorescence intensity after adding thiamethoxam. Based on the change in the fluorescence intensity of FLA@OSA at 400 nm before and after adding thiamethoxam, according to the fluorescence quenching rate I0 and I are the fluorescence intensities of the blank sample and the sample after adding thiamethoxam, respectively. It can be calculated that the fluorescence quenching rate is 47.83%, indicating that FLA@OSA has a good detection effect on thiamethoxam in acetonitrile. The fluorescence emission spectra of the FLA@OSA and FLA@OSA + TH acetonitrile suspensions are shown in Figure 7 .

[0088] Example 8

[0089] 5 mg of FLA@OSA prepared in Example 1 was added to 10 mL of ethyl acetate to prepare a 0.5 mg / mL FLA@OSA ethyl acetate suspension. 2 mL of it was taken and placed in a four-way fluorescence cuvette, and its emission spectrum was measured using a fluorescence spectrometer (measurement conditions: λ ex = 350 nm, λ em = 525 nm, slit width = 3.10 nm), the fluorescence intensity of the blank sample was obtained. Then 1 mg of thiamethoxam (TH) was added, and after ultrasonic treatment at 40 KHz for 30 min, the emission spectrum was measured under the same conditions to obtain the fluorescence intensity after adding thiamethoxam. Based on the change in the fluorescence intensity of FLA@OSA at 408 nm before and after adding thiamethoxam, according to the fluorescence quenching rate I0 and I are the fluorescence intensities of the blank sample and the sample after adding thiamethoxam, respectively. It can be calculated that the fluorescence quenching rate is 57.40%, indicating that FLA@OSA has a good detection effect on thiamethoxam in ethyl acetate. The fluorescence emission spectra of the FLA@OSA and FLA@OSA + TH ethyl acetate suspensions are shown in Figure 8 .

[0090] Example 9

[0091] 5 mg of FLA@OSA prepared in Example 1 was added to 10 mL of dichloromethane. 2 mL of the 0.5 mg / mL FLA@OSA dichloromethane suspension was taken and placed in a four-way fluorescence cuvette, and its emission spectrum was measured using a fluorescence spectrometer (measurement conditions: λ ex = 350 nm, λ em= 525 nm, slit width = 3.10 nm), the fluorescence intensity of the blank sample was obtained. Then, a 0.005 mol / L thiamethoxam dichloromethane solution was gradually added dropwise. After mixing evenly, its emission spectrum was measured under the same conditions, and the fluorescence intensity of FLA@OSA at different thiamethoxam concentrations was obtained. A standard curve in the concentration range of 0.025 - 0.055 mM could be obtained, y = -1.204×10 7 x + 1.963×10 6 , R 2 = 0.9963. According to the standard curve, the thiamethoxam concentration was quantified by measuring the fluorescence intensity of the unknown solution. The standard curve of the FLA@OSA dichloromethane suspension in the concentration range of 0.025 - 0.055 mM is shown in Figure 9 .

[0092] Example 10

[0093] 5 mg of FLA@OSA prepared in Example 1 was added to 10 mL of dichloromethane. 2 mL of a 0.5 mg / mL FLA@OSA dichloromethane suspension was placed in a four-way fluorescence cuvette, and its emission spectrum was measured using a fluorescence spectrometer (measurement conditions: λ ex = 350 nm, λ em = 525 nm, slit width = 3.10 nm), the fluorescence intensity of the blank sample was obtained. Then, a 0.005 mol / L thiamethoxam dichloromethane solution was gradually added dropwise. After mixing evenly, its emission spectrum was measured under the same conditions, and the fluorescence intensity of FLA@OSA at different thiamethoxam concentrations was obtained. A standard curve in the concentration range of 0.06 - 0.10 mM could be obtained, y = -7.130×10 6 x + 1.754×10 6 , R 2 = 0.9953. According to the standard curve, the thiamethoxam concentration was quantified by measuring the fluorescence intensity of the unknown solution. The standard curve of the FLA@OSA dichloromethane suspension in the concentration range of 0.06 - 0.10 mM is shown in Figure 10 .

[0094] Example 11

[0095] 5 mg of FLA@OSA prepared in Example 1 was added to 10 mL of dichloromethane. 2 mL of a 0.5 mg / mL FLA@OSA dichloromethane suspension was placed in a four-way fluorescence cuvette, and its emission spectrum was measured using a fluorescence spectrometer (measurement conditions: λ ex = 350 nm, λ em(λex = 525 nm, slit width = 3.10 nm), the fluorescence intensity of the blank sample was obtained. Then, a 0.005 mol / L thiamethoxam dichloromethane solution was gradually added dropwise. After mixing evenly, its emission spectrum was measured under the same conditions. In the low concentration range, the detection limit was calculated to be 7.73 μM through the formula.

[0096] Example 12

[0097] 5 mg of FITC@Ch prepared in Example 1 was added to 10 mL of water to prepare a 0.5 mg / mL FITC@Ch water suspension. 2 mL was taken and placed in a four-way fluorescence cuvette. The emission spectrum was measured using a fluorescence spectrometer (measurement conditions: λex = 490 nm, λem = 520 nm, slit width = 1.35 nm) to obtain the fluorescence intensity of the blank sample. Then, 1 mg of emamectin benzoate (EB) was added. After ultrasonic treatment at 40 KHz for 30 min, the emission spectrum was measured under the same conditions to obtain the fluorescence intensity after adding emamectin benzoate. According to the fluorescence quenching rate I0 and I are the fluorescence intensities of the blank sample and the sample after adding thiamethoxam, respectively. After calculation, the fluorescence quenching rate was 11.85%. The fluorescence emission spectra of FITC@Ch and FITC@Ch + EB water suspensions are shown in Figure 11 .

[0098] Example 13

[0099] 5 mg of FITC@Ch prepared in Example 1 was added to 10 mL of ethanol to prepare a 0.5 mg / mL FITC@Ch ethanol suspension. 2 mL was taken and placed in a four-way fluorescence cuvette. The emission spectrum was measured using a fluorescence spectrometer (measurement conditions: λex = 490 nm, λem = 520 nm, slit width = 1.35 nm) to obtain the fluorescence intensity of the blank sample. Then, 1 mg of emamectin benzoate (EB) was added. After ultrasonic treatment at 40 KHz for 30 min, the emission spectrum was measured under the same conditions to obtain the fluorescence intensity after adding emamectin benzoate. After calculation, the fluorescence quenching rate was 5.908%. The fluorescence emission spectra of FITC@Ch and FITC@Ch + EB ethanol suspensions are shown in Figure 12 .

[0100] Example 14

[0101] Take 5 mg of FITC@Ch prepared in Example 1 and add it to 10 mL of methanol to prepare a 0.5 mg / mL FITC@Ch methanol suspension. Take 2 mL and place it in a four-way fluorescence cuvette, and use a fluorescence spectrometer to measure its emission spectrum (measurement conditions: λex = 490 nm, λem = 520 nm, slit width = 1.35 nm) to obtain the fluorescence intensity of the blank sample. Then add 1 mg of emamectin benzoate (EB), and after sonication at 40 KHz for 30 min, measure its emission spectrum under the same conditions to obtain the fluorescence intensity after adding emamectin benzoate. According to the fluorescence quenching rate I0 and I are the fluorescence intensities of the blank sample and the sample after adding thiamethoxam, respectively. After calculation, the fluorescence quenching rate is 19.13%. The fluorescence emission spectra of the FITC@Ch and FITC@Ch+EB methanol suspensions are shown in Figure 13 .

[0102] Example 15

[0103] Take 5 mg of FITC@Ch prepared in Example 1 and add it to 10 mL of dichloromethane to prepare a 0.5 mg / mL FITC@Ch dichloromethane suspension. Take 2 mL and place it in a four-way fluorescence cuvette, and use a fluorescence spectrometer to measure its emission spectrum (measurement conditions: λex = 490 nm, λem = 520 nm, slit width = 1.35 nm) to obtain the fluorescence intensity of the blank sample. Then add 1 mg of emamectin benzoate (EB), and after sonication at 40 KHz for 30 min, measure its emission spectrum under the same conditions to obtain the fluorescence intensity after adding emamectin benzoate. After calculation, the fluorescence quenching rate is 98.97%. The fluorescence emission spectra of the FITC@Ch and FITC@Ch+EB dichloromethane suspensions are shown in Figure 14 .

[0104] Example 16

[0105] Take 5 mg of FITC@Ch prepared in Example 1 and add it to 10 mL of acetonitrile to prepare a 0.5 mg / mL FITC@Ch acetonitrile suspension. Take 2 mL and place it in a four-way fluorescence cuvette, and use a fluorescence spectrometer to measure its emission spectrum (measurement conditions: λex = 490 nm, λem = 520 nm, slit width = 1.35 nm) to obtain the fluorescence intensity of the blank sample. Then add 1 mg of emamectin benzoate (EB), and after sonication at 40 KHz for 30 min, measure its emission spectrum under the same conditions to obtain the fluorescence intensity after adding emamectin benzoate. According to the fluorescence quenching rate I0 and I are the fluorescence intensities of the blank sample and the sample after adding thiamethoxam, respectively. After calculation, the fluorescence quenching rate is 26.24%. The fluorescence emission spectra of the FITC@Ch and FITC@Ch+EB acetonitrile suspensions are shown in Figure 15 .

Claims

1. A method for in-situ fluorescence detection of nano-pesticides, characterized in that, Comprising the following steps in sequence: 1) Dissolve the fluorescent biopolysaccharide for in-situ fluorescence detection of nano-pesticides in a dispersant to obtain a blank sample suspension; 2) Take the blank sample suspension and place it in a four-way fluorescence cuvette. Detect its emission spectrum under the conditions of an excitation wavelength of 300 - 550 nm, an emission wavelength of 480 - 600 nm, a slit width of 1 - 4 nm, and a fluorescence peak wavelength of 300 - 600 nm to obtain the fluorescence intensity of the blank sample; 3) Then add the sample to be tested to the four-way fluorescence cuvette containing the blank sample suspension in step 2). After ultrasonic treatment, test its emission spectrum under the same conditions as in step 2) to obtain the fluorescence intensity after adding the sample to be tested; 4) Calculate the fluorescence quenching rate ; Wherein: I 0 and I are the fluorescence intensities of the blank sample and the fluorescence intensity after adding the nano-pesticide, respectively; The fluorescent biopolysaccharide for in-situ fluorescence detection of nano-pesticides is prepared by the following method: 1) Dissolve sodium alginate in distilled water, then add sodium periodate, and stir for 4 - 8 h at room temperature in the dark; 2) After the reaction is completed, add ethylene glycol to terminate the reaction, and continue to stir for 0.5 - 1.5 h; finally, add ethanol to obtain a white precipitate, filter and freeze-dry to obtain white powder oxidized sodium alginate; 3) Dissolve the oxidized sodium alginate obtained in step 1) in distilled water, then dissolve fluoresceinamine in tetrahydrofuran, and then mix it with the oxidized sodium alginate solution. The mixture is stirred at room temperature for 10 - 14 h; 4) Remove the tetrahydrofuran solvent by rotary evaporation, and freeze-dry the fluoresceinamine-labeled sodium alginate to obtain a red powder, which is fluoresceinamine-labeled oxidized sodium alginate; Wherein: the mass ratio of the sodium alginate to sodium periodate and fluoresceinamine is 2 - 4:1:

1.

2. The in-situ fluorescence detection method for nano-pesticide according to claim 1, wherein The dispersant described in step 1) is one of water, ethanol, methanol, dichloromethane, acetonitrile, and ethyl acetate.

3. A method for in-situ fluorescence detection of nano-pesticides according to claim 1, characterized in that, In the fluorescence spectrometer test conditions in step 2), the excitation wavelength is 450 - 550 nm, the emission wavelength is 480 - 600 nm, the slit width is 1 - 4 nm, and the fluorescence peak wavelength is 300 - 600 nm.

4. A method for in-situ fluorescence detection of nano-pesticides according to claim 1, characterized in that The concentration of the blank sample suspension is 0.2 - 1 mg / mL.

5. A method for in-situ fluorescence detection of nano-pesticides, characterized in that, Comprising the following steps in sequence: 1) Dissolve the fluorescent biopolysaccharide for in-situ fluorescence detection of nano-pesticides in a dispersant to obtain a blank sample suspension; 2) Take the blank sample suspension and place it in a four-way fluorescence cuvette. Detect its emission spectrum under the conditions of an excitation wavelength of 300 - 550 nm, an emission wavelength of 480 - 600 nm, a slit width of 1 - 4 nm, and a fluorescence peak wavelength of 300 - 600 nm to obtain the fluorescence intensity of the blank sample; 3) Then add the sample to be tested to the four-way fluorescence cuvette containing the blank sample suspension in step 2). After ultrasonic treatment, test its emission spectrum under the same conditions as in step 2) to obtain the fluorescence intensity after adding the sample to be tested; 4) Calculate the fluorescence quenching rate ; Wherein: I 0 and I are the fluorescence intensities of the blank sample and the fluorescence intensity after adding the nano-pesticide, respectively; The fluorescent biopolysaccharide for in-situ fluorescence detection of nano-pesticides is prepared by the following method: 1) Add 5 - 15 mL of anhydrous methanol to 15 - 25 mL of 0.1 mol / L acetic acid aqueous solution containing 400 - 600 mg of chitosan, and stir to dissolve; 2) Add 4 - 6 mL of fluorescein isothiocyanate methanol solution with a concentration of 1.5 - 2.5 mg / mL, and stir and react for 2 - 6 h in the dark at room temperature to allow the amino group on chitosan to react with the isothiocyanate group on fluorescein isothiocyanate to form a thiourea bond; 3) Add 50 - 150 mL of NaOH solution with a concentration of 0.15 - 0.25 mol / L to precipitate the labeled product. Centrifuge the mixture for 12 - 18 min, and wash the obtained precipitate with deionized water several times until no fluorescence is detected in the supernatant. Collect the precipitate to obtain fluorescein isothiocyanate-labeled chitosan.

6. A method for in-situ fluorescence detection of nano-pesticides according to claim 5, characterized in that, The dispersant described in step 1) is one of water, ethanol, methanol, dichloromethane, acetonitrile, and ethyl acetate.

7. A method for in-situ fluorescence detection of nano-pesticides according to claim 5, characterized in that, In step 2), the test conditions of the fluorescence spectrometer are as follows: the excitation wavelength is 450 - 550 nm, the emission wavelength is 480 - 600 nm, the slit width is 1 - 4 nm, and the fluorescence peak wavelength is 300 - 600 nm.

8. A method for in-situ fluorescence detection of nano-pesticides according to claim 5, characterized in that The concentration of the blank sample suspension is 0.2 - 1 mg / mL.

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