A Fluorescent Sensing System and Its Application in the Detection of Pyrethroid Pesticides

Abstract

The present invention discloses a fluorescence sensing system, which includes serum protein and a probe. The structural formula of the probe is that the serum protein can catalyze the hydrolysis of the lactone bond of the probe and has an inhibitory effect on the twisted intramolecular charge transfer effect of the hydrolyzed probe. Furthermore, a significant fluorescence emission peak appears at 510 nm in the fluorescence sensing system. Pyrethroid pesticides can reduce the fluorescence intensity at 510 nm in the system, showing a significant inhibitory effect on the fluorescence effect of the fluorescence sensing system, while organophosphorus and carbamate pesticides have no significant inhibitory effect on the fluorescence effect of this fluorescence sensing system. The fluorescence sensing system provided by the present invention can specifically recognize pyrethroid pesticides. Using this fluorescence sensing system to detect pyrethroids does not require large-scale instruments, has simple operation and high efficiency, and can achieve on-site batch rapid detection of pyrethroid pesticide residues.

Description

Technical Field

[0001] The present invention relates to the technical field of pesticide residue detection, and particularly relates to a fluorescence sensing system and its application in the detection of pyrethroid pesticides. Technical Background

[0002] Pesticides have made great contributions to the improvement of the quality of fruits and vegetables, but excessive pesticide residues seriously endanger human health and ecological balance. Currently, relatively mature pesticide residue detection methods include gas chromatography, liquid chromatography, gas chromatography-mass spectrometry, liquid chromatography-mass spectrometry, etc. However, these methods all require expensive large-scale instruments and professional operators, and cannot achieve on-site detection. The rapid pesticide residue detection method widely circulated in the market is the rapid test card method (paper strip method), and its detection principle is as follows: cholinesterase can catalyze the hydrolysis of indophenol acetate (red) into acetic acid and indophenol (blue). Organophosphorus or carbamate pesticides have an inhibitory effect on cholinesterase, changing the process of catalysis, hydrolysis, and color change, thereby judging whether there are high-dose organophosphorus or carbamate pesticides in the sample; this method can achieve on-site inspection, but the cholinesterase used in this method is expensive and has harsh storage conditions, and this method can only detect organophosphorus and carbamate pesticides, and has a poor response effect on pyrethroid pesticides. However, compared with organophosphorus and carbamate pesticides, pyrethroid pesticides are less toxic and pose less harm to the environment, animals, and humans, and are widely used in the growth process of crops. Therefore, developing a new method for specific and rapid detection of pyrethroid pesticides can make up for the deficiencies in the current field of rapid pesticide residue detection, provide a more comprehensive reference basis for the market supervision of pesticide residues, and more effectively protect human health and the ecological environment. Summary of the Invention

[0003] The present invention proposes a fluorescence sensing system, which can be used for specific and rapid detection of pyrethroid pesticides. Using this fluorescence sensing system to detect pyrethroid pesticides is simple in operation, strong in specificity, high in sensitivity, not limited by instruments and professionals, and can achieve on-site and high-throughput rapid detection of pyrethroid pesticides.

[0004] The technical solution to achieve the present invention is as follows:

[0005] A fluorescence sensing system, comprising serum protein and a probe, and the structural formula of the probe is

[0006] The serum protein is human serum albumin (HSA) or bovine serum albumin (BSA).

[0007] Application of the fluorescence sensing system in the detection of pyrethroid pesticides. The probe itself has no obvious fluorescence at 510 nm. Serum protein in the fluorescence sensing system can catalyze the hydrolysis of the lactone bond of the probe and has an inhibitory effect on the twisted intramolecular charge transfer effect of the hydrolyzed probe, thereby causing significant fluorescence to appear in the system at 510 nm. Pyrethroids have an inhibitory effect on serum protein, causing changes in catalysis, hydrolysis, and the fluorescence intensity of the system at 510 nm. Therefore, whether pyrethroids exist in the sample can be judged according to the inhibition rate of the fluorescence effect of the sample on the system.

[0008] The inhibition efficiency of the fluorescence effect is calculated by the following formula:

[0009] Inhibition efficiency(%)=(F0 - F S ) / F0×100%

[0010] Wherein, F0 is the fluorescence emission intensity of the blank group solution without pyrethroids at 510 nm; F s is the fluorescence emission intensity of each solution in the test group at 510 nm.

[0011] The fluorescence response mechanism of the fluorescence sensing system for detecting pyrethroids is as follows:

[0012]

[0013] The method for detecting pyrethroids by the fluorescence sensing system in the above application includes the following steps:

[0014] (1) Prepare a phosphate buffer solution with a concentration of 10.0 mM and a pH of 7.0, and dissolve the probe in DMSO to prepare a probe stock solution with a concentration of 2 mM;

[0015] (2) Take the phosphate buffer solution, add serum protein and the probe stock solution to the phosphate buffer solution so that the final concentrations of serum protein and the probe are both 5.0 μM, incubate at 37 °C. When the serum protein is HSA, the incubation time is 35 min; when the serum protein is BSA, the incubation time is 20 min. After the incubation is completed, measure the fluorescence emission spectrum of the solution under the excitation light of 425 nm;

[0016] (3) Take phosphate buffer, add serum protein to the phosphate buffer, and then add pyrethroid pesticides at different concentrations and incubate them once at 37°C for 15 min. Then add the probe stock solution to make the final concentrations of both serum protein and the probe 5.0 μM, and incubate them a second time at 37°C. When the serum protein is HSA, the second incubation time is 35 min; when the serum protein is BSA, the second incubation time is 20 min. After the incubation is completed, measure the fluorescence emission spectra of each solution under an excitation light of 425 nm.

[0017] (4) Observe the change in fluorescence intensity of the system before and after the action of pyrethroid pesticides, and calculate the fluorescence effect inhibition rate of the pesticides on the system in step (3).

[0018] Change in fluorescence intensity: After the probe is incubated with serum protein, significant fluorescence appears at 510 nm. When the serum protein is first incubated with pyrethroid pesticides and then with the probe, the fluorescence intensity of the system at 510 nm decreases. Pyrethroid pesticides have a significant inhibitory effect on the fluorescence sensing system, and the ratio I of the fluorescence intensity of the solution at 650 nm to that at 510 nm 650nm / I 510nm has a linear relationship with the concentration of pyrethroids.

[0019] The detection object of the above application is lake water or agricultural products.

[0020] The beneficial effects of the present invention are as follows:

[0021] (1) The fluorescence sensing system provided by the present invention can specifically recognize pyrethroids, making up for the deficiency of existing rapid detection techniques that can only recognize organophosphorus and carbamate pesticides.

[0022] (2) The present invention uses low-cost serum protein and fluorescence probes to replace the cholinesterase used in traditional rapid detection of pesticide residues, with lower costs, more convenient storage, and simpler operation.

[0023] (3) The present invention does not require expensive instruments, is not affected by the professional knowledge of the detection personnel, and is more popularizable. Description of the Drawings

[0024] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for use in the description of the embodiments or the prior art. Obviously, the following drawings are only some embodiments of the present invention. For those of ordinary skill in the art, other drawings can be obtained based on these drawings without creative efforts.

[0025] Figure 1Bar chart of the inhibitory effect of pesticides on the fluorescence effect of the fluorescence sensing system; (a) Bar chart of the inhibitory effect of the fluorescence effect of the system when the serum protein is HSA; (b) Bar chart of the inhibitory effect of the fluorescence effect of the system when the serum protein is BSA.

[0026] Figure 2 When the serum protein is HSA, the fluorescence emission spectra of the system before and after the action with pyrethroid pesticides at different concentrations and the linear relationship diagram of I 650nm / I 510nm and the pyrethroid concentration.

[0027] Figure 3 When the serum protein is BSA, the fluorescence emission spectra of the system before and after the action with pyrethroid pesticides at different concentrations and the linear relationship diagram of I 650nm / I 510nm and the pyrethroid concentration. Detailed implementation manners

[0028] Next, the technical solutions of the present invention will be clearly and completely described in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all of the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0029] Example 1 Inhibitory effect of different types of pesticides on the fluorescence effect of the fluorescence sensing system

[0030] (1) Prepare a phosphate buffer solution with a concentration of 10.0 mM and a pH of 7.0, and dissolve the probe in DMSO to prepare a probe stock solution with a concentration of 2 mM; the probe is obtained by the method described in the paper (Chen, Jianhong, et al. "Coumarin-and rhodamine-fused deep red fluorescent dyes: synthesis, photophysical properties, and bioimaging in vitro." Journal of Organic Chemistry 78.12 (2013): 6121-6130).

[0031] (2) Fluorescence spectrum determination of the blank group: Take phosphate buffer, add serum protein and probe stock solution to the phosphate buffer, the final volume of the solution is 2 mL, and the concentrations of serum protein and probe in the final solution are both 5.0 μM. Incubate at 37 °C. When the serum protein is HSA, the incubation time is 35 min; when the serum protein is BSA, the incubation time is 20 min. After incubation, measure the fluorescence emission spectrum of the solution under the excitation light of 425 nm.

[0032] (3) Fluorescence spectrum determination of the test group: Take phosphate buffer, add serum protein to the phosphate buffer, and then add pesticides with different concentrations respectively. Incubate once at 37 °C, and the incubation time for one time is 15 min. Add probe stock solution to the solution after one incubation, and incubate twice at 37 °C. When the serum protein is HSA, the incubation time for the second time is 35 min; when the serum protein is BSA, the incubation time for the second time is 20 min. The final volume of the system solution is 2 mL, and the concentrations of serum protein and probe in the final solution are both 5.0 μM. The final concentrations of pesticides are 1 μM, 2 μM, 3 μM, 4 μM and 5 μM respectively. After the second incubation, measure the fluorescence emission spectra of each solution under the excitation light of 425 nm.

[0033] Among them, the pesticides include organophosphorus pesticides, carbamate pesticides and pyrethroid pesticides; the organophosphorus pesticides are represented by Malathion, Dimethoate, Dichlorvos, Phosfolan, Fenitrothion, Famphur; the carbamate pesticides are represented by Methomyl, Propoxur, Isoprocarb, Carbofuran, Carbaryl, Chlorpropham; the pyrethroid pesticides are represented by Flucythrinate, Cypermethrin, Deltamethrin, Fenvalerate, Flumethrin, Tau-fluvalinate.

[0034] (4) Calculate the inhibition rate (Inhibition efficiency) of the fluorescence effect of pesticides on the system in each solution of the test group. The calculation formula is as follows: Inhibition efficiency (%) = (F0 - F S ) / F0 × 100%

[0035] Among them, F0 is the fluorescence emission intensity of the blank group solution at 510 nm; Fs To test the fluorescence emission intensity of each solution in the test group at 510 nm.

[0036] It can be seen from Figure 1 that organophosphorus and carbamate pesticides have no significant inhibitory effect on the fluorescence sensing system, while pyrethroid pesticides have a significant inhibitory effect on the fluorescence sensing system. This indicates that the fluorescence sensing system provided by the present invention can specifically identify pyrethroid pesticides, and whether pyrethroid pesticides exist in the sample can be quickly judged through the fluorescence effect inhibition rate, making up for the deficiency of only being able to identify organophosphorus and carbamate pesticides in the current rapid detection methods, perfecting the defects in the field of pesticide rapid detection, and providing a more comprehensive supervision basis for relevant regulatory departments.

[0037] I of the fluorescence sensing system in Example 2 650nm / I 510nm Linear relationship with pyrethroid pesticides

[0038] In a phosphate buffer solution with a concentration of 10.0 mM and a pH of 7.0, serum protein and pyrethroid pesticides with different concentrations were added, incubated at 37 °C for 15 min once, and then the probe stock solution was added to make the final concentrations of serum protein and the probe both 5.0 μM, and incubated at 37 °C for the second time. When the serum protein is HSA, the second incubation time is 35 min, and when the serum protein is BSA, the second incubation time is 20 min. After the second incubation, the fluorescence emission spectra of each solution were measured under an excitation light of 425 nm. With the concentration of pyrethroid pesticides as the abscissa and the ratio I of the fluorescence emission intensities of the solution at 650 nm and 510 nm after the second incubation 650nm / I 510nm as the ordinate, a linear relationship graph was plotted.

[0039] Figures 2-3 are the fluorescence spectra of the fluorescence sensing system before and after interacting with pyrethroid pesticides with different concentrations and the linear relationship graph of I 650nm / I 510nm and the concentration of pyrethroid pesticides; Table 1 summarizes the relevant parameters of the interaction between the fluorescence sensing system and pyrethroid pesticides. Within a certain range, the fluorescence sensing system has a good linear relationship with various pyrethroid pesticides, and the detection limit ranges from 0.009 to 0.107 mg / L, indicating that the fluorescence sensing system designed by the present invention has high sensitivity to pyrethroid pesticides.

[0040] Table 1 Relevant parameters of the interaction between the fluorescence sensing system and pyrethroid pesticides

[0041]

[0042] Note: Y in Table 1 represents I650nm / I 510nm , where X represents the concentration of pyrethroid pesticides.

[0043] Application of the fluorescence sensing system in detecting pyrethroid pesticides in lake water and agricultural products The content of cypermethrin in lake water and lettuce was detected by the fluorescence sensing system constructed with HSA and the probe.

[0044] (a) Preparation of the test solution

[0045] Lake water test solution: Take the lake water of Zhengda Meihu, filter it through a 0.22 μm filter membrane, and adjust the pH to 7 with HCL or NaOH solution to obtain the test solution of lake water;

[0046] Lettuce test solution: Crush 25 g of lettuce and soak it in 200 mL of phosphate buffer (10 mM, pH = 7.0), ultrasonically extract for 10 min, filter to remove the residue with a 0.22 μm filter membrane, heat the filtrate in a water bath at 95 °C for 10 min, and cool to obtain the test solution.

[0047] (b) Testing

[0048] Take the above test solutions, add HSA to each test solution, incubate at 37 °C for 15 min, then add the probe stock solution, and continue to incubate at 37 °C for 35 min. The concentrations of serum protein and the probe in the final solution are both 5.0 μM. Measure the fluorescence emission spectra of each solution under an excitation light of 425 nm. No pyrethroid pesticides were detected in the lake water test solution and the lettuce test solution.

[0049] (c) Standard addition recovery experiment

[0050] Take the above test solutions, add different amounts of known-concentration cypermethrin stock solutions (the cypermethrin stock solution is a DMSO solution of cypermethrin. The concentration increments of cypermethrin in the lake water test solution are 2.0 μM and 3.0 μM respectively, and the concentration increments of cypermethrin in the lettuce test solution are 0.5 μM and 1.0 μM respectively) to each test solution, add HSA and incubate at 37 °C for 15 min, then add the probe stock solution, and continue to incubate at 37 °C for 35 min. The concentrations of serum protein and the probe in the final solution are both 5.0 μM. Measure the fluorescence emission spectra of each solution under an excitation light of 425 nm, and calculate the standard addition recovery rates of cypermethrin in the lake water test solution and the lettuce test solution. The results are shown in Table 2.

[0051] As can be seen from Table 2, the fluorescence sensing system constructed with HSA and the probe has good standard addition recovery rates for cypermethrin in the lake water and lettuce extracts, indicating that the fluorescence sensing system designed in the present invention can be used for the accurate and rapid detection of pyrethroid pesticides in actual samples such as the environment and agricultural products, and is expected to be popularized and applied in practical applications.

[0052] Table 2 Spike recovery rates of pyrethroids in lake water and lettuce

[0053]

[0054] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principles of the present invention shall be included within the protection scope of the present invention.

Claims

1. Application of a fluorescence sensing system in the detection of pyrethroid pesticides, characterized in that, Comprising a serum protein and a probe, the structural formula of the probe is .

2. Use of the fluorescence sensing system according to claim 1 in the detection of pyrethroid pesticides, characterized in that, The detection method includes the following steps: (1) Prepare a phosphate buffer solution and a probe stock solution; (2) Take the phosphate buffer solution, add serum protein and the probe stock solution thereto for incubation. After the incubation ends, measure the fluorescence emission spectrum of the solution under an excitation light of 425 nm; (3) Take the phosphate buffer solution, add serum protein thereto, then add pyrethroid pesticides with different concentrations for a primary incubation, and then add the probe stock solution for a secondary incubation. After the incubation ends, measure the fluorescence emission spectra of the respective solutions under an excitation light of 425 nm; (4) Observe the change in the fluorescence intensity of the system before and after the action of pyrethroid pesticides.

3. Use of the fluorescence sensing system according to claim 2 in the detection of pyrethroid pesticides, characterized in that, In the step (1), the concentration of the phosphate buffer solution is 10.0 mM and the pH is 7.0; the probe stock solution is a 2 mM DMSO solution.

4. Use of the fluorescence sensing system according to claim 2 in the detection of pyrethroid pesticides, characterized in that, In the step (2), the final concentrations of the serum protein and the probe are both 5.0 μM; in the step (3), the final concentrations of the serum protein and the probe are both 5.0 μM.

5. Use of the fluorescence sensing system according to claim 2 in the detection of pyrethroid pesticides, characterized in that, The serum protein in the step (2) and the step (3) is human serum protein; the incubation temperature in the step (2) is 37 °C and the incubation time is 35 min; the primary incubation temperature and the secondary incubation temperature in the step (3) are both 37 °C, the primary incubation time is 15 min, and the secondary incubation time is 35 min.

6. Use of the fluorescence sensing system according to claim 2 in the detection of pyrethroid pesticides, characterized in that, The serum protein in the step (2) and the step (3) is bovine serum protein; the incubation temperature in the step (2) is 37 °C and the incubation time is 20 min; the primary incubation temperature and the secondary incubation temperature in the step (3) are both 37 °C, the primary incubation time is 15 min, and the secondary incubation time is 20 min.

7. Use of the fluorescence sensing system according to claim 2 in the detection of pyrethroid pesticides, characterized in that, After the probe was incubated with serum protein, significant fluorescence appeared at 510 nm; after the serum protein was incubated with pyrethroid pesticides and then with the probe, the fluorescence intensity of the system at 510 nm decreased, and the ratio I 650nm / I 510nm of the fluorescence intensity at 650 nm to the fluorescence intensity at 510 nm of the solution was linearly related to the concentration of pyrethroid.

8. Use of the fluorescence sensing system according to claim 2 in the detection of pyrethroid pesticides, characterized in that, The detection object is lake water or agricultural products.

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