A fluorescent probe and a preparation method and application thereof

Abstract

The application discloses a fluorescent probe and a preparation method and application thereof, and belongs to the technical field of fluorescent detection and rapid detection of pesticide residues. The fluorescent probe provided by the application takes hydroxyl semi-florocyanine as a fluorescent mother nucleus and acetyl as a recognition site; an intermediate NS-OH is subjected to a substitution reaction with acetyl bromide; the obtained fluorescent probe NS-A can detect acetylcholinesterase and can also be combined with an enzyme inhibition method to detect pesticide residues in traditional Chinese medicinal materials; a standard curve is drawn by a fluorescence spectrum method to quantitatively determine the pesticides, and the result shows that the standard curve error of the pesticide chlorpyrifos is small, the pesticide standard addition recovery rate in the traditional Chinese medicinal materials is between 96.03% and 102.57%, and the detection lower limit of the pesticide can reach the required detection standard; the fluorescent probe has the advantages of low cost, high sensitivity, good selectivity and the like, and has a good application prospect.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of fluorescent detection and rapid detection of pesticide residues, and particularly relates to a fluorescent probe and a preparation method and application thereof. BACKGROUND

[0002] In the cultivation process of traditional Chinese medicinal materials, a large amount of pesticides are usually used to prevent and control various diseases and pests. At present, the most widely used and largest amount of pesticides is organophosphorus pesticides, which are difficult to remove from traditional Chinese medicinal materials through simple treatment, and pose a great hidden danger to the safe use of traditional Chinese medicinal materials. In addition, excessive or non-standard use of pesticides can also lead to serious pesticide residue problems, which not only affect the quality and efficacy of traditional Chinese medicinal materials, but also pose a threat to human health, such as causing cerebrovascular diseases, cardiovascular diseases, and infertility, and teratogenic problems. In order to ensure the safety of traditional Chinese medicinal materials, the 2020 edition of Chinese Pharmacopoeia has made clear regulations on the limits of 33 kinds of prohibited pesticides in plant medicinal materials (not more than the quantitative limit). Therefore, it is of great significance to rapidly, sensitively and accurately detect pesticide residues in traditional Chinese medicinal materials.

[0003] At present, the methods for detecting pesticide residues include high performance liquid chromatography, liquid chromatography-mass spectrometry, gas chromatography, enzyme-linked immunoassay, etc. These methods have accurate detection results, but have high detection cost, long time consumption and require professional technical personnel, which is not conducive to large-scale deployment. Compared with traditional methods, the detection method based on enzyme inhibition principle combined with optical probe technology has the advantages of fast response speed, high sensitivity and good selectivity, and has attracted much attention. Moreover, such optical probes have been successfully developed into a qualitative and quantitative tool for detecting pesticide residues.

[0004] Commonly used pesticide residues (such as organophosphorus pesticides) in traditional Chinese medicinal materials can inhibit the activity of acetylcholinesterase (AChE) under certain conditions, and the inhibition rate is positively correlated with the concentration of pesticides. If there is no or little pesticide residue in the traditional Chinese medicinal material sample, the activity of acetylcholinesterase will not be significantly inhibited. If there is more pesticide residue in the traditional Chinese medicinal material sample, the activity of acetylcholinesterase will be inhibited. This characteristic can be combined with fluorescent probe technology to achieve the purpose of detecting pesticide residues.

[0005] At present, there are few reports on the use of acetylcholinesterase inhibition principle combined with fluorescent probe technology to detect pesticide residues, and the types of fluorescent probes are few. Therefore, it is necessary to develop new fluorescent probes that can directly detect acetylcholinesterase, and provide a powerful tool for rapid and simple detection of pesticide residues in traditional Chinese medicinal materials. SUMMARY

[0006] In view of the above problems, the purpose of the present application is to provide a fluorescent probe and a preparation method and application thereof. The specific technical solutions are as follows:

[0007] One of the purposes of the present application is to provide a fluorescent probe, the molecular structure of which is as follows:

[0008]

[0009] The second purpose of the present application is to provide a preparation method of the fluorescent probe, comprising the following steps:

[0010] The compound NS-OH and triethylamine are added to anhydrous acetonitrile or dimethylformamide, and acetyl bromide is added under stirring at room temperature, and the molar ratio of the compound NS-OH and acetyl bromide is 1:2-4;

[0011] After the acetyl bromide is completely added, the reaction is concentrated by rotary evaporation after 1 h, and then column chromatography separation is performed to obtain the fluorescent probe;

[0012] The structural formula of the compound NS-OH is as follows:

[0013]

[0014] Further, the molar ratio of the compound NS-OH and acetyl bromide is 1:3.

[0015] Further, the reaction temperature after the acetyl bromide is completely added is 50 DEG C.

[0016] The third purpose of the present application is to provide the application of the fluorescent probe in detecting acetylcholinesterase and in detecting pesticide residues in traditional Chinese medicinal materials.

[0017] Further, the pesticide residues in the traditional Chinese medicinal materials are organophosphorus residues.

[0018] The application mode in detecting pesticide residues in traditional Chinese medicinal materials is to combine the fluorescent probe with the inhibition of acetylcholinesterase by pesticide residues to detect pesticide residues in traditional Chinese medicinal materials.

[0019] Specifically, the following steps are included:

[0020] The acetylcholinesterase, the fluorescent probe and different concentrations of organophosphorus are reacted at 37 DEG C for 20 min, and the fluorescence intensity corresponding to different concentrations of organophosphorus after the reaction is recorded by using a fluorescence spectrometer;

[0021] The organophosphorus concentration and the fluorescence intensity are fitted to establish an equation of the organophosphorus concentration and the fluorescence intensity;

[0022] The acetylcholinesterase, the fluorescent probe and different concentrations of organophosphorus are reacted at 37 DEG C for 20 min, and the fluorescence intensity corresponding to different concentrations of organophosphorus after the reaction is recorded by using a fluorescence spectrometer;

[0023] ​​According to the fluorescence intensity corresponding to the reacted sample solution of the to-be-tested traditional Chinese medicinal material and the equation, the concentration of the organophosphorus in the to-be-tested traditional Chinese medicinal material is determined.

[0024] Further, the organophosphorus includes chlorpyrifos, phorate, phorate, phorate and phorate.

[0025] The beneficial effects of the present application are:

[0026] The fluorescent probe provided in the present application takes hydroxyl hemicyanine as a fluorescent mother nucleus and acetyl as a recognition site. The intermediate NS-OH is subjected to a substitution reaction with acetyl bromide, and the obtained fluorescent probe NS-A can detect acetylcholinesterase and indirectly detect common organophosphorus pesticide residues in traditional Chinese medicinal materials, and has low cost, high sensitivity, good selectivity and good application prospect.

[0027] The fluorescent probe provided in the present application has the advantages of easy raw material, simple synthesis, easy operation, single selective recognition of acetylcholinesterase in MeOH / PBS (pH = 7.4) (v / v = 3:7) system, high sensitivity and low detection limit (0.88 mU / mL), and the fluorescence intensity and the concentration of acetylcholinesterase in the range of 0-1.6 U / mL have a good linear relationship. The fluorescent probe combined with enzyme inhibition method can be used for pesticide detection in traditional Chinese medicinal materials, and the lower limit of pesticide detection can reach the required detection standard. The fluorescent probe provided in the present application can be used for quantitative determination of pesticides by fluorescence spectroscopy. The standard curve error for pesticide chlorpyrifos is small, R 2 >0.97, and the pesticide recovery rate in traditional Chinese medicinal materials is between 96.03% and 102.57%, which shows that the present application can be used for quantitative detection of organophosphorus pesticide residues in traditional Chinese medicinal materials.

[0028] Other features and advantages of the present application will be set forth in the following description, and in part will become apparent to those skilled in the art from the description, or can be learned by practice of the present application. The objects and other advantages of the present application can be realized and obtained by the structure indicated in the specification, claims and drawings. BRIEF DESCRIPTION OF DRAWINGS

[0029] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the following will briefly introduce the drawings needed to be used in the embodiments or prior art description. Obviously, the drawings in the following description are some embodiments of the present application, and those skilled in the art can also obtain other drawings according to these drawings without creative labor.

[0030] Figure 1 The nuclear magnetic resonance hydrogen spectrum of the fluorescent probe NS-A prepared in the embodiment of the present application is shown;

[0031] Figure 2 The carbon NMR spectrum of the fluorescent probe NS-A prepared in an embodiment of the present invention is shown.

[0032] Figure 3 The diagram shows the changes in the UV spectrum of the fluorescent probe (20 μM) prepared in the embodiments of the present invention as the concentration of acetylcholinesterase (0-2.0 U / mL) increases.

[0033] Figure 4 The diagram shows the fluorescence spectrum of the fluorescent probe (20 μM) prepared in the embodiments of the present invention as the concentration of acetylcholinesterase (0-2.0 U / mL) increases. The inset is a linear relationship between the fluorescence intensity and the concentration of acetylcholinesterase when different concentrations of acetylcholinesterase are added to the fluorescent probe (20 μM).

[0034] Figure 5 The fluorescence spectrum changes of the fluorescent probe (20 μM) prepared in the embodiments of the present invention after the addition of different cations, anions, amino acids and enzymes are shown; the concentration of all enzymes is 2.0 U / mL, and the concentration of other analytes is 200 μM.

[0035] Figure 6 The fluorescence spectrum changes of the fluorescent probe (20 μM) prepared in the embodiments of the present invention after the addition of acetylcholinesterase in the presence of different cations, anions, amino acids and enzymes are shown; the concentration of all enzymes is 2.0 U / mL, and the concentration of other analytes is 200 μM.

[0036] Figure 7 The changes in fluorescence intensity of the fluorescent probe (20 μM) prepared in the embodiments of the present invention with different concentrations of acetylcholinesterase over time are shown.

[0037] Figure 8 The fluorescence spectrum changes of the fluorescent probe (20 μM) prepared in the embodiments of the present invention before and after the addition of acetylcholinesterase (2.0 U / mL) under different pH conditions are shown.

[0038] Figure 9A The fluorescent response of the fluorescent probe (20 μM) prepared in the embodiments of the present invention to different concentrations of chlorpyrifos (0-1.4 μM) after reacting with acetylcholinesterase (2.0 U / mL);

[0039] Figure 9B The illustration shows the linear relationship between fluorescence intensity and chlorpyrifos concentration for the fluorescent probe prepared in the embodiments of the present invention, wherein the inset is a standard curve of fluorescence intensity versus chlorpyrifos concentration (0-0.9 μM). Detailed Implementation

[0040] In order to make the purposes, technical solutions and advantages of the embodiments of the present application clearer, the technical solutions in the embodiments of the present application will be clearly and completely described below with reference to the drawings in the embodiments of the present application. Obviously, the described embodiments are only some of the embodiments of the present application, but not all the embodiments of the present application. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative work belong to the scope of protection of the present application.

[0041] The present application provides a fluorescent probe (named as NS-A) which can be used for detecting pesticide residues in traditional Chinese medicinal materials and acetylcholinesterase, and the molecular structural formula of the fluorescent probe is as follows:

[0042]

[0043] The molecular formula is: C 24 H 20 INO3S.

[0044] The present application further provides a preparation method of the fluorescent probe, and the preparation steps are as follows:

[0045] The compound NS-OH and triethylamine are added into anhydrous acetonitrile, acetyl bromide is slowly added under stirring at room temperature, the reaction is carried out at 55 DEG C for 1 h, the product is concentrated by rotary evaporation after the reaction solution is cooled to room temperature, and then the fluorescent probe NS-A is separated by column chromatography.

[0046] The synthetic route is as follows:

[0047]

[0048] The solvents used in the embodiments of the present application are commercially available chemical pure or analytical pure. The structure of the compound is determined by nuclear magnetic resonance (NMR). The NMR is measured by using a Bruker nuclear magnetic resonance instrument, and the solvent used for measurement is DMSO- d 6, and the internal standard is TMS.

[0049] Example 1: Preparation of fluorescent probe

[0050] In the present embodiment, the feeding ratio of the compound NS-OH and acetyl bromide is analyzed, and it is found that under the same conditions, when the molar ratio of the compound NS-OH and acetyl bromide is 1:2, 1:3 and 1:4 respectively, the obtained weights are about 31 mg, 83 mg and 67 mg respectively. Therefore, the molar ratio of the compound NS-OH and acetyl bromide is selected as 1:3 for subsequent experiments. The specific preparation process is as follows:

[0051] Compound NS-OH (0.2 g, 0.41 mmol) and three drops of triethylamine were added to 10 mL of anhydrous acetonitrile, and acetyl bromide (0.1 mL, 1.23 mmol) was slowly added under stirring at room temperature. The reaction was carried out at 55 ℃ for 1 h. After the reaction solution was cooled to room temperature, it was concentrated by rotary evaporation, and then column chromatography was performed with DCM / MeOH (v / v = 90:1) as the eluent to obtain a purple solid powder (83 mg), which was fluorescent probe NS-A, with a yield of 39%. The compound NS-OH was prepared by the method described in the patent with publication number CN117567456A, Example 1.

[0052] In other embodiments, the anhydrous acetonitrile can also be replaced by dimethylformamide.

[0053] The obtained purple powder solid was subjected to nuclear magnetic analysis, and the obtained nuclear magnetic resonance hydrogen spectrum is as shown in Figure 1 1 H NMR (600 MHz, DMSO- d 6) δ 8.29 (d, J = 8.1 Hz, 1H), 8.10 (d, J = 8.4 Hz, 1H), 7.95(d, J = 14.7 Hz, 1H), 7.79 (t, J = 7.9 Hz, 1H), 7.69 (t, J = 7.6 Hz, 1H), 7.46(d, J = 8.4 Hz, 1H), 7.26 (d, J = 2.4 Hz, 1H), 7.08 (s, 1H), 7.04 (d, J = 8.4Hz, 1H), 6.89 (d, J = 14.7 Hz, 1H), 4.18 (s, 3H), 2.96–2.86 (m, 4H), 2.30 (s,3H).

[0054] The nuclear magnetic resonance carbon spectrum is as shown in Figure 2 13 C NMR (151 MHz, DMSO- d ​​6) δ 170.04, 169.01, 160.63, 151.90, 151.32, 142.11, 139.93, 138.14, 129.73, 127.92, 127.62, 127.03, 123.91, 122.96, 120.43, 119.00, 117.64, 116.10, 110.20, 108.30, 35.51, 25.54, 24.26, 20.99.

[0055] Its molecular formula is: C 24 H 20 INO3S

[0056] Example 2 Spectral response of fluorescent probe NS-A to acetylcholinesterase

[0057] The detection method steps are as follows:

[0058] 1. UV / fluorescence spectral changes of fluorescent probe NS-A to acetylcholinesterase

[0059] The fluorescent probe obtained in Example 1 was used, and a probe stock solution with a concentration of 1 mM was prepared using DMSO; acetylcholinesterase was dissolved in deionized water to prepare an enzyme stock solution with a concentration of 100 U / mL; the pesticide chlorpyrifos was dissolved in deionized water to prepare a stock solution with a concentration of 1 mM; a spectral solution of MeOH / PBS (pH = 7.4) (v / v = 3:7) was prepared; in order to maintain the enzyme activity of acetylcholinesterase, the temperature 37 ℃ was selected as the condition for the probe NS-A to detect acetylcholinesterase.

[0060] Accurately take 3 mL of the spectral solution into a cuvette, take 60 μL of the probe stock solution into the cuvette, and then add the enzyme stock solution in the following order: 0, 6, 12, 18, 24, 30, 36, 42, 48, 54, and 60 μL, respectively; after 20 min of reaction at 37 ℃, the spectral graphs of the probe and the probe plus acetylcholinesterase solution were tested using a UV-visible spectrometer and a fluorescence spectrometer.

[0061] From Figure 3 and Figure 4As can be seen from the arrows in the graph, the initial concentration of AChE was 0 U / ml. With each additional curve, the concentration increased by 0.2 U / ml. In the absence of acetylcholinesterase, the fluorescent probe exhibited a strong absorption peak at 555 nm, but almost no fluorescence emission at 718 nm. With increasing enzyme concentration, the absorption peak at 555 nm gradually weakened, and a new absorption peak appeared and strengthened at 695 nm. Simultaneously, the fluorescence intensity at 718 nm also gradually increased. Then, through data processing, such as... Figure 4 As shown, fluorescence intensity exhibits a good linear relationship with enzyme concentration in the range of 0-1.6 U / mL, with a fitting constant R0. 2 = 0.9697. The experimental data above show that the probe does indeed have a good recognition effect on acetylcholinesterase.

[0062] 2. Selectivity and anti-interference experiments

[0063] Accurately transfer 3 mL of the spectral solution into a cuvette. After adding 60 μL of the probe stock solution to the cuvette, add other different cations, anions, amino acids, and enzymes sequentially: Na+. + K + Ca 2+ CO3 2- SO4 2- NO2 - ,ClO - The concentrations of H2O2, AA, DHA, Cys, Hcy, GSH, Glu, His, Collagenase, α-Amylase, Trypsin, β-Gal, and AChE were measured at 37 °C.

[0064] The results are as follows Figure 5 As shown in the figure, the bars corresponding to 1-20 represent Na in sequence. + K + Ca 2+ CO3 2- SO4 2- NO2 - ,ClO - The fluorescence intensity changes were observed with the addition of H2O2, AA, DHA, Cys, Hcy, GSH, Glu, His, Collagenase, α-Amylase, Trypsin, β-Gal, and AChE. It can be seen that the fluorescence intensity changes very little with the addition of other ions, while the fluorescence intensity changes significantly with the addition of acetylcholinesterase. This indicates that the fluorescent probe has good single selectivity for recognizing acetylcholinesterase.

[0065] Accurately pipette 3 mL of the spectrum solution into a cuvette, add 60 μL of the fluorescent probe mother liquor into the cuvette, and then add other different cations, anions, amino acids, and enzymes (Na + , K + , Ca 2+ , CO3 2- , SO4 2- , NO2 - , ClO - , H2O2, AA, DHA, Cys, Hcy, GSH, Glu, His, Collagenase, α-Amylase, Trypsin, and β-Gal) each 60 μL, and then continue to add AChE (60 μL). Perform the determination at 37 ℃, and the results are shown in Figure 6 . In the figure, 1-19 correspond to Na + , K + , Ca 2+ , CO3 2- , SO4 2- , NO2 - , ClO - , H2O2, AA, DHA, Cys, Hcy, GSH, Glu, His, Collagenase, α-Amylase, Trypsin, and β-Gal. It can be seen that the addition of other potential interfering substances to the probe has little effect on the fluorescence signal change of acetylcholinesterase, indicating that the fluorescent probe has good anti-interference ability for detecting acetylcholinesterase. Figure 6

[0066] 3. Time response experiment

[0067] Incubate the probe (20 μM) with acetylcholinesterase at 37 ℃ for 60 min, record the fluorescence spectrum once every three minutes for the first 25 min, and then record it once every 5 min. The results are shown in Figure 7 . The probe and acetylcholinesterase (2.0 U / mL) respond completely at about 20 minutes.

[0068] 4. Effect of different pH on the fluorescent probe

[0069] Incubate the probe (20 μM) with acetylcholinesterase (2.0 U / mL) at 37 ℃ under different pH conditions for 20 min to record the spectrum. As shown in Figure 8 , the fluorescent probe has almost no fluorescence intensity at pH 2-8. After adding acetylcholinesterase, the fluorescence intensity significantly increases in the range of pH 7-11, indicating that the probe can effectively detect acetylcholinesterase in this pH range.

[0070] ​Example 3: Establishing the curve of the concentration of organophosphorus and the fluorescence intensity, this example takes chlorpyrifos as an example to illustrate, in other examples, the corresponding curve of organophosphorus and fluorescence intensity can be established according to the detection needs, for example, it can be phorate, phorate, imine phosphorus and dibromophos, etc.

[0071] After different concentrations of chlorpyrifos (0-1.4 μM), acetylcholinesterase (2.0 U / mL) and probe were reacted at 37 ℃ for 20 min, the spectral changes were recorded by fluorescence spectrometer, as shown in Figure 9A With the increase of chlorpyrifos concentration, the fluorescence intensity becomes weaker and weaker, because chlorpyrifos inhibits the activity of acetylcholinesterase, hinders the combination of probe and acetylcholinesterase, resulting in fluorescence reduction, so that the concentration of chlorpyrifos cannot be calculated according to the fluorescence intensity. As shown in Figure 9B The concentration of chlorpyrifos and fluorescence intensity was nonlinearly fitted, and the inset was the linear fitting curve of chlorpyrifos (0-0.9 μM) and fluorescence intensity, it can be seen that chlorpyrifos (0-0.9 μM) and fluorescence intensity showed good linear relationship, the equation was:

[0072] y = -202.2354x+276.3576, R 2 =0.9743.

[0073] Example 4: Chlorpyrifos pesticide in traditional Chinese medicine sample recovery experiment

[0074] A certain amount of jiegeng, baishao, huangjing, mudanpi and heshan shihuchong were weighed, washed with water, dried, crushed and then wet digestion was carried out to obtain traditional Chinese medicinal material sample solution. Chlorpyrifos (0.3, 0.6, 0.9 μM) was added to the traditional Chinese medicinal material sample solution, and the corresponding fluorescence of chlorpyrifos in the traditional Chinese medicinal material was detected by the method of combining enzyme inhibition principle and fluorescence probe, and the spectral data was substituted into the equation y = -202.2354x+276.3576 to calculate, and the results are shown in Table 1:

[0075] Table 1

[0076]

[0077] From Table 1, it can be seen that the experimental recovery rate is between 96.03% and 102.57%, and the relative standard deviation is less than 4.01%, so the detection method established in this experiment has good recovery rate.

[0078] In other embodiments of the present application, a plurality of organophosphorus such as phorate, phorate, imine phosphorus and dibromophos are detected by the method in the above examples, and the results are similar to those of Example 4, which can prove that the fluorescence probe proposed in the present application can be used for detection and analysis of organophosphorus residues in traditional Chinese medicinal materials.

[0079] Although the present application has been described in detail with reference to the foregoing embodiments, it should be understood that modifications can be made to the foregoing embodiments, or additional implementations can be implemented, without departing from the spirit and scope of the inventive subject matter. Accordingly, the present application is not limited to the implementations described herein, but is intended to be defined by the claims set forth below, and equivalents thereof.

Claims

1. Application of a fluorescent probe in detecting organic phosphorus residues in traditional Chinese medicinal materials, characterized in that, The fluorescence detection is carried out at an excitation wavelength of 555 nm and an emission wavelength of 718 nm. The fluorescent probe is prepared in the following steps: Compound NS-OH is added to dry acetonitrile or dimethylformamide, acetyl bromide is added with stirring at room temperature, the molar ratio of compound NS-OH and acetyl bromide is 1:2-4; the structural formula of compound NS-OH is as follows: After the complete addition of acetyl bromide, the reaction is concentrated by rotary evaporation after 1 hour, and then column chromatography separation is performed to obtain the fluorescent probe; the reaction temperature after the complete addition of acetyl bromide is 50℃, and the molecular structural formula of the fluorescent probe is as follows: 。 2. The use of the fluorescent probe according to claim 1, characterized in that, The molar ratio of the compound NS-OH to acetyl bromide is 1:

3.

3. Use of a fluorescent probe in the preparation of a reagent for detecting acetylcholinesterase, characterized in that, The molecular structure of the fluorescent probe is as follows: .

4. Use according to claim 1, characterized in that, The application mode is to combine the fluorescent probe with the inhibition effect of pesticide residues on acetylcholinesterase to detect organic phosphorus pesticide residues in Chinese herbal medicines.

5. The use according to claim 1, characterized in that, The combination of the fluorescent probe and the inhibition effect of pesticide residues on acetylcholinesterase to detect organic phosphorus pesticide residues in Chinese herbal medicines includes the following steps: Acetylcholinesterase, the fluorescent probe and different concentrations of organic phosphorus are reacted at 37℃ for 20 min, and the fluorescence intensity corresponding to different concentrations of organic phosphorus after the reaction is recorded by using a fluorescence spectrometer; The organic phosphorus concentration and the fluorescence intensity are fitted to establish an equation of the organic phosphorus concentration and the fluorescence intensity; The sample solution of the Chinese herbal medicine to be tested, acetylcholinesterase and the fluorescent probe are reacted at 37℃ for 20 min, and the fluorescence intensity corresponding to the sample solution of the Chinese herbal medicine to be tested after the reaction is recorded by using a fluorescence spectrometer; The organic phosphorus concentration in the Chinese herbal medicine to be tested is determined according to the fluorescence intensity corresponding to the sample solution of the Chinese herbal medicine to be tested after the reaction and the equation.

6. Use according to claim 5, characterized in that, The organic phosphorus includes chlorpyrifos, imidacloprid, phorate, phosalone and bromophos.

Images