A novel camphor-based fluorescent probe for detecting organophosphorus pesticides dichlorvos and trichlorfon, and its preparation method and application

Abstract

The present invention discloses a novel camphor-based fluorescent probe for detecting the organophosphorus pesticides dichlorvos and trichlorfon, as well as its preparation method and application. The fluorescent probe is methyl 4′-(7,8,8-trimethyl-3-(pyridin-4-yl)-4,5,6,7-tetrahydro-2H-4,7-methyleneindazole-2-yl)-[1,1′-biphenyl]-4-carboxylate (CP-CM). The compound CP-CM is obtained by esterification of 4′-(7,8,8-trimethyl-3-(pyridin-4-yl)-4,5,6,7-tetrahydro-2H-4,7-methyleneindazole-2-yl)-[1,1′-biphenyl]-4-carboxylate (CP-CH). The compound CP-CM undergoes enzymatic hydrolysis with carboxylesterase. Under 380nm ultraviolet light, the solution's fluorescence changes from dark green to bright yellow-green. Since dichlorvos and trichlorfon effectively inhibit carboxylesterase activity, when these compounds are added to the solution, the solution's fluorescence gradually changes from bright yellow-green to dark green under 380nm ultraviolet light. Therefore, this compound can be used as a fluorescent probe for the detection of dichlorvos and trichlorfon. It has many advantages, including low detection limits (43pg / L and 120pg / L), good selectivity, and high stability, and has great application prospects.

Description

Technical Field

[0001] The present invention belongs to the technical field of fluorescence detection, and relates to a novel camphor-based fluorescent probe for detecting organophosphorus pesticides dichlorvos and trichlorfon, and a preparation method and application thereof. Background Art

[0002] The use of pesticides can effectively control the invasion of crop diseases, pests and bacteria, which not only improves the yield and quality of crops, but also ensures the safety and health of food in agricultural production. Organophosphorus pesticides are a class of broad-spectrum, highly effective and popular agricultural insecticides that are widely used in the fields of insecticide, fungicide and weed control of crops. Dichlorvos and trichlorfon are common organophosphorus pesticides that play an important role in fruit and vegetable cultivation and agriculture. However, if used improperly, pesticide residues will be produced, which will inevitably lead to environmental degradation and pose a huge threat to human health. Excessive inhalation of these pesticides will inevitably cause a series of diseases such as respiratory failure, gastrointestinal discomfort and nervous system disorders. Therefore, it is of great significance to develop an analytical method for the detection of dichlorvos and trichlorfon.

[0003] Currently, there are multiple methods for detecting dichlorvos and trichlorfon, including immunoassays, electrochemical methods, gas chromatography-mass spectrometry, molecularly imprinted polymers, and enzyme-linked immunosorbent assays. However, these techniques suffer from shortcomings such as poor sensitivity, high cost, and complex sample preparation, significantly limiting their ability to detect pesticides in real time. In recent years, fluorescent probe technology has gained favor in clinical medicine, drug analysis, and food safety due to its advantages such as ease of operation, high sensitivity, good membrane permeability, and strong temporal and spatial resolution. However, reports on the use of fluorescence technology to detect dichlorvos and trichlorfon residues have almost all been based on microfluidic paper chips, CdS / CdTe quantum dots, CRISPR-Cas12a systems, and copper nanoparticles, which suffer from drawbacks such as poor reproducibility, high equipment requirements, and complex synthesis. Combining the principles of enzyme inhibition with fluorescent probe technology offers a new approach for achieving rapid, high-throughput, low-limit-of-detection, and highly sensitive detection of pesticides. Therefore, the development of an analytical method for the detection of dichlorvos and trichlorfon based on the principles of enzyme inhibition and fluorescent probe technology is of great significance. Summary of the Invention

[0004] In view of the deficiencies in the prior art, the technical problem to be solved by the present invention is to provide a new camphor-based fluorescent probe for detecting the organophosphorus pesticides dichlorvos and trichlorfon, which can meet the usage requirements.

[0005] In order to solve the above technical problems, the technical solution adopted by the present invention is:

[0006] A new camphor-based fluorescent probe for detecting the organophosphorus pesticides dichlorvos and trichlorfon has the following structural formula:

[0007]

[0008] The molecular formula of this fluorescent probe is C 30 H 29 N3O2, named 4′-(7,8,8-trimethyl-3-(pyridin-4-yl)-4,5,6,7-tetrahydro-2H-4,7-endomethylindazol-2-yl)-[1,1′-biphenyl]-4-carboxylic acid methyl ester, abbreviated as CP-CM.

[0009] The method for preparing a novel camphor-based fluorescent probe for detecting the organophosphorus pesticides dichlorvos and trichlorfon comprises the following steps:

[0010] Compound CP-CM was obtained by esterification reaction with 4′-(7,8,8-trimethyl-3-(pyridin-4-yl)-4,5,6,7-tetrahydro-2H-4,7-endomethylindazol-2-yl)-[1,1′-biphenyl]-4-carboxylic acid (abbreviated as CP-CH) as raw material.

[0011] The specific preparation method of compound CP-CM includes:

[0012] (1) In a 100 mL flask equipped with a stirrer and a reflux condenser, add 0.45-0.67 g (1-1.5 mmol) of CP-CH, 50-60 mL of methanol, and 2-3 mL of concentrated sulfuric acid, and react at 60-65°C for 3-4 h.

[0013] (2) After removing methanol from the reaction solution by distillation, 40-50 mL of ethyl acetate was added to dissolve the distillation residue, which was washed with saturated brine until neutral. The crude product was obtained by drying and distillation. The crude product was further purified by silica gel column chromatography (petroleum ether / ethyl acetate = 3 / 1) to obtain CP-CM.

[0014] The compound CP-CM can specifically undergo an enzymatic hydrolysis reaction with carboxylesterase. Under ultraviolet light with a wavelength of 380 nm, the fluorescent color of the solution changes from dark green to bright yellow-green. Dichlorvos and trichlorfon can inhibit the activity of carboxylesterase. Under 380 nm ultraviolet light, the fluorescent color of the probe solution changes from bright yellow-green to dark green.

[0015] Beneficial Effects: Compared with existing technologies, the compound CP-CM of the present invention can simultaneously detect dichlorvos and trichlorfon, and can sensitively detect the dichlorvos and trichlorfon content in solution. The detection ranges for dichlorvos and trichlorfon are 0-200 μg / L and 0-350 μg / L, respectively, with detection limits of 43 pg / L and 120 pg / L, respectively. As a fluorescent probe for the detection of dichlorvos and trichlorfon, CP-CM has many advantages, including easy synthesis, good selectivity, and high sensitivity, and has promising application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 This is the fluorescence spectrum of the compound CP-CM interacting with different pesticides;

[0017] Figure 2 This is the fluorescence spectrum of the compound CP-CM interacting with different concentrations of dichlorvos and trichlorfon. DETAILED DESCRIPTION

[0018] The present invention will be further described below with reference to specific embodiments.

[0019] Example 1

[0020] Preparation of compound CP-CM

[0021] The reaction formula is as follows:

[0022]

[0023] The specific steps are as follows:

[0024] To a 100 mL flask equipped with a stirrer and a reflux condenser, 0.45-0.67 g (1-1.5 mmol) of CP-CH, 50-60 mL of methanol, and 2-3 mL of concentrated sulfuric acid were added and reacted at 60-65°C for 3-4 hours. After distilling the reaction solution to remove the methanol, 40-50 mL of ethyl acetate was added to dissolve the distillation residue, which was then washed with saturated brine until neutral. The crude product was dried and distilled to obtain a crude product. The crude product was further purified by silica gel column chromatography (petroleum ether / ethyl acetate = 3 / 1) to obtain the yellow powder compound CP-CM with a yield of 49% and a purity of 98.5%. 1 H NMR (600MHz, DMSO-d6) δ8.63 (s, 2H), 8.05 (d, J=8.0Hz, 2H), 7.92-7.86 (m, 4H), 7.76 (s, 2H), 7.66 (d, J=8.1Hz, 2H), 3.88 (s, 3H), 3.25 (d, J=3.7Hz, 1H ), 2.16 (ddd, J=12.4, 8.3, 4.1Hz, 1H), 1.89 (ddd, J=12.4, 9.2, 3.5Hz, 1H), 1.41 (ddd, J=12.3, 8.9, 3.5Hz, 1H), 1.16 (s, 3H), 0.93 (s, 3H), 0.78 (s, 3H). 13C NMR (150MHz, DMSO-d6) δ166.47, 155.69, 150.68, 143.99, 142.12, 141.00, 139.76, 138.64, 130.36, 129.58, 129.20 , 128.19, 127.49, 125.08, 120.43, 63.89, 53.64, 52.67, 48.08, 33.43, 27.53, 20.61, 19.90, 12.44.HRMS: m / z[M+H] + calcd for C 30 H 29 N3O2464.2333; found464.2352.

[0025] Example 2

[0026] Compound CP-CM was prepared into 1×10 -5 M PBS buffer solution (pH = 7.4, DMSO / PBS = 1 / 9, 10mM), and chlorpyrifos, phoxim, trichlorfon, glufosinate, diazinon, acetamiprid, methyl parathion, dimethoate, chlorothiazide, and dichlorvos were dissolved in DMSO to prepare solutions with a concentration of 100ng / L respectively. The fluorescence emission spectra of compound CP-CM in the presence of different pesticides were measured on a fluorescence spectrophotometer using fluorescence spectrometry titration. Figure 1 The results showed that the fluorescence emission intensity was significantly weakened in the presence of dichlorvos and trichlorfon. This indicates that compound CP-CM can be used as a fluorescent probe to detect dichlorvos and trichlorfon by utilizing the enzyme inhibition principle.

[0027] Example 3

[0028] Compound CP-CM was prepared into 1×10 -5 M PBS buffer solution (pH = 7.4, DMSO / PBS = 1 / 9, 10mM), and dichlorvos and trichlorfon were dissolved in DMSO to prepare solutions with concentrations of 0-200μg / L and 0-350μg / L, respectively. The fluorescence emission spectrum of compound CP-CM in the presence of different concentrations of dichlorvos and trichlorfon was measured on a fluorescence spectrophotometer using fluorescence spectrometry. Figure 2 The results showed that as the concentration of dichlorvos in the solution gradually increased from 0 μg / L to 200 μg / L, and the concentration of trichlorfon gradually increased from 0 μg / L to 350 μg / L, the fluorescence emission intensity of compound CP-CM at 530 nm gradually decreased. This indicates that the compound can be used as a fluorescent probe for the sensitive detection of dichlorvos and trichlorfon.

Claims

1. A novel camphor-based fluorescent probe for detecting organophosphorus pesticides dichlorvos and trichlorfon, characterized in that: The fluorescent probe is 4′-(7,8,8-trimethyl-3-(pyridin-4-yl)-4,5,6,7-tetrahydro-2H-4,7-endomethylindazol-2-yl)-[1,1′-biphenyl]-4-carboxylic acid methyl ester (abbreviated as CP-CM), and its chemical structure is:

2. The method for preparing a novel camphor-based fluorescent probe for detecting the organophosphorus pesticides dichlorvos and trichlorfon according to claim 1, characterized in that: The steps include: Compound CP-CM was obtained by esterification reaction of 4′-(7,8,8-trimethyl-3-(pyridin-4-yl)-4,5,6,7-tetrahydro-2H-4,7-endomethylindazol-2-yl)-[1,1′-biphenyl]-4-carboxylic acid (abbreviated as CP-CH) as raw material.

3. The method for preparing a novel camphor-based fluorescent probe for detecting the organophosphorus pesticides dichlorvos and trichlorfon according to claim 2, wherein: The specific preparation method of compound CP-CM includes: (1) Add 0.45-0.67 g (1-1.5 mmol) of CP-CH, 50-60 mL of methanol, and 2-3 mL of concentrated sulfuric acid to a 100 mL flask equipped with a stirrer and a reflux condenser, and react at 60-65°C for 3-4 h. (2) After removing methanol from the reaction solution by distillation, 40-50 mL of ethyl acetate was added to dissolve the distillation residue, which was washed with saturated brine until neutral. The crude product was obtained by drying and distillation. The crude product was purified by silica gel column chromatography using a 3 / 1 petroleum ether / ethyl acetate as eluent to obtain CP-CM.

4. Use of the compound CP-CM according to claim 1 in detecting dichlorvos and trichlorfon, wherein the purpose of the use is not to diagnose or treat a disease.

5. The use according to claim 4, characterized in that The compound CP-CM can undergo enzymatic hydrolysis reaction with carboxylesterase. Under ultraviolet light with a wavelength of 380nm, the fluorescent color of the solution changes from dark green to bright yellow-green. Since dichlorvos and trichlorfon can effectively inhibit the activity of carboxylesterase, under 380nm ultraviolet light, the fluorescent color of the compound solution changes from bright yellow-green to dark green.

6. The use according to claim 4, characterized in that The compound CP-CM can simultaneously identify the organophosphorus pesticides dichlorvos and trichlorfon, and detect the content of dichlorvos and trichlorfon in the solution. The detection concentration ranges are 0-200μg / L and 0-350μg / L, respectively, and the detection limits are 43pg / L and 120pg / L, respectively.

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