A method for specific detection of sarin nerve agent mimic diethyl chlorophosphate using fluorescent probe reagent

Abstract

The present application relates to a kind of fluorescent probe reagent specific detection sarin nerve agent analog diethyl chlorophosphate, the chemical name of the probe molecule is (2Z,2'Z)-2,2'-(1,4-phenylene) bis (3-(pyridine-4-yl) acrylonitrile), organic solvent is methanol composition.Due to recognition site pyridyl and sarin nerve agent analog diethyl chlorophosphate occur nucleophilic addition reaction, realize the rapid, naked eye, high sensitivity and super specificity identification to sarin nerve agent analog diethyl chlorophosphate, detection process does not need complex analysis equipment.Probe molecule solution is excited under 395 nm, from no fluorescence to green fluorescence emission, detection limit is 16.4 nM.At the same time, the probe reagent has superior selectivity, especially for the interference of common acid (nitric acid, hydrochloric acid, phosphoric acid, hydrofluoric acid, acetic acid), solve the fatal problem in diethyl chlorophosphate detection field.And, the reagent response speed is fast, fluorescence signal is obvious, naked eye identification is without barrier, provide very effective theoretical basis and technical means for sarin nerve agent analog diethyl chlorophosphate detection.

Description

Technical Field

[0001] This invention belongs to the field of nerve agent detection and provides a method for the specific detection of the sarin nerve agent simulant diethyl chlorophosphate using a fluorescent probe reagent. Background Technology

[0002] Nerve agents are a class of highly toxic organophosphorus compounds. Their high toxicity stems from the strong electrophilicity of the phosphorus atoms in their structure. These atoms can be nucleophilically attacked by the hydroxyl groups on the serine residues of acetylcholinesterase, causing the enzyme to lose activity. This leads to paralysis of the central nervous system, resulting in impaired musculoskeletal function, respiratory distress, convulsions, and ultimately death within minutes. Therefore, developing rapid and highly sensitive methods for detecting nerve agents is essential.

[0003] To date, various detection methods for nerve agents have been developed, such as mass spectrometry, ion mobility chromatography, bioenzyme sensors, and surface-enhanced Raman scattering, some of which exhibit good selectivity and repeatability. However, these methods may suffer from drawbacks such as being cumbersome, time-consuming, having low sensitivity, and poor portability, making them unsuitable for on-site detection. Fluorescence sensing technology, on the other hand, offers advantages such as portability, speed, and high sensitivity, and is widely used in the field of nerve agent detection. Due to the high hazard of nerve agents, their acquisition and use are strictly restricted, and researchers generally cannot use actual nerve agents to test the sensing performance of materials. Therefore, compounds with similar chemical structures to nerve agents but lower toxicity are typically used as nerve agent simulants to test material performance. Sarin, a colorless and odorless liquid with high volatility, has an average lethal dose of 75-100 mg / min. 3 Its rapid life- and health hazard value is 7 ppb (Archives of Toxicology, 2016, 90, 2131-2145; Chemical Stockpile Disposal Program: Final Programmatic Environmental Impact Statement. Aberdeen Proving Ground, Maryland, Program Executive Officer-Program Manager for Chemical Demilitarization, 1988). Rapid and highly sensitive detection of sarin nerve agent has attracted widespread attention; its commonly used mimic is diethyl chlorophosphate (DCP).

[0004] Currently, some studies have been conducted on the rapid and highly sensitive detection of diethyl chlorophosphate (DCP), a nerve agent mimicking sarin, using probe molecules for nerve agent detection (Analytica Chimica Acta, 2019, 1076, 125-130; Sensors and Actuators: B. Chemical 2020, 323, 128698; Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy, 2021, 263, 120206; Journal of Materials Chemistry C, 2016, 4, 10105-10110). While rapid and highly sensitive detection of DCP can be achieved, the inherent limitations of its molecular structure, especially the susceptibility of probe molecules using pyridine as the recognition site to acid interference, affect the specificity of DCP detection. Therefore, there is an urgent need to develop a specific probe molecule that also possesses rapid and highly sensitive properties to meet the practical requirements for efficient detection of DCP. Summary of the Invention

[0005] The purpose of this invention is to provide a fluorescent probe reagent for the specific detection of diethyl chlorophosphate, a mimic of sarin nerve agent. This reagent consists of a probe molecule and a methanol solvent. This fluorescent probe reagent enables highly sensitive and interference-resistant fluorescence detection of diethyl chlorophosphate, a mimic of sarin nerve agent. The detection principle is as follows: pyridine acts as the recognition site, undergoing a nucleophilic addition reaction with diethyl chlorophosphate, followed by hydrolysis leading to protonation of the pyridine group, thereby altering the molecular structure and causing a change in fluorescence intensity. Therefore, after the reagent reacts with diethyl chlorophosphate, the fluorescence changes from blue to green fluorescence at an excitation wavelength of 365 nm; and from no fluorescence to green fluorescence at an excitation wavelength of 395 nm. Further evaluation of its detection performance at an excitation wavelength of 395 nm shows a detection limit of 16.4 nM. It can distinguish more than 10 interfering substances, including common volatile organic solvents, diethyl chlorophosphate structural analogs, and various acids, exhibiting superior selectivity, especially addressing the problem of acid interference in the detection of diethyl chlorophosphate (DCP). When nitric acid, phosphoric acid, acetic acid, and hydrofluoric acid are added to this reagent, its fluorescence intensity does not change significantly. When hydrochloric acid is added, a blue fluorescence signal appears, which is different from the green fluorescence signal of diethyl chlorophosphate.

[0006] This invention discloses a fluorescent probe reagent for the specific detection of the sarin nerve agent mimic, diethyl chlorophosphate. The reagent consists of a probe molecule and an organic solvent. The probe molecule is chemically named (2Z,2'Z)-2,2'-(1,4-phenylene)bis(3-(pyridin-4-yl)acrylonitrile), and the organic solvent is methanol. The specific operation is performed according to the following steps:

[0007] a. At room temperature, weigh the probe (2Z,2'Z)-2,2'-(1,4-phenylene)bis(3-(pyridin-4-yl)acrylonitrile), dissolve it in methanol solvent, the probe concentration in the solution is 0.1 mM, and sonicate until completely dissolved to obtain the fluorescent probe reagent for detecting the sarin nerve agent mimic diethyl chlorophosphate;

[0008] b. Aspirate the fluorescent probe reagent obtained in step a into a small centrifuge tube, add the analyte, and if the analyte contains diethyl chlorophosphate, the fluorescence will change from no fluorescence to green fluorescence when the excitation wavelength is 395 nm.

[0009] The present invention discloses a method for specifically detecting the sarin nerve agent mimic diethyl chlorophosphate using a fluorescent probe reagent. The preparation method of the probe molecule of this reagent is carried out according to the following steps:

[0010] a. Add 2 mmol of 1,4-phenylacetonitrile solid and 4.4 mmol of 4-pyridinecarboxaldehyde liquid to a round-bottom flask containing 40 mL of dichloromethane. When the solid is completely dissolved, the solution is brownish-yellow, and a mixture is obtained.

[0011] b. Heat and stir the mixture in a water bath at 50°C, and add 0.45 mL of tetrabutylammonium hydroxide (TBAH, 40% methanol solution) using a syringe. The solution changes from brownish-yellow to dark green.

[0012] c. Stir in a water bath at 50°C for 2 hours, cool to room temperature, then evaporate the solvent using a rotary evaporator. Wash the solid with water, filter three times, and then filter and wash with ethyl acetate to obtain purified (2Z,2'Z)-2,2'-(1,4-phenylene)bis(3-(pyridin-4-yl)acrylonitrile) probe molecule solid.

[0013] The present invention describes a method for specifically detecting the sarin nerve agent mimic, diethyl chlorophosphate (DCP), using a fluorescent probe reagent. The specific method for applying this reagent to the detection of DCP is as follows:

[0014] Diethyl chlorophosphate (DCP) pure solution was mixed with methanol solution to prepare an alcohol solution with a concentration of 0.05M;

[0015] Measure 2 mL of the fluorescent probe detection reagent into the cuvette using a pipette, and add 0, 1, 2, 3, 4, 5, 6, 8, 10, 12, 14, 18, 20, and 30 μL of 0.05 M diethyl chlorophosphate (DCP) methanol solution, respectively. Perform fluorescence emission spectroscopy using a fluorescence spectrometer to measure the fluorescence emission spectra of the reagent after detecting different concentrations of diethyl chlorophosphate (DCP) under 390 nm excitation light. Then, plot the fluorescence emission intensity at 510 nm as the ordinate and the diethyl chlorophosphate (DCP) concentration as the abscissa to obtain a linear curve, thereby determining the fluorescence detection limit of the detection reagent.

[0016] By conducting a nucleophilic addition reaction between pyridine and diethyl chlorophosphate (DCP) at the pyridine recognition site, resulting in pyridine protonation and altering the molecular structure, a rapid, highly sensitive, and interference-resistant detection of the nerve agent mimic DCP has been achieved, providing an excellent research foundation for the field of nerve agent detection and monitoring.

[0017] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0018] This invention describes a fluorescent probe reagent for the specific detection of diethyl chlorophosphate (DCP), a nerve agent mimic, using a method that achieves detection of DCP by adjusting the excitation wavelength based on fluorescence ratio and fluorescence illumination, without background fluorescence interference. Furthermore, this probe reagent exhibits superior selectivity, particularly against interference from common acids (nitric acid, hydrochloric acid, phosphoric acid, acetic acid, and hydrofluoric acid), solving a critical problem in DCP probe design. Moreover, the reagent provides a fast response, a clear fluorescence signal, and is easily identifiable with the naked eye, offering an excellent theoretical foundation and technical means for the field detection of DCP, a nerve agent mimic, and providing a valuable foundation for fundamental research in the field of nerve agent safety detection and monitoring. Attached Figure Description

[0019] Figure 1 The one-dimensional proton NMR spectrum of the probe molecule in Example 1 of this invention, where the horizontal axis represents chemical shift and the vertical axis represents signal intensity;

[0020] Figure 2 The photographs and fluorescence spectral changes between the probe molecule (0.1 mM) methanol solution and different concentrations of the nerve agent mimic diethyl chlorophosphate (DCP) in Example 2 of this invention; (a) is an image of the probe molecule detecting different concentrations of diethyl chlorophosphate (DCP), (b) is the fluorescence spectral data, and (c) is the linear fitting of the fluorescence intensity at 519 nm and the corresponding color change graph.

[0021] Figure 3The graphs of the probe molecule (0.1 mM) methanol solution and the nerve agent mimic diethyl chlorophosphate (DCP) in Example 3 of this invention are shown, where (a) is the response time spectrum and (b) is the fluorescence intensity at 519.02 nm as DCP is added.

[0022] Figure 4 The selective detection and recognition diagram of the nerve agent mimic diethyl chlorophosphate (DCP) by the probe molecule (0.1 mM) methanol solution in Example 4 of this invention is shown in (a) as an image of the probe molecule detecting different selective substances or DCP, (b) as fluorescence spectral data, and (c) as a linear fitting of the fluorescence intensity at 519 nm and the corresponding color change diagram. Detailed Implementation

[0023] The present invention will be further described in detail below through specific embodiments.

[0024] Example 1

[0025] Synthesis of fluorescent probes:

[0026] a. Add 2 mmol of 1,4-phenylacetonitrile solid and 4.4 mmol of 4-pyridinecarboxaldehyde liquid to a round-bottom flask containing 40 mL of dichloromethane. When the solid is completely dissolved, the solution is brownish-yellow.

[0027] b. Heat and stir the mixture in a water bath at 50°C, and add 0.45 mL of tetrabutylammonium hydroxide (TBAH, 40% methanol solution) using a syringe. The solution changes from brownish-yellow to dark green.

[0028] c. Stir in a water bath at 50°C for 2 hours, cool to room temperature, then evaporate the solvent using a rotary evaporator. Wash the solid with water, filter once, filter twice, filter three times, and then filter and wash with ethyl acetate to obtain purified (2Z,2'Z)-2,2'-(1,4-phenylene)bis(3-(pyridin-4-yl)acrylonitrile) probe molecule solid.

[0029] Figure 1 For the 1H NMR spectrum of the probe molecule: 1 H NMR (400MHz, DMSO-d6) δ 8.79 (d, J = 6.1 Hz, 4H), 8.26 (s, 2H), 8.00 (s, 4H), 7.85 (d, J = 7.7 Hz, 4H).

[0030] Example 2

[0031] a. At room temperature, weigh the probe (2Z,2'Z)-2,2'-(1,4-phenylene)bis(3-(pyridin-4-yl)acrylonitrile) from Example 1, dissolve it in methanol solvent, the probe concentration in the solution is 0.1 mM, and sonicate until completely dissolved to obtain a fluorescent probe reagent for detecting the sarin nerve agent mimic diethyl chlorophosphate;

[0032] b. Using a pipette, measure 2 mL of the fluorescent probe detection reagent obtained in step a into the colorimetric cell, and add 0, 1, 2, 3, 4, 5, 6, 8, 10, 12, 14, 18, 20, and 30 μL of 0.05 M diethyl chlorophosphate methanol solution, respectively, to obtain mixed solutions with diethyl chlorophosphate (DCP) concentrations ranging from 0 to 750 μM; From the optical images captured under 390 nm excitation light (… Figure 2 a) Its fluorescence intensity increases with increasing diethyl chlorophosphate concentration; fluorescence emission spectroscopy scans using a fluorescence spectrometer show that the fluorescence emission intensity of this reagent after detecting different concentrations of diethyl chlorophosphate under 390 nm excitation light increases with increasing diethyl chlorophosphate concentration. Figure 2 b); By establishing a linear relationship between the fluorescence intensity at 519 nm and the concentration of diethyl chlorophosphate solution, the linear equation y = 548.4x + 11956.2 can be obtained. Figure 2 c) According to the calculation equation of the detection limit: detection limit = 3σ / k, where σ is the standard deviation of the fluorescence spectrometer used, the standard deviation of the fluorescence spectrometer used in this invention is 3, and k is the slope of the fitted linear equation, i.e. k = 548.4. The fluorescence detection limit of the reagent can be calculated to be 16.4 nM.

[0033] Example 3

[0034] The response time of the probe molecule to diethyl chlorophosphate was evaluated using the 0.1 mM fluorescent probe molecule reagent obtained in Example 2. Figure 3 The fluorescence spectrum change of the fluorescent probe when diethyl chlorophosphate analyte is added to methanol solution is shown. The fluorescence intensity of the probe molecule shows a significant change within 1 second with the addition of the nerve agent mimic diethyl chlorophosphate, and the fluorescence intensity remains basically unchanged, indicating that the probe molecule responds rapidly to diethyl chlorophosphate.

[0035] Example 4

[0036] The selectivity of the probe molecule to diethyl chlorophosphate was evaluated using the 0.1 mM fluorescent probe molecule reagent obtained in Example 2. Figure 4The images show (a) optical images, (b) fluorescence spectra, and (c) corresponding fluorescence intensities under 395 nm excitation when various common volatile organic solvents, diethyl chlorophosphate structural analogs, and acids are added to the fluorescent probe reagent. Common volatile organic solvents include acetone, dimethyl sulfoxide, ethanol, tetrahydrofuran, toluene, ethyl acetate, and n-hexane. Diethyl chlorophosphate structural analogs include dimethyl methylphosphonate, diisopropyl methylphosphate, tributyl phosphate, and triethyl phosphate. Acids include hydrofluoric acid, acetic acid, phosphoric acid, hydrochloric acid, and nitric acid. When structural analogs of the aforementioned common nerve agent mimics or common volatile organic solvents or acids (except hydrochloric acid) were added, the fluorescence intensity or spectrum of the fluorescent probe solution showed almost no change. After the addition of diethyl chlorophosphate, obvious green fluorescence appeared and the fluorescence spectrum changed drastically. When hydrochloric acid was added, although there was also an enhancement of the fluorescence spectrum, its fluorescence color was blue, and the spectral characteristic peaks were inconsistent with the fluorescence characteristic peaks of diethyl chlorophosphate, indicating that the fluorescent probe molecule has a good specific recognition function for diethyl chlorophosphate.

[0037] Example 5

[0038] The anti-interference performance of the 0.1 mM fluorescent probe molecule obtained in Example 2 was evaluated using the 0.1 mM fluorescent probe molecule reagent. When various common volatile organic solvents, diethyl chlorophosphate structural analogs, and acids (except hydrochloric acid) were added to the fluorescent probe reagent, the fluorescence intensity or spectrum of the fluorescent probe solution was basically consistent with that of diethyl chlorophosphate alone, showing obvious green fluorescence. When a mixture of hydrochloric acid and diethyl chlorophosphate was added, the fluorescence color was blue, and the spectral characteristic peak was at 480 nm. Common volatile organic solvents included acetone, dimethyl sulfoxide, ethanol, tetrahydrofuran, toluene, ethyl acetate, and n-hexane. Diethyl chlorophosphate structural analogs included dimethyl methylphosphonate, diisopropyl methyl phosphate, tributyl phosphate, and triethyl phosphate. Acids included hydrofluoric acid, acetic acid, phosphoric acid, hydrochloric acid, and nitric acid.

[0039] Example 6

[0040] A methanol solution of (2Z,2'Z)-2,2'-(1,4-phenylene)bis(3-(pyridin-4-yl)acrylonitrile) probe molecule with a concentration of 0.1 mM was prepared to obtain a fluorescent probe molecule reagent for detecting diethyl chlorophosphate.

[0041] When an unknown colorless and transparent liquid is dropped into a 2 mL centrifuge tube containing the reagent, no characteristic green fluorescence signal is observed under 395 nm excitation light, proving that the analyte does not contain diethyl chlorophosphate.

[0042] Example 7

[0043] A methanol solution of (2Z,2'Z)-2,2'-(1,4-phenylene)bis(3-(pyridin-4-yl)acrylonitrile) probe molecule with a concentration of 0.1 mM was prepared to obtain a fluorescent probe molecule reagent for detecting diethyl chlorophosphate.

[0044] When an unknown colorless and transparent liquid is dropped into a 2 mL centrifuge tube containing the reagent, the solution exhibits a characteristic green fluorescence signal under 395 nm excitation light, proving that the sample contains diethyl chlorophosphate.

[0045] Example 8

[0046] A methanol solution of (2Z,2'Z)-2,2'-(1,4-phenylene)bis(3-(pyridin-4-yl)acrylonitrile) probe molecule with a concentration of 0.1 mM was prepared to obtain a fluorescent probe molecule reagent for detecting diethyl chlorophosphate.

[0047] When an unknown colorless and transparent liquid is dropped into a 2 mL centrifuge tube containing the reagent, the solution exhibits a characteristic blue fluorescence signal under 395 nm excitation light, proving that the sample contains hydrochloric acid.

Claims

1. A method for specifically detecting the sarin nerve agent mimic diethyl chlorophosphate using a fluorescent probe reagent, characterized in that... This reagent consists of a probe molecule and an organic solvent. The chemical name of the probe molecule is (2Z,2'Z)-2,2'-(1,4-phenylene)bis(3-(pyridin-4-yl)acrylonitrile), and the organic solvent is methanol. The specific operation is carried out according to the following steps: a. At room temperature, weigh the probe (2Z,2'Z)-2,2'-(1,4-phenylene)bis(3-(pyridin-4-yl)acrylonitrile), dissolve it in methanol solvent, the probe concentration in the solution is 0.1 mM, and sonicate until completely dissolved to obtain the fluorescent probe reagent for detecting the sarin nerve agent mimic diethyl chlorophosphate; b. Take the fluorescent probe reagent obtained in step a into a small centrifuge tube, add the analyte, and if the analyte contains diethyl chlorophosphate, the fluorescence will change from no fluorescence to green fluorescence when the excitation wavelength is 395 nm.

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