CN110981821A - Fluorescent probes and their use for detecting nerve agents - Google Patents
Fluorescent probes and their use for detecting nerve agents Download PDFInfo
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- C07D249/00—Heterocyclic compounds containing five-membered rings having three nitrogen atoms as the only ring hetero atoms
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
- G01N21/643—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" non-biological material
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- C09K2211/1059—Heterocyclic compounds characterised by ligands containing three nitrogen atoms as heteroatoms
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
- G01N2021/6432—Quenching
Abstract
The invention relates to a fluorescent probe represented by the following general formula I or II and application thereof in detecting nerve agents. The fluorescent probe can efficiently detect G series of nervonic toxicants such as sarin, soman, tabun, ethyl sarin and V series of VX, VG, VM, VE and the like based on a hybridization local-charge transfer excited state and a dehybridization mechanism, has an excellent detection limit, high sensitivity and good selectivity, and can be recycled.
Description
Technical Field
The invention relates to the technical field of fluorescent probes, in particular to a fluorescent probe and application thereof in detecting nerve agents.
Background
Nerve toxicants such as Sarin (Sarin), Soman (Soman), Tabun (Tabun) and the like can rapidly destroy the nerve conduction function of a human body under very low concentration, cause serious poisoning symptoms and even death of the human body, and even can be directly used as chemical weapons, thereby seriously endangering public safety and even international safety. Although the international society has already proposed documents such as "rule for prohibiting chemical weapons from using" and the like to prevent the application of nerve agents as chemical weapons, the rapid and efficient detection of nerve agents is still required for malignant criminal events such as underground ferricerin poison gas events and jinzheng male concealed killing events in japan for the last three decades.
The implications of the efficiency of detection of nerve agents include not only the low detection limit of the method, but also the detection time, recoverability of the method, and even the portability of the probe device, etc. At present, the conventional nerve agent detection methods include mass spectrometry, electrochemical methods and the like, but the methods have high detection limit, limited detection precision and long detection time, and more importantly, related instruments are not portable and are difficult to meet the requirements of conventional anti-virus detection, for example, real-time and rapid anti-virus detection is realized in public places such as stations, airports and other personnel intensive places.
The fluorescence detection technology has the advantages of low cost, easy operation, portable instruments and the like, and has important potential in the field of real-time rapid detection. However, the existing fluorescence detection is based on a resonance energy transfer mechanism, a photoinduced electron transfer mechanism or an energy trap mechanism, and the substrate selectivity is limited (more interferents) due to the mechanisms, and the detection limit of the fluorescence probe based on the traditional mechanisms still cannot meet the practical application requirement.
Disclosure of Invention
The technical purpose of the invention is to provide a fluorescent probe for real-time detection of nerve agents and application thereof, wherein the fluorescent probe has the advantages of low detection limit, high sensitivity, good selectivity, strong anti-interference property, recyclability and the like in nerve agent detection.
In one aspect, the present invention provides a fluorescent probe represented by the following general formula I or II:
in the above general formulae I and II,
R1、R2and R3May each independently be selected from one of the following structures:
in particular, R1And R2Have the same structure;
b may be selected from H, phenyl, pyridyl, cyano-substituted phenyl, cyano-substituted pyridyl, triazinyl, phenyl-substituted triazinyl, pyrimidinyl, pyridazinyl, pyrazinyl:
in particular, B may be selected from H, phenyl,
C may be selected from pyridyl, cyano-substituted phenyl, cyano-substituted pyridyl, triazinyl, phenyl-substituted triazinyl, pyrimidinyl, pyridazinyl, pyrazinyl:
in particular, C may be selected from
In the definition of the above-mentioned substituents,
indicating that the substituent is attached to the parent structure from here.
In some embodiments, the fluorescent probe is selected from one of the following fluorescent probes:
the method for synthesizing the above-mentioned fluorescent probe of the present invention is not particularly limited, and it can be synthesized by referring to the synthesis method of the analogous compound in the prior art or the synthesis method of the compound of the following example.
In another aspect, the invention provides the use of the fluorescent probe represented by formula I or formula II for detecting nerve agents.
In specific embodiments, the nerve agent is selected from Sarin (Sarin, GB), Soman (Soman, GD), Tabun (GA), ethylsarin (GE), and the V series of VX, VG, VM, VE.
In still another aspect, the present invention provides a nerve agent detecting element comprising the fluorescent probe represented by the general formula I or the general formula II.
In particular embodiments, the nerve agent detection element is a test strip or film.
Advantageous effects
In the invention, a hybrid local-charge transfer excited state is formed based on a donor-acceptor structure, and compound structures formed by substituting different donors have large pi conjugated structures, so that the donor-acceptor structure with a detection function is formed. Therefore, the fluorescent probe can efficiently detect the nerve agent based on the hybridization local-charge transfer excited state and the dehybridization mechanism, has excellent detection limit, high sensitivity and good selectivity, and can be recycled, thereby having practical application prospect.
In addition, the detection mechanism of the fluorescent probe is different from the mechanisms based on Photoinduced Electron Transfer (PET) and Forster Resonance Energy Transfer (FRET) of most of DCP detection fluorescent probes at present, and the sensitive signal conversion is realized directly by changing the excitation state property of the fluorescent probe, so that on one hand, the faster response of the nerve toxin can be realized, and on the other hand, the specific identification of the nerve toxin can be realized in the hybridization-hybridization conversion.
In addition, the detection reaction mechanisms of the nerve agents reported at present, including G series sarin, soman, tabun, ethylsarin and the like and V series VX, VG, VM, VE and the like, are similar to those in the application, and the reaction activities thereof are higher than that of DCP, so that it can be concluded that the fluorescent probe of the present invention will have a more sensitive response to the nerve agents than to DCP.
Drawings
FIG. 1: the reaction mechanism of the fluorescent probe molecule prepared by the method and the DCP is disclosed.
FIG. 2: the principle of detecting DCP by the fluorescent probe prepared by the application.
FIG. 3: the test strip of the fluorescent probes 1-4 (TPA-HTAZ, TPA-BTAZ, TPA-CNTAZ and TPA-2CNTAZ respectively) prepared by the method has a schematic diagram of the detection effect of DCP gas.
FIG. 4 is a fluorescence spectrum of a test strip of a fluorescent probe 2-4 (TPA-BTAZ, TPA-CNTAZ, TPA-2CNTAZ, respectively) prepared in the present application under different DCP concentrations, wherein FIG. 4a is a fluorescence spectrum of TPA-BTAZ under different DCP concentrations, FIG. 4b is a fluorescence spectrum of TPA-CNTAZ under different DCP concentrations, and FIG. 4c is a fluorescence spectrum of TPA-2CNTAZ under different DCP concentrations.
FIG. 5: the fluorescence spectra of the fluorescent probe 1(TPA-HTAZ) test strips prepared in the application are shown under different concentrations of DCP.
FIG. 6: the reciprocating cycle recovery experiment maps of the fluorescent probes 1-4 (TPA-HTAZ, TPA-BTAZ, TPA-CNTAZ and TPA-2CNTAZ respectively) prepared by the method are shown in the figure 6a, the figure 6b, the figure 6c and the figure 6d, wherein the figure is the reciprocating cycle recovery experiment map of TPA-HTAZ, the figure 6b is the reciprocating cycle recovery experiment map of TPA-BTAZ, the figure 6c is the reciprocating cycle recovery experiment map of TPA-CNTAZ, and the figure 6d is the reciprocating cycle recovery experiment map of TPA-2 CNTAZ.
FIG. 7: the test strips of the fluorescent probes 1-4 (TPA-HTAZ, TPA-BTAZ, TPA-CNTAZ and TPA-2CNTAZ respectively) prepared by the method fit curves under the condition of low-concentration DCP, wherein a graph in figure 7a shows that the test strip of TPA-HTAZ fits curves under the condition of low-concentration DCP, a graph in figure 7b shows that the test strip of TPA-BTAZ fits curves under the condition of low-concentration DCP, a graph in figure 7c shows that the test strip of TPA-CNTAZ fits curves under the condition of low-concentration AZP, and a graph in figure 7d shows that the test strip of TPA-2CNT fits curves under the condition of low-concentration DCP.
FIG. 8: the interference tests of the fluorescent probes 1-4 (TPA-HTAZ, TPA-BTAZ, TPA-CNTAZ and TPA-2CNTAZ respectively) prepared by the method are provided.
Detailed Description
The present invention will be described in detail below with reference to specific examples, which, however, are only illustrative and not intended to limit the scope of the present invention.
Preparation example 1 Synthesis of TPA-HTAZ (fluorescent Probe 1)
Step (1): synthesis of intermediate bromobenzoyl hydrazine (Br-2O-Br)
Weighing 4-bromobenzoyl chloride, hydrazine hydrate and triethylamine according to the molar ratio of about 2-3:1: 2-3. Dissolving 4-bromobenzoyl chloride in chloroform at 0 ℃, adding triethylamine by a dropper after the 4-bromobenzoyl chloride is completely dissolved, slowly dropwise adding hydrazine hydrate into the mixed solution by an injector, wherein white precipitate can be observed in the dropwise adding process, stirring for 0.5-1 hour under the condition, then turning to room temperature, and continuously stirring for 3-5 hours. After the reaction is finished, the product is filtered, and the petroleum ether and the deionized water are respectively and fully washed and dried to obtain the catalyst. The structure is characterized as follows: mass Spectrometry MW 398.05, M/z 398.05(M +).1H NMR(500MHz,DMSO)δ10.64(s,2H),7.87(d,J＝8.6Hz,4H),7.76(d,J＝8.5Hz,4H)。
Step (2): synthesis of intermediate chloro-azo-bromotoluene (Br-2Cl-Br)
Br-2O-Br and phosphorus pentachloride were weighed in about 1:10-15 molar ratio, i.e., a large excess of phosphorus pentachloride was required. Mixing the two solids at 0-5 deg.C in a volumetric flask, immediately mounting a spherical condenser tube, adding about 30-50mL of refined toluene along the condenser tube, and connecting the balloon device with a Y-tubeUsed for collecting the tail gas, and refluxing for 3-4 hours at 110-120 ℃ under the protection of nitrogen, and the water vapor in the air is isolated to prevent the system from exploding. After the mL reaction is finished, about 10-15mL of distilled water is dripped to quench the reaction, the reaction is kept still for 10-15 minutes, toluene is removed by evaporation, and the product is obtained by recrystallization through absolute ethyl alcohol and drying. The structure is characterized as follows: mass Spectrometry MW 433.61, M/z 434.94(M +).1H NMR(500MHz,DMSO)δ8.03–7.99(m,2H),7.89–7.85(m,2H),7.85–7.80(m,2H),7.75–7.70(m,2H).
And (3): synthesis of intermediate Br-TAZ-H
Weighing Br-2Cl-Br and ammonia water according to the molar ratio of 1: 1-1.5. The weighed raw materials are dissolved in degassed N-methyl pyrrolidone (or N, N-dimethylformamide), and stirred for 10 to 14 hours under the protection of nitrogen at the temperature of 130-. And after the reaction is finished, cooling, adding 10-15mL of 2M dilute hydrochloric acid, observing that a precipitate appears in the process of dropwise adding the dilute hydrochloric acid, continuously stirring at room temperature for 30-60 minutes, filtering the product, washing with a large amount of deionized water, and drying to obtain the catalyst. The structure is characterized as follows: mass Spectrometry MW379.60, M/z 378.91(M +).1H NMR(500MHz,DMSO)δ8.11–8.08(m,4H),7.89–7.85(m,4H).
And (4): synthesis of final product TPA-HTAZ
Weighing 4-triphenylamine borate and Br-TAZ-H according to the molar ratio of 2-3: 1. Mixing with 2-4mol/L anhydrous potassium carbonate water solution, toluene and tetrahydrofuran, adding 20-100mg of catalyst palladium tetratriphenylphosphine under the protection of nitrogen, and refluxing at 85-100 ℃ for more than 3 days under anhydrous and oxygen-free conditions. After the reaction is finished, the product is extracted by dichloromethane and then purified by column chromatography to obtain the final pure product. The structure is characterized as follows: mass Spectrometry MW 707.70, M/z 707.88(M +).1H NMR(500MHz,Chloroform-d)δ8.26–8.19(m,4H),7.80–7.74(m,4H),7.60–7.54(m,4H),7.36–7.28(m,8H),7.22–7.15(m,12H),7.13–7.06(m,4H).13C NMR(500MHz,CDCl3)164.52,148.13,147.45,143.88,133.17,129.39,127.81,127.41,127.03,124.77,123.37,122.11 elemental analysis: calculated value C50H37N5C, 84.84; h, 5.27; n, 9.89; experimental values C, 84.27; h, 5.07; and N, 7.63.
Preparation example 2 Synthesis of TPA-BTAZ (fluorescent Probe 2)
The intermediates of steps (1) and (2) were prepared as in steps (1) and (2) of example 1, respectively.
And (3): synthesis of intermediate Br-TAZ-B
The chloro-azo-bromotoluene and aniline are weighed according to the molar ratio of 1: 1-1.8. The weighed raw materials are dissolved in degassed N-methyl pyrrolidone (or N, N-dimethylformamide), and stirred for 10 to 14 hours under the protection of nitrogen at the temperature of 130-. After cooling, about 10-15mL of 2M dilute hydrochloric acid was added, and precipitation was observed during the addition of the dilute hydrochloric acid. And (3) continuously stirring for 30-60 minutes at room temperature, carrying out suction filtration on the product, washing with a large amount of deionized water, and drying to obtain the product. The structure is characterized as follows: mass Spectrometry MW454.97, M/z 454.95(M +).1H NMR(500MHz,DMSO)δ7.62–7.59(m,2H),7.59–7.58(m,2H),7.55–7.42(m,5H),7.35–7.32(m,2H),7.32–7.30(m,2H).
And (4): final product TPA-BTAZ: weighing the 4-triphenylamine borate and the Br-TAZ-B according to the molar ratio of 2-3: 1. Mixing with 2-4mol/L anhydrous potassium carbonate water solution, toluene and tetrahydrofuran, adding 20-100mg of catalyst palladium tetratriphenylphosphine under the protection of nitrogen, and refluxing at 85-100 ℃ for more than 3 days under anhydrous and oxygen-free conditions. After the reaction is finished, the product is extracted by dichloromethane and then purified by column chromatography to obtain the final pure product. The structure is characterized as follows: mass Spectrometry MW 783.11, M/z 783.98(M +).1H NMR(500MHz,CDCl3)δ7.55–7.53(m,2H),7.51(t,J＝5.7Hz,8H),7.47(d,J＝8.7Hz,4H),7.32–7.24(m,16H),7.14(t,J＝3.0Hz,6H),7.13(d,J＝2.0Hz,2H),7.07(s,2H),7.05(s,1H).13CNMR(500MHz,CDCl3)154.60,147.77,147.52,141.72,135.37,133.50,130.11,129.73,129.33,129.14,127.96,127.66,126.42,124.82,124.61,123.57,123.17 elemental analysis: calculated value C56H41N5C, 85.8; h, 5.27; n, 8.93; experimental values of C, 81.94; h, 5.52; and N, 8.22.
Preparation example 3 Synthesis of TPA-CNTAZ (fluorescent Probe 3)
The synthesis of the intermediates of steps (1) and (2) was the same as in steps (1) and (2) of example 1, respectively.
And (3): synthesis of intermediate Br-TAZ-CN
The chloro-azo-bromotoluene and the 4-aminobenzonitrile are weighed according to the molar ratio of 1: 1-1.8. The weighed raw materials are dissolved in degassed N-methyl pyrrolidone (or N, N-dimethylformamide), and stirred for 10 to 14 hours under the protection of nitrogen at the temperature of 130-. After cooling, about 10-15mL of 2M dilute hydrochloric acid was added, and precipitation was observed during the addition of the dilute hydrochloric acid. And (3) continuously stirring for 30-60 minutes at room temperature, carrying out suction filtration on the product, washing with a large amount of deionized water, and drying to obtain the product. Mass Spectrometry MW808.67, M/z 808.99(M +).1H NMR(500MHz,Chloroform-d)δ7.81–7.75(m,2H),7.60–7.53(m,4H),7.52–7.40(m,8H),7.37(d,J＝8.5Hz,2H),7.32–7.29(m,6H),7.18–7.12(m,12H),7.08(tt,J＝7.2,1.2Hz,4H).13C NMR(500MHz,CDCl3)154.33,147.99,147.46,142.27,139.12,133.90,133.06,129.37,129.30,128.85,127.69,126.67,124.70,123.68,123.47,123.29,117.38,113.75 elemental analysis: calculated value C53H33N5C, 84.63; h, 4.98; n, 10.39; experimental values of C, 84.41; h, 4.93; n, 10.32.
And (4): final product ofThe substance TPA-CNTAZ: weighing 4-triphenylamine borate and Br-TAZ-CN according to a molar ratio of 2-3: 1. Mixing the weighed raw materials with 2-4mol/L anhydrous potassium carbonate aqueous solution, toluene and tetrahydrofuran, adding 20-100mg of catalyst tetratriphenylphosphine palladium under the protection of nitrogen, and refluxing for more than 3 days at 85-100 ℃ under anhydrous and oxygen-free conditions. After the reaction is finished, the product is extracted by dichloromethane and then purified by column chromatography to obtain the final pure product. The structure is characterized as follows: mass spectrum: MW 590.71, M/z 590.69(M +).1H NMR(500MHz,CDCl3)δ7.74(d,J＝8.1Hz,2H),7.64(s,8H),7.54(d,J＝8.7Hz,2H),7.32(t,J＝7.9Hz,4H),7.24(d,J＝8.1Hz,2H),7.18(dd,J＝11.5,8.2Hz,6H),7.11(t,J＝7.4Hz,2H).13C NMR(500MHz,CDCl3)153.75,148.65,147.26,143.03,132.37,132.33,131.41,130.70,129.47,129.10,128.29,127.74,124.89,123.63,123.24,117.99,113.76 elemental analysis: calculated value C40H26N6: c,81.34,; h, 4.44; n, 14.23; the experimental value is C, 81.8; h, 4.58; n, 14.04.
Preparation example 4 Synthesis of TPA-2CNTAZ (fluorescent Probe 4)
Step (1): synthesis of intermediate 4, 4' -dicyanobenzoyl hydrazine (CN-2O-CN)
Weighing 4-cyanobenzoyl chloride, hydrazine hydrate and triethylamine according to the molar ratio of about 2-3:1: 2-3. Dissolving 4-cyanobenzoyl chloride in chloroform at 0 ℃, adding triethylamine by a dropper after the 4-cyanobenzoyl chloride is completely dissolved, then slowly dripping hydrazine hydrate into the triethylamine by an injector, wherein a precipitate appears in the dripping process, stirring for 1 to 2 hours under the condition, then turning to room temperature, and continuing to stir for 3 to 5 hours. After the reaction is finished, the product is filtered, and the petroleum ether and the deionized water are respectively and fully washed and dried to obtain the catalyst. The structure is characterized as follows: mass spectrum: MW 290.08, M/z 290.08(M +).1H NMR(500MHz,DMSO)δ8.11(d,J＝8.4Hz,4H),8.02(d,J＝8.3Hz,4H).
Step (2): intermediate 4, 4' -dicyano chloroazotoluene (CN-2 Cl-CN): the CN-2O-CN and the phosphorus pentachloride are weighed according to the molar ratio of about 1:10-15, namely the phosphorus pentachloride needs to be in large excess. Mixing the two solids in a volumetric flask at 0-5 ℃, immediately putting a spherical condenser tube on the flask, adding about 30-50mL of refined toluene into the volumetric flask along the condenser tube, collecting tail gas by using a Y-tube connected balloon device, refluxing for 3-4 hours under the protection of nitrogen at 110-120 ℃, and taking attention to isolate water vapor in the air to prevent the system from exploding. After the reaction is finished, 10-15mL of distilled water is carefully dripped into the product to quench the reaction, the mixture is kept stand for 10-15 minutes, toluene is removed by evaporation, and the product is obtained by recrystallization and drying. The structure is characterized as follows: mass spectrum: MW 325.9, M/z 326.01(M +).1H NMR(500MHz,DMSO)δ10.08(s,2H),8.11(d,J＝1.7Hz,4H),8.01(d,J＝1.8Hz,4H).
And (3): synthesis of intermediate 2CN-TAZ-Br
Weighing CN-2Cl-CN and 4-bromoaniline according to a molar ratio of 1: 1-1.5. The weighed raw materials are dissolved in degassed N-methyl pyrrolidone (or N, N-dimethylformamide), and stirred for 10 to 14 hours under the protection of nitrogen at the temperature of 130-. After the reaction was completed, the reaction mixture was cooled, about 10 to 15mL of 2M diluted hydrochloric acid was added, and a precipitate was observed during the addition of the diluted hydrochloric acid. And continuously stirring for 30-60 minutes, carrying out suction filtration on the product, washing with a large amount of deionized water, and drying to obtain the catalyst. Mass spectrum: MW 426.28, M/z 426.28(M +).1H NMR(500MHz,DMSO)δ8.16–8.11(m,1H),7.93–7.87(m,3H),7.75–7.71(m,2H),7.61–7.55(m,4H),7.51–7.43(m,2H).
And (4): synthesis of final product TPA-2CNTAZ
Weighing 4-triphenylamine borate and 2CN-TAZ-Br according to the molar ratio of 1.2-2: 1. Mixing the weighed raw material with 2-4 mol-Mixing L anhydrous potassium carbonate water solution, toluene and tetrahydrofuran, adding 20-100mg of catalyst palladium tetratriphenylphosphine under the protection of nitrogen, and refluxing at 85-95 ℃ for more than 3 days under anhydrous and oxygen-free conditions. After the reaction is finished, the product is extracted by dichloromethane and then purified by column chromatography to obtain the final pure product. The structure is characterized as follows: mass spectrum: MW 590.71, M/z 590.69(M +).1H NMR(500MHz,CDCl3)δ7.74(d,J＝8.1Hz,2H),7.64(s,8H),7.54(d,J＝8.7Hz,2H),7.32(t,J＝7.9Hz,4H),7.24(d,J＝8.1Hz,2H),7.18(dd,J＝11.5,8.2Hz,6H),7.11(t,J＝7.4Hz,2H).13C NMR(500MHz,CDCl3)153.75,148.65,147.26,143.03,132.37,132.33,131.41,130.70,129.47,129.10,128.29,127.74,124.89,123.63,123.24,117.99,113.76 elemental analysis: calculated value C40H26N6: c,81.34,; h, 4.44; n, 14.23; the experimental value is C, 81.8; h, 4.58; n, 14.04.
Example 1: fluorescent probe test strip preparation
Commercial qualitative analysis filter paper was cut to 0.5 x 3cm format for use. Weighing a certain mass of fluorescent probe compound, dissolving the fluorescent probe compound in a dichloromethane solvent to prepare a dilute solution with the concentration of 0.2-0.5mg/mL, fully soaking the filter paper strip in the dilute solution, taking out and airing to obtain the fluorescent probe test strip.
Example 2: preparation of fluorescent Probe film
Film formation: the diluted solutions of the four fluorescent probe compounds prepared above were prepared according to the method and ratio of example 1, a soda-lime glass substrate with a specification of about 12.5mm was placed on a spin-coating machine, 50 μ L of the above fluorescent probe solution was taken with a liquid-transfer gun, and spin-coated at 1000-.
Test examples:
because of the highly toxic nature of nerve agents, their use is regulated, and thus during the course of the experiment, Diethyl Chlorophosphite (DCP) with similar chemical activity but relatively weak risk can be selected as a nerve agent substitute for detection. As shown in fig. 1 and 2, sp2 hybridized nitrogen atom containing lone pair of electrons can rapidly undergo nucleophilic substitution reaction with DCP, and further hydrolysis of the intermediate product can effectively protonate the nitrogen atom, so that the whole group containing sp2 nitrogen atom becomes a very strong electron acceptor, and the whole fluorescent molecule is induced to have strong CT state, resulting in hybridization of HLCT state, and fluorescence is efficiently and completely quenched or changed in light color. The chemical reactions are all fast reactions, and the fluorescent response can be realized within seconds.
Test example 1: detection of DCP saturated vapor by fluorescent probe test strip
A small amount of DCP was placed in a clean, transparent screw reagent bottle in a dark room environment and the vapor saturated above it with moderate heat (the saturated vapor concentration of DCP at room temperature was 132 ppm). The screw bottle filled with saturated DCP vapor was opened, the probe strip prepared in example 1 was fixed on the bottle cap and sealed in the bottle, and the color change of the fluorescent probe strip before and after addition was observed, taking note that liquid DCP could not be contacted.
Taking TPA-HTAZ, TPA-BTAZ, TPA-CNTAZ and TPA-2CNTAZ as examples, in the time of <1s, the four compounds have quick and obvious fluorescent response to DCP steam, wherein a TPA-HTAZ test strip can observe the phenomenon of quick recovery after color change; TPA-2CNTAZ test strips exhibit a fluorescence quenching phenomenon; TPA-BTAZ and TPA-CNTAZ test strips showed discoloration (FIG. 3).
It is emphasized here that it is reasonable to test in this application with DCP as a substitute for nerve agents, since it is clearly more efficient to test than DCP if, according to a number of reports, a more chemically active nerve agent is actually tested.
Test example 2: detection of DCP vapor with different concentrations by fluorescent probe film
Preparing DCP gas with different concentrations: selecting a 0.5-1L gas sampling bag, injecting 2-3mL of DCP solution into the sampling bag, and standing for 5-7 days. A certain amount of gas is taken out from the sealed sampling bag through a syringe, and the gas is injected into 10mL, 50mL, 100mL, 500mL, 1L and 1.5L gas storage bags filled with air, and the gas is kept stand overnight to be uniformly mixed, so that the DCP gas with different concentrations can be obtained.
The fluorescent probe film prepared in example 2 was placed in a cuvette, prepared DCP gas was introduced into the cuvette, and spectral data were collected to determine the response of different concentrations of DCP vapor to the fluorescent probe, as shown in fig. 4 and 5.
Taking TPA-HTAZ, TPA-BTAZ, TPA-CNTAZ and TPA-2CNTAZ as examples, the emission intensity of the original fluorescence peak position is gradually reduced and the emission intensity of the newly generated emission peak is gradually enhanced along with the increase of the steam concentration of DCP, and the same fluorescence discoloration phenomenon as that in test example 1 can be observed in the experiment. TPA-2CNTAZ only shows the phenomenon that the emission intensity is continuously reduced along with the increase of the steam concentration of DCP. The same phenomenon of fluorescence quenching as in test example 1 was observed in the experiment.
Referring to test example 1, the TPA-HTAZ probe film having a fast recovery from discoloration was tested by placing the TPA-HTAZ probe film in a cuvette and introducing DCP gas into the cuvette during the experiment, as shown in fig. 5, the TPA-HTAZ initially collected only the spectrum where its emission intensity was reduced, taking the cuvette and adding a spin-on film to seal it in order to detect its true spectrum of change, keeping the film in saturated DCP vapor, and testing to obtain a 132ppm discoloration curve, as shown in fig. 5.
FIG. 7 shows the curves fitted to TPA-HTAZ, TPA-BTAZ, TPA-CNTAZ and TPA-2CNTAZ test strips under low DCP concentration conditions.
In addition, the detection limits of the DCP by TPA-HTAZ, TPA-BTAZ, TPA-CNTAZ and TPA-2CNTAZ are measured by steam detection of DCP with different concentrations, and the results are shown in the following table 1.
TABLE 1
As shown in Table 1, the detection limit of the four fluorescent probes is lower than 1ppb, which is far superior to other reported materials. More importantly, the detection limits for these four compounds are well below the 7ppb concentration requirement set by sandrin poison, which is Immediately Life-threatening or Health (IDLH). Moreover, the reactivity of sarin poison gas is higher than that of DCP, and the detection sensitivity of the detection reagent in the process of actually applying to the detection of sarin, soman, tabun and other poison gases is stronger than that of DCP. Therefore, it can be determined that the fluorescent probe of the present application has higher detection sensitivity for toxic gases such as sarin, soman, tabun and the like.
Test example 3: cyclic test experiment of fluorescent probe film
Placing the fluorescent probe film prepared in the example 2 into a cuvette, and carrying out fluorescence intensity detection after saturated DCP steam treatment; and introducing saturated ammonia gas into the cuvette to reduce the probe film, and performing fluorescence detection again. And repeating the steps for a plurality of cycles, observing experimental phenomena and evaluating the reversible test performance of the fluorescent film.
Taking TPA-HTAZ, TPA-BTAZ, TPA-CNTAZ and TPA-2CNTAZ as examples, the cycle test curves of the four probe compounds are shown in figure 6, and it can be seen that the fluorescent probe compounds have good cycle performance in a DCP test and have the characteristic of being capable of being used repeatedly.
Test example 4: interference test of fluorescent probe film
The fluorescent probe films prepared in example 2 were placed in cuvettes and vapors of Volatile Organic Compounds (VOCs) and other nerve agent mimics were separately bubbled as interference test substances. In this example, the selected interference test substances include hydrochloric acid (HCl), acetic acid (HAc), trifluoroacetic acid (TFA), Aniline (Aniline), Ethyl Acetate (EA), 2-chloroethylethyl sulfide (2-CESS), dimethyl methylphosphonate (DMMP), Diethyl Cyanate (DCNP), Pyridine (Pyridine), Benzene (Benzene), toluene (Tol), triethyl phosphate (TEP), and the like, in combination with the actual use environment.
Taking TPA-HTAZ, TPA-BTAZ, TPA-CNTAZ, and TPA-2CNTAZ as examples, low concentrations of DCP vapor can result in significant fluorescence quenching of the fluorescent probe film, but the compound molecular film is hardly quenched for all the listed interferents (even at very high concentrations) (see fig. 8), indicating that the fluorescent probe film is not interfered by other substances in DCP detection, and has single selectivity for DCP.
In conclusion, the fluorescent probe prepared by the method has an extremely low detection limit for nerve agents, so that the fluorescent probe has high sensitivity, has the advantage of good selectivity, can resist the interference of various interferents, and can be recycled. Therefore, the fluorescent probe prepared by the method has a great application prospect in the high-efficiency, real-time and reversible detection of nerve agents.
Claims (9)
1. A fluorescent probe represented by the following general formula I or II:
in the above general formulae I and II,
R1、R2and R3Each independently is selected from one of the following structures:
b is selected from H, phenyl, pyridyl, cyano-substituted phenyl, cyano-substituted pyridyl, triazinyl, phenyl-substituted triazinyl, pyrimidinyl, pyridazinyl, pyrazinyl:
c is selected from pyridyl, cyano-substituted phenyl, cyano-substituted pyridyl, triazinyl, phenyl-substituted triazinyl, pyrimidinyl, pyridazinyl, pyrazinyl.
2. The fluorescent probe of claim 1, wherein R1And R2Have the same structure.
3. The fluorescent probe according to claim 1 or 2,
b is selected from H, phenyl,
4. The fluorescent probe according to claim 1,
c is selected from
5. The fluorescent probe of claim 1, wherein the fluorescent probe is selected from the group consisting of:
6. use of a fluorescent probe according to any of claims 1 to 5 for the detection of nerve agents.
7. The use according to claim 6, wherein the nerve agent is selected from the G series of sarin, soman, tabun, ethylsarin, and the V series of VX, VG, VM, VE.
8. A nerve agent detection element comprising the fluorescent probe of any one of claims 1-5.
9. The nerve agent detection element of claim 8, which is a test strip or film.
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