Fluorescent probe for detecting nitrosamine as well as preparation method and application of fluorescent probe

Abstract

The invention belongs to the technical field of chemical detection, and particularly relates to a fluorescent probe for detecting nitrosamine as well as a preparation method and application of the fluorescent probe. The detection method comprises the following steps: by taking a fluorescent probe P1 as a fluorescent detection reagent, carrying out reductive coupling reaction on the probe P1 in a DMSO-Tris-HCl solution system by utilizing the oxidability of nitrosamine NNK, reacting the NNK with a compound containing triaryl phosphorus to generate an intermediate containing phosphorus and nitrogen ylide, and detecting the intermediate containing phosphorus and nitrogen ylide. According to the intermediate, a new compound containing sulfinic acid amine and a fluorophore with exposed hydroxyl are generated through intramolecular acetyl conversion and hydrolysis, and strong fluorescence is released, so that specific detection of NNK is realized. The detection method shows relatively high sensitivity to nitrosamine NNK in tobacco, the lowest detection limit is as low as 1.5 * 10 <-6 > mol / L, the detection process is simple and convenient, the anti-interference capability is strong, the method is rapid and sensitive, and the detection result is accurate.

Description

Technical Field

[0001] The invention belongs to the technical field of chemical detection, and in particular relates to a method for detecting nitrosamines. Background Art

[0002] Tobacco-specific nitrosamines (TSNAs) are a group of carcinogens found only in tobacco, tobacco products and smoke. TSNAs are mainly present in the form of 4-N-nitrosomethylamino-1-(3-pyridyl)butanone (NNK), which is highly carcinogenic and has been confirmed to be related to the formation of tumors in the lungs, mouth, esophagus, stomach, pancreas, liver and other parts. At present, there are about 1.3 billion smokers worldwide, and about 5 to 6 million people die from smoking-related diseases each year. Therefore, the current research on the formation, pathogenic mechanism and detection technology of TSNAs has always been the focus and hotspot of research at home and abroad.

[0003] The earliest detection of TSNAs was to analyze N-nitrosamines in cigarette smoke using thin layer chromatography, but due to limitations of instrument conditions and other factors, the results of TSNA analysis have always been far from ideal. It was not until the emergence of thermal energy analyzer (TEA) in 1975 that the detection of TSNAs was improved. At present, the more commonly used analytical methods for detecting TSNAs in tobacco and smoke are gas chromatography-thermal energy analyzer (GC-TEA), gas chromatography-nitrogen phosphorus detection (GC-NPD), gas chromatography-mass spectrometry (GC-MS), liquid chromatography-tandem mass spectrometry (HPLC-MS / MS) and ultra-high performance liquid chromatography-electrospray tandem mass spectrometry (HPLC-MS / MS). These methods have the disadvantages of complicated operation, long detection time and high detection cost, and are highly dependent on professional technicians and instruments and equipment, making it difficult to achieve rapid detection of trace amounts. In addition, the prior art CN115453013A discloses a method for enriching and detecting 4-methylnitrosamino-1-(3-pyridyl)-1-butanone in tobacco, but it first uses a magnetic dendritic molecular imprinting recognition adsorption material to separate and enrich NNK in a methanol solution, and finally uses high performance liquid chromatography to detect NNK. Therefore, a simple, portable, and low-cost detection technology is urgently needed for rapid and sensitive detection of tobacco-specific nitrosamine content in tobacco, tobacco products, and smoke.

[0004] Fluorescent probe technology developed in recent years has the advantages of specific selectivity, high sensitivity, simple and fast operation, and less damage to the detected object. It has been widely used in the detection of common heavy metals, anions, and active small molecules such as active sulfur, active oxygen and active sulfur in the environment and biological systems. The fluorescent probe detection method not only makes up for the shortcomings of traditional large-scale instrumental analysis methods, such as large sample reagent requirements, difficulty in real-time online analysis, cumbersome steps, and unsuitability for biological and toxicological research, but also shows many advantages compared with traditional detection methods. First, it has specific selectivity and specificity, which can achieve specific identification of certain small molecules and ions; second, the fluorescent probe method has high sensitivity, so that the minimum detection limit of this method is better than that of traditional detection methods, and trace detection of the detected object is achieved; fourth, compared with traditional large-scale instrumental detection methods, the fluorescent probe method has the advantages of simple operation, and can realize real-time online detection, avoiding the loss of traditional samples in the pretreatment stage and other interference factors. Therefore, the development of TSNAs rapid detection methods based on fluorescent probes in tobacco and tobacco products is of great practical value and scientific significance. Summary of the invention

[0005] In view of the above technical problems, the present invention proposes a fluorescent probe for detecting nitrosamines and a preparation method and application thereof.

[0006] To achieve the above object, the technical solution of the present invention is implemented as follows:

[0007] A fluorescent probe for detecting nitrosamines, the structural formula of which is as follows:

[0008]

[0009] The preparation method of the fluorescent probe for detecting nitrosamines is as follows:

[0010]

[0011] The specific preparation steps are as follows: 2-(diphenylphosphino)benzoic acid, N,N'-diisopropylcarbodiimide (DIC) and 4-dimethylaminopyridine (DMAP) are dissolved in dichloromethane (DCM), and then a dichloromethane solution containing fluorescein is added dropwise, and the fluorescent probe P1 is prepared after stirring at room temperature.

[0012] Furthermore, the molar ratio of the above-mentioned 2-(diphenylphosphino)benzoic acid to fluorescein is (2-8):1.

[0013] The above-mentioned fluorescent probe P1 for detecting nitrosamines is used in detecting nitrosamines in tobacco.

[0014] Mechanism of fluorescent probe P1 in identifying NNK: NNK solution is added to the probe P1 solution. Due to the strong oxidizing property of -NN=O in the NNK structure, -NN=O in the NNK structure can react with compound 1 containing triaryl phosphorus to generate an intermediate containing phosphorus nitrogen ylide (compound 2). Compound 2 undergoes rapid intramolecular acetyl conversion and hydrolysis to generate compound 3 containing sulfenic acid amine and simultaneously releases fluorescent groups, thereby achieving rapid and efficient identification of NNK.

[0015] A method for detecting nitrosamines in tobacco, comprising the following steps:

[0016] (1) dissolving the fluorescent probe P1 in dimethyl sulfoxide to prepare a probe solution; mixing a Tris-HCl buffer solution with dimethyl sulfoxide to obtain a mixed solution A; and preparing a 4-N-nitrosomethylamino-1-(3-pyridyl)butanone solution with dimethyl sulfoxide;

[0017] (2) Add the probe solution to the mixed solution A, then add the 4-N-nitrosomethylamino-1-(3-pyridyl)butanone solution, and ultrasonically vibrate the solution in an ultrasonic cleaning machine to obtain a test solution; use 490 nm as the excitation wavelength, and use a fluorescence spectrometer to measure the fluorescence emission intensity at 520 nm to obtain a working curve F between the concentration and fluorescence intensity of 4-N-nitrosomethylamino-1-(3-pyridyl)butanone. 520 nm =16.66C+2889.01, the unit of C is μmol / L;

[0018] (3) Mixing the mixed solution A with the probe solution, adding the sample solution to be tested; using 490 nm as the excitation wavelength, using a fluorescence spectrometer to measure the fluorescence emission intensity at 520 nm and bringing it into the working curve of step (2), the nitrosamines in the sample solution to be tested can be quantitatively detected.

[0019] The concentration of the probe solution in the above step (1) is 1-5mmol / L. For example, the concentration of the probe solution is 1-4mmol / L; the concentration of the probe solution is 2-5mmol / L; the concentration of the probe solution is 1mmol / L, 2mmol / L, 3mmol / L, 4mmol / L or 5mmol / L.

[0020] In the above step (1), the concentration of the Tris-HCl buffer solution is 20-50mmol / L, and the pH is 7.4. For example, the concentration of the Tris-HCl buffer solution is 20-40mmol / L; the concentration of the Tris-HCl buffer solution is 30-50mmol / L; the concentration of the Tris-HCl buffer solution is 20-30mmol / L; the concentration of the Tris-HCl buffer solution is 30-40mmol / L; the concentration of the Tris-HCl buffer solution is 40-50mmol / L; the concentration of the Tris-HCl buffer solution is 20mmol / L, 30mmol / L, 40mmol / L or 50mmol / L.

[0021] In the above step (1), the volume ratio of the Tris-HCl buffer solution to dimethyl sulfoxide in the mixed solution A is (5-8):(2-5). For example, the volume ratio of the Tris-HCl buffer solution to dimethyl sulfoxide in the mixed solution A is (5-7):(2-4); the volume ratio of the Tris-HCl buffer solution to dimethyl sulfoxide in the mixed solution A is (6-8):(3-5); the volume ratio of the Tris-HCl buffer solution to dimethyl sulfoxide in the mixed solution A is (5-8):2; the volume ratio of the Tris-HCl buffer solution to dimethyl sulfoxide in the mixed solution A is (5-8):3; the volume ratio of the Tris-HCl buffer solution to dimethyl sulfoxide in the mixed solution A is (5-8):4. The volume ratio of methyl sulfoxide is (5-8):7; the volume ratio of Tris-HCl buffer solution to dimethyl sulfoxide in mixed solution A is 5:(2-5); the volume ratio of Tris-HCl buffer solution to dimethyl sulfoxide in mixed solution A is 7:(2-5); the volume ratio of Tris-HCl buffer solution to dimethyl sulfoxide in mixed solution A is 8:(2-5); the volume ratio of Tris-HCl buffer solution to dimethyl sulfoxide in mixed solution A is 1:1, 5:2, 4:1, 8:5, 7:3 or 2:1.

[0022] In the above step (1), the concentration of the 4-N-nitrosomethylamino-1-(3-pyridyl)butanone solution is 2-15mmol / L. For example, the concentration of the 4-N-nitrosomethylamino-1-(3-pyridyl)butanone solution is 2-5mmol / L; the concentration of the 4-N-nitrosomethylamino-1-(3-pyridyl)butanone solution is 5-10mmol / L; the concentration of the 4-N-nitrosomethylamino-1-(3-pyridyl)butanone solution is 10-15mmol / L; the concentration of the 4-N-nitrosomethylamino-1-(3-pyridyl)butanone solution is 2mmol / L, 4mmol / L, 5mmol / L, 6mmol / L, 7mmol / L, 8mmol / L, 9mmol / L, 10mmol / L, 11mmol / L, 12mmol / L, 13mmol / L, 14mmol / L, or 15mmol / L.

[0023] The final concentration of the fluorescent probe in the above steps (2) and (3) is 10 μmol / L.

[0024] In the above step (2), the final concentration of 4-N-nitrosomethylamino-1-(3-pyridyl)butanone in the detection solution is 0-45 μmol / L.

[0025] Furthermore, the ultrasonic oscillation time of the ultrasonic cleaning machine is 30-60 minutes. For example, the ultrasonic oscillation time of the ultrasonic cleaning machine is 30-40 minutes; the ultrasonic oscillation time of the ultrasonic cleaning machine is 40-50 minutes; the ultrasonic oscillation time of the ultrasonic cleaning machine is 50-60 minutes; the ultrasonic oscillation time of the ultrasonic cleaning machine is 30-45 minutes; the ultrasonic oscillation time of the ultrasonic cleaning machine is 45-60 minutes; the ultrasonic oscillation time of the ultrasonic cleaning machine is 30 minutes, 35 minutes, 40 minutes, 45 minutes, 50 minutes or 60 minutes.

[0026] The beneficial effects produced by the present invention are:

[0027] The fluorescent probe P1 of the present invention is a fluorescence-enhanced fluorescent probe, the first one used for NNK detection. Compared with the fluorescence-quenched probe, the fluorescence-enhanced probe has better specificity, anti-interference and higher sensitivity; the linear range of the fluorescence-enhanced probe for NNK detection is 0-45 μmol / L, and the minimum detection limit is 1.5×10 -6 mol / L, the detection process is simple, anti-interference ability is strong, fast, sensitive, the detection result is accurate, and it has strong practical application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0029] Figure 1 This is the fluorescence selectivity diagram of the fluorescent probe P1 of the present invention, with an excitation wavelength of 490 nm.

[0030] Figure 2 This is the anti-interference diagram of the fluorescent probe P1 of the present invention in identifying NNK, with an excitation wavelength of 490 nm and an emission wavelength of 520 nm.

[0031] Figure 3 This is the fluorescence titration diagram of the fluorescent probe P1 of the present invention recognizing NNK, with an excitation wavelength of 490 nm.

[0032] Figure 4 This is the minimum detection limit diagram of the fluorescent probe P1 of the present invention for identifying NNK, with an excitation wavelength of 490 nm and an emission wavelength of 520 nm.

[0033] Figure 5 This is a diagram showing the mechanism of the fluorescent probe P1 of the present invention recognizing NNK.

[0034] Figure 6 This is a high-resolution mass spectrum diagram that verifies the mechanism of the fluorescent probe P1 of the present invention in recognizing NNK. DETAILED DESCRIPTION

[0035] The technical solution of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0036] The chemical reagents, solvents, metal ions, etc. used in the process of preparing the fluorescent probe P1 in the present invention were purchased from Aladdin Reagent Co., Ltd. In the confirmation and performance test process of the fluorescent probe P1, a DTX-300 nuclear magnetic resonance spectrometer from Bruke was used, the solvent was deuterated chloroform, and TMS was used as the internal standard to record the nuclear magnetic resonance hydrogen and carbon spectra; the Q-Exactive HR-MS mass spectrometer from Thermo was used to record high-resolution mass spectrometry data.

[0037] Example 1

[0038] The preparation method of the fluorescent probe for detecting nitrosamines in this embodiment comprises the following steps:

[0039] In a 100 mL round-bottom flask, 2-(diphenylphosphino)benzoic acid (918.9 mg, 3 mmol), N,N'-diisopropylcarbodiimide (574.25 mg, 3 mmol), and 4-dimethylaminopyridine (122 mg, 1 mmol) were added and dissolved in 50 mL of dry dichloromethane. After stirring at room temperature for 10 minutes, 10 mL of dichloromethane solution containing fluorescein (332.3 mg, 1 mmol) was added dropwise. After the addition was complete, the mixture was stirred at room temperature overnight. The reaction was completed by TLC detection. The solution was washed three times with saturated brine, and the organic phase was dried over anhydrous magnesium sulfate, filtered, concentrated, and column chromatography was performed to obtain 350 mg of a white foamy solid with a yield of 38.5%. 31 P NMR (122MHz, CDCl3) δ-2.88; 1 H NMR (300MHz, CDCl3) δ6.62-6.63(d,J=3.0Hz,1H),6.65-6.66(d,J=3.0Hz,1H),6.74(s,1H),6.77(s,1H),6.91-6.92(d,J =3.0Hz,2H),7.0(m,2H),7.12-7.15(t,J=4.5Hz,1H),7.30(m,21H),7.47(m,4H),7.64(m,2H),8.01(m,1H),8.25(m,2H); 13 C NMR (75MHz, CDCl3) δ169.5,164.9,164.9,153.3,152.1,151.1,151.6,142.0,141.6,137.6,134.7,1 34.4,134.1,133.0,129.1,129.1,128.9,128.8,128.6,118.0,116.6,110.7,81.9.MS(ESI)m / zcalcd for C 58 H 38 O7P2 908.209, found 909.3[M+H + ] + ,931.3[M+Na + ] + ,1840.0[2M+Na + ] + .MP125 -127℃.

[0040] Example 2

[0041] The preparation method of the fluorescent probe for detecting nitrosamines in this embodiment comprises the following steps:

[0042] In a 100 mL round-bottom flask, 2-(diphenylphosphino)benzoic acid (612.6 mg, 2 mmol), N,N'-diisopropylcarbodiimide (574.25 mg, 3 mmol), and 4-dimethylaminopyridine (122 mg, 1 mmol) were added and dissolved in 50 mL of dry dichloromethane. After stirring at room temperature for 10 minutes, 10 mL of dichloromethane solution containing fluorescein (332.3 mg, 1 mmol) was added dropwise. After the addition was complete, the mixture was stirred at room temperature overnight. The reaction was completed by TLC detection. The solution was washed three times with saturated brine, and the organic phase was dried over anhydrous magnesium sulfate, filtered, concentrated, and column chromatography was performed to obtain 277.3 mg of a white foamy solid with a yield of 30.5%. 31 P NMR (122MHz, CDCl3) δ-2.88; 1 H NMR (300MHz, CDCl3) δ6.62-6.63(d,J=3.0Hz,1H),6.65-6.66(d,J=3.0Hz,1H),6.74(s,1H),6.77(s,1H),6.91-6.92(d,J =3.0Hz,2H),7.0(m,2H),7.12-7.15(t,J=4.5Hz,1H),7.30(m,21H),7.47(m,4H),7.64(m,2H),8.01(m,1H),8.25(m,2H); 13 CNMR (75MHz, CDCl3) δ169.5,164.9,164.9,153.3,152.1,151.1,151.6,142.0,141.6,137.6,134. 7,134.4,134.1,133.0,129.1,129.1,128.9,128.8,128.6,118.0,116.6,110.7,81.9.MS(ESI)m / z calcd for C 58 H 38 O7P2 908.209, found 909.3[M+H + ] + ,931.3[M+Na + ] + ,1840.0[2M+Na + ] + .MP125 -127℃.

[0043] Example 3

[0044] The preparation method of the fluorescent probe for detecting nitrosamines in this embodiment comprises the following steps:

[0045] In a 100 mL round-bottom flask, 2-(diphenylphosphino)benzoic acid (1.225 g, 4 mmol), N,N'-diisopropylcarbodiimide (574.25 mg, 3 mmol), and 4-dimethylaminopyridine (122 mg, 1 mmol) were added and dissolved in 50 mL of dry dichloromethane. After stirring at room temperature for 10 minutes, 10 mL of dichloromethane solution containing fluorescein (332.3 mg, 1 mmol) was added dropwise. After the addition was complete, the mixture was stirred at room temperature overnight. The reaction was completed by TLC detection. The solution was washed three times with saturated brine, and the organic phase was dried over anhydrous magnesium sulfate, filtered, concentrated, and column chromatography was performed to obtain 376.3 mg of a white foamy solid with a yield of 41.4%. 31 P NMR (122MHz, CDCl3) δ-2.88; 1 H NMR (300MHz, CDCl3) δ6.62-6.63(d,J=3.0Hz,1H),6.65-6.66(d,J=3.0Hz,1H),6.74(s,1H),6.77(s,1H),6.91-6.92(d,J =3.0Hz,2H),7.0(m,2H),7.12-7.15(t,J=4.5Hz,1H),7.30(m,21H),7.47(m,4H),7.64(m,2H),8.01(m,1H),8.25(m,2H); 13 C NMR (75MHz, CDCl3) δ169.5,164.9,164.9,153.3,152.1,151.1,151.6,142.0,141.6,137.6,134.7,1 34.4,134.1,133.0,129.1,129.1,128.9,128.8,128.6,118.0,116.6,110.7,81.9.MS(ESI)m / zcalcd for C 58 H 38 O7P2 908.209, found 909.3[M+H + ] + ,931.3[M+Na + ] + ,1840.0[2M+Na + ] + .MP125 -127℃.

[0046] Example 4

[0047] The preparation method of the fluorescent probe for detecting nitrosamines in this embodiment comprises the following steps:

[0048] In a 100mL round-bottom flask, 2-(diphenylphosphino)benzoic acid (1.838g, 6mmol), N,N'-diisopropylcarbodiimide (574.25mg, 3mmol), and 4-dimethylaminopyridine (122mg, 1mmol) were added and dissolved in 50mL dry dichloromethane. After stirring at room temperature for 10 minutes, 10mL dichloromethane solution containing fluorescein (332.3mg, 1mmol) was added dropwise. After the addition was complete, the mixture was stirred at room temperature overnight. The reaction was completed by TLC detection. The solution was washed three times with saturated brine, and the organic phase was dried over anhydrous magnesium sulfate, filtered, concentrated, and column chromatography was performed to obtain 392.7mg of a white foamy solid with a yield of 43.2%. 31 P NMR (122MHz, CDCl3) δ-2.88; 1 H NMR (300MHz, CDCl3) δ6.62-6.63(d,J=3.0Hz,1H),6.65-6.66(d,J=3.0Hz,1H),6.74(s,1H),6.77(s,1H),6.91-6.92(d,J =3.0Hz,2H),7.0(m,2H),7.12-7.15(t,J=4.5Hz,1H),7.30(m,21H),7.47(m,4H),7.64(m,2H),8.01(m,1H),8.25(m,2H); 13 C NMR (75MHz, CDCl3) δ169.5,164.9,164.9,153.3,152.1,151.1,151.6,142.0,141.6,137.6,134.7,1 34.4,134.1,133.0,129.1,129.1,128.9,128.8,128.6,118.0,116.6,110.7,81.9.MS(ESI)m / zcalcd for C 58 H 38 O7P2 908.209, found 909.3[M+H + ] + ,931.3[M+Na + ] + ,1840.0[2M+Na + ] + .MP125 -127℃.

[0049] Example 5

[0050] The preparation method of the fluorescent probe for detecting nitrosamines in this embodiment comprises the following steps:

[0051] In a 100mL round-bottom flask, 2-(diphenylphosphino)benzoic acid (2.450g, 8mmol), N,N'-diisopropylcarbodiimide (574.25mg, 3mmol), and 4-dimethylaminopyridine (122mg, 1mmol) were added and dissolved in 50mL dry dichloromethane. After stirring at room temperature for 10 minutes, 10mL dichloromethane solution containing fluorescein (332.3mg, 1mmol) was added dropwise. After the addition was complete, the mixture was stirred at room temperature overnight. The reaction was completed by TLC detection. The solution was washed three times with saturated brine, and the organic phase was dried over anhydrous magnesium sulfate, filtered, concentrated, and column chromatography was performed to obtain 453.6mg of a white foamy solid with a yield of 49.9%. 31 P NMR (122MHz, CDCl3) δ-2.88; 1 H NMR (300MHz, CDCl3) δ6.62-6.63(d,J=3.0Hz,1H),6.65-6.66(d,J=3.0Hz,1H),6.74(s,1H),6.77(s,1H),6.91-6.92(d,J =3.0Hz,2H),7.0(m,2H),7.12-7.15(t,J=4.5Hz,1H),7.30(m,21H),7.47(m,4H),7.64(m,2H),8.01(m,1H),8.25(m,2H); 13 C NMR (75MHz, CDCl3) δ169.5,164.9,164.9,153.3,152.1,151.1,151.6,142.0,141.6,137.6,134.7,1 34.4,134.1,133.0,129.1,129.1,128.9,128.8,128.6,118.0,116.6,110.7,81.9.MS(ESI)m / zcalcd for C 58 H 38 O7P2 908.209, found 909.3[M+H + ] + ,931.3[M+Na + ] + ,1840.0[2M+Na + ] + .MP125 -127℃.

[0052] Implementation effect example

[0053] (1) Selectivity of probe for detecting NNK

[0054] A Tris-HCl buffer solution with a pH of 7.4 and a concentration of 40 mM was prepared, and a mixed solution A with a volume ratio of 7:3 was prepared using the above buffer solution and DMSO, and a probe P1 solution with a concentration of 1 mM was prepared using DMSO. The selectivity of probe P1 for NNK in mixed solution A was tested using a fluorescence spectrometer. Figure 1 As shown, under the excitation condition at 490nm, the single probe P1 (10μM) has a weak fluorescence emission intensity at 520nm in the mixed solution A. When NNK (100μM) is added, the fluorescence emission intensity at 520nm is significantly enhanced. However, when other substances (100μM) are added, the fluorescence emission intensity of the solution system does not change significantly compared with the fluorescence emission intensity of the single probe system.

[0055] The above experimental results show that probe P1 has good fluorescence specific selectivity for NNK in mixed solution A.

[0056] The mechanism of the fluorescent probe P1 of the present invention for identifying NNK is as follows: NNK solution is added to the probe P1 solution. Due to the strong oxidizing property of -NN=O in the NNK structure, -NN=O in the NNK structure can react with the triarylphosphorus-containing compound 1 to generate an intermediate (compound 2) containing phosphorus nitrogen ylide. Compound 2 undergoes rapid intramolecular acetyl conversion and hydrolysis to generate compound 3 containing sulfinic acid amine and simultaneously releases a fluorescent group, thereby realizing rapid and efficient identification of NNK. The structure of the product R'OH was confirmed by high-resolution mass spectrometry (HR-MS) (see attached figure). Figure 6 ). The experimental results show that the theoretical calculated value of R'OH is 331.0612, and the HR-MS result shows 331.0620. This data proves Figure 5 The mechanism of action is shown.

[0057] (2) Anti-interference of probe detection of NNK

[0058] Prepare a Tris-HCl buffer solution with a pH of 7.4 and a concentration of 40mM. Use the above buffer solution and DMSO to prepare a mixed solution A with a volume ratio of 7:3, and use DMSO to prepare a probe P1 solution with a concentration of 1mM. Add 3000μL of mixed solution A and 30μL of probe P1 solution to 22 clean fluorescent cuvettes, respectively, and then add 10 molar equivalents of NNK and 10 molar equivalents of other analytes (GSSG, Arg, Cys, Leu, Gly, SO3 2- ,I - ,K + ,Na + ,HPO4 2- ,NO3 - Mg 2+ ,SO42- ,Cl - ,Fe 3+ ,Li + ,Pb 2+ ,TBHP,NO,NO2 - ,ClO - ), detected on a fluorescence spectrometer, and plotted a histogram of the 520nm fluorescence intensity corresponding to different analytes to obtain a fluorescence emission histogram ( Figure 2 ).

[0059] Experiments have shown that the recognition of NNK by probe P1 in mixed solution A is not interfered by the above-mentioned other analytes and has good anti-interference ability.

[0060] (3) Working curve of probe detection of NNK

[0061] A Tris-HCl buffer solution with a pH of 7.4 and a concentration of 40 mM was prepared, a mixed solution A with a volume ratio of 7:3 was prepared using the above buffer solution and DMSO, and a probe P1 solution with a concentration of 1 mM was prepared using DMSO. A 5 mmol / L 4-N-nitrosomethylamino-1-(3-pyridyl)butanone (NNK) solution was prepared with DMSO. 3 mL of the mixed solution A was added to a clean fluorescence cuvette, and the probe P1 solution was added. Then, the NNK solution was added in sequence. The added volumes were: 0 μL, 3 μL, 6 μL, 9 μL, 12 μL, 15 μL, 18 μL, 21 μL, 24 μL, and 27 μL. After the addition was completed, the ultrasonic cleaner was ultrasonically vibrated for a period of time. With 490 nm as the excitation wavelength, the fluorescence emission intensity at 520 nm was measured on a fluorescence spectrometer. With the concentration of NNK as the horizontal coordinate and the fluorescence intensity at 520 nm as the vertical coordinate, the working curve of NNK concentration was obtained. The linear regression equation was: F 520 nm =16.66C+2889.01, the unit of C is μmol / L ( Figure 3 ).

[0062] (4) Minimum detection limit of NNK by probe detection

[0063] A good detection limit is one of the criteria for testing whether a probe molecule has application value. Prepare a Tris-HCl buffer solution with a pH of 7.4 and a concentration of 40mM, use the above buffer solution and DMSO to prepare a mixed solution A with a volume ratio of 7:3, and use DMSO to prepare a probe P1 solution with a concentration of 1mM. The concentration of probe P1 was fixed at 10μmol / L, and its response intensity to different concentrations of NNK was measured. With the increase of NNK concentration, the fluorescence emission intensity of the system continued to increase at 520nm. The study found that the fluorescence emission intensity of the solution was linear in the range of NNK concentration from 0 to 45μmol / L (R2 =0.993), and the minimum detection limit of the probe molecule for NNK was calculated to be 1.5×10 -6 mol / L(see Figure 4 ).

[0064] Application Examples

[0065] The fluorescent probe P1 prepared in Example 1 was applied to the detection of NNK in tobacco, and the specific steps were as follows:

[0066] The tobacco leaf sample was placed in an oven, dried at 40℃ for 6h, ground into powder, passed through a 60-mesh sieve, 1.0g of tobacco sample was weighed, placed in a 50mL cone, 30mL of 100mmol / L ammonium acetate aqueous solution was added, and an oscillator (200r / min) was used for 1 hour at room temperature. The extract was centrifuged at high speed and used for NNK content determination. 100μL of 300μmol / L fluorescent probe P1 solution was added to the cuvette, and then 2850μL of mixed solution A (Tris-HCl buffer solution with a pH of 7.4 and a concentration of 40mM, and the mixed solution A with a volume ratio of 7:3 was prepared with the above buffer solution and DMSO) and 50μL of tobacco extract were added to the above cuvette. The blank control was replaced with the buffer solution for tobacco extract, and the mixture was fully mixed and reacted at room temperature for 30min. All were repeated three times, and the fluorescence spectrometer with a wavelength of 490nm as the excitation wavelength and 520nm as the emission wavelength was selected for detection, and the NNK content of the measured tobacco leaf was calculated according to the measured values ​​​​into the working curve.

[0067] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A fluorescent probe for detecting nitrosamines, characterized in that: The structural formula of the fluorescent probe is as follows: 。 2. The method for preparing the fluorescent probe for detecting nitrosamines according to claim 1, characterized in that: The steps are as follows: 2-(diphenylphosphino)benzoic acid, N,N'-diisopropylcarbodiimide and 4-dimethylaminopyridine are dissolved in dichloromethane, and then a dichloromethane solution containing fluorescein is added dropwise, and the fluorescent probe is prepared after stirring at room temperature for reaction.

3. The method for preparing a fluorescent probe for detecting nitrosamines according to claim 2, characterized in that: The molar ratio of 2-(diphenylphosphino)benzoic acid to fluorescein is (2-8):

1.

4. Use of the fluorescent probe for detecting nitrosamines according to claim 1 in detecting nitrosamines in tobacco.

5. A method for detecting nitrosamines in tobacco, characterized in that: Here are the steps: (1) Dissolving the fluorescent probe described in claim 1 in dimethyl sulfoxide to prepare a probe solution; mixing a Tris-HCl buffer solution with dimethyl sulfoxide to obtain a mixed solution A; and using dimethyl sulfoxide to prepare a 4-N-nitrosomethylamino-1-(3-pyridyl)butanone solution; (2) Add the probe solution to the mixed solution A, and then add the 4-N-nitrosomethylamino-1-(3-pyridyl)butanone solution to obtain a detection solution; use a fluorescence spectrometer to measure the fluorescence emission intensity at 520 nm to obtain a working curve F between the concentration and fluorescence intensity of 4-N-nitrosomethylamino-1-(3-pyridyl)butanone 520 nm =16.66C+2889.01, the unit of C is μmol / L; (3) Mix the mixed solution A with the probe solution and add the sample solution to be tested. Use a fluorescence spectrometer to measure the fluorescence emission intensity at 520 nm and substitute it into the working curve of step (2) to quantitatively detect the nitrosamines in the sample solution to be tested.

6. The method for detecting nitrosamines in tobacco according to claim 5, characterized in that: The concentration of the probe solution in step (1) is 1-5 mmol / L; the concentration of the Tris-HCl buffer solution is 20-50 mmol / L, and the pH is 7.

4.

7. The method for detecting nitrosamines in tobacco according to claim 6, characterized in that: The volume ratio of Tris-HCl buffer solution to dimethyl sulfoxide in the mixed solution A of step (1) is (5-8):(2-5); the concentration of 4-N-nitrosomethylamino-1-(3-pyridyl)butanone solution is 2-15 mmol / L.

8. The method for detecting nitrosamines in tobacco according to claim 7, characterized in that: The final concentration of the fluorescent probe in step (2) and step (3) is 10 µmol / L.

9. The method for detecting nitrosamines in tobacco according to claim 8, characterized in that: In the step (2), the final concentration of 4-N-nitrosomethylamino-1-(3-pyridyl)butanone in the detection solution is 0-45 µmol / L.

10. The method for detecting nitrosamines in tobacco according to claim 9, characterized in that: When the fluorescence spectrometer is used for measurement in steps (2) and (3), the excitation wavelength is 490 nm.

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