A fluorescent probe for detecting nitrosamines and its preparation method and application

Abstract

The present invention belongs to the field of chemical detection technology, and in particular relates to a fluorescent probe for detecting nitrosamines, and its preparation method and application. The structural formula of the fluorescent probe of the present invention is: The NNK detection method is: using fluorescent probe P1 as a fluorescent detection reagent, in a DMSO-Tris-HCl solution system, utilizing the oxidizing property of nitrosamine NNK to cause probe P1 to undergo a reduction coupling reaction, NNK reacts with a compound containing triaryl phosphorus to generate an intermediate containing phosphorus nitrogen ylide, and the intermediate is converted into a new compound containing sulfinic acid amine and a hydroxyl-exposed fluorophore through intramolecular acetyl conversion and hydrolysis, releasing strong fluorescence, thereby achieving specific detection of NNK. The detection method shows high sensitivity to nitrosamine NNK in tobacco, with a minimum detection limit as low as 1.5×10 ‑6 mol / L, the detection process is simple, anti-interference ability is strong, fast, sensitive, and the test results are 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 tobacco smoke. TSNAs, primarily in the form of 4-N-nitrosomethylamino-1-(3-pyridyl)butanone (NNK), are highly carcinogenic and have been linked to tumor formation in the lungs, mouth, esophagus, stomach, pancreas, liver, and other organs. Currently, there are approximately 1.3 billion smokers worldwide, and approximately 5 to 6 million people die annually from smoking-related illnesses. Therefore, research into the formation, pathogenic mechanisms, and detection technologies of TSNAs has been a key focus and hot topic both domestically and internationally.

[0003] TSNAs were first detected using thin-layer chromatography (TLC) to analyze N-nitrosamines in cigarette smoke. However, due to limitations in instrumentation and other factors, the results were far from ideal. TSNA detection improved only with the advent of the thermal energy analyzer (TEA) in 1975. Currently, the most commonly used analytical methods for detecting TSNAs in tobacco and cigarette smoke include 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 drawbacks such as complex procedures, lengthy testing times, and high costs. Furthermore, they rely heavily on specialized technicians and instrumentation, making rapid detection of trace amounts difficult. Furthermore, prior art CN115453013A discloses a method for enriching and detecting 4-methylnitrosamino-1-(3-pyridyl)-1-butanone in tobacco. However, this method first uses a magnetic dendrimer molecular imprinting adsorption material to separate and enrich NNK from a methanol solution, and then finally uses high-performance liquid chromatography to detect NNK. Therefore, there is an urgent need for simple, portable, and low-cost detection technologies for rapid and sensitive detection of tobacco-specific nitrosamines in tobacco, tobacco products, and smoke.

[0004] Fluorescent probe technology, developed in recent years, boasts unique selectivity, high sensitivity, simple and rapid operation, and minimal damage to the target. It has been widely used to detect common heavy metals, anions, and reactive small molecules such as active sulfur, reactive oxygen, and activated sulfur in environmental and biological systems. Fluorescent probe detection not only overcomes the shortcomings of traditional instrumental analysis methods, such as high sample and reagent requirements, difficulty in real-time online analysis, cumbersome procedures, and unsuitability for biological and toxicological studies, but also offers numerous advantages over traditional detection methods. First, it offers unparalleled selectivity and specificity, enabling the specific identification of certain small molecules and ions. Second, its high sensitivity results in lower detection limits (LDLs) that surpass those of traditional methods, enabling trace detection of the target substances. Fourth, compared to traditional instrumental methods, fluorescent probes offer the advantages of simple operation and real-time online detection, avoiding sample loss and other interfering factors during pretreatment. Therefore, the development of rapid fluorescent probe-based detection methods for TSNAs in tobacco and tobacco products holds great practical value and scientific significance. Summary of the Invention

[0005] In response to the above technical problems, the present invention proposes a fluorescent probe for detecting nitrosamines, a preparation method thereof, and an 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. After stirring at room temperature, the fluorescent probe P1 is prepared.

[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 to detect nitrosamines in tobacco.

[0014] The 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 form an intermediate containing phosphorus nitrogen ylide (compound 2). Compound 2 undergoes rapid intramolecular acetyl conversion and hydrolysis to form compound 3 containing sulfinic acid amine and simultaneously releases a fluorescent group, 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 detection 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 of 4-N-nitrosomethylamino-1-(3-pyridyl)butanone and the fluorescence intensity. 520 nm =16.66C+2889.01, the unit of C is μmol / L;

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

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

[0020] In the above step (1), the concentration of the Tris-HCl buffer solution is 20-50 mmol / L, and the pH is 7.4. For example, the concentration of the Tris-HCl buffer solution is 20-40 mmol / L; the concentration of the Tris-HCl buffer solution is 30-50 mmol / L; the concentration of the Tris-HCl buffer solution is 20-30 mmol / L; the concentration of the Tris-HCl buffer solution is 30-40 mmol / L; the concentration of the Tris-HCl buffer solution is 40-50 mmol / L; the concentration of the Tris-HCl buffer solution is 20 mmol / L, 30 mmol / L, 40 mmol / L, or 50 mmol / 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):3. The volume ratio of methyl sulfoxide is (5-8):7; the volume ratio of Tris-HCl buffer solution to dimethyl sulfoxide in the mixed solution A is 5:(2-5); the volume ratio of Tris-HCl buffer solution to dimethyl sulfoxide in the mixed solution A is 7:(2-5); the volume ratio of Tris-HCl buffer solution to dimethyl sulfoxide in the mixed solution A is 8:(2-5); and the volume ratio of Tris-HCl buffer solution to dimethyl sulfoxide in the 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 2-15 mmol / L. For example, the concentration of the 4-N-nitrosomethylamino-1-(3-pyridyl)butanone solution is 2-5 mmol / L; the concentration of the 4-N-nitrosomethylamino-1-(3-pyridyl)butanone solution is 5-10 mmol / L; the concentration of the 4-N-nitrosomethylamino-1-(3-pyridyl)butanone solution is 10-15 mmol / L; and the concentration of the 4-N-nitrosomethylamino-1-(3-pyridyl)butanone solution is 2 mmol / L, 4 mmol / L, 5 mmol / L, 6 mmol / L, 7 mmol / L, 8 mmol / L, 9 mmol / L, 10 mmol / L, 11 mmol / L, 12 mmol / L, 13 mmol / L, 14 mmol / L, or 15 mmol / 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 of its kind for NNK detection. Compared to fluorescence-quenched probes, fluorescence-enhanced probes have better specificity, better interference resistance, and higher sensitivity. The linear range of NNK concentration for the fluorescence-enhanced probe used for NNK detection is 0-45 μmol / L, with a minimum detection limit of 1.5×10 -6 mol / L, the detection process is simple, anti-interference ability is strong, fast, sensitive, the detection results are 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 following briefly introduces the drawings required for use in the embodiments or the description of the prior art. 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 any 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 in recognizing NNK.

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

[0035] The following will clearly and completely describe the technical solutions of the present invention in conjunction with the embodiments of the present invention. Obviously, the embodiments described are only some embodiments of the present invention, not all 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, and metal ions used in the preparation of fluorescent probe P1 were purchased from Aladdin Reagents. During the validation and performance testing of fluorescent probe P1, a Bruke DTX-300 nuclear magnetic resonance spectrometer was used, using deuterated chloroform as the solvent and TMS as the internal standard to record H and C NMR spectra. High-resolution mass spectrometric data were recorded using a Thermo Scientific Q-Exactive HR-MS mass spectrometer.

[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, a 10 mL 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 to obtain 350 mg of a white foamy solid with a yield of 38.5%. 31 P NMR (122 MHz, 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, a 10 mL 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 detected by TLC. The solution was washed three times with saturated brine, and the organic phase was dried over anhydrous magnesium sulfate, filtered, concentrated, and purified by column chromatography to obtain 277.3 mg of a white foamy solid with a yield of 30.5%. 31 P NMR (122 MHz, 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, a 10 mL 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 to obtain 376.3 mg of a white foamy solid with a yield of 41.4%. 31 P NMR (122 MHz, 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 100 mL round-bottom flask, 2-(diphenylphosphino)benzoic acid (1.838 g, 6 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, a 10 mL 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 to obtain 392.7 mg of a white foamy solid with a yield of 43.2%. 31 P NMR (122 MHz, 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 100 mL round-bottom flask, 2-(diphenylphosphino)benzoic acid (2.450 g, 8 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, a 10 mL 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 detected by TLC. The solution was washed three times with saturated brine, and the organic phase was dried over anhydrous magnesium sulfate, filtered, concentrated, and purified by column chromatography to obtain 453.6 mg of a white foamy solid with a yield of 49.9%. 31 P NMR (122 MHz, 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 examples

[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. A mixed solution A with a volume ratio of 7:3 was prepared using the above buffer solution and DMSO. A probe P1 solution with a concentration of 1 mM was also prepared using DMSO. The selectivity of probe P1 for NNK in mixed solution A was tested using a fluorescence spectrometer. Figure 1 As shown in the figure, under the excitation condition of 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 in 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 triarylphosphine-containing compound 1 to generate an intermediate (compound 2) containing a phosphorus nitrogen ylide. Compound 2 undergoes rapid intramolecular acetyl conversion and hydrolysis to generate compound 3 containing a sulfenic acid amine and simultaneously releases a fluorescent group, thereby achieving rapid and efficient identification of NNK. The structure of the product R'OH was confirmed by high-resolution mass spectrometry (HR-MS) (see Appendix). 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 performance 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. Use DMSO to prepare a probe P1 solution with a concentration of 1mM. In 22 clean fluorescence cuvettes, add 3000μL of mixed solution A and 30μL of probe P1 solution, 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 a histogram of the 520 nm fluorescence intensity corresponding to different analytes was drawn 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 other analytes mentioned above 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, and a mixed solution A with a volume ratio of 7:3 was prepared using the above buffer solution and DMSO. A probe P1 solution with a concentration of 1 mM was also 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 the following order: 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 solution was ultrasonically vibrated in an ultrasonic cleaning machine for a period of time. The fluorescence emission intensity at 520 nm was measured on a fluorescence spectrometer with an excitation wavelength of 490 nm. The NNK concentration was used as the horizontal axis and the fluorescence intensity at 520 nm was used as the vertical axis to obtain a working curve of the NNK concentration. 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

[0063] A good detection limit is one of the criteria for testing whether a probe molecule has application value. A Tris-HCl buffer solution with a pH of 7.4 and a concentration of 40mM was prepared. A mixed solution A with a volume ratio of 7:3 was prepared using the above buffer solution and DMSO. A probe P1 solution with a concentration of 1mM was prepared using DMSO. The concentration of the fixed probe P1 was 10μmol / L, and its response intensity to different concentrations of NNK was measured. As the concentration of NNK increased, the fluorescence emission intensity of the system at 520nm continued to increase. 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] Tobacco leaf samples were placed in an oven at 40°C for 6 hours with exhaust air, ground into powder, and passed through a 60-mesh sieve. 1.0 g of tobacco sample was weighed and placed in a 50 mL conical tube. 30 mL of 100 mmol / L ammonium acetate solution was added and shaken at room temperature (200 rpm) for 1 hour. The extract was then centrifuged at high speed and used for NNK determination. 100 μL of a 300 μmol / L fluorescent probe P1 solution was added to a cuvette. Then, 2850 μL of mixed solution A (pH 7.4, 40 mM Tris-HCl buffer, prepared with this buffer and DMSO in a 7:3 volume ratio) and 50 μL of tobacco extract were added to the cuvette. A blank control was replaced with the buffer solution. The mixture was thoroughly mixed and allowed to react at room temperature for 30 minutes. Three replicates were performed. Fluorescence spectrometer measurements were performed using an excitation wavelength of 490 nm and an emission wavelength of 520 nm. The measured values ​​were plugged into the working curve to calculate the NNK content of the tobacco leaves.

[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 principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A fluorescent probe for detecting 4-N-nitrosomethylamino-1-(3-pyridyl)butanone in tobacco, characterized in that: The structural formula of the fluorescent probe is as follows: 。 2. A method for detecting 4-N-nitrosomethylamino-1-(3-pyridyl)butanone in tobacco, characterized in that: Here are the steps: (1) The fluorescent probe is dissolved in dimethyl sulfoxide to prepare a probe solution; a Tris-HCl buffer solution is mixed with dimethyl sulfoxide to obtain a mixed solution A; and a 4-N-nitrosomethylamino-1-(3-pyridyl)butanone solution is prepared with dimethyl sulfoxide; wherein the structural formula of the fluorescent probe is as follows: ; (2) Add the probe solution to the mixed solution A, and then add the 4-N-nitrosomethylamino-1-(3-pyridyl)butanone solution to obtain the detection solution; use a fluorescence spectrometer to measure the fluorescence emission intensity at 520 nm to obtain the working curve F of the 4-N-nitrosomethylamino-1-(3-pyridyl)butanone concentration and fluorescence intensity. 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.

3. The method for detecting 4-N-nitrosomethylamino-1-(3-pyridyl)butanone in tobacco according to claim 2, wherein: 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.

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

5. The method for detecting 4-N-nitrosomethylamino-1-(3-pyridyl)butanone in tobacco according to claim 4, characterized in that: The final concentration of the fluorescent probe in step (2) and step (3) is 10 µmol / L.

6. The method for detecting 4-N-nitrosomethylamino-1-(3-pyridyl)butanone in tobacco according to claim 5, characterized in that: In step (2), the final concentration of 4-N-nitrosomethylamino-1-(3-pyridyl)butanone in the detection solution is 0-45 µmol / L.

7. The method for detecting 4-N-nitrosomethylamino-1-(3-pyridyl)butanone in tobacco according to claim 6, characterized in that: When measuring with a fluorescence spectrometer in steps (2) and (3), the excitation wavelength is 490 nm.

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