Sulfur dioxide detection method based on fluorine nuclear magnetic resonance

Abstract

The invention relates to a sulfur dioxide detection method based on fluorine nuclear magnetic resonance, which comprises the following steps: firstly, providing a high-sensitivity fluorine probe P1-CF3 which can quickly react with sulfur dioxide, performing nuclear magnetic resonance on the formed product, collecting a < 19 > F nuclear magnetic spectrum, and generating signal peaks at chemical shifts of-61.09 ppm and-61.30 ppm; furthermore, the product yield can be measured according to the signal peak area so as to judge the sulfur dioxide content in the sample. Through the fluorine probe P1-CF3 and a nuclear magnetic resonance detection method, the content of sulfur dioxide in an environment sample or a food sample can be qualitatively and quantitatively detected, and the probe can also be used for detecting sulfur dioxide in cells.

Description

Technical Field

[0001] The invention belongs to the field of detection technology, and in particular relates to a sulfur dioxide detection method based on fluorine nuclear magnetic resonance. Background Art

[0002] Reactive sulfur species (RSS) are a class of sulfur-containing compounds in biological systems (including biothiols, sulfur dioxide, persulfides, etc.), which play an important role in organisms as antioxidants and signal transducers. 2 ) is generally considered as an air pollutant, but it is widely used in the food processing industry as a bleaching agent, preservative, antioxidant, etc. In recent years, sulfur dioxide has been revealed to be a possible successor to nitric oxide (NO), hydrogen sulfide (H 2 Sulfur dioxide is the fourth gaseous signal molecule after sulfur (S) and carbon monoxide (CO), and is involved in regulating a variety of physiological functions and pathological processes. In mammalian cells, endogenous sulfur dioxide is mainly produced by the metabolism of sulfur-containing amino acids. In addition, hydrogen sulfide can also produce sulfur dioxide, which can be directly oxidized to sulfur dioxide by reduced coenzyme II oxidase, or first oxidized to thiosulfate under the catalysis of sulfide oxidase, and then converted to sulfur dioxide by thiosulfate sulfotransferase and glutathione. Sulfur dioxide is easily soluble in water and exists mainly in the form of sulfite and bisulfite. At present, studies have reported that sulfur dioxide is involved in a variety of physiological processes such as cell proliferation, apoptosis, oxidative stress, inflammatory response, and endoplasmic reticulum stress. Therefore, accurate and rapid quantitative detection of sulfur dioxide is of great significance for environmental protection, food safety and human health.

[0003] Current methods for detecting sulfur dioxide include titration, flow injection, capillary electrophoresis, chromatography, and fluorescence detection. Although each has its own advantages, it still faces many challenges. Among them, fluorescent probes are the only method that can detect sulfur dioxide in cells. However, the presence of a variety of biothiol molecules in cells and background signal interference may affect the accurate detection of sulfur dioxide by fluorescent probes. Therefore, it is of great significance to develop a new method that can detect sulfur dioxide in the environment and in cells at the atomic resolution level. Summary of the invention

[0004] In order to solve the above technical problems, the present invention provides a sulfur dioxide detection method based on fluorine nuclear magnetic resonance.

[0005] The technical solution adopted by the present invention is: a method for detecting sulfur dioxide based on fluorine nuclear magnetic resonance, wherein a fluorine probe with a structure as shown in Formula 1 is added to a sample to be tested, and the sample is collected. 19 F NMR spectrum, by analyzing the signal peak with chemical shift between 61-62ppm, it is determined whether the sample contains sulfur dioxide;

[0006]

[0007] Preferably, the signal peaks at the chemical shifts of -61.09 ppm and -61.30 ppm are analyzed.

[0008] Preferably, the sulfur dioxide content is quantified based on the signal peak area.

[0009] Preferably, the specific steps are as follows:

[0010] Step 1: Add the liquid to be tested into a phosphate buffer at pH 7.5 and add a fluorine probe at a final concentration of 0.15-0.5 mM;

[0011] Step 2: Transfer the sample to the NMR tube and collect the NMR signal;

[0012] Step 3: Observe the signal peak intensity at the chemical shifts of -61.09ppm and -61.30ppm to determine the sulfur dioxide content.

[0013] Preferably, in the fluorine spectrum collection conditions, the spectrum width is 40 ppm and the spectrum center position is -65 ppm.

[0014] Preferably, the sulfur dioxide content in the sample to be tested is calculated based on the signal peak intensity and the standard curve.

[0015] Preferably, the sample to be tested is an environmental sample or a food sample.

[0016] Application of sulfur dioxide detection method based on fluorine nuclear magnetic resonance in the detection of endogenous sulfur dioxide in cells.

[0017] Preferably, one or more samples of cell culture supernatant, cell suspension and cell lysate are tested.

[0018] The advantages and positive effects of the present invention are: the fluorine probe can react quickly with sulfur dioxide, the combined product has high stability, the method can accurately quantify sulfur dioxide with a concentration as low as micromolar, and has high repeatability;

[0019] The fluorine probe has high selectivity for sulfur dioxide, has no background signal interference, and can more accurately reflect the true concentration of sulfur dioxide in the sample; therefore, this method is not only suitable for the detection of sulfur dioxide in environmental samples and foods, but can also realize the quantitative detection of endogenous sulfur dioxide in cells, and has wide applicability. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 Fluorine NMR spectrum of sulfur dioxide quantitatively detected by fluorine probe; (a) Analysis of the reaction product between fluorine probe and sodium sulfite; (b) Linear fitting curve of product peak integrated area and sodium sulfite concentration;

[0021] Figure 2 Selective analysis of fluorine probe and sulfur dioxide; (a) Comparison of fluorine NMR spectra after the reaction of fluorine probe with different active sulfur species; (b) Comparison of relative product peak intensities in Figure a;

[0022] Figure 3 Results of sulfur dioxide detection in commercial beer and red wine; (a) Comparison of fluorine nuclear magnetic resonance spectra after the reaction of fluorine probe with different brands of red wine and beer samples; (b) Determination of sulfur dioxide content in different food samples;

[0023] Figure 4 The fluorine probe detects the fluorine spectrum of intracellular sulfur dioxide. DETAILED DESCRIPTION

[0024] The embodiments of the present invention are described below with reference to the accompanying drawings.

[0025] The present invention relates to a method for detecting sulfur dioxide based on fluorine nuclear magnetic resonance. Firstly, a high-sensitivity fluorine probe P1-CF is provided. 3 , the structure is shown in Formula 1, and it can react rapidly with sulfur dioxide to form a stable addition product. The reaction process is shown in the following formula;

[0026]

[0027] The obtained product was collected by nuclear magnetic resonance. 19 F NMR spectrum can produce signal peaks at chemical shifts of -61.09ppm and -61.30ppm; the product concentration can be further measured based on the signal peak area to determine the sulfur dioxide content in the sample. 3 The nuclear magnetic resonance detection method can realize the qualitative and quantitative analysis of the sulfur dioxide content in the sample. The probe can also be used to detect sulfur dioxide in cells.

[0028] During the test, the test object is added to a phosphate buffer with a pH of 7.5, and the reaction is carried out at room temperature after the fluorine probe is added. The reaction mixture is transferred to a nuclear magnetic resonance tube for nuclear magnetic resonance signal collection. The number of scans for fluorine spectrum collection is adjusted according to the sample concentration (the number of scans in the buffer is 64 or 128), the spectrum width is 40ppm, and the center position of the spectrum is -65ppm. The sulfur dioxide in the sample to be tested is qualitatively and / or quantitatively determined based on the nuclear magnetic resonance spectrum. It has been verified by multiple experiments that the fluorine probe and sulfur dioxide complex has high stability, and the fluorine probe has high selectivity for sulfur dioxide. Compared with other sulfur-containing compounds, the fluorine probe has high selectivity for sulfur dioxide, and the adduct has a unique chemical shift, with product signal peaks at -61.09ppm and -61.30ppm.

[0029] The sulfur dioxide detection method based on fluorine nuclear magnetic resonance is applicable to a variety of environmental samples or food samples, such as red wine, beer, river water, seawater and other liquid samples; it can also be used to detect endogenous sulfur dioxide in organisms. Based on the high selectivity of fluorine probes for sulfur dioxide, it can avoid the influence of other types of biological thiols in cells. Through the high selectivity of highly sensitive fluorine probes for sulfur dioxide and rapid reaction kinetics, it can also be used to track the dynamic changes of endogenous sulfur dioxide in cells.

[0030] The scheme of the present invention is described below in conjunction with the accompanying drawings, wherein the experimental methods without specific operating steps are all carried out in accordance with the corresponding product instructions, and the instruments, reagents, and consumables used in the examples can all be purchased from commercial companies unless otherwise specified.

[0031] Example 1: Highly sensitive fluorine probe P1-CF 3 Preparation

[0032]

[0033] Prepared according to the following reaction formula;

[0034]

[0035] Dissolve 2-trifluoromethylnicotinic acid in dichloromethane, connect a drying tube, add oxalyl chloride and 0.1mL N'N-dimethylformamide at room temperature; the molar ratio of 2-trifluoromethylnicotinic acid: oxalyl chloride is 1:2; stir for 4 hours, concentrate the reaction solution, and remove the excess oxalyl chloride by vacuum drying. Under ice bath conditions, slowly add 30mL of methanol to the reaction bottle (the reaction is highly exothermic), and the addition is completed in 10 minutes. Stir under this condition for 0.5h. TLC shows that the reaction is complete, and the reaction solution is dried by spin drying; add 100mL of ethyl acetate, wash the organic phase with saturated sodium bicarbonate and water (3×20mL), separate the liquids, dry the organic phase with anhydrous sodium sulfate, filter, concentrate the filtrate, and purify by column to obtain compound 2.

[0036] Compound 2 was dissolved in anhydrous methanol, and sodium borohydride was added in batches under stirring in an ice bath, with a molar ratio of compound 2:sodium borohydride of 1:5; after the addition, the ice bath was removed, and the temperature was raised to room temperature for reaction for about 1 h. When TLC showed that the reaction was complete, 2 mL of water was added to quench the reaction; the reaction solution was concentrated (to remove methanol), 100 mL of ethyl acetate was added, and the organic phase was washed with saturated brine (3×20 mL), separated, the organic phase was dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated and passed through a column to obtain compound 3.

[0037] Dissolve compound 3 in anhydrous dichloromethane, add manganese dioxide at room temperature, the molar ratio of compound 3 to manganese dioxide is 1:10; raise the temperature to 45°C and stir to react; after 24 hours, TLC shows that the reaction is complete, stop heating and stirring, and cool. Filter with diatomaceous earth, wash the filter cake with dichloromethane, and collect the filtrate.

[0038] The product was concentrated and passed through a column to obtain compound 4.

[0039] Dissolve compound 4 in 10 mL of anhydrous ethanol, then add cyanoacetamide and 1-methylpiperazine respectively, with the molar ratio of compound 4: cyanoacetamide: 1-methylpiperazine being 1:1.5:1; stir and react at room temperature. After about 2 hours, TLC shows that the reaction is complete, and stirring is stopped. Concentrate the reaction solution, add 50 mL of ethyl acetate, wash the organic phase with saturated brine (3×10 mL), separate the liquids, dry the organic phase with anhydrous sodium sulfate, filter, concentrate the filtrate, and pass it through a column to obtain P1-CF 3 .P1-CF 3 : 1 H NMR (400 MHz, CDCl 3 )δ8.85(d,J=3.6Hz,1H),8.72(s,1H),8.39(d,J=7.7Hz,1H),7.79–7.62(m,1H),6.47(d,J=13.6Hz,2H). 13 C NMR (101MHz, DMSO-d6) δppm: 160.87, 150.66, 145.86, 143.75(q), 138.80, 127.67, 127.53, 121.53(q), 114.76, 114.63.

[0040] Example 2: Analysis of the reaction products of fluorine probe and sulfur dioxide

[0041] Sulfur dioxide is easily soluble in water and forms HSO 3 - and SO 3 2- , sodium sulfite can be used instead of sulfur dioxide to prepare the standard curve.

[0042] The fluorine probe prepared in Example 1 was dissolved in DMF to prepare a probe mother solution with an initial concentration of 100 mM; sodium sulfite was then dissolved in ultrapure water to prepare a sodium sulfite mother solution with a concentration of 100 mM. Sodium sulfite with final concentrations of 0 μM, 5 μM, 10 μM, 20 μM, 50 μM and 100 μM was respectively prepared in 20 mM phosphate buffer (pH 7.5), 0.15 mM probe was added thereto, and fluorine nuclear magnetic resonance spectra were collected. Figure 1As shown in the figure, after the probe reacts with sodium sulfite, product signal peaks are generated at -61.09ppm and -61.30ppm; and as the concentration of sodium sulfite increases, the product peak intensity gradually increases. Furthermore, the fluorine nuclear magnetic resonance spectra collected at different times did not change the product peak intensity, indicating that the product of the combination of the probe and sulfur dioxide has strong stability.

[0043] Example 3: Selectivity experiment of fluorine probe and sulfur dioxide

[0044] In order to explore the selectivity between the fluorine probe and sulfur dioxide, the fluorine probe and other active sulfur species were also used for detection. Among them, other active sulfur species selected were cysteine, homocysteine, cysteinylglycine, glutathione, sodium sulfide, cysteine ​​sulfinic acid, taurine or hypotaurine. The other active sulfur species were dissolved in ultrapure water to prepare a mother solution with a concentration of 100mM. Then, active sulfur species with a final concentration of 0.5mM were prepared in 20mM phosphate buffer, pH 7.5, and 0.5mM probe was added thereto, and fluorine nuclear magnetic resonance spectra were collected. If the probe reacts with the active sulfur species, a signal other than the probe will be observed in the fluorine spectrum. The experimental results are as follows Figure 2 As shown, at -61.09ppm and -61.30ppm, only the sodium sulfite sample showed corresponding signal peaks. The reactions of other active sulfur species, such as biothiol and sodium sulfide with the probe were reversible and the chemical shifts of the generated products were significantly different from those of sulfur dioxide. However, no corresponding signal peaks were generated when cysteine ​​sulfinic acid, taurine or hypotaurine reacted with the fluorine probe. It can be seen that the probe has a high selectivity for sulfur dioxide, and the chemical shifts of the generated products are significantly different from those of other active sulfur species.

[0045] Example 4: Detection of sulfur dioxide in commercial beer and red wine using fluorine probe

[0046] Two types of beer and three types of red wine were purchased as test samples, diluted to appropriate concentrations and added to 20 mM phosphate buffer (pH 7.5). A fluorine probe with a final concentration of 0.2 mM was added to each sample, and the fluorine NMR spectrum was collected. The experiment was repeated three times, and the sulfur dioxide content was determined based on the peak intensity at the chemical shifts of -61.09 ppm and -61.30 ppm. The results are as follows: Figure 3 As shown, the sulfur dioxide contents in these five samples were determined to be: red wine 1 (produced in 2022): 444.8±9.5μM; red wine 2 (produced in 2020): 728.5±18.6μM; red wine 3 (produced in 2016): 13.5±0.6μM; beer 1 (produced in 2024): 4.9±0.1μM; beer 2 (produced in 2024): 24.6±3.1μM.

[0047] Example 5: Detection of intracellular sulfur dioxide using fluorine probe

[0048] HEK293T cells were taken and the sulfur dioxide content in HEK293T cells was detected by fluorine probe. HEK293T cells were cultured in DMEM medium containing 10% FBS, and the culture dish diameter was 10 cm. When the cell density reached more than 90%, the cells were digested with trypsin, collected, washed once with PBS, and counted. The cells were resuspended in DMEM and 1×10 7 Each cell was resuspended in 150 μL solution, to which the fluorine probe was added to a final concentration of 0.2 mM, and the sample was transferred to a 3 mm NMR tube for NMR signal collection.

[0049] The results are as follows Figure 4 As shown, a product signal similar to that in vitro was observed in the sample. The collected cell samples were centrifuged at 200g for 3 minutes, and the supernatant and cell pellet were collected separately. The supernatant and cell pellet were resuspended in 150μL DMEM, and the cell pellet was repeatedly frozen and thawed to obtain cell lysate. The signals at -61.09 and -61.30ppm in the supernatant and lysate were collected respectively. The results are shown in Figure 4 As shown, this probe can be successfully used for the simultaneous quantitative detection of intracellular and extracellular sulfur dioxide.

[0050] The embodiments of the present invention are described in detail above, but the contents described are only preferred embodiments of the present invention and cannot be considered to limit the scope of implementation of the present invention. All equivalent changes and improvements made according to the scope of application of the present invention should still fall within the scope of the patent coverage of the present invention.

Claims

1. A method for detecting sulfur dioxide based on fluorine nuclear magnetic resonance, characterized in that: Add the fluorine probe with the structure shown in Formula 1 to the sample to be tested, and collect 19 F NMR spectrum, by analyzing the signal peak with chemical shift between 61-62ppm, it is determined whether the sample contains sulfur dioxide; 2. The method for detecting sulfur dioxide based on fluorine nuclear magnetic resonance according to claim 1, characterized in that: Analyze the signal peaks at chemical shifts -61.09 ppm and -61.30 ppm.

3. The method for detecting sulfur dioxide based on fluorine nuclear magnetic resonance according to claim 2, characterized in that: The sulfur dioxide content was quantified based on the signal peak area.

4. The method for detecting sulfur dioxide based on fluorine nuclear magnetic resonance according to any one of claims 1 to 3, characterized in that: The specific steps are as follows: Step 1: Add the liquid to be tested into a phosphate buffer at pH 7.5 and add a fluorine probe at a final concentration of 0.15-0.5 mM; Step 2: Transfer the sample to the NMR tube and collect the NMR signal; Step 3: Collect the signal peaks at the chemical shifts of -61.09ppm and -61.30ppm to determine the sulfur dioxide content.

5. The method for detecting sulfur dioxide based on fluorine nuclear magnetic resonance according to claim 4, characterized in that: The fluorine spectrum acquisition conditions were a spectrum width of 40 ppm and a spectrum center position of -65 ppm.

6. The method for detecting sulfur dioxide based on fluorine nuclear magnetic resonance according to claim 4, characterized in that: According to the signal peak intensity and the standard curve, the sulfur dioxide content in the sample to be tested is calculated.

7. The method for detecting sulfur dioxide based on fluorine nuclear magnetic resonance according to any one of claims 1 to 3, 5 and 6, characterized in that: The samples to be tested are environmental samples or food samples.

8. Use of the sulfur dioxide detection method based on fluorine nuclear magnetic resonance according to any one of claims 1 to 6 in the detection of endogenous sulfur dioxide in cells.

9. The use according to claim 8, characterized in that: One or more samples of cell culture supernatant, cell suspension and cell lysate are tested.

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