A method for detecting sulfur dioxide based on fluorine nuclear magnetic resonance

Abstract

The application relates to a fluorine nuclear magnetic resonance-based sulfur dioxide detection method, which first provides a high-sensitivity fluorine probe P1-CF3, which can rapidly react with sulfur dioxide, and the product formed is collected by nuclear magnetic resonance 19 F nuclear magnetic spectrum, can produce signal peaks at chemical shifts of -61.09ppm and -61.30ppm; further, the product yield can be measured according to the signal peak area, so as to judge the sulfur dioxide content in the sample. The fluorine probe P1-CF3 and the nuclear magnetic resonance detection method can realize qualitative and quantitative detection of the sulfur dioxide content in environmental samples or food samples, and the probe can also be used for detecting intracellular sulfur dioxide.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of detection, and particularly relates to a sulfur dioxide detection method based on fluorine nuclear magnetic resonance. BACKGROUND

[0002] Reactive sulfur species (RSS) is a kind of sulfur-containing compounds (including biological thiols, sulfur dioxide, persulfide, etc.) in biological systems, which plays an important role in the body as an antioxidant and a signal transduction agent. Sulfur dioxide (SO2) is generally considered to be an atmospheric pollutant, but it is widely used as a bleaching agent, preservative, antioxidant, etc. in the field of food processing. In recent years, sulfur dioxide has been revealed to be the fourth gas signal molecule after nitric oxide (NO), hydrogen sulfide (H2S) and carbon monoxide (CO), which participates in the regulation of various physiological functions and pathological processes. In mammalian cells, the endogenous generation of sulfur dioxide is mainly produced by the metabolism of sulfur-containing amino acids. In addition, hydrogen sulfide oxidation 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 the action of thiosulfate sulfur transferase and glutathione. Sulfur dioxide is easily soluble in water and mainly exists in the form of sulfite and bisulfite. Current studies have reported that sulfur dioxide is involved in cell proliferation, apoptosis, oxidative stress, inflammatory response, endoplasmic reticulum stress and other physiological processes. Therefore, accurate and rapid quantitative detection of sulfur dioxide is of great significance for environmental protection, food safety and human health.

[0003] Current sulfur dioxide detection methods include titration, flow injection, capillary electrophoresis, chromatography and fluorescence detection method, etc., although each has its own advantages, but still faces many challenges. Among them, the fluorescence probe is the only method that can detect intracellular sulfur dioxide. However, the presence of various biological thiols in cells and background signal interference may affect the accurate detection of sulfur dioxide by the fluorescence probe. Therefore, it is of great significance to develop a new method for detecting sulfur dioxide in the environment and cells at the atomic resolution level. SUMMARY

[0004] To solve the above technical problems, the application provides a sulfur dioxide detection method based on fluorine nuclear magnetic resonance.

[0005] The technical scheme adopted by the application is that a sulfur dioxide detection method based on fluorine nuclear magnetic resonance is provided, a fluorine probe with a structure as shown in formula 1 is added to a sample to be detected, and a F nuclear magnetic spectrum is collected. 19 The signal peak between chemical shifts 61-62ppm is analyzed to determine whether sulfur dioxide is contained in the sample to be detected.

[0006]

[0007] Preferably, analyze the signal peak condition at chemical shift -61.09ppm and -61.30ppm.

[0008] Preferably, the sulfur dioxide content is quantified according to the signal peak area.

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

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

[0011] Step two: transfer the sample into a nuclear magnetic tube and collect the nuclear magnetic signal;

[0012] Step three: observe the signal peak intensity at chemical shift -61.09ppm and -61.30ppm, and judge the sulfur dioxide content.

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

[0014] Preferably, the sulfur dioxide content in the sample to be tested is calculated according to 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 the fluorine nuclear magnetic resonance-based sulfur dioxide detection method in intracellular endogenous sulfur dioxide detection.

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

[0018] The present application has the advantages and positive effects that the fluorine probe can rapidly react with sulfur dioxide, the combination 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 real concentration of sulfur dioxide in the sample; therefore, the method is not only suitable for sulfur dioxide detection in environmental samples and food, but also can realize quantitative detection of intracellular endogenous sulfur dioxide, and has wide applicability. BRIEF DESCRIPTION OF DRAWINGS

[0020] Figure 1 Fluorine nuclear magnetic resonance spectrum of fluorine probe for quantitative detection of sulfur dioxide; (a) analysis of reaction product of fluorine probe and sodium sulfite; (b) linear fitting curve of product peak integral area and sodium sulfite concentration;

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

[0022] Figure 3 Results of sulfur dioxide detection in commercially available beer and wine; (a) Comparison of fluorine NMR spectra of different brands of wine and beer samples after reaction with fluorine probe; (b) Determination of sulfur dioxide content in different food samples;

[0023] Figure 4 Fluorine probes were used to detect the fluorine spectrum of sulfur dioxide in cells. Detailed Implementation

[0024] The embodiments of the present invention will now be described with reference to the accompanying drawings.

[0025] This invention relates to a method for detecting sulfur dioxide based on fluorine nuclear magnetic resonance. First, it provides a highly sensitive fluorine probe P1-CF3, with the structure shown in Formula 1, which 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 products were collected by nuclear magnetic resonance. 19 The F-NMR spectrum can produce signal peaks at chemical shifts of -61.09 ppm and -61.30 ppm; further, the product concentration can be measured based on the peak area, thereby determining the sulfur dioxide content in the sample. Qualitative and quantitative analysis of sulfur dioxide content in samples can be achieved using the fluorine probe P1-CF3 and NMR detection methods. This probe can also be used for the detection of intracellular sulfur dioxide.

[0028] During detection, the analyte was added to a phosphate buffer solution at pH 7.5, followed by the addition of a fluorine probe. The reaction was carried out at room temperature. The reaction mixture was transferred to an NMR tube for NMR signal acquisition. The number of scans for the fluorine spectrum was adjusted according to the sample concentration (64 or 128 scans in the buffer solution), with a spectral width of 40 ppm and a spectral center position of -65 ppm. The sulfur dioxide in the sample was qualitatively and / or quantitatively analyzed based on the NMR spectrum. Multiple experiments verified that the fluorine probe-sulfur dioxide conjugate exhibits high stability and high selectivity for sulfur dioxide. Compared with other sulfur-containing compounds, this fluorine probe shows higher selectivity for sulfur dioxide, and the adduct exhibits a unique chemical shift, with product signal peaks at -61.09 ppm and -61.30 ppm.

[0029] The fluorine nuclear magnetic resonance-based sulfur dioxide detection method is suitable for various environmental samples or food samples, such as liquid samples such as red wine, beer, river water and seawater; and can also be used for detecting endogenous sulfur dioxide in organisms, and based on the high selectivity of the fluorine probe to sulfur dioxide, the influence of other types of biological thiols in cells can be avoided. Through the high selectivity and fast reaction kinetics of the high-sensitivity fluorine probe to sulfur dioxide, the dynamic changes of endogenous sulfur dioxide in cells can also be tracked.

[0030] The present application will be described below in conjunction with the accompanying drawings, wherein the experimental methods of the operation steps are not specifically described, and are performed according to the corresponding product instructions. The instruments, reagents and consumables used in the examples can be purchased from commercial companies unless otherwise specified.

[0031] Example 1: Preparation of high-sensitivity fluorine probe P1-CF3

[0032]

[0033] Prepared according to the following reaction formula;

[0034]

[0035] 2-trifluoromethyl nicotinic acid was dissolved in dichloromethane, a drying tube was connected, and oxalyl chloride and 0.1 mL of N'N-dimethylformamide were added at room temperature; the molar ratio of 2-trifluoromethyl nicotinic acid to oxalyl chloride was 1:2; stirring for 4 h, concentrating the reaction solution, and vacuum drying to remove excess oxalyl chloride. Under ice bath conditions, 30 mL of methanol was slowly added to the reaction bottle (the reaction was exothermic), and the addition was completed in 10 minutes. Under this condition, stirring was performed for 0.5 h, TLC showed that the reaction was complete, and the reaction solution was rotary evaporated; 100 mL of ethyl acetate was added, the organic phase was washed with saturated sodium bicarbonate and water (3×20 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated and purified by column to obtain compound 2.

[0036] Compound 2 was dissolved in anhydrous methanol, and sodium borohydride was added in batches under ice bath stirring; the molar ratio of compound 2 to sodium borohydride was 1:5; after the addition was completed, the ice bath was removed, and the reaction was carried out at room temperature for about 1 h; TLC showed that the reaction was complete, and 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, the organic phase was washed with saturated brine (3×20 mL), the organic phase was separated, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated and purified by column to obtain compound 3.

[0037] Compound 3 was dissolved in anhydrous dichloromethane, and manganese dioxide was added at room temperature; the molar ratio of compound 3 to manganese dioxide was 1:10; the temperature was increased to 45°C for stirring reaction; after 24 h, TLC showed that the reaction was complete, the heating and stirring were stopped, and the reaction was cooled. Filtration was performed with diatomite, the filter cake was washed with dichloromethane, the filtrate was collected,

[0038] Concentrate and purify by column chromatography to give compound 4.

[0039] Dissolve compound 4 in 10 mL of anhydrous ethanol, and then add cyanacetamide and 1-methylpiperazine in a molar ratio of 1:1.5:1, respectively. Stir the reaction at room temperature. After about 2 h, TLC shows that the reaction is complete, and stop stirring. Concentrate the reaction solution, add 50 mL of ethyl acetate, wash the organic phase with saturated brine (3 x 10 mL), dry the organic phase over anhydrous sodium sulfate, filter, concentrate the filtrate, and purify by column chromatography to give P1-CF3. 1 H NMR (400 MHz, CDC13) δ 8.85 (d, J = 3.6 Hz, 1H), 8.72 (s, 1H), 8.39 (d, J = 7.7 Hz, 1H), 7.79 - 7.62 (m, 1H), 6.47 (d, J = 13.6 Hz, 2H). 13 C NMR (101 MHz, DMSO-d6) δ ppm: 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 product of fluorine probe and sulfur dioxide

[0041] Sulfur dioxide is easily soluble in water and forms HSO3 - and SO3 2- in water. Sodium sulfite can be used instead of sulfur dioxide to prepare the standard curve.

[0042] Dissolve the fluorine probe prepared in Example 1 in DMF to prepare a probe stock solution with an initial concentration of 100 mM; and dissolve sodium sulfite in ultrapure water to prepare a sodium sulfite stock solution with a concentration of 100 mM. Prepare sodium sulfite with final concentrations of 0 μM, 5 μM, 10 μM, 20 μM, 50 μM, and 100 μM in 20 mM phosphate buffer (pH 7.5), respectively, and add 0.15 mM of the probe thereto, and collect fluorine nuclear magnetic resonance spectra. As shown in FIG. 1, after the reaction of the probe with sodium sulfite, product signal peaks are generated at -61.09 ppm and -61.30 ppm; and as the concentration of sodium sulfite increases, the intensity of the product peaks gradually increases. Further, fluorine nuclear magnetic resonance spectra are collected at different times, and the intensity of the product peaks does not change, indicating that the product of the combination of the probe and sulfur dioxide has strong stability. Figure 1

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

[0044] ​To explore the selectivity of the fluorine probe to sulfur dioxide, the fluorine probe was used to detect other active sulfur species. Among them, other active sulfur species were selected as cysteine, homocysteine, cysteine glycine, glutathione, sodium sulfide, cysteine sulfinic acid, taurine or hypotaurine. Other active sulfur species were dissolved in ultrapure water to prepare a stock solution with a concentration of 100 mM. Then, active sulfur species with a final concentration of 0.5 mM were prepared in 20 mM phosphate buffer, pH 7.5, and 0.5 mM probe was added to each sample, respectively, for fluorine nuclear magnetic resonance spectrum acquisition. If the probe reacts with active sulfur species, a signal other than the probe will be observed in the fluorine spectrum. The experimental results are shown in Figure 2 -61.09 ppm and -61.30 ppm, only the sodium sulfite sample showed corresponding signal peaks. Other active sulfur species, such as biological sulfhydryl, sodium sulfide, reacted reversibly with the probe and generated products with different chemical shifts from sulfur dioxide. Cysteine sulfinic acid, taurine or hypotaurine did not produce corresponding signal peaks when reacted with the fluorine probe. It can be seen that the probe has high selectivity for sulfur dioxide, and the chemical shift of the generated product is significantly different from other active sulfur species.

[0045] Example 4: Detection of sulfur dioxide in commercially available beer and wine by fluorine probe

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

[0047] Example 5: Detection of sulfur dioxide in cells by fluorine probe

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

[0049] The results are as follows Figure 4 As shown, product signals similar to those observed in vitro were observed in the samples. The collected cell samples were centrifuged at 200g for 3 min, and the supernatant and cell pellet were collected separately. Both the supernatant and cell pellet were resuspended in 150 μL DMEM. Cell lysates were obtained from the cell pellet using a repeated freeze-thaw method. Signals at -61.09 and -61.30 ppm positions were collected in the supernatant and lysates, respectively. The results are shown below. Figure 4 As shown, this indicates that the probe can be successfully used for the simultaneous quantitative detection of sulfur dioxide inside and outside cells.

[0050] The embodiments of the present invention have been described in detail above, but the content described is only a preferred embodiment of the present invention and should not be considered as limiting the scope of the present invention. All equivalent changes and improvements made within the scope of the present invention should still fall within the patent coverage of the present invention.

Claims

1. A method for detecting sulfur dioxide based on fluorine nuclear magnetic resonance, characterized by: The fluorine probe with structure as shown in formula 1 is added into the sample to be tested, and the signal peaks of the sample to be tested are collected 19 F nuclear magnetic spectrum, whether the sample to be tested contains sulfur dioxide is judged by analyzing the signal peaks between chemical shifts 61-62ppm; 2. The method of claim 1, wherein the method is a fluorine nuclear magnetic resonance based sulfur dioxide detection method. The signal peaks at chemical shift -61.09 ppm and -61.30 ppm are analyzed.

3. The method of claim 2, wherein the method is a fluorine nuclear magnetic resonance based sulfur dioxide detection method. The sulfur dioxide content is quantified according to the signal peak area.

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

5. The method of claim 4, wherein the method is a fluorine nuclear magnetic resonance based sulfur dioxide detection method. The spectrum width in the fluorine spectrum collection condition is 40 ppm, and the spectrum center position is -65 ppm.

6. The method of claim 4, wherein the method is a fluorine nuclear magnetic resonance based sulfur dioxide detection method. 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 according to any one of claims 1 to 3, 5 and 6, wherein: The sample to be tested is an environmental sample or a food sample.

8. The fluorine nuclear magnetic resonance-based sulfur dioxide detection method according to any one of claims 1-6 is applied to the detection of endogenous sulfur dioxide in cells.

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

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