CN113150771A - Chitosan-based fluorescent probe for detecting sulfur dioxide and preparation method and application thereof - Google Patents
Chitosan-based fluorescent probe for detecting sulfur dioxide and preparation method and application thereof Download PDFInfo
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Abstract
The invention discloses a chitosan-based fluorescent probe for detecting sulfur dioxide and a preparation method and application thereof, wherein the molecular structural formula of the probe is as follows:
Description
Technical Field
The invention belongs to the technical field of analysis and detection, and particularly relates to a chitosan-based fluorescent probe for detecting sulfur dioxide, and a preparation method and application thereof.
Background
Sulfur dioxide (SO)2) Is widely considered as one of the main gases causing air pollution, and has been found to participate in many physiological activities and some biological functions of the human body in recent years. In living beingsIn the organic body, SO2Is a 4 th gas signal molecule, endogenous SO, other than nitric oxide, carbon monoxide and hydrogen sulfide2Can be produced by biosynthesis of sulfur-containing amino acids such as cysteine and glutathione to maintain biological sulfur balance. Toxicological and epidemiological studies have demonstrated that endogenous SO2Is closely related to various diseases such as respiratory diseases, nervous system diseases, cardiovascular diseases and cancers, for example, SO2As a gas signaling molecule, studies have shown that it is involved in vasodilation, regulating blood insulin levels, maintaining redox balance. When sulfur dioxide is inhaled into human body and passes through respiratory system, the sulfur dioxide exists in the form of Sulfite (SO)3 2-) And bisulfite ion (HSO)3 -)。
In various tests of SO2Among the methods of (1), fluorescence spectroscopy is attracting attention because of its advantages such as low cost, high sensitivity, and good selectivity. Therefore, various fluorescent probes are developed by using different fluorescent groups such as rhodamine, BODIPY, anthocyanin and carbazole and the like and ICT recognition mechanisms and the like. There are some disadvantages, such as poor donor diversity, short Stokes shift, and exposure to GSH, Hcy, Cys, or H2Interference of S, etc. When detecting SO2When the fluorescent probe is applied to organisms, the defects of poor biocompatibility and poor stability of the fluorescent probe exist, and most of artificially synthesized fluorescent or colorimetric probes are organic compounds which have the characteristic of low solubility in water.
Chitosan is a rich natural biopolymer, and is a deacetylated product of chitin, a large molecular chain of the chitosan has a plurality of hydroxyl and carboxyl, and the chitosan contains hydrophilic groups and hydrophobic groups, and meanwhile, hydrogen bonds among skeleton chains form a secondary structure of a large molecule. The structural particularity of the chitosan and the physical characteristics of the macromolecular compound ensure that the chitosan and a plurality of ions, organic matters, biological molecules and the like have the functions of ion exchange, chelation, adsorption and the like. Chitosan has not only excellent properties such as versatility, biocompatibility, biodegradability and low cytotoxicity, but also unique ability of crossing cell membrane, and is attracting attention. However, chitosan is only soluble in dilute acid and some specific solvents, so that the application of the chitosan is greatly limited. Active hydroxyl and amino in the main chain of the chitosan can form various multifunctional structures through simple chemical modification, and the chitosan can be enzymolyzed in organisms into micromolecular substances which are easy to be absorbed by organisms and have no toxic or side effect, can not remain in the organisms, and is a biodegradable and absorbable high polymer material. Primary amine groups in chitosan chains easily react with biomolecules, such as proteins, DNA, enzymes, antigen antibodies, biotin, fluorescent molecule isothiocyanate (FITC) and the like, and are likely to be good substrates for coupling fluorescent molecules and biological recognition molecules.
In summary, the research and development of the method for detecting SO in the living body with good selectivity, high sensitivity, high stability and good biocompatibility2The fluorescent probe has important significance.
Disclosure of Invention
Aiming at detecting SO in the prior art2The invention provides a chitosan-based fluorescent probe for detecting sulfur dioxide, a preparation method and application thereof, and the prepared fluorescent probe has good selectivity, high stability and good biocompatibility and can be used for detecting SO in organisms2And carrying out effective detection.
The invention is realized by the following technical scheme:
a chitosan-based fluorescent probe for detecting sulfur dioxide, wherein the structure of the ratio-type fluorescent probe is shown as CS-MAE:
in the invention, the preparation method of the chitosan-based fluorescent probe for detecting sulfur dioxide comprises the following steps:
(1) adding chitosan into NaOH aqueous solution, stirring and heating to 50-70 ℃, reacting for 20-30 h, filtering reaction liquid, repeatedly washing to be neutral, and freeze-drying to obtain deacetylated chitosan;
(2) adding deacetylated chitosan into an acetic acid aqueous solution, stirring and heating to 45-60 ℃, adding hydrogen peroxide in batches after complete dissolution, stopping heating, filtering to remove insoluble impurities, adjusting the pH value to be more than 10 by using sodium hydroxide, adding ethanol with the same volume, standing overnight, centrifuging a lower-layer precipitate, cleaning, and freeze-drying to obtain degraded chitosan;
(3) adding the degraded chitosan into acetic acid aqueous solution for dissolving, diluting with methanol after complete dissolution, adding succinic anhydride acetone solution, stirring for reaction for 1.5-3 h, standing overnight, performing rotary evaporation, dialyzing until dialysate is neutral, and performing freeze drying to obtain succinylated chitosan;
(4) adding EDC and NHS into a DMSO solution in which a compound MAE is dissolved, reacting for 3-5 h in a dark place, adding into a succinylated chitosan hydrochloric acid solution, reacting for 10-14h in a dark place at room temperature, dialyzing the reaction product with water for 2-3 days, and freeze-drying to obtain a chitosan-based fluorescent probe for detecting sulfur dioxide;
the MAE in the step (4) is prepared by the following method:
a) dissolving a compound a2 in acetonitrile, adding KI, carrying out heating reflux reaction at 110-130 ℃ for 0.5-2 h, then adding an acetonitrile solution of a compound a1, carrying out heating reflux at 80-100 ℃ for 20-30 h, cooling to room temperature after the reaction is finished, filtering, carrying out rotary evaporation on filtrate, separating and purifying to obtain a compound a;
b) adding chloroform to a compound b1 for dissolving, adding a compound b2 and triethylamine, heating and refluxing at 75-90 ℃ for 4-6 h, cooling to room temperature after the reaction is finished, extracting saturated sodium bicarbonate solution and dichloromethane, performing rotary evaporation, separating and purifying to obtain a compound b;
c) heating and refluxing the compound a and the compound b in an acetonitrile solution at 80-100 ℃ for 12-20 h, performing rotary evaporation, and separating and purifying to obtain a ratio type fluorescent probe MAE for detecting sulfur dioxide;
the synthesis route of the chitosan-based fluorescent probe for detecting sulfur dioxide is as follows:
the synthesis route of the compound MAE is as follows:
further, the NaOH in the step (1) is 50% NaOH aqueous solution; the acetic acid aqueous solution in the step (2) is a 2% acetic acid aqueous solution, and the NaOH is a 10% NaOH aqueous solution; the acetic acid in the step (3) is a 3% acetic acid solution, and the concentration of the succinic anhydride acetone solution is 0.47 mol/L; the hydrochloric acid solution in the step (4) is a 5% hydrochloric acid solution.
Further, 25-30 mLNaOH solution is added into each gram of chitosan in the step (1); adding 30-50mL of acetic acid aqueous solution into each gram of deacetylated chitosan in the step (2), and adding 0.8-1 mL of hydrogen peroxide into each gram of deacetylated chitosan; adding 80-120 mL of acetic acid solution into each gram of degraded chitosan in the step (3), adding 80-120 mL of methanol into each gram of degraded chitosan, and adding 5-8 mL of succinic anhydride acetone solution into each gram of degraded chitosan; adding 10mL of DMSO solution into each gram of MAE in the step (4), wherein the molar ratio of the MAE to EDC to NHS is 1: 1.2; 1.2, the mass ratio of the MAE to the succinylated chitosan is 1: 5.
further, the molar ratio of compound a1 to compound a2 in step a) is 1: 1.2; the molar ratio of compound a2 to KI was 1.3: 1.2; the molar ratio of compound b1 to compound b2 in step b) is 1: 1.2; the molar ratio of the compound b1 to triethylamine in the step b) is 1: 0.8; the molar ratio of compound a to compound b in step c) is 1: 1.3.
Further, the separation and purification method in the step a) is that the solid after rotary evaporation is dissolved by dichloromethane, and column chromatography separation is carried out by using a mixed solvent of dichloromethane and methanol with the volume ratio of 10: 1; the separation and extraction method in the step b) is that the solid after rotary evaporation is dissolved by methanol, and the volume ratio of the solid is 50:1, performing column chromatography separation on the mixed solvent of the petroleum ether and the dichloromethane; the separation and purification method of the step c) is that the solid after rotary evaporation is dissolved by methanol, and the volume ratio is 30:1, performing column chromatography separation by using a mixed solvent of dichloromethane and methanol.
In the invention, the method for detecting sulfur dioxideDetection of SO by using chitosan-based fluorescent probe2The use of (1).
Further, the SO2Is endogenous SO2Or exogenous SO2。
The chitosan-based fluorescent probe provided by the invention detects sulfur dioxide in a manner of fluorescence change and obvious color change. The chitosan-based fluorescent probe can detect endogenous sulfur dioxide and exogenous sulfur dioxide, and when sulfur dioxide is added into the chitosan-based fluorescent probe, the fluorescence intensity rises and the color becomes lighter along with the increase of the concentration of sulfur dioxide. Therefore, the chitosan-based fluorescent probe synthesized by the invention can specifically identify and detect sulfur dioxide. The chitosan carrier provides protection and barrier effects for the small-molecule fluorescent probe, and is used as a better bedding for a medicine carrier in the future.
Advantageous effects
According to the invention, chitosan is used as a carrier, and MAE is modified on the surface of chitosan by utilizing the characteristic of high fluorescence property of MAE, so that a novel chitosan-based fluorescent probe is obtained, wherein the probe has the characteristics of high fluorescence intensity, good stability, good water solubility and high fluorescence quantum yield; in addition, the fluorescent probe is a polysaccharide matrix, has the characteristics of good biocompatibility, small toxicity and good cell compatibility, can be suitable for detecting sulfur dioxide in organisms, and can also provide a hydrophobic environment of chitosan in the organisms so as to achieve a more stable effect.
Drawings
FIG. 1 shows MAE and chitosan-based fluorescent probes1H NMR spectrum;
FIG. 2 shows fluorescence spectra of chitosan-based fluorescent probes under different concentrations of sodium bisulfite (excitation wavelength of the fluorescence spectra is 360nm, emission wavelengths are 420nm and 440nm, and detection wavelengths are 420nm and 440 nm);
FIG. 3 is a graph showing fluorescence intensity values of chitosan-based fluorescent probes after different biomolecules are added;
fig. 4 is a photograph of cell imaging.
Detailed Description
For a further understanding of the invention, reference will now be made to the preferred embodiments of the invention in conjunction with the following examples, but it will be understood that the description is intended to illustrate the features and advantages of the invention further, and not to limit the invention.
Example 1
A chitosan-based fluorescent probe for detecting sulfur dioxide has a structure shown as a formula (CS-MAE):
a preparation method of a chitosan-based fluorescent probe for detecting sulfur dioxide comprises the following steps:
(1) dissolving 2g of compound a2 (3-bromopropionic acid) in 50mL of acetonitrile, adding 2g of KI, refluxing for 1h at 120 ℃, adding 10mL of compound a1 acetonitrile solution (in which 2g of compound a1 is dissolved), refluxing for 24h at 90 ℃, cooling the reaction solution to room temperature after the reaction is completed, filtering, carrying out rotary evaporation on the filtrate, removing the solvent, dissolving the solid with dichloromethane, and carrying out separation and purification by using a mixed solvent column chromatography method of dichloromethane and methanol with the volume ratio of 10:1 to obtain 2.5g of a final reaction product, namely compound a, wherein the yield is 78%; chemical characterization data for compound a are:1H NMR(400MHz,Methanol-d4)：δ8.33(d,J＝ 8.5Hz,1H),8.25(d,J＝9.0Hz,1H),8.17(d,J＝8.2Hz,1H),8.03(d,J＝8.9Hz,1H),7.81(t,J＝ 7.7Hz,1H),7.72(t,J＝7.6Hz,1H),3.31(m,4H),3.16(t,J＝6.5Hz,2H),1.84(s,6H)；13C NMR (101MHz,Methanol-d4)：δ199.41,173.07,139.41,138.61,135.13,132.37,131.03,129.69, 129.07,128.68,124.40,113.96,57.41,45.60,32.24,22.35,14.44(m)；HRMS(ESI)：calcd. C18H20NO2 +[M]+:282.15,found:282.1475；
(2) weighing a compound b 1186 mg, adding the compound b 1186 mg into 30mL of chloroform, stirring the mixture till the compound b2 is completely dissolved, adding 1mL of triethylamine, carrying out reflux reaction at 80 ℃ for 5 hours, cooling the mixture to room temperature after the reaction is finished, extracting the mixture by using a saturated sodium bicarbonate solution and dichloromethane in sequence, removing the solvent by rotary evaporation, dissolving the solid by using methanol, carrying out column chromatography separation by using a mixed solvent of petroleum ether and dichloromethane with the reference ratio of 50:1 to obtain a final reaction product, namely 98mg of yellow solid, namely a chemical reaction productCompound b, yield 75%, chemical characterization data as1H NMR(400MHz,DMSO-d6)：δ10.15(m,1H),8.95(m,1H), 8.67(d,J＝8.0Hz,1H),8.47(d,J＝7.9Hz,1H),8.34(d,J＝8.0Hz,1H),8.05(d,J＝7.8Hz,1H), 7.88(m,1H),7.38(d,J＝7.8Hz,1H)；13C NMR(101MHz,DMSO-d6)：δ192.73,170.31, 154.17(d,J＝19.4Hz),142.09,139.94,137.01,134.00(d,J＝40.6Hz),132.82,130.13,129.72, 128.62,123.60,121.96,120.79(d,J＝6.7Hz),116.36,59.73,20.72,14.05；HRMS(ESI)：calcd. for C17H10N2O6 +[M]+:338.05,found:338.28；
(3) Dissolving 0.5g of the compound a and 0.8g of the compound b in acetonitrile solution, refluxing at 90 ℃ for 18h, removing the solvent from the reaction solution by rotary evaporation after the reaction is finished, dissolving the solid in methanol, and performing column chromatography separation and purification by using a mixed solvent of dichloromethane and methanol with a volume ratio of 30:1 to obtain a final reaction product, namely 0.4g of orange solid, namely MAE, with the yield of 80%. The chemical characterization data are:1H NMR(400MHz,Methanol-d4)：δ8.94(d,J＝2.6Hz,1H),8.66(d,J＝16.7Hz,2H), 8.49(d,J＝9.4Hz,1H),8.30(d,J＝8.7Hz,1H),8.24(dd,J＝8.9,4.9Hz,2H),8.05(t,J＝8.1Hz, 2H),7.82(t,J＝7.6Hz,1H),7.73(s,2H),7.51(m,1H),7.37(d,J＝9.2Hz,1H),5.06(m,9H), 2.92(t,J＝6.6Hz,2H),2.16(s,7H),1.29(s,1H)；13C NMR(101MHz,DMSO-d6)：δ171.55, 166.95,154.10(d,J＝37.4Hz),151.84,142.24,140.12,138.41(d,J＝22.2Hz),136.02,134.01, 133.19,131.01,130.47,129.91(d,J＝22.2Hz),126.69,123.20,122.01,120.96(d,J＝32.0Hz), 116.08,113.41,69.76,67.39,65.00,54.90,53.97(d,J＝2.3Hz),43.06,32.39,29.77,28.33,25.46, 23.22,22.36,18.62,13.86,10.77；HRMS(ESI)：calcd.for C35H28N3O7 +[M]+:602.19,found: 602.1910。
the method for synthesizing the chitosan-based fluorescent probe for detecting sulfur dioxide by using the synthesized MAE as a raw material comprises the following steps:
a) accurately weighing 20g of chitosan powder as a raw material, adding the raw material into a three-necked bottle containing 550mL of 50% NaOH aqueous solution, heating to 60 ℃ in the stirring process, keeping the temperature, stirring for 24 hours, taking out the three-necked bottle, filtering, repeatedly washing the three-necked bottle with triple distilled water to be neutral, freezing and drying in a freeze dryer to finally obtain 18g of deacetylated chitosan powder;
b) weighing 10g of chitosan powder after deacetylation treatment, adding the chitosan powder into a three-neck flask containing 400mL of 2% acetic acid aqueous solution, stirring and heating to 50 ℃, adding 3mL of hydrogen peroxide every 3 hours after chitosan is completely dissolved, reacting for 9 hours, stopping heating, filtering to remove insoluble impurities, dropwise adding a 10% sodium hydroxide solution prepared in advance under stirring until the pH value is 10.5, completely separating out chitosan at the moment, adding ethanol with the same volume, standing overnight, fully settling chitosan, removing a supernatant, centrifuging a lower layer precipitate, washing with tri-distilled water, and freeze-drying to finally obtain 6g of degraded chitosan powder;
c) putting 500mg of degraded chitosan powder into a round-bottom flask, adding 50mL of 3% acetic acid solution, stirring until the chitosan powder is completely dissolved, diluting with 50mL of methanol, adding an acetone solution of succinic anhydride (the succinic anhydride is 140mg and 1.4mmol is dissolved in 3mL of acetone), stirring vigorously for 2h, standing overnight, removing an organic solvent and part of water by rotary evaporation, dialyzing in distilled water by using a 3500 dialysis bag until the dialyzate is neutral, and freeze-drying in a freeze-drying machine to obtain succinylated chitosan (CS-COOH) with the mass of 480 mg;
d) adding 7.2mg of EDC and 4.5mg of NHS into 20mg of compound MAE dissolved in 10mL of DMSO, stirring for 4h under a dark condition, then dropping into 100mg of CS-COOH solution dissolved in 5% hydrochloric acid, reacting for 12h at room temperature in the dark, dialyzing the product with distilled water, and freeze-drying after three days of dialysis to obtain a product CS-MAE;
synthetic route to compound MAE:
the synthesis route of the chitosan-based fluorescent probe comprises the following steps:
as shown in FIG. 1, when H spectra of chitosan and chitosan-based derivatives are compared, chemical shifts of 12.5ppm of carboxylic acid and 7-9 ppm of benzene ring show peaks, and the product CS-MAE has been successfully synthesized.
Example 2
Titration experiment for detecting sulfur dioxide by using chitosan-based fluorescent probe
In Hepes buffer (pH 7.4), chitosan-based ratiometric fluorescent probe was added at an initial concentration of 1mM so that the concentration of the fluorescent probe in the solution was 10 μ M. Then, different amounts of sodium bisulfite with an initial concentration of 1.00mM were added in order to make the concentrations of sodium bisulfite in the solution 5. mu.M, 20. mu.M, 50. mu.M, 80. mu.M, 100. mu.M, 180. mu.M, 250. mu.M, 300. mu.M, respectively, and left to stand for 0.5h without adding sodium bisulfite as a control to sufficiently react the sodium bisulfite with the chitosan-based ratiometric fluorescent probe.
The fluorescence spectra under the conditions of different concentrations of sodium bisulfite were measured by a fluorescence spectrometer, the excitation wavelength of the fluorescence spectra was 360nm, the emission wavelengths were 420nm and 440nm, and the detection wavelengths were 420nm and 440nm, the results are shown in fig. 2, respectively. As can be seen from FIG. 2, the fluorescence intensity at 420nm wavelength gradually increased with the increase of the sulfur dioxide concentration, indicating that the chitosan-based fluorescent probe of the present invention can also respond to sulfur dioxide.
Example 3
Chitosan-based fluorescent Probe for Sulfur dioxide Selectivity test prepared in example 1:
in Hepes buffer (pH 7.4), chitosan-based fluorescent probe was added at an initial concentration of 1mM to give a concentration of 10 μ M of fluorescent probe in solution, and an excess of other bioactive small molecules (S, respectively) was added to the solution2O6 2-、SO4 2-、 NO2-、NO3-、Zn2+、Fe3+、Cu2+、Mg2+、OH-、GSH、Cys、ClO4 -、O2 2-、S2 -、·O2 -、Vc、 (CH3)3COO-、ONOO-、SO3 2-、HSO3-) Measure and measureThe fluorescence spectra after adding different bioactive small molecules are tried, the excitation wavelength is 360nm, the emission wavelength is 440nm, the detection wavelength is 440nm, different bioactive small molecules are used as abscissa, the fluorescence intensity at the detection wavelength of 440nm is used as ordinate, the result is shown in fig. 3, as can be seen from fig. 3, the strong light intensity is obviously enhanced after adding sodium hydrosulfide, and other bioactive small molecules do not interfere with the detection result, which indicates that the chitosan-based fluorescence probe prepared by the invention has higher selectivity to sulfur dioxide.
Example 4
Cell experiment of Chitosan-based fluorescent Probe for detecting Sulfur dioxide prepared in example 1
10 mu L of chitosan-based fluorescent probe with the concentration of 10g/mL is added into a culture dish containing cultured HeLa cells with the concentration of 10g/mL, a confocal microscope is used for shaking uniformly, the excitation wavelength is 405nm, the emission wavelength is 440nm, the detection wavelength is 440nm, photographing observation is carried out every 5min, 10min, 30min and 60min, and the image of the cells after 30min is shown in figure 4 (a).
Similarly, 10. mu.L of 10g/mL chitosan-based fluorescent probe and 10mg/mL sodium bisulfite were added to a culture dish containing 10g/mL cultured HeLa cells, shaken uniformly, photographed and observed by a confocal microscope at an excitation wavelength of 405nm, an emission wavelength of 440nm, a detection wavelength of 440nm, every 5min, 10min, 30min, and 60min, and a cell imaging picture after 30min is shown in FIG. 4 (b).
Similarly, 10 μ L of 10g/mL chitosan-based fluorescent probe and 5mL of sulfur dioxide endogenous stimulator were added to a culture dish containing 10g/mL cultured HeLa cells, shaken uniformly, photographed and observed by a confocal microscope at an excitation wavelength of 405nm, an emission wavelength of 440nm, and a detection wavelength of 440nm every 5min, 10min, 30min, and 60min, and a cell imaging picture after 30min is shown in fig. 4 (c).
Through comparative observation, as can be seen in fig. 4, (in the figure, abc is respectively a figure a showing that the chitosan-based fluorescent probe is imaged, a figure b showing that the chitosan-based fluorescent probe is imaged after sodium hydrosulfide is added, and a figure c showing that the chitosan-based fluorescent probe is imaged after sulfur dioxide endogenous stimulant is added.
It will be apparent to those skilled in the art that various changes and modifications may be made in the present invention without departing from the spirit and scope of the invention. Thus, if such modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include such modifications and variations.
Claims (8)
1. A chitosan-based fluorescent probe for detecting sulfur dioxide is characterized in that the structure of the ratio-type fluorescent probe is shown as CS-MAE:
2. the method for preparing the chitosan-based fluorescent probe for detecting sulfur dioxide according to claim 1, characterized by comprising the following steps:
(1) pretreatment of chitosan: adding chitosan into NaOH aqueous solution, stirring and heating to 50-70 ℃, reacting for 20-30 h, filtering reaction liquid, repeatedly washing to be neutral, and freeze-drying to obtain deacetylated chitosan;
(2) adding deacetylated chitosan into an acetic acid aqueous solution, stirring and heating to 45-60 ℃, adding hydrogen peroxide in batches after complete dissolution, stopping heating, filtering to remove insoluble impurities, adjusting the pH value to be more than 10 by using sodium hydroxide, adding ethanol with the same volume, standing overnight, centrifuging a lower-layer precipitate, cleaning, and freeze-drying to obtain degraded chitosan;
(3) adding the degraded chitosan into acetic acid aqueous solution for dissolving, diluting with methanol after complete dissolution, adding succinic anhydride acetone solution, stirring for reaction for 1.5-3 h, standing overnight, performing rotary evaporation, dialyzing until dialysate is neutral, and performing freeze drying to obtain succinylated chitosan;
(4) adding EDC and NHS into a DMSO solution in which a compound MAE is dissolved, reacting for 3-5 h in a dark place, adding into a succinylated chitosan hydrochloric acid solution, reacting for 10-14h in a dark place at room temperature, dialyzing the reaction product with water for 2-3 days, and freeze-drying to obtain a chitosan-based fluorescent probe for detecting sulfur dioxide;
the MAE in the step (4) is prepared by the following method:
a) dissolving a compound a2 in acetonitrile, adding KI, carrying out heating reflux reaction at 110-130 ℃ for 0.5-2 h, then adding an acetonitrile solution of a compound a1, carrying out heating reflux at 80-100 ℃ for 20-30 h, cooling to room temperature after the reaction is finished, filtering, carrying out rotary evaporation on filtrate, separating and purifying to obtain a compound a;
b) adding chloroform to a compound b1 for dissolving, adding a compound b2 and triethylamine, heating and refluxing at 75-90 ℃ for 4-6 h, cooling to room temperature after the reaction is finished, extracting saturated sodium bicarbonate solution and dichloromethane, performing rotary evaporation, separating and purifying to obtain a compound b;
c) heating and refluxing the compound a and the compound b in an acetonitrile solution at 80-100 ℃ for 12-20 h, performing rotary evaporation, and separating and purifying to obtain a ratio type fluorescent probe MAE for detecting sulfur dioxide;
the synthesis route of the chitosan-based fluorescent probe for detecting sulfur dioxide is as follows:
the synthesis route of the compound MAE is as follows:
3. the method according to claim 1, wherein the NaOH in step (1) is 50% NaOH aqueous solution; the acetic acid aqueous solution in the step (2) is a 2% acetic acid aqueous solution, and the NaOH is a 10% NaOH aqueous solution; the acetic acid in the step (3) is a 3% acetic acid solution, and the concentration of the succinic anhydride acetone solution is 0.47 mol/L; the hydrochloric acid solution in the step (4) is a 5% hydrochloric acid solution.
4. The preparation method according to claim 3, wherein 25-30 ml of NaOH solution is added to each gram of chitosan in step (1); adding 30-50mL of acetic acid aqueous solution into each gram of deacetylated chitosan in the step (2), and adding 0.8-1 mL of hydrogen peroxide into each gram of deacetylated chitosan; adding 80-120 mL of acetic acid solution into each gram of degraded chitosan in the step (3), adding 80-120 mL of methanol into each gram of degraded chitosan, and adding 5-8 mL of succinic anhydride acetone solution into each gram of degraded chitosan; adding 10mL of DMSO solution into each gram of MAE in the step (4), wherein the molar ratio of the MAE to EDC to NHS is 1: 1.2; 1.2, the mass of the MAE and the succinylated chitosan is 1: 5.
5. the process according to claim 3, wherein the molar ratio of compound a1 to compound a2 in step a) is 1: 1.2; the molar ratio of compound a2 to KI was 1.3: 1.2; the molar ratio of compound b1 to compound b2 in step b) is 1: 1.2; the molar ratio of the compound b1 to triethylamine in the step b) is 1: 0.8; the molar ratio of compound a to compound b in step c) is 1: 1.3.
6. The preparation method of claim 2, wherein the separation and purification method in step a) is that the rotary evaporated solid is dissolved by dichloromethane, and the column chromatography separation is carried out by using a mixed solvent of dichloromethane and methanol with a volume ratio of 10: 1; the separation and extraction method in the step b) is that the solid after rotary evaporation is dissolved by methanol, and the volume ratio of the solid is 50:1, performing column chromatography separation on the mixed solvent of the petroleum ether and the dichloromethane; the separation and purification method of the step c) is that the solid after rotary evaporation is dissolved by methanol, and the volume ratio is 30:1, performing column chromatography separation by using a mixed solvent of dichloromethane and methanol.
7. The chitosan-based fluorescent probe for detecting sulfur dioxide in the detection of SO according to claim 12The use of (1).
8. Use according to claim 7, wherein said SO is2Is endogenous SO2Or exogenous SO2。
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Non-Patent Citations (2)
| Title |
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| WANG HAO等: "A dual-site fluorescent probe for separate detection of hydrogen sulfide and bisulfite", 《DYES AND PIGMENTS》 * |
| WANG YONGFEI等: "A ratiometric fluorescence probe for imaging sulfur dioxide derivatives in the mitochondria of living cells", 《ORGANIC & BIOMOLECULAR CHEMISTRY》 * |
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