Fluorescent probe for identifying bisulfite and preparation method and detection method thereof
阅读说明：本技术 一种识别亚硫酸氢根的荧光探针及其制备方法和检测方法 (Fluorescent probe for identifying bisulfite and preparation method and detection method thereof ) 是由 张奇龙 余昌金 吴受婷 张兴 徐红 黄亚励 于 2019-10-25 设计创作，主要内容包括：本发明公开了一种识别亚硫酸氢根的荧光探针及其制备方法和检测方法。其制备是(1)将4-乙烯基吡啶、1,4-二溴-2,3,5,6-四甲基苯和醋酸钯加入三乙胺中混合,反应,得A品；(2)将A品用二氯甲烷完全溶解成黑色悬液,得B品；(3)将B品洗涤,得C品；(4)将C品静置分层,将最下层的二氯甲烷层分离出来后,干燥,旋干,重结晶,得D品；(5)将D品加入N,N-二甲基甲酰胺中溶解,然后加入1-溴癸烷,加热反应,反应液中加入乙醚至其完全沉淀,抽滤,用乙醚洗涤两次,烘干,得到探针。是在水溶液中检测亚硫酸氢根。本发明的荧光探针能够对亚硫酸氢根进行识别,且识别成本低,操作简单,结果可视,灵敏度高,选择性好。(The invention discloses a bisulfite-identifying fluorescent probe and a preparation method and a detection method thereof. The preparation method comprises (1) adding 4-vinylpyridine, 1, 4-dibromo-2, 3, 5, 6-tetramethylbenzene and palladium acetate into triethylamine, mixing, and reacting to obtain product A; (2) completely dissolving product A in dichloromethane to obtain black suspension to obtain product B; (3) washing the product B to obtain product C; (4) standing and layering the product C, separating the dichloromethane layer at the lowest layer, drying, spin-drying, and recrystallizing to obtain product D; (5) and adding the product D into N, N-dimethylformamide for dissolving, then adding 1-bromodecane, heating for reaction, adding diethyl ether into the reaction solution until the diethyl ether is completely precipitated, performing suction filtration, washing twice with diethyl ether, and drying to obtain the probe. Is to detect bisulfite in aqueous solution. The fluorescent probe can identify the bisulfite, and has the advantages of low identification cost, simple operation, visual result, high sensitivity and good selectivity.)
1. A bisulfite-recognizing fluorescent probe is characterized in that the molecular formula of the probe is as follows: c44H66Br2N2Structural formula is：
2. The method for preparing bisulfite-labeled fluorescent probe according to claim 1, comprising the steps of:
(1) adding 4-vinylpyridine, 1, 4-dibromo-2, 3, 5, 6-tetramethylbenzene and palladium acetate into triethylamine for mixing, sealing the mixture in N2Reacting for 18-32h at the temperature of 100-120 ℃ in a high-pressure reaction bottle under the atmosphere to obtain a product A;
(2) completely dissolving product A in dichloromethane to obtain black suspension to obtain product B;
(3) washing the product B with water and saturated saline solution sequentially to obtain product C;
(4) standing and layering the product C, separating the dichloromethane layer at the lowest layer, drying with anhydrous magnesium sulfate, spin-drying, and recrystallizing with ethanol to obtain product D;
(5) and (3) adding the D product into N, N-dimethylformamide for dissolving, then adding 1-bromodecane, heating to 75-85 ℃, reacting for 20-36h, adding diethyl ether into the reaction solution until the diethyl ether is completely precipitated, performing suction filtration, washing twice with diethyl ether, and drying to obtain the probe.
3. The method for preparing bisulfite-labeled fluorescent probes according to claim 2, wherein the method comprises: in the step (1), 150mg of 4-vinylpyridine, 150mg of 1, 4-dibromo-2, 3, 5, 6-tetramethylbenzene and 30mg of palladium acetate were added to each 10mL of triethylamine.
4. The method for preparing bisulfite-labeled fluorescent probes according to claim 2, wherein the method comprises: in the step (3), the product B is washed for 3 times by water and saturated salt solution in sequence.
5. The method for preparing bisulfite-labeled fluorescent probes according to claim 2, wherein the method comprises: in the step (4), anhydrous ethanol is adopted for recrystallization.
6. The method for preparing bisulfite-labeled fluorescent probes according to claim 2, wherein the method comprises: in the step (5), the mass ratio of the D product to the 1-bromodecane is 1:2, the reaction temperature is 80 ℃, and the reaction time is 24 hours.
7. A method for detecting bisulfite using the bisulfite-recognizing fluorescent probe according to claim 1, characterized in that: is to detect bisulfite in aqueous solution.
8. The method for detecting bisulfite with bisulfite-recognizing fluorescent probe according to claim 7, wherein: dissolving a fluorescent probe in methanol, diluting with secondary water to obtain a fluorescent reagent, dripping a sample to be identified into the reagent to obtain a sample solution, carrying out fluorescence excitation on the sample solution, and testing and analyzing the fluorescence wavelength excited by fluorescence.
9. The method for detecting bisulfite with bisulfite-recognizing fluorescent probe according to claim 8, wherein the method is characterized in that: the concentration of the probe in the fluorescent reagent is 10-5mol.L-1The fluorescence excitation wavelength used is 348nm, and when the sample to be identified is added and bisulfite is identified, the fluorescence emission maximum wavelength of the reagent is red shifted from 510nm to 544 nm.
Technical Field
The invention relates to a fluorescent probe and a preparation method and application thereof, in particular to a fluorescent probe for identifying bisulfite and a preparation method and a detection method thereof.
Background
Sulfur dioxide (SO)2) Is one of the most widely distributed atmospheric pollutants in the living environment of human beings, and has great harm to the health of human beings and other living bodies. SO (SO)2Together with hydrocarbons, carbon monoxide, nitrogen oxides and particulate matter, are recognized as the five largest pollutants in air that are most harmful to humans. As a toxic and harmful gas, it can cause acid rain, thus damaging the environment and ecosystem. It can enter into plant through leaf stomatal aperture, enter human or animal body through respiratory system, and then react with water to decompose into its derivative Sulfite (SO)3 2-) And bisulfite (HSO)3 -). In addition, since sulfite prevents bacterial growth and inhibits enzymatic and non-enzymatic browning, sulfite is generally used as an enzyme inhibitor, an antibacterial agent and a food additive in foods, medicines, drinks. Therefore, it is very interesting to develop a highly selective and sensitive method for detecting sulfur dioxide and its derivatives.
At present, various methods are used for detecting sulfur dioxide and derivatives thereof, mainly including titration analysis, chromatography, electrochemical method, capillary electrophoresis, enzyme technology method, spectrophotometry and the like. However, there are a number of limitations common to these methods: the sample pretreatment is troublesome, the operation of the detection tool is complex, the cost is high, and the method is not suitable for biological tissue samples. However, compared with these conventional detection methods, the fluorescent probe analysis method has the advantages of simple operation, low cost, high selectivity, high sensitivity, low detection limit, good biocompatibility and the like, so that the method can be applied to the imaging and detection of organisms. The design of fluorescent probes with good synthesis selectivity, high sensitivity, low detection limit and rapid response is of great significance.
Disclosure of Invention
The invention aims to provide a bisulfite-recognizing fluorescent probe, and a preparation method and a detection method thereof. The fluorescent probe can identify the bisulfite, and has the advantages of low identification cost, simple operation, visual result, high sensitivity and good selectivity.
The technical scheme of the invention is as follows: a fluorescent probe for identifying bisulfite has the molecular formula: c44H66Br2N2The structural formula is shown in figure 1.
The preparation method of the bisulfite-recognizing fluorescent probe comprises the following steps:
(1) adding 4-vinylpyridine, 1, 4-dibromo-2, 3, 5, 6-tetramethylbenzene and palladium acetate into triethylamine for mixing, sealing the mixture in N2Reacting for 18-32h at the temperature of 100-120 ℃ in a high-pressure reaction bottle under the atmosphere to obtain a product A;
(2) completely dissolving product A in dichloromethane to obtain black suspension to obtain product B;
(3) washing the product B with water and saturated saline solution sequentially to obtain product C;
(4) standing and layering the product C, separating the dichloromethane layer at the lowest layer, drying with anhydrous magnesium sulfate, spin-drying, and recrystallizing with ethanol to obtain product D;
(5) and (3) adding the D product into N, N-dimethylformamide for dissolving, then adding 1-bromodecane, heating to 75-85 ℃, reacting for 20-36h, adding diethyl ether into the reaction solution until the diethyl ether is completely precipitated, performing suction filtration, washing twice with diethyl ether, and drying to obtain the probe.
In the preparation method of the bisulfite-recognizing fluorescent probe, in the step (1), 150mg of 4-vinylpyridine, 150mg of 1, 4-dibromo-2, 3, 5, 6-tetramethylbenzene, and 30mg of palladium acetate are added to each 10mL of triethylamine.
In the method for preparing the bisulfite-recognizing fluorescent probe, in the step (3), the product B is washed with water and saturated saline solution 3 times in this order.
In the preparation method of the bisulfite-recognizing fluorescent probe, in the step (4), the recrystallization is performed by using absolute ethanol.
In the preparation method of the bisulfite-based fluorescent probe, in the step (5), the ratio of the amounts of the D substance and the 1-bromodecane substance is 1:2, the reaction temperature is 80 ℃, and the reaction time is 24 h.
A method for detecting bisulfite by using the fluorescent probe for identifying bisulfite is to detect bisulfite in an aqueous solution.
The method for detecting the bisulfite by the fluorescent probe for identifying the bisulfite comprises the steps of dissolving the fluorescent probe in methanol, diluting the methanol with secondary water to obtain a fluorescent reagent, dripping a sample to be identified into the reagent to obtain a sample solution, carrying out fluorescence excitation on the sample solution, and testing and analyzing the fluorescence wavelength excited by the fluorescence.
In the method for detecting bisulfite by using the bisulfite-recognizing fluorescent probe, the concentration of the probe in the fluorescent reagent is 10-5mol.L-1The fluorescence excitation wavelength used is 348nm, and when the sample to be identified is added and bisulfite is identified, the fluorescence emission maximum wavelength of the reagent is red shifted from 510nm to 544 nm.
The invention has the advantages of
The fluorescent probe can identify and detect the bisulfite, and has the advantages of low cost in the detection process, simple operation, direct visualization of the result through the change of fluorescence and visualization; in addition, the probe of the invention has the advantages of strong anti-interference capability, high detection sensitivity and good selectivity.
To further illustrate the beneficial effects of the present invention, the inventors made the following experiments:
first, qualitative analysis test
1. The characteristics are marked as follows: in the concentration range of 10-6～10-4In mol/L fluorescent probe water solution, when the excitation wavelength is 348nm, the maximum emission wavelength of the fluorescent probe is 510nm, and when HSO is added into the fluorescent probe water solution3 -Then, the maximum emission wavelength of the fluorescent probe is from 51The 0nm red shifts to 544nm, which appears as yellow fluorescence.
Second, quantitative analysis test
1. The preparation method of the fluorescent probe solution comprises the following steps: 7.082mg of the probe was weighed, dissolved in methanol, and prepared to a volume of 10mL and a concentration of 1.0
2. Weighing superior pure potassium thiocyanate to prepare 10mL of aqueous solution with the concentration of 1.0 multiplied by 10-1mol·L-1And diluting with secondary water step by step according to the requirement.
3. Taking fluorescent reagent 1.0 × 10-5mol·L-1The standard solution was prepared by adding 3mL of a fluorescent reagent to a cuvette and adding 1.0
4. Respectively with HSO3 -The ion concentration is the abscissa, and the fluorescence intensity is the ordinate, to obtain the working curve.
5. And (3) sample determination: two 10.0mL volumetric flasks were taken and added with a fluorescent reagent of 1.0
Drawings
FIG. 1 is a chemical structural formula of a fluorescent probe molecule;
FIG. 2 is a nuclear magnetic hydrogen spectrum of a fluorescent probe molecule;
FIG. 3 is a fluorescence spectrum of the interaction of fluorescent probe molecules with different anions;
FIG. 4 is a graph showing the change in fluorescence of fluorescent probe molecules in water with bisulfite addition;
FIG. 5 is a fitting graph of detection limits of bisulfite detection by fluorescent probe molecules.
Detailed Description
The present invention is further illustrated by the following examples, which are not to be construed as limiting the invention.
Examples of the invention