阅读说明：本技术 基于hbt的甲基衍生物荧光探针及制备与应用 (HBT-based methyl derivative fluorescent probe and preparation and application thereof ) 是由 王素华 余龙 李鑫 袁亚茹 孙明泰 于 2021-08-10 设计创作，主要内容包括：本发明通过对HBT分子苯环上的不同位置上引入甲基的研究提供一种荧光探针5-HBT及制备与其荧光检测的应用,从而实现了现场快速可视化检测水体中残留次氯酸钠。通过本发明研究证明了苯环上的一个甲基基团的位置,对某些荧光分子能产生巨大的影响,主要包括荧光的强度和荧光的稳定性,其中最明显的是5-HBT荧光大大减弱,所得到的5-HBT探针在固体状态下可以发射荧光,在制作纸质荧光传感器时有很大的优势,荧光效果明显,可以实现现场快速可视化检测次氯酸钠溶液。(The invention provides a fluorescent probe 5-HBT, preparation and application of fluorescence detection thereof through research of introducing methyl groups to different positions on a benzene ring of HBT molecules, thereby realizing rapid visual detection of residual sodium hypochlorite in a water body on site. The research of the invention proves that the position of a methyl group on a benzene ring can have great influence on some fluorescent molecules, mainly comprises the intensity of fluorescence and the stability of fluorescence, wherein the most obvious is that the fluorescence of the 5-HBT is greatly weakened, the obtained 5-HBT probe can emit fluorescence in a solid state, and the invention has great advantages in manufacturing a paper fluorescent sensor, has obvious fluorescence effect and can realize the rapid visual detection of sodium hypochlorite solution on site.)

1. HBT-based methyl derivative fluorescent probe, named 5-HBT, has the following structural formula:

2. the method for preparing a methyl derivative fluorescent probe based on HBT of claim 1, which comprises dissolving 5-methyl salicylaldehyde and 2-aminothiophenol in ethanol, vacuumizing, introducing nitrogen, reacting at normal temperature for 20-40 min under nitrogen atmosphere, adding hydrochloric acid and hydrogen peroxide solution, reacting for 10-12 hr under nitrogen atmosphere, adding hydrochloric acid under ice water bath to remove unreacted 2-aminothiophenol, and post-treating and purifying.

3. The method of claim 2, wherein the post-treatment purification is performed by adding saturated saline solution, extracting with ethyl acetate, rotary evaporating to obtain a primary purified product, and further separating by column chromatography purification to obtain a pure sample.

4. Use of the fluorescent probe obtained by the preparation method according to claim 2 or 3 in hypochlorite detection.

5. Use according to claim 4, characterized in that the following method steps are used: dissolving the fluorescent probe in methanol to obtain a probe stock solution, and then carrying out chlorite detection.

6. The use of claim 5, wherein the probe stock solution is dissolved in acetonitrile and sodium hydroxide solution is added prior to hypochlorite detection.

7. The use of claim 4, wherein 0.01mmol of 5-HBT is dissolved in 10mL of methanol to obtain a stock solution with a sample concentration of 1 mM; dissolving 20 μ L of 5-HBT stock solution in 1.5mL acetonitrile, and adding 20 μ L of 0.04M sodium hydroxide solution; then, a sodium hypochlorite solution was added for fluorescence detection.

Technical Field

The invention belongs to the technical field of preparation and fluorescence analysis of organic compounds, and mainly relates to fluorescence difference analysis of methyl derivatives based on 2- (2-Hydroxyphenyl) Benzothiazole (HBT) and detection of sodium hypochlorite.

Background

The sodium hypochlorite solution has strong oxidizing ability and excellent sterilization and disinfection effects, however, excessive sodium hypochlorite poses potential threats to the health of human beings and animals, and can cause diseases such as kidney and lung injuries, cancers and the like. Therefore, the method has important significance for the detection of the sodium hypochlorite.

The addition or deletion of one methyl group on the phenyl ring generally has no significant effect on the fluorescent properties of the molecule. 2- (2-Hydroxyphenyl) Benzothiazole (HBT) is widely applied to fluorescence analysis and detection due to excellent fluorescence performance and an excited state intramolecular proton transfer mechanism.

Chinese patent publication No. CN107602504A discloses a fluorescent probe for detecting hypochlorous acid, a preparation method and a use method thereof. The invention utilizes 2- (2 '-hydroxyphenyl) benzothiazole to construct a classic ESIPT system, and introduces an azide part directly at a 5' -position, so that the ESIPT system has more biological and optical stability. The ESIPT effect of the probe is inhibited by the azide group and no fluorescence is emitted, but under the condition that hypochlorous acid exists, the azide group is oxidized by the hypochlorous acid, so that the inhibition effect disappears, and the probe molecule emits strong fluorescence. The on-off type hypochlorous acid probe of the 2- (2' -hydroxyphenyl) benzothiazole-azide dye provided by the invention has good response to hypochlorous acid solution, can realize sensitive quantitative detection of trace hypochlorous acid in a sample, and has the advantages of simple and convenient operation, low cost, sensitive response, easy popularization and application and the like.

Chinese patent publication No. CN105441065A discloses a fluorescent probe for detecting hypochlorite ions, a preparation method and a use method thereof. The invention uses 2- (2 '-hydroxyphenyl) benzothiazole to construct a classic ESIPT system, and introduces an azophenyl part directly at the 5' -position, so that the ESIPT system has better biological and optical stability. When hypochlorite exists, azophenyl is oxidized, and then probe molecules emit strong fluorescence, the on-off type hypochlorite probe of the 2- (2' -hydroxyphenyl) benzothiazole dye and the special detection kit thereof have good response to hypochlorite solution, can realize the detection of intracellular hypochlorite, and have the advantages of simple and convenient operation, low cost, sensitive response, easy popularization and application and the like.

Chinese patent publication No. CN110372632A discloses a fluorescent probe molecule for rapidly identifying hypochlorite ions, and a preparation method and application thereof. The structural formula of the fluorescent probe molecule BS is obtained by reacting 2-benzothiazolyl-4-methylphenol with N, N-dimethyl amino thiocarbonyl chloride. When the fluorescent probe molecule BS is added into a test sample containing hypochlorite ions, the fluorescence of the test sample is obviously changed, and the fluorescence intensity of the fluorescent probe molecule BS at 482nm has a linear positive correlation with the concentration of the hypochlorite ions; meanwhile, the probe molecule BS has good selectivity on hypochlorite ions, and the test process is simple to operate and has the advantages of high speed, high efficiency and high sensitivity.

It can be seen that, in the published related research results, the research contents are all that new recognition groups are introduced on HBT molecules so as to realize the detection of hypochlorous acid/hypochlorite in the environment, and few researches are carried out on adding a methyl group on HBT and the influence of the substitution position transformation of the methyl group on the fluorescence detection of the hypochlorous acid/hypochlorite.

Disclosure of Invention

The invention discovers that the introduction of methyl groups at different positions on the benzene ring of molecules of HBT and derivatives thereof has great influence on the fluorescence intensity and the fluorescence stability of the molecules, and discovers that the molecules are also greatly different in different solvents, thereby realizing the fluorescence detection of sodium hypochlorite in a specific solvent. This provides a new idea for organic molecule fluorescence analysis.

The invention aims to provide a fluorescent probe 5-HBT, preparation and application of fluorescence detection thereof through research on introduction of methyl groups at different positions on a benzene ring of HBT molecules, so that rapid visual detection of residual sodium hypochlorite in a water body on site is realized.

The methyl derivative fluorescent probe based on HBT is named as 5-HBT, and the structural formula is as follows:

the preparation method of the methyl derivative fluorescent probe based on the HBT comprises the steps of dissolving 5-methyl salicylaldehyde and 2-aminothiophenol in ethanol, vacuumizing, introducing nitrogen, reacting for 20-40 minutes at normal temperature in the nitrogen atmosphere, adding hydrochloric acid and hydrogen peroxide solution, continuing to react for 10-12 hours in the nitrogen atmosphere, adding hydrochloric acid in an ice water bath, removing the unreacted 2-aminothiophenol, and performing post-treatment and purification to obtain the methyl derivative fluorescent probe.

In the preparation method, the post-treatment purification refers to adding saturated saline solution, extracting with ethyl acetate, performing rotary evaporation to obtain a primarily purified product, and further separating by column chromatography purification to obtain a pure sample. Specifically, the post-treatment can be carried out by transferring the reaction solution into a beaker with a suitable size, adding a small amount of ethyl acetate into a reaction bottle to dissolve a sample attached to the bottle wall, transferring the dissolved sample into the beaker, adding a proper amount of saturated saline solution into the beaker, extracting the sample for three times by using ethyl acetate, and evaporating the extracted ethyl acetate by using a rotary evaporator to obtain a crude product; in order to further purify the crude product, the primarily purified product can be further separated and purified by adopting a column chromatography method, petroleum ether and ethyl acetate in a certain proportion can be used as eluent to finally obtain a pure product, then a sample is evaporated to dryness by a rotary evaporator and is put into a refrigerator, and a small amount of sample is taken for high-resolution mass spectrometry.

The HBT-based methyl derivative fluorescent probe can be applied to hypochlorite detection, and the application can adopt the following method steps: dissolving the fluorescent probe in methanol to obtain a probe stock solution, and then carrying out chlorite detection.

In the above application, preferably, the probe stock solution is dissolved in acetonitrile, and a sodium hydroxide solution is added, followed by hypochlorite detection. Since the probe has an increased fluorescence intensity under alkaline conditions, the amount of NaOH added was measured, and the initial fluorescence intensity of the probe was maximized when 20. mu.L of a 0.04M NaOH solution was added.

As a preferred application, the following method steps can be used: dissolving 0.01mmol of 5-HBT in 10mL of methanol solution to obtain a stock solution with the sample concentration of 1 mM; dissolving 20 μ L of 5-HBT stock solution in 1.5mL acetonitrile, and adding 20 μ L of 0.04M sodium hydroxide solution; sodium hypochlorite solution was then added, followed by fluorescence photography under an ultraviolet lamp.

The invention researches the fluorescence properties of HBTs substituted by different methyl positions in different organic solvents, and fully compares the differences of different methyl derivatives in different solvents, namely the methyl derivatives with highly similar structures show different results in different solvents. Solvents tested included water, methanol, ethanol, isopropanol, N-dimethylformamide, acetonitrile, dimethyl sulfoxide, and acetone. As can be seen from the fluorescence photograph, 3-HBT is easily quenched by sodium hypochlorite in methanol solvent, and 5-HBT is easily quenched by sodium hypochlorite in acetonitrile solvent. And the fluorescence of HBT and 4-HBT in various organic solvents is not easy to quench, and the fluorescence of 6-HBT is weak, so that the further research is not needed. Then, the fluorescent response effect of the 3-HBT and the 5-HBT to sodium hypochlorite in methanol and acetonitrile solvents is respectively tested, and the experimental result shows that the 3-HBT can be completely quenched after the methanol solution is added with the sodium hypochlorite and the fluorescence can be completely quenched after the methanol solution is incubated for 15 minutes. But in the acetonitrile solution of 5-HBT, the fluorescence can be completely quenched in only 1 minute, and the detection efficiency is greatly improved. Therefore, the 5-HBT can be used for manufacturing the sodium hypochlorite test paper sensor and for rapidly and visually detecting the sodium hypochlorite in the water body on site.

According to the invention, the ultraviolet absorption spectrum shows that after the sodium hypochlorite solution is added, the ultraviolet absorption peak near 350-450nm disappears, and the material cannot be excited by a 365nm light source, so that the fluorescence is quenched.

The research of the invention proves that the position of a methyl group on a benzene ring can have great influence on some fluorescent molecules, mainly comprises the intensity of fluorescence and the stability of fluorescence, wherein the most obvious is that the fluorescence of the 5-HBT is greatly weakened, the obtained 5-HBT probe can emit fluorescence in a solid state, and the invention has great advantages in manufacturing a paper fluorescence sensor, has obvious fluorescence effect and can realize the on-site quick visual semi-quantitative detection of the sodium hypochlorite solution.

Drawings

FIG. 1 shows a high-resolution mass spectrum of 2- (2-Hydroxyphenyl) Benzothiazole (HBT).

Figure 2 is a high resolution mass spectrum of a 3-HBT.

Figure 3 is a high resolution mass spectrum of a 4-HBT.

Figure 4 is a high resolution mass spectrum of a 5-HBT.

FIG. 5 is a high-resolution mass spectrum of the 6-HBT.

Figure 6 is a photograph of HBT in 1. water, 2. methanol, 3. ethanol, 4. isopropanol, 5.N, N-dimethylformamide, 6. acetonitrile, 7. dimethyl sulfoxide and 8. acetone. (a) Shooting under natural light; (b) shooting under a 365nm ultraviolet lamp; (c) after adding an excess of sodium hypochlorite, the film was photographed under a 365nm UV lamp.

Figure 7 is a picture of 3-HBT in 1. water, 2. methanol, 3. ethanol, 4. isopropanol, 5.N, N-dimethylformamide, 6. acetonitrile, 7. dimethyl sulfoxide, and 8. acetone. (a) Shooting under natural light; (b) shooting under a 365nm ultraviolet lamp; (c) after adding an excess of sodium hypochlorite, the film was photographed under a 365nm UV lamp.

Figure 8 is a picture of 4-HBT in 1. water, 2. methanol, 3. ethanol, 4. isopropanol, 5.N, N-dimethylformamide, 6. acetonitrile, 7. dimethyl sulfoxide, and 8. acetone. (a) Shooting under natural light; (b) shooting under a 365nm ultraviolet lamp; (c) after adding an excess of sodium hypochlorite, the film was photographed under a 365nm UV lamp.

Figure 9 is a picture of 5-HBT in 1. water, 2. methanol, 3. ethanol, 4. isopropanol, 5.N, N-dimethylformamide, 6. acetonitrile, 7. dimethyl sulfoxide, and 8. acetone. (a) Shooting under natural light; (b) shooting under a 365nm ultraviolet lamp; (c) after adding an excess of sodium hypochlorite, the film was photographed under a 365nm UV lamp.

FIG. 10 is a graph showing the UV absorption spectrum of 5-HBT before and after the addition of sodium hypochlorite.

FIG. 11 is a graph showing fluorescence detection of semi-quantitatively detected sodium hypochlorite.

Detailed Description

The following examples are further illustrative of the present invention as to the technical content of the present invention, but the essence of the present invention is not limited to the following examples, and one of ordinary skill in the art can and should understand that any simple changes or substitutions based on the essence of the present invention should fall within the protection scope of the present invention.

Example 1

3mmol of salicylaldehyde, 3-methylsalicylaldehyde, 2-hydroxy-4-methylbenzaldehyde, 5-methylsalicylaldehyde and 2-hydroxy-6-methylbenzaldehyde were added to a round-bottomed flask containing 6mL of ethanol solvent, respectively, with 3.2mmol of 2-aminothiophenol. The flask was evacuated and purged with nitrogen through a glass three-way valve with a balloon, and magnetically stirred at room temperature for 30 min. After reacting for 30min, 0.34mL of H was added rapidly₂O₂(30% by mass) and 0.17mL of HCl (37% by mass). The reaction was continued for 12h under nitrogen atmosphere. After completion of the reaction, 0.05mL of HCl (37%) was added under ice-water conditions, and the solution was transferred to a 100mL beaker, followed by addition of 30mL of saturated brine. The extract was extracted 3 times with 50mL of ethyl acetate and the sample was evaporated to dryness by rotary evaporator to give a primary purified sample. Then analyzing and purifying by column chromatography, wherein the eluent used is petroleum ether: ethyl acetate 5:1, v/v. The preparation equation is as follows (refer to the preparation process for HBT in Long Yu. A fluorine protocol for high selectivity and sensitive detection of gaseous ozone based on exposed-state intracellular molecular proton transfer. Sensors and activators B266 (2018)717 723):

and (3) respectively dissolving a small amount of solid in a methanol solvent to perform high-resolution mass spectrometry on the sample after analysis and purification, and determining the molecular weight of the product. As shown in fig. 1-5, respectively. ESI-MS M/z, HBT, [ M + H ]]⁺:228.04738；3-HBT,[M-H]^-:240.04826；4-HBT,[M+H]⁺:242.06303；5-HBT,[M-H]^-:240.04852；6-HBT,[M-H]^-:240.04834。

Example 2

0.01mmol of HBT, 3-HBT, 4-HBT and 5-HBT obtained in example 1 were dissolved in 10mL of methanol, respectively, to obtain a stock solution having a sample concentration of 1 mM.

20 mu L of stock solutions of HBT, 3-HBT, 4-HBT and 5-HBT are respectively dissolved in 2mL centrifuge tubes filled with 1.5mL of water, methanol, ethanol, isopropanol, N-dimethylformamide, acetonitrile, dimethyl sulfoxide and acetone solvents, and then 20 mu L of 0.04M sodium hydroxide solution is added. At the moment, the fluorescence intensity reaches the maximum, and the pictures are respectively taken under natural light and a 365nm ultraviolet lamp. An excess of sodium hypochlorite solution was then added and the picture taken again under a 365nm UV lamp. It is found from fig. 6 to 9 that the stock solutions of HBT, 3-HBT, 4-HBT and 5-HBT are colorless under natural light, and are all clear green or blue-green fluorescence under ultraviolet lamp, and after adding excessive sodium hypochlorite solution, detection under ultraviolet lamp finds that the fluorescence of 3-HBT in methanol is quenched by sodium hypochlorite, the fluorescence of HBT and 4-HBT in various organic solvents is not quenched easily, and the fluorescence of 5-HBT in acetonitrile is quenched by sodium hypochlorite most significantly. It was also found that the methanol solution of 3-HBT required incubation for 15 minutes after the addition of sodium hypochlorite before fluorescence could be completely quenched. But in the acetonitrile solution of the 5-HBT, the fluorescence can be completely quenched in only 1 minute, the reaction time of the acetonitrile solution of the 5-HBT and sodium hypochlorite is shortest, and the quenching effect is best. Thus, the 5-HBT is selected as a sodium hypochlorite fluorescent probe, and ultraviolet absorption spectra before and after the reaction of the 5-HBT and sodium hypochlorite are measured, as shown in FIG. 10, after the sodium hypochlorite solution is added, an ultraviolet absorption peak near 350-450nm disappears, and the fluorescence is quenched. The 5-HBT can emit fluorescence in a solid state, so that the design of the test paper sensor is facilitated, and the test paper sensor can be conveniently used for rapidly and visually detecting the residual sodium hypochlorite in tap water and lakes on site.

Example 3

0.01mmol of 5-HBT probe was dissolved in 10mL of methanol to obtain a 1mM probe stock solution. mu.L of the probe stock was taken and added to a 2mL centrifuge tube containing 1.5mL acetonitrile, followed by the gradual addition of 5,10,15,20 and 25. mu.L of 0.04M sodium hydroxide solution. When added to 20. mu.L, the fluorescence intensity did not substantially increase. Therefore, the solvent environment is preferably 20. mu.L of NaOH solution with a concentration of 0.04M added under the same conditions.

Example 4

Manufacturing test paper: a piece of paper made of polytetrafluoroethylene material is made into a piece of paper round paper through a puncher, then the paper round paper is immersed in 5-HBT with the concentration of 1mM, and then the paper is dried to obtain the fluorescent test paper for detecting sodium hypochlorite. The concentration of the sodium hypochlorite mother liquor is about 1M, 1mL of sodium hypochlorite is taken and diluted to 10mL, and the concentration is 0.1M. Then 5, 25, 50, 75, 100 and 200. mu.L of sodium hypochlorite solution are respectively taken to be made to 10 mL. The concentration of sodium hypochlorite at this time was 3.7, 18.5, 37, 55.5, 74 and 148ppm in this order. Then 10 mu L of sodium hypochlorite solutions with different ppm are dripped on the test paper, and a photo is taken under a 365nm ultraviolet lamp to obtain a fluorescence detection picture for semi-quantitatively detecting the sodium hypochlorite. As shown in fig. 11. In the figure, group A is a control group, and each round test paper shows green fluorescence; and the group B is a testing group, green fluorescence is quenched after the sodium hypochlorite solution is dripped, and the fluorescence quenching is more obvious along with the increase of the concentration of the sodium hypochlorite solution.

It should be noted that the technical contents described above are only explained and illustrated to enable those skilled in the art to know the technical spirit of the present invention, and therefore, the technical contents are not to limit the scope of the present invention. The scope of the invention is defined by the appended claims. It should be understood by those skilled in the art that any modification, equivalent replacement, and improvement made based on the spirit of the present invention should be considered to be within the spirit and scope of the present invention.