阅读说明：本技术 基于芘和香豆素衍生物的亚硫酸氢根荧光探针、制备方法及应用 (Pyrene and coumarin derivative-based bisulfite fluorescent probe, and preparation method and application thereof ) 是由 高光芹 郑昕 谢普会 赵鹏飞 申丽婕 郭冰洁 凡雨鑫 李明旻 于 2020-06-16 设计创作，主要内容包括：本发明提供了一种基于芘和香豆素衍生物的亚硫酸氢根荧光探针、制备方法及应用,荧光探针的结构式如下：<Image he="95" wi="139" file="DEST_PATH_IMAGE002.GIF" imgContent="drawing" imgFormat="GIF" orientation="portrait" inline="no"></Image>,将1-乙酰基芘和4-(二乙氨基)水杨醛溶解于甲烷磺酸中,90℃反应一段时间,反应完全后,将反应液倒入冰水中,加入高氯酸,有大量沉淀产生,抽滤得固态粗产品,固态粗产品经硅胶柱层析分离得到荧光探针(B1)。该探针在CH<Sub>3</Sub>CN-HEPES(v/v＝6:4,v/v,pH＝7.4)缓冲溶液中与SO<Sub>2</Sub>发生特异性亲核反应,从而实现对水溶液中亚硫酸氢根的特异性识别检测。(The invention provides a pyrene and coumarin derivative-based bisulfite fluorescent probe, a preparation method and application thereof, wherein the structural formula of the fluorescent probe is as follows: dissolving 1-acetylpyrene and 4- (diethylamino) salicylaldehyde in methanesulfonic acid, reacting for a period of time at 90 ℃, pouring the reaction solution into ice water after the reaction is completed, adding perchloric acid, generating a large amount of precipitates, performing suction filtration to obtain a solid crude product, and performing silica gel column chromatography separation on the solid crude product to obtain the fluorescent probe (B1). The probe is in CH 3 Reaction with SO in CN-HEPES (v/v: 6:4, v/v, pH 7.4) buffer solution 2 A specific nucleophilic reaction is carried out, thereby realizing the sulfurous acid in the aqueous solutionSpecific recognition and detection of hydrogen radicals.)

1. A pyrene and coumarin derivative-based bisulfite fluorescent probe is characterized in that the structural formula of the fluorescent probe is as follows:

2. the method for preparing a bisulfite fluorescent probe based on pyrene and coumarin derivatives as claimed in claim 1, characterized by the following steps: dissolving 1-acetylpyrene and 4- (diethylamino) salicylaldehyde in methanesulfonic acid, reacting for a period of time at 90 ℃, pouring the reaction liquid into ice water after complete reaction, adding perchloric acid, generating a large amount of precipitates, performing suction filtration to obtain a solid crude product, and performing silica gel column chromatography separation on the solid crude product to obtain the fluorescent probe.

3. The method for preparing a bisulfite fluorescent probe based on pyrene and coumarin derivatives according to claim 2, wherein the method comprises the following steps: the molar ratio of the 1-acetylpyrene to the 4- (diethylamino) salicylaldehyde is 1: (1-6) the reaction time is 8-18 h.

4. The method for preparing a bisulfite fluorescent probe based on pyrene and coumarin derivatives according to claim 2, wherein the method comprises the following steps: the mol ratio of the 1-acetylpyrene to the methane sulfonic acid is 1: (25-50), wherein the molar ratio of the 1-acetylpyrene to the perchloric acid is 1: (6-16).

5. The method for preparing a bisulfite fluorescent probe based on pyrene and coumarin derivatives according to claim 2, wherein the method comprises the following steps: the silica gel column chromatography separation adopts the eluent of methanol and dichloromethane with the volume ratio of 1 (20-40), and the yield is 45-85%.

6. Use of the fluorescent probe according to claim 1 in the field of detecting bisulfite.

Technical Field

The invention relates to the field of bisulfite detection agents, and in particular relates to a pyrene and coumarin derivative-based bisulfite fluorescent probe, a preparation method and application.

Background

Sulfur dioxide (SO)₂) Can cause atmospheric pollution, sulfur dioxide is easy to dissolve in water, and the bisulphite (HSO) derivative thereof can be used in water₃ ^-) Or Sulfite (SO)₃ ^2-) Exist in the form of (1). Excessive contact with sulfur dioxide can be detrimental to the health of the organism. The long-term exposure in sulfur dioxide gas can bring harm to the respiratory system of people, and the risks of cardiovascular diseases and respiratory diseases are greatly increased. For example, excessive sulfur dioxide intake can cause gastrointestinal damage in humans. Therefore, it is very necessary to detect the residual amount of sulfur dioxide. Several conventional techniques, including electrochemical method, chromatography and capillary electrophoresis, have been developed for detecting sulfur dioxide derivatives, but most of these methods take a long time and damage the structure of the substance to be detected.

The fluorescent probe detection method has the outstanding advantages of high sensitivity, simplicity in operation, non-invasiveness, non-destructiveness, high selectivity, high time sequence, high spatial resolution, good biocompatibility and the like, and is particularly suitable for real-time monitoring. Fluorescent probes can detect analytes both in the environment and in vivo. As such, it has very general applications in chemistry, environment, medicine, and biology. As such, methods for detecting the residual amount of sulfur dioxide using fluorescent probes have been receiving increasing attention. The fluorescent probe can obtain results in a short time, has good timeliness, and cannot damage detected substances.

Disclosure of Invention

The invention provides a pyrene and coumarin derivative-based bisulfite fluorescent probe, a preparation method and application, and solves the technical problems that a conventional bisulfite detection agent in the prior art cannot be applied to actual medical treatment, the preparation method is complex and the cytotoxicity is high.

This application is based on SO₂The nucleophilicity of the probe designs a fluorescent probe containing pyrene and coumarin structures, and the probe is on CH₃Reaction with SO in CN-HEPES (v/v: 6:4, v/v, pH 7.4) buffer solution₂And carrying out specific nucleophilic reaction, thereby realizing specific recognition and detection of the bisulfite in the aqueous solution.

The technical scheme for realizing the invention is as follows:

a pyrene and coumarin derivative-based bisulfite fluorescent probe has the following structural formula:。

the preparation method of the bisulfite fluorescent probe based on pyrene and coumarin derivatives comprises the following steps: dissolving 1-acetylpyrene and 4- (diethylamino) salicylaldehyde in methanesulfonic acid, reacting for a period of time at 90 ℃, pouring the reaction solution into ice water after the reaction is completed, adding perchloric acid, generating a large amount of precipitates, performing suction filtration to obtain a solid crude product, and performing silica gel column chromatography separation on the solid crude product to obtain the fluorescent probe (B1).

The synthetic route is as follows:

the molar ratio of the 1-acetylpyrene to the 4- (diethylamino) salicylaldehyde is 1: (1-6) the reaction time is 8-18 h.

The mol ratio of the 1-acetylpyrene to the methane sulfonic acid is 1: (25-50), wherein the molar ratio of the 1-acetylpyrene to the perchloric acid is 1: (6-16).

The silica gel column chromatography separation adopts the eluent of methanol and dichloromethane with the volume ratio of 1 (20-40), and the yield of the fluorescent probe is 45-85%.

The fluorescent probe is applied to the field of detecting bisulfite.

The recognition mechanism of the fluorescent probe based on pyrene and coumarin derivatives on bisulfite is as follows:

the invention has the beneficial effects that:

(1) the fluorescent probe has the advantages of high fluorescence quantum yield, simple raw materials, simple synthetic method, high yield, easiness in obtaining and the like.

(2) The fluorescence emission wavelength (630 nm) of the fluorescent probe to which the present application relates is in the near infrared region. Some endogenous fluorophores in the environment and organisms and tissues generate 'autofluorescence', the emission wavelength of the autofluorescence is in a visible light region (400-600 nm), and meanwhile, the biological organism tissues have strong scattering on visible light, so that the fluorescence signals of the exogenous fluorescent probes are often seriously interfered. Background fluorescence in the near infrared region (600-900 nm) in the environment and organism tissues is obviously weakened, the absorption coefficient is minimum, scattering of near infrared light is less, and near infrared light energy can have strong tissue penetrability in organisms. The fluorescent probes of the present application greatly reduce the associated background interference and thus increase the sensitivity and penetration of the fluorescent technology.

(3) The fluorescent probe has high-efficiency and specific recognition performance on the bisulfite, and has the advantages of strong anti-interference performance, high response speed (the response time is 30 s) and high sensitivity (the minimum detection limit of the bisulfite is 39 nM).

(4) The fluorescent probe is mainly used for identifying the bisulfite by utilizing the nucleophilicity of the bisulfite, and the probe and the bisulfite generate specific nucleophilic addition. The recognition mechanism is confirmed by high-resolution mass spectrometry. The high resolution mass spectrometry data of the probe alone in the positive ion mode was 402.1850 (theoretical value of 402.1852), and the high resolution mass spectrometry data of the probe after the specific recognition reaction with bisulfite emission in the negative ion mode was 482.1434 (theoretical value of 482.1432) (FIG. 10). High-resolution mass spectrometry data verifies the identification mechanism of the bisulfite to be identified by the fluorescent probe, and lays a foundation for further developing more bisulfite fluorescent probes.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, it is obvious that the drawings in the following description are only some embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to the drawings without creative efforts.

FIG. 1 shows the NMR spectrum of fluorescent probe B1 (DMSO is the solvent) in example 1 of the present invention.

FIG. 2 shows the NMR spectrum of fluorescent probe B1 (DMSO is the solvent) in example 1 of the present invention.

FIG. 3 is a high resolution mass spectrum of fluorescent probe B1 (solvent is CH) in example 1 of the present invention₃OH）。

FIG. 4 is a graph showing fluorescence selectivity of the fluorescent probe B1 of the present invention, with an excitation wavelength of 550 nm.

FIG. 5 is a graph showing the fluorescence interference resistance of bisulfite being recognized by the fluorescent probe B1 of the present invention, wherein the excitation wavelength is 550 nm and the emission wavelength is 630 nm.

FIG. 6 is a graph showing the fluorescence titration of bisulfite being recognized by the fluorescent probe B1 of the present invention, with an excitation wavelength of 550 nm.

FIG. 7 is a diagram showing the lowest detection limit of bisulfite being recognized by fluorescent probe B1 of the present invention, wherein the excitation wavelength is 550 nm and the emission wavelength is 630 nm.

FIG. 8 is a pH adaptation graph for bisulfite identification by fluorescent probe B1 of the present invention, with an excitation wavelength of 550 nm and an emission wavelength of 630 nm.

FIG. 9 is a fluorescence kinetic diagram of bisulfite identification by fluorescent probe B1 of the present invention, with an excitation wavelength of 550 nm and an emission wavelength of 630 nm.

FIG. 10 is a high-resolution mechanism verification diagram of bisulfite discrimination by the fluorescent probe B1 of the present invention (solvent CH)₃OH, negative ion mode).

Detailed Description

The technical solutions of the present invention will be described clearly and completely with reference to the following embodiments of the present invention, and it should be understood that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be obtained by a person skilled in the art without inventive effort based on the embodiments of the present invention, are within the scope of the present invention.