阅读说明：本技术 一种用于亚硫酸氢根检测的比率型荧光探针及其制备方法及应用 (Ratio type fluorescent probe for detecting bisulfite and preparation method and application thereof ) 是由 *** 王川川 陈晓培 刘俊桃 刘志景 吴金松 吕全建 于 2019-09-24 设计创作，主要内容包括：本发明公开了一种用于亚硫酸氢根检测的比率型荧光探针的制备方法及应用,其分子式为C<Sub>29</Sub>H<Sub>29</Sub>N<Sub>6</Sub>O<Sub>4</Sub><Sup>+</Sup>,简称为：CBP-N,通过荧光光谱仪研究了探针CBP-N在CH<Sub>3</Sub>CN-PBS溶液中与金属离子、常见氨基酸、活性氧、活性硫等小分子物质的识别特性。结果表明：探针CBP-N对亚硫酸氢根具有高效专一的选择性,具有较强的抗干扰能力,其最低检出限为18 nM。探针CBP-N与亚硫酸氢根响应后,荧光发射光谱中,在549 nm处荧光强度明显增强,631 nm处荧光强度显著降低,可以实现对亚硫酸氢根的比率型检测,该特性可以克服环境中常见因素的干扰,具有较好的实际应用价值。(The invention discloses a preparation method and application of a ratio type fluorescent probe for detecting bisulfite, wherein the molecular formula of the ratio type fluorescent probe is C 29 H 29 N 6 O 4 + For short: CBP-N, the probe CBP-N is studied in CH by fluorescence spectrometer 3 And the CN-PBS solution has the identification characteristics with metal ions, common amino acids, active oxygen, active sulfur and other small molecular substances. The results show that: the probe CBP-N has high-efficiency and specific selectivity on bisulfite, has strong anti-interference capability, and has the lowest detection limit of 18 nM. After the probe CBP-N responds to the bisulfite, in a fluorescence emission spectrum, the fluorescence intensity at 549 nm is obviously enhanced, the fluorescence intensity at 631 nm is obviously reduced, the ratio type detection of the bisulfite can be realized, and the characteristic can be realizedThe interference of common factors in the environment is overcome, and the method has good practical application value.)

1. A ratio-type fluorescent probe for detecting bisulfite, which is characterized in that the structural formula of the fluorescent probe is as follows:

。

2. the method for preparing a ratiometric fluorescent probe for bisulfite detection according to claim 1, characterized by the steps of:

(1) dissolving the intermediate CBP and triethylamine in acetonitrile, mixing and stirring, adding 4-chloro-7-nitrobenzofurazan, and stirring and reacting for 6-18 hours at room temperature;

(2) and (2) spin-drying the reaction liquid obtained in the step (1) to remove acetonitrile, re-dissolving the reaction liquid by using dichloromethane, washing the reaction liquid by using a saturated sodium chloride solution, separating an organic phase, drying the organic phase by using anhydrous sodium sulfate, filtering, concentrating under reduced pressure, and finally performing column chromatography separation to obtain a dark blue solid, namely the ratio type fluorescent probe for detecting the bisulfite.

3. The method for preparing a ratiometric fluorescent probe for bisulfite detection according to claim 2, wherein in step (1), the intermediate CBP is prepared by:

synthesis of CBP-1: dissolving 69 mmol of piperazine in 30 mL of DMF, adding 69 mmol of potassium carbonate, stirring for 15 minutes, adding 2.298 mmol of p-fluoroacetophenone in the mixed solution, refluxing overnight at 80 ℃, removing the solvent under reduced pressure after the reaction is completed, dissolving dichloromethane, washing with saturated sodium chloride, drying with anhydrous sodium sulfate, and removing the solvent under reduced pressure to obtain CBP-1;

synthesis of CBP: weighing 1mmol of 4-diethylamino salicylaldehyde and 1mmol of CBP-1 in a 25 mL round-bottom flask, adding 3mL of concentrated sulfuric acid, and heating and stirring at 90 ℃ for 6 hours; and cooling to room temperature after the reaction is finished, dropwise adding the reaction solution into 30 mL of ice water, dropwise adding 7-8 drops of perchloric acid, separating out solids, filtering out the solids, washing, drying, and then performing column chromatography separation and purification to obtain a green solid, namely the intermediate CBP.

4. The method for preparing a ratiometric fluorescent probe for bisulfite detection according to claim 2, wherein in the step (1), the molar ratio of the intermediate CBP, triethylamine and 4-chloro-7-nitrobenzofurazan is 1: (1-8): (1-10), and the mixing and stirring time is 5-25 minutes.

5. The method for preparing a ratiometric fluorescent probe for bisulfite detection according to claim 2, characterized in that: the eluent adopted by the column chromatography separation in the step (2) is CH₂Cl₂And CH₃OH is 1 according to the volume ratio (30-50).

6. The use of the ratiometric fluorescent probe for bisulfite detection according to claim 1 as a specific, highly sensitive detection of bisulfite in food and in the environment.

Technical Field

The invention belongs to the field of organic small-molecule fluorescent probes, and particularly relates to a ratio type fluorescent probe for detecting bisulfite and a preparation method and application thereof.

Background

Sulfur dioxide is a common atmospheric pollutant and is readily hydrolyzed to bisulfite and sulfite in neutral aqueous solutions. In daily production and life, sulfur dioxide and derivatives are widely applied to enzyme inhibitors, antibacterial agents, preservatives and the like. In the food industry, sulfites are widely used as preservatives for food and beverages to prevent food oxidation and bacterial growth, and to inhibit enzymatic and non-enzymatic browning from occurring during production and storage. However, high doses of sulfite may cause adverse reactions and acute symptoms, and the world health organization dictates that threshold levels of sulfite in food products be tightly controlled. Therefore, it is of great significance to develop a detection technique for rapidly and effectively detecting bisulfite and derivatives.

In recent years, organic small molecule fluorescent probes have attracted much attention for their application in the detection of specific target analytes. After the fluorescent probe and the specific target analyte are changed, the fluorescent signal is obviously changed, so that the target analyte is identified and detected. The fluorescence analysis method using the fluorescent probe has the advantages of high specificity selectivity, high sensitivity, fast response time, low detection limit and the like, and is widely applied to the fields of environmental science and biomedicine at present. Patent 108129428 discloses a ratiometric fluorescent probe for detecting bisulfite and its application, wherein the probe is a ratiometric fluorescent probe for detecting bisulfite based on fluorescence resonance energy transfer mechanism, which is mainly applied in intracellular lipid titration site imaging and has low sensitivity, and is prepared by using dansyl fluorophore as donor, (E) -2- (3-cyano-4- (4- (dimethylamino) styrene) -5, 5-dimethylfuran-2 (5H) -ylidene) malononitrile fluorophore as acceptor, and piperazine coupling structure; patent CN201310213806.4 discloses a bisulfite ion fluorescent probe using a product obtained by condensing benzindoline and aromatic aldehyde as a probe molecule and a technical scheme of a preparation method, but the probe only shows a specificity indicator of the existence of bisulfite ions in an aqueous solution; use limitations and low sensitivity; therefore, it is very interesting to develop efficient and sensitive fluorescent probes for bisulfite detection.

Disclosure of Invention

In order to solve the technical problems, the invention provides a ratio type fluorescent probe for detecting bisulfite and a preparation method and application thereof, wherein the fluorescent probe CBP-N has high-efficiency and specific selectivity to bisulfite, has strong anti-interference capability, and has a minimum detection limit of 18 nM.

The technical scheme of the invention is realized as follows:

a ratio-type fluorescent probe for bisulfite detection, the structural formula of the fluorescent probe is as follows:

。

the synthetic route of the ratio type fluorescent probe for detecting the bisulfite is as follows:

。

the preparation method of the ratio type fluorescent probe comprises the following steps:

(1) dissolving the intermediate CBP and triethylamine in acetonitrile, mixing and stirring, adding 4-chloro-7-nitrobenzofurazan (NBD-Cl), and stirring and reacting for 6-18 hours at room temperature;

(2) and (2) spin-drying the reaction liquid obtained in the step (1) to remove acetonitrile, re-dissolving the reaction liquid by using dichloromethane, washing the reaction liquid by using a saturated sodium chloride solution, separating an organic phase, drying the organic phase by using anhydrous sodium sulfate, filtering, concentrating under reduced pressure, and finally performing column chromatography separation to obtain a dark blue solid, namely the ratio type fluorescent probe for detecting the bisulfite.

In the step (1), the preparation route and method of the intermediate CBP are as follows:

synthesis of CBP-1: dissolving piperazine (6 g, 69 mmol) in 30 mL DMF, adding potassium carbonate (9.51 g, 69 mmol), stirring for 15 minutes, adding p-fluoroacetophenone (3.2 g, 2.298 mmol) to the mixture, refluxing at 80 ℃ overnight, after the reaction is complete, removing the solvent under reduced pressure, dissolving dichloromethane, washing with saturated sodium chloride, drying over anhydrous sodium sulfate, removing the solvent under reduced pressure to obtain CBP-1, yield: 55 percent;

synthesis of CBP: weighing 4-diethylamino salicylaldehyde (193.1 mg, 1 mmol) and CBP-1 (204.1 mg, 1 mmol) in a 25 mL round-bottom flask, adding 3mL concentrated sulfuric acid, and heating and stirring at 90 ℃ for 6 h; after the reaction is finished, cooling to room temperature, dropwise adding the reaction liquid into 30 mL of ice water, dropwise adding 7-8 drops of perchloric acid, separating out a solid, filtering out the solid, washing, drying, and then performing column chromatography separation and purification (dichloromethane: methanol =15:1, v/v) to obtain a green solid with the yield of 71%.

The molar ratio of the intermediate CBP, triethylamine and 4-chloro-7-nitrobenzofurazan in the step (1) is 1: (1-8): (1-10), and the mixing and stirring time is 5-25 minutes.

The eluent adopted by the column chromatography separation in the step (2) is CH₂Cl₂And CH₃OH is 1 according to the volume ratio (30-50).

The application of the ratio type fluorescent probe for detecting the bisulfite is used for specifically and highly sensitively detecting the bisulfite in foods and environments.

The invention has the following beneficial effects:

1. the preparation method of the ratio-type fluorescent probe is simple and high in yield. Analysis and test results show that the probe CBP-N has high-efficiency and specific selectivity on the bisulfite, has strong anti-interference capability, has the lowest detection limit of 18 nM, and after the probe CBP-N responds to the bisulfite, the fluorescence intensity at 549 nM in a fluorescence emission spectrum is obviously enhanced, and the fluorescence intensity at 631 nM is obviously reduced, so that the ratio type detection on the bisulfite can be realized, the characteristic can overcome the interference of common factors in the environment, and the probe CBP-N has good practical application value.

2. Conventional fluorescent probes for bisulfite detection are of the simple fluorescence enhancing or quenching type, the action mechanism is based on the fluorescence enhancement or quenching of the probe, the probe is easily influenced by the pH value, the probe concentration, the temperature, the solution polarity and the like in the detection environment, and the probe also has the defect of difficult quantification, the identification mechanism of the ratio type bisulfite detection method of the invention is Fluorescence Resonance Energy Transfer (FRET), the mechanism is based on the quantitative determination of the ratio of fluorescence intensities measured at two different wavelengths, can carry out probe intramolecular self-calibration, can eliminate or effectively eliminate the interference of external factors such as environmental temperature, solution polarity, pH value, detector efficiency and the like on the fluorescence intensity measurement, thereby realizing the real-time qualitative and quantitative detection of the content of the trace bisulfite, and having great superiority in biological analysis and environmental detection.

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 the fluorescent probe CBP-N of the present invention.

FIG. 2 is a nuclear magnetic resonance carbon spectrum of the fluorescent probe CBP-N of the present invention.

FIG. 3 is a high resolution mass spectrum of the fluorescent probe CBP-N of the present invention.

FIG. 4 is a diagram of fluorescence selectivity (common anions and active small molecules) of the fluorescent probe CBP-N of the present invention, with an excitation wavelength of 450 nm.

FIG. 5 is a histogram of fluorescence selectivity (common anions and active small molecules) of the fluorescent probe CBP-N of the present invention, with an excitation wavelength of 450 nm, emission wavelengths of 549 nm and 631 nm.

FIG. 6 is a histogram of fluorescence selectivity (common amino acids) of the fluorescent probe CBP-N of the present invention, with an excitation wavelength of 450 nm, emission wavelengths of 549 nm and 631 nm.

FIG. 7 is a fluorescence titration chart of bisulfite identification by the fluorescent probe CBP-N of the present invention, with an excitation wavelength of 450 nm.

FIG. 8 is a graph showing the results of the fluorescent probe CBP-N of the present invention identifying bisulfite, and the emission wavelengths are 549 nm and 631 nm.

FIG. 9 is the pH value experiment chart of bisulfite identification by fluorescent probe CBP-N of the present invention, with emission wavelengths of 549 nm and 631 nm.

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.