CN108101867B

CN108101867B - Preparation method and application technology of fluorescent probe for detecting glutathione

Info

Publication number: CN108101867B
Application number: CN201711343255.8A
Authority: CN
Inventors: 刘恒; 陈逢灶; 张修华; 王升富
Original assignee: Hubei University
Current assignee: Hubei University
Priority date: 2017-12-15
Filing date: 2017-12-15
Publication date: 2020-05-19
Anticipated expiration: 2037-12-15
Also published as: CN108101867A

Abstract

The invention belongs to the technical field of fluorescent probe sensing in analytical chemistry, and relates to a preparation method of a novel anthraquinone structure-based fluorescent probe and an application technology of the novel anthraquinone structure-based fluorescent probe in Glutathione (GSH) detection. The preparation method comprises the step of carrying out chlorination reaction on thiadiazole anthraquinone derivatives and potassium chlorate to obtain probe molecules, and the preparation method is simple and easy to implement, low in cost and stable in structure of the prepared probe. Under mild conditions, the probe can be applied to high-sensitivity, high-selectivity and rapid visual detection of glutathione. In addition, the probe has been successfully applied to the detection of glutathione in biological cells. The invention provides a preparation method and an application technology of a novel fluorescent probe for detecting glutathione, and the preparation method and the application technology have good application prospects.

Description

Preparation method and application technology of fluorescent probe for detecting glutathione

Technical Field

The invention relates to the technical field of fluorescent probe sensing in analytical chemistry, in particular to a preparation method and an application technology of a novel fluorescent probe for quickly and visually detecting glutathione with high selectivity and high sensitivity.

Background

Glutathione (GSH) is a small peptide composed of three amino acids, cysteine, glutamic acid, and glycine, which is present in almost every cell of the body. GSH plays a very important role in living organisms, and helps them maintain normal immune system functions, including antioxidant action and integrated detoxification. The physiological level of GSH content is helpful for eliminating free radicals in the organism, not only has the functions of enhancing immunity, resisting oxidation and resisting aging, but also plays an irreplaceable role in the aspects of protecting the liver, protecting damaged organs, resisting radiation and treating certain diseases, and abnormal GSH concentration change is related to various serious diseases, such as AIDS, cardiovascular diseases, cirrhosis, cancer and the like. In addition, GSH has wide applications in the fields of pharmaceuticals, foods, functional beverages, and cosmetics. Therefore, it is important to accurately detect the concentration of GSH. However, the detection of the GSH concentration in a complex actual sample is often limited by various factors, so that a good detection result cannot be obtained, and the traditional GSH concentration detection methods mainly comprise a spectrophotometry method, an enzyme circulation method, a colorimetric method, a liquid chromatography method and the like. Although the methods can realize qualitative or quantitative detection of GSH, the methods have certain defects, such as complex sample preparation, expensive instruments, complex operation, long detection period, poor selectivity, low sensitivity, difficulty in popularization and application and the like. Therefore, it is very important to develop a method for rapidly and conveniently detecting the concentration of GSH with high selectivity and high sensitivity, which will have very important significance for the initial clinical diagnosis and treatment of some serious diseases. The fluorescent probe analysis method has the advantages of simplicity, convenience, rapidness, high sensitivity, good selectivity, low cost, easiness in popularization and the like, so that the realization of accurate detection of GSH by using the fluorescent probe also becomes a target pursued by researchers. Because the structures and the reaction activities of cysteine (Cys) and homocysteine (Hcy) are similar to GSH, the GSH is difficult to distinguish from Cys/Hcy, and only a few fluorescent probes can realize rapid, high-selectivity and high-sensitivity detection of the GSH at present. In view of this, the development of fluorescent probes capable of highly selectively differentiating GSH and Cys/Hcy is an urgent issue to be solved at present.

The invention relates to a preparation method and an application technology of a fluorescent probe for detecting glutathione. The thiadiazole anthraquinone derivative and potassium chlorate are adopted to obtain the fluorescent probe ATD-Cl through chlorination reaction, the preparation method is simple and easy to implement, the cost is low, and the prepared probe is stable in structure. Under mild conditions, the fluorescent probe can be applied to high-sensitivity, high-selectivity and rapid visual detection of glutathione. In addition, the fluorescent probe has been successfully applied to the detection of glutathione in biological cells. Therefore, the fluorescent probe has good application prospect.

Disclosure of Invention

Aiming at the defects of the prior art, the invention aims to provide a fluorescence-enhanced probe for detecting glutathione and application thereof.

According to the invention, the fluorescent probe is obtained by carrying out chlorination reaction on the thiadiazole anthraquinone derivative and potassium chlorate, and the synthesis steps specifically comprise: (1) dissolving 1, 2-diaminoanthraquinone in tetrahydrofuran under the environment of anhydrous and nitrogen protection, then slowly dropwise adding thionyl chloride and triethylamine, stirring overnight at room temperature, and reacting to obtain purple solid. After the reaction is finished, pouring the reaction liquid into crushed ice, then filtering, carrying out vacuum drying at 40 ℃ to obtain a crude product, and recrystallizing with ethanol to obtain a pure product thiadiazole anthraquinone derivative (ATD); (2) will be provided withATD is dissolved in glacial acetic acid, heated and stirred to 90 ℃, and then concentrated hydrochloric acid is added. Then adding KClO in small amount for multiple times₃And then continuously reacting at 90 ℃ for 4 hours, stopping heating after the reaction is finished, cooling to room temperature, pouring the reaction liquid into ice water, filtering to obtain a crude product, and recrystallizing glacial acetic acid to obtain a target probe molecule ATD-Cl, namely the fluorescent probe for detecting glutathione.

The synthetic route of ATD-Cl is as follows:

the application of the probe ATD-Cl of the invention is as follows: the fluorescent probe can be used for high-selectivity detection of glutathione in water environment and biological cell lines in a pH 7.4 system. We performed fluorescence titration, probe-target response time exploration, interference assays and pH optimization on the fluorescent probes.

Compared with the prior art, the patent technology has the following advantages:

1. the selectivity is good, the fluorescent probe has very good specificity to glutathione, and other related substances have little influence on the fluorescent probe;

2. the sensitivity is high, and the fluorescent probe can realize the detection of trace glutathione;

3. the fluorescent probe can realize rapid and real-time detection;

3. simple synthesis, low cost and no pollution.

Drawings

FIG. 1a is the nuclear magnetic hydrogen spectrum diagram of fluorescent probe ATD-Cl

FIG. 1b is a high-resolution mass spectrum of fluorescent probe ATD-Cl

FIG. 2 is a fluorescence titration chart of the fluorescence probe for detecting glutathione

FIG. 3 is a fluorescence spectrum diagram of the response time of the fluorescent probe for detecting glutathione

FIG. 4 is a fluorescence spectrum of the fluorescence probe for selective experiment

FIG. 5 is a pH optimized fluorescence spectrum of a detection environment

FIG. 6 is a diagram showing the intracellular fluorescence imaging test of the fluorescent probe

Detailed Description

The present invention will be further described with reference to the following examples, which are only for illustrating the technical solutions of the present invention and are not to be construed as limiting the present invention.

Example 1

1, 2-diaminoanthraquinone (0.6g,2.5mmol) was dissolved in tetrahydrofuran (15mL) under anhydrous nitrogen, and then thionyl chloride (0.7mL,9.7mmol) and 1.5mL triethylamine were added dropwise slowly and stirred at room temperature overnight to give a purple solid. After the reaction is finished, pouring the reaction liquid into 100g of crushed ice, deeply cooling, filtering to obtain a crude product, recrystallizing glacial acetic acid, and recrystallizing ethanol to obtain the product ATD (0.73g,2.3 mmol). ATD (0.5g,1.6mmol) was dissolved in 18mL of glacial acetic acid, heated to 90 ℃ with stirring, and 8mL of concentrated hydrochloric acid was added. Then adding KClO in small amount for multiple times₃(3.0g,22.4 mmol), continuing to react at 90 ℃ for 4 hours, stopping heating after the reaction is finished, cooling to room temperature, pouring the reaction liquid into ice water, filtering to obtain a crude product, recrystallizing glacial acetic acid to obtain the target probe molecule (0.18g,0.59mmol) with the yield of 37%, and carrying out nuclear magnetic characterization data:¹HNMR(400MHz,CDCl₃) δ 8.61(s,1H),8.38(dd, J ═ 7.0,1.7Hz,1H),8.31(dd, J ═ 7.2,1.6Hz,1H),7.91-7.84(m,2H) (fig. 1 a); HRMS Calcd for C₁₄H₅ClN₂O₂S[M+H]⁺300.9833; found:300.9828 (FIG. 1 b).

Application example 1: as shown in FIG. 2, the probe detects a fluorescence titration graph of glutathione. When a fluorescent probe (10 mu M) is added into a PBS (10mM, pH 7.4) solution containing 1mM hexadecyltrimethylammonium bromide (CTAB), the original fluorescence of the fluorescent probe is extremely weak under the excitation of light at 465nm, however, when the fluorescent probe responds to glutathione added at different concentrations, the fluorescence intensity at 558nm changes remarkably, the fluorescence intensity at 558nm is enhanced along with the increment of the concentration of the added glutathione, and a good linear relation exists in the range of the concentration of the glutathione being 0-20 mu M (R)²0.993), glutathione concentration reachedThe reaction reaches saturation at 60 mu M, the fluorescence intensity is stable and does not change any more, and the detection limit of the probe reaches 89 nM. This example demonstrates that the fluorescent probe can be applied to glutathione detection with high sensitivity.

Application example 2: as shown in FIG. 3, the fluorescent probe detects the response time fluorescence spectrum of glutathione. To a solution of 1mM cetyltrimethylammonium bromide (CTAB) in PBS (10mM, pH 7.4) was added a fluorescent probe (10 μ M), followed by glutathione (60 μ M). Under 465nm light excitation, the fluorescence intensity at 558nm began to increase gradually with time and the response was complete within 30 minutes. The application example proves that the fluorescent probe can realize the rapid detection of glutathione.

Application example 3: FIG. 4 shows the fluorescence spectrum of the fluorescence probe in the selectivity experiment. Different interferents such as various amino acids were added separately under the same test system and test conditions: cysteine, homocysteine, alanine, arginine, aspartic acid, glycine, glutamic acid, histidine, leucine, isoleucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, valine, and various cationic and reactive oxygen species such as: HS^-、Br^-、Cl^-、F^-、I^-、HPO₄ ^2-、NO₃ ^-、SO₄ ^2-、Al³⁺、Fe³⁺、 Mg²⁺、Na⁺、Zn²⁺、ClO^-、H₂O₂). The experimental result proves that the fluorescent probe has extremely high selectivity to the glutathione and shows that the fluorescence intensity at 558nm is obviously enhanced, but the fluorescent probe hardly responds to other related interferents and shows that the fluorescence intensity at 558nm is almost kept unchanged.

Application example 4: FIG. 5, pH optimized fluorescence spectra of the detection environment. And (3) respectively adding the fluorescent probe and the glutathione into PBS (phosphate buffer solution) with different pH (pH 3-12), keeping a tested system and conditions consistent, and measuring the response condition of the fluorescent probe and the glutathione of the fluorescent probe under different pH values. As can be seen from the figure, the fluorescence intensity of the fluorescent probe itself maintains high stability in the environment with different pH, and the fluorescence intensity of the probe is increased significantly in the range of pH 5 to 12 when glutathione is added to the system, wherein the fluorescence intensity reaches the maximum value in the system with pH 8. The comprehensive experimental data show that: the fluorescent probe has stability in different pH environments, can better respond to glutathione in a wide pH range, achieves the best measurement effect in a neutral environment, and is beneficial to application of the fluorescent probe in biological sample detection.

Application example 5: as shown in FIG. 6, MCF-7 was tested for intracellular fluorescence imaging. In order to prove that the ATD-Cl probe can identify GSH and Cys/Hcy in cells with high selectivity, 5 groups of cell imaging experiments are performed in total. A first group: incubating the probe (10. mu.M concentration) with MCF-7 cells for 30 min at 37 ℃; second group: adding N-ethylmaleimide (NEM, concentration of 0.5mM) into a culture dish of MCF-7 cells, incubating for 30 minutes, adding a probe (concentration of 10 μ M), and continuing to incubate for 30 minutes; third group: adding N-ethylmaleimide (NEM, concentration of 0.5mM) into a culture dish of MCF-7 cells, incubating for 30 minutes, adding a probe (concentration of 10 μ M), incubating for 30 minutes, and adding glutathione (concentration of 100 μ M), and incubating for 30 minutes; and a fourth group: adding N-ethylmaleimide (NEM, concentration of 0.5mM) into a culture dish of MCF-7 cells, incubating for 30 minutes, adding a probe (concentration of 10 μ M), incubating for 30 minutes, and adding cysteine (concentration of 100 μ M), and incubating for 30 minutes; n-ethylmaleimide (NEM, 0.5mM) was added to the culture dish of MCF-7 cells and incubated for 30 minutes, followed by addition of the probe (10. mu.M) for 30 minutes and finally addition of homocysteine (100. mu.M) for 30 minutes. Five experimental MCF-7 cells were then imaged using fluorescence confocal, excitation wavelength 488nm, and green channel (530-590nm) collected. The results are shown in fig. 6, and only in the presence of the probe, the green channel has strong fluorescence, which indicates that the probe can image GSH endogenous to the cell; when almost no fluorescence was observed in the green channel after addition of the thiol blocker NEM, and further addition of GSH, Cys, Hcy, respectively, we found that the addition of GSH alone caused a significant increase in the fluorescence of the green channel. The experimental results show that the probe ATD-Cl can identify GSH with high selectivity and has potential application prospect.

Claims

1. The new application of the fluorescent probe is characterized in that the fluorescent probe is used for detecting glutathione in a water environment with pH 7.4 and a biological cell system, and the molecular structural formula of the fluorescent probe is as follows:

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

a: dissolving 1, 2-diaminoanthraquinone in tetrahydrofuran under the environment of anhydrous and nitrogen protection, then slowly dropwise adding thionyl chloride and triethylamine, stirring at room temperature overnight to generate purple solid, after the reaction is finished, pouring the reaction liquid into crushed ice, then filtering, drying in vacuum at 40 ℃ to obtain a crude product, and recrystallizing ethanol to obtain a pure product, namely the thiadiazole anthraquinone derivative;

b: dissolving the thiadiazole anthraquinone derivative in the step a in glacial acetic acid, heating and stirring to 90 ℃, adding concentrated hydrochloric acid, and then adding KClO for a plurality of times in small amount₃And then continuously reacting at 90 ℃ for 4 hours, stopping heating after the reaction is finished, cooling to room temperature, pouring the reaction liquid into ice water, filtering to obtain a crude product, and recrystallizing glacial acetic acid to obtain the fluorescent probe.

2. The novel use of the fluorescent probe as claimed in claim 1, wherein the stoichiometric molar ratio of the 1, 2-diaminoanthraquinone as the raw material to thionyl chloride in step a is 1: 0.1-10.

3. The novel use of the fluorescent probe as claimed in claim 2, wherein the thiadiazole anthraquinone derivative and KClO in step b₃The stoichiometric molar ratio of (A) to (B) is 1:0.1 to 20.