阅读说明：本技术 一种小分子荧光HClO探针、制备方法及其应用 (Small-molecule fluorescent HClO probe, preparation method and application thereof ) 是由 丁峰 戴立上 张积太 孙鹏 陈洪 于 2021-06-10 设计创作，主要内容包括：本发明公开了一种小分子荧光HClO探针、制备方法及其应用。该小分子荧光探针由7-(二乙氨基基)香豆素-3-甲醛和2-肼基苯并噻唑基团合成而来,其中二者连接部分易被次氯酸氧化后导致2-肼基苯并噻唑基团脱落,从而生成另一香豆素衍生物,与此同时探针荧光也由绿色转为蓝色,因此可实现比色和荧光比率式、精确和快速地检测次氯酸或次氯酸根离子,可用于检测活细胞中外源性及内源性的次氯酸或次氯酸根离子。此外,它还可以检测溶液、植物豆芽、检测试纸中外源性的次氯酸或次氯酸根离子。本发明合成方法简单,操作方便,并不需要苛刻的条件,而且合成产率和纯度都很高。因此,它在次氯酸或次氯酸根离子检测方面具有良好的应用前景。(The invention discloses a small-molecule fluorescent HClO probe, a preparation method and application thereof. The micromolecule fluorescent probe is synthesized by 7- (diethylamino) coumarin-3-formaldehyde and 2-hydrazino benzothiazole groups, wherein the connecting part of the two groups is easy to be oxidized by hypochlorous acid to cause the 2-hydrazino benzothiazole groups to fall off, so that another coumarin derivative is generated, and meanwhile, the fluorescence of the probe is converted from green to blue, so that a colorimetric and fluorescent ratio type can be realized, the hypochlorous acid or hypochlorite ions can be accurately and quickly detected, and the micromolecule fluorescent probe can be used for detecting exogenous and endogenous hypochlorous acid or hypochlorite ions in living cells. In addition, it can also detect exogenous hypochlorous acid or hypochlorite ions in solution, plant bean sprouts and detection test paper. The synthesis method is simple, convenient to operate, does not need harsh conditions, and has high synthesis yield and purity. Therefore, the method has good application prospect in the aspect of hypochlorous acid or hypochlorite ion detection.)

1. A small molecule fluorescent HClO probe is characterized in that: the molecular formula of the small molecular fluorescent probe is C₂₁H₂₁N₄O₂S, the structural formula is as follows:

2. a method for preparing the small molecule fluorescent HClO probe of claim 1, wherein the method comprises the steps of: the compound is synthesized by reacting 7- (diethylamino) coumarin-3-formaldehyde and 2-hydrazinobenzothiazole in a solvent.

3. The method of claim 2, wherein: the solvent is ethanol.

4. The method of claim 2, wherein: and (2) stirring the 7- (diethylamino) coumarin-3-formaldehyde and the 2-hydrazinobenzothiazole in a solvent to react to generate a solid, filtering and washing with ethanol after the reaction is finished, and drying to obtain the small-molecule fluorescent HClO probe.

5. Use of the small molecule fluorescent HClO probe of claim 1 to detect, identify hypochlorous acid or hypochlorite ions in an environment or in a biological sample.

6. A reagent or kit for detecting, identifying hypochlorous acid or hypochlorite ions in an environment or in a biological sample, characterized in that: comprising a small molecule fluorescent HClO probe according to claim 1.

7. A method for detecting, identifying hypochlorous acid or hypochlorite ions in an environment or in a biological sample, comprising: the method comprises the following steps:

(1) mixing the small-molecule fluorescent HClO probe of claim 1 with a sample to be tested;

(2) hypochlorous acid or hypochlorite ions in the environment or in the biological sample can be detected and identified by judging whether the color of the solution changes before and after mixing, and the hypochlorous acid or hypochlorite ions in the environment or in the biological sample can be judged if the fluorescence of the solution changes to blue after mixing.

8. The method of claim 7, wherein: the step (2) is to measure the absorbance of the solution before and after mixing in the wavelength range of 300 nm-700 nm by an ultraviolet spectrophotometry; hypochlorous acid or hypochlorite ion levels in the environment or in the biological sample are identified from the color change of the solution before and after mixing.

9. The method of claim 7, wherein: specifically, the step (2) is to measure the fluorescence emission wavelength of the mixed solution in the wavelength range of 420nm to 720nm by using 395nm as the excitation wavelength through a fluorescence spectrophotometry; fluorescence emission intensity ratios at emission wavelengths of 478 and 528nm are used to identify hypochlorous acid or hypochlorite ion level changes in the environment or in biological samples.

10. The use according to claim 5 or the method according to any of claims 7-9, wherein: the biological sample is living cells, plant bean sprouts or detection test paper.

Technical Field

The invention belongs to the field of fluorescent imaging molecular probes, and particularly relates to a small molecular fluorescent probe, a preparation method and application thereof.

Background

HClO or its anionic form (ClO) together with hydroxyl radical, singlet oxygen, nitric oxide, superoxide, etc^-) Constitute the Reactive Oxygen Species (ROS) family. ROS play an important role in almost all types of life processes, from cell signaling to cancer progression to autophagy. HClO is an important participant in various ROS and also helps to keep our immune system well functioning. Low concentrations of HClO can kill invading microorganisms or pathogens. On the other hand, excessive amounts of HClO have been shown to cause DNA damage and to cause a variety of diseases. These complex situations require us to develop highly sensitive and selective HClO probes to monitor their subtle changes in all systems.

For many years, several HClO probes developed based on different building blocks and sensing strategies have been reported. Among them, fluorescence-based HClO probes account for the vast majority of current reports. Generally, the presently reported HClO probes are mainly built on Coumarin (CM) or rhodamine derivative blocks. Coumarin has been widely used for constructing small molecule fluorescent probes due to its excellent physical and chemical properties, and the present applicant has previously developed several CM-derived fluorescent probes that can detect various types of ions in vitro and in vivo in various model systems, see chinese patent application No. 202110292304.X, 202110297631.4.

In addition, the currently available HClO probe moieties suffer from complex synthetic procedures, unsatisfactory sensitivity and/or selectivity, poor water solubility, insufficient exploration of potential for applications in biological or environmental assays, and the like. Moreover, most HClO probes rely on fluorescence enhancement or quenching of the fluorophore unit to achieve detection, which is clearly inferior to those that rely on a ratiometric fluorescent response, since it is not susceptible to other environmental factors. Together, these deficiencies motivate us to continue to develop new HClO probes.

Disclosure of Invention

The invention aims to overcome the defects of the prior art and provide a small-molecule fluorescent HClO probe, a preparation method and application thereof.

In order to achieve the above object, the first aspect of the present invention provides a small molecule fluorescent HClO probe, wherein the molecular formula of the small molecule fluorescent probe is C₂₁H₂₁N₄O₂S, the structural formula is as follows:

the second aspect of the invention provides a preparation method of the small-molecule fluorescent HClO probe, which is synthesized by reacting 7- (diethylamino) coumarin-3-formaldehyde and 2-hydrazinobenzothiazole in a solvent.

The further setting is that the solvent is ethanol.

The method is further provided with the steps that 7- (diethylamino) coumarin-3-formaldehyde and 2-hydrazinobenzothiazole are stirred in a solvent to react to generate a solid, and after the reaction is finished, the solid is filtered, washed by ethanol and dried to obtain the small-molecule fluorescent HClO probe.

In addition, the invention also provides an application of the small-molecule fluorescent HClO probe in detecting and identifying hypochlorous acid or hypochlorite ions in an environment or a biological sample.

The invention also provides a reagent or a kit for detecting, identifying hypochlorous acid or hypochlorite ions in an environment or a biological sample, which comprises the small-molecule fluorescent HClO probe.

The invention also provides a method for detecting and identifying hypochlorous acid or hypochlorite ions in an environment or a biological sample, which comprises the following steps:

(1) mixing the small-molecule fluorescent HClO probe of claim 1 with a sample to be tested;

(2) hypochlorous acid or hypochlorite ions in the environment or in the biological sample can be detected and identified by judging whether the color of the solution changes before and after mixing, and the hypochlorous acid or hypochlorite ions in the environment or in the biological sample can be judged if the fluorescence of the solution changes to blue after mixing.

Further setting that the step (2) specifically measures the absorbance of the solution before and after mixing in the wavelength range of 300 nm-700 nm by an ultraviolet spectrophotometry; hypochlorous acid or hypochlorite ion levels in the environment or in the biological sample are identified from the color change of the solution before and after mixing.

Further setting that the step (2) specifically measures the fluorescence emission wavelength of the mixed solution in the wavelength range of 420nm to 720nm by using 395nm as an excitation wavelength through a fluorescence spectrophotometry; fluorescence emission intensity ratios at emission wavelengths of 478 and 528nm are used to identify hypochlorous acid or hypochlorite ion level changes in the environment or in biological samples.

In the above technical scheme, the biological sample is living cells, plant bean sprouts or test paper.

The invention has the following beneficial effects: the small molecular fluorescent probe provided by the invention is synthesized on the basis of 7- (diethylamino) coumarin-3-formaldehyde and 2-hydrazinobenzothiazole, has green fluorescence, is easy to be oxidized by hypochlorous acid to cause the shedding of a 2-hydrazinobenzothiazole group, so that a coumarin derivative is generated, and meanwhile, the fluorescence of the probe is changed from green to blue. The small molecular probe can realize the colorimetric and fluorescent ratio type, can accurately and quickly detect hypochlorous acid or hypochlorite ions, and can be used for detecting exogenous hypochlorite ions in solution, plant bean sprouts and biological test paper and exogenous and endogenous hypochlorite ions in living cells. The experimental result is not easily affected by the surrounding environment due to the adoption of ratio type detection, the probe has rapid reaction (<100s) and low detection limit (545nM), and belongs to a leading level in the similar probes, and the application of the probe in detection of HClO in different systems such as cells, plant bean sprouts, developed simple test paper and the like is explored, so the probe has very good application prospect in the aspect of hypochlorous acid or hypochlorite ion detection. Meanwhile, the synthesis method is simple, convenient to operate and free of harsh conditions.

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 introduced below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and it is within the scope of the present invention for those skilled in the art to obtain other drawings based on the drawings without inventive exercise.

FIG. 1 is a schematic diagram of the synthesis route of the fluorescent small molecule probe in example 1;

FIG. 2 is a mass spectrum of the synthesized small molecule fluorescent probe of example 1;

FIG. 3 is nuclear magnetic carbon spectrum of the synthesized small molecule fluorescent probe of example 1;

FIG. 4 is the UV and fluorescence spectra of the small molecule fluorescent probe for hypochlorous acid recognition in example 2;

FIGS. 5 and 6 show the selectivity, speed response and pH adaptability of the fluorescent probe for hypochlorous acid in example 3;

FIG. 7 is the detection of exogenous and endogenous hypochlorous acid in cancer cells by the small molecule fluorescent probe of example 4;

FIG. 8 is a graph showing the detection of exogenous hypochlorous acid in the plant bean sprouts by the small molecule fluorescent probe of example 5;

FIG. 9 is the detection of exogenous hypochlorous acid in the test paper by the small molecule fluorescent probe in example 6;

FIG. 10 is the density functional theory calculation of hypochlorous acid recognition by the small molecule fluorescent probe in example 7.

Detailed Description

In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention will be described in further detail with reference to the accompanying drawings.

Example 1: synthesis of Small molecule fluorescent probes

The chemical formula of the synthesized small molecule fluorescent probe is shown in figure 1, wherein EtOH is ethanol, and RT is room temperature.

The specific synthetic process is as follows: dissolving 7- (diethylamino) coumarin-3-carbaldehyde (500mg,2.04mmol) in 30mL of ethanol solution, adding 2-hydrazinobenzothiazole (336mg,2.04mmol) at room temperature, stirring at room temperature for reaction for 16h, filtering the reaction product through a Buchner funnel, washing with ethanol and drying to obtain a green target probe solid. As shown in fig. 2 and 3, the product can be determined to be the target small molecule fluorescent probe by mass spectrometry, nuclear magnetism and spectroscopy.

Example 2: ultraviolet and fluorescence spectrum of small-molecule fluorescent probe responding to hypochlorous acid and fluorescence colorimetric analysis test HClO

Preparation of 0.5mL Small molecule fluorescent Probe (10.0X 10)^-6mol/L) in EtOH/PBS (v/v-2/8, pH-7.4). 0-30. mu.M hypochlorous acid solution was added dropwise to the probe solution, as shown in FIG. 4(A), and after hypochlorous acid was added to the probe solution, the main absorption band at 450nm gradually decreased, and a new absorption peak at-550 nm was observed, and the absorption intensity gradually increased with the HClO ion concentration. The inner graph shows that the color of the fluorescent probe solution is changed from original green to colorless after hypochlorous acid is added, so that colorimetric detection of hypochlorous acid can be realized.

In a fluorescence titration experiment, 3mL of small molecule probe (10.0X 10) was prepared^-6mol/L) in EtOH/PBS (v/v-2/8, pH-7.4). 0-30. mu.M hypochlorous acid solution was dropped into the probe solution, and the fluorescence value of the probe from 420nm to 720nm was measured at 395nm as the excitation wavelength, and the experimental result is shown in FIG. 4 (B). It was observed that the fluorescence emission of the probe varied with increasing hypochlorous acid concentration, with the original 528nm emission peak intensity decreasing and the new 478nm emission peak intensity increasing. FIG. 4(C, D) shows the fluorescence F obtained by analyzing the above-mentioned change in the fluorescence emission ratio and the concentration of hypochlorous acid added and performing linear simulation in the range of linear response₄₇₈/F₅₂₈The ratio has good linear response relation with the hypochlorous acid concentration (14-28 mu M), and the probe can realize the fluorescence ratio type detection of the hypochlorous acid level.

Example 3 verification of Small molecule fluorescent probes for hypochlorous acid Selectivity, response speed and pH Adaptation

5mL of molecular probe (10.0X 10) was prepared^-6mol/L) in EtOH/PBS (v/v-2/8, pH-7.4). Preparation of each by dissolving the corresponding salt in deionized waterInterfering medium solution (1: HNAP-hdbt,2: OH,3: AcO^-,4:SO₃ ^2-,5:O₂ ^-,6:SO₄ ^2-,7:HClO,8:NO₃ ^-,9:H₂O₂,10:Cys,11:GSH,12:PO₄ ^2-，30.0×10^-6mol/L). Subsequently, an equivalent amount of hypochlorous acid solution and these interfering agents were added to the probe solution separately. Detecting by fluorescence spectroscopy, and taking fluorescence emission wavelength ratio (F)₄₇₈/F₅₂₈) And (6) carrying out comparison. The results show (FIG. 5A) that none of these interfering agents produced as significant a change in fluorescence of the probe as hypochlorous acid, and that they belong to the ROS series OH, H₂O₂Nor have any significant effect. Only after the addition of hypochlorous acid, the fluorescence ratio of the probe solution changes significantly. Meanwhile, the probe solution can be observed by naked eyes to have obvious color change: from green to approximately colorless (fig. 5B left); under UV irradiation, the probe solution changed color from green to blue upon exposure to hypochlorous acid, with no other change (FIG. 5B, right).

FIG. 6A is a graph in which the response speed of the probe solution to hypochlorous acid was examined, and the results showed that the fluorescence ratio reached a stable equilibrium value already at around 100S, indicating that the probe responded extremely rapidly to hypochlorous acid. FIG. 6B shows the effect of pH change on the response of the probe to hypochlorous acid, and the result shows that the fluorescence ratio is not affected to a certain extent in the pH range of 4-8, which indicates the wide application range of the probe.

Example 4: imaging effect of small-molecule fluorescent probe in cancer cells

In the cancer cell imaging system, a control group (a single probe is used for treating cells) and an experimental group (after the probe treatment, hypochlorite ions with different concentrations are added for treatment or LPS treatment is used for detecting endogenous hypochlorous acid), and finally, photographing recording is carried out through a green channel and a blue channel in a fluorescence imaging system. The results of the experiment are shown in FIG. 7.

As shown in FIG. 7, only green fluorescence appeared in the cancer cells treated with the probe alone in the absence of hypochlorous acid, whereas blue fluorescence appeared in the cells gradually with the addition of exogenous hypochlorous acid, and the intensity was gradually increased while the green fluorescence was gradually decreased. When the concentration of hypochlorite ions added is 30. mu.M, the green fluorescence of the probe solution almost disappears, and only blue fluorescence is shown, and these results indicate that the probe can detect endogenous hypochlorous acid in the cancer cell body. Also after LPS (2mg/ml) stimulates the cells, green and blue fluorescence can be observed in the cells, and the surface probe can also detect endogenous hypochlorous acid or hypochlorite ions, so that the probe can realize imaging detection of the exogenous and endogenous hypochlorous acid or hypochlorite ions in the living cells.

Example 5: imaging effect of small-molecule fluorescent probe in plant bean sprouts

In the plant bean sprout imaging system, a control group (the probe treats the bean sprouts first and then treats the bean sprouts with PBS) and an experimental group (the probe treats the bean sprouts first and then adds hypochlorous acid for treatment) are set, and then photographing recording is carried out through green and blue channels in a fluorescence imaging system, and the experimental result is shown in figure 8.

The results in fig. 8 show that only green fluorescence appears in the bean sprouts treated with the probe alone under irradiation of an ultraviolet lamp (365nm) in the absence of hypochlorous acid. With the addition of hypochlorous acid (30. mu.M), only blue fluorescence of the probe in the bean sprouts appeared. These results also indicate that the probe can detect exogenous hypochlorous acid or hypochlorite ions in the plant bean sprouts.

Example 6: imaging effect of small molecular fluorescent probe in test paper

In the test paper system, a control group (a single probe is used for treating the test paper) and an experimental group (the probe is treated firstly and then hypochlorous acid is added for treating) are set, and finally, photographing and recording are respectively carried out through a blue channel and a green channel in a fluorescence imaging system, wherein the experimental result is shown in fig. 9.

The results in FIG. 9 show that only green fluorescence appears in the strip treated with probe alone in the absence of hypochlorous acid. With the addition of 30. mu.M hypochlorous acid, only blue fluorescence appeared on the probe in the test paper, while green fluorescence disappeared. These results also indicate that the probe can detect exogenous hypochlorous acid or hypochlorite ions in the strip.

Example 7: density functional theory calculation of small molecule fluorescence probe for hypochlorous acid recognition

FIG. 10 is a density functional theory calculation of hypochlorous acid identification by the small molecule fluorescent probe in examples 4, 5 and 6;

a process of change of fluorescence from green to blue in the presence of the probe alone and after binding hypochlorite, wherein the specific cause of the change is essentially explained by calculating the energy level transition of the molecular fluorescent probe before and after binding, and calculating whether there is a difference in energy required for the transition between the two. The results of the experiment are shown in FIG. 10.

As shown in FIG. 10, in the presence of the probe alone, the energy of the highest occupied orbital (HOMO) of the molecular probe was-5.0462 ev, the energy of the lowest unoccupied orbital (LUMO) was-1.8937 ev, and the energy difference between them was: 3.1525 ev. The probe had a HOMO value of-5.7321 ev and a LUMO value of-2.0943 ev after binding to hypochlorous acid, wherein the difference in energy levels of the two orbitals is: 3.6378 ev; thus, it was found that the difference in level between the probe and hypochlorous acid became large, which corresponded to the blue shift exhibited by the fluorescence spectrum.

The small molecule fluorescent probe can detect hypochlorous acid or hypochlorite ions in a solution by a fluorescence spectrum technology.

The micromolecule fluorescent probe has obvious changes of ultraviolet absorption main peak (red shift of 450nm to 550nm) and fluorescence emission peak (blue shift of 528nm to 478nm) in the presence of hypochlorous acid or hypochlorite ions, so that quantitative detection of the hypochlorous acid or hypochlorite ions can be realized according to the solution color change after ultraviolet absorption and the ratio change of the fluorescence emission peak.

The invention has the following advantages: the micromolecular fluorescent probe synthesized by the preparation method can also realize accurate sensing of hypochlorous acid or hypochlorite ions by an ultraviolet colorimetric method and a fluorescence spectroscopy method, and can quickly and accurately detect the hypochlorous acid or hypochlorite ions in cancer cells, plant bean sprouts and detection test paper. Therefore, the method has good application prospect in the aspect of hypochlorous acid or hypochlorite ion detection. Meanwhile, the synthesis method is simple, convenient to operate and free of harsh conditions.

The above disclosure is only for the purpose of illustrating the preferred embodiments of the present invention, and it is therefore to be understood that the invention is not limited by the scope of the appended claims.