CN115677592B

CN115677592B - Amino coordination type high-selectivity mercury ion fluorescent probe, preparation method and application

Info

Publication number: CN115677592B
Application number: CN202211379660.6A
Authority: CN
Inventors: 史沫苒; 李泽佶; 于谦; 李筱; 张晓琪; 盛文龙; 李�灿; 于妙慧; 付婷婷; 李文斋; 靳梦; 李晓彬; 夏青
Original assignee: Biology Institute of Shandong Academy of Sciences
Current assignee: Biology Institute of Shandong Academy of Sciences
Priority date: 2022-11-04
Filing date: 2022-11-04
Publication date: 2023-05-02
Anticipated expiration: 2042-11-04
Also published as: CN115677592A

Abstract

The invention relates to an amino coordination type high-selectivity mercury ion fluorescent probe, in particular to a probe which can be used for measuring, detecting or screening mercury ions and living cell fluorescent imaging. The probe of the invention takes the amino compound as a mercury ion coordination unit, can rapidly, sensitively and selectively identify mercury ions through the fluorescent probe coordinated by the amino group and mercury, and can detect the mercury ions in living cells and zebra fish bodies in an ultrasensitive and highly selective manner through a fluorescence analysis method.

Description

Amino coordination type high-selectivity mercury ion fluorescent probe, preparation method and application

Technical Field

The invention belongs to the field of fluorescent probes, and particularly relates to an amino coordination type high-selectivity mercury ion fluorescent probe and application thereof in a mercury ion measurement, detection or screening and living cell fluorescent imaging method; the invention also provides a method for preparing the fluorescent probe.

Background

Mercury (Hg) ²⁺ ) As a metal element having serious physiological toxicity, it is one of the most attractive environmental pollutants at present due to its durability, easy migration and high bioaccumulation. Inorganic mercury ions in the environment can be converted into highly toxic methyl mercury by organisms under certain conditions. Inorganic mercury affects mainly the kidneys, while methylmercury affects mainly the nervous system, especially the central nervous system, after entering the human body. Both can be highly enriched in biological tissues through food chains, thereby causing great harm to people and nature. Mercury poisoning can have an extremely adverse effect on the whole society, and mercury is now preferentially listed on the global environmental monitoring system list, so that selective recognition of mercury ions, especially in-situ, real-time and online monitoring of mercury ions, has great significance for medicine, biology and environmental science.

At present, reported analysis methods for detecting mercury ions include atomic absorption-emission spectrometry, high performance liquid chromatography, inductively coupled plasma mass spectrometry, nuclear magnetic resonance, electrochemical methods, and fluorescent probe analysis, among which fluorescent probe methods have been receiving attention because of their unique advantages. However, the methods reported at present have certain defects such as poor selectivity, low sensitivity, complex synthesis, long response time and the like. Therefore, the development of a fluorescent probe for detecting mercury ions, which is rapid, highly selective, highly sensitive, and simple to synthesize, is a subject to be solved by those skilled in the art.

Disclosure of Invention

In view of the above, the invention provides a novel high-selectivity fluorescent probe for detecting amino coordination type mercury ions, which has the advantages of simple synthesis, good selectivity and rapid response, and can realize rapid and sensitive detection of mercury ions in aqueous solution, cells and zebra fish.

Specifically, the invention provides a fluorescent probe for measuring, detecting or screening mercury ions, which has a structure shown in a formula (I):

in the formula (I), R ₁ ，R ₂ ，R ₃ ，R ₄ ，R ₅ ，R ₆ ，R ₇ ，R ₈ ，R ₉ ，R ₁₀ ，R ₁₁ And R is ₁₂ Is independently selected from the group consisting of a hydrogen atom, a linear or branched alkyl group, a linear or branched alkoxy group, a sulfonic acid group, an ester group, and a carboxyl group; and wherein R is ₁ ，R ₂ ，R ₃ ，R ₄ ，R ₅ ，R ₆ ，R ₇ ，R ₈ ，R ₉ ，R ₁₀ ，R ₁₁ And R is ₁₂ May be the same or different.

In some embodiments of the invention, the fluorescent probe of the invention is R ₁ ，R ₂ ，R ₃ ，R ₄ ，R ₅ ，R ₆ ，R ₇ ，R ₈ ，R ₉ ，R ₁₀ ，R ₁₁ And R is ₁₂ The compounds of formula (IV) which are all hydrogen atoms have the following structural formula:

the invention also provides a preparation method of the compound shown in the formula (I), which comprises the following steps:

dissolving a compound of a formula (II) and a compound of a formula (III) in absolute ethyl alcohol, carrying out reflux reaction at 90 ℃ under nitrogen atmosphere, cooling to room temperature after the reaction is finished, and carrying out vacuum filtration under reduced pressure to obtain a solid, thereby obtaining a crude product containing the compound of the formula (I). Dissolving the crude product with methylene chloride to remove soluble impurities, and obtaining a pure compound of formula (I), wherein the reaction formula is as follows:

in the formulae (I) - (III): r is R ₁ ，R ₂ ，R ₃ ，R ₄ ，R ₅ ，R ₆ ，R ₇ ，R ₈ ，R ₉ ，R ₁₀ ，R ₁₁ And R is ₁₂ Is independently selected from the group consisting of a hydrogen atom, a linear or branched alkyl group, a linear or branched alkoxy group, a sulfonic acid group, an ester group, and a carboxyl group; and wherein R is ₁ ，R ₂ ，R ₃ ，R ₄ ，R ₅ ，R ₆ ，R ₇ ，R ₈ ，R ₉ ，R ₁₀ ，R ₁₁ And R is ₁₂ May be the same or different.

Specifically: dissolving a compound of a formula (II) and a compound of a formula (III) in absolute ethyl alcohol, carrying out reflux reaction at 90 ℃ under nitrogen atmosphere, cooling to room temperature after the reaction is finished, and carrying out vacuum filtration under reduced pressure to obtain a solid, thereby obtaining a crude product containing the compound of the formula (I). The crude product is dissolved in methylene chloride to remove soluble impurities, and a pure compound of formula (I) can be obtained.

In some embodiments of the invention, the molar ratio of the compound of formula (II) to the compound of formula (III) is from 1:1 to 1:2.

In some embodiments of the invention, the reaction time for the preparation of the compound of formula (I) step (1) and step (2) is from 6 to 8 hours.

The invention also provides a fluorescent probe composition for measuring, detecting or screening mercury ions, comprising the compound of formula (I) according to the invention.

In some embodiments of the invention, the compound of formula (I) has the following structure:

in some embodiments of the invention, the fluorescent probe composition further comprises a solvent, an acid, a base, a buffer solution, or a combination thereof.

The invention also provides a method for detecting the presence of mercury ions in a sample or measuring the mercury ion content in a sample, comprising:

a) Contacting the compound of formula (I) or formula (iv) with a sample to form a fluorescent compound;

b) Determining the fluorescent properties of the fluorescent compound.

In some embodiments of the invention, the sample is a chemical sample or a biological sample.

In some embodiments of the invention, the sample is a biological sample including water, blood, a microorganism, or an animal cell or tissue.

The invention also provides a kit for detecting the presence of mercury ions in a sample or determining the content of mercury ions in a sample, comprising the compound of formula (I) or formula (IV).

The invention also provides application of the compound shown in the formula (I) or the formula (IV) in cell fluorescence imaging.

The invention also provides a reagent for detecting mercury ions, which is prepared from the compound of the formula (I) or the formula (IV).

Compared with the prior art, the invention has the following remarkable advantages and effects:

(1) High selectivity and high anti-interference ability

The mercury ion fluorescent probe can selectively react with mercury ions specifically to generate products with fluorescence change, and compared with other common metal ions including but not limited to nickel ions, copper ions, potassium ions, lead ions, chromium ions, ferric ions, silver ions, zinc ions, calcium ions, magnesium ions, aluminum ions, ferrous ions, sodium ions and the like, the fluorescent probe has higher selectivity and strong anti-interference capability.

(2) High sensitivity and quick response

The mercury ion fluorescent probe reacts with mercury ions very sensitively and responds rapidly, thereby being beneficial to the detection of mercury ions.

(3) Can be applied under physiological level

The mercury ion fluorescent probe can be applied under the physiological level condition, and the common metal ions in organisms have small interference on the mercury ion fluorescent probe, so that the mercury ion fluorescent probe can be applied to fluorescent imaging of living cells and zebra fish.

(4) Good stability

The mercury ion fluorescent probe has good stability, and can be stored for a long time.

(5) Simple synthesis

The mercury ion fluorescent probe is simple to synthesize and is favorable for commercialization popularization and application.

Drawings

In order to more clearly illustrate the embodiments of the invention or the technical solutions in the prior art, the drawings that are required in the embodiments or the description of the prior art will be briefly described, it being obvious that the drawings in the following description are only some embodiments of the invention, and that other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.

FIG. 1 is an absorption spectrum of a probe (5. Mu.M) before and after addition of mercury ions (0-20. Mu.M);

FIG. 2 is a time kinetic profile of probe (5. Mu.M) versus mercury ions (20. Mu.M) at 570 nm;

FIG. 3 (a) is a fluorescence spectrum of the probe (5. Mu.M) before and after addition of mercury ions (0-20. Mu.M);

FIG. 3 (b) is a graph of the fluorescence intensity of the probe (5. Mu.M) at 570nm versus mercury ions (0-6. Mu.M);

FIG. 4 (a) is the effect of mercury ions (20. Mu.M) and other different ionic analytes (50. Mu.M) on the fluorescence intensity of the probe (5. Mu.M);

FIG. 4 (b) is the fluorescence intensity of probe (5. Mu.M) after recognition of mercury ions (20. Mu.M) in the presence of different ionic analytes (50. Mu.M);

FIG. 5 (a) is a fluorescence microscopy image of the probe (10. Mu.M) for exogenous mercury ions in HeLa cells;

FIG. 5 (b) shows the fluorescence intensity of the probe (10. Mu.M) before and after addition of different concentrations of mercury ions (20, 50. Mu.M);

FIG. 6 (a) is a fluorescence microscopy image of the probe (10. Mu.M) for exogenous mercury ions in zebra fish;

FIG. 6 (b) is the fluorescence intensity of the probe (10. Mu.M) before and after addition of mercury ions (20. Mu.M);

Detailed Description

The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it should be apparent that the described embodiments are only some of the embodiments of the present invention and should not be used to limit the protection scope of the present invention. All other embodiments, which can be made by those skilled in the art based on the embodiments of the present invention without making any inventive effort, are intended to fall within the scope of the present invention.

EXAMPLE 1 Synthesis of Compound of formula (IV)

The synthetic route is as follows:

the specific operation steps are as follows:

embodiment 1: the first reaction step is to add phenanthrenequinone, terephthalaldehyde and ammonium acetate into glacial acetic acid and reflux the mixture for 2 hours under nitrogen atmosphere. The product of the first step (322 mg,1 mmol) and diamino Ma Laijing (216 mg,2 mmol) were dissolved in 15ml of absolute ethanol, and the mixture was refluxed at 90℃for 6 hours under nitrogen atmosphere, and after the reaction was completed, cooled to room temperature, and suction filtration was performed under reduced pressure to obtain a solid, thereby obtaining a crude product containing the compound of formula (IV). The crude product was dissolved in methylene chloride to remove soluble impurities, thus giving 359mg of the pure compound of formula (IV) in 87.1% yield.

Embodiment 2: the first reaction step is to add phenanthrenequinone, terephthalaldehyde and ammonium acetate into glacial acetic acid and reflux the mixture for 2 hours under nitrogen atmosphere. The product of the first step (322 mg,1 mmol) and diamino Ma Laijing (108 mg,1 mmol) were dissolved in 15ml of absolute ethanol, and the mixture was refluxed at 90℃for 6 hours under nitrogen atmosphere, cooled to room temperature after the reaction was completed, and suction-filtered under reduced pressure to obtain a solid, thereby obtaining a crude product containing the compound of formula (IV). The crude product was dissolved in methylene chloride to remove soluble impurities, yielding 340mg of pure compound of formula (IV) in 82.5% yield.

Embodiment 3: the first reaction step is to add phenanthrenequinone, terephthalaldehyde and ammonium acetate into glacial acetic acid and reflux the mixture for 2 hours under nitrogen atmosphere. The product of the first step (322 mg,1 mmol) and diamino Ma Laijing (130 mg,1.2 mmol) were dissolved in 15ml of absolute ethanol, and the mixture was refluxed at 90℃for 8 hours under nitrogen atmosphere, cooled to room temperature after the reaction was completed, and suction-filtered under reduced pressure to obtain a solid, thereby obtaining a crude product containing the compound of formula (IV). The crude product was dissolved in methylene chloride to remove soluble impurities, which gave 378mg of the pure compound of formula (IV) in 91.7% yield.

Embodiment 4: the first reaction step is to add phenanthrenequinone, terephthalaldehyde and ammonium acetate into glacial acetic acid and reflux the mixture for 2 hours under nitrogen atmosphere. The product of the first step (322 mg,1 mmol) and diamino Ma Laijing (130 mg,1.2 mmol) were dissolved in 10ml of absolute ethanol, and the mixture was refluxed at 90℃for 8 hours under nitrogen atmosphere, cooled to room temperature after the reaction was completed, and suction-filtered under reduced pressure to obtain a solid, thereby obtaining a crude product containing the compound of formula (IV). The crude product was dissolved in methylene chloride to remove soluble impurities, yielding 348mg of pure compound of formula (IV) in 84.5% yield.

Embodiment 5: the first reaction step is to add phenanthrenequinone, terephthalaldehyde and ammonium acetate into glacial acetic acid and reflux the mixture for 2 hours under nitrogen atmosphere. The product of the first step (322 mg,1 mmol) and diamino Ma Laijing (162 mg,1.5 mmol) were dissolved in 15ml absolute ethanol, and the mixture was refluxed at 90℃for 8 hours under nitrogen atmosphere, cooled to room temperature after the reaction was completed, and suction-filtered under reduced pressure to obtain a solid, thereby obtaining a crude product containing the compound of formula (IV). The crude product was dissolved in methylene chloride to remove soluble impurities, yielding 365mg of pure compound of formula (IV) in 88.6% yield.

Example 2 testing absorbance spectra of fluorescent probes before and after addition of different concentrations of mercury ions

A plurality of parallel samples with the probe concentration of 5 mu M are arranged in a 10mL colorimetric tube, mercury ions (0-20 mu M) with different concentrations are added into a test system, and the test system is uniformly shaken and then measured by an ultraviolet absorption spectrometer. The above assay was performed in a HEPES buffer solution (5 mM HEPES, pH 7.4) system using the probe prepared in example 1, and the absorbance spectrum test was performed at 25℃and the test results are shown in FIG. 1.

As is clear from FIG. 1, the absorption peak of the probe changes significantly when mercury ions (0-20. Mu.M) are added.

Example 3: testing of time dynamics of fluorescent probes

A10 mL test system with a probe concentration of 5. Mu.M was prepared, and then 20. Mu.M of mercury ions was added to the test system, and immediately after shaking, the change in fluorescence intensity was measured by a fluorescence spectrometer. The above assay was performed in a HEPES buffer solution (5 mM HEPES, pH 7.4) system using the probe prepared in example 1, and the fluorescence spectrum was measured at 25℃and the test results are shown in FIG. 2.

As is clear from fig. 2, when the mercury ions are added, the fluorescence intensity reaches a minimum value instantaneously and remains unchanged, which indicates that the probe reacts rapidly with the mercury ions, and can provide a rapid analysis method for the determination of the mercury ions.

Example 4: testing concentration gradient of fluorescent probe to Mercury ion

A plurality of parallel samples with the probe concentration of 5 mu M are arranged in a 10mL colorimetric tube, mercury ions (0-20 mu M) with different concentrations are added into a test system, and after uniform shaking, a fluorescence spectrometer is used for testing the change of fluorescence intensity. The above assay was performed in a HEPES buffer solution (5 mM HEPES, pH 7.4) system using the probe prepared in example 1, and fluorescence spectra were measured at 25℃and the test results are shown in FIG. 3 (a) and FIG. 3 (b).

As is clear from fig. 3 (a), the fluorescence intensity at 570nm gradually decreases as the mercury ion concentration increases. Also, as can be seen from FIG. 3 (b), after the probe (5. Mu.M) was added to the mercury ions (0-6. Mu.M), a good linear relationship was exhibited between the fluorescence intensity at 570nm and the concentration of the mercury ions, which demonstrated that quantitative analysis of the mercury ions was possible with the aid of the fluorescent probe.

Example 5: testing the selectivity of fluorescent probes

A plurality of parallel samples with the probe concentration of 5 mu M are prepared in a 10mL colorimetric tube, then different analytes (the analytes are respectively blank, nickel ion, copper ion, potassium ion, lead ion, chromium ion, ferric ion, silver ion, zinc ion, calcium ion, magnesium ion, aluminum ion, ferrous ion, sodium ion and mercury ion, the concentration of other analytes except for 20 mu M is 50 mu M) are added into a test system, and a fluorescence spectrometer is used for testing the fluorescence intensity change after the analytes are uniformly shaken. The above assay was performed in a HEPES buffer solution (5 mM HEPES, pH 7.4) system using the probe prepared in example 1, and the fluorescence spectrum was measured at 25℃and the test results are shown in FIG. 4 (a).

As is clear from fig. 4 (a), only when mercury ions are added can a strong change in the fluorescence intensity of the probe be induced, while the effect of other analytes is almost negligible. Experiments prove that the probe has higher selectivity on mercury ions, and is favorable for detection and analysis of the mercury ions.

Example 6: testing the tamper resistance of fluorescent probes

A plurality of parallel samples with the probe concentration of 5 mu M are arranged in a 10mL colorimetric tube, then different analytes (the analytes are respectively blank, nickel ion, copper ion, potassium ion, lead ion, chromium ion, ferric ion, silver ion, zinc ion, calcium ion, magnesium ion, aluminum ion, ferrous ion and sodium ion, the analyte concentration is 50 mu M except the blank) are added into a test system, after shaking is uniform, mercury ions (the concentration is 20 mu M) are respectively added except the first blank group, and after shaking is uniform, a fluorescence spectrometer is used for testing the fluorescence intensity change. The above assay was performed in a HEPES buffer solution (5 mM HEPES, pH 7.4) system using the probe prepared in example 1, and the fluorescence spectrum was measured at 25℃and the test results are shown in FIG. 4 (b).

As can be clearly seen from fig. 4 (b), the addition of other metal ions hardly interferes with the detection of mercury ions by the fluorescent probe, and experiments prove that the probe has higher anti-interference capability on mercury ions and is beneficial to the detection and analysis of mercury ions.

Example 7 fluorescence microscopy imaging of exogenous Mercury ions in HeLa cells by fluorescent probes HeLa cells were divided into three groups, group A incubated with probes (10 μM) for 30min; group B was incubated with probe (10. Mu.M) for 30min followed by mercuric ions (20. Mu.M) for 30min; group C was incubated with probe (10. Mu.M) for 30min followed by 30min with addition of mercury ions (50. Mu.M). Finally, confocal microscopy imaging was performed on each of the three groups of cells, and the test results are shown in fig. 5 (a) and 5 (b).

As can be seen from FIG. 5 (a), the probe can detect exogenous mercury ions in HeLa cells. Further, as can be seen from FIG. 5 (b), after the probe (10. Mu.M) and the mercury ions (20, 50. Mu.M) were added to the cells, the fluorescence intensity was decreased with the increase of the mercury ion concentration, and it was confirmed that the fluorescent probe was capable of detecting the mercury ions in the HeLa cells.

Example 8 fluorescent microscopy imaging of exogenous Mercury ions in zebra fish with fluorescent probes

Dividing zebra fish cells into three groups, wherein group A is used as a blank control group, and group B is incubated with a probe (10 mu M) for 30min; group C was incubated with probe (10. Mu.M) for 30min followed by 30min with addition of mercury ions (20. Mu.M). Finally, confocal microscopy imaging was performed on each of the three groups of cells, and the test results are shown in fig. 6 (a) and 6 (b).

As can be seen from fig. 6 (a), the probe can detect exogenous mercury ions in zebra fish cells; further, as can be seen from fig. 6 (b), after the probe (10 μm) and the mercury ion (20 μm) were added to the zebra fish, the fluorescence intensity thereof was decreased with the increase of the mercury ion concentration, and it was confirmed that the fluorescence probe was capable of detecting the mercury ion in the zebra fish. Experiments prove that the probe can be applied to mercury ion detection in biological samples.

While the invention has been described with reference to the above embodiments, it will be understood that the invention is capable of further modifications and variations without departing from the spirit of the invention, and these modifications and variations are within the scope of the invention.

Claims

1. A fluorescent probe for measuring, detecting or screening mercury ions, characterized by: it has the following structure:

2. a method of preparing the fluorescent probe of claim 1, comprising the steps of: reacting a compound of formula (V) with diaminomaleonitrile to produce a compound of formula (IV) having the following reaction formulae:

3. the method of manufacturing according to claim 2, comprising the steps of:

dissolving a compound of a formula (V) and diamino Ma Laijing in absolute ethyl alcohol, carrying out reflux reaction at 90 ℃ in a nitrogen atmosphere, cooling to room temperature after the reaction is finished, and carrying out vacuum filtration to obtain a solid, thereby obtaining a crude product containing the compound of the formula (IV); dissolving the crude product with methylene dichloride to remove soluble impurities, and obtaining the pure compound of the formula (IV).

4. A fluorescent probe composition for measuring, detecting or screening mercury ions comprising the compound of claim 1.

5. The fluorescent probe composition of claim 4, further comprising a solvent, an acid, a base, a buffer solution, or a combination thereof.

6. A method for detecting the presence of mercury ions in a sample or determining the mercury ion content in a sample for non-therapeutic or diagnostic purposes, comprising:

a) Contacting the fluorescent probe of claim 1 with a sample to form a fluorescent compound;

b) Determining the fluorescent properties of the fluorescent compound.

7. The method of claim 6, wherein the sample is a chemical sample or a biological sample.

8. Use of a fluorescent probe as claimed in claim 1 for fluorescence imaging of cells for non-therapeutic or diagnostic purposes.

9. The use of a fluorescent probe according to claim 1, wherein: the fluorescent probes are used for preparing reagents for detecting mercury ions for non-therapeutic or diagnostic purposes.