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

Abstract

The invention relates to an amino coordination type highly selective mercury ion fluorescent probe, specifically, the probe of the invention can be used for measuring, detecting or screening mercury ions and living cell fluorescence imaging. The probe of the present invention uses amino compounds as mercury ion coordination units, and can quickly, sensitively and highly selectively identify mercury ions through the fluorescent probe coordinated by amino groups and mercury, and can be detected ultrasensitively and highly selectively by fluorescence analysis methods Mercury ions in living cells and in zebrafish.

Description

An amino-coordination type highly selective mercury ion fluorescent probe, preparation method and application

technical field

The invention belongs to the field of fluorescent probes, and in particular relates to an amino-coordination type highly selective mercury ion fluorescent probe and its application in measuring, detecting or screening mercury ions and live cell fluorescence imaging methods; the invention also provides a method for preparing The fluorescent probe method.

Background technique

Mercury (Hg ²⁺ ), as a metal element with serious physiological toxicity, has become one of the most concerned environmental pollutants due to its persistence, easy migration and high bioaccumulation. Inorganic mercury ions in the environment can be converted by organisms into highly toxic methylmercury under certain conditions. Inorganic mercury mainly affects the kidneys, while methylmercury mainly damages the nervous system after entering the human body, especially the central nervous system. Both can be highly enriched in biological tissues through the food chain, thus causing great harm to humans and nature. Mercury poisoning will have an extremely bad impact on the whole society. Mercury is now prioritized on the list of the global environmental monitoring system. Therefore, the selective identification of mercury ions, especially the in-situ, real-time and online monitoring of mercury ions is very important for medicine, Biology and environmental science are both important.

At this stage, the reported analytical 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. , the fluorescent probe method has attracted extensive attention due to its unique advantages. However, the reported methods still have certain defects, such as poor selectivity, low sensitivity, complex synthesis, and long response time. Therefore, developing a rapid, highly selective, highly sensitive, and easy-to-synthesize fluorescent probe for detecting mercury ions has become an urgent problem to be solved by those skilled in the art.

Contents of the invention

In view of this, the present invention provides a novel highly selective fluorescent probe for the detection of amino-coordination mercury ions, which is simple in synthesis, good in selectivity, and rapid in response, and can be rapidly and sensitively detected in aqueous solution, cells, and zebrafish. detection of mercury ions.

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

In 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 ₁₂ are independently selected from hydrogen atom, straight chain or branched chain alkyl, straight chain or branched chain alkoxy group, sulfonic acid group, ester group and carboxyl group; and wherein R ₁ , R ₂ , R ₃ , R ₄ , R ₅ , R ₆ , R ₇ , R ₈ , R ₉ , R ₁₀ , R ₁₁ and R ₁₂ may be the same or different.

In some specific embodiments of the present invention, the fluorescent probes of the present invention are R ₁ , R ₂ , R ₃ , R ₄ , R ₅ , R ₆ , R 7 , R ₈ , R ₉ , R ₁₀ , R _{11 and} R ₁₂ is the formula (IV) compound of hydrogen atom, and its structural formula is as follows:

The present invention also provides a preparation method for the compound of formula (I), comprising the following steps:

Dissolve the compound of formula (II) and compound of formula (III) in absolute ethanol, and react under reflux at 90°C under a nitrogen atmosphere. After the reaction, cool to room temperature, and filter under reduced pressure to obtain a solid, thereby obtaining the compound containing formula (I) The crude product of the compound. The crude product is dissolved with dichloromethane to remove soluble impurities, and the pure compound of formula (I) can be obtained, and its reaction formula is as follows:

In formula (I)-(III): R ₁ , R ₂ , R ₃ , R ₄ , R ₅ , R ₆ , R ₇ , R ₈ , R ₉ , R ₁₀ , R ₁₁ and R ₁₂ are independently selected from A group consisting of hydrogen atom, straight chain or branched chain alkyl group, straight chain or branched chain alkoxy group, sulfonic acid group, ester group and carboxyl group; and wherein R ₁ , R ₂ , R ₃ , R ₄ , R ₅ , R ₆ , R ₇ , R ₈ , R ₉ , R ₁₀ , R ₁₁ and R ₁₂ may be the same or different.

Specifically: dissolve the compound of formula (II) and compound of formula (III) in absolute ethanol, and react under reflux at 90°C under a nitrogen atmosphere. After the reaction, cool to room temperature, and filter under reduced pressure to obtain a solid, thereby obtaining Crude product of compound of formula (I). The crude product is dissolved in dichloromethane to remove soluble impurities, and the pure compound of formula (I) can be obtained.

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

In some specific embodiments of the present invention, the reaction time in step (1) and step (2) of the preparation method of the compound of formula (I) is 6-8 hours.

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

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

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

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

a) contacting said compound of formula (I) or formula (IV) with a sample to form a fluorescent compound;

b) Determining the fluorescent properties of said fluorescent compound.

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

In some embodiments of the invention, the sample is a biological sample including water, blood, microorganisms, or animal cells or tissues.

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

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

The present invention also provides a reagent prepared from the compound of formula (I) or formula (IV) and used for detecting mercury ions.

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

(1) High selectivity, strong anti-interference ability

The mercury ion fluorescent probe of the present invention can selectively and specifically react with mercury ions to generate fluorescently changed products, 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, etc., the fluorescent probe of the present invention shows high selectivity, and anti-interference strong ability.

(2) High sensitivity and quick response

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

(3) It can be applied under the condition of physiological level

The mercury ion fluorescent probe of the present invention can be applied under the condition of physiological level, and the common metal ions in the living body interfere less with it, and can be applied to the fluorescence imaging of living cells and zebrafish.

(4) good stability

The mercury ion fluorescent probe of the present invention has good stability and can be stored and used for a long time.

(5) Simple synthesis

The mercury ion fluorescent probe of the invention is simple to synthesize and is beneficial to commercial popularization and application.

Description of drawings

In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings that need to be used in the description of the embodiments or the prior art. Obviously, the accompanying drawings in the following description are only These are some embodiments of the present invention. Those skilled in the art can also obtain other drawings based on these drawings without creative work.

Fig. 1 is the absorption spectrum before and after adding mercury ion (0-20 μ M) to probe (5 μ M);

Fig. 2 is the time kinetic spectrum of probe (5 μ M) to mercury ion (20 μ M) at 570nm;

Figure 3(a) is the fluorescence spectrum before and after adding mercury ions (0-20 μM) to the probe (5 μM);

Fig. 3 (b) is the linear relationship diagram between the fluorescence intensity of the probe (5 μM) and the mercury ion (0-6 μM) at 570 nm;

Figure 4(a) is the effect of mercury ions (20 μM) and other different ion analytes (50 μM) on the fluorescence intensity of the probe (5 μM);

Figure 4(b) is the fluorescence intensity of the mercury ion (20 μM) recognized by the probe (5 μM) in the presence of different ion analytes (50 μM);

Figure 5(a) is the fluorescence microscopic imaging of probe (10 μM) to exogenous mercury ions in HeLa cells;

Figure 5(b) is the fluorescence intensity of the cells before and after adding different concentrations of mercury ions (20, 50 μM) to the probe (10 μM);

Figure 6(a) is the fluorescent microscopic imaging of the probe (10 μM) to exogenous mercury ions in zebrafish;

Figure 6(b) is the fluorescence intensity of the probe (10 μM) before and after adding mercury ions (20 μM);

Detailed ways

The technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention and should not be used to limit the scope of the present invention. protected range. Based on the embodiments of the present invention, all other embodiments obtained by persons of ordinary skill in the art without making creative efforts belong to the protection scope of the present invention.

Synthesis of embodiment 1 formula (Ⅳ) compound

The synthetic route is as follows:

The specific operation steps are as follows:

Embodiment 1: The first step reaction is that phenanthrenequinone, terephthalaldehyde and ammonium acetate are added to glacial acetic acid, and refluxed for 2 hours under nitrogen atmosphere. Dissolve the product of the first step (322mg, 1mmol) and diaminomaleonitrile (216mg, 2mmol) in 15ml of absolute ethanol, under a nitrogen atmosphere, reflux at 90°C for 6 hours, after the reaction, cool to room temperature, and depressurize The solid was obtained by suction filtration, thereby obtaining a crude product containing the compound of formula (IV). The crude product was dissolved in dichloromethane to remove soluble impurities to obtain 359 mg of pure compound of formula (IV) with a yield of 87.1%.

Embodiment 2: The first step reaction is that phenanthrenequinone, terephthalaldehyde and ammonium acetate are added to glacial acetic acid, and refluxed for 2 hours under nitrogen atmosphere. Dissolve the product of the first step (322mg, 1mmol) and diaminomaleonitrile (108mg, 1mmol) in 15ml of absolute ethanol, and react under reflux at 90°C for 6 hours under a nitrogen atmosphere. After the reaction is completed, cool to room temperature and depressurize The solid was obtained by suction filtration, thereby obtaining a crude product containing the compound of formula (IV). The crude product was dissolved in dichloromethane to remove soluble impurities to obtain 340 mg of pure compound of formula (IV) with a yield of 82.5%.

Embodiment 3: The first step reaction is that phenanthrenequinone, terephthalaldehyde and ammonium acetate are added to glacial acetic acid, and refluxed for 2 hours under nitrogen atmosphere. The first step product (322mg, 1mmol) and diaminomaleonitrile (130mg, 1.2mmol) were dissolved in 15ml of absolute ethanol, under a nitrogen atmosphere, 90 ° C reflux reaction for 8 hours, after the reaction was completed, cooled to room temperature, reduced The solid was obtained by suction filtration, thereby obtaining a crude product containing the compound of formula (IV). The crude product was dissolved in dichloromethane to remove soluble impurities to obtain 378mg of pure compound of formula (IV) with a yield of 91.7%.

Embodiment 4: The first step reaction is that phenanthrenequinone, terephthalaldehyde and ammonium acetate are added to glacial acetic acid, and refluxed for 2 hours under nitrogen atmosphere. The first step product (322mg, 1mmol) and diaminomaleonitrile (130mg, 1.2mmol) were dissolved in 10ml of absolute ethanol, under a nitrogen atmosphere, refluxed at 90 ° C for 8 hours, after the reaction was completed, cooled to room temperature, reduced The solid was obtained by suction filtration, thereby obtaining a crude product containing the compound of formula (IV). The crude product was dissolved in dichloromethane to remove soluble impurities to obtain 348mg of pure compound of formula (IV) with a yield of 84.5%.

Embodiment 5: The first step reaction is that phenanthrenequinone, terephthalaldehyde and ammonium acetate are added to glacial acetic acid, and refluxed for 2 hours under nitrogen atmosphere. The first step product (322mg, 1mmol) and diaminomaleonitrile (162mg, 1.5mmol) were dissolved in 15ml of absolute ethanol, under a nitrogen atmosphere, 90 ° C reflux reaction for 8 hours, after the reaction was completed, cooled to room temperature, reduced The solid was obtained by suction filtration, thereby obtaining a crude product containing the compound of formula (IV). The crude product was dissolved in dichloromethane to remove soluble impurities to obtain 365 mg of pure compound of formula (IV) with a yield of 88.6%.

Example 2 Test the absorbance spectrum of the fluorescent probe before and after adding different concentrations of mercury ions

Configure multiple parallel samples with a probe concentration of 5 μM in a 10 mL colorimetric tube, then add different concentrations of mercury ions (0-20 μM) into the test system, shake it evenly, and measure it with an ultraviolet absorption spectrometer. Above-mentioned mensuration is carried out in HEPES buffer solution (5mMHEPES, pH 7.4) system, the probe used is the probe prepared in embodiment 1, and absorption spectrum test is recorded at 25 ℃, test result is as follows Figure 1 shows.

It can be clearly seen from Figure 1 that when mercury ions (0-20 μM) are added, the absorption peak of the probe changes significantly.

Example 3: Testing the Time Kinetics of Fluorescent Probes

Prepare a 10mL test system with a probe concentration of 5 μM, then add 20 μM mercury ions into the test system, shake it evenly, and immediately measure the change in fluorescence intensity with a fluorescence spectrometer. The above-mentioned determination is carried out in the HEPES buffer solution (5mM HEPES, pH 7.4) system, the probe used is the probe prepared in Example 1, and the fluorescence spectrum is measured at 25 ℃, and the test results are as follows: Figure 2 shows.

It can be clearly seen from Figure 2 that when mercury ions are added, the fluorescence intensity reaches the minimum value instantaneously and remains unchanged, which indicates that the probe reacts rapidly with mercury ions and can provide a fast analytical method for the determination of mercury ions.

Embodiment 4: Test the concentration gradient of fluorescent probes for mercury ions

Configure multiple parallel samples with a probe concentration of 5 μM in a 10 mL colorimetric tube, then add different concentrations of mercury ions (0-20 μM) into the test system, shake evenly, and measure the change in fluorescence intensity with a fluorescence spectrometer. The above-mentioned determination is carried out in the HEPES buffer solution (5mM HEPES, pH 7.4) system, the probe used is the probe prepared in Example 1, and the fluorescence spectrum is measured at 25 ℃, and the test results are as follows: Figure 3 (a) and Figure 3 (b) shown.

It can be clearly seen from Figure 3(a) that the fluorescence intensity at 570nm decreases gradually with the increase of mercury ion concentration. And, as can be seen from Fig. 3 (b) after the probe (5μM) is added with mercury ions (0-6μM), there is a good linear relationship between the fluorescence intensity at its 570nm place and the concentration of mercury ions, which proves that by means of The fluorescent probe can quantitatively analyze mercury ions.

Embodiment 5: Test the selectivity of fluorescent probe

Configure multiple parallel samples with a probe concentration of 5 μM in a 10mL colorimetric tube, and then separate different analytes (the analytes are blank, nickel ions, copper ions, potassium ions, lead ions, chromium ions, and ferric ions) , silver ions, zinc ions, calcium ions, magnesium ions, aluminum ions, ferrous ions, sodium ions, mercury ions; except for mercury ions at 20 μM, the concentration of other analytes is 50 μM) into the test system, shake evenly Fluorescence intensity changes were measured with a fluorescence spectrometer. The above-mentioned determination is carried out in the HEPES buffer solution (5mM HEPES, pH 7.4) system, the probe used is the probe prepared in Example 1, and the fluorescence spectrum is measured at 25 ℃, and the test results are as follows: Figure 4(a) shows.

It can be clearly seen from Figure 4(a) that only the addition of mercury ions can cause a strong change in the fluorescence intensity of the probe, while the effects of other analytes are almost negligible. Experiments have proved that the probe has high selectivity to mercury ions, which is beneficial to the detection and analysis of mercury ions.

Embodiment 6: Test the anti-interference ability of fluorescent probe

Configure multiple parallel samples with a probe concentration of 5 μM in a 10mL colorimetric tube, and then separate different analytes (the analytes are blank, nickel ions, copper ions, potassium ions, lead ions, chromium ions, and ferric ions) , silver ions, zinc ions, calcium ions, magnesium ions, aluminum ions, ferric ions, sodium ions; except for the blank, the concentration of the analyte is 50 μM) into the test system, after shaking evenly, except for the first blank Outside the group, mercury ions (concentration: 20 μM) were added to the other groups, and after shaking evenly, the changes in fluorescence intensity were tested with a fluorescence spectrometer. The above-mentioned determination is carried out in the HEPES buffer solution (5mM HEPES, pH 7.4) system, the probe used is the probe prepared in Example 1, and the fluorescence spectrum is measured at 25 ℃, and the test results are as follows: Figure 4(b) shows.

It can be clearly seen from Figure 4(b) that the addition of other metal ions hardly interferes with the detection of mercury ions by the fluorescent probe. Detection analysis.

Example 7: Fluorescence microscopic imaging of exogenous mercury ions in HeLa cells by fluorescent probes. HeLa cells were divided into three groups. Group A was incubated with probe (10 μM) for 30 min; Add mercury ions (20 μM) and incubate for 30 minutes; group C first incubate with probe (10 μM) for 30 minutes, then add mercury ions (50 μM) for 30 minutes. Finally, confocal microscopy imaging was performed on the three groups of cells, and the test results are shown in Figures 5(a) and 5(b).

It can be seen from Figure 5(a) that the probe can detect exogenous mercury ions in HeLa cells. Moreover, it can be seen from Figure 5(b) that after the probe (10 μM) and mercury ions (20, 50 μM) are added to the cells, the fluorescence intensity decreases with the increase of the concentration of mercury ions, which proves that the fluorescent probe can Detection of mercury ions in Hela cells.

Example 8: Fluorescence microscopy imaging of exogenous mercury ions in zebrafish by fluorescent probes

The zebrafish cells were divided into three groups, group A served as blank control group, group B was incubated with probe (10 μM) for 30 min; group C was first incubated with probe (10 μM) for 30 min, and then incubated with mercury ions (20 μM) for 30 min. Finally, confocal microscopy imaging was performed on the three groups of cells, and the test results are shown in Figures 6(a) and 6(b).

As can be seen from Figure 6(a), the probe can detect exogenous mercury ions in zebrafish cells; 20 μM), its fluorescence intensity decreased with the increase of mercury ion concentration, which proved that the fluorescent probe could detect mercury ion in zebrafish. Experiments show that the probe can be applied to the detection of mercury ions in biological samples.

Although the present invention has been described with the above embodiments, it should be understood that, without departing from the spirit of the present invention, the present invention can be further modified and changed, and these modifications and changes are within the protection scope of the present invention .

Claims (9)

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

2. a method for preparing the fluorescent probe described in claim 1, is characterized in that, comprises the steps: make formula (Ⅴ) compound and diaminomaleonitrile reaction prepare formula (Ⅳ) compound, its reaction formula is respectively as follows :

3. preparation method according to claim 2, is characterized in that, comprises the steps: Dissolve the compound of formula (Ⅴ) and diaminomaleonitrile in absolute ethanol, and react under reflux at 90°C under a nitrogen atmosphere. After the reaction is completed, cool to room temperature and filter under reduced pressure to obtain a solid, thereby obtaining the compound containing formula (Ⅳ). The crude product of the compound; the crude product is dissolved in dichloromethane to remove soluble impurities, and the pure compound of formula (IV) can be obtained. 4. A fluorescent probe composition for measuring, detecting or screening mercury ions, comprising the compound described in claim 1. 5. The fluorescent probe composition according to claim 4, wherein the fluorescent probe composition further comprises solvent, acid, alkali, buffer solution or a combination thereof. 6. A method for detecting the presence or determining the amount of mercury ions 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 said fluorescent compound. 7. The method of claim 6, wherein the sample is a chemical sample or a biological sample. 8. The use of the fluorescent probe described in claim 1 in cell fluorescence imaging for non-therapeutic or diagnostic purposes. 9. The application of the fluorescent probe according to claim 1, characterized in that: the fluorescent probe is prepared as a reagent for detecting mercury ions for non-therapeutic or diagnostic purposes.

Images