阅读说明：本技术 检测铜离子和汞离子的荧光探针及其制备方法和应用 (Fluorescent probe for detecting copper ions and mercury ions and preparation method and application thereof ) 是由 董明 李春举 于 2019-11-06 设计创作，主要内容包括：本发明公开了一种检测铜离子和汞离子的荧光探针及其制备方法和应用,荧光探针的制备方法,包括以下步骤：将罗丹明B内酰肼、4-二乙胺基-2-乙烯氧基苯甲醛和乙醇混合,于90～110℃回流反应3～6小时,通过柱色谱分离,得淡黄色固体为Probe 1,本发明的荧光探针具有双识别功能,可以分别对汞离子和铜离子进行检测,在铜离子和汞离子共存的环境下,该荧光探针可以给出第三种荧光响应信号。(The invention discloses a fluorescent probe for detecting copper ions and mercury ions, a preparation method and application thereof, wherein the preparation method of the fluorescent probe comprises the following steps: mixing rhodamine B lactohydrazide, 4-diethylamino-2-vinyloxybenzaldehyde and ethanol, carrying out reflux reaction at 90-110 ℃ for 3-6 hours, and separating by column chromatography to obtain a light yellow solid, namely Probe 1.)

1. A fluorescent Probe is characterized in that the fluorescent Probe is Probe-1, and the molecular formula of the Probe-1 is as follows:

2. the method for preparing the fluorescent probe according to claim 1, comprising the steps of: mixing rhodamine B lactohydrazide (1), 4-diethylamino-2-vinyloxy benzaldehyde (2) and ethanol, carrying out reflux reaction at 90-110 ℃ for 3-6 hours, and separating by using column chromatography to obtain a light yellow solid as Probe 1, wherein the ratio of the rhodamine B lactohydrazide to the 4-diethylamino-2-vinyloxy benzaldehyde is 1:1 by mass.

3. The preparation method according to claim 2, wherein the ratio of the parts by weight of the substance of the rhodamine B internal hydrazide (1) to the parts by volume of the ethanol is 1 (10-15), and when the parts by weight of the substance is mmol, the parts by volume is mL;

and (2) taking a mixture of petroleum ether and ethyl acetate as a first eluent, and purifying by using the column chromatography to obtain a light yellow solid as the Probe 1, wherein the volume ratio of the petroleum ether to the ethyl acetate in the first eluent is (3-4): 1.

4. the method for preparing the rhodamine B internal hydrazide according to claim 2, wherein the method for preparing the rhodamine B internal hydrazide is as follows: mixing rhodamine B, hydrazine hydrate and ethanol, carrying out reflux reaction at 100-110 ℃ for 6-8 h, cooling to room temperature of 20-25 ℃ after the reaction is finished to obtain reaction liquid, mixing the reaction liquid with water, and filtering to obtain precipitate as rhodamine B internal hydrazide, wherein the ratio of the mass part of the rhodamine B, the volume part of the hydrazine hydrate and the volume part of the ethanol is 1: 1: (20-40), when the unit of the mass part is g, the unit of the volume part is mL.

5. The method according to claim 2, wherein the method for preparing 4-diethylamino-2-vinyloxybenzaldehyde comprises: mixing 2-bromoethoxy-4-diethylaminobenzaldehyde, potassium tert-butoxide and dimethyl sulfoxide, stirring at room temperature of 20-25 ℃ for reaction for 2-3 h, adding water, extracting with ethyl acetate, concentrating an organic phase, purifying by column chromatography with a mixture of petroleum ether and ethyl acetate as a second eluent to obtain a brown oily product, namely 4-diethylamino-2-vinyloxybenzaldehyde, wherein the ratio of the mass parts of the 2-bromoethoxy-4-diethylaminobenzaldehyde to the mass parts of potassium tert-butoxide to the volume parts of dimethyl sulfoxide to the volume parts of water is 0.3: 0.12: (20-40): (100-200), when the unit of the mass part is g, the unit of the volume part is mL;

the preparation method of the 2-bromoethoxy-4-diethylaminobenzaldehyde comprises the following steps: mixing 4-diethylamino salicylaldehyde, 1, 2-dibromoethane, potassium carbonate and acetonitrile, carrying out reflux reaction at 100-110 ℃ for 5-6 h, cooling to room temperature of 20-25 ℃, and purifying by column chromatography by using a mixture of petroleum ether and ethyl acetate as a second eluent to obtain a white solid product, namely the 2-bromoethoxy-4-diethylamino benzaldehyde, wherein the ratio of the mass fraction of the 4-diethylamino salicylaldehyde to the volume fraction of the 1, 2-dibromoethane to the mass fraction of the potassium carbonate to the volume fraction of the acetonitrile is 0.193: 0.5: 0.276: (30-60), when the unit of the mass part is g, the unit of the volume part is mL;

the volume ratio of petroleum ether to ethyl acetate in the second eluent is (10-15): 1.

6. the method for detecting mercury ions and/or copper ions by using the fluorescent probe as claimed in claim 1, which comprises the following steps: and directly or indirectly adding the fluorescent probe into a solution to be detected, respectively taking light with the wavelength of 420nm and light with the wavelength of 530nm as exciting light, and comparing fluorescent signals excited by different exciting lights before and after the fluorescent probe is added into the solution to be detected, wherein the fluorescence probe is indirectly dissolved in an organic solvent mutually soluble with water and then added into the solution to be detected.

7. The method of claim 6, wherein the water-miscible organic solvent is a mixture of one or more of methanol, ethanol, acetonitrile, dimethyl sulfoxide, and N, N-dimethylformamide.

8. The method according to claim 6, wherein the fluorescent probe is directly added to the solution to be tested when the fluorescent probe is capable of dissolving in the solution to be tested and the solution to be tested is tested; and when the fluorescent probe cannot be dissolved in the solution to be detected, the fluorescent probe is indirectly added into the solution to be detected when the solution to be detected is detected.

9. The method according to claim 6, wherein the step of comparing the fluorescence signals of the fluorescence probe excited by different excitation lights before and after the fluorescence probe is added into the solution to be tested comprises:

compared with the method that before the fluorescent probe is added into the solution to be detected, after the fluorescent probe is added into the solution to be detected:

when the fluorescent material is excited by 420nm, the reduction range of the fluorescence intensity at 515nm reaches 70-85%, and the reduction range of the fluorescence intensity at 580nm is 50-60%; when the solution is excited at 530nm, the fluorescence intensity changes by-10%, and at the moment, the mercury ions in the solution to be detected are judged;

when the fluorescent material is excited by 420nm, the reduction amplitude of the fluorescence intensity at 515nm reaches 65-70%, the fluorescence intensity at 580nm is enhanced, and a strongest emission peak is formed; when the solution is excited at 530nm, the fluorescence intensity at 580nm is enhanced and the strongest emission peak is formed, and at the moment, the solution to be detected is judged to contain copper ions;

when the fluorescent material is excited by 420nm, the reduction amplitude of the fluorescence intensity at 515nm reaches 85-95%, and the fluorescence intensity at 580nm is reduced; when the solution is excited at 530nm, the fluorescence intensity at 580nm is enhanced and the strongest emission peak is formed, and at the moment, the solution to be detected is judged to contain copper ions and mercury ions.

10. Use of the fluorescent probe according to claim 1 for detecting mercury ions and/or copper ions.

Technical Field

The invention belongs to the technical field of organic small-molecule fluorescent probes, and particularly relates to a fluorescent probe for detecting copper ions and mercury ions, and a preparation method and application thereof.

Background

Soluble mercury salts are a highly toxic environmental pollutant, and mercury in the atmosphere and in water can be generated by various industrial activities, such as combustion of sulfur-containing coal, chemical production and the like. In human body, mercury ions can make protein containing sulfhydryl lose activity and lose function; can also combine with amino, carboxyl and hydroxyl in enzyme and phosphoryl in cell membrane to cause corresponding damage and even death. Copper is one of the important trace elements in the human body, and the lack of copper in the human body causes anemia, abnormal hair, abnormal bones and arteries, and even brain disorders. However, excess causes cirrhosis, diarrhea, vomiting, dyskinesia, and sensory neuropathy. The detection methods of mercury ions and copper ions mainly include inductively coupled plasma spectrometers, spectrophotometry, electrochemical detection methods, gas chromatography, liquid chromatography, sensor methods and the like. However, each detection method is applicable to different samples, and has advantages and limitations. Therefore, the invention of the fluorescent probe capable of simultaneously detecting mercury ions and copper ions in a solution sample has important practical significance for environmental monitoring and disease research related to mercury and copper elements. At present, few bifunctional fluorescent probes for simultaneously identifying mercury ions and copper ions are reported.

Disclosure of Invention

In view of the defects of the prior art, the invention aims to provide a fluorescent probe which can simultaneously detect mercury ions and copper ions in a solution through fluorescence spectrum change, and two detection paths do not interfere with each other.

Another object of the present invention is to provide a method for preparing the above fluorescent probe.

The purpose of the invention is realized by the following technical scheme.

A fluorescent Probe is Probe-1, and the molecular formula of the Probe-1 is as follows:

the preparation method of the fluorescent probe comprises the following steps: mixing rhodamine B lactohydrazide (1), 4-diethylamino-2-vinyloxy benzaldehyde (2) and ethanol, carrying out reflux reaction at 90-110 ℃ for 3-6 hours, and separating by using column chromatography to obtain a light yellow solid as Probe 1, wherein the ratio of the rhodamine B lactohydrazide to the 4-diethylamino-2-vinyloxy benzaldehyde is 1:1 by mass.

In the technical scheme, the ratio of the parts by weight of the substance of the rhodamine B internal hydrazide (1) to the parts by volume of the ethanol is 1 (10-15), and when the unit of the parts by weight of the substance is mmol, the unit of the parts by volume is mL.

In the technical scheme, a mixture of petroleum ether and ethyl acetate is used as a first eluent, and the mixture is purified by the column chromatography to obtain a light yellow solid which is the Probe 1, wherein the volume ratio of the petroleum ether to the ethyl acetate in the first eluent is (3-4): 1.

in the technical scheme, the preparation method of the rhodamine B internal hydrazide comprises the following steps: mixing rhodamine B, hydrazine hydrate and ethanol, carrying out reflux reaction at 100-110 ℃ for 6-8 h, cooling to room temperature of 20-25 ℃ after the reaction is finished to obtain reaction liquid, mixing the reaction liquid with water, and filtering to obtain precipitate as rhodamine B internal hydrazide, wherein the ratio of the mass part of the rhodamine B, the volume part of the hydrazine hydrate and the volume part of the ethanol is 1: 1: (20-40), when the unit of the mass part is g, the unit of the volume part is mL.

In the technical scheme, the preparation method of the 4-diethylamino-2-vinyloxy benzaldehyde comprises the following steps: mixing 2-bromoethoxy-4-diethylaminobenzaldehyde, potassium tert-butoxide and dimethyl sulfoxide, stirring at room temperature of 20-25 ℃ for reaction for 2-3 h, adding water, extracting with ethyl acetate, concentrating an organic phase, purifying by column chromatography with a mixture of petroleum ether and ethyl acetate as a second eluent to obtain a brown oily product, namely 4-diethylamino-2-vinyloxybenzaldehyde, wherein the ratio of the mass parts of the 2-bromoethoxy-4-diethylaminobenzaldehyde to the mass parts of potassium tert-butoxide to the volume parts of dimethyl sulfoxide to the volume parts of water is 0.3: 0.12: (20-40): (100-200), when the unit of the mass part is g, the unit of the volume part is mL.

In the technical scheme, the preparation method of the 2-bromoethoxy-4-diethylaminobenzaldehyde comprises the following steps: mixing 4-diethylamino salicylaldehyde, 1, 2-dibromoethane, potassium carbonate and acetonitrile, carrying out reflux reaction at 100-110 ℃ for 5-6 h, cooling to room temperature of 20-25 ℃, and purifying by column chromatography by using a mixture of petroleum ether and ethyl acetate as a second eluent to obtain a white solid product, namely the 2-bromoethoxy-4-diethylamino benzaldehyde, wherein the ratio of the mass fraction of the 4-diethylamino salicylaldehyde to the volume fraction of the 1, 2-dibromoethane to the mass fraction of the potassium carbonate to the volume fraction of the acetonitrile is 0.193: 0.5: 0.276: (30-60), when the unit of the mass part is g, the unit of the volume part is mL.

In the technical scheme, the volume ratio of petroleum ether to ethyl acetate in the second eluent is (10-15): 1.

the method for detecting mercury ions and/or copper ions by using the fluorescent probe comprises the following steps: and directly or indirectly adding the fluorescent probe into a solution to be detected, respectively taking light with the wavelength of 420nm and light with the wavelength of 530nm as exciting light, and comparing fluorescent signals excited by different exciting lights before and after the fluorescent probe is added into the solution to be detected, wherein the fluorescence probe is indirectly dissolved in an organic solvent mutually soluble with water and then added into the solution to be detected.

In the technical scheme, the organic solvent mutually soluble with water is one or a mixture of more of methanol, ethanol, acetonitrile, dimethyl sulfoxide and N, N-dimethylformamide.

In the technical scheme, when the fluorescent probe can be dissolved in a solution to be detected, the fluorescent probe is directly added into the solution to be detected when the solution to be detected is detected; and when the fluorescent probe cannot be dissolved in the solution to be detected, the fluorescent probe is indirectly added into the solution to be detected when the solution to be detected is detected.

In the above technical scheme, the specific steps of comparing fluorescence signals excited by different excitation lights before and after the fluorescent probe is added into the solution to be measured are as follows:

compared with the method that before the fluorescent probe is added into the solution to be detected, after the fluorescent probe is added into the solution to be detected:

when the fluorescent material is excited by 420nm, the reduction range of the fluorescence intensity at 515nm reaches 70-85%, and the reduction range of the fluorescence intensity at 580nm is 50-60%; when the solution is excited at 530nm, the fluorescence intensity changes by-10%, and at the moment, the mercury ions in the solution to be detected are judged;

when the fluorescent material is excited by 420nm, the reduction amplitude of the fluorescence intensity at 515nm reaches 65-70%, the fluorescence intensity at 580nm is enhanced, and a strongest emission peak is formed; when the solution is excited at 530nm, the fluorescence intensity at 580nm is enhanced and the strongest emission peak is formed, and at the moment, the solution to be detected is judged to contain copper ions;

when the fluorescent material is excited by 420nm, the reduction amplitude of the fluorescence intensity at 515nm reaches 85-95%, and the fluorescence intensity at 580nm is reduced; when the solution is excited at 530nm, the fluorescence intensity at 580nm is enhanced and the strongest emission peak is formed, and at the moment, the solution to be detected is judged to contain copper ions and mercury ions.

The fluorescent probe is applied to detection of mercury ions and/or copper ions.

The invention has the following beneficial effects:

(1) the single fluorescent probe has double recognition functions and can be used for respectively detecting mercury ions and copper ions;

(2) under the coexistence environment of copper ions and mercury ions, the fluorescent probe can give a third fluorescent response signal;

(3) the preparation method of the fluorescent probe is simple and easy to operate.

Drawings

FIG. 1 is a scheme for synthesizing Probe-1 obtained in example 1;

FIG. 2 shows nuclear magnetic hydrogen spectrum (CDCl) of Probe-1 obtained in example 1₃,400MHz)；

FIG. 3 shows the fluorescence response (420nm excitation) of Probe-1 to different ions in example 2;

FIG. 4 shows the fluorescence response (excitation at 530nm) of Probe-1 to different ions in example 3;

FIG. 5 shows the fluorescence titration detection of Probe-1 with different concentrations of mercury ions in example 4;

FIG. 6 shows the fluorescence titration detection of Probe-1 with different concentrations of copper ions in example 5;

FIG. 7 shows the fluorescence response (420nm excitation) of Probe-1 to other metal ions in example 6.

Detailed Description

The technical scheme of the invention is further explained by combining specific examples.

The following examples used analytical reagents, where high purity water was obtained from Hangzhou Wahaha purified water, rhodamine B, hydrazine hydrate, 4-diethylamino salicylaldehyde, 1, 2-dibromoethane, and potassium tert-butoxide was obtained from Yinakai reagent, the desired cationic salt: magnesium chloride, calcium chloride, strontium chloride, barium chloride, chromium chloride, nickel perchlorate, manganese perchlorate, lead perchlorate, cobalt perchlorate, zinc perchlorate, cadmium perchlorate, copper perchlorate, ferrous perchlorate, and iron perchlorate are purchased from warfarin reagents corporation, other reagents: ethanol, dimethyl sulfoxide, acetonitrile, ethyl acetate, petroleum ether, potassium carbonate, potassium dihydrogen phosphate, disodium hydrogen phosphate, sodium chloride and potassium chloride are all purchased from the institute of optometry and Fine chemical engineering in Tianjin.

In the following examples the mercury salt is mercury perchlorate and the copper salt is copper perchlorate.

The following examples used characterization instruments including: bruker 400MHz liquid nuclear magnetism, Hitachi F-4500 fluorescence spectrometer, Saedolis PB-10 type pH meter.

The preparation method of the PBS buffer solution comprises the following steps: 0.27g of potassium dihydrogen phosphate, 1.42g of disodium hydrogen phosphate, 8g of sodium chloride, 0.2g of potassium chloride and 800mL of deionized water are taken to be fully stirred and dissolved, then concentrated hydrochloric acid is added to adjust the pH value to 7.4, and finally the solution is fixed to 1L and stored in a refrigerator at 4 ℃.

Example 1

A fluorescent Probe for detecting copper ions and mercury ions is Probe-1, and the molecular formula of the Probe-1 is as follows:

the preparation method of the fluorescent probe comprises the following steps: in a 50mL round-bottom flask, 0.456g (0.001mol) of rhodamine B internal hydrazide (1), 0.219g (0.001mol) of 4-diethylamino-2-vinyloxybenzaldehyde (2) and 10mL of ethanol are mixed, reflux reaction is carried out at 100 ℃ for 5 hours, a mixture of petroleum ether and ethyl acetate is used as a first eluent, column chromatography is carried out, and a light yellow solid is obtained as Probe 1, wherein the volume ratio of the petroleum ether to the ethyl acetate in the first eluent is 3: 1. the structure of Probe 1 is characterized by nuclear magnetic hydrogen spectrum, as shown in FIG. 2. Yield: 79 percent. The specific reaction equation is as follows:

the preparation method of the rhodamine B lactohydrazide comprises the following steps: mixing 0.479g of rhodamine B, 0.5mL of hydrazine hydrate and 30mL of ethanol in a 100mL round-bottom flask, carrying out reflux reaction at 100 ℃ for 6 hours, cooling to room temperature of 20-25 ℃ after the reaction is finished to obtain a reaction solution, mixing the reaction solution with 200mL of water, filtering to obtain precipitate rhodamine B internal hydrazide with the yield of 92%.

The preparation method of the 4-diethylamino-2-vinyloxybenzaldehyde (2) comprises the following steps: in a 50mL round bottom flask, 0.3g of 2-bromoethoxy-4-diethylaminobenzaldehyde, 0.12g of potassium tert-butoxide and 20mL of dimethyl sulfoxide are stirred at room temperature of 20-25 ℃ for reaction for 2 hours, then 100mL of water is added, after extraction with ethyl acetate, the organic phase is concentrated and purified by column chromatography using a mixture of petroleum ether and ethyl acetate as a second eluent to obtain a brown oily product 4-diethylamino-2-vinyloxybenzaldehyde (2), yield: 82 percent. The volume ratio of the petroleum ether to the ethyl acetate in the second eluent is 10: 1.

the preparation method of the 2-bromoethoxy-4-diethylaminobenzaldehyde comprises the following steps: mixing 0.193g of 4-diethylamino salicylaldehyde, 0.5mL of 1, 2-dibromoethane, 0.276g of potassium carbonate and 30mL of acetonitrile in a 100mL round-bottom flask, carrying out reflux reaction at 100 ℃ for 5 hours, cooling to room temperature of 20-25 ℃ after the reaction is finished, and purifying by column chromatography by using a mixture of petroleum ether and ethyl acetate as a second eluent to obtain a white solid product, namely 2-bromoethoxy-4-diethylamino benzaldehyde, wherein the yield is as follows: 52 percent. The volume ratio of the petroleum ether to the ethyl acetate in the second eluent is 10: 1.

the fluorescent probe needs to achieve detection effects in a dissolved state. If the solution to be detected can dissolve the fluorescent probe, the fluorescent probe is directly or indirectly added into the solution to be detected, so that the detection effect can be realized, and the fluorescent probe is indirectly added: dissolving the fluorescent probe in an organic solvent which is mutually soluble with water, and then adding the solution to be detected. Water-miscible organic solvent: methanol, ethanol, acetonitrile, dimethyl sulfoxide, N-dimethylformamide and the like. If the solution to be detected cannot dissolve the fluorescent probe, the detection effect cannot be achieved (for example, the solvent of the solution to be detected is pure water or petroleum ether), and at this time, the fluorescent probe needs to be dissolved in an organic solvent which is miscible with water, and then the solution to be detected is added.

Example 2

Preparing probe mother liquor: the fluorescent probe prepared in example 1 was added to acetonitrile to obtain a probe mother liquor, and the concentration of the fluorescent probe in the probe mother liquor was 10 mM.

Preparing a mercury metal ion solution: mercury salt was added to acetonitrile to give a mercury metal ion solution with a concentration of mercury salt of 10 mM.

Preparing a copper metal ion solution: and adding copper salt into acetonitrile to obtain copper metal ion solution, wherein the concentration of the copper salt in the copper metal ion solution is 10 mM.

Probe-1 selectivity to different ions under 420nm excitation: 0.6mL of acetonitrile, 2. mu.L of the probe stock solution and X were added to 1.4mL of PBS aqueous solution, and after shaking for 5 minutes, fluorescence detection was performed (wavelength. lambda. of excitation light; light intensity. lambda. of excitation light)_ex420nm), a fluorescence emission spectrum was plotted against each ion, and the results are shown in fig. 3. X is shown in Table 1.

TABLE 1

From FIG. 3, it can be seen that the addition of mercury ions significantly reduces the fluorescence intensity of Probe-1 at 515nm compared to the blank, and the addition of copper ions reduces the fluorescence intensity of Probe-1 at 515nm and simultaneously leads to an increase in fluorescence intensity at 580nm, i.e., a new maximum fluorescence emission peak is generated at 580 nm. After mercury ions and copper ions are added simultaneously, the fluorescence intensity of Probe-1 at 515nm is also obviously reduced, which is similar to the phenomenon of adding mercury ions separately, but is different from the phenomenon of adding copper ions separately.

Example 3

Preparing probe mother liquor: the fluorescent probe prepared in example 1 was added to acetonitrile to obtain a probe mother liquor, and the concentration of the fluorescent probe in the probe mother liquor was 10 mM.

Preparing a mercury metal ion solution: mercury salt was added to acetonitrile to give a mercury metal ion solution with a concentration of mercury salt of 10 mM.

Preparing a copper metal ion solution: and adding copper salt into acetonitrile to obtain copper metal ion solution, wherein the concentration of the copper salt in the copper metal ion solution is 10 mM.

Selectivity of Probe-1 to different ions under excitation of 530 nm: 0.6mL of acetonitrile, 2. mu.L of the probe stock solution and Y were added to 1.4mL of PBS aqueous solution, and after shaking for 5 minutes, fluorescence detection was performed (wavelength. lambda. of excitation light; light intensity. lambda.)_ex530nm), a fluorescence emission spectrum was plotted against each ion, and the results are shown in fig. 4. Y is shown in Table 2.

TABLE 2

From FIG. 4, it can be seen that Probe-1 has little response to the fluorescence of mercury ions, and only the addition of copper ions increases the fluorescence intensity of the compound Probe-1 at 580nm, resulting in the maximum fluorescence emission peak. After mercury ions and copper ions are added simultaneously, the fluorescence intensity of the compound Probe-1 at 580nm is also obviously enhanced, which is similar to the phenomenon of adding copper ions separately, but is different from the phenomenon of adding mercury ions separately.

When the copper ions and the mercury ions coexist in the solution to be detected, the fluorescence signals expressed by the fluorescence probe are different from the fluorescence signals which individually act with the copper ions or the mercury ions, and the change conditions of the fluorescence intensities at three positions, namely the excitation wavelength of 420nm, the emission wavelength of 515nm and 580nm, the excitation wavelength of 530nm and the emission wavelength of 580nm, can be observed to judge that the fluorescence intensity in the solution to be detected is: (1) copper ions and mercury ions are not present; (2) copper ions are present but no mercury ions; (3) mercury ions are present but no copper ions; (4) copper ions and mercury ions are present simultaneously.

As compared with the fluorescence intensity of Probe-1 of the present invention, 0 indicates no significant change in fluorescence intensity, "+" indicates significant increase in fluorescence intensity, and "-" indicates significant decrease in fluorescence intensity, and the correspondence between the specific sample components and the change in fluorescence intensity is shown in Table 3.

TABLE 3

Example 4

Preparing probe mother liquor: the fluorescent probe prepared in example 1 was added to acetonitrile to obtain a probe mother liquor, and the concentration of the fluorescent probe in the probe mother liquor was 10 mM.

Preparing a mercury metal ion solution: adding mercury salt into acetonitrile to obtain mercury metal ion solution with concentration of mercury salt C₁ mM。C₁The values are shown in Table 4.

TABLE 4

C1(Unit: μ M)	Numbered in FIG. 5
		0	Probe-1+0μM Hg2+
1	Probe-1+5μM Hg2+
		2	Probe-1+10μM Hg2+
3	Probe-1+15μM Hg2+
		4	Probe-1+20μM Hg2+
5	Probe-1+25μM Hg2+
		6	Probe-1+30μM Hg2+
8	Probe-1+40μM Hg2+
		20	Probe-1+100μM Hg2+

0.6mL of acetonitrile, 2. mu.L of the probe stock solution and 20. mu.L of the mercury metal ion solution were added to 1.4mL of the PBS aqueous solution, and after shaking for 5 minutes, fluorescence detection was performed (wavelength. lambda. of excitation light. lambda_ex420 nm). As shown in FIG. 5, the fluorescence intensity of the reaction system gradually decreases with the increase of the concentration of mercury ions, and when the concentration of mercury ions reaches 100. mu.M, the fluorescence intensity of the reaction system decreases to approach the saturation state.

Example 5:

preparing probe mother liquor: the fluorescent probe prepared in example 1 was added to acetonitrile to obtain a probe mother liquor, and the concentration of the fluorescent probe in the probe mother liquor was 10 mM.

Preparing a copper metal ion solution: adding copper salt into acetonitrile to obtain copper metal ion solution, wherein the concentration of the copper salt in the copper metal ion solution is C₂ mM。C₂The values are shown in Table 5.

TABLE 5

C2(Unit: μ M)	Numbered in FIG. 6
		0	Probe-1+0μM Cu2+
0.5	Probe-1+2.5μM Cu2+
		1.0	Probe-1+5.0μM Cu2+
1.5	Probe-1+7.5μM Cu2+
		2	Probe-1+10μM Cu2+
3	Probe-1+15μM Cu2+
		4	Probe-1+20μM Cu2+
8	Probe-1+40μM Cu2+

0.6mL of acetonitrile, 2. mu.L of the probe stock solution and 20. mu.L of the copper metal ion solution were added to 1.4mL of the PBS aqueous solution, and after shaking for 5 minutes, fluorescence detection (wavelength. lambda. of excitation light. lambda_ex420 nm). As shown in FIG. 6, the original fluorescence of the reaction system gradually decreases and new fluorescence is generated as the concentration of copper ions increasesAnd (3) light emission, wherein when the concentration of copper ions reaches 40 mu M, the fluorescence intensity of the reaction system is enhanced to a saturation state.

The fluorescent Probe Probe-1 for detecting mercury ions and copper ions has strong fluorescence, and after the Probe and the mercury ions act, the Probe-1 and the mercury ions undergo vinyl ether hydrolysis reaction to generate 4-diethylamino-2-hydroxyphenyl rhodamine B acylhydrazone (3), so that the original fluorescence intensity of the fluorescent Probe at 515nm is reduced (excitation light is 420 nm).

The recognition mechanism is as follows:

after the fluorescent Probe disclosed by the invention is reacted with copper ions, Probe-1 and the copper ions induce ring opening of a rhodamine structure through coordination, so that a fluorescent rhodamine complex (4) is generated, and the fluorescence of the fluorescent Probe is enhanced at 580nm (excitation light is 420nm or 530 nm).

The recognition mechanism is as follows:

after the fluorescent Probe of the invention acts with copper ions and mercury ions simultaneously, Probe-1 not only has hydrolysis reaction with the mercury ions, but also induces ring opening of a rhodamine structure through coordination with the copper ions to generate a fluorescent rhodamine complex (5), so that the Probe shows fluorescence enhancement at 580 (excitation light is 530 nm).

The recognition mechanism is as follows:

example 6

Preparing probe mother liquor: the fluorescent probe prepared in example 1 was added to acetonitrile to obtain a probe mother liquor, and the concentration of the fluorescent probe in the probe mother liquor was 10 mM.

Respectively preparing 15 metal ions(Mg²⁺,Ca²⁺,Ba²⁺,Sr²⁺,Ni²⁺,Cr³⁺,Mn²⁺,Co²⁺,Zn²⁺,Cd²⁺,Pb²⁺,Fe²⁺,Fe³⁺,Cu²⁺And Hg²⁺) The solution (2): one of the above metal ion salts was added to acetonitrile to obtain the metal ion solution, and the concentration of the corresponding metal salt in the metal ion solution was 10 mM.

Probe-1 selectivity to different ions under 420nm excitation: to 1.4mL of an aqueous PBS solution, 0.6mL of acetonitrile, 2 μ L of a probe stock solution, and 8 μ L of a metal ion solution were added, and after shaking for 5 minutes, fluorescence detection was performed (wavelength λ ex of excitation light is 420nm), and histograms of fluorescence emission intensity at 515 and 580nm and each ion were established, and the results are shown in fig. 7. The metal ions contained in the metal ion solution are shown in Table 6.

TABLE 6

From FIG. 7, it can be seen that the addition of mercury ions significantly reduces the fluorescence intensity of Probe-1 at 515nm and 580nm as compared with the blank; the addition of copper ions enables the fluorescence intensity of Probe-1 at 515nm to be obviously reduced and simultaneously leads the fluorescence intensity at 580nm to be obviously increased; after the addition of other metals, the fluorescence intensity of Probe-1 at 515 and 580nm is not obviously increased or reduced, and is very close to the blank, i.e. the fluorescence emission of Probe-1 is not obviously interfered.

The invention has been described in an illustrative manner, and it is to be understood that any simple variations, modifications or other equivalent changes which can be made by one skilled in the art without departing from the spirit of the invention fall within the scope of the invention.