CN112409330B

CN112409330B - Novel Hg detection method 2+ The preparation method of the fluorescent molecular probe and the application thereof in the environment and the organism

Info

Publication number: CN112409330B
Application number: CN201910767662.4A
Authority: CN
Inventors: 不公告发明人
Original assignee: Hunan Chaoji Testing Technology Co ltd
Current assignee: Hunan Chaoji Testing Technology Co ltd
Priority date: 2019-08-20
Filing date: 2019-08-20
Publication date: 2022-11-11
Anticipated expiration: 2039-08-20
Also published as: CN112409330A

Abstract

The invention discloses a preparation method of a specific mercury ion fluorescent molecular probe and application thereof in environment and organisms, wherein the chemical structural formula is as follows:

the fluorescent molecular probe provided by the invention has the advantages of good water solubility, quick response and high specificity, can avoid the interference of other metal ions, and is beneficial to Hg in the environment ²⁺ Rapid detection of (2); in addition, the probe also has stronger red light emission, can effectively avoid the interference of biological autofluorescence, has good cell membrane permeability and low cytotoxicity, and can be used for Hg ²⁺ The biological imaging has strong practical application value in the fields of environment and life science.

Description

Novel Hg detection method 2+ The preparation method of the fluorescent molecular probe and the application thereof in the environment and the organism

Technical Field

The invention belongs to the technical field of analytical chemistry, and relates to a novel Hg detection method ²⁺ The preparation method of the fluorescent molecular probe and the application thereof in environment and organism.

Background

Mercury (Mercury) is one of the most toxic heavy metal pollutants, widely present in water, air and soil, and has a total Mercury emission of nearly 7500 tons per year in the united states Environmental Protection Agency (EPA) report. In nature, mercury exists mainly in 3 forms of zero-valent mercury, inorganic mercury and organic mercury. Among them, mercury in the environment can enter into the organism through food chain, and combine with mercapto (-SH) in protein molecule to form mercaptol salt which is not easy to degrade, so that it can be accumulated and enriched in the body of animal and human body, and can produce chronic poisoning, and finally can produce several diseases mainly containing nerve damage, such as Japanese water yeast disease. Therefore, the development of a high-sensitivity and high-specificity rapid detection method for realizing the conventional detection of mercury ions in the environment and organisms has very important significance.

At present, various traditional detection methods including electrochemical method, atomic absorption spectrometry, plasma emission spectrometry (ICP), gas chromatography and the like can realize Hg in the environment ²⁺ The method has the advantages of accurate determination, wide measurement range and the like. However, these techniques generally require complicated sample pretreatment steps and expensive laboratory equipment, and are not suitable for real-time monitoring of Hg in a biological environment ²⁺ The detection of (2) severely restricts the great extent of the technologiesAnd (4) large-scale application.

The fluorescence spectroscopy based on the fluorescent probe has the advantages of high sensitivity, good specificity, high response speed, simple operation, real-time detection and the like, and becomes a research hotspot in recent years and is widely applied to the fields of environment, food, medicine and the like. The Shiraishi group reported a fluorescent molecular probe based on coumarin fluorescent nuclei that could achieve Hg in aqueous solution at pH =2-12 ²⁺ Specific detection of (3). The probe was prepared by a method based on Intramolecular Charge Transfer (ICT) in acetonitrile: water =1, an aqueous solution exhibiting strong blue fluorescence (maximum excitation wavelength of 445 nm), with Hg added ²⁺ Thereafter, ICT effect is weakened, leading to gradual reduction of 445 nm fluorescence intensity, thereby quantitatively detecting Hg through fluorescence quenching ²⁺ (Shiraishi Y, sumiya S, hirai T, A coumarins-thiourea conjugate as a fluorogenic probe for Hg (II) in an aqueous medium with a branched pH range of 2-12. Org. Biomol. Chem., 2010, 8 (6): 1310-4.). The probe has the advantages of good specificity, strong pH stability and the like, but is caused by Hg ²⁺ The fluorescent quenching molecular probe is a heavy metal ion and can cause fluorescent quenching, so that the fluorescent quenching molecular probe is easily interfered by various factors during detection and has poor accuracy. Therefore, the fluorescence-enhanced probe is more suitable for Hg ²⁺ Detection of (3). CN 102268249B reports Hg with specific recognition effect and good sensitivity ²⁺ Fluorescent molecular probe, hg in probe solution ²⁺ After that, the yellow fluorescence intensity at 560 nm is dependent on Hg ²⁺ The concentration is increased and enhanced, has good linear relation, can realize quantitative detection, and can detect Hg with the concentration more than 0.1 mu M ²⁺ . However, the probe has poor water solubility and short emission wavelength, and Hg in a water quality environment and a biological environment is difficult to realize ²⁺ The detection of (3). Compared with the short-wavelength emitted light, the long-wavelength emitted light can reduce the photodamage of the biological sample in the organism, has stronger tissue penetration capability and less background fluorescence interference, and therefore has better application prospect. Therefore, a red light emitting fluorescent molecular probe with high sensitivity, high specificity and good water solubility is developed to realize mercury dissociation in environment and organismsThe routine detection of the son has very important significance.

Disclosure of Invention

Aiming at the existing Hg ²⁺ The invention aims to provide Hg which has good water solubility, high specificity and red light emission ²⁺ A fluorescent molecular probe.

The second purpose of the invention is to provide a high-efficiency preparation method of the fluorescent molecular probe.

The third purpose of the invention is to provide Hg in water solution and organism by the fluorescent molecular probe ²⁺ The detection application of (1).

In order to achieve the above object, the present invention provides a fluorescent molecular probe, having a structure of formula I:

formula I

The preparation method of the fluorescent probe is preferably as follows:

4-bromo-1, 8-naphthalic anhydride and n-hexylamine are used as raw materials, heated and refluxed at 120 ℃ in absolute ethyl alcohol, poured into ice water after reaction is finished, and precipitates are filtered, washed and dried by column chromatography to obtain light yellow powder. Dissolving in DMSO, adding N-hydroxysuccinimide and anhydrous potassium carbonate, stirring, and stirring to obtain solution 90% ^o Heating and refluxing to complete reaction at C, cooling to room temperature, pouring into ice water, adjusting pH to be nearly neutral by using dilute HCl solution, extracting by ethyl acetate, drying, and purifying by silica gel column chromatography. Dissolving the purified product in trifluoroacetic acid, adding hexamethylene tetramine, 90% ^o And C, heating and refluxing to completely react, cooling to room temperature, dropwise adding into ice water, adjusting the pH value to be nearly neutral by using a dilute NaOH solution, performing suction filtration, drying, and purifying by silica gel column chromatography. Dissolving the purified product in ethanol, adding 2-methylpyridine salt and 1 drop of piperidine, 40 ^o C, heating and refluxing in water bath, after the reaction is finished, concentrating in vacuum, and separating and purifying by using silica gel column chromatography. Dissolving the purified product in DMF solution under nitrogen protectionAdding K to the system in batches ₂ CO ₃ And 1, 2-dibromoethane, placing at 80 ℃ for heating and reacting till the reaction is complete, pouring into ice water, and extracting by using an ethyl acetate solution. The organic layers were combined, washed with water and NaCl solution, and then with anhydrous Na ₂ SO ₄ Drying, evaporating the solvent under reduced pressure, and purifying by silica gel column chromatography. Dissolving the purified product and potassium tert-butoxide in DMSO solution, adding into normal temperature, stirring to react completely, extracting with ethyl acetate, mixing the organic layers, washing with water and NaCl solution respectively, and removing anhydrous Na ₂ SO ₄ Drying, decompressing and evaporating the solvent, and purifying by silica gel column chromatography to obtain the target molecular probe.

The synthesis of the invention is as follows:

the invention provides application of the fluorescent probe, which can be applied to Hg in environment and organisms ²⁺ The detection principle is as follows: utilizes the vinyl to block the ICT function of the probe, so that the probe only emits weak fluorescence and reacts with Hg ²⁺ After the reaction, hydroxyl is recovered, ICT function is recovered, so that the fluorescence of the probe at 597 nm is obviously enhanced, and Hg is finally treated ²⁺ And (4) carrying out quantitative detection. The detection mechanism is shown in the figure:

the invention provides a method for measuring Hg by using the fluorescent probe ²⁺ The method of (1). The specific determination method comprises the following steps: the fluorescent probe was dissolved in a PBS buffer solution (10 mM, pH = 7.4) at room temperature, and was set to a concentration of 5. Mu.M-20. Mu.M. HgCl was added to the system at different concentrations ₂ The fluorescence intensity of the aqueous solution was measured and determined from the fluorescence intensity and Hg ²⁺ Linear relationship of concentration to Hg ²⁺ And (4) carrying out quantitative detection.

In the above detection method, preferably, the solvent system is PBS buffer solution.

Preferably, the pH of the detection method is 7.4.

In the above detection method, the concentration of the fluorescent probe is preferably 10. Mu.M.

The fluorescent probe can be applied to intracellular Hg ²⁺ Detection of (3). The specific detection method comprises the following steps: adding 10 μ M fluorescent probe solution into HeLa medium, and placing at 37 ^o C, 5% CO ₂ After incubation in an incubator for 30 minutes, the cells were washed three times with 0.1M PBS buffer (10 mM, pH = 7.4) to remove probe molecules that did not enter the cells, and then the medium was replaced with HgCl ₂ After incubation in buffer (25. Mu.M) for 30 min, the cells fluoresce strongly red. Experiments show that fluorescent probes target Hg in cells ²⁺ Has good imaging effect, and can be used for detecting Hg in organisms ²⁺ 。

Compared with the prior art, the technical scheme of the invention has the beneficial technical effects that:

the invention relates to Hg detection ²⁺ The fluorescent molecular probe has the following advantages:

(1) The fluorescent probe has the advantage of high sensitivity, and the fluorescence intensity of the probe solution and Hg ²⁺ The concentration of (A) was well linear in the range of 0-10. Mu.M, and the detection line was as low as 0.53. Mu.M.

(2) The fluorescent probe has the advantages of good water solubility, quick response and high specificity, can avoid the interference of other metal ions, and is beneficial to Hg in the environment ²⁺ The rapid detection of the protein has strong practical application value in the field of environmental science.

(3) The probe has stronger red light emission, can effectively avoid the interference of biological autofluorescence, has good cell membrane permeability and low cytotoxicity, and can be used for Hg ²⁺ The biological imaging has stronger practical application value in the field of life science.

Drawings

FIG. 1 shows fluorescence intensity of fluorescent probe according to Hg in the practice of the present invention ²⁺ Emission spectrum of concentration change;

FIG. 2 shows fluorescence intensity and Hg of fluorescent probe in the practice of the present invention ²⁺ A linear plot of concentration;

FIG. 3 shows a fluorescent probe pair for Hg in the practice of the present invention ²⁺ A selectivity profile of (a);

FIG. 4 is a confocal image of fluorescence of a fluorescent probe in HeLa cells in the practice of the present invention.

Detailed Description

The following embodiments are intended to further illustrate the invention and are not intended to limit the invention.

Example 1

Synthesis of Compound 1:

4-bromo-1, 8-naphthalic anhydride (2770 mg,10 mmol) was weighed out and dissolved in 30 mL of absolute ethanol, and n-hexylamine (1262.5 mg,12.5 mmol) was added to the system and the reaction was heated at 120 ℃ and monitored by TLC until the reaction was complete. Pouring the reaction system into ice water, precipitating yellow floccule, performing suction filtration, collecting and washing a filter cake, purifying by silica gel column chromatography, and removing the solvent by rotary evaporation to obtain light yellow powder of 3186 mg with the yield of 88.5%.

Synthesis of Compound 2:

compound 1 (2280 mg,8 mmol), N-hydroxysuccinimide (1012mg, 8.8 mmol) and anhydrous potassium carbonate (3864 mg,4 mmol) were added to a round-bottomed flask, respectively, and dissolved by adding an appropriate amount of DMSO solution, reacted at 90 ℃ and monitored by TLC until the reaction was completed. The system was poured into ice water, pH adjusted to near neutral with dilute HCl solution, extracted with ethyl acetate, dried and purified by silica gel column chromatography, and solvent removed by rotary evaporation to give 1908 mg of a pale yellow solid in 80.3% yield. ¹ H-NMR(400 MHz, DMSO-d6, TMS): δ = 0.92(s, 3H), 1.33 (m, 2H), 1.57 (m, 4H), 2.21 (t, 2H, J=7.41 Hz), 3.99 (t, 2H, J=7.48 Hz), 7.16 (d, 1H, J=8.18 Hz), 7.74 (t, 1H, J=7.86 Hz), 8.33 (d, 1H, J=8.32 Hz), 8.44 (d, 1H, J=6.89 Hz), 8.51 (d, 1H, J=8.32 Hz), 11.98 (s, 1H). HR-MS (ESI, negative), m/z: calculated [M-H] ^- : 296.11067, found [M-H] ^- : 296.09415.

Synthesis of Compound 3

Separately, compound 2 (1782) was added to the round bottom flaskmg,6 mmol), and hexamethylenetetramine (4200 mg,30 mmol), dissolved by addition of 15 mL of trifluoroacetic acid, the reaction is refluxed at 90 ℃ and monitored by TLC until completion. Pouring the system into ice water, adjusting the pH value to be nearly neutral by using NaOH solution, performing suction filtration and drying, performing silica gel column chromatography purification, and removing the solvent by rotary evaporation to obtain 1362 mg of yellow solid with the yield of 69.8%. ¹ H-NMR (400 MHz, DMSO-d6, TMS): δ =0.93 (s, 3H), 1.33 (m, 2H), 1.57 (m, 4H), 2.22 (t, 2H, J=7.40 Hz), 3.95 (t, 2H, J=7.56 Hz), 7.79 (t, 1H, J=7.81 Hz), 8.47 (d, 1H, J=7.39 Hz), 8.55 (s, 1H), 8.58 (d, 1H, J=8.42 Hz). HR-MS (ESI, negative), calculated [M-H] ^- : 324.10259, found [M-H] ^- : 324.09979.

Synthesis of Compound 4

Compound 3 (1300 mg,4 mmol), 2-methylpyridine salt (1147.2 mg, 4.8 mmol) was weighed out and dissolved in absolute ethanol, and 1 drop of piperidine was added thereto, followed by reaction in a water bath at 40 ℃ until completion. Evaporating the system to dryness under reduced pressure, purifying by silica gel column chromatography, and evaporating the solvent to dryness under reduced pressure to obtain mauve solid powder 1698 mg with yield of 76.1%. ¹ H-NMR (400 MHz, DMSO-d6, TMS): δ = 0.99 (s, 3H), 1.32 (m, 2H), 1.57 (m, 4H), 2.22 (t, 2H, J=7.12 Hz), 3.81 (s, 3H), 4.01 (t, 2H, J=7.23 Hz), 7.37 (t, 1H, J=7.61 Hz), 7.48 (t, 1H, J=7.55 Hz), 7.55 (t, 1H, J=7.55 Hz), 7.57 (d, 1H, J=7.79 Hz), 7.70 (d, 1H, J=7.34 Hz), 8.33 (d, 1H, J=7.79 Hz), 8.48 (d, 1H, J=7.63 Hz), 8.64 (s, 1H). HR-MS (ESI, positive), m/z: calculated [M] ⁺ : 557.22275, found [M] ⁺ : 557.22485.

Synthesis of Compound 5

Compound 4 (1674 mg,3 mmol) was dissolved in an appropriate amount of DMF under a nitrogen atmosphere, and K was added to the system in portions ₂ CO ₃ (1243 mg,9 mmol) and 1, 2-dibromoethane (5610 mg,30 mmol). After the reaction was completed by heating the system at 80 ℃, the reaction mixture was poured into ice water and extracted with ethyl acetate. The organic layers were combined, washed with water and NaCl solution, respectively, and then anhydrous Na ₂ SO ₄ Drying, evaporating solvent under reduced pressure, purifying by silica gel column chromatography, and rotary evaporating to remove solvent to obtain compound 5 (1386 mg)，69.5 %）。 ¹ H-NMR (400 MHz, DMSO-d6, TMS): δ = 0.99 (s, 3H), 1.32 (m, 2H), 1.57 (m, 4H), 2.22 (t, 2H, J=7.12 Hz), 3.81 (s, 3H), ,3.93 (t, 2H, J=7.34), 4.01 (t, 2H, J=7.23 Hz), 4.12 (t, 2H, J=6.3), 7.36 (t, 1H, J=7.61 Hz), 7.49 (t, 1H, J=7.55 Hz), 7.55 (t, 1H, J=7.55 Hz), 7.57 (d, 1H, J=7.79 Hz), 7.70 (d, 1H, J=7.34 Hz), 8.33 (d, 1H, J=7.79 Hz), 8.48 (d, 1H, J=7.63 Hz). HR-MS (ESI, positive), m/z: calculated [M] ⁺ : 664.19727, found [M] ⁺ : 664.20328.

Synthesis of target molecular probe:

compound 5 (1330 mg,2 mmol) was dissolved in dimethyl sulfoxide solution, and potassium tert-butoxide (340 mg,3 mmol) was added. Stirring at room temperature until the reaction is complete, extracting with ethyl acetate, combining organic layers, washing with water and NaCl solution respectively, and removing anhydrous Na ₂ SO ₄ Drying, evaporating the solvent under reduced pressure, and purifying by silica gel column chromatography to obtain 796 mg of target molecular probe with a yield of 68.2%. ¹ H-NMR (400 MHz, DMSO-d6, TMS): δ = 0.99 (s, 3H), 1.32 (m, 2H), 1.57 (m, 4H), 2.22 (t, 2H, J=7.12 Hz), 3.81 (s, 3H), , 4.01 (t, 2H, J=7.23 Hz), 4.12 (d, 2H, J=6.5), 4.27 (dd, 1H), 7.36 (t, 1H, J=7.61 Hz), 7.49 (t, 1H, J=7.55 Hz), 7.55 (t, 1H, J=7.55 Hz), 7.57 (d, 1H, J=7.79 Hz), 7.70 (d, 1H, J=7.34 Hz), 8.33 (d, 1H, J=7.79 Hz), 8.48 (d, 1H, J=7.63 Hz).

Example 2

Preparation of fluorescent probe mother liquor

The product isolated above and having a purity of 99% is weighed accurately at 5.84 mg and carefully transferred into a 50 mL volumetric flask, where CH is added at room temperature ₃ CN solution is dissolved completely, and the volume is determined to the scale mark, thus obtaining the probe mother liquor with the concentration of 1 mM. During the test, 20. Mu.L of the above solution was taken out by a micro-injector each time, and dissolved in the test system so that the total volume per test was 2 mL, at which time the concentration of the fluorescent probe was 10. Mu.M.

Example 3

Hg ²⁺ Preparation of mother liquor

Hg ²⁺ Buffered with PBSThe solutions were prepared as 5 mL stock solutions with different concentration gradients (0.1 mM, 0.2 mM, 0.3 mM, 0.5 mM, 0.7 mM, 1 mM, 1.5 mM, 2 mM, 3.0 mM, 4.0 mM). The other tests required the use of metal ions, respectively, in PBS buffer solution to make 3 mM stock solution.

Example 4

Fluorescence intensity of fluorescent probe and Hg ²⁺ Relation of concentration

4.900 mL of PBS buffer solution was measured, 50. Mu.L of 1 mM probe stock solution was dissolved therein, and 50. Mu.L of Hg at different concentrations was transferred ²⁺ In the mother liquor, the concentration of the probe of the whole detection system is 10 mu M and Hg is finally obtained ²⁺ The concentrations of (A) are 1. Mu.M, 2. Mu.M, 3. Mu.M, 5. Mu.M, 7. Mu.M, 10. Mu.M, 15. Mu.M, 20. Mu.M, 30. Mu.M and 40. Mu.M, respectively. After incubation at room temperature for 20 min, the fluorescence spectra of the different systems were tested in 10 mm cuvettes, respectively (FIG. 1). The results show that Hg is related to Hg ²⁺ The concentration is gradually increased, and the fluorescence emission intensity of the system at 597 nm is gradually increased.

Example 5

Fluorescent probe for Hg ²⁺ Detection limit of response

The fluorescence intensity in each detection system in example 4 was calculated to establish the fluorescence intensity and Hg ²⁺ The standard curve of concentration, shown in figure 2, when Hg is ²⁺ In the range of 0-10 μ M, the fluorescence emission intensity at 597 nm and Hg ²⁺ The concentration of (b) has a good linear relation, the regression linear equation is y =10.291x +9.9767, and the linear correlation coefficient R ² = 0.9973. By the formula, the detection limit can be calculated to be 0.52 mu M, and the result shows that the fluorescent probe has good sensitivity.

Example 6

Fluorescent probe for Hg ²⁺ Selectivity of detection

50. Mu.L of 1 mM probe stock solution was dissolved in 4.900 mL PBS buffer solution, and 50. Mu.L of 3 mM NaCl, KCl, mgCl was pipetted ₂ ，CaCl ₂ ，BaCl ₂ ，MnCl ₂ ， ZnCl ₂ ，Pb(NO ₃ ) ₂ ，FeCl ₂ ，CuCl ₂ ，AgNO ₃ And FeCl ₃ And respectively adding the mother liquor into the system, incubating for 20 min at room temperature, respectively measuring the fluorescence spectra, and recording the fluorescence intensity value of 597 nm. The results show that only Hg was added ²⁺ When the fluorescent probe is added with other test metal ions, the fluorescence of the fluorescent probe is obviously enhanced, and no or only weak fluorescence change exists. The fluorescent probe is shown to have good selectivity.

Example 7

Fluorescent probe for Hg ²⁺ Kinetic study of the response

Low (15. Mu.M) and high (30. Mu.M) concentrations of Hg were added separately ²⁺ Measuring the change of fluorescence intensity at 597 nm along with time in a detection system in solution, wherein the fluorescence probe is used for detecting high-concentration Hg ²⁺ The response can be completed in 20 seconds, and the response is quick.

Example 8

Fluorescent probe for Hg in cells ²⁺ Response to (2)

Adding 10 μ M fluorescent probe solution into HeLa medium, and placing at 37 ^o C, 5% CO ₂ After incubation in an incubator for 30 minutes, the cells were washed three times with 0.1M PBS buffer (10 mM, pH = 7.4) to remove probe molecules that did not enter the cells, and then the medium was replaced with HgCl ₂ The buffer solution (25. Mu.M) was incubated for 30 minutes, washed three times with 0.1M PBS buffer (10 mM, pH = 7.4), and the change in fluorescence was observed under a fluorescence microscope, and the results are shown in FIG. 4. Experiments show that probe molecules and Hg enter cells ²⁺ In response, an intense red fluorescence is emitted, and the fluorescent probe therefore targets Hg in the cell ²⁺ Has good imaging effect, and can be used for detecting Hg in organism ²⁺ 。

Although the present invention has been described with reference to the specific embodiments shown in the drawings, it is not intended to limit the scope of the present invention, and various modifications or variations can be made by those skilled in the art from the disclosure of the present invention without inventive efforts.

Claims

1. Can be used for specificityIdentifying Hg ²⁺ The fluorescent molecular probe of (1), characterized by having a structure represented by formula (I):

formula I.

2. The method for preparing a fluorescent molecular probe according to claim 1, comprising the steps of:

(1) Weighing 4-bromo-1, 8-naphthalic anhydride and n-hexylamine, dissolving in absolute ethyl alcohol, heating at 120 ℃ for reflux reaction, monitoring by TLC (thin layer chromatography) until complete reaction is achieved, pouring the system into ice water, separating out yellow floccule, performing suction filtration, collecting and washing a filter cake, performing silica gel column chromatography purification after drying, and performing rotary evaporation to remove a solvent to obtain a light yellow powder compound 1, wherein the compound 1 has the following structural formula:

(2) Dissolving a compound 1 in a proper amount of DMSO (dimethylsulfoxide) solution, respectively adding N-hydroxysuccinimide and anhydrous potassium carbonate, heating and refluxing at 90 ℃, monitoring by TLC (thin layer chromatography) until complete reaction, pouring the system into ice water, adjusting the pH to 6 to 7 by using a dilute HCl solution, extracting by using an ethyl acetate solution, combining organic layers, drying, purifying by silica gel column chromatography, and removing a solvent by rotary evaporation to obtain a light yellow solid compound 2, wherein the compound 2 has the following structural formula:

(3) Dissolving a compound 2 and hexamethylenetetramine in a proper amount of trifluoroacetic acid, carrying out reflux reaction at 90 ℃, monitoring by TLC (thin layer chromatography) until the reaction is completed, pouring the system into ice water, adjusting the pH to 6 to 7 by using NaOH solution, carrying out suction filtration and drying, carrying out silica gel column chromatography purification, and carrying out rotary evaporation to remove a solvent to obtain a yellow solid compound 3, wherein the structural formula of the compound 3 is as follows:

(4) Weighing a compound 3 and 2-methylpyridine salt, dissolving in absolute ethyl alcohol, adding 1 drop of piperidine, reacting in a water bath at 40 ℃ until the reaction is complete, evaporating the system to dryness under reduced pressure, purifying by silica gel column chromatography, and evaporating the solvent to dryness under reduced pressure to obtain a mauve solid powder compound 4, wherein the structural formula of the compound 4 is as follows:

(5) Dissolving the compound 4 in DMF solution under the protection of nitrogen, and adding K to the system in batches ₂ CO ₃ And 1, 2-dibromoethane, heating the system at 80 ℃ for reaction to be complete, pouring the reaction product into ice water, and extracting the reaction product by using an ethyl acetate solution;

the organic layers were combined, washed with water and NaCl solution, and then with anhydrous Na ₂ SO ₄ Drying, evaporating the solvent to dryness under reduced pressure, purifying by silica gel column chromatography, and removing the solvent by rotary evaporation to obtain a compound 5, wherein the structural formula of the compound 5 is as follows:

(6) Dissolving compound 5 and potassium tert-butoxide in DMSO solution, adding into the solution at room temperature, stirring to react completely, extracting with ethyl acetate, mixing the organic layers, washing with water and NaCl solution respectively, and adding anhydrous Na ₂ SO ₄ Drying, decompressing and evaporating the solvent, and purifying by silica gel column chromatography to obtain the target molecular probe.

3. The method for preparing a fluorescent probe according to claim 2, characterized in that:

in the step (1), the molar ratio of the 4-bromo-1, 8-naphthalic anhydride to the n-hexylamine is 1 to 1.5;

in the step (2), the molar ratio of the compound 2 to the N-hydroxysuccinimide to the anhydrous potassium carbonate is 1: 1.1 to 1.25: 3.5 to 4;

in the step (3), the molar ratio of the compound 2 to hexamethylenetetramine is 1: 5 to 8;

in the step (4), the molar ratio of the compound 3 to the 2-methylpyridine salt is 1: 1.1 to 2;

in the step (5), the compound 4, K ₂ CO ₃ The molar ratio of the ethylene glycol to the 1, 2-dibromoethane is 1: 2 to 4: 8 to 12;

in the step (6), the molar ratio of the compound 5 to potassium tert-butoxide is 1: 1.2 to 2.5.

4. Use of the fluorescent probe of claim 1 in a detection reagent for non-disease diagnosis and treatment, for Hg in the environment and cells ²⁺ Detection of (3).

5. Use of the fluorescent probe according to claim 4 in a detection reagent for non-disease diagnosis and treatment, characterized in that Hg ²⁺ The detection conditions of (a) are as follows: the excitation wavelength is 360 nm, the fluorescence emission spectrum detection is carried out in the range of 400-700 nm, and the solvent of the detection system is PBS buffer solution.

6. The use of the fluorescent probe in a reagent for the detection of non-disease diagnosis and treatment according to claim 5, wherein the sample to be tested is added to the detection solution of the fluorescent probe, and if the fluorescence of the solution is red, it indicates that Hg is contained in the sample to be tested ²⁺ If no obvious red fluorescence exists, the Hg is not contained in the sample to be detected ²⁺ And the measured fluorescence intensity of the solution can be used as Hg ²⁺ Evaluation index of concentration.