Ratio-type fluorescent probe for instantly detecting mercury ions as well as preparation method and application of ratio-type fluorescent probe

Abstract

The invention relates to a ratio type fluorescent probe for instantly detecting mercury ions as well as a preparation method and application of the ratio type fluorescent probe, the ratio type fluorescent probe is used for detecting the fluorescence intensity ratio of a mercury ion solution at 516nm and 794nm, and the concentration of the mercury ions to be detected can be obtained according to a linear equation of the fluorescence intensity ratio and the concentration of the mercury ions; compared with a conventional detection method, the ratio fluorescent probe is used for detecting the mercury ions, has the advantages of high detection sensitivity, quick response (1min), large Stokes displacement (358nm), capability of monitoring the concentration of the mercury ions in real time by using a smart phone and the like, is successfully used for detecting the mercury ions in organisms such as cells and the like, and is high in use value and good in application prospect.

Description

Technical Field

[0001] The invention belongs to the technical field of fluorescent probes, in particular to a ratiometric fluorescent probe for real-time detection of mercury ions and a preparation method and application thereof. Background Art

[0002] Among heavy metals and toxic metal ions, mercury is known to have a good affinity for both amino and sulfhydryl groups of proteins, peptides and nucleic acids, which can lead to many health problems, such as myocardial infarction, brain damage, kidney failure, cognitive and movement disorders, etc. At the same time, mercury is also considered to be one of the most dangerous substances among heavy metals and transition metal ions. In the list of carcinogens published by the International Agency for Research on Cancer (IARC) of the World Health Organization (WHO), mercury and inorganic mercury compounds are listed as Class III carcinogens. Therefore, it is necessary to develop a rapid, convenient and effective method to prevent and detect Hg 2+ Technology is of great significance to environmental safety and human health.

[0003] The traditional Hg 2+ In order to overcome these shortcomings, many methods for detecting Hg 2+ New fluorescent probes have been developed. However, most of these probes still have shortcomings such as long response time, poor specificity, and poor biocompatibility, which make these fluorescent probes unable to be fully applied.

[0004] Based on the above considerations, we invented a new ratiometric fluorescent probe that can be used to detect Hg 2+ , and can also perform biological imaging in living cells and zebrafish, using smartphones to monitor Hg 2+ Conduct convenient testing. Summary of the invention

[0005] The purpose of the present invention is to overcome the shortcomings of existing mercury ion fluorescent probes and to provide a ratiometric fluorescent molecular probe with large Stokes shift, fast response time and high specificity, as well as a preparation method and application thereof.

[0006] The present invention solves the technical problem by adopting the following technical solutions:

[0007] A ratiometric fluorescent probe for real-time detection of mercury ions has a structure shown in Formula I:

[0008]

[0009] A method for preparing a ratiometric fluorescent probe for real-time detection of mercury ions comprises the following steps:

[0010]

[0011] S1, dissolving 2-(2-methyl-4H-thiobenzopyran-4-ylidene)malononitrile and 6-hydroxy-2-naphthaldehyde in toluene, adding glacial acetic acid and piperidine, heating and refluxing for 10-14h, after the reaction, spin-drying the reaction solution, and purifying the crude product by silica gel column to obtain compound 1;

[0012] S2, compound 1 and hexamethylamine were added to trifluoroacetic acid solution, the temperature was raised to reflux until the reaction was completed, 1 mol / L hydrochloric acid solution was added and stirred, and the mixture was adjusted to near neutrality with NaHCO3 solution, extracted with dichloromethane, and the organic phase was washed with distilled water, dried over anhydrous magnesium sulfate, filtered, and concentrated under reduced pressure to obtain compound 2;

[0013] S3. Dissolve compound 2 and 1,3-propanedithiol in anhydrous dichloromethane, stir in an ice-water bath for 1-2 hours, then add a dichloromethane solution of boron trifluoride ether to the reaction solution, react at room temperature for 3-5 hours, concentrate under reduced pressure after the reaction is completed, and purify by silica gel column chromatography to obtain the ratiometric fluorescent probe described in Formula I.

[0014] Moreover, in step S1, the molar ratio of the compound 2-(2-methyl-4H-thiobenzopyran-4-ylidene)malononitrile, 6-hydroxy-2-naphthaldehyde, glacial acetic acid and piperidine is 0.8-1.2 mmol: 0.8-1.2 mmol: 4-4.2 mmol: 4-4.5 mmol.

[0015] Moreover, in step S2, the molar ratio of compound 1 to hexamethylamine is 0.8-1.2 mmol:1.5-2 mmol.

[0016] Moreover, in step S3, the molar ratio of compound 2, 1,3-propanedithiol, and boron trifluoride ether is 1-1.5 mmol: 1.5-2 mmol: 0.2-0.8 mmol.

[0017] Moreover, in step S1, during the purification by silica gel column chromatography, the developing solvent used is petroleum ether:ethyl acetate=1:0-2:1.

[0018] Furthermore, in step S3, during the purification by silica gel column chromatography, the developing solvent used is dichloromethane:methanol=100:1-50:1.

[0019] The invention discloses an application of a ratiometric fluorescent probe for real-time detection of mercury ions in detecting mercury ions.

[0020] A detection method of a ratio-type fluorescent probe for real-time detection of mercury ions includes a detection method for measuring mercury ions in a water environment and a detection method for measuring mercury ions in a biological cell system.

[0021] Moreover, the detection method for determining mercury ions in a water environment is as follows: dissolving the ratiometric fluorescent probe described in Formula I in dimethyl sulfoxide to obtain a fluorescent probe solution, adding the fluorescent probe solution to a buffer solution having a volume ratio of dimethyl sulfoxide to PBS of 7:3, detecting its ultraviolet absorption and fluorescence emission wavelength, and comparing with other interfering ions;

[0022] The detection method for measuring mercury ions in a biological cell system is as follows: adding the fluorescent probe solution to the culture medium of A549 cells, placing the cells in an incubator containing CO2 for incubation, then washing with PBS buffer to remove the probe molecules that have not entered the cells, then replacing the culture medium, and then culturing with mercury ion solutions of different concentrations to measure the fluorescence thereof respectively;

[0023] The culture medium of A549 cells is 1640 containing 10% fetal bovine serum and 1% penicillin-streptomycin mixture.

[0024] The advantages and positive effects of the present invention are:

[0025] 1. The present invention successfully synthesized a ratiometric fluorescent probe that can be used for rapid detection of mercury ions, which has the advantages of high chemical stability and high optical stability and is more suitable for further modification.

[0026] 2. The fluorescent probe of the present invention can realize ratiometric identification of mercury ions and has the characteristics of dual-wavelength emission (or excitation). The change of the wavelength ratio value is independent of the probe concentration and the light source intensity, which can greatly reduce the interference of other detection conditions and improve the detection accuracy.

[0027] 3. The fluorescent probe of the present invention has the advantages of good water solubility, rapid response and high specificity. It can avoid interference from other analytes, is conducive to the rapid detection of mercury ions in the environment, and has strong practical application value in the field of environmental science.

[0028] 4. The fluorescent probe of the present invention has a large Stokes shift, can effectively avoid the interference of biological spontaneous fluorescence, has good cell membrane permeability, and has low cytotoxicity. It can be used for biological imaging of mercury ions and has strong practical application value in the field of life sciences.

[0029] 5. The fluorescent probe of the present invention can detect color changes through a smartphone to identify different concentrations of the detected object. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 This is the hydrogen nuclear magnetic resonance spectrum of the fluorescent probe TM-ND-Hg of the present invention.

[0031] Figure 2 This is the carbon nuclear magnetic resonance spectrum of the fluorescent probe TM-ND-Hg of the present invention.

[0032] Figure 3 This is the mass spectrum of the fluorescent probe TM-ND-Hg of the present invention.

[0033] Figure 4 The fluorescent probe TM-ND-Hg of the present invention is 2+ UV absorption spectrum of the response.

[0034] Figure 5 The fluorescent probe TM-ND-Hg of the present invention is 2+ Response fluorescence emission spectra.

[0035] Figure 6 This is the absorption-emission spectrum of the fluorescent probe TM-ND-Hg of the present invention.

[0036] Figure 7 The fluorescent probe TM-ND-Hg of the present invention is 2+ UV absorption spectrum of concentration changes.

[0037] Figure 8 The fluorescent probe TM-ND-Hg of the present invention is 2+ Fluorescence emission spectra of concentration changes.

[0038] Fig. 9 is the fluorescence intensity ratio of the fluorescent probe TM-ND-Hg of the present invention (I 794 / I 516 ) and Hg 2+ Linear relationship with concentration.

[0039] Fig.10 The fluorescent probe TM-ND-Hg of the present invention is 2+ Time response UV absorption spectrum.

[0040] Fig.11 The fluorescent probe TM-ND-Hg of the present invention is 2+ Fluorescence emission spectra of the time response.

[0041] Fig.12 This is the colorimetric diagram of the fluorescent probe TM-ND-Hg of the present invention for different analytes.

[0042] Fig.13 This is a histogram showing the fluorescence specificity of the fluorescent probe TM-ND-Hg of the present invention to different analytes.

[0043] Fig.14 This is a simulation diagram of smartphone recognition of the fluorescent probe TM-ND-Hg of the present invention detecting solutions at different concentrations.

[0044] Fig.15 This is the cytotoxicity of the fluorescent probe TM-ND-Hg of the present invention.

[0045] Fig.16 This is the confocal imaging of A549 cells using the fluorescent probe TM-ND-Hg of the present invention.

[0046] Fig.17 This is the zebrafish confocal imaging of the fluorescent probe TM-ND-Hg of the present invention. DETAILED DESCRIPTION

[0047] The present invention is further described in detail below through specific examples. The following examples are only illustrative and not restrictive, and the protection scope of the present invention cannot be limited thereto.

[0048] A ratiometric fluorescent probe for real-time detection of mercury ions has a structure shown in Formula I:

[0049]

[0050] A method for preparing a ratiometric fluorescent probe for real-time detection of mercury ions comprises the following steps:

[0051]

[0052] S1, dissolving 2-(2-methyl-4H-thiobenzopyran-4-ylidene)malononitrile and 6-hydroxy-2-naphthaldehyde in toluene, adding glacial acetic acid and piperidine, heating and refluxing for 12 h, after the reaction, spin-drying the reaction solution, and purifying the crude product by silica gel column to obtain compound 1;

[0053] S2, compound 1 and hexamethylamine were added to trifluoroacetic acid solution, the temperature was raised to reflux until the reaction was completed, 1 mol / L hydrochloric acid solution was added and stirred, and the mixture was adjusted to near neutrality with NaHCO3 solution, extracted with dichloromethane, and the organic phase was washed with distilled water, dried over anhydrous magnesium sulfate, filtered, and concentrated under reduced pressure to obtain compound 2;

[0054] S3. Dissolve compound 2 and 1,3-propanedithiol in anhydrous dichloromethane, stir in an ice-water bath for 2 h, then add a dichloromethane solution of boron trifluoride ether to the reaction solution, react at room temperature for 6 h, concentrate under reduced pressure after the reaction is completed, and purify by silica gel column chromatography to obtain the ratiometric fluorescent probe described in Formula I.

[0055] Example 1

[0056] A method for preparing a ratiometric fluorescent probe for real-time detection of mercury ions comprises the following steps:

[0057] S1. Add 500 mg of 2-(2-methyl-4H-thiobenzopyran-4-ylidene)malononitrile and 422 mg of 6-hydroxy-2-naphthaldehyde into a reaction bottle, add 20 mL of toluene to dissolve the reactants, add 0.5 mL of glacial acetic acid and 0.5 mL of piperidine to the mixed solution, reflux the reaction for 12 h, and detect the reaction by TLC. The volume ratio of the developing solvent is petroleum ether: ethyl acetate = 1:1. After the raw materials react completely, the temperature is lowered to room temperature, the reaction solution is spin-dried, and the crude product is purified by silica gel column (petroleum ether: ethyl acetate = 2:1) to obtain compound 1 as a red solid (375 mg, 44%).

[0058] Among them, the product characterization data are as follows:

[0059] 1 H NMR (400MHz, DMSO-d6) δ10.08(s,1H),8.68(d,J=8Hz,1H),8.01(s,1H),7.91(d,J=8Hz,1H),7.82(d,J=12 Hz,1H),7.73(d,J=8Hz,2H),7.67(d,J=8Hz,3H),7.54(s,1H),7.41(d,J=16Hz,1H),7.11(d,J=12Hz,2H). 13 CNMR(100MHz,DMSO-d6)δ157.36,155.97,148.90,138.05,135.98,134.90,133.02,130.77,130.15,130.10,128.85,128. 41,128.06,127.92,127.27,125.09,124.98,124.57,121.24,119.79,117.93,116.44,109.57,66.83.HRMS(ESI)m / z:[MH] - Calculate for C 24 H 13 N2OS:377.0754,found:377.0719.

[0060] S2. Add 300 mg of compound 1 and 222 mg of hexamethylamine to 10 mL of trifluoroacetic acid solution, heat to reflux, and detect the reaction by TLC. The volume ratio of the developing solvent is petroleum ether: ethyl acetate = 1:1. After the reaction, add 1 mol / L hydrochloric acid solution and stir for one hour. Adjust to nearly neutral with NaHCO3 solution, extract the reaction solution with dichloromethane (3×50 mL), wash the organic phase with distilled water, remove water from the organic phase with anhydrous magnesium sulfate, filter, and spin-dry the organic phase to obtain compound 2.

[0061] S3. Add 150 mg of compound 2 and 0.6 mL of 1,3-propanedithiol to a reaction flask, add 5 mL of anhydrous dichloromethane to dissolve the reactants, and place the reaction flask in an ice-water bath and stir for 2 h. After completion, slowly drop a solution of boron trifluoride ether (74 μL) dissolved in 2 mL of anhydrous dichloromethane into the reaction flask and stir at room temperature for 6 h. Detect the reaction by TLC, and the volume ratio of the developing solvent is dichloromethane: methanol = 100: 1. After the raw materials react completely, spin-dry the reaction solution. Purify the crude product by silica gel column (dichloromethane: methanol = 100: 1) to obtain the fluorescent probe of formula I as a red solid (87 mg, 48%).

[0062] Among them, the product characterization data are as follows:

[0063] 1 H NMR (400MHz, DMSO-d6) δ10.64(s,1H),8.94(d,J=8Hz,1H),8.07(s,1H),7.92-7.85(m,2H),7.79-7.95(m,2H),7.62(t,J=16Hz,1H),7. 55(s,2H),7.25(d,J=8Hz,1H),7.13(s,1H),6.30(s,1H),3.17-3.20(m,2H),2.95-2.98(m,2H),2.18-2.23(m,1H),1.83-1.87(m,1H). 13 C NMR (100MHz, DMSO-d6) δ180.14,167.44,152.95,149.82,136.36,135.91,133.82,132.74,132.17,131.02,129.99,129.79,129. 55,129.13,128.39,128.04,127.54,126.94,125.78,124.12,123.43,117.12,67.86,38.54,30.26,23.71.HRMS(ESI)m / z:[M+H] + Calculate for C 28 H 21 N2OS3:497.3311,found:497.3363.

[0064] Example 2

[0065] A detection method of a ratiometric fluorescent probe for real-time detection of mercury ions, comprising:

[0066] The spectral test conditions of this embodiment are as follows: the fluorescent probe TM-ND-Hg is dissolved in 10 mL of analytical grade dimethyl sulfoxide to prepare a 1 mM solution. For each test, 20 μL of the test solution is added to 2 mL of dimethyl sulfoxide: PBS = 7:3 (0.M, pH = 7.4) buffer solution for dilution, and then the optical test is performed. The solutions used in other tests are all prepared with deionized water (all at a concentration of 1 mM). The UV absorption spectrum and fluorescence emission spectrum of the complete reaction are recorded at room temperature.

[0067] In order to explore the effect of this fluorescent probe TM-ND-Hg on Hg 2+ To study the optical properties of the fluorescent probe TM-ND-Hg, 20 μL of the solution was added into 2 mL of dimethyl sulfoxide: PBS = 7:3 (0.1 M, pH = 7.4) buffer solution for dilution (concentration of 10 μM) to study its optical properties in the presence or absence of Hg. 2+ Changes in UV-visible absorption spectrum and fluorescence emission spectrum under the conditions of Figure 4 As shown, without adding Hg 2+ Before, the probe solution was dark green, and the maximum UV absorption wavelength was 611nm; 2+ Afterwards, the probe solution showed orange-yellow color, the absorption peak at 611nm disappeared, and the absorption peak at 436nm increased significantly. Figure 5 As shown in the fluorescence emission spectrum, with 436nm as the excitation wavelength, in the absence of Hg 2+ Before, the emission peak appeared at 516nm; after adding Hg 2+ Afterwards, the probe solution showed obvious changes, the emission peak at 516nm disappeared, and a new emission peak appeared at 794nm. This shows that the fluorescent probe is sensitive to Hg 2+ Has obvious response. Figure 6 As can be seen from the absorption-emission spectrum of the probe shown, the probe has a huge Stokes shift (358nm).

[0068] In order to study the effect of the fluorescent probe TM-ND-Hg on Hg 2+ The response sensitivity of the probe was tested for its concentration dependence. Figure 7 UV absorption spectrum shows that without adding Hg 2+ Previously, an absorption peak appeared at 611 nm, and Hg 2+ The solution was added to the test solution in a concentration gradient of 0.2, 0.4, 0.6, 0.8, 1, 2, 3...6, 7eq. 2+ As the concentration of increases, the absorption peak at 611nm gradually decreases until it disappears, while the absorption peak at 436nm increases significantly and reaches saturation when 7eq is added. Figure 8 The fluorescence emission spectrum shows that with the increase of Hg 2+The concentration of Hg increases with 436nm as the excitation wavelength. 2+ Previously, an emission peak appeared at 516 nm. 2+ With the increase of the concentration, the emission peak at 516nm gradually decreases until it disappears, and a new emission peak appears at 794nm and shows a regular upward trend. The fluorescence intensity also increases and reaches saturation when 7eq is added. It can be seen from the figure that the fluorescent probe is a ratiometric probe.

[0069] When Hg 2+ When the concentration is lower than 60 μM, the fluorescence intensity of the fluorescent probe TM-ND-Hg solution tends to be saturated. It can be concluded that the optimal concentration range of the probe for detecting mercury ions is 0-60 μM, and a linear relationship diagram between the fluorescence signal enhancement factor and the mercury ion concentration within this range is drawn ( Fig. 9 ), R 2 =0.99009. According to the formula LOD=3σ / k, the detection limit is 0.39μM.

[0070] For the detection of Hg by the fluorescent probe TM-ND-Hg 2+ To study the response time, 2eq of Hg was added to the test solution. 2+ Solution. Fig.10 As shown in the figure, at a fixed time interval of 30 seconds, the absorption peak at 611nm decreases rapidly, and then the absorption peak at 563nm increases, reaching saturation at 1 minute. Fig.11 As shown in the figure, at a fixed time interval of 30 seconds, the fluorescence intensity at 516nm drops rapidly, and the fluorescence intensity at 794nm rises rapidly. At 1 minute, the fluorescence intensity stops increasing and the fluorescence emission peak reaches saturation. 2+ The response time is 1in, which means that the probe has a fast response time.

[0071] In order to test the fluorescence probe TM-ND-Hg for Hg 2+ Whether it is specific, add Ag + 、Al 3+ , Ba 2+ , Ca 2 + 、Co 2+ Cr 2+ , Cu 2+ , Fe 2+ , Fe 3+ , K + Mg 2+ , Mn 2+ 、Na + 、Ni + , Pb 2+ 、Zn 2+As an interferent of the probe. Fig.12 As shown in Figure 2, when other ions are added to the probe solution, the solution color remains dark green without any change, while when Hg 2+ The solution color changes to orange-yellow. Fig.13 As shown in Figure 2, when other ions are added to the probe solution, the fluorescence intensity ratio of the probe does not change significantly, while when Hg is added 2+ The fluorescence intensity of the probe solution increased significantly. Therefore, it can be known that the fluorescence probe is 2+ It has better specificity.

[0072] Example 3

[0073] A smart phone detection experiment, including:

[0074] The spectral test conditions of this embodiment are as follows: the fluorescent probe TM-ND-Hg is dissolved in 10 mL of analytical grade dimethyl sulfoxide to prepare a 1 mM solution. For each test, 20 μL of the test solution is added to 2 mL of dimethyl sulfoxide: PBS = 7:3 (0.1 M, pH = 7.4) buffer solution for dilution, and then optical testing is performed. The solutions used in other tests are all prepared with deionized water (all at a concentration of 1 mM). The colors of the test solutions of different concentrations under visible light conditions with complete reaction are recorded at room temperature.

[0075] like Fig.14 As shown in the simulation diagram of smartphone detection, without adding Hg 2+ Before, the test solution was dark green, and Hg 2+ The solution was added to the test solution in a concentration gradient of 1, 2, 3, ..., 6, 7eq. 2+ With the increase of concentration, the detection solution changes from dark green to brown. By detecting the RGB value of the detection solution color through the color recognition software of the smart phone, the linear relationship between its R value, B value and different concentrations can be obtained, among which the linear relationship between concentration and R value is y=0.66956x+148.5487(R 2 =0.99125), the linear relationship between concentration and B value is y=-0.49762x+126.93047(R 2 =0.99476).

[0076] Example 4

[0077] A detection method of a ratiometric fluorescent probe for real-time detection of mercury ions, comprising:

[0078] Cell confocal imaging: The fluorescent probe TM-ND-Hg was tested for cytotoxicity in A549 cells. Fig.15As shown in the figure, when the probe concentration is 10 μM, the cell survival rate can still reach more than 80%. This shows that the probe itself has low cytotoxicity and is suitable for cell imaging research. In the cell confocal imaging experiment of this fluorescent probe, A549 cells were inoculated into two 3.5 mm cell culture dishes in advance and cultured overnight. The cells in the two culture dishes were pretreated with the fluorescent probe TM-ND-Hg for 30 minutes. After 30 minutes, the probe solution in the culture dish was washed three times with PBS, and one of the boxes was treated with different concentrations of Hg 2+ The (0.5eq, 1eq, 2eq) solution was treated for another 30 min, and then the culture dish was washed three times with PBS. The two boxes of cells were scanned by laser confocal microscopy to obtain fluorescence images. Fig.16 As shown in the figure, under the excitation wavelength of 470nm, in the culture dish with only the probe solution added, there is no fluorescence in the mixed field, 525nm fluorescence field (Red) and bright field, and obvious blue fluorescence can be seen in the 470nm fluorescence field (Blue); 2+ As the concentration increases, the blue fluorescence at 470nm (Blue) gradually weakens, and there is no obvious fluorescence at 2eq. At the same time, the red fluorescence at 525nm (Red) gradually increases. Therefore, it can be shown that this probe can be used to detect Hg in cells. 2+ ratiometric fluorescent probe.

[0079] Zebrafish Confocal Imaging:

[0080] Newborn zebrafish cultured for two days were divided into two groups for pretreatment. The first group was only added with the fluorescent probe TM-ND-Hg and cultured for 30 minutes. After the culture, they were washed three times with deionized water and anesthetics were added for laser confocal imaging experiments. Fig.17 As shown, no fluorescence was generated in the mixed field, 525nm fluorescence field and bright field under the excitation wavelength of 470nm. The second group added the fluorescent probe TM-ND-Hg and cultured for 30min. After the culture was completed, it was washed three times with deionized water and then 2eq Hg was added. 2+ The solution was cultured for 30 minutes, washed three times with deionized water, and anesthesia was added for laser confocal imaging experiments. Red fluorescence appeared in the zebrafish in both the mixed field and the 525nm fluorescence field. Therefore, this fluorescent probe has good tissue penetration and can be used to detect Hg in organisms. 2+ .

[0081] Although the embodiments of the present invention are disclosed for illustrative purposes, those skilled in the art will appreciate that various substitutions, changes and modifications are possible without departing from the spirit and scope of the present invention and the appended claims. Therefore, the scope of the present invention is not limited to the contents disclosed in the embodiments.

Claims

1. A ratiometric fluorescent probe for real-time detection of mercury ions, characterized in that: It has the structure shown in formula I:

2. The method for preparing a ratiometric fluorescent probe for real-time detection of mercury ions according to claim 1, characterized in that: The steps include: S1, dissolving 2-(2-methyl-4H-thiobenzopyran-4-ylidene)malononitrile and 6-hydroxy-2-naphthaldehyde in toluene, adding glacial acetic acid and piperidine, heating and refluxing for 10-14h, after the reaction, spin-drying the reaction solution, and purifying the crude product by silica gel column to obtain compound 1; S2, compound 1 and hexamethylamine were added to trifluoroacetic acid solution, the temperature was raised to reflux until the reaction was completed, 1 mol / L hydrochloric acid solution was added and stirred, and the mixture was adjusted to near neutrality with NaHCO3 solution, extracted with dichloromethane, and the organic phase was washed with distilled water, dried over anhydrous magnesium sulfate, filtered, and concentrated under reduced pressure to obtain compound 2; S3. Dissolve compound 2 and 1,3-propanedithiol in anhydrous dichloromethane, stir in an ice-water bath for 1-2 hours, then add a dichloromethane solution of boron trifluoride ether to the reaction solution, react at room temperature for 3-5 hours, concentrate under reduced pressure after the reaction is completed, and purify by silica gel column chromatography to obtain the ratiometric fluorescent probe described in Formula I.

3. The method for preparing a ratiometric fluorescent probe for real-time detection of mercury ions according to claim 2, characterized in that: In step S1, the molar ratio of the compound 2-(2-methyl-4H-thiobenzopyran-4-ylidene)malononitrile, 6-hydroxy-2-naphthaldehyde, glacial acetic acid and piperidine is 0.8-1.2 mmol: 0.8-1.2 mmol: 4-4.2 mmol: 4-4.5 mmol.

4. The method for preparing a ratiometric fluorescent probe for real-time detection of mercury ions according to claim 2, characterized in that: In step S2, the molar ratio of compound 1 to hexamethylamine is 0.8-1.2 mmol:1.5-2 mmol.

5. The method for preparing a ratiometric fluorescent probe for real-time detection of mercury ions according to claim 2, characterized in that: In step S3, the molar ratio of compound 2, 1,3-propanedithiol and boron trifluoride ether is 1-1.5 mmol: 1.5-2 mmol: 0.2-0.8 mmol.

6. The method for preparing a ratiometric fluorescent probe for real-time detection of mercury ions according to claim 2, characterized in that: In step S1, during the purification by silica gel column chromatography, the developing solvent used is petroleum ether:ethyl acetate=1:0-2:

1.

7. The method for preparing a ratiometric fluorescent probe for real-time detection of mercury ions according to claim 2, characterized in that: In step S3, during the purification by silica gel column chromatography, the developing solvent used is dichloromethane:methanol=100:1-50:

1.

8. Use of the ratiometric fluorescent probe for real-time detection of mercury ions according to claim 1 in detecting mercury ions.

9. The detection method of a ratiometric fluorescent probe for real-time detection of mercury ions according to claim 1, characterized in that: The invention comprises a detection method for determining mercury ions in a water environment and a detection method for determining mercury ions in a biological cell system.

10. The detection method of the ratiometric fluorescent probe for real-time detection of mercury ions according to claim 9, characterized in that: The detection method for determining mercury ions in a water environment is as follows: dissolving the ratiometric fluorescent probe of formula I in dimethyl sulfoxide to obtain a fluorescent probe solution, adding the fluorescent probe solution into a buffer solution having a volume ratio of dimethyl sulfoxide to PBS of 7:3, detecting its ultraviolet absorption and fluorescence emission wavelength, and comparing with other interfering ions; The detection method for measuring mercury ions in a biological cell system is as follows: adding the fluorescent probe solution to the culture medium of A549 cells, placing the cells in an incubator containing CO2 for incubation, then washing with PBS buffer to remove the probe molecules that have not entered the cells, then replacing the culture medium, and then culturing with mercury ion solutions of different concentrations to measure the fluorescence thereof respectively; The culture medium of A549 cells is 1640 containing 10% fetal bovine serum and 1% penicillin-streptomycin mixture.

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