A ratiometric fluorescent probe for the instantaneous detection of mercury ions, its preparation method and application

Abstract

This invention relates to a ratiometric fluorescent probe for real-time detection of mercury ions, its preparation method, and its application. By using the ratiometric fluorescent probe to detect the fluorescence intensity ratio of a mercury ion solution at 516 nm and 794 nm, the concentration of the mercury ions to be measured can be obtained according to the linear equation between the fluorescence intensity ratio and the mercury ion concentration. Compared with conventional detection methods, this invention utilizes a ratiometric fluorescent probe to detect mercury ions, which has advantages such as high detection sensitivity, rapid response (1 min), large Stokes shift (358 nm), and the ability to monitor mercury ion concentration in real-time using a smartphone. It has been successfully applied to the detection of mercury ions in cells and other biological organisms, demonstrating high practical value and promising application prospects.

Description

Technical Field

[0001] This invention belongs to the field of fluorescent probe technology, and in particular to a ratiometric fluorescent probe for instantaneous detection of mercury ions, its preparation method and application. Background Technology

[0002] Among heavy metals and toxic metal ions, mercury is known to have a strong affinity for the amino and sulfhydryl groups of proteins, peptides, and nucleic acids, leading to numerous health problems such as myocardial infarction, brain damage, kidney failure, and cognitive and motor impairments. Furthermore, mercury is considered one of the most dangerous 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 classified as Group 3 carcinogens. Therefore, developing a rapid, convenient, and effective method for the prevention and detection of mercury (Hg) is crucial. 2+ Technology is of great importance to environmental safety and human health.

[0003] High cost, complex operation, long detection time, inability to perform simple and convenient detection, and difficulty in sample preparation are the main drawbacks of traditional Hg testing. 2+ These shortcomings are unavoidable in detection techniques. To overcome these limitations, many methods for detecting Hg have been developed. 2+ Novel fluorescent probes have been developed. However, most of these probes still have shortcomings such as long response time, poor specificity, and poor biocompatibility, which prevent them from being fully utilized.

[0004] Based on the above considerations, we have invented a novel ratiometric fluorescent probe that can be used to detect Hg. 2+ It can also perform bioimaging on living cells and zebrafish, and can use a smartphone to visualize Hg. 2+ Convenient testing. Summary of the Invention

[0005] The purpose of this 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 its preparation method and application.

[0006] The technical problem solved by this invention is achieved through the following technical solution:

[0007] A ratiometric fluorescent probe for the instantaneous detection of mercury ions has the structure shown in Formula I:

[0008]

[0009] A method for preparing a ratiometric fluorescent probe for the instantaneous detection of mercury ions includes the following steps:

[0010]

[0011] S1. Dissolve 2-(2-methyl-4H-thiobenzopyran-4-yl)malononitrile and 6-hydroxy-2-naphthaldehyde in toluene, add glacial acetic acid and piperidine, heat under reflux for 10-14 h, after the reaction is complete, evaporate the reaction solution to dryness, and purify the crude product by silica gel column chromatography to obtain compound 1.

[0012] S2. Compound 1 and hexamethylamine were added to trifluoroacetic acid solution, and the mixture was heated to reflux until the reaction was complete. 1 mol / L hydrochloric acid solution was added and stirred. The mixture was adjusted to near neutral with NaHCO3 solution, extracted with dichloromethane, washed with distilled water, dried over anhydrous magnesium sulfate, filtered, and concentrated under reduced pressure to obtain compound 2.

[0013] S3. Compound 2 and 1,3-propanedithiol were dissolved in anhydrous dichloromethane and stirred in an ice-water bath for 1-2 hours. Then, a dichloromethane solution of boron trifluoride diethyl ether was added to the reaction solution and reacted at room temperature for 3-5 hours. After the reaction was completed, the solution was concentrated under reduced pressure and purified by silica gel column chromatography to obtain the ratiometric fluorescent probe described in Formula I.

[0014] Furthermore, in step S1, the molar ratio of compound 2-(2-methyl-4H-thiobenzopyran-4-yl)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] Furthermore, in step S2, the molar ratio of compound 1 to hexamethylamine is 0.8–1.2 mmol: 1.5–2 mmol.

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

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

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

[0019] Application of a ratiometric fluorescent probe for instantaneous detection of mercury ions in the detection of mercury ions.

[0020] A detection method for a ratiometric fluorescent probe for the instantaneous detection of mercury ions includes a detection method for measuring mercury ions in an aquatic environment and a detection method for measuring mercury ions in a biological cell system.

[0021] Furthermore, the detection method for mercury ions in an aquatic environment is as follows: the ratiometric fluorescent probe described in Formula I is dissolved in dimethyl sulfoxide to obtain a fluorescent probe solution, the fluorescent probe solution is added to a buffer solution in which the volume ratio of dimethyl sulfoxide to PBS is 7:3, its ultraviolet absorption and fluorescence emission wavelengths are detected, and compared with other interfering ions.

[0022] The detection method for mercury ions in biological cell systems is as follows: the fluorescent probe solution is added to the culture medium of A549 cells, and the cells are incubated in an incubator containing CO2. Then, the cells are washed with PBS buffer to remove probe molecules that have not entered the cells. The culture medium is then replaced, and the cells are cultured with mercury ion solutions of different concentrations. The fluorescence of the cells is then measured.

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

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

[0025] 1. This invention successfully synthesizes a ratiometric fluorescent probe 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 ratio-based recognition 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, fast response and high specificity. It can avoid interference from other analytes and is conducive to the rapid detection of mercury ions in the environment. It has strong practical application value in the field of environmental science.

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

[0029] 5. The fluorescent probe of the present invention can detect color changes via a smartphone, thereby enabling the identification of different concentrations of the analyte. Attached Figure Description

[0030] Figure 1 This is the proton NMR spectrum of the fluorescent probe TM-ND-Hg of this invention.

[0031] Figure 2 This is the carbon NMR spectrum of the fluorescent probe TM-ND-Hg of this invention.

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

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

[0034] Figure 5 The fluorescent probe TM-ND-Hg of this invention is for Hg 2+ Fluorescence emission spectrum of the response.

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

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

[0037] Figure 8 The fluorescent probe TM-ND-Hg of this invention is for Hg 2+ Fluorescence emission spectrum with varying concentration.

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

[0039] Figure 10 The fluorescent probe TM-ND-Hg of this invention is for Hg 2+ Time-response UV absorption spectrum.

[0040] Figure 11 The fluorescent probe TM-ND-Hg of this invention is for Hg 2+ Fluorescence emission spectrum of time response.

[0041] Figure 12 This is a colorimetric diagram of the fluorescent probe TM-ND-Hg of this invention against different analytes.

[0042] Figure 13 This is a bar chart showing the fluorescence specificity of the fluorescent probe TM-ND-Hg of this invention for different analytes.

[0043] Figure 14 This is a simulation of smartphone recognition of the solution detected by the fluorescent probe TM-ND-Hg of this invention at different concentrations.

[0044] Figure 15 This invention relates to the cytotoxicity of the fluorescent probe TM-ND-Hg.

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

[0046] Figure 17 This is a confocal imaging of zebrafish using the fluorescent probe TM-ND-Hg of this invention. Detailed Implementation

[0047] The present invention will be further described in detail below through specific embodiments. The following embodiments are merely descriptive and not limiting, and should not be used to limit the scope of protection of the present invention.

[0048] A ratiometric fluorescent probe for the instantaneous detection of mercury ions has the structure shown in Formula I:

[0049]

[0050] A method for preparing a ratiometric fluorescent probe for the instantaneous detection of mercury ions includes the following steps:

[0051]

[0052] S1. Dissolve 2-(2-methyl-4H-thiobenzopyran-4-yl)malononitrile and 6-hydroxy-2-naphthaldehyde in toluene, add glacial acetic acid and piperidine, heat under reflux for 12 h, after the reaction is complete, evaporate the reaction solution to dryness, and the crude product is purified by silica gel column chromatography to obtain compound 1.

[0053] S2. Compound 1 and hexamethylamine were added to trifluoroacetic acid solution, and the mixture was heated to reflux until the reaction was complete. 1 mol / L hydrochloric acid solution was added and stirred. The mixture was adjusted to near neutral with NaHCO3 solution, extracted with dichloromethane, washed with distilled water, dried over anhydrous magnesium sulfate, filtered, and concentrated under reduced pressure to obtain compound 2.

[0054] S3. Compound 2 and 1,3-propanedithiol were dissolved in anhydrous dichloromethane and stirred in an ice-water bath for 2 hours. Then, a dichloromethane solution of boron trifluoride diethyl ether was added to the reaction solution and reacted at room temperature for 6 hours. After the reaction was completed, the solution was concentrated under reduced pressure and purified 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 the instantaneous detection of mercury ions includes the following steps:

[0057] S1. Add 500 mg of 2-(2-methyl-4H-thiobenzopyran-4-yl)malononitrile and 422 mg of 6-hydroxy-2-naphthaldehyde to the reaction flask. 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. Detect the reaction by TLC. The volume ratio of the developing solvent is petroleum ether:ethyl acetate = 1:1. After the reactants have reacted completely, lower the temperature to room temperature and evaporate the reaction solution to dryness. Purify the crude product by silica gel column chromatography (petroleum ether:ethyl acetate = 2:1) to obtain compound 1, a red solid (375 mg, 44%).

[0058] 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] - calcd 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 is complete, add 1 mol / L hydrochloric acid solution and stir for one hour. Adjust to near 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 evaporate the organic phase to dryness to obtain compound 2.

[0061] S3. Add 150 mg of compound 2 and 0.6 mL of 1,3-propanedithiol to the 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 the reaction is complete, slowly add 74 μL of boron trifluoride diethyl ether solution dissolved in 2 mL of anhydrous dichloromethane to the reaction flask and stir at room temperature for 6 h. Detect the reaction by TLC, with the developing solvent volume ratio of dichloromethane:methanol = 100:1. After the reactants have reacted completely, evaporate the reaction solution to dryness. Purify the crude product by silica gel column chromatography (dichloromethane:methanol = 100:1) to obtain the fluorescent probe of Formula I, a red solid (87 mg, 48%).

[0062] 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] + calcd for C 28 H 21 N2OS3:497.3311,found:497.3363.

[0064] Example 2

[0065] A detection method using a ratiometric fluorescent probe for the instantaneous detection of mercury ions, comprising:

[0066] The spectral testing conditions in this embodiment were as follows: The fluorescent probe TM-ND-Hg was dissolved in 10 mL of analytical grade dimethyl sulfoxide (DMSO) to prepare a 1 mM solution. For each test, 20 μL of the sample solution was diluted in 2 mL of a DMSO:PBS buffer solution at a ratio of 7:3 (0 M, pH 7.4) before optical testing. All solutions used in other tests were prepared with deionized water (concentration 1 mM). The UV absorption and fluorescence emission spectra were recorded at room temperature after the reaction was complete.

[0067] To investigate the effect of the fluorescent probe TM-ND-Hg on Hg 2+ To investigate the optical properties, 20 μL of the fluorescent probe TM-ND-Hg solution was diluted in 2 mL of a dimethyl sulfoxide:PBS (0.1 M, pH 7.4) buffer solution (concentration 10 μM) to study its performance in the presence or absence of Hg. 2+ The changes in UV-Vis absorption and fluorescence emission spectra under certain conditions. For example... Figure 4 As shown, without the addition of Hg 2+ Previously, the probe solution was dark green with a maximum UV absorption wavelength of 611 nm; after adding Hg... 2+ Subsequently, the probe solution turned orange-yellow, the absorption peak at 611 nm disappeared, and the absorption peak at 436 nm significantly increased. For example... Figure 5 The fluorescence emission spectrum shows that, with an excitation wavelength of 436 nm, without the addition of Hg... 2+ Previously, an emission peak appeared at 516 nm; after adding Hg... 2+ Subsequently, the probe solution showed significant changes: the emission peak at 516 nm disappeared, and a new emission peak appeared at 794 nm. This indicates that the fluorescent probe is responsive to Hg. 2+ It has a clear response. For example... Figure 6 The absorption-emission spectrum of the probe shown indicates that the probe has a large Stokes shift (358 nm).

[0068] To investigate the effect of this fluorescent probe TM-ND-Hg on Hg 2+ The response sensitivity of the probe was assessed using a concentration-dependent test. Figure 7 The ultraviolet absorption spectrum shows that, without the addition of Hg 2+ Previously, an absorption peak appeared at 611 nm, which was related to Hg. 2+ The solution was added sequentially to the test solution at concentration gradients of 0.2, 0.4, 0.6, 0.8, 1, 2, 3...6, 7 eq, with Hg... 2+ With increasing concentration, the absorption peak at 611 nm gradually decreased until it disappeared, while the absorption peak at 436 nm increased significantly, reaching saturation at 7 eq. Figure 8 As shown in the fluorescence emission spectrum, with Hg 2+The concentration of Hg was increased, with an excitation wavelength of 436 nm, without the addition of Hg. 2+ Previously, an emission peak appeared at 516nm, and with Hg 2+ As the concentration of the fluorescent probe increases, the emission peak at 516 nm gradually decreases until it disappears, and a new emission peak appears at 794 nm, showing a regular upward trend. The fluorescence intensity also increases accordingly, reaching saturation when added to 7 eq. This figure shows that the fluorescent probe is a ratiometric probe.

[0069] When Hg 2+ When the concentration of mercury ions is below 60 μM, the fluorescence intensity of the TM-ND-Hg solution tends to saturate. Therefore, the optimal concentration range for detecting mercury ions by this probe is 0-60 μM. A linear relationship between the fluorescence signal enhancement factor and the mercury ion concentration within this range was then plotted. Figure 9 ), R 2 =0.99009. Based on the formula LOD = 3σ / k, the detection limit is 0.39 μM.

[0070] For the detection of Hg using the fluorescent probe TM-ND-Hg 2+ The response time study involved adding 2 eq of Hg to the test solution. 2+ Solution. For example... Figure 10 As shown, with a fixed time interval of 30 seconds, the absorption peak at 611 nm rapidly decreases, followed by an increase in the absorption peak at 563 nm, reaching saturation at 1 minute. Figure 11 As shown, with a fixed time interval of 30 seconds, the fluorescence intensity decreases rapidly at 516 nm and increases rapidly at 794 nm. After 1 minute, the fluorescence intensity stops increasing, indicating that the fluorescence emission peak has reached saturation. Therefore, this probe is effective for Hg. 2+ The response time is 1 minute, which indicates that the probe has a rapid response time.

[0071] To test the fluorescent probe TM-ND-Hg for Hg 2+ Whether it has specificity depends on the addition of 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 a distraction for the probe. For example... Figure 12 As shown, when other ions are added to the probe solution, the solution color remains dark green and does not change. However, when Hg is added... 2+ The solution color turns orange-yellow. For example... Figure 13 As shown, when other ions are added to the probe solution, the fluorescence intensity of the probe does not change significantly, but when Hg is added... 2+ The fluorescence intensity of the probe solution showed a significant increase. Therefore, it can be concluded that this fluorescent probe is effective against Hg. 2+ It has relatively good specificity.

[0072] Example 3

[0073] A smartphone testing experiment includes:

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

[0075] like Figure 14 As shown in the smartphone detection simulation diagram, without the addition of Hg 2+ Previously, the test solution was dark green, and Hg was detected. 2+ The solution was added sequentially to the test solution at concentration gradients of 1, 2, 3...6, 7 eq, with Hg... 2+ As the concentration of the active ingredient increases, the detection solution changes from dark green to brown. By using color recognition software on a smartphone to detect the RGB values ​​of the detection solution, the linear relationship between the R and B values ​​and different concentrations can be obtained. The linear relationship between concentration and the 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 = 0.99125), 2 =0.99476).

[0076] Example 4

[0077] A detection method using a ratiometric fluorescent probe for the instantaneous detection of mercury ions, comprising:

[0078] Cellular confocal imaging: Cytotoxicity assays were performed on A549 cells using the fluorescent probe TM-ND-Hg, such as... Figure 15As shown, when the probe concentration is 10 μM, the cell viability can still reach over 80%. This indicates that the probe itself has low cytotoxicity and is suitable for cell imaging studies. To perform confocal cell imaging experiments using this fluorescent probe, A549 cells were pre-seeded into two 3.5 mm cell culture dishes and cultured overnight. The cells in both dishes were pretreated with the fluorescent probe TM-ND-Hg for 30 min. After 30 min, the probe solution in the culture dishes was washed three times with PBS, one of which was treated with different concentrations of Hg. 2+ The cells were treated with (0.5 eq, 1 eq, 2 eq) solutions for another 30 min, followed by washing the culture dishes three times with PBS. Fluorescence images were obtained by scanning both boxes of cells using a laser confocal microscope. Figure 16 As shown, under excitation at a wavelength of 470 nm, culture dishes containing only the probe solution showed no fluorescence in the mixed field, the 525 nm fluorescence field (Red), and the bright field, but exhibited clear blue fluorescence in the 470 nm fluorescence field (Blue); while with Hg... 2+ As the concentration increases, the blue fluorescence at 470 nm (Blue) gradually weakens, and no obvious fluorescence is observed at 2 eq, while the red fluorescence at 525 nm (Red) gradually strengthens. Therefore, this probe can be used to detect intracellular Hg. 2+ Ratio-dependent fluorescent probes.

[0079] Zebrafish confocal imaging:

[0080] Newborn zebrafish cultured for two days were divided into two groups for pretreatment. The first group was cultured for 30 minutes with only the fluorescent probe TM-ND-Hg. After culture, the zebrafish were washed three times with deionized water and then subjected to laser confocal imaging experiments with the addition of an anesthetic. Figure 17 As shown, no fluorescence was generated in the mixed field, the fluorescence field, and the bright field at an excitation wavelength of 470 nm. The second group was incubated with the fluorescent probe TM-ND-Hg for 30 min, washed three times with deionized water after incubation, and then 2 eq Hg was added. 2+ The solution was incubated for 30 min, followed by washing three times with deionized water. Anesthesia was then added for laser confocal imaging experiments. Red fluorescence was observed in both the mixed field and the 525 nm fluorescence field within the zebrafish. Therefore, this fluorescent probe exhibits good tissue penetration and can be used to detect Hg in organisms. 2+ .

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

Claims

1. A ratiometric fluorescent probe for instant detection of mercury ions, characterized in that: having the structure of Formula I: 。 2. The method for preparing a ratiometric fluorescent probe for instant detection of mercury ions according to claim 1, characterized in that: comprising the following steps: ; S1 dissolving 2- (2-methyl-4H-thiobenzopyran-4-ylidene) malonitrile and 6-hydroxy-2-naphthaldehyde in toluene, adding acetic acid and piperidine thereto, heating to reflux for 10-14h, after the reaction is completed, the reaction solution is spin-dried, the crude product is purified by silica gel column to obtain compound 1; S2 adding compound 1 and hexamethyliminium to a trifluoroacetic acid solution, heating to reflux until the reaction is completed, adding 1 mol / L hydrochloric acid solution and stirring, adjusting to near neutral with NaHCO3 solution, extracting with dichloromethane, washing the organic phase with distilled water, drying over anhydrous magnesium sulfate, filtering, and concentrating under reduced pressure to obtain compound 2; S3 dissolving compound 2 and 1.3-propanedithiol in anhydrous dichloromethane, stirring in an ice water bath for 1-2h, then adding a dichloromethane solution of boron trifluoride etherate to the reaction solution, reacting at room temperature for 3-5h, after the reaction is completed, concentrating under reduced pressure, and purifying by silica gel column chromatography to obtain the ratiometric fluorescent probe of Formula I; In step S1, the molar ratio of the feedings of compound 2- (2-methyl-4H-thiobenzopyran-4-ylidene) malonitrile, 6-hydroxy-2-naphthaldehyde, acetic acid and piperidine is 0.8-1.2mmol: 0.8-1.2mmol: 4-4.2mmol: 4-4.5mmol; In step S2, the molar ratio of the feedings of compound 1 and hexamethyliminium is 0.8-1.2mmol: 1.5-2mmol; In step S3, the molar ratio of the feedings of compound 2, 1,3-propanedithiol and boron trifluoride etherate is 1-1.5mmol: 1.5-2mmol: 0.2-0.8mmol; In step S1, when purified by silica gel column chromatography, the developing agent used is petroleum ether: ethyl acetate = 1:0-2:1 In step S3, when purified by silica gel column chromatography, the developing agent used is dichloromethane: methanol = 100:1-50:

1.

3. Use of the ratiometric fluorescent probe for instant detection of mercury ions according to claim 1 in the detection of mercury ions for non-therapeutic and diagnostic purposes.

4. A detection method for instant detection of mercury ions, characterized by: Use of the compound according to claim 1 as a ratiometric fluorescent probe to measure mercury ions in an aqueous environment and in a biological cell system.

5. The method of claim 4, wherein the method is a point-of- care test for mercury ions. The detection method for measuring mercury ions in an aqueous environment is to dissolve the ratiometric fluorescent probe of Formula I in dimethyl sulfoxide to obtain a fluorescent probe solution, add the fluorescent probe solution to a buffer solution of dimethyl sulfoxide and PBS in a volume ratio of 7:3, detect the ultraviolet absorption and fluorescence emission wavelength, and compare with other interfering ions; The detection method for measuring mercury ions in a biological cell system is to add the fluorescent probe solution to the culture medium of A549 cells, incubate in a culture box containing CO2, then wash with PBS buffer to remove the probe molecules that have not entered the cells, then replace the culture medium with a mercury ion solution of different concentrations, and measure the fluorescence respectively. The culture medium of A549 cells is 1640 containing 10% fetal bovine serum and 1% penicillin-streptomycin mixture.

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