A method for detecting Hg 2+ On-off fluorescent probe and preparation method and detection application thereof

Abstract

The invention discloses a method for detecting Hg 2+ The on-off fluorescent probe and its preparation method and detection application are characterized in that the chemical structure of the on-off fluorescent probe is shown in the following formula (I). The preparation method comprises the steps of mixing N-ethylcarbazole and ZnCl2, adding dichloromethane and acetyl chloride as reaction solvents under nitrogen atmosphere protection, reacting completely to obtain compound 1, and then adding ammonium acetate and malononitrile to react completely to obtain compound 2; adding compound 2 and p-dihydroxybenzaldehyde to ethanol, and adding piperidine as a reaction catalyst to react completely to obtain compound 3, and adding Dess-Martin reagent to react completely to obtain compound 4; finally, dissolving compound 4 in dichloromethane, and adding ethanedithiol and boron trifluoride ether to react completely to obtain the on-off fluorescent probe. The advantages are that the difficulty of synthesis can be reduced and the selectivity, sensitivity and accuracy are high.

Description

Technical Field

[0001] The present invention relates to an on-off type fluorescent probe, in particular to a fluorescent probe for detecting Hg 2+ On-off fluorescent probe and its preparation method and detection application. Background Art

[0002] Mercury (Hg) is a neurotoxic persistent environmental pollutant and bioaccumulative heavy metal that can lead to a decrease in soil microbial communities, reduced crop yields, and excessive levels of heavy metals in food. Mercury stays in the atmosphere for 0.8-1.7 years, with a permissible limit of 0.002 mg / L. Mercury compounds are very harmful to humans. A small amount of mercury compounds can damage the human central nervous system, causing symptoms such as visual and auditory impairment and movement disorders, and can also affect the human reproductive system. 2+ The aquaculture environmental pollution and aquatic product safety issues caused by Hg 2+ At present, atomic absorption spectrometry, cold vapor atomic fluorescence spectrometry and inductively coupled plasma mass spectrometry are widely used in Hg 2+ However, these techniques require complex sample preparation, time-consuming procedures and expensive instrumentation.

[0003] An on-off fluorescent probe is a chemical sensor that can detect the presence or absence of a target substance by changes in the fluorescent signal. This probe turns on the fluorescent signal when the target substance is present, and turns off when the target substance is absent, hence the name on-off fluorescent probe. Its working principle is based on the interaction between the fluorophore and the quencher. When the target substance binds to the fluorophore, the quencher is removed or shielded, causing the fluorescent signal to turn on; when the target substance is not present, the quencher is close to the fluorophore, causing the fluorescent signal to turn off.

[0004] On-off fluorescent probes have excellent performance in practical applications, especially in the detection of heavy metal ions. They are easy to operate and do not require expensive instruments. They are very suitable for real-time or in-situ detection of heavy metal ions. However, on-off fluorescent probes usually require complex synthesis steps and even involve dozens of intermediates, which increases the difficulty of preparing the probes. In addition, on-off fluorescent probes have poor stability and selectivity and may be interfered with in some complex samples or extreme pH. Summary of the invention

[0005] The technical problem to be solved by the present invention is to provide a method for detecting Hg with reduced synthesis difficulty, good selectivity, high sensitivity and high accuracy. 2+On-off fluorescent probe and its preparation method and detection application.

[0006] The technical solution adopted by the present invention to solve the above technical problems is: a method for detecting Hg 2+ An on-off fluorescent probe, wherein the chemical structure of the on-off fluorescent probe is as shown in the following formula (I):

[0007] (I).

[0008] The present invention also provides a method for detecting Hg 2+ The method for preparing an on-off fluorescent probe comprises the following steps:

[0009] Step 1: N-ethylcarbazole and ZnCl2 are mixed and then, under the protection of nitrogen atmosphere, dichloromethane and acetyl chloride are added as reaction solvents. After the reaction is complete, compound 1 is obtained, and its structural formula is shown in the following formula (II):

[0010] (II);

[0011] Step 2: Compound 1 is dissolved in acetic acid, and then ammonium acetate and malononitrile are added to react completely to obtain a light yellow solid, namely compound 2, whose structural formula is shown in the following formula (III):

[0012] (III);

[0013] Step 3: Compound 2 and p-dihydroxybenzaldehyde are added to ethanol, and piperidine is added as a reaction catalyst. After the reaction is complete, compound 3 is obtained, and its structural formula is shown in the following formula (IV):

[0014] (IV);

[0015] Step 4: Compound 3 is dissolved in dichloromethane, and a Dess-Martin reagent is added to react completely to obtain compound 4, whose structural formula is shown in the following formula (V):

[0016] (V);

[0017] Step 5: Dissolve compound 4 in dichloromethane, add ethanedithiol and boron trifluoride ether, and after complete reaction, obtain a light yellow solid compound for detecting Hg. 2+ An on-off fluorescent probe, whose chemical structure is shown in formula (I),

[0018] (I).

[0019] Further, the specific steps are as follows:

[0020] Step 1: Weigh 5.5-6.5 g of N-ethylcarbazole and 8-9.5 g of ZnCl2 and place them in a three-necked bottle. After evacuating the three-necked bottle to a vacuum degree of 0.2-0.4 MPa, fill the three-necked bottle with nitrogen. Under the protection of a nitrogen atmosphere, add 60-80 mL of dichloromethane and 2-4 mL of acetyl chloride as a reaction solvent into the bottle. After heating and refluxing at 40°C for 6-8 h, the reaction product is poured into an HCl solution to terminate the reaction. The product is extracted three times with dichloromethane, and the organic layer solution is spin-dried at 37-46°C to obtain compound 1, whose structural formula is shown in formula (II);

[0021] Step 2: Weigh 1-1.6 g of compound 1 and dissolve it in 10-15 mL of acetic acid at a temperature of 0-2°C, stir and dissolve at 40°C, after compound 1 is completely dissolved, add 0.65-0.9 g of ammonium acetate and 0.4-1 mL of malononitrile, heat and reflux for 12-24 h. After the reaction is completed, pour the reaction product liquid into pure water to terminate the reaction, then add 200 mL of dichloromethane to extract three times, take the organic layer, add 10-15 g of desiccant and shake for 10 min, then use a mixed solution of petroleum ether and ethyl acetate in a volume ratio of 20:1 as a chromatographic solution, perform silica gel column chromatography, take the yellow part of the solution in the silica gel column, and then spin dry at 32-37°C to obtain a light yellow solid, which is compound 2, and its structural formula is shown in formula (III);

[0022] Step 3: Weigh 160-180 mg of compound 2 and 60-75 mg of p-dihydroxybenzaldehyde into 10 mL of ethanol, dropwise add 50-80 μL of piperidine as a reaction catalyst, heat and reflux for 8-10 h. After the reaction is completed, cool the reaction product to room temperature and filter to remove the solvent to obtain compound 3, whose structural formula is shown in formula (IV);

[0023] Step 4: Weigh 300-320 mg of compound 3 and dissolve it in 10 mL of dichloromethane, add 300-320 mg of Dess-Martin reagent, stir and react at room temperature for 2-6 h, then add saturated sodium thiosulfate solution to terminate the reaction, and extract three times with 200 mL of dichloromethane, take the organic layer and add 20-40 mL of a mixed solution of saturated sodium bicarbonate and saturated sodium chloride, shake, wash and filter, add 10-15 g of desiccant to the filtered solution and shake for 10 min, then spin dry at 37-45°C to obtain compound 4, whose structural formula is shown in formula (V);

[0024] Step 5: Weigh 500-545 mg of compound 4 and dissolve it in 10 mL of dichloromethane, add 150 μL of ethanedithiol, react in an ice bath at -2 to 0°C for 30 to 45 min, then add 150 μL of boron trifluoride ether, react in an ice bath for 10 min, transfer to room temperature, and continue to react for 12 to 16 h. After the reaction is completed, add saturated sodium bicarbonate solution to the reaction product to terminate the reaction, then add 200 mL of dichloromethane to extract three times, take the organic layer, add 10-15 g of desiccant and shake for 10 min, then use a mixture of petroleum ether and ethyl acetate in a volume ratio of 3:1 as a chromatographic liquid, perform silica gel column chromatography, take the yellow solution in the silica gel column and spin dry at 32 to 42°C to obtain a light yellow solid compound, which is used to detect Hg 2+ An on-off fluorescent probe, whose chemical structure is shown in formula (I).

[0025] Furthermore, the desiccant is any one of calcium sulfate, sodium sulfate and magnesium sulfate.

[0026] The present invention also provides a method for detecting Hg using the on-off fluorescent probe. 2+ The method comprises the following steps: dissolving an on-off fluorescent probe in a solvent system composed of methanol and HEPES buffer in a volume ratio of (4-6): (6-4) to obtain an on-off fluorescent probe solution with a concentration of 10 μmol / L; adjusting the pH of the on-off fluorescent probe solution to 5.5-6.5; adding the on-off fluorescent probe solution and a test solution in a volume ratio of 9:1 into a cuvette, reacting for 30 seconds; setting the excitation wavelength of the fluorescence spectrophotometer to 390 nm; measuring the fluorescence intensity of the cuvette at an emission wavelength of 590 nm; and comparing the fluorescence intensity value with the Hg 2+ The linear relationship between the concentrations is calculated to obtain the Hg content in the test solution. 2+ concentration.

[0027] Preferably, the volume ratio of methanol to HEPES buffer in the solvent system is 5:5.

[0028] Compared with the prior art, the advantages of the present invention are: 2+ On-off fluorescent probe and its preparation method and detection application, Hg 2+ It has thiophilicity and can coordinate with groups containing thio substituents (such as thioamide, thiourea, thioketone, etc.) to undergo ring opening and mercury dithizone reaction to form mercury dithizone complex, thereby changing the fluorescence intensity of the probe to detect Hg 2+The purpose of this study is to use the fluorescence characteristics of 2-[1-(9-ethylcarbazole-3-yl)ethylidene]malononitrile and the recognition effect of 4-(1,3-dithiolane)benzaldehyde to construct an "on-off" fluorescent probe, which can not only reduce the difficulty of synthesis and the generation of by-products, but also has excellent stability and selectivity, and can be used for low-concentration Hg in actual water samples. 2+ Identification detection. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 is a synthetic route diagram of probe I in specific embodiment 1;

[0030] Figure 2 is a high-resolution mass spectrum of probe I in specific embodiment 1;

[0031] Figure 3 The effect of the ratio of methanol to HEPES buffer solution in the solvent system in the specific embodiment 3 on the fluorescence intensity of probe I, wherein a is a volume ratio of methanol to HEPES buffer solution of 6:4, b is a volume ratio of methanol to HEPES buffer solution of 5:5, and c is a volume ratio of methanol to HEPES buffer solution of 4:6;

[0032] Figure 4 The detection effect of probe I on mercury ions at different pH values ​​in specific embodiment 3;

[0033] Figure 5 : is the result of the selectivity determination of probe I in specific embodiment 4, wherein a is the ultraviolet absorption intensity value of probe I to various ions, b is the fluorescence intensity value of probe I to various ions, and c is the color change diagram of the probe I solution under 365nm ultraviolet light after adding various ions;

[0034] Figure 6 The probe I in the specific embodiment 5 is Hg 2+ The sensitivity test results of probe I with Hg 2+ The fluorescence intensity value of probe I changes with the concentration of Hg 2+ Linear curve of concentration change;

[0035] Figure 7 The probe I in the specific embodiment 6 and the addition of Hg 2+ Imaging diagram of probe I in cells;

[0036] Figure 8 The results of the cell toxicity test of probe I in specific embodiment 6, wherein a is the relationship between different concentrations of probe I and cell half inhibition rate, and b is the LC of probe I on cells. 50 Half lethal concentration curve. DETAILED DESCRIPTION

[0037] The present invention is further described in detail below with reference to the accompanying drawings.

[0038] The thin layer chromatography silica gel plate used for mass spectrometry (MS) determination uses Yantai Huanghai HSGF254 or Qingdao GF254 silica gel plate. The silica gel plate used in thin layer chromatography adopts a specification of 0.20 mm-0.25 mm. Column chromatography generally uses Yantai Huanghai 200-300 mesh silica gel as a carrier.

[0039] Specific embodiment 1: a method for detecting Hg 2+ The on-off fluorescent probe (hereinafter referred to as probe I) has a chemical structure as shown in the following formula (I):

[0040] (I).

[0041] The preparation method of the on-off fluorescent probe is as follows Figure 1 As shown, the following steps are included:

[0042] Step 1: Weigh 5.5-6.5 g of N-ethylcarbazole and 8-9.5 g of ZnCl2 and place them in a 250 mL three-necked bottle. Use a vacuum pump to evacuate for 5 minutes until the vacuum degree in the three-necked bottle reaches 0.2-0.4 MPa. Then, fill the three-necked bottle with nitrogen for 5 minutes. Under the protection of nitrogen atmosphere, add 60-80 mL of dichloromethane and 2-4 mL of acetyl chloride as reaction solvents into the bottle. Heat and reflux at 40° C. for 6-8 h. Use thin layer chromatography to track the reaction progress. When the silica gel plate shows only one band under 365 nm ultraviolet light, the reaction is completed. Pour the reaction product into 10 mL of 0.1 mol / L HCl solution to terminate the reaction. Use 200 mL of dichloromethane to extract three times. Take the organic layer solution and spin dry it at 37-46° C. to obtain compound 1 with a yield of 55-65%. Its chemical structure is shown in the following formula (II):

[0043] (II);

[0044] Step 2: Weigh 1-1.6 g of compound 1 and dissolve it in 10-15 mL of acetic acid at a temperature of 0-2°C, stir and dissolve at 40°C, add 0.65-0.9 g of ammonium acetate and 0.4-1 mL of malononitrile after compound 1 is completely dissolved, heat and reflux for 12-24 h, and use thin layer chromatography to track the progress of the reaction. When the silica gel plate shows only one band under 365 nm ultraviolet light, the reaction is completed. Pour the reaction product liquid into pure water to terminate the reaction, extract it three times with 200 mL of dichloromethane, take the organic layer solution, add 10-15 g of desiccant (the desiccant is any one of calcium sulfate, sodium sulfate and magnesium sulfate), and shake for 10 min, remove water from the organic layer solution, and then use a mixture of petroleum ether and ethyl acetate in a volume ratio of 20:1 as a chromatographic solution for silica gel column chromatography, take the yellow part of the silica gel column solution, and then spin dry at 32-37°C to obtain a light yellow solid, which is compound 2, with a yield of 51-67%. Its structural formula is shown in the following formula (III):

[0045] (III);

[0046] Step 3: Weigh 160-180 mg of compound 2 and 60-75 mg of p-dihydroxybenzaldehyde into 10 mL of ethanol, drop 50-80 μL of piperidine as a reaction catalyst, heat and reflux for 8-10 h, and track the reaction progress by thin layer chromatography. When the silica gel plate shows only one band under 365 nm ultraviolet light, the reaction is completed. After the reaction, the reaction product is cooled to room temperature and filtered to remove the solvent to obtain compound 3 with a yield of 45-56%. Its structural formula is shown in the following formula (IV):

[0047] (IV);

[0048] Step 4: Weigh 300-320 mg of compound 3 and dissolve it in 10 mL of dichloromethane, add 300-320 mg of Dess-Martin reagent, stir the reaction at room temperature for 2-6 h, then add saturated sodium thiosulfate solution to terminate the reaction, and extract three times with 200 mL of dichloromethane, take the organic layer and add 20-40 mL of a mixed solution of saturated sodium bicarbonate and saturated sodium chloride, shake, wash and filter, add 10-15 g of desiccant (the desiccant is any one of calcium sulfate, sodium sulfate and magnesium sulfate) to the filtered solution, shake for 10 min, filter and dry to remove water in the organic layer, and then spin dry at 37-45°C to obtain compound 4 with a yield of 48-65%, whose structural formula is shown in the following formula (V),

[0049] (V);

[0050] Step 5: Weigh 500-545 mg of compound 4 and dissolve it in 10 mL of dichloromethane, add 150 μL of ethanedithiol, react in an ice bath at -2 to 0°C for 30 to 45 min, then add 150 μL of boron trifluoride ether, react in an ice bath for 10 min, then transfer to room temperature, continue the reaction for 12 to 16 h, track the progress of the reaction by thin layer chromatography, and the reaction is completed when the silica gel plate shows only one band under 365 nm ultraviolet light, then add saturated sodium bicarbonate solution to the reaction product to terminate the reaction, add 200 mL of dichloromethane to extract three times, take the organic layer, add 10 to 15 g of desiccant (the desiccant is any one of calcium sulfate, sodium sulfate and magnesium sulfate), shake for 10 min, filter and dry, use a mixture of petroleum ether and ethyl acetate in a volume ratio of 3:1 as a chromatographic solution, perform silica gel column chromatography, take the yellow solution in the silica gel column and spin dry at 32 to 42°C to obtain a light yellow solid compound, which is used to detect Hg 2+ The on-off fluorescent probe, abbreviated as probe I, has a yield of 44-57%. Its structure was verified by high-resolution mass spectrometry. Figure 2 As shown, high resolution mass spectrometry gives the standard structure HR-MS (m / z): calcd. forC 29 H 24 N3S2, 478.14, indicating that probe I was successfully prepared, and its chemical structure is shown in the above formula (I).

[0051] Specific Example 2: Detection of Hg using the on-off fluorescent probe prepared by the method of Specific Example 1 2+ The method comprises the following steps:

[0052] Step 1, instrument parameter setting: The detection instrument uses a fluorescence spectrophotometer model F-4600, set the excitation wavelength to 390 nm, the emission wavelength to 590 nm, the photomultiplier voltage to 650 V, the slit width to 10 nm, and a quartz cuvette with four sides transparent to light, with a specification of length × width × height = 1 cm × 1 cm × 3 cm;

[0053] Step 2: Hg 2+ Concentration determination: Probe I was dissolved in a solvent system composed of methanol and HEPES buffer in a volume ratio of 5:5 to obtain a probe I solution with a concentration of 10 μmol / L. The pH of the probe I solution was adjusted to 5.5-6.5 with hydrochloric acid, and 900 μL of the probe I solution was mixed with 100 μL of the test solution and added to a cuvette. The reaction was continued for 30 seconds, and the fluorescence intensity of the cuvette at 590 nm was measured. The fluorescence intensity value was compared with the Hg 2+ The linear relationship between the concentrations is calculated to obtain the Hg content in the test solution. 2+ concentration.

[0054] Specific embodiment 3: Using the method of specific embodiment 2 to carry out Hg 2+ Optimization of detection conditions.

[0055] 1. Screening of solvent system: The volume ratio of methanol and HEPES buffer in the solvent system of step 2 of specific embodiment 2 was set to 4:6, 5:5, and 6:4, respectively, and then probe I was dissolved in the solvent system of different ratios to make the final concentration of probe I 10 μmol / L, and then the fluorescence intensity of probe I solution at 590 nm was measured by fluorescence spectrophotometer, and 990 μL of probe I solution was added to 10 μL of Hg solution with a concentration of 100 μmol / L. 2+ The standard solution was shaken for 5 min, and the fluorescence intensity at 590 nm was measured again. 2+ The degree of fluorescence quenching was then used to screen the optimal solvent system composition ratio.

[0056] The results are as follows Figure 3 As shown in FIG. 1 , as the amount of HEPES buffer in the solvent system increases, the fluorescence intensity of probe I gradually decreases. Figure 3 As shown in a and b, when the volume ratio of methanol to HEPES buffer is 6:4 and 5:5, the fluorescence intensity of probe I is higher, which is suitable for Hg 2+ Determination of Figure 3 As shown in Figure c, when the volume ratio of methanol to HEPES buffer is 4:6, the fluorescence intensity of probe I is almost reduced to the minimum. This is because the presence of too much HEPES will inhibit the reaction between probe I and Hg 2+ In addition, at the same Hg 2+ At the concentration of 5:5, when the volume ratio of methanol to HEPES buffer was 5:5, the probe I 2+ The fluorescence response of the sample was the best and the fluorescence quenching degree was the largest. Therefore, a solvent system with a volume ratio of methanol to HEPES buffer of 5:5 was selected as the detection solvent.

[0057] 2. pH optimization setting: Probe I was dissolved in a solvent system composed of methanol and HEPES buffer in a volume ratio of 5:5 to obtain a probe I solution with a concentration of 10 μmol / L. The pH values ​​of the probe I solution were then set to 3, 4, 5, 5.5, 6, 6.5, 7, 8, 9, 10, and 11, respectively. The fluorescence intensity of probe I at 590 nm was then measured using a fluorescence spectrophotometer. Subsequently, 990 μL of probe I solution was mixed with 10 μL of Hg-containing 2+The aqueous solution with a concentration of 100 μmol / L was mixed and reacted for 5 min. The fluorescence intensity of probe I at 590 nm was measured again using a fluorescence spectrophotometer, and the fluorescence intensity value at 590 nm was taken to draw a dot-line graph. The results are shown in Figure 4 As shown in Figure 2, the fluorescence intensity of probe I is relatively stable within the pH range, but probe I binds to Hg 2+ The fluorescence intensity of probe I decreased significantly in the pH range of 5.0-6.5, while under other pH conditions, the fluorescence intensity of probe I was affected by Hg 2+ The effect is not significant, indicating that when the pH is in the range of 5.0-6.5, probe I has no significant effect on Hg 2+ The strongest response.

[0058] Specific Example 4: The selectivity of probe I was determined using the method of Specific Example 2.

[0059] Experimental method: Weigh 0.1 mmol of ferric chloride tetrahydrate (FeCl2·4H2O), ferric chloride hexahydrate (FeCl3·6H2O), magnesium chloride hexahydrate (MgCl2·6H2O), potassium chloride (KCl), chromium chloride hexahydrate (CrCl3·6H2O), cadmium chloride (CdCl2), zinc chloride (ZnCl2), calcium chloride (CaCl2), aluminum chloride hexahydrate (AlCl3·6H2O), sodium chloride (NaCl), mercuric sulfate (HgSO4), cobalt sulfate heptahydrate (CoSO4·7H2O), nickel sulfate hexahydrate (NiSO4·6H2O), copper sulfate pentahydrate (CuSO4·5H2O), manganese sulfate monohydrate (MnSO4·H2O), ammonium chloride (NH4Cl), barium oxalate [(CH3COO)2Ba], silver nitrate (AgNO3) and mercuric sulfate (HgSO4) were dissolved in deionized water and mixed to obtain ion solutions with a concentration of 100 μmol / L.

[0060] The ultraviolet absorption intensity at 380 nm and the fluorescence intensity at 590 nm of the probe I solution with a pH of 6 and a concentration of 10 μmol / L were measured using a fluorescence spectrophotometer. Then, 10 μL of the pre-prepared ion solutions were added to 990 μL of the probe I solution. The reaction lasted for 5 min. The ultraviolet absorption intensity at 380 nm and the fluorescence intensity at 590 nm of the probe I solution with different ions added were measured respectively. At the same time, pictures of the probe I solutions with different ions added were taken under a 365 nm ultraviolet lamp. The results are shown in the figure. Figure 5 shown.

[0061] like Figure 5 As shown in a and b, after adding other ions to the probe I solution, the UV absorption peak of probe I at 380 nm and the fluorescence intensity at 590 nm did not change significantly, while adding Hg2+ Afterwards, the UV absorption peak of probe I blue-shifted from 380 nm to 350 nm; the fluorescence intensity at 590 nm also decreased significantly. Figure 5 As shown in Figure c, the color of the probe I solution under 365 nm ultraviolet light changes from orange to colorless. The reason for the decrease in fluorescence intensity may be that after adding Hg 2+ Afterwards, the sulfur-containing groups in the probe react with Hg 2+ The metal chelation reaction was generated, and the dithioacetal was transformed into an aldehyde group, which inhibited the normal flow of electrons inside probe I, resulting in a significant decrease in the fluorescence intensity of probe I and a slight blue shift, indicating that probe I was effective for Hg 2+ There is good selectivity.

[0062] Specific Example 5: Using the method of Specific Example 2 to carry out Hg 2+ Sensitivity determination.

[0063] Hg 2+ Sensitivity determination method: prepare Hg at concentrations of 0, 1 μmol / L, 5 μmol / L, 10 μmol / L, 15 μmol / L, 30 μmol / L, 45 μmol / L, 50 μmol / L, and 100 μmol / L. 2+ 10 μL of Hg standard solution with different concentrations were added 2+ The standard solution was added to 990 μL of probe I solution with pH = 6 and a concentration of 10 μmol / L, and the reaction was continued for 5 min. 2+ Fluorescence intensity value of probe I solution at 590 nm.

[0064] result Figure 6 As shown in a, with the increase of Hg 2+ As the concentration of Hg increases, the fluorescence intensity of probe I at 590 nm gradually decreases. 2+ When the concentration is 100 mmol / L, the fluorescence intensity of probe I tends to be stable. Figure 6 As shown in b, the fluorescence intensity value is related to Hg 2+ The concentration of is linearly related to each other, and the linear equation is: y=1746.46-24.48x (R 2 =0.9970), the detection range was 10~55umol / L, and the detection limit was 9.15 nmol / L.

[0065] Specific Example 6: The cell imaging and toxicity experiments of probe I were carried out using the method of Specific Example 2.

[0066] Cell imaging experiment: 20 μL of probe I solution with pH=6 and concentration of 10 μmol / L was mixed with 100 μL of 5×10 4 cells / mL of Hela cell solution was added to each well of a 96-well culture plate and incubated in a 37°C, 5% CO2 incubator for 24 hours. Cell fluorescence images were collected at an excitation wavelength of 380 nm, and the cell status was observed under a laser confocal microscope and the fluorescence intensity was recorded. Subsequently, 20 μL of 10 μmol / L Hg 2+ The solution was incubated for 1 hour, and the state of the cells was observed under a laser confocal microscope and the fluorescence intensity was recorded. Figure 7 As shown in the figure, the cell structure is intact under bright field. Under the excitation wavelength of 380 nm, the cell emits red fluorescence, proving that probe I can penetrate the cell membrane and enter the cell. 2+ After 1 h of incubation, the red fluorescence of the cells weakened, indicating that probe I can be used to detect Hg in cells. 2+ Detection.

[0067] Cytotoxicity experiment: 20 μL of probe I solution with a concentration of 2.5 μmol / L, 5 μmol / L, 10 μmol / L, 25 μmol / L, 50 μmol / L and 100 μmol / L (pH 6) were mixed with 100 μL of 5×10 4 cells / mL Hela cell solution was cultured, and after incubation for 24 h, the cell fluorescence images were collected under an excitation wavelength of 380 nm, the survival status of the cells was observed and recorded, and the half inhibition rate was calculated using the prism drawing software. Figure 8 As shown in Figure a, as the concentration of probe I increases, the cell survival rate gradually decreases. Figure 8 As shown in b, the LC of probe I was calculated by logarithmic inversion. 50 The half-lethal concentration of probe I was 29.34 μmol / L. When the concentration of probe I was 10 μmol / L, the inhibition rate of probe I on cells was less than 20%, and the cytotoxicity was relatively small. 2+ It is feasible to select the concentration of probe I as 10 μmol / L in the process of detection. At the same time, this concentration has low cytotoxicity and can be used for intracellular Hg 2+ Detection and imaging.

[0068] Specific Example 7: Using the method of Specific Example 2 to measure Hg in actual water samples 2+ Carry out determination.

[0069] Deionized water, tap water and reservoir water were selected as samples to explore the Hg content of probe I in actual water samples. 2+ The feasibility of the test,results are shown in Table 1.

[0070] Table 1 Probe I for Hg in water samples 2+ The spike recovery

[0071]

[0072] As shown in Table 1, the probe I has a 2+ The recovery rates of pure water were between 85.00% and 122.90%, tap water was between 78.60% and 102.90%, and reservoir water was between 95.25% and 118.40%, all within the appropriate range, indicating that the detection method has high accuracy and probe I has the ability to detect Hg in actual samples. 2+ potential application value.

[0073] The above description is not intended to limit the present invention, and the present invention is not limited to the above examples. Any changes, modifications, additions or substitutions made by a person skilled in the art within the spirit and scope of the present invention shall also fall within the protection scope of the present invention.

Claims

1. A method for detecting Hg 2+ An on-off fluorescent probe characterized by The chemical structure of the on-off fluorescent probe is shown in the following formula (I): （I）。 2. A method for detecting Hg according to claim 1 2+ A method for preparing an on-off fluorescent probe, characterized in that The following steps are involved: Step 1: N-ethylcarbazole and ZnCl2 are mixed and then, under the protection of nitrogen atmosphere, dichloromethane and acetyl chloride are added as reaction solvents. After the reaction is complete, compound 1 is obtained, and its structural formula is shown in the following formula (II): （Ⅱ）； Step 2: Compound 1 is dissolved in acetic acid, and then ammonium acetate and malononitrile are added to react completely to obtain a light yellow solid, namely compound 2, whose structural formula is shown in the following formula (III): （Ⅲ）； Step 3: Compound 2 and p-hydroxymethylbenzaldehyde are added to ethanol, and piperidine is added as a reaction catalyst. After the reaction is complete, compound 3 is obtained, and its structural formula is shown in the following formula (IV): （Ⅳ）； Step 4: Compound 3 is dissolved in dichloromethane, and a Dess-Martin reagent is added to react completely to obtain compound 4, whose structural formula is shown in the following formula (V): （Ⅴ）； Step 5: Dissolve compound 4 in dichloromethane, add ethanedithiol and boron trifluoride ether, and after complete reaction, obtain a light yellow solid compound for detecting Hg. 2+ An on-off fluorescent probe, whose chemical structure is shown in formula (I), （I）。 3. A method for detecting Hg according to claim 2. 2+ A method for preparing an on-off fluorescent probe, characterized in that The specific steps are as follows: Step 1: Weigh 5.5-6.5 g of N-ethylcarbazole and 8-9.5 g of ZnCl2 and place them in a three-necked bottle. After evacuating the three-necked bottle to a vacuum degree of 0.2-0.4 MPa, fill the three-necked bottle with nitrogen. Under the protection of a nitrogen atmosphere, add 60-80 mL of dichloromethane and 2-4 mL of acetyl chloride as a reaction solvent into the bottle. After heating and refluxing at 40°C for 6-8 h, the reaction product is poured into an HCl solution to terminate the reaction. The product is extracted three times with dichloromethane, and the organic layer solution is spin-dried at 37-46°C to obtain compound 1, whose structural formula is shown in formula (II); Step 2: Weigh 1-1.6 g of compound 1 and dissolve it in 10-15 mL of acetic acid at a temperature of 0-2°C, stir and dissolve at 40°C, after compound 1 is completely dissolved, add 0.65-0.9 g of ammonium acetate and 0.4-1 mL of malononitrile, heat and reflux for 12-24 h. After the reaction is completed, pour the reaction product liquid into pure water to terminate the reaction, then add 200 mL of dichloromethane to extract three times, take the organic layer, add 10-15 g of desiccant and shake for 10 min, then use a mixed solution of petroleum ether and ethyl acetate in a volume ratio of 20:1 as a chromatographic solution, perform silica gel column chromatography, take the yellow part of the solution in the silica gel column, and then spin dry at 32-37°C to obtain a light yellow solid, which is compound 2, and its structural formula is shown in formula (III); Step 3: Weigh 160-180 mg of compound 2 and 60-75 mg of p-hydroxymethylbenzaldehyde and add them into 10 mL of ethanol, dropwise add 50-80 μL of piperidine as a reaction catalyst, heat and reflux for 8-10 h. After the reaction is completed, cool the reaction product to room temperature and filter to remove the solvent to obtain compound 3, whose structural formula is shown in formula (IV); Step 4: Weigh 300-320 mg of compound 3 and dissolve it in 10 mL of dichloromethane, add 300-320 mg of Dess-Martin reagent, stir and react at room temperature for 2-6 h, then add saturated sodium thiosulfate solution to terminate the reaction, and extract three times with 200 mL of dichloromethane, take the organic layer and add 20-40 mL of a mixed solution of saturated sodium bicarbonate and saturated sodium chloride, shake, wash and filter, add 10-15 g of desiccant to the filtered solution and shake for 10 min, then spin dry at 37-45°C to obtain compound 4, whose structural formula is shown in formula (V); Step 5: Weigh 500-545 mg of compound 4 and dissolve it in 10 mL of dichloromethane, add 150 μL of ethanedithiol, react in an ice bath at -2 to 0°C for 30 to 45 min, then add 150 μL of boron trifluoride ether, react in an ice bath for 10 min, transfer to room temperature, and continue to react for 12 to 16 h. After the reaction is completed, add saturated sodium bicarbonate solution to the reaction product to terminate the reaction, then add 200 mL of dichloromethane to extract three times, take the organic layer, add 10-15 g of desiccant and shake for 10 min, then use a mixed solution of petroleum ether and ethyl acetate in a volume ratio of 3:1 as the chromatographic solution, perform silica gel column chromatography, take the yellow solution in the silica gel column and spin dry at 32 to 42°C to obtain a light yellow solid compound, which is used to detect Hg 2+ An on-off fluorescent probe, whose chemical structure is shown in formula (I).

4. A method for detecting Hg according to claim 3. 2+ A method for preparing an on-off fluorescent probe, characterized in that: The desiccant is any one of calcium sulfate, sodium sulfate and magnesium sulfate.

5. A method for detecting Hg using the on-off fluorescent probe described in claim 1 2+ The method is not intended for disease diagnosis or treatment, and is characterized in that The method comprises the following steps: dissolving an on-off fluorescent probe in a solvent system composed of methanol and HEPES buffer in a volume ratio of (4-6): (6-4) to obtain an on-off fluorescent probe solution with a concentration of 10 μmol / L; adjusting the pH of the on-off fluorescent probe solution to 5.5-6.5; adding the on-off fluorescent probe solution and a test solution in a volume ratio of 9:1 into a cuvette, reacting for 30 seconds; setting the excitation wavelength of the fluorescence spectrophotometer to 390 nm; measuring the fluorescence intensity of the cuvette at an emission wavelength of 590 nm; and comparing the fluorescence intensity value with the Hg 2+ The linear relationship between the concentrations is calculated to obtain the Hg content in the test solution. 2+ concentration.

6. The on-off fluorescent probe according to claim 5 detects Hg 2+ The method is not intended for disease diagnosis or treatment, and is characterized in that The following steps are involved: The volume ratio of methanol to HEPES buffer in the solvent system is 5:5.

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