A mercury fluorescent probe based on carbazole boronic acid and its preparation method and application

Abstract

The present invention belongs to the field of detection and analysis technology, and discloses a mercury fluorescent probe based on carbazole boronic acid and a preparation method thereof. 9H-carbazole-3-boronic acid pinacol ester and compound Y (any one of KOH, K2CO3, Cs2CO3, NaH, triethylamine, DBU, DMAP, and DABCO) are dissolved in a polar aprotic solvent, and then alkylene oxide is added. After heating and stirring for a period of time, material A is obtained; the organic phase in material A is extracted with EA, and then compound B is obtained by column chromatography purification; compound B is dissolved with a solution of acetone / water or THF / water, and then a mixture of NaIO4 and CH3COONH4 or a mixture of NaIO4 and hydrochloric acid is added. After reacting for a period of time, the organic phase is extracted with EA, and then the fluorescent probe is obtained by column chromatography purification. The fluorescent probe provided by the present invention can be used for quantitative detection of mercury ions and methylmercury in water samples, and has good sensitivity and selectivity. The present invention further provides the application of two other compounds as fluorescent probes in mercury detection.

Description

Technical Field

[0001] The invention belongs to the technical field of detection and analysis, and particularly relates to a fluorescent probe for detecting mercury and a preparation method and application thereof. Background Art

[0002] Mercury is a highly toxic and bio-non-essential element naturally present in the environment. Mercury's toxicity depends on its form, which includes metallic mercury, inorganic mercury, and organic mercury. Organic mercury is much more toxic than inorganic mercury. Due to its high lipid solubility, organic mercury easily penetrates cell membranes and crosses the blood-brain barrier. When it accumulates in brain tissue, it can cause severe brain damage. For example, Minamata disease in Japan was caused by mercury contamination in Minamata Bay. Methylmercury, the most well-known form of organic mercury, poses the greatest threat to the human body. Mercury contaminants can be converted to methylmercury by microorganisms. Therefore, the detection of mercury ions and methylmercury is of great significance.

[0003] Traditional fluorescent probes consist of three main components: a fluorescent group, which converts the binding of the receptor to the analyte into an information signal; a recognition group, which binds or reacts with the analyte; and a linker, which connects the fluorescent group and the recognition group. When the analyte binds to the recognition group, a change in the fluorescence signal is observed.

[0004] Existing fluorescent probe molecules have complicated preparation processes and complex structures, and only a few can be applied to the detection of actual water samples. Therefore, providing a simple fluorescent probe for the detection of mercury ions and methylmercury in actual water samples is of great research significance. Summary of the Invention

[0005] In view of the problems existing in the prior art, one of the objectives of the present invention is to provide a fluorescent probe. A second objective of the present invention is to provide a method for preparing the fluorescent probe. A third objective of the present invention is to provide applications of the fluorescent probe.

[0006] To achieve the above objectives, the present invention provides the following specific technical solutions.

[0007] First, the present invention provides a fluorescent probe, the structural formula of which is

[0008] Secondly, the present invention provides a method for preparing the above fluorescent probe, comprising the following steps:

[0009] Step S1, dissolving 9H-carbazole-3-boronic acid pinacol ester and compound Y in a polar aprotic solvent, then adding alkylene oxide, heating and stirring for a period of time to obtain material A; the compound Y is any one of KOH, K2CO3, Cs2CO3, NaH, triethylamine, DBU (1,8-diazabicyclo[5.4.0]undec-7-ene), DMAP (4-dimethylaminopyridine), and DABCO (triethylenediamine);

[0010] Step S2, extracting the organic phase from material A with EA (ethyl acetate), and then purifying by column chromatography to obtain compound B;

[0011] Step S3, dissolving compound B in an acetone / water or THF (tetrahydrofuran) / water solution, then adding a mixture of NaIO4 and CH3COONH4, or a mixture of NaIO4 and hydrochloric acid, reacting for a period of time, extracting the organic phase with EA, and then purifying by column chromatography to obtain the fluorescent probe.

[0012] Furthermore, in some preferred embodiments of the above preparation method, the polar aprotic solvent is selected from any one of DMSO (dimethyl sulfoxide), DMF (dimethylformamide), THF, and acetonitrile.

[0013] Furthermore, in some preferred embodiments of the above preparation method, the molar ratio of 9H-carbazole-3-boronic acid pinacol ester to compound Y is 1:1.25-2.

[0014] Furthermore, in some preferred embodiments of the above preparation method, the amount of polar aprotic solvent added is 5 to 15 times the total mass of the solute.

[0015] Furthermore, in some preferred embodiments of the above preparation method, the molar ratio of 9H-carbazole-3-boronic acid pinacol ester to alkylene oxide is 1:1.25-2.

[0016] Furthermore, in some preferred embodiments of the above preparation method, the alkylene oxide is selected from at least one of ethylene oxide, propylene oxide, methylpropylene oxide, and butylene oxide.

[0017] Furthermore, in some preferred embodiments of the above preparation method, in step S1, the heating temperature is 100-130° C., and the stirring time is 6-8 h.

[0018] Furthermore, in some preferred embodiments of the above preparation method, in step S2, the volume ratio of PE (petroleum ether) to EA in the column chromatography is 3 to 6:1.

[0019] Furthermore, in some preferred embodiments of the above preparation method, in the acetone / water or THF / water solution, the volume ratio of acetone, THF and water is 2 to 4:1, respectively.

[0020] Furthermore, in some preferred embodiments of the above preparation method, in step S3, the amount of NaIO4 added is 2 to 3 times the theoretical amount for reacting with compound B, and the amount of CH3COONH4 or hydrochloric acid added is 2 to 3 times the theoretical amount for reacting with compound B.

[0021] Furthermore, the organic phase obtained by EA extraction is dried before column chromatography purification, and preferably dried over anhydrous MgSO4.

[0022] Based on the same inventive concept, the present invention further provides the use of the above fluorescent probe in mercury detection.

[0023] In addition, the inventors creatively discovered during the research of the above fluorescent probe that other structures containing carbazole boronic acid moieties can also be used as fluorescent probes and for detecting mercury. Therefore, the present invention provides a structural formula of Application of the compound as a fluorescent probe in mercury detection, where R is an arbitrary substituent.

[0024] Furthermore, the present invention provides a structural formula Application of the compounds as fluorescent probes in mercury detection.

[0025] Compared with the prior art, the present invention has the following obvious beneficial technical effects:

[0026] (1) The fluorescent probe provided by the present invention can be used to quantitatively detect mercury ions and methylmercury in water samples with good sensitivity and selectivity.

[0027] (2) The preparation method of the fluorescent probe provided by the present invention has a simple route, strong practicality, and high prospects for industrial application.

[0028] (3) Based on the specific reaction between aromatic (such as phenyl) boronic acid and mercury and the strong fluorescence emission characteristics of the carbazole skeleton, it was confirmed for the first time that carbazole boronic acid can be used as a highly sensitive and selective fluorescent probe for the detection of divalent mercury or methylmercury. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 is the chemical reaction equation for synthesizing fluorescent probes.

[0030] Figure 2 This is the hydrogen nuclear magnetic resonance spectrum of the fluorescent probe prepared in Example 1.

[0031] Figure 3The fluorescence titration diagram of the fluorescent probe prepared in Example 1 and mercury, wherein a is the fluorescence titration diagram of the fluorescent probe and Hg 2+ Fluorescence titration diagram of the fluorescent probe and MeHg + Fluorescence titration graph.

[0032] Figure 4 The response time curve of the fluorescent probe and Hg prepared in Example 1, wherein a is the response time curve of the fluorescent probe and Hg 2+ The response time curve of the fluorescent probe and MeHg + Response time curve.

[0033] Figure 5 This is a diagram showing the selectivity effect of the fluorescent probe prepared in Example 1.

[0034] Figure 6 is the fluorescence titration diagram of the compound described in Example 6 and mercury, wherein a is the fluorescence titration diagram of the compound and Hg 2+ Fluorescence titration diagram of the compound and MeHg + Fluorescence titration graph.

[0035] Figure 7 The response time curve of the compound described in Example 6 and mercury, wherein a is the response time curve of the fluorescent probe and Hg 2+ The response time curve of the fluorescent probe and MeHg + Response time curve.

[0036] Figure 8 This is a diagram showing the selectivity effect of the compound described in Example 6 as a fluorescent probe.

[0037] Figure 9 is the fluorescence titration diagram of the compound described in Example 7 and mercury, wherein a is the fluorescence titration diagram of the compound and Hg 2+ Fluorescence titration diagram of the compound and MeHg + Fluorescence titration graph.

[0038] Figure 10 The response time curve of the compound described in Example 7 and mercury, wherein a is the response time curve of the fluorescent probe and Hg 2+ The response time curve of the fluorescent probe and MeHg + Response time curve.

[0039] Figure 11 This is a diagram showing the selectivity of the compound described in Example 7 as a fluorescent probe for detecting mercury. DETAILED DESCRIPTION

[0040] To facilitate understanding of the present invention, the present invention will be described in more comprehensive and detailed form below in conjunction with the accompanying drawings and preferred embodiments. However, the protection scope of the present invention is not limited to the following specific embodiments.

[0041] Unless otherwise defined, all technical terms used hereinafter have the same meanings as those generally understood by those skilled in the art. The technical terms used herein are only for the purpose of describing specific embodiments and are not intended to limit the scope of protection of the present invention.

[0042] Unless otherwise specified, various raw materials, reagents, instruments and equipment used in the present invention can be purchased from the market or prepared by existing methods.

[0043] The structural formula of the fluorescent probe provided in Examples 1-5 of the present invention is pass Figure 1 The chemical reaction equation shown can be synthesized.

[0044] like Figure 1 As shown, compound 1a (9H-carbazole-3-boronic acid pinacol ester) reacts with a polar aprotic solvent and other substances to obtain compound 1b.

[0045] The polar aprotic solvent is preferably DMSO (dimethyl sulfoxide), DMF, THF or acetonitrile. DMSO is further preferred as the polar aprotic solvent. When DMSO is selected as the polar aprotic solvent, the reaction yield is higher.

[0046] Other substances are any one of KOH, K2CO3, Cs2CO3, NaH, triethylamine, DBU, DMAP (4-dimethylaminopyridine), and DABCO (triethylenediamine). KOH is more preferred in terms of the wide range of raw material sources and cost.

[0047] Compound 1b is further reacted with a mixture of NaIO4 and CH3COONH4, or a mixture of NaIO4 and hydrochloric acid to obtain a fluorescent probe.

[0048] The following specific examples 1-5 are provided for further explanation.

[0049] Example 1

[0050] 1.760 g (6 mmol) of compound 1a (9H-carbazole-3-boronic acid pinacol ester) and 336 mg (9 mmol) of KOH were added to a reaction flask, dissolved in 25 mL of DMSO, and then 396 mg (9 mmol) of ethylene oxide was added. The mixture was heated to 100°C and stirred for 6 h. After the reaction, the mixture was diluted with water, and the organic phase was extracted with EA. After drying over anhydrous MgSO4, the mixture was purified by column chromatography (PE:EA = 6:1) to obtain compound 1b in a 56% yield.

[0051] 277 mg (0.81 mmol) of compound 1b was added to a reaction flask and dissolved in 20 mL of a mixture of acetone and water (v / v = 2:1). 486 mg (2.4 mmol) of NaIO4 and 185 mg (2.4 mmol) of CH3COONH4 were then added and allowed to react at room temperature for 20 h. After completion of the reaction, the mixture was concentrated under reduced pressure, and the organic phase was extracted with EA. After drying over anhydrous MgSO4, the resulting product was purified by column chromatography (PE:EA = 4:1) to obtain the fluorescent probe in a 23% yield.

[0052] Figure 2 This is the hydrogen nuclear magnetic resonance spectrum of the fluorescent probe prepared in Example 1.

[0053] (1) Fluorescence titration of the probe: A fluorescence spectrophotometer was used in the actual detection.

[0054] The fluorescent probe prepared in Example 1 was dissolved in a mixed solution containing HEPES buffer solution (10 mM, pH = 7.4): DMSO (V / V = 99:1), wherein the probe concentration was 1 μM. An aqueous solution containing mercury ions or methylmercury was added to the probe solution, and the fluorescence emission signal was detected. The selected excitation wavelength was 270 nm, and the emission intensity at 353 nm in the fluorescence emission spectrum was negatively correlated with the concentration of mercury ions or methylmercury ions. The results are shown in FIG. Figure 3 As shown, the fluorescence intensity of the probe gradually decreases with increasing mercury or methylmercury concentrations. The probe's detection limits for mercury and methylmercury are 0.47 ppb and 0.55 ppb, respectively (calculated using the formula LOD = 3σ / k, where σ is the standard deviation of the blank measurement and k is the slope of the fluorescence intensity at 353 nm versus mercury or methylmercury concentration).

[0055] (2) Response time of fluorescent probe

[0056] The probe prepared in Example 1 was dissolved in a mixed solution of HEPES buffer (10 mM, pH = 7.4): DMSO (V / V = 99:1) at a probe concentration of 1 μM. An aqueous solution containing mercury ions or methylmercury was added to the probe solution, and the fluorescence excitation wavelength was fixed at 270 nm and the fluorescence emission wavelength was fixed at 353 nm. The change in fluorescence emission intensity at 353 nm over time was observed. The results are shown in FIG. Figure 4 The probe has a relatively fast response time of about 5 minutes for mercury ion detection and less than 10 minutes for methylmercury detection.

[0057] (3) Selectivity of probe detection

[0058] The probe prepared in Example 1 was dissolved in a mixed solution of HEPES buffer solution (10 mM, pH = 7.4): DMSO (V / V = 99:1) to prepare a probe solution with a concentration of 1 μM. 2 mL of the probe solution (1 μM) was taken out from the probe solution and added to a cuvette, and then 20 μL (0.01 M) of the metal ion solution was added. Using an excitation wavelength of 270 nm, the probe was tested with a fluorescence spectrophotometer against different metal ions (BaO) at the same concentration. 2+ , Ca 2+ 、Cd 2+ 、Co 2+ Cr 3+ 、Fe 3+ , K + Mg 2+ 、Mn 2+ 、Na + 、Ni + 、Zn 2+ ) to observe the changes in the fluorescence spectrum. Then take 20 μL of mercury ion solution (0.1 mM) and add it to the cuvette containing the probe solution and metal ions. Use a fluorescence spectrophotometer to measure the fluorescence emission spectrum of the probe and mercury ions in the presence of other metal ions. The results are as follows. Figure 5 As shown, other metal ions have little effect on the fluorescence intensity of the probe, and the probe has good selectivity.

[0059] Example 2

[0060] 1.173 g (4 mmol) of compound 1a (9H-carbazole-3-boronic acid pinacol ester) and 224 mg (6 mmol) of KOH were added to a reaction flask, dissolved in 15 mL of DMSO, and then 264 mg (6 mmol) of ethylene oxide was added. The mixture was heated to 100°C and stirred for 6 h. After the reaction, the mixture was diluted with water, and the organic phase was extracted with EA. After drying over anhydrous MgSO4, the mixture was purified by column chromatography (PE:EA = 6:1) to obtain compound 1b in a 59% yield.

[0061] 185 mg (0.54 mmol) of compound 1b was added to a reaction flask and dissolved in 15 mL of acetone / water (v / v = 2:1). 324 mg (1.6 mmol) of NaIO4 and 123 mg (1.6 mmol) of CH3COONH4 were then added and allowed to react at room temperature for 20 h. After completion of the reaction, the mixture was concentrated under reduced pressure, and the organic phase was extracted with EA. After drying over anhydrous MgSO4, the resulting product was purified by column chromatography (PE:EA = 4:1) to obtain the fluorescent probe in a 25% yield.

[0062] Example 3

[0063] 1.466 g (5 mmol) of compound 1a (9H-carbazole-3-boronic acid pinacol ester) and 336 mg (7.5 mmol) of KOH were added to a reaction flask, dissolved in 20 mL of DMSO, and then 396 mg (6 mmol) of ethylene oxide was added. The mixture was heated to 100°C and stirred for 6 h. After the reaction, the mixture was diluted with water, and the organic phase was extracted with EA. After drying over anhydrous MgSO4, the mixture was purified by column chromatography (PE:EA = 6:1) to obtain compound 1b in a 55% yield.

[0064] 451 mg (1.34 mmol) of compound 1b was added to a reaction flask and dissolved in 30 mL of acetone / water (v / v = 2:1). 859 mg (4 mmol) of NaIO4 and 310 mg (4 mmol) of CH3COONH4 were then added and allowed to react at room temperature for 20 h. After completion of the reaction, the mixture was concentrated under reduced pressure, and the organic phase was extracted with EA. After drying over anhydrous MgSO4, the resulting product was purified by column chromatography (PE:EA = 4:1) to obtain the fluorescent probe in a 23% yield.

[0065] Furthermore, the present invention provides the application of fluorescent probes in mercury detection, which can be seen in Examples 4 and 5 for details.

[0066] Example 4

[0067] A fluorescent probe solution is provided, comprising a fluorescent probe and a solvent. The solvent is any solvent that can dissolve the fluorescent probe and is suitable for detecting mercury ions or methylmercury, including but not limited to a mixed solution containing HEPES buffer and an organic solvent. The organic solvent may be acetonitrile, DMSO, methanol, or the like. The HEPES buffer has a concentration of 10 mM and a pH of 7.4. The volume ratio of HEPES buffer to organic solvent is 99:1.

[0068] The mercury content in the water sample is detected by the following steps.

[0069] The prepared fluorescent probe is prepared into a probe solution with a concentration of 1 μM using the corresponding solvent. A series of mercury ion or methylmercury standard solutions with a concentration gradient (0-50 ppb) are prepared for use. Take 1 mL of the fluorescent probe solution and add it to a fluorescence cuvette. Then, add 1 mL of mercury ion or methylmercury standard solutions with different concentration gradients. After mixing, record the fluorescence emission spectrum using a fluorescence spectrophotometer. Then, use the fluorescence intensity at the corresponding emission wavelength as the ordinate and the mercury ion or methylmercury concentration as the abscissa to fit a standard curve of fluorescence intensity and mercury ion or methylmercury concentration. Take 1 mL of the fluorescent probe solution and add it to a fluorescence cuvette. Then, add 1 mL of the water sample to be tested. After mixing, test its fluorescence emission spectrum. Then, substitute its fluorescence intensity as the ordinate into the standard curve. The corresponding abscissa is the mercury ion or methylmercury concentration in the sample to be tested.

[0070] Example 5

[0071] The probe is used to detect mercury ions and methylmercury in actual water samples: the probe is dissolved in a mixed solution of HEPES buffer solution (10mM, pH=7.4):DMSO (V / V=99:1) to prepare a probe solution with a concentration of 1μM. Groundwater, Xiangjiang River water and tap water are used as actual water samples to detect mercury ions and methylmercury (groundwater, Xiangjiang River water and tap water do not contain mercury ions or methylmercury). The water sample is filtered with a 0.22μm water filter membrane to remove insoluble impurities in the water sample, and then mercury ions or methylmercury are added to prepare test solutions with different mercury addition amounts. Take 1mL of the probe solution and 1mL of the test solution into a fluorescence cuvette, mix them evenly and measure their fluorescence intensity. The concentration of mercury in the test sample is obtained according to the determination method of Example 4. The average value is obtained by three parallel experiments for each spiked amount. The results are shown in Tables 1 and 2. The data in Table 1 show that the recoveries of the fluorescent probe solution in actual water samples ranged from 91.49% to 110.31% when the mercury ion concentrations were 5, 10, and 15 ppb, respectively. The data in Table 2 show that the recoveries of the fluorescent probe solution in actual water samples ranged from 92.52% to 115.81% when the methylmercury concentrations were 5, 10, and 15 ppb, respectively.

[0072] Table 1 Recovery rate of mercury ion detection in actual water samples

[0073]

[0074] Table 2 Recovery of methylmercury in actual water samples

[0075]

[0076] During the research on fluorescent probes, the inventors discovered that other compounds containing carbazole boronic acid units can also be used as fluorescent probes for detecting mercury ions or methylmercury. The present invention is specifically illustrated by Examples 6 and 7.

[0077] Example 6

[0078] The inventor discovered It can be used as a fluorescent probe to detect mercury ions or methylmercury. This example specifically illustrates the use of the compound as a fluorescent probe.

[0079] The above compounds were purchased from Bidex Pharmaceuticals.

[0080] (1) Fluorescence titration of probe

[0081] The fluorescent probe was dissolved in a mixed solution containing HEPES buffer solution (10mM, pH=7.4):DMSO (V / V=99:1), where the probe concentration was 1μM. An aqueous solution containing mercury ions or methylmercury was added to the probe solution, and the fluorescence emission signal was detected. The selected excitation wavelength was 271 nanometers, and the emission intensity at 365 nanometers in the fluorescence emission spectrum was negatively correlated with the concentration of mercury ions or methylmercury ions. The results are shown in Figure 2. Figure 6 As shown, the fluorescence intensity of the probe gradually decreases with increasing mercury or methylmercury concentrations. The probe's detection limits for mercury and methylmercury are 0.45 ppb and 0.95 ppb, respectively (calculated using the formula LOD = 3σ / k, where σ is the standard deviation of the blank measurement and k is the slope of the fluorescence intensity at 365 nm versus mercury or methylmercury concentration).

[0082] (2) Response time of fluorescent probe

[0083] The probe was dissolved in a mixed solution containing HEPES buffer (10 mM, pH = 7.4): DMSO (V / V = 99:1) at a probe concentration of 1 μM. An aqueous solution containing mercury ions or methylmercury was added to the probe solution. The fluorescence excitation wavelength was fixed at 271 nm and the fluorescence emission wavelength was fixed at 365 nm. The change in fluorescence emission intensity at 365 nm over time was observed. The results are shown in Figure 2. Figure 7 The probe has a relatively fast response time of about 3 minutes for mercury ion detection and less than 10 minutes for methylmercury detection.

[0084] (3) Selectivity of probe detection

[0085] The probe was dissolved in a mixed solution containing HEPES buffer solution (10mM, pH=7.4):DMSO (V / V=99:1) to prepare a probe solution with a concentration of 1μM. 2mL of the probe solution (1μM) was taken out from the probe solution and added to a cuvette, and then 20μL (0.01M) of metal ion solution was added. Using an excitation wavelength of 271nm, the probe was tested with a fluorescence spectrophotometer with different metal ions (BaO) at the same concentration. 2+ , Ca 2+ 、Cd 2+ 、Co 2+ Cr 3+ 、Fe 3+ , K + Mg 2+ 、Mn 2+ 、Na + 、Ni + 、Zn 2+) to observe the changes in the fluorescence spectrum. Then take 20 μL of mercury ion solution (0.1 mM) and add it to the cuvette containing the probe solution and metal ions. Use a fluorescence spectrophotometer to measure the fluorescence emission spectrum of the probe and mercury ions in the presence of other metal ions. The results are as follows. Figure 8 As shown, other metal ions have little effect on the fluorescence intensity of the probe, and the probe has good selectivity.

[0086] (4) For the detection of mercury ions and methylmercury in actual water samples: the probe was dissolved in a mixed solution of HEPES buffer solution (10mM, pH=7.4):DMSO (V / V=99:1) to prepare a probe solution with a concentration of 1μM. Groundwater, Xiangjiang River water and tap water were used as actual water samples to detect mercury ions and methylmercury. The water samples were filtered with a 0.22μm water filter membrane to remove insoluble impurities in the water samples, and then mercury ions or methylmercury were added to prepare test solutions with different mercury addition amounts. 1mL of probe solution and 1mL of test solution were taken into a fluorescent cuvette, mixed evenly and then the fluorescence intensity was measured. The concentration of mercury in the test sample was obtained according to the determination method of Example 4. Each spiked amount was tested by three parallel experiments to obtain the average value. The results are shown in Tables 3 and 4. It can be seen from the data in Table 3 that when the mercury ion concentration was 5, 10 and 15ppb respectively, the recovery rate of the fluorescent probe solution in the actual water sample was 92.53% to 106.49%. From the data in Table 4, it can be seen that when the methylmercury concentrations are 5, 10, and 15 ppb, respectively, the recovery rates of the fluorescent probe solution in the actual water samples are 88.71% to 109.11%.

[0087] Table 3 Recovery of mercury ion detection in actual water samples

[0088]

[0089] Table 4 Recovery of methylmercury in actual water samples

[0090]

[0091] Example 7

[0092] The inventor discovered It can be used as a fluorescent probe to detect mercury ions or methylmercury. This example specifically illustrates the use of the compound as a fluorescent probe.

[0093] The above compounds were purchased from Bidex Pharmaceuticals.

[0094] (1) Fluorescence titration of probe

[0095] The fluorescent probe was dissolved in a mixed solution containing HEPES buffer solution (10mM, pH=7.4):DMSO (V / V=99:1), where the probe concentration was 1μM. An aqueous solution containing mercury ions or methylmercury was added to the probe solution, and the fluorescence emission signal was detected. The selected excitation wavelength was 270 nanometers, and the emission intensity at 355 nanometers in the fluorescence emission spectrum was negatively correlated with the concentration of mercury ions or methylmercury ions. The results are shown in Figure 2. Figure 9 As shown, the fluorescence intensity of probe 1 gradually decreases with increasing mercury or methylmercury concentrations. The detection limits of the probe for mercury and methylmercury are 0.91 ppb and 1.12 ppb, respectively (calculated using the formula LOD = 3σ / k, where σ is the standard deviation of the blank measurement and k is the slope of the fluorescence intensity at 355 nm versus mercury or methylmercury concentration).

[0096] (2) Response time of fluorescent probe

[0097] The probe was dissolved in a mixed solution containing HEPES buffer (10 mM, pH = 7.4): DMSO (V / V = 99:1) at a probe concentration of 1 μM. An aqueous solution containing mercury ions or methylmercury was added to the probe solution, and the fluorescence excitation wavelength was fixed at 270 nm and the fluorescence emission wavelength was 355 nm. The change in fluorescence emission intensity at 355 nm over time was observed. The results are shown in Figure 2. Figure 10 The probe has a relatively fast response time of about 5 minutes for mercury ion detection and less than 8 minutes for methylmercury detection.

[0098] (3) Selectivity of probe detection

[0099] The probe was dissolved in a mixed solution containing HEPES buffer solution (10mM, pH=7.4):DMSO (V / V=99:1) to prepare a probe solution with a concentration of 1μM. 2mL of the probe solution (1μM) was taken out from the probe solution and added to a cuvette, and then 20μL (0.01M) of metal ion solution was added. Using an excitation wavelength of 270nm, the probe was tested with a fluorescence spectrophotometer with different metal ions (BaO) at the same concentration. 2+ , Ca 2+ 、Cd 2+ 、Co 2+ Cr 3+ 、Fe 3+ , K + Mg 2+ 、Mn 2+ 、Na + 、Ni + 、Zn 2+) to observe the changes in the fluorescence spectrum. Then take 20 μL of mercury ion solution (0.1 mM) and add it to the cuvette containing the probe solution and metal ions. Use a fluorescence spectrophotometer to measure the fluorescence emission spectrum of the probe and mercury ions in the presence of other metal ions. The results are as follows. Figure 11 As shown, other metal ions have little effect on the fluorescence intensity of the probe, and the probe has good selectivity.

[0100] 4) Detection of mercury ions and methylmercury in actual water samples: The probe was dissolved in a mixture of HEPES buffer (10 mM, pH 7.4):DMSO (v / v = 99:1) to prepare a probe solution with a concentration of 1 μM. Groundwater, Xiangjiang River water, and tap water were used as actual water samples for detection of mercury ions and methylmercury. The water samples were filtered through a 0.22 μm water filter to remove insoluble impurities. Then, mercury ions or methylmercury were added to prepare test solutions with varying mercury additions. 1 mL of the probe solution and 1 mL of the test solution were added to a fluorescence cuvette, mixed thoroughly, and the fluorescence intensity was measured. The mercury concentration in the test sample was determined according to the determination method in Example 4. Three replicate experiments were performed for each spiked amount, and the average value was obtained. The results are shown in Tables 5 and 6. As shown in Table 5, the recoveries of the fluorescent probe solution in actual water samples ranged from 90.71% to 107.95% when the mercury ion concentrations were 5, 10, and 15 ppb, respectively. From the data in Table 6, it can be seen that when the methylmercury concentrations are 5, 10, and 15 ppb, respectively, the recovery rates of the fluorescent probe solution in the actual water samples are 93.96% to 107.64%.

[0101] Table 5 Recovery of mercury ion detection in actual water samples

[0102]

[0103] Table 6 Recovery of methylmercury in actual water samples

[0104]

[0105] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. A fluorescent probe, characterized in that The structural formula of the fluorescent probe is .

2. A method for preparing a fluorescent probe according to claim 1, characterized in that: The following steps are involved: Step S1, dissolving 9H-carbazole-3-boronic acid pinacol ester and compound Y in a polar aprotic solvent, then adding ethylene oxide, heating and stirring for a period of time to obtain material A; the compound Y is any one of KOH, K2CO3, Cs2CO3, NaH, triethylamine, DBU, DMAP, and DABCO; Step S2, extracting the organic phase from material A with EA, and then purifying by column chromatography to obtain compound B; Step S3, dissolving compound B in an acetone / water or THF / water solution, then adding a mixture of NaIO4 and CH3COONH4, or a mixture of NaIO4 and hydrochloric acid, reacting for a period of time, extracting the organic phase with EA, and then purifying by column chromatography to obtain the fluorescent probe.

3. The preparation method according to claim 2, wherein The polar aprotic solvent is selected from any one of DMSO, DMF, THF or acetonitrile; the amount of the polar aprotic solvent added is 5 to 15 times the total mass of the solute; the molar ratio of 9H-carbazole-3-boronic acid pinacol ester and compound Y is 1:1.25 to 2; the molar ratio of 9H-carbazole-3-boronic acid pinacol ester to ethylene oxide is 1:1.25 to 2.

4. The preparation method according to claim 2, wherein In step S1, the heating temperature is 100-130° C., and the stirring time is 6-8 hours.

5. The preparation method according to claim 2, wherein In step S2, the volume ratio of PE to EA in the column chromatography is 3 to 6:

1.

6. The preparation method according to claim 2, wherein In the acetone / water or THF / water solution, the volume ratio of acetone, THF and water is 2 to 4:1, respectively.

7. The preparation method according to claim 2, wherein In step S3, the amount of NaIO4 added is 2 to 3 times the theoretical amount for reaction with compound B, and the amount of CH3COONH4 or hydrochloric acid added is 2 to 3 times the theoretical amount for reaction with compound B.

8. Use of the fluorescent probe according to claim 1 in preparing a mercury detection reagent.

9. The structural formula is or The application of the compound as a fluorescent probe in the preparation of mercury detection reagents.

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