A novel reactive fluorescent probe for specific recognition of mercury ions, its preparation method, and its application in detecting mercury ions

Abstract

The present invention relates to a novel reaction type fluorescent probe for specific identification of mercury ions and its preparation method and application in detecting mercury ions. The preparation method of the fluorescent probe RhB-M comprises the following steps: taking an acetonitrile solution of compound N-(6-(diethylamino)-9-(2-isocyanophenyl)-3H-anthracene-3-ylidene)-N-ethylethylamine, adding an acetonitrile solution of compound 1-(2-aminoethyl)-3-phenylthiourea thereto, stirring at room temperature for 1h, removing the solvent by reduced pressure distillation, and purifying the crude product by silica gel column chromatography with dichloromethane and methanol as developing agents to obtain the target product. The present invention can be applied to the qualitative and quantitative detection of mercury ions in water quality, obtains good test results, does not require complex equipment, is easy to operate, is low in price, has obvious response effect, and has good application prospects in the detection of mercury ions in organisms and the environment.

Description

Technical Field

[0001] The present invention belongs to the technical field of chemical synthesis and analytical detection, and particularly relates to a novel reaction-type fluorescent probe for specifically identifying mercury ions, a preparation method thereof, and application in detecting mercury ions. Background Art

[0002] Compared with other heavy metals such as copper, chromium, lead and cadmium, mercury is more toxic. Mercury (Hg) is one of the most common deadly toxins on the earth. It is a toxic heavy metal that seriously harms the environment and life and health. Mercury is produced from many sources, such as metallurgy and coal burning. It is widely present in water and soil and mainly comes from industrial wastewater discharge. The continuous acceleration of the industrialization process has led to a large amount of Hg 2+ ions are emitted into the environment. Hg 2+ The ion toxicity is persistent, difficult to degrade, and has a high bioaccumulation capacity, making it one of the most serious pollutants in the world. 2+ ) can accumulate in aquatic organisms and be converted by bacteria and microorganisms into the more toxic methylmercury. Methylmercury is not easily metabolized and ultimately enters the human body through the food chain, causing cellular dysfunction and damaging the central nervous system and endocrine system, leading to a range of diseases, including acne, Hunter-Russell syndrome, Alzheimer's disease, and Menkes disease. The World Health Organization (WHO) stipulates that the content of inorganic mercury in drinking water should not exceed 6 ppb (3 nM). Therefore, rapid and effective detection of mercury ions is of great significance to human health and ecological protection.

[0003] Rhodamine compounds are basic dyes based on xanthenes, characterized by a wide wavelength range, high fluorescence quantum yield, high molar absorptivity, and excellent photostability. Due to their unique structure and corresponding fluorescence properties, rhodamine fluorescent dyes are widely used in chemical and biological analysis, including pharmacology, physiology, molecular biology, cell biology, molecular genetics, environmental chemistry, single molecule detection, information science, fluorescent labeling, and laser dye research.

[0004] At present, although many Hg 2+ Fluorescent probes, but many have problems such as poor anti-interference ability and poor selectivity, and few probes can be used in engineering practice. Therefore, it is necessary to develop high-sensitivity and strong anti-interference ability that can be used to detect Hg in actual water samples. 2+ The development of a fluorescent probe that can be made into a visual test paper for the efficient detection of various mercury ions is an urgent problem to be solved by researchers in this field. Summary of the Invention

[0005] In order to overcome the defects of the prior art, the present invention provides a method for preparing a reactive fluorescent probe for specifically recognizing mercury ions, which is fatigue-resistant, has a long open-loop life, high fluorescence quantum efficiency, good selectivity, high sensitivity, is easy to use, has strong anti-interference ability, and does not fade significantly, and its application in detecting mercury ions.

[0006] The present invention is achieved through the following technical solutions:

[0007] A new type of reactive fluorescent probe RhB-M that specifically recognizes mercury ions, with the molecular formula C 37 H 42 N6O2S has the structure shown in (I):

[0008]

[0009] The preparation method of the novel reactive fluorescent probe RhB-M for specifically recognizing mercury ions comprises the following steps: taking an acetonitrile solution of the compound N-(6-(diethylamino)-9-(2-isocyanophenyl)-3H-anthracene-3-ylidene)-N-ethylethylamine, adding an acetonitrile solution of the compound 1-(2-aminoethyl)-3-phenylthiourea thereto, stirring at room temperature for 1 hour, removing the solvent by reduced pressure distillation, and purifying the crude product by silica gel column chromatography using dichloromethane and methanol as developing solvents to obtain the novel reactive fluorescent probe RhB-M for specifically recognizing mercury ions.

[0010] Furthermore, in the above preparation method, the molar ratio of the compound N-(6-(diethylamino)-9-(2-isocyanophenyl)-3H-anthracene-3-ylidene)-N-ethylethylamine:1-(2-aminoethyl)-3-phenylthiourea is 1:0.5-1.

[0011] Furthermore, in the above preparation method, the volume ratio of dichloromethane:methanol is 30:1.

[0012] The application of the above-mentioned new reactive fluorescent probe RhB-M that specifically recognizes mercury ions in the visual detection of mercury ions in water.

[0013] The above-mentioned new reactive fluorescent probe RhB-M for specific recognition of mercury ions is used in the visual detection of mercury ions in agricultural products.

[0014] Furthermore, in any of the above applications, the detection method comprises the following steps:

[0015] 1) Prepare the probe stock solution: Dissolve 12.696 mg of the novel reactive fluorescent probe RhB-M that specifically recognizes mercury ions in dichloromethane and dilute to a volume of 10 mL in a volumetric flask to obtain a concentration of 2 × 10 -3 mol / L probe stock solution;

[0016] 2) Prepare the test solution: 1 mL of a solution with a concentration of 2×10 -3 The probe stock solution was added to a 100 mL volumetric flask, the dichloromethane was blown dry, and then dissolved in acetonitrile / HEPES buffer solution to prepare a concentration of 2 × 10 -5 mol / L, pH 7.4 test solution;

[0017] 3) Drawing a standard curve: Take the test solution and a mercury ion solution of known concentration, mix them evenly, perform UV-visible absorption spectroscopy and fluorescence spectroscopy tests respectively, and draw standard curves of the UV-visible absorption spectrum and fluorescence spectrum of mercury ions;

[0018] 4) Testing: Take the test solution and the test solution containing mercury ions, mix them evenly, and then perform UV-visible absorption spectroscopy and fluorescence spectroscopy tests respectively. Use the standard curves of the UV-visible absorption spectrum and fluorescence spectrum of mercury ions to calculate the concentration of mercury ions in the test solution containing mercury ions.

[0019] Furthermore, in the above application, the volume ratio of acetonitrile to HEPES in the acetonitrile / HEPES buffer solution is 4:6.

[0020] The novel reactive fluorescent probe RhB-M for specifically recognizing mercury ions is used to prepare a test paper capable of visually detecting mercury ions in water.

[0021] The novel reactive fluorescent probe RhB-M for specifically recognizing mercury ions is used to prepare a kit capable of visually detecting mercury ions in water.

[0022] The beneficial effects of the present invention are:

[0023] 1. The reactive fluorescent probe RhB-M for specific recognition of mercury ions of the present invention has readily available raw materials, mild reaction conditions, low synthesis cost, high yield, and stable structure. The probe with this structure was placed at room temperature for 150 days, and no structural change was observed by thin layer plate monitoring.

[0024] 2. The probe of the present invention is used in combination with Hg 2+ After the reaction occurs, the fluorescence intensity of the system is significantly enhanced, and the solution changes from colorless and non-fluorescent to visually obvious pink and strong fluorescence. It has good selectivity and can be used for qualitative and quantitative detection of mercury ions in water quality, achieving good test results.

[0025] 3. The probe of the present invention is sensitive to Hg 2+ The detection process is convenient and fast, does not require large instruments and equipment, is easy to operate, low in price, and has obvious response effect, achieving Hg 2+ Visual detection can be made into a convenient Hg 2+Detection kits, visual test strips, etc. are used for water quality monitoring, cell imaging, etc. They are stable in nature, have good glossiness, and are non-toxic, harmless, and environmentally friendly. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 The fluorescence probe RhB-M prepared in Example 1 is used to detect Hg before and after adding mercury ions. 2+ Absorption and fluorescence color response diagram, where a: UV-visible absorption color change; b: fluorescence color change.

[0027] Figure 2 The selectivity of the fluorescent probe RhB-M prepared in Example 1 for the ultraviolet-visible absorption spectrum of metal cations.

[0028] Figure 3 The fluorescence spectrum selectivity of the fluorescent probe RhB-M prepared in Example 1 to metal cations.

[0029] Figure 4 This is the trend of change of the UV-visible absorption peak along with the mercury ion concentration in the titration of mercury ion concentration by the fluorescent probe RhB-M prepared in Example 2 (absorption-standard curve).

[0030] Figure 5 This is the changing trend of the fluorescence peak along with the mercury ion concentration in the mercury ion concentration titration of the fluorescent probe RhB-M prepared in Example 2 (fluorescence-standard curve).

[0031] Figure 6 This is a physical detection colorimetric card for detecting mercury ions using the fluorescent probe RhB-M prepared in Example 1. DETAILED DESCRIPTION

[0032] Example 1 Preparation method of a novel reactive fluorescent probe RhB-M for specific recognition of mercury ions

[0033] The reaction formula is as follows:

[0034]

[0035] The preparation method comprises the following steps:

[0036] 1) Synthesis steps of the compound N-(6-(diethylamino)-9-(2-isocyanophenyl)-3H-anthracen-3-ylidene)-N-ethylethanamine: 0.3 g of rhodamine B (0.6 mmol), 0.18 mL of phosphorus oxychloride (1.8 mmol), and 100 mL of anhydrous 1,2-dichloroethane were accurately weighed, mixed, reacted at 90° C. for 4 h, cooled to room temperature, and the solvent was removed by distillation under reduced pressure; the resulting solid was dissolved in 100 mL of dry acetonitrile, and 51 mg of a saturated aqueous solution of sodium azide (0.78 mmol) was added, and the mixture was stirred at room temperature for 12 h. The resulting reaction solution was dried over anhydrous magnesium sulfate and filtered; the filtrate was reacted at 82° C. for 40 min, and cooled to room temperature to obtain the compound N-(6-(diethylamino)-9-(2-isocyanophenyl)-3H-anthracen-3-ylidene)-N-ethylethanamine.

[0037] 2) Take the compound N-(6-(diethylamino)-9-(2-isocyanophenyl)-3H-anthracene-3-ylidene)-N-ethylethylamine (79.30 mg, 0.18 mmol), dissolve it in a small amount of acetonitrile, add the compound 1-(2-aminoethyl)-3-phenylthiourea acetonitrile solution (0.035 g, 0.18 mmol), stir at room temperature for 1 hour, and remove the solvent by distillation under reduced pressure. The crude product is purified by silica gel column chromatography using dichloromethane and methanol as developing solvents (30:1, v:v) to obtain a novel reactive fluorescent probe RhB-M that specifically recognizes mercury ions. Example 2 Application of the novel reactive fluorescent probe RhB-M that specifically recognizes mercury ions in the detection of mercury ions

[0038] (1) Qualitative testing

[0039] 1) Preparation of RhB-M probe stock solution: 12.696 mg of the novel reactive fluorescent probe RhB-M that specifically recognizes mercury ions was dissolved in dichloromethane and diluted to a volume of 10 mL in a volumetric flask to obtain a concentration of 2 × 10 -3 mol / L probe stock solution;

[0040] 2) Prepare RhB-M test solution: 1 mL of 2×10 -3 The probe stock solution was added to a 100 mL volumetric flask, the dichloromethane was blown dry, and then dissolved in acetonitrile / HEPES buffer solution (acetonitrile:HEPES=4:6 by volume) to a concentration of 2×10 -5 mol / L, pH 7.4 RhB-M test solution;

[0041] 3) Take 3.0 mL of RhB-M test solution and add 30 μL of 2×10 -2 mol / L of different cations Na + , K + , Mg2+ , Ca 2+ , Fe 2+ , Fe 3+ , Al 3+ , Mn 2+ , Zn 2+ , Hg 2+ , Pb 2+ , Cr 3+ , Cd 2+ , Ag + , Cu 2+ , and UV-visible spectroscopy and fluorescence spectroscopy were performed respectively.

[0042] like Figure 1 As shown in (a), only when 30 μL of 2×10 -2 mol / L metal ion Hg 2+ After that, the color of the solution changes from colorless to red. This is because the solution of RhB-M changes from colorless to pink after being irradiated with 313nm light. Other cations have no effect on the UV-visible absorption spectrum, such as Figure 2 shown.

[0043] like Figure 1 As shown in (b), only when 30 μL of 2×10 -2 mol / L metal ion Hg 2+ After that, the fluorescence color of the solution changes from colorless to red. This is due to the fluorescence intensity of the solution after the RhB-M fluorescent probe is illuminated by a 365nm fluorescent lamp; other cations have no effect on the fluorescence spectrum, such as Figure 3 shown.

[0044] The mechanism by which RhB-M detects mercury ions is that mercury ions cause the six-membered spiro ring to open, generating bright red, highly fluorescent rhodamine, thereby enabling rapid detection of RhB-M. RhB-M enables visible, rapid, and highly sensitive detection of mercury ions. Other metal cations did not cause changes in the UV-visible and fluorescence spectra.

[0045] (2) Quantitative detection

[0046] 1) Preparation of RhB-M probe stock solution: 12.696 mg of the novel reactive fluorescent probe RhB-M that specifically recognizes mercury ions was dissolved in dichloromethane and diluted to a volume of 10 mL in a volumetric flask to obtain a concentration of 2 × 10 -3 mol / L probe stock solution;

[0047] 2) Prepare RhB-M test solution: 1 mL of 2×10 -3The probe stock solution was added to a 100 mL volumetric flask, the dichloromethane was blown dry, and then dissolved in acetonitrile / HEPES buffer solution (acetonitrile:HEPES=4:6 by volume) to a concentration of 2×10 -5 mol / L, pH 7.4 RhB-M test solution;

[0048] 3) Draw a standard curve: Take 3.0 mL of RhB-M test solution and 30 μL of mercury ion solution with a known concentration in the range of 0-0.3 mg / L, mix them evenly, and perform UV-visible spectroscopy and fluorescence spectroscopy detection, respectively.

[0049] Select the absorbance at the maximum absorption peak of 565nm in the UV-visible spectrum as the vertical axis and the mercury ion concentration as the horizontal axis to draw a standard curve. Figure 4 As shown in the figure, the mercury ion concentration in the absorption standard curve is linear in the range of 0-0.3 mol / L.

[0050] The intensity of the maximum fluorescence emission peak at 585nm in the fluorescence spectrum was selected as the ordinate and the mercury ion concentration was selected as the abscissa to draw a standard curve. Figure 5 As shown in the fluorescence standard curve, the mercury ion concentration is linear in the range of 0-0.3 mol / L.

[0051] 4) Testing: Mix 3.0 mL of RhB-M test solution and 30 μL of a test solution containing mercury ions of unknown concentration. Measure the absorbance at the maximum absorption peak of 565 nm in the UV-visible spectrum and the intensity at the maximum fluorescence emission peak of 585 nm in the fluorescence spectrum. Calculate the mercury ion concentration in the test solution using the standard curve.

[0052] like Figure 4 As shown, a standard curve was drawn using the absorbance at the maximum absorption peak of 565 nm in the UV-visible spectrum as the ordinate and the mercury ion concentration as the abscissa. The mercury ion concentration in the absorption standard curve exhibited a linear relationship within the range of 0-0.3 mg / L. The mercury ion concentration in the test solution was calculated using the standard curve.

[0053] like Figure 5 As shown in the figure, a standard curve was drawn with the intensity of the maximum fluorescence emission peak at 585 nm in the fluorescence spectrum as the ordinate and the mercury ion concentration as the abscissa. The mercury ion concentration in the fluorescence standard curve showed a linear relationship in the range of 0-0.3 mg / L. The mercury ion concentration in the test solution was calculated using the standard curve.

[0054] like Figure 6 As shown, according to the different concentrations of Hg 2+The color development of RhB-M under the same concentration of RhB-M (i.e., the above 2×10 -5 mol / L probe detection solution), add 30μL of Hg 2+ The concentrations are 0.005, 0.01, 0.05, 0.10, 0.15, 0.2, 0.25, and 0.3 mg / L, respectively.

[0055] The above description is a specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any person skilled in the art can easily conceive of various equivalent modifications or substitutions within the technical scope disclosed in the present invention, and such modifications or substitutions are intended to be within the scope of protection of the present invention. Therefore, the scope of protection of the present invention shall be subject to the scope of protection of the claims.

Claims

1. A reactive fluorescent probe RhB-M for specific recognition of mercury ions, characterized in that: The molecular formula is C 37 H 42 N6O2S has the structure shown in (I):

2. The method for preparing the reactive fluorescent probe RhB-M for specific recognition of mercury ions according to claim 1, characterized in that: The method comprises the following steps: taking an acetonitrile solution of the compound N-(6-(diethylamino)-9-(2-isocyanophenyl)-3H-anthracene-3-ylidene)-N-ethylethylamine, adding an acetonitrile solution of the compound 1-(2-aminoethyl)-3-phenylthiourea thereto, stirring at room temperature for 1 hour, removing the solvent by reduced pressure distillation, and purifying the crude product by silica gel column chromatography using dichloromethane and methanol as developing solvents to obtain a reactive fluorescent probe RhB-M that specifically recognizes mercury ions.

3. The preparation method according to claim 2, characterized in that In terms of molar ratio, the compound N-(6-(diethylamino)-9-(2-isocyanophenyl)-3H-anthracen-3-ylidene)-N-ethylethylamine:1-(2-aminoethyl)-3-phenylthiourea=1:0.5-1.

4. The preparation method according to claim 2, characterized in that By volume ratio, dichloromethane:methanol=30:

1.

5. Use of the reactive fluorescent probe RhB-M for specific recognition of mercury ions according to claim 1 in visual detection of mercury ions in water.

6. Use of the reactive fluorescent probe RhB-M for specific recognition of mercury ions according to claim 1 in visual detection of mercury ions in agricultural products.

7. The use according to claim 5 or 6, characterized in that The detection method includes the following steps: 1) Prepare the probe stock solution: Dissolve 12.696 mg of the reactive fluorescent probe RhB-M that specifically recognizes mercury ions in dichloromethane and dilute to a volume of 10 mL in a volumetric flask to obtain a concentration of 2 × 10 -3 mol / L probe stock solution; 2) Prepare the test solution: 1 mL of a solution with a concentration of 2×10 -3 The probe stock solution was added to a 100 mL volumetric flask, the dichloromethane was blown dry, and then dissolved in acetonitrile / HEPES buffer solution to prepare a concentration of 2 × 10 -5 mol / L, pH 7.4 test solution; 3) Drawing a standard curve: Take the test solution and a mercury ion solution of known concentration, mix them evenly, perform UV-visible absorption spectroscopy and fluorescence spectroscopy tests respectively, and draw standard curves of the UV-visible absorption spectrum and fluorescence spectrum of mercury ions; 4) Testing: Take the test solution and the test solution containing mercury ions, mix them evenly, and then perform UV-visible absorption spectroscopy and fluorescence spectroscopy tests respectively. Use the standard curves of the UV-visible absorption spectrum and fluorescence spectrum of mercury ions to calculate the concentration of mercury ions in the test solution containing mercury ions.

8. The use according to claim 7, characterized in that In the acetonitrile / HEPES buffer solution, the volume ratio of acetonitrile to HEPES is 4:

6.

9. The reactive fluorescent probe RhB-M for specifically recognizing mercury ions according to claim 1 is used to prepare a test paper capable of visually detecting mercury ions in water.

10. The reactive fluorescent probe RhB-M for specifically recognizing mercury ions according to claim 1 is used for preparing a kit for visually detecting mercury ions in water.

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