Quinoline matrix-based mercury ion detection fluorescent probe as well as preparation method and application thereof

Abstract

The invention discloses a mercury ion detection fluorescent probe based on quinoline as well as a preparation method and application of the mercury ion detection fluorescent probe. The fluorescent probe and a structural formula of the fluorescent probe are shown as a formula I in the specification. A quinoline derivative, namely 2-methyl-7-hydroxyquinoline, is used as a fluorescent chromophore, a thioester group is used as a recognition group, and the prepared fluorescent probe shows high sensitivity and high selectivity on Hg < 2 + > in a solution. The preparation method of the fluorescent probe is simple in steps, raw materials are easy to obtain, and the obtained product is a white flocculent solid, is easy to store, has a wide application prospect and can be produced and applied on a large scale.

Description

Technical Field

[0001] This invention belongs to the field of fluorescent probe technology, specifically relating to an aluminum ion detection fluorescent probe NHO, its preparation method, and its application. Background Technology

[0002] Mercury (Hg) plays a vital role in many biological and chemical transformations within ecosystems. However, as a non-essential trace element for humans, excessive exposure can cause various diseases. Furthermore, when mercury concentrations in plant irrigation water exceed safe levels, it can lead to stunted growth, weak stunted plants, and pale leaves. In rice, in particular, excessive mercury levels can cause poor root development, increased empty grains, and yield reductions of over 20%. The general limit for total mercury in grains is 0.02 mg / kg.

[0003] Currently reported analytical methods for detecting mercury ions include atomic absorption-emission spectroscopy, high-performance liquid chromatography, nuclear magnetic resonance, colorimetric methods (such as the traditional dithizone method), and electrochemical methods. However, these methods require expensive instruments, rigorous experimental conditions, complex sample pretreatment, and relatively long measurement times. In contrast, fluorescent probes have attracted considerable attention due to their excellent selectivity, high sensitivity, short detection time, simple operation, and low detection cost. Therefore, the design and development of highly sensitive and selective Hg... 2+ The probe has important practical significance. Summary of the Invention

[0004] Objective of the invention: To address the problems existing in the prior art, the present invention provides a fluorescent probe for mercury ion detection based on quinoline parent compounds. This fluorescent probe compound has specific recognition of mercury ions, short response time, and high detection sensitivity.

[0005] This invention also provides a method for preparing and applying a quinoline-based fluorescent probe for mercury ion detection.

[0006] Technical solution: To achieve the above-mentioned objectives, this invention provides a fluorescent probe for mercury ion detection based on quinoline precursor, the structure of which is shown in Formula I below: .

[0007] The method for preparing the mercury ion detection fluorescent probe based on quinoline parent compound includes the following steps:

[0008] First, m-aminophenol and crotonaldehyde were substituted to obtain intermediate II; then, intermediate II was used as the starting material to undergo a one-step condensation reaction with 2-methyl-7-hydroxyquinoline to obtain fluorescent probe I based on quinoline.

[0009] The reaction route for preparing the mercury ion detection fluorescent probe based on the quinoline precursor is shown below:

[0010]

[0011] In this process, m-aminophenol, tetrachlorobenzoquinone, and hydrochloric acid are added to an organic solvent. After dissolution, crotonaldehyde solution is added dropwise while the mixture is refluxed and stirred. After the reaction is complete, the mixture is cooled to room temperature and then extracted and dried to obtain a crude product. After purification, intermediate II is obtained.

[0012] In this process, m-aminophenol, tetrachlorobenzoquinone, and hydrochloric acid are added to an organic solvent. After dissolution, the temperature is raised to 105-110℃ and crotonaldehyde solution is added dropwise. The mixture is then refluxed and stirred for 4-5 hours.

[0013] The crotonaldehyde solution is a n-butanol solution of crotonaldehyde, and the dripping time is not less than 30 min.

[0014] In this process, intermediate II and phenyl thiochloroformate were dissolved in an organic solvent, and triethylamine was added and stirred at room temperature. After the reaction was complete, the solvent was removed by vacuum distillation to obtain the crude product. The crude product was then purified to obtain fluorescent probe I.

[0015] The mixture after adding triethylamine was stirred at room temperature for 10-12 hours.

[0016] Preferably, the m-aminophenol, tetrachlorobenzoquinone, and hydrochloric acid are added to an organic solvent. After dissolution, the mixture is heated to 105°C, and a n-butanol solution of crotonaldehyde is added dropwise while the mixture is refluxed and stirred. After the reaction is complete, the mixture is cooled to room temperature, and a saturated NaCl solution is added. The resulting extract is dried over anhydrous sodium sulfate, and the organic solvent is removed by rotary evaporation to obtain the crude product. The crude product is purified by silica gel column chromatography and dried to obtain the pure product intermediate II.

[0017] Preferably, the intermediate of type II, phenyl thiochloroformate, is dissolved in an organic solvent, and triethylamine is added and refluxed at room temperature with stirring. After the reaction is complete, the solvent is removed by vacuum distillation to obtain a crude product. The crude product is purified by silica gel column chromatography and dried to obtain fluorescent probe I.

[0018] As a preferred option, the synthetic route is shown in the figure below:

[0019]

[0020] Among them, the intermediate of Formula II is 2-methyl-7-methylquinoline, and the compound of Formula I, O-(2-methylquinoline-7-yl)-O-phenylthiocarbonate, is the fluorescent probe compound for detecting mercury ions described in this invention.

[0021] Furthermore, the preparation process includes:

[0022] (1) Preparation of Type II intermediate

[0023] m-Aminophenol and tetrachlorobenzoquinone were added to n-butanol. After dissolution, hydrochloric acid was added. The mixture was heated to 105 °C, and a n-butanol solution of crotonaldehyde was added dropwise over a period of at least 30 min. The mixture was then refluxed and stirred for 5 h. After the reaction was complete and cooled to room temperature, a saturated NaCl solution was added. The mixture was extracted with petroleum ether, and the aqueous layer was neutralized to pH 7-8 with 10% NaOH, followed by extraction with ethyl acetate. The combined organic phases were dried over anhydrous sodium sulfate, and the organic solvent was removed by rotary evaporation to obtain the crude product. The crude product was purified by silica gel column chromatography (V... PE V EA V MeOH V 三乙胺 =1:5:2%:1%), dried, and the solvent was removed by vacuum distillation to obtain pure product II intermediate.

[0024] (2) Preparation of mercury ion fluorescent probe compound I based on quinoline parent compound

[0025] Intermediate II and phenyl thiochloroformate were added to dichloromethane. After dissolution, triethylamine was added, and the mixture was refluxed and stirred at room temperature for 12 h. After the reaction was complete, the solvent was removed by vacuum distillation to obtain the crude product, which was then purified by silica gel column chromatography (V). PE V EA (8:1), dried, to obtain fluorescent probe I.

[0026] The present invention relates to the application of a quinoline-based mercury ion probe in the detection of mercury ions.

[0027] The present invention relates to the application of a quinoline-based mercury ion probe in the detection of mercury ions.

[0028] Furthermore, this invention relates to the application of a quinoline-based mercury ion probe in the preparation of reagents and tools for detecting mercury ions.

[0029] Compared to other Hg 2+ Fluorescent probe, Hg prepared in this invention 2+ Fluorescent probes have three major advantages.

[0030] First, the probe molecule synthesis process is simple, and the raw materials are readily available. While ensuring high sensitivity and selectivity, the synthesis process is simple, requiring only two steps. The preparation method uses readily available raw materials, is simple, and the product is a solid powder that is easy to store and has good stability.

[0031] Second, the probe molecule exhibits high sensitivity, high selectivity, and a low detection limit. This probe molecule is related to Hg. 2+ The concentrations all showed a good linear relationship, R 2 =0.99129, with a detection limit as low as 41.25 μmol / L, a value lower than most quinoline-based Hg...2+ Fluorescent probe. Compared with other related species, including reactive oxygen species, amino acids, and inorganic salts, this probe is highly reactive to Hg. 2+ It exhibits excellent selective recognition.

[0032] Third, the Hg prepared by this invention 2+ The fluorescent probe, 2-methyl-7-hydroxyquinoline, possesses advantages such as good optical stability, rigid planar structure, low biotoxicity, excellent water solubility, and suitable lipid solubility. These characteristics make this fluorescent probe a potential detection method in biological models such as cells.

[0033] Beneficial effects: Compared with the prior art, the present invention has the following advantages:

[0034] 1. The Hg prepared by this invention 2+ Fluorescent probes are prepared using readily available raw materials and simple methods. The resulting product is a solid powder that is easy to store and has good stability.

[0035] 2. The Hg prepared by this invention 2+ The fluorescent probe uses 2-methyl-7-hydroxyquinoline as a chromophore and thioester as a recognition group, giving it high selectivity and specificity. When the probe recognizes Hg... 2+ When this occurs, it directly triggers a change in signal (fluorescence), which can be observed with the naked eye. This probe molecule interacts with Hg... 2+ The concentrations all showed a good linear relationship, R 2 =0.99129, with a detection limit as low as 41.25 μmol / L, a value lower than most quinoline-based Hg... 2+ Fluorescent probe. Compared with other related species, including reactive oxygen species, amino acids, and inorganic salts, this probe is highly reactive to Hg. 2+ It exhibits excellent selective recognition.

[0036] 3. The Hg prepared by this invention 2+ The fluorescent probe, 2-methyl-7-hydroxyquinoline, possesses advantages such as good optical stability, rigid planar structure, low biotoxicity, excellent water solubility, and suitable lipid solubility. These characteristics make this fluorescent probe a potential detection method in biological models such as cells.

[0037] 4. The Hg prepared by this invention 2+ The fluorescent probe has specific recognition of mercury ions and a short response time (40 min). Attached Figure Description

[0038] Figure 1 The mercury ion fluorescent probe prepared in Example 1 was tested in PBS (pH=7.2) solution for different concentrations of mercury ions (Hg). 2+ The fluorescence spectrum response diagram of )

[0039] Figure 2 The fluorescence spectra of the mercury ion fluorescent probe prepared in Example 1 for different metal ions in PBS (pH=7.2) solution are shown.

[0040] Figure 3 The image shows the fluorescence response of the mercury ion fluorescent probe prepared in Example 1 to the detection of interference from different metal ions in PBS (pH=7.2) solution.

[0041] Figure 4 The graph shows the response time of the mercury ion fluorescent probe prepared in Example 1 when detecting mercury ions in PBS (pH=7.2) solution;

[0042] Figure 5 The fluorescence spectrum response of the mercury ion fluorescent probe prepared in Example 1 in PBS solutions with different pH values ​​for detecting mercury ions is shown.

[0043] Figure 6 The detection limit diagram of mercury ions in PBS (pH=7.2) solution is shown for the mercury ion fluorescent probe prepared in Example 1.

[0044] Figure 7 Nuclear magnetic resonance (NMR) of the mercury ion fluorescent probe prepared in Example 1 1 H-NMR spectrum;

[0045] Figure 8 Nuclear magnetic resonance (NMR) of the mercury ion fluorescent probe prepared in Example 1 13 C-NMR spectrum. Detailed Implementation

[0046] The present invention will be further described below with reference to the accompanying drawings and embodiments.

[0047] Unless otherwise specified, the experimental methods used in this invention are conventional. All materials and reagents used in the experiments are commercially available unless otherwise specified. All reagents used in the examples are commercially available analytical grade or chemically pure.

[0048] In the examples, the various ionic solutions are prepared by adding deionized water to salt chemical reagents with a purity of over 99%, such as copper sulfate pentahydrate, sodium chloride, and anhydrous aluminum chloride.

[0049] Example 1

[0050] The mercury ion fluorescent probe compound based on quinoline was prepared using the following method:

[0051] (1) Preparation of intermediate II (2-methyl-7-hydroxyquinoline)

[0052] In a round-bottom flask, 2 mL of 12 mol / L hydrochloric acid was added to a 5 mL solution of 3-aminophenol (1.1 g, 10 mmol) and tetrachlorobenzoquinone (2.5 g, 10 mmol) in n-butanol. The reaction mixture was then heated to 105 °C. At this temperature, a 5 mL solution of crotonaldehyde (0.9 g, 13 mmol) in n-butanol was added dropwise over approximately 30 min. The final mixture was refluxed at 105 °C for 5 hours. After cooling to room temperature, 50 mL of saturated NaCl solution was added. The mixture was extracted with petroleum ether (3 × 20 mL), and the aqueous layer was neutralized to pH 7 with 10% NaOH, followed by extraction with ethyl acetate (3 × 20 mL). The combined organic phases were dried over anhydrous sodium sulfate, and the organic solvent was removed by rotary evaporation to obtain the crude product. The crude product was purified by silica gel column chromatography (VPE:VEA:VMeOH:Vtriethylamine = 1:5:2%:1%), dried, and the pure product intermediate II was obtained as a brownish-green solid powder (683.7 mg, yield 43%).

[0053] The structural formula of intermediate II is:

[0054]

[0055] (2) Preparation of mercury ion fluorescent probe compounds based on quinoline parent compound

[0056] In a pressure-resistant tube, triethylamine (208 μL, 1.5 mmol) and phenyl thiochloroformate (289 μL, 2.25 mmol) were added to a solution of intermediate II (238.5 mg, 1.5 mmol) in dichloromethane (5 mL). The mixture was reacted at room temperature for 12 hours. After the reaction was complete, the solvent was evaporated to obtain the crude product. The crude product was purified by silica gel column chromatography (V... PE V EA =8:1), dried, to obtain pure product quinoline-based mercury ion fluorescent probe compound I, as a white flocculent solid (1389.6 mg, yield 89%).

[0057] The structural formula of the obtained fluorescent probe compound is:

[0058]

[0059] The mercury ion fluorescent probe compound prepared in this invention 1H NMR (400 MHz, CHLOROFORM-D) δ8.07-8.00 (m, 1H), 7.83-7.77 (m, 2H), 7.47-7.39 (m, 2H), 7.35 (dd, J = 8.8,2.3 Hz, 1H), 7.32-7.26 (m, 2H), 7.23-7.19 (m, 2H), 2.71 (s, 3H). 13 C NMR (101MHz, CHLOROFORM-D) δ 194.6, 160.3, 153.9, 153.6, 148.4, 136.2, 129.8, 129.0,127.0, 125.1, 122.5, 121.9, 121.1, 120.4, 25.5.

[0060] The fluorescent probe nuclear magnetic resonance obtained in Example 1 1 H-NMR spectrum, nuclear magnetic resonance 13 The C-NMR spectra are as follows: Figure 6 , 7 As shown, the fluorescent probe of the present invention was successfully synthesized.

[0061] Example 2

[0062] The mercury ion detection fluorescent probe prepared in Example 1 was prepared into a 1 mM probe stock solution using DMSO.

[0063] Weigh anhydrous FeCl3, FeSO4·7H2O, CuSO4·5H2O, CuCl, anhydrous AlCl3, CoCl3, MnCl2, ZnCl2, CaCl2, NaCl, AgNO3, KCl, CdCl2·5H2O, CrCl3, PbCl2, MgCl2, and NiCl2 and dissolve them in deionized water to prepare 1 mmol / L metal ion stock solutions respectively.

[0064] Mercuric trifluoromethanesulfonate, as a mercury ion stock solution, has the core advantage of long-term stability; its concentration does not change due to hydrolysis or precipitation. The mercury in the solution exists almost entirely in the form of free Hg²⁺, avoiding interference from anionic complexes on the detection signal. This makes it suitable for most modern analytical techniques that require Hg²⁺ standard solutions, such as inductively coupled plasma mass spectrometry (ICP-MS), atomic absorption / emission spectroscopy (AAS / AES), atomic fluorescence spectroscopy (AFS), and electrochemical analysis. Therefore, mercuric trifluoromethanesulfonate is dissolved in deionized water to prepare a 1 mmol / L mercury ion stock solution.

[0065] Add 30 μL of probe stock solution and 90 μL of mercury ion stock solution to 3 mL of blank PBS (pH=7.2), let stand for 30 min, and then detect with a fluorescence spectrometer. The test results showed that the maximum excitation wavelength of the fluorescent probe was 368 nm and the maximum emission wavelength was 420 nm.

[0066] Add 30 μL of probe stock solution and 0–90 μL (0, 10, 20, 30, 40, 50, 60, 70, 80, 90 μL) of mercury ion solution (1 mM mercury ion stock solution) to 3 mL of blank PBS (pH=7.2). Figure 1 The fluorescent probe for mercury ion detection shown above detects different concentrations of mercury ions (Hg). 2+ The fluorescence spectrum response diagram shows that the fluorescent probe itself has almost no fluorescence in solution, but the fluorescence at 502 nm continuously increases with the increase of mercury ion concentration, indicating that the fluorescence intensity increases linearly and regularly with the increase of mercury ion concentration.

[0067] Add 90 μL of various metal ion solutions and 30 μL of probe stock solution to 3 mL of blank PBS (pH=7.2), let stand for 30 min, and then perform fluorescence spectroscopy. Figure 2 The fluorescence spectra of the mercury ion detection fluorescent probe added to various metal solutions are shown below. The results indicate that the fluorescence intensity at 502 nm is higher than that of Hg ions. 2+ Almost no change in fluorescence was detected with the addition of other metal ions, but with the addition of Hg 2+ The fluorescence intensity then significantly increased, a phenomenon that can be observed under a handheld UV lamp. This indicates that the fluorescent probe of this invention exhibits excellent selectivity for mercury ions.

[0068] like Figure 3 The following are bar charts showing the fluorescence intensity at 502 nm before and after the mercury ion detection fluorescent probe reacts with mercury ions in the presence of different interfering metal ions (1-18: Ag + K + Cu + Na + Pb 2+ Ni 2+ Mn 2+ Cd 2+ Mg 2+ Cu 2+ Co 2+ Ca 2+ Zn 2+ Fe 2+ Fe 3+ Cr 3+ Al 3+ Hg 2+To 3 mL of blank PBS (pH=7.2), 90 μL of various metal ion solutions, 90 μL of mercury ion stock solution, and 30 μL of probe stock solution were added respectively. After standing for 30 min, fluorescence spectroscopy was performed. The results showed that the presence of other metal ions did not significantly interfere with the recognition of mercury ions by the mercury ion fluorescent probe compound of this invention.

[0069] Add 90 μL of mercury ion stock solution and 30 μL of probe stock solution to 3 mL of blank PBS (pH=7.2), and measure the fluorescence intensity sequentially from 0 to 60 minutes. Figure 4 As shown, it was found that adding Hg... 2+ The fluorescence intensity of the probe increased rapidly afterward, and then stabilized after 30 minutes. This indicates that the probe is effective against Hg. 2+ It has a fast response time.

[0070] Take PBS buffer solutions with pH values ​​of 3–8, add 90 μL of mercury ion stock solution and 30 μL of probe stock solution respectively, let stand for 30 min, and then perform fluorescence spectroscopy testing. Figure 5 As shown, the results indicate that pH values ​​of 3–8 are suitable for the action of Hg. 2+ The response was relatively stable. However, when the pH value was greater than 9, the probe accelerated hydrolysis, especially at pH 10, without the addition of Hg. 2+ Under these conditions, the probe has already undergone rapid hydrolysis. This indicates that the overly alkaline reaction system has significant background fluorescence interference, which will affect the probe's response to Hg. 2+ The detection.

[0071] Compared to other Hg 2+ Fluorescent probe, Hg prepared in this invention 2+ Fluorescent probes have three major advantages.

[0072] First, the probe molecule synthesis process is simple, and the raw materials are readily available. While ensuring high sensitivity and selectivity, the synthesis process is simple, requiring only two steps. The preparation method uses readily available raw materials, is simple, and the product is a solid powder that is easy to store and has good stability.

[0073] Second, the probe molecule exhibits high sensitivity, high selectivity, and a low detection limit. This probe molecule is related to Hg. 2+ The concentrations all showed a good linear relationship, R 2 =0.99129, with a detection limit as low as 41.25 μmol / L, a value lower than most quinoline-based Hg... 2+ Fluorescent probe. Compared with other related species, including reactive oxygen species, amino acids, and inorganic salts, this probe is highly reactive to Hg. 2+ It exhibits excellent selective recognition.

[0074] Third, the Hg prepared by this invention 2+ The fluorescent probe, 2-methyl-7-hydroxyquinoline, possesses advantages such as good optical stability, rigid planar structure, low biotoxicity, excellent water solubility, and suitable lipid solubility. These characteristics make this fluorescent probe a potential detection method in biological models such as cells.

Claims

1. A fluorescent probe for mercury ion detection based on quinoline precursor, characterized in that, Its structure is shown in Equation I below: 。 2. A method for preparing a quinoline-based mercury ion detection fluorescent probe as described in claim 1, characterized in that, Includes the following steps: First, m-aminophenol and crotonaldehyde were substituted to obtain intermediate II; then, intermediate II was used as the starting material to undergo a one-step condensation reaction with 2-methyl-7-hydroxyquinoline to obtain fluorescent probe I based on quinoline.

3. The preparation method according to claim 2, characterized in that, The preferred reaction route for preparing the quinoline-based mercury ion detection fluorescent probe is shown below: 。 4. The preparation method according to claim 2, characterized in that, The m-aminophenol, tetrachlorobenzoquinone, and hydrochloric acid were added to an organic solvent. After dissolution, the mixture was heated and crotonaldehyde solution was added dropwise while the mixture was refluxed and stirred. After the reaction was complete, the mixture was cooled to room temperature and then extracted and dried to obtain the crude product. After purification, intermediate II was obtained.

5. The preparation method according to claim 4, characterized in that, The m-aminophenol, tetrachlorobenzoquinone, and hydrochloric acid are added to an organic solvent. After dissolution, the temperature is raised to 105-110℃ and crotonaldehyde solution is added dropwise. The mixture is then refluxed and stirred for 4-5 hours.

6. The preparation method according to claim 4, characterized in that, The crotonaldehyde solution is a n-butanol solution of crotonaldehyde, and the dropping time is not less than 30 min.

7. The preparation method according to claim 2, characterized in that, The intermediate of type II and phenyl thiochloroformate were dissolved in an organic solvent, and triethylamine was added and stirred at room temperature. After the reaction was completed, the solvent was removed by vacuum distillation to obtain the crude product. The crude product was then purified to obtain fluorescent probe I.

8. The preparation method according to claim 7, characterized in that, The mixture after adding triethylamine was stirred at room temperature for 10-12 hours.

9. The application of the quinoline-based mercury ion probe of claim 1 in the detection of mercury ions.

10. The application of the quinoline-based mercury ion probe of claim 1 in the detection of mercury ions in solution.

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