A fluorescent probe for detecting divalent mercury, and a preparation method and application thereof

Abstract

This invention belongs to the field of fluorescence detection and molecular probe technology, specifically relating to a fluorescent probe for detecting divalent mercury, its preparation method, and its application. The fluorescent probe for detecting divalent mercury of this invention is a compound with the structure shown in Formula I. The fluorescent probe of this invention uses benzopyran salt as the fluorescent core and N,N-dimethylaminothiocarbamate as the recognition site, exhibiting a novel structure. When this fluorescent probe is used for the detection of divalent mercury, it exhibits single-selective recognition, high sensitivity, and a low detection limit. Furthermore, application experiments have verified that the fluorescent probe of this invention can simultaneously and effectively detect divalent mercury in traditional Chinese medicine materials, cells, live plants, and live animals. Therefore, the fluorescent probe of this invention has broad application prospects in the fields of divalent mercury detection reagents and rapid heavy metal detection.

Description

Technical Field

[0001] This invention belongs to the field of fluorescence detection and molecular probe technology, specifically relating to a fluorescent probe for detecting divalent mercury, its preparation method, and its application. Background Technology

[0002] Mercury, commonly known as quicksilver, is a non-essential heavy metal with extremely high biotoxicity. Mercury has no essential physiological functions in living organisms; instead, it extensively disrupts the structure and activity of various metalloenzymes and functional proteins by displacing essential metal ions such as iron, copper, and zinc, and by covalently binding to protein sulfhydryl groups. Furthermore, the accumulation of mercury ions can induce severe neurotoxicity, nephrotoxicity, and immunotoxicity, and is closely related to the development of various serious diseases such as Minamata disease, cognitive decline, and motor disorders. Meanwhile, with the continued growth in industrial demand for mercury and its compounds, the widespread use of mercury in metallurgy, chemical engineering, electronics, and medical devices has exacerbated pollution problems in water bodies, soil, and the atmosphere. Moreover, the bioaccumulation effect of mercury makes it one of the most concerning environmental pollutants globally.

[0003] Currently, traditional methods for mercury ion detection include atomic absorption spectrometry (AAS), inductively coupled plasma mass spectrometry (ICP-MS), and electrochemical techniques. These methods often rely on large, sophisticated instruments and involve complex sample pretreatment processes, making real-time in-situ detection difficult and significantly limiting their application in complex biological systems. In contrast, fluorescent probe technology, with its significant advantages of non-destructive detection, rapid response, and high spatiotemporal resolution, provides an effective means for dynamic and visual analysis of metal ions within living cells. While fluorescent probes currently lag behind traditional methods in terms of quantitative accuracy and detection limits, their unique advantages in biological systems (such as cells and living organisms) are irreplaceable. Therefore, developing novel fluorescent probes for mercury ions has become an important research direction for supplementing and improving existing mercury ion detection systems.

[0004] Furthermore, in recent years, with the increasing global efforts to control heavy metal pollution and the continued vigorous development of my country's traditional Chinese medicine industry, further strengthening the detection of heavy metals in Chinese medicinal materials can not only effectively monitor their safety and quality, ensure compliance with national or international standards, and promote the sustainable development of traditional Chinese medicine, but also relates to public health and safety. It is of great significance for protecting consumer health and promoting the healthy development of the traditional Chinese medicine industry.

[0005] Therefore, the technical problem to be solved by this invention is how to provide a novel fluorescent probe for the detection of divalent mercury, and how to use this fluorescent probe for the detection of divalent mercury in cells, Chinese medicinal materials and living organisms, so as to achieve rapid, simple and sensitive detection of divalent mercury. Summary of the Invention

[0006] To overcome the shortcomings of existing technologies, the present invention aims to provide a fluorescent probe for detecting divalent mercury, its preparation method, and its application. The fluorescent probe for detecting divalent mercury provided by the present invention uses benzopyran salt as the fluorescent core and N,N-dimethylaminothiocarbamate as the recognition site, enabling rapid, sensitive, and specific detection of divalent mercury in cells, traditional Chinese medicine materials, and living plants and animals.

[0007] To achieve the above objectives, a first aspect of the present invention provides a fluorescent probe for detecting divalent mercury, which is a compound with the structure shown in Formula I:

[0008] .

[0009] A second aspect of the present invention provides a method for preparing a fluorescent probe for detecting divalent mercury as described above, comprising the following steps:

[0010] (1) 4-Fluoro-2-hydroxybenzaldehyde was reacted with piperidine and triethylamine in solvent A, and then purified to obtain the intermediate compound shown in formula II;

[0011] (2) The intermediate compound shown in Formula II was reacted with 6-hydroxy-1-tetrahydronaphthone and perchloric acid in solvent B to obtain the intermediate compound shown in Formula III;

[0012] (3) The intermediate compound shown in Formula III is reacted with N,N-dimethylaminothiocarbamate chloride and triethylamine in solvent C, and then purified to obtain the fluorescent probe for detecting divalent mercury as described in Formula I.

[0013] The structural formula of the intermediate compound shown in Formula II is as follows: The structural formula of the intermediate compound shown in Formula III is: .

[0014] As a preferred embodiment, in step (1), the molar ratio of 4-fluoro-2-hydroxybenzaldehyde to piperidine and triethylamine is 1:(1.0~1.8):(0.8~1.2), the temperature is 45~55℃, and the time is 20~30h.

[0015] As a preferred embodiment, in step (2), the molar ratio of the intermediate compound shown in Formula II to 6-hydroxy-1-tetrahydronaphthone and perchloric acid is 1:(0.8~1.2):(4~5), the temperature is 85~95℃, and the time is 1~5h.

[0016] As a preferred embodiment, in step (3), the molar ratio of the intermediate compound shown in Formula III to N,N-dimethylaminothiocarbamate chloride and triethylamine is 1:(1.8~2.2):(1.8~2.2), the temperature is 20~30℃, and the time is 10~15min.

[0017] As a preferred embodiment, in step (1), solvent A is N,N-dimethylformamide; in step (2), solvent B is acetic acid; and in step (3), solvent C is anhydrous acetonitrile.

[0018] As a preferred embodiment, in steps (1) and (3), the purification is performed by column chromatography.

[0019] A third aspect of the present invention provides the application of the fluorescent probe for detecting divalent mercury as described above in the detection of divalent mercury.

[0020] In a fourth aspect, the present invention provides a kit for detecting divalent mercury, the kit comprising a fluorescent probe for detecting divalent mercury as described above and auxiliary reagents.

[0021] As a preferred embodiment, the auxiliary reagents include an organic solvent and a phosphate buffer solution; the organic solvent is dimethyl sulfoxide. The organic solvent is used to dissolve the fluorescent probe to prepare the fluorescent probe solution; the phosphate buffer solution is used to maintain the pH stability of the detection system.

[0022] A fifth aspect of the present invention provides a method for detecting divalent mercury using the fluorescent probe described above or the kit described above, comprising the following steps:

[0023] The fluorescent probe is prepared into a fluorescent probe solution; the sample to be tested is mixed with the fluorescent probe solution to obtain a mixture; the mixture is incubated and then fluorescence detection is performed, thereby realizing the detection of divalent mercury in the sample to be tested.

[0024] As a preferred embodiment, the concentration of the fluorescent probe solution is 20 μM to 1 mM; the incubation time is 10 to 480 min; and the sample to be tested is one or more of cells, traditional Chinese medicine materials, live plants, and live animals.

[0025] As a further preferred embodiment, when the sample to be tested is cells, the fluorescence detection involves performing cell fluorescence imaging on the cells and determining the content of divalent mercury in different cells by observing changes in fluorescence intensity during cell fluorescence imaging; when the sample to be tested is a traditional Chinese medicine, the fluorescence detection involves measuring the fluorescence intensity of the mixture at 620-630 nm and substituting the fluorescence intensity into a pre-established standard curve to obtain the content of divalent mercury in the traditional Chinese medicine; when the sample to be tested is a living plant or animal, the fluorescence detection involves performing in vivo fluorescence imaging on the living plant or animal and determining the content of divalent mercury in different living plants or animals by observing changes in fluorescence intensity during in vivo fluorescence imaging.

[0026] As a preferred embodiment, the medicinal materials are one or more of the following: peony bark, salvia miltiorrhiza, atractylodes lancea, atractylodes macrocephala, and honeysuckle.

[0027] As a preferred embodiment, the standard curve is a standard curve of divalent mercury concentration versus fluorescence intensity.

[0028] The technical solution of the present invention has the following advantages and beneficial effects:

[0029] The fluorescent probe for detecting divalent mercury provided by this invention uses benzopyran salt as the fluorescent core and N,N-dimethylaminothiocarbamate as the recognition site. It has a novel structure and can achieve rapid, sensitive and specific detection of divalent mercury in different samples.

[0030] The present invention provides a method for preparing a fluorescent probe for detecting divalent mercury. The method involves reacting 4-fluoro-2-hydroxybenzaldehyde, piperidine, and triethylamine to obtain an intermediate compound as shown in Formula II. This intermediate compound is then reacted with 6-hydroxy-1-tetrahydronaphthone and perchloric acid to obtain an intermediate compound as shown in Formula III. Finally, this intermediate compound is reacted with N,N-dimethylthioformyl chloride to prepare the fluorescent probe. The process of this invention is characterized by readily available raw materials, a simple route, and easy operation.

[0031] Experiments have confirmed that the fluorescent probe of this invention exhibits unique selectivity, high sensitivity, and a low detection limit (75 nM) for the detection of divalent mercury. Furthermore, its fluorescence intensity shows a good linear relationship with the concentration of divalent mercury within the range of 0-280 μM. Application experiments have also verified that when the fluorescent probe of this invention is used to detect divalent mercury in traditional Chinese medicine, a standard curve is plotted using fluorescence spectroscopy for quantitative determination. The results show that the standard curve error for divalent mercury detection is small, and the recovery rate is between 96.5% and 107.7%. Therefore, this confirms that the fluorescent probe of this invention can be used for the quantitative detection of divalent mercury in traditional Chinese medicine. Further, cell, plant, and animal in vivo imaging experiments also show a correlation between fluorescence intensity and mercury ion content, indicating that the fluorescent probe of this invention can detect divalent mercury ions in cells and living organisms in real time.

[0032] Therefore, the fluorescent probe of the present invention can simultaneously detect divalent mercury in cells, Chinese medicinal materials, living plants and living animals, and has the advantages of low cost, high sensitivity and good selectivity. It has broad application prospects in the fields of divalent mercury detection reagents and rapid detection of heavy metals. Attached Figure Description

[0033] Figure 1 The 1H NMR spectrum of the intermediate compound RDP-OH prepared in Example 1 of this invention is shown.

[0034] Figure 2 The carbon NMR spectrum of the intermediate compound RDP-OH prepared in Example 1 of this invention is shown.

[0035] Figure 3 The hydrogen nuclear magnetic resonance spectrum of the fluorescent probe RDP prepared in Example 1 of this invention is shown.

[0036] Figure 4 The carbon NMR spectrum of the fluorescent probe RDP prepared in Example 1 of this invention is shown.

[0037] Figure 5 The diagram shows the changes in the ultraviolet spectrum of the fluorescent probe prepared in Example 1 of the present invention as the concentration of divalent mercury increases when different concentrations of divalent mercury are added.

[0038] Figure 6 The graph shows the change in fluorescence spectrum of the fluorescent probe prepared in Example 1 of the present invention as the concentration of divalent mercury increases when different concentrations of divalent mercury are added.

[0039] Figure 7 The following diagram shows the fluorescence spectrum changes of the fluorescent probe prepared in Example 1 of the present invention after the addition of different cations, anions and biothiols for a selective experiment.

[0040] Figure 8The diagram shows the fluorescence spectrum changes of the fluorescent probe prepared in Example 1 of the present invention after adding divalent mercury in the presence of different cations, anions and biothiols, and the anti-interference experiment.

[0041] Figure 9 The graph shows the fluorescence intensity changes of the fluorescent probe prepared in Example 1 of the present invention with different concentrations of divalent mercury over reaction time;

[0042] Figure 10 The following diagram shows the fluorescence spectrum changes of the fluorescent probe prepared in Example 1 of the present invention before and after the addition of divalent mercury under different pH conditions;

[0043] Figure 11 The graph shows the linear relationship between fluorescence intensity and divalent mercury concentration when different concentrations of divalent mercury are added to the fluorescent probe prepared in Example 1 of the present invention.

[0044] Figure 12 The fluorescence image and fluorescence intensity of the fluorescent probe prepared in Example 1 of the present invention when detecting intracellular divalent mercury are shown.

[0045] Figure 13 The fluorescence image and fluorescence intensity of the fluorescent probe prepared in Example 1 of the present invention when detecting divalent mercury in Atractylodes lancea in vivo are shown.

[0046] Figure 14 The fluorescence detection results of the fluorescent probe prepared in Example 1 of the present invention when detecting divalent mercury in mice are shown; wherein, a is the fluorescence distribution image of each group of live mice; b is the fluorescence distribution image of each group of isolated organs; c is the average fluorescence intensity corresponding to a; d is the average fluorescence intensity of each organ in c. Detailed Implementation

[0047] The present invention will now be clearly and completely described in conjunction with the accompanying drawings and specific embodiments. It should be noted that the following embodiments are merely further illustrative of the present invention and not intended to limit it. Without conflict, the various embodiments or technical features described below can be arbitrarily combined to form new embodiments. Unless otherwise specified, the reagents and materials used in the following embodiments are commercially available.

[0048] In this invention, all solvents used were commercially available chemically pure or analytically pure. The structure of the fluorescent probe was determined using nuclear magnetic resonance (NMR) technology. The NMR measurements were performed using a Bruker NMR spectrometer, with DMSO-d6 (dimethyl sulfoxide-d6) as the solvent and TMS (dimethylsilane) as the internal standard.

[0049] Example 1

[0050] This embodiment provides a fluorescent probe for detecting divalent mercury, named RDP, with the molecular formula C0. 25 H 27 N2O2S + The structural formula is:

[0051] .

[0052] The synthetic route for preparing the fluorescent probe for detecting divalent mercury described above is shown below:

[0053] ;

[0054] The specific preparation steps of the fluorescent probe for detecting divalent mercury are as follows:

[0055] (1) Dissolve 3.5 g of 4-fluoro-2-hydroxybenzaldehyde in 20 mL of N,N-dimethylformamide. Then, under stirring at 50 °C, add piperidine (2.58 g) and triethylamine (3 mL) and mix. Reflux the mixture and stir for 24 h. After cooling to room temperature (20~30 °C), concentrate the reactants by rotary evaporation. Purify the product using a silica gel column chromatography column (eluent is a mixture of petroleum ether and ethyl acetate, PE / EA=1:1, v / v) to obtain a white solid powder, which is designated as intermediate compound 1.

[0056] (2) Dissolve intermediate compound 1 (0.1 g) and 6-hydroxy-1-tetrahydronaphthone (0.08 g) in pure acetic acid (4 mL), and continue to add perchloric acid (0.25 mL). The resulting mixture is refluxed and stirred at 90 °C for 2 h. After cooling, add 2 mL of a mixture of petroleum ether and ethyl acetate (PE / EA=1:1, v / v) and stir for 30 min. Then filter and collect the reactants to obtain a purple solid powder, which is designated as intermediate compound RDP-OH.

[0057] The intermediate compound RDP-OH obtained above was analyzed by nuclear magnetic resonance (NMR) analysis. The resulting proton NMR spectrum is shown below. Figure 1 As shown, the carbon NMR spectrum is as follows: Figure 2 As shown. Figure 1 and Figure 2 The nuclear magnetic resonance characterization results are as follows: 1HNMR (600 MHz, DMSO-d6) δ 11.13 (s, 1H), 8.60 (s, 1H), 8.11 (d, J = 8.7 Hz,1H), 7.89 (d, J = 9.4 Hz, 1H), 7.59 (dd, J = 9.4, 2.5 Hz, 1H), 7.47 (d, J =3.1 Hz, 1H), 6.95 (dd, J = 8.7, 2.4 Hz, 1H), 6.87 (d, J = 2.5 Hz, 1H), 3.79 -3.74 (m, 4H), 3.01 (s, 4H), 1.75 - 1.70 (m, 2H), 1.68 - 1.63 (m, 4H). 13 C NMR(151 MHz, DMSO-d6) δ 163.76, 163.42, 157.38, 155.08, 147.07, 145.24, 130.89,128.21, 119.98, 117.05, 116.88, 116.73, 115.16, 115.11, 96.11, 47.72, 25.89,24.99, 24.05, 23.20.

[0058] (3) The intermediate compound RDP-OH (0.93 g) and N,N-dimethylaminothiocarbamate chloride (0.23 g) were dissolved in anhydrous acetonitrile (10 mL), and then an anhydrous acetonitrile solution of triethylamine (0.258 mL) (10 mL) was added dropwise. The resulting mixture was stirred at room temperature for 10 min, and then the resulting reactant was concentrated by rotary evaporation. The product was purified by silica gel column chromatography (eluents were dichloromethane and methanol, DCM / MeOH=30:1, v / v) to obtain a purple solid powder, which is the fluorescent probe for detecting divalent mercury in this example, denoted as RDP.

[0059] The fluorescent probe RDP for detecting divalent mercury obtained above was analyzed by nuclear magnetic resonance (NMR) analysis. The resulting proton NMR spectrum is shown below. Figure 3 As shown, the carbon NMR spectrum is as follows: Figure 4 As shown. Figure 3 and Figure 4 The nuclear magnetic resonance characterization results are as follows: 1H NMR (600 MHz, DMSO-d6) δ 8.71 (s, 1H), 8.21 (d, J = 8.5 Hz, 1H), 7.97(d, J = 9.4 Hz, 1H), 7.70 (dd, J = 9.5, 2.5 Hz, 1H), 7.56 (d, J = 2.5 Hz,1H), 7.33 (dd, J = 8.5, 2.4 Hz, 1H), 7.28 (d, J = 2.4 Hz, 1H), 3.86 - 3.82(m, 4H), 3.39 (s, 3H), 3.35 (s, 3H), 3.08 (q, J = 3.8 Hz, 4H), 1.74 (q, J =6.3 Hz, 2H), 1.68 (d, J = 3.8 Hz, 4H). 13 C NMR (151 MHz, DMSO-d6) δ 185.71, 162.02, 159.26, 158.42, 156.78, 148.94, 143.81, 132.53, 127.09, 124.25, 123.78, 123.13, 121.73, 119.74, 119.35, 96.94, 49.16, 43.36, 39.24, 26.54, 26.32, 24.82, 24.22. The above analytical results show that the structure of the fluorescent probe in this invention has been confirmed using nuclear magnetic resonance analysis.

[0060] Experimental Example 1: Changes in the UV / fluorescence spectrum of divalent mercury by the fluorescent probe RDP

[0061] The detection steps for ultraviolet / fluorescence spectroscopy are as follows:

[0062] (i) The fluorescent probe (RDP) prepared in Example 1 was dissolved in dimethyl sulfoxide (DMSO) to obtain a probe stock solution with a concentration of 1 mM. A 10 mM divalent mercury stock solution was prepared by dissolving divalent mercury in pure water. A spectral solution was also prepared; the spectral solution consisted of methanol and phosphate buffer, pH=7.4, methanol / PBS=3:7, v / v.

[0063] (ii) Accurately transfer 3 mL of the spectral solution into a cuvette, then add 60 μL of the probe stock solution to the cuvette (the probe concentration after mixing is 20 μM), and add divalent mercury stock solution sequentially. The volumes of divalent mercury stock solution added are 0, 12, 24, 36, 48, 60, 72, 84, 96, 108, 120, 132, and 144 μL (the mercury concentration after mixing is 0~480 μM). After reacting for 10 min, use a UV-Vis spectrometer and a fluorescence spectrometer to test the spectra of the probe and the probe plus divalent mercury stock solution.

[0064] Figure 5 The graph shows the change in the ultraviolet spectrum of the fluorescent probe prepared in Example 1 of this invention as the concentration of divalent mercury increases when different concentrations of divalent mercury are added. Figure 5 The illustration shows the color change of sunlight before and after the reaction of the fluorescent probe with divalent mercury. (Example:) Figure 5 As shown, the fluorescent probe (RDP) prepared in Example 1 exhibits a characteristic absorption peak at 455 nm, which decreases with increasing Hg. 2+ As the concentration increased from 0 μM to 480 μM, the absorption peak gradually weakened, while a new absorption peak appeared at 580 nm, and the solution color changed from orange-yellow to pink.

[0065] Figure 6 The graph shows the change in fluorescence spectrum of the fluorescent probe prepared in Example 1 of this invention as the concentration of divalent mercury increases when different concentrations of divalent mercury are added. Figure 6 The illustration shows the change in fluorescence color before and after the fluorescent probe reacts with divalent mercury. (Example:) Figure 6 As shown, the fluorescent probe RDP itself emits weakly, and the addition of Hg... 2+ Subsequently, its fluorescence intensity at 625 nm was significantly enhanced, and the solution changed from a dark fluorescence state to a bright red fluorescence state.

[0066] The experimental results above show that the fluorescent probe of the present invention has a good recognition effect on divalent mercury.

[0067] Experiment Example 2: Selectivity and Anti-interference Test

[0068] The selective test method is as follows: Accurately transfer 3 mL of the spectral solution (same as in Experiment 1) into a cuvette, then take 60 μL of the probe stock solution (same as in Experiment 1) into the cuvette, and add the following different cations, anions, and biothiols (0. pure water control; 1. Ag) respectively. + ;2. Al 3+ 3. Ca 2+ 4. Cd 2+ 5. Co 2+ 6. Cr 3+ 7. Cu 2+ 8. Fe 2+ 9. K+ 10. Li + 11. Mg 2+ 12. Mn 2+ 13. Na + 14. Pb 2+ 15. Zn 2+ 16. HCO3 - ;17. CN - 18. SO4 2- ;19.GSH;20.Cys;21.Hcy;22.Hg 2+ 144 μL of each of the two solutions were added, and after reacting for 10 min, the fluorescence intensity at 625 nm was measured using a fluorescence spectrometer. GSH represents reduced glutathione, Cys represents cysteine, and Hcy represents homocysteine.

[0069] Figure 7 The fluorescence spectrum changes of the fluorescent probe prepared in Example 1 of this invention after the addition of different cations, anions and biothiols were performed in a selective experiment. Figure 7 In the middle, the corresponding columns from left to right are: 0. Pure water control; 1. Ag + ;2. Al 3+ 3. Ca 2+ 4. Cd 2+ 5. Co 2+ 6. Cr 3+ 7. Cu 2+ 8. Fe 2+ 9. K + 10. Li + 11. Mg 2+ 12. Mn 2+ 13. Na + 14. Pb 2+ 15. Zn 2+ 16. HCO3 - ;17. CN - 18. SO4 2- ;19.GSH;20.Cys;21.Hcy;22.Hg 2+ .like Figure 7 As shown, the fluorescence intensity changes very little after adding other metal ions to the fluorescent probe of Example 1, while the fluorescence intensity increases significantly after adding divalent mercury, indicating that the fluorescent probe of the present invention has good single selectivity for recognizing divalent mercury.

[0070] The test method for the anti-interference experiment is as follows: Accurately transfer 3 mL of the spectral solution (same as in Experiment 1) into a cuvette, then take 60 μL of the probe stock solution (same as in Experiment 1) into the cuvette, and then add the following different cations, anions, and biothiols (1. Ag) respectively. + ;2. Al 3+ 3. Ca 2+ 4. Cd 2+ 5. Co 2+ 6. Cr 3+ 7. Cu 2+ 8. Fe 2+ 9. K + 10. Li + ;11.Mg 2+ 12. Mn 2+ 13. Na + 14. Pb 2+ 15. Zn 2+ 16. HCO3 - ;17. CN - 18. SO4 2- After adding 144 μL each of the following ions (19. GSH; 20. Cys; 21. Hcy; 22. Pure water control), 144 μL of Hg was added to each ion in the system containing the interfering ions. 2 + After reacting for 10 min, the fluorescence intensity at 625 nm was measured using a fluorescence spectrometer.

[0071] Figure 8 The fluorescence spectrum changes of the fluorescent probe prepared in Example 1 of this invention after adding divalent mercury in the presence of different cations, anions and biothiols were tested for interference resistance. Figure 8 In the middle, the corresponding columns from left to right are: 1. Ag + ;2. Al 3+ 3. Ca 2+ 4. Cd 2+ 5. Co 2+ 6. Cr 3+ 7. Cu 2+ 8. Fe 2+ 9. K + 10. Li + 11. Mg 2+ 12. Mn 2+ 13. Na + 14. Pb 2+ 15. Zn 2+ 16. HCO3 - ;17. CN- 18. SO4 2- 19. GSH; 20. Cys; 21. Hcy; 22. Pure water control. (e.g.) Figure 8 As shown, after adding other potential interfering substances to the fluorescent probe of Example 1, the change in the fluorescence signal of the probe recognizing divalent mercury was very small, indicating that the fluorescent probe of the present invention has good anti-interference ability in detecting divalent mercury.

[0072] Experiment Example 3: Time Response Experiment

[0073] The time response experiment was conducted as follows: 3 mL of spectral solution (same as in Experiment 1) was accurately transferred into a cuvette, followed by 60 μL of probe stock solution (same as in Experiment 1) and then 0, 48, 96, and 144 μL of divalent mercury stock solution (same as in Experiment 1) were added. The concentrations of divalent mercury after addition were 0, 160, 320, and 480 μM, respectively. Fluorescence spectra were recorded every 1 min, and the fluorescence intensity at 625 nm was recorded.

[0074] Figure 9 The fluorescence intensity change of the fluorescent probe prepared in Example 1 of this invention with divalent mercury over reaction time. Figure 9 It can be seen that the fluorescent probe of Example 1 of the present invention responds completely to divalent mercury within 10 minutes.

[0075] Experimental Example 4: The Effect of Different pH Values ​​on Fluorescent Probes

[0076] The test procedure for the effect of different pH values ​​on the fluorescent probe was as follows: 3 mL of the spectral solution (same as in Example 1) was accurately transferred to a cuvette, followed by 60 μL of the probe stock solution (same as in Example 1), and then 144 μL of divalent mercuric stock solution (same as in Example 1). The resulting mixture was incubated for 10 min under different pH conditions, and the fluorescence spectra were recorded, including the fluorescence intensity at 625 nm. A control group was also set up using an equal volume of the spectral solution instead of the divalent mercuric stock solution.

[0077] Figure 10 The image shows the fluorescence spectrum changes of the fluorescent probe prepared in Example 1 of this invention before and after the addition of divalent mercury under different pH conditions. Figure 10 As shown, the fluorescent probe RDP of the present invention remains stable within this pH range, and the fluorescence signal does not fluctuate significantly. However, when Hg is added... 2+ Subsequently, the system exhibited significant fluorescence enhancement within the pH range of 6.00–8.00. These results demonstrate that the fluorescent probe RDP possesses good response characteristics in a pH environment close to physiological conditions.

[0078] Experimental Example 5: Establishment of Curves for Divalent Mercury Concentration versus Fluorescence Intensity

[0079] The method for establishing the curve of divalent mercury concentration versus fluorescence intensity is as follows: accurately transfer 3 mL of spectral solution (same as in Experiment 1) into a cuvette, then add 60 μL of probe stock solution (same as in Experiment 1) into the cuvette (the probe concentration after mixing is 20 μM), and then add divalent mercury stock solution sequentially. The volumes of divalent mercury added are 0, 12, 24, 36, 48, 60, 72, and 84 μL (the concentrations of divalent mercury are 0, 40, 80, 120, 160, 200, 240, and 280 μM, respectively). After reacting for 10 min, record the spectral changes using a fluorescence spectrometer and record the fluorescence intensity at 625 nm.

[0080] Figure 11 The graph shows the linear relationship between fluorescence intensity and divalent mercury concentration when different concentrations of divalent mercury are added to the fluorescent probe prepared in Example 1 of this invention. Figure 11 As shown, this invention performs linear fitting on the concentration of divalent mercury and fluorescence intensity. It can be seen that divalent mercury exhibits a good linear relationship with fluorescence intensity in the concentration range of 0-280 μM. The established equation is: y = 1.93474x + 36.75687, R0 2 =0.9827, thus the detection limit for divalent mercury is determined to be 75 nM.

[0081] Experimental Example 6: Application Experiment for the Detection of Divalent Mercury in Traditional Chinese Medicine Samples

[0082] Weigh 0.3 g of each of the following traditional Chinese medicines (Paeonia suffruticosa, Salvia miltiorrhiza, Atractylodes lancea, Atractylodes macrocephala, and Lonicera japonica), wash, dry, and pulverize (100 mesh). Place them in a digestion vessel, add 3 mL of nitric acid, and let stand overnight. Then, add 0.2 mL of perchloric acid and 0.3 mL of sulfuric acid sequentially, and heat to perform acidification digestion. After the digestion solution becomes clear, continue heating to remove the acid until the remaining volume is less than 0.5 mL. Adjust the pH of the solution to 7.4, and make up to 25 mL to obtain the sample solutions of each traditional Chinese medicine. Mix the sample solutions of the traditional Chinese medicines with methanol at a volume ratio of 7:3 to construct a detection system. Then, add the fluorescent probe RDP of Example 1 of this invention (the concentration of the fluorescent probe after addition is 20 μM) to the detection system, and then add Hg at concentrations of 20, 40, and 60 μM respectively. 2+ The standard solution was used to record the spectral signal at 625 nm. The spectral data was substituted into the equation y = 1.93474x + 36.75687 to calculate the spiked recovery rate of divalent mercury in the traditional Chinese medicine sample, as shown in Table 1.

[0083] Table 1. Results of spiked recovery of divalent mercury in traditional Chinese medicine samples

[0084] As shown in Table 1, when the fluorescent probe RDP of the present invention is used for the detection of divalent mercury in traditional Chinese medicine, the recovery rate is between 96.5% and 107.7%, and the relative standard deviation is between 2.12% and 6.09%. This demonstrates that the fluorescent probe of the present invention exhibits good recovery rate when used for the detection of divalent mercury in traditional Chinese medicine, confirming its application potential in this field.

[0085] Experiment Example 7: Cell Fluorescence Imaging Experiment

[0086] The procedure for the cell fluorescence imaging experiment was as follows: 4T1 cells (mouse breast cancer cell line) were cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS) at 37°C in an atmosphere of 5% carbon dioxide and 95% air. The cells were then placed in culture dishes and cultured for another 24 h. After culturing, the cells were washed three times with PBS (pH=7.2), pretreated with the fluorescent probe RDP (20 μM) from Example 1 for 30 min, and then different concentrations (0, 20, 40, and 60 μM) of Hg were added. 2+ The cells were cultured for 30 min. Finally, the cells were washed three times with PBS, fixed with paraformaldehyde, and images were taken using an inverted fluorescence microscope. Fluorescence intensity was recorded and significance analysis was performed. * indicates P < 0.05, ** indicates P < 0.01, and *** indicates P < 0.001, all indicating significant differences between groups.

[0087] Figure 12 The fluorescence image and fluorescence intensity of the fluorescent probe prepared in Example 1 of this invention when detecting intracellular divalent mercury. Figure 12 As shown, in this invention, 4T1 cells exhibited only weak basal fluorescence signals (a1–a3) after pretreatment with the 20 μM fluorescent probe RDP. In contrast, cells exposed to Hg... 2+ Intracellular fluorescence intensity significantly increased with increasing concentrations (20, 40, and 60 μM) (b1–b3, c1–c3, d1–d3). Statistical results of fluorescence intensity also yielded consistent conclusions. These results collectively demonstrate that RDP can sensitively and persistently detect Hg in live cells. 2+ .

[0088] Experiment Example 8: In vivo fluorescence imaging experiment of plants

[0089] The procedure for the in vivo fluorescence imaging experiment of plants was as follows: Atractylodes lancea seedlings were exposed to different concentrations (0, 20, 40, 60 μM) of Hg. 2+The seedlings were immersed in the solution for 12 hours to establish mercury-containing conditions, and then cultured with 100 μM of the fluorescent probe RDP from Example 1 for 8 hours. After rinsing with distilled water, fluorescence images of the entire seedling were captured using a Vilber Bio imaging system (λex = 530 nm, λem = 600 nm), and fluorescence intensity was recorded and significance analyzed.

[0090] Figure 13 The fluorescence image and fluorescence intensity of the fluorescent probe prepared in Example 1 of this invention when detecting divalent mercury in Atractylodes lancea in vivo. Figure 13 In the case of Atractylodes lancea seedlings being immersed in Hg with a pollution level exceeding 0.002 mg / L, 2+ The cells were immersed in solutions (0, 20, 40, 60 μM) for 12 h to establish contamination conditions, and then cultured with the 100 μM fluorescent probe RDP for 8 h. With Hg... 2+ With increasing concentration, the probe in Atractylodes lancea reacts with Hg. 2+ The fluorescence signal gradually increased. Further comparison of light flux revealed that the increasing trend of fluorescence signal in living plants was consistent with the results in cells. This indicates that the fluorescent probe RDP of this invention can monitor the level of mercury pollution in living plants.

[0091] Experiment Example 9: Animal In vivo fluorescence imaging experiment

[0092] To investigate the practical biological applicability of the fluorescent probe RDP of Example 1 of this invention, fluorescence imaging was performed on experimental mice. The mice were divided into three groups of three, and were injected with 100 μL of physiological saline (blank group), 100 μL of PBS solution (RDP group), and 100 μL of 0.5 mmol mercury ion solution (RDP+Hg group), respectively. 2+ After incubation for 10 minutes, the RDP group and the RDP+Hg group were then incubated. 2+ The groups were injected with 100 μL and 1 mmol of RDP solution, respectively, and incubated for 10 min. The administration route was intraperitoneal injection. Subsequently, fluorescence imaging was performed using an in vivo small animal imaging system. The excitation wavelength for fluorescence imaging was λ. ex The emission wavelength is 540nm, λ em The fluorescence imaging results for each group of mice were obtained at 620 nm. Subsequently, the mice were euthanized, and key internal organs were isolated and subjected to in vitro fluorescence imaging.

[0093] Figure 14 The fluorescence detection results of the fluorescent probe prepared in Example 1 of this invention when detecting divalent mercury in mice are shown; where a is the fluorescence distribution image of each group of live mice; b is the fluorescence distribution image of each group of isolated organs; c is the average fluorescence intensity corresponding to a; and d is the average fluorescence intensity of each organ in c. Figure 14As shown, no obvious fluorescence signal was observed in the blank group and the RDP group. In contrast, RDP+Hg 2+ Significant fluorescent signals were detected in the peritoneal cavity of the mice. Ex vivo imaging of internal organs of the mice showed that the fluorescence signal originated from RDP+Hg. 2+ A significant fluorescent signal was observed in the peritoneal cavity of the mice in the control group, while almost no such signal was detected in the blank control group and the RDP group alone. This confirms that the fluorescent probe of the present invention can track mercury ions in living animals and has promising prospects for the detection of mercury ions in living organisms.

[0094] In summary, the fluorescent probe provided by this invention exhibits unique selectivity, high sensitivity, and a low detection limit (75 nM) for the detection of divalent mercury. Furthermore, its fluorescence intensity shows a good linear relationship with the concentration of divalent mercury within the range of 0-280 μM. Application experiments have verified that when the fluorescent probe of this invention is used to detect divalent mercury in traditional Chinese medicine, quantitative determination of divalent mercury is achieved by plotting a standard curve using fluorescence spectroscopy. The results show that the standard curve error for divalent mercury detection is small, and the spiked recovery rate is between 96.5% and 107.7%. Further, when using the fluorescent probe of this invention to detect divalent mercury ions in cell, plant, and animal in vivo imaging, its fluorescence intensity increases with increasing divalent mercury ion concentration. Therefore, this confirms that the fluorescent probe of this invention can be used simultaneously for the quantitative detection of divalent mercury in traditional Chinese medicine, cells, plants, and animals.

[0095] The preferred embodiments of the present invention have been described in detail above. It should be understood that those skilled in the art can make numerous modifications and variations based on the concept of the present invention without creative effort. Therefore, all technical solutions that can be obtained by those skilled in the art based on the concept of the present invention through logical analysis, reasoning, or limited experimentation on the basis of existing technology should be within the protection scope of the present invention.

Claims

1. A fluorescent probe for detecting divalent mercury, characterized in that, Compounds with the structure shown in Formula I: 。 2. A method for preparing a fluorescent probe for detecting divalent mercury as described in claim 1, characterized in that, Includes the following steps: (1) 4-Fluoro-2-hydroxybenzaldehyde was reacted with piperidine and triethylamine in solvent A, and then purified to obtain the intermediate compound shown in formula II; (2) The intermediate compound shown in Formula II was reacted with 6-hydroxy-1-tetrahydronaphthone and perchloric acid in solvent B to obtain the intermediate compound shown in Formula III; (3) The intermediate compound shown in Formula III is reacted with N,N-dimethylaminothiocarbamate chloride and triethylamine in solvent C, and then purified to obtain the fluorescent probe for detecting divalent mercury as described in Formula I. The structural formula of the intermediate compound shown in Formula II is as follows: The structural formula of the intermediate compound shown in Formula III is: .

3. The method for preparing a fluorescent probe for detecting divalent mercury according to claim 2, characterized in that, In step (1), the molar ratio of 4-fluoro-2-hydroxybenzaldehyde to piperidine and triethylamine is 1:(1.0~1.8):(0.8~1.2), the temperature is 45~55℃, and the time is 20~30h.

4. The method for preparing a fluorescent probe for detecting divalent mercury according to claim 2, characterized in that, In step (2), the intermediate compound shown in Formula II reacts with 6-hydroxy-1-tetrahydronaphthone and perchloric acid in a molar ratio of 1:(0.8~1.2):(4~5), at a temperature of 85~95℃, for a time of 1~5h.

5. The method for preparing a fluorescent probe for detecting divalent mercury according to claim 2, characterized in that, In step (3), the molar ratio of the intermediate compound shown in Formula III to N,N-dimethylaminothiocarbamate chloride and triethylamine is 1:(1.8~2.2):(1.8~2.2), the temperature is 20~30℃, and the time is 10~15min.

6. The method for preparing a fluorescent probe for detecting divalent mercury according to any one of claims 2 to 5, characterized in that, In step (1), solvent A is N,N-dimethylformamide; in step (2), solvent B is acetic acid; and in step (3), solvent C is anhydrous acetonitrile.

7. The application of a fluorescent probe for detecting divalent mercury as described in claim 1 in the detection of divalent mercury.

8. A reagent kit for detecting divalent mercury, characterized in that, The kit includes the fluorescent probe for detecting divalent mercury as described in claim 1, as well as auxiliary reagents.

9. A method for detecting divalent mercury using the fluorescent probe as described in claim 1 or the kit as described in claim 8, characterized in that, Includes the following steps: The fluorescent probe is prepared into a fluorescent probe solution; the sample to be tested is mixed with the fluorescent probe solution to obtain a mixture; the mixture is incubated and then fluorescence detection is performed, thereby realizing the detection of divalent mercury in the sample to be tested.

10. The method for detecting divalent mercury using a fluorescent probe or kit according to claim 9, characterized in that, The concentration of the fluorescent probe solution is 20 μM to 1 mM; the incubation time is 10 to 480 min; and the sample to be tested is one or more of cells, traditional Chinese medicine, live plants, and live animals.

Images