A coumarin-based Hg 2+ Detection fluorescent probes, their preparation methods and applications

Abstract

This invention discloses a Hg based on dicoumarin. 2+ This invention relates to the field of biodetection technology, specifically a fluorescent probe for detecting Hg, its preparation method, and its applications. 2+ The detection fluorescent probe uses dicumarol as the parent fluorophore, and its photophysical properties are modulated by introducing different electronegative substituents (F, Cl, Br). Thiocarbonate is used as the Hg... 2+ Based on specific recognition groups, three novel fluorescent probes, DCF-Hg, DCCl-Hg, and DCBr-Hg, were designed and synthesized. This invention relates to Hg. 2+ This provides new molecular tools and strategies for convenient detection and bioimaging.

Description

Technical Field

[0001] This invention relates to the field of biodetection technology, and more specifically to a Hg-based method using dicumarol. 2+ Detection fluorescent probes, their preparation methods, and applications. Background Technology

[0002] Mercury is a highly toxic heavy metal element widely used in industrial production. However, with the intensification of industrial activities and natural processes (such as volcanic eruptions and metamorphic transformations), mercury and its compounds have caused global environmental pollution. Mercury easily accumulates in soil, water bodies, and organisms, and is passed down through the food chain, posing a serious threat to ecosystems and human health. Therefore, the development of a sensitive and rapid detection method for Hg in environmental and biological samples is crucial. 2+ The analytical methods described are of great significance for environmental monitoring and toxicological research.

[0003] Fluorescent probe technology, with its advantages of high sensitivity, ease of operation, rapid response, and the ability to achieve in-situ real-time imaging, has shown broad application potential in the detection of heavy metal ions. Among many fluorescent dyes, coumarin has become a popular choice for designing Hg-related fluorescent dyes due to its small molecular weight, good biocompatibility, and simple synthesis. 2+ An ideal molecular framework for fluorescent probes.

[0004] Therefore, a Hg based on dicoumarin is provided. 2+ The detection of fluorescent probes, their preparation methods, and applications are problems that urgently need to be solved by those skilled in the art. Summary of the Invention

[0005] In view of this, the present invention provides a Hg based on dicoumarin. 2+ Detection fluorescent probes, their preparation methods, and applications.

[0006] This invention uses dicumarol as the parent fluorophore and modulates its photophysical properties by introducing different electronegative substituents (F, Cl, Br), and uses thiocarbonate as Hg. 2+ Three novel fluorescent probes, DCF-Hg, DCCl-Hg, and DCBr-Hg, were designed and synthesized to specifically recognize Hg groups. The optical response performance of the probes was systematically evaluated using UV-Vis absorption and fluorescence spectroscopy, and DCF-Hg was selected as the best-performing probe for further research. This probe not only recognizes Hg in environmental samples but also... 2+ The detection also enabled convenient on-site visual analysis through test strip loading. Furthermore, using inverted fluorescence microscopy and laser confocal microscopy, DCF-Hg was successfully applied to Hg levels in plant root tips, live cells, and zebrafish models. 2+ Fluorescence imaging revealed its role in Hg in complex biological systems. 2+Application prospects in tracking and distribution research. This invention is for Hg 2+ This provides new molecular tools and strategies for convenient detection and bioimaging.

[0007] To achieve the above objectives, the present invention adopts the following technical solution:

[0008] A coumarin-based Hg 2+ The detection fluorescent probe has the following structural formula:

[0009] Furthermore, the aforementioned Hg based on dicoumarin 2+ The method for preparing the detection fluorescent probe includes the following steps: (1) Synthesis of Cou-F, Cou-Cl, and Cou-Br: 569 mg of 4-fluoro-2-hydroxybenzaldehyde, 634 mg of 4-chloro-2-hydroxybenzaldehyde, and 812 mg of 4-bromo-2-hydroxybenzaldehyde were weighed and placed in a reaction flask. 20 mL of anhydrous ethanol was added, and 961 mg of diethyl malonate was added while stirring. Then, 40 μL of acetic acid and 140 μL of piperidine were added, and the mixture was refluxed at 80 °C for 5 h. After the reaction was completed, the solvent was removed by rotary evaporation, and the compounds were purified by rapid column chromatography to obtain Cou-F, Cou-Cl, and Cou-Br. (2) Synthesis of DCou-F, DCou-Cl, and DCou-Br: 236.2 mg Cou-F, 253 mg Cou-Cl, 297 mg Cou-Br, 220.2 mg resorcinol, and 6.1 mg 4-dimethylaminopyridine were weighed and added to a reaction flask. After stirring at 140 °C for 3 h, 20 mL of methanol was added, and the mixture was sonicated for 15 min. The mixture was then filtered, washed with dichloromethane (DCM) and methanol, and dried to obtain DCou-F, DCou-Cl, and DCou-Br. (3) Synthesis of DCF-Hg, DCCl-Hg, and DCBr-Hg: 149.1 mg DCou-F, 315 mg DCou-Cl, and 359 mg DCou-Br were weighed into reaction flasks, 20 mL of ultra-dry dichloromethane was added, and the mixture was stirred. Then, 20 μL of N,N-diisopropylethylamine was added, and the mixture was stirred for 15 min. Then, 103.8 μL of phenyl thiochloroformate was added, and the mixture was reacted at room temperature for 30 min. After the reaction was completed, the solvent was removed by vortexing, and 5 mL of DCM and 20 mL of MeOH were added. The mixture was sonicated for 5 min, filtered, and washed with methanol to obtain DCF-Hg, DCCl-Hg, and DCBr-Hg.

[0010] Furthermore, the aforementioned Hg based on dicoumarin 2+ Detection of fluorescent probes in the detection of Hg 2+ Applications in [the context of the text].

[0011] Furthermore, the aforementioned Hg based on dicoumarin 2+ Detection of fluorescent probes in the visual detection of Hg 2+ Applications in [the context of the text].

[0012] As can be seen from the above technical solution, compared with the prior art, the present invention discloses a Hg based on dicoumarin. 2+ This paper describes the detection of fluorescent probes, their preparation methods, and applications. Based on a dicumarin fluorescent core, three probes for detecting Hg were successfully constructed by introducing different electron-withdrawing groups (F, Cl, Br) for structural modification and using thiocarbonate as a recognition group. 2+ The fluorescent probes are DCF-Hg, DCCl-Hg, and DCBr-Hg. The optical properties of these probes were tested using a UV spectrophotometer and a fluorescence spectrometer. DCF-Hg exhibited a relatively high fluorescence enhancement factor (33-fold), a low detection limit (43.7 nmol / L), and high selectivity, demonstrating excellent in vitro and in vivo detection capabilities.

[0013] In applied research, DCF-Hg has demonstrated good practicality in both environmental monitoring and bioimaging. In environmental monitoring, test strips loaded with this probe can achieve concentrations of 0-50 μmol / L Hg. 2+ Semi-quantitative detection with naked eye, and the probe is effective for detecting Hg in environmental water samples. 2+ It also exhibits good detection results. In terms of bioimaging, this probe has low cytotoxicity, making it suitable for bioimaging applications. Furthermore, it can clearly visualize Hg levels in the roots of cabbage using an inverted fluorescence microscope. 2+ The enrichment level of Hg was observed; simultaneously, using laser confocal microscopy and inverted fluorescence microscopy, respectively, the enrichment of Hg was successfully achieved in cells and zebrafish models. 2+ Real-time dynamic monitoring was conducted, and the detoxification effects of dimercaptosuccinic acid (DMSA) and glutathione (GSH) on Hg in the environment were evaluated. 2+ This provides a simple method for rapid detection and also for Hg 2+ It provides a reliable fluorescence tool for in vivo visualization monitoring and detoxification studies. Attached Figure Description

[0014] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on the provided drawings without creative effort.

[0015] Figure 1 Synthetic routes for probes DCF-Hg, DCCl-Hg, and DCBr-Hg.

[0016] Figure 2 For compound DCF-Hg 1 H NMR (600 MHz).

[0017] Figure 3 For compound DCF-Hg 13 C NMR (600 MHz).

[0018] Figure 4 For compound DCF-Hg 19 F NMR (600 MHz).

[0019] Figure 5 HRMS of compound DCF-Hg.

[0020] Figure 6 For the compound DCCl-Hg 1 H NMR (400 MHz).

[0021] Figure 7 For the compound DCCl-Hg 13 C NMR (400 MHz).

[0022] Figure 8 For the compound DCBr-Hg 1 H NMR (400 MHz).

[0023] Figure 9 For the compound DCBr-Hg 13 C NMR (400 MHz).

[0024] Figure 10 Adding Hg to different pH systems 2+ Fluorescence responses before and after: (a) DCF-Hg, (b) DCCl-Hg, (c) DCBr-Hg.

[0025] Figure 11 For probe against Hg 2+ The ultraviolet absorption spectra of (a) DCF-Hg, (b) DCCl-Hg, and (c) DCBr-Hg.

[0026] Figure 12 For probe and Hg 2+ The kinetic curves of the reaction are shown in the graphs: (a) DCF-Hg, (b) DCCl-Hg, and (c) DCBr-Hg.

[0027] Figure 13 For probe and Hg 2+ The fluorescence titration spectra of the reaction are shown in (a) DCF-Hg, (b) DCCl-Hg, and (c) DCBr-Hg.

[0028] Figure 14 The fluorescence intensity of the probe is related to different concentrations of Hg. 2+ Linear relationship diagrams between (a) DCF-Hg, (b) DCCl-Hg, and (c) DCBr-Hg.

[0029] Figure 15 For probe against Hg 2+ Selectivity test diagrams: (a) DCF-Hg, (b) DCCl-Hg, (c) DCBr-Hg; test substances 1-17 are blank test system (PBS:DMSO=1:1), Ag2SO4, CoCl2, CuCl2, FeCl3, BaCl2, CrCl3, Pb(CH3COO)2, CdSO4, MnCl2, MgCl2, KCl, ZnCl2, Cys, GSH, H2O2, Hg 2+ .

[0030] Figure 16 For probe against Hg 2+ The recognition mechanism diagram (a) and probe pair Hg 2+ The mass spectrometry mechanism verification diagram (b) is shown.

[0031] Figure 17 To detect Hg by loading test paper with probes of different concentrations of DCF-Hg. 2+ Schematic diagram of post-fluorescence color development.

[0032] Figure 18 For probes DCF-Hg and Hg in different environmental samples 2+ Linearity graphs of concentrations (0-20 µmol / L); (a) tap water, (b) lake water, (c) soil leachate.

[0033] Figure 19 The cell viability of three cell types after co-incubating with different concentrations of probe DCF-Hg for 24 h was determined.

[0034] Figure 20 For different concentrations of Hg 2+ Cell survival rate after co-incubation with HeLa cells, HepG2 cells, and A549 cells for 24 h.

[0035] Figure 21 For use containing Hg 2+Fluorescence imaging of Chinese cabbage cultured in culture medium (a); (A) 0, (B) 20 μmol / L, (C) 100 μmol / L; and fluorescence intensity corresponding to figure (a) (b).

[0036] Figure 22 For Hg 2+ Imaging images of HeLa, A549, and HepG2 (a) and the corresponding fluorescence intensity (b).

[0037] Figure 23 First, use different concentrations of Hg 2+ Cell imaging (a) after incubation with a 20 μmol / L probe; and fluorescence intensity (b) corresponding to Figure (a).

[0038] Figure 24 First use 20 μmol / L Hg 2+ Cell imaging after incubation with probes of different concentrations (a); and the corresponding fluorescence intensity (b) of Figure (a).

[0039] Figure 25 For DCF-Hg and Hg 2+ The fluorescence intensity changes over time after cell incubation (a); and the corresponding fluorescence intensity (b) for (a).

[0040] Figure 26 Image (a) showing HeLa cells incubated with different therapeutic drugs; and fluorescence intensity (a) corresponding to image (a).

[0041] Figure 27 Image (a) showing zebrafish incubated with different therapeutic drugs; and fluorescence intensity (b) corresponding to image (a). Detailed Implementation

[0042] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0043] Experimental materials required for synthesis: Petroleum ether (PE, AR), acetonitrile (AR), dichloromethane (DCM, AR), methanol (AR), ethyl acetate (EA, AR), anhydrous ethanol (AR), and acetic acid (AR) were purchased from Comio Reagent Company; 4-dimethylaminopyridine and acetic acid were purchased from Anaiji; 2-hydroxybenzaldehyde, resorcinol, and ultra-dry dichloromethane were purchased from Inokai Reagent Company; phenyl thiocarbamate was purchased from Shanghai Dipo Biotechnology Co., Ltd.; GSH and diethyl malonate were purchased from Sinopharm Chemical Reagent Co., Ltd.; 4-fluoro-2-hydroxybenzaldehyde, 4-chloro-2-hydroxybenzaldehyde, and 4-bromo-2-hydroxybenzaldehyde were all purchased from Jiangsu Aikon Reagent Company; sodium chloride and piperidine were purchased from Shanghai Test Reagent Company; filter paper was purchased from Nantong Bangjieman Filtration Material Co., Ltd.; mercuric nitrate solution standard was purchased from Weiye Metrology Institute; the nutrient solution used for cabbage cultivation was Stanley Plant Nutrient Solution; and the Grade IV creamy bok choy used in the experiment was purchased from the local farmers' market. All water used in the experiment was ultrapure water.

[0044] The human cervical cancer cells (HeLa), human lung cancer cells (A549), and human liver cancer cells (HepG2) used in the cell and zebrafish experiments were all purchased from Wuhan Pronosai Life Science Technology Co., Ltd.; DMEM medium, 0.25% trypsin, fetal bovine serum, CCK-8 kit, PBS, 1% penicillin and streptomycin solution were all purchased from Dalian Meilun Biotechnology Co., Ltd.; zebrafish embryos and E3 culture medium (containing phenylthiourea PTU) were all purchased from Nanjing Yishu Lihua Biotechnology Co., Ltd.

[0045] The experimental instruments are shown in Table 1.

[0046] Table 1. Instruments and related information involved in the experiment

[0047] Example 1: Synthesis and Characterization of Probes DCF-Hg, DCCl-Hg, and DCBr-Hg The synthetic routes for probes DCF-Hg, DCCl-Hg, and DCBr-Hg are shown below. Figure 1 .

[0048] 1) Synthesis of Cou-F, Cou-Cl, and Cou-Br: 4-Fluoro-2-hydroxybenzaldehyde (569 mg, 4 mmol), 4-chloro-2-hydroxybenzaldehyde (634 mg, 4 mmol), and 4-bromo-2-hydroxybenzaldehyde (812 mg, 4 mmol) were weighed and placed in a reaction flask. 20 mL of anhydrous ethanol was added, followed by the addition of diethyl malonate (961 mg, 6 mmol) while stirring. Then, 40 μL of acetic acid and 140 μL of piperidine were added, and the mixture was refluxed at 80 °C for 5 h. After the reaction was complete, the solvent was removed by rotary evaporation, and the mixture was purified by rapid column chromatography (VDCM:VMeOH = 200:1).

[0049] Cou-F is a white solid, 430.8 mg, yield 45.6%. 1 H NMR (400 MHz, DMSO-d6) δ 8.79 (s,1H),8.03(dd,J=8.8,6.4Hz,1H),7.45(dd,J=9.7,2.5Hz,1H),7.33(td,J=8.7,2.5Hz,1H),4.31(q,J=7.1Hz,2H),1.33(t,J=7.1Hz,3H). 13 CNMR (101MHz, DMSO-d6) δ 167.14, 164.61, 162.99, 156.59, 156.46, 156.16, 148.89, 133.19, 133.08, 116.86, 116.83, 115.50, 113.62, 113.39, 104.51, 104.25, 61.74, 14.57. Cou-Cl is a white solid, 583.0 mg, yield 57.7%. 1 H NMR (400 MHz, DMSO-d6) δ 8.78(s,1H),7.96 (d, J =8.4 Hz,1H),7.66(d, J=2.0Hz,1H),7.50(dd,J=8.3,2.0Hz,1H),4.32(q,J=7.1Hz,2H),1.07(t,J=7.0Hz,3H). 13 C NMR (101MHz, DMSO-d6) δ 162.90,155.97,155.45,148.52,139.30,132.11,125.69,118.14,117.40,116.86, 61.82,14.56. Cou-Br is a white solid, 702.3 mg, with a yield of 59.1%. 1H NMR (400 MHz, Chloroform-d) δ 8.51(s, 1H),7.54(s, 1H), 7.52–7.47(m,2H),4.42(q,J=7.1Hz,2H), 1.42 (t,J=7.1Hz,3H). 13 CNMR (101MHz, Chloroform-d) δ 162.79,156.02,155.24,148.00,130.43,128.91,128.51,120.14,118.33,116.81, 62.19,14.28. 2) Synthesis of DCou-F, DCou-Cl, and DCou-Br: Cou-F (236.2 mg, 1 mmol), Cou-Cl (253 mg, 1 mmol), Cou-Br (297 mg, 1 mmol), resorcinol (220.2 mg, 2 mmol), and 4-dimethylaminopyridine (6.1 mg, 0.05 mmol) were weighed and added to a reaction flask. After stirring at 140 °C for 3 h, the reactants turned into an orange molten state. 20 mL of methanol was added, and the mixture was sonicated for 15 min. The mixture was then filtered, washed with a large amount of DCM and methanol, and dried.

[0050] DCou-F is an orange solid, 161.0 mg, with a yield of 54.0%. 1 H NMR (400 MHz, DMSO-d6) δ 8.44(dd, J =9.2,6.1 Hz,1H),8.24(d, J =9.0Hz,1H),7.51(dd,J=9.4,2.7Hz,1H),7.36(td,J=8.7,2.6Hz,1H),6.95(d,J=9.1Hz,1H),6.81(s,1H). 13 CNMR (101MHz, DMSO-d6) δ 166.89,164.89, 164.35,157.45,156.51,156.38,155.84,155.52,152.40,132.52,132.41,131.55,114.83,113.27,113.04,112.92,107.53,105.47,105.22,103.39,102.55. DCou-Cl is an orange-yellow solid, 194.2 mg, yield 62.3%. 1H NMR (400 MHz, DMSO-d6) δ 8.36(d, J = 8.8 Hz, 1H), 8.22 (d, J = 9.1 Hz, 1H), 7.70 (d, J = 2.2 Hz, 1H), 7.51(dd, J = 8.8, 2.2 Hz, 1H), 6.94 (dd, J = 9.0, 2.5 Hz, 1H), 6.80 (d, J = 2.4Hz, 1H). 13 C NMR (101 MHz, DMSO-d6) δ 164.79, 157.44, 155.75, 155.34, 155.27, 152.21, 139.46, 131.49, 131.37, 125.37, 117.93, 114.91, 114.79, 107.57, 103.40. DCou-Br is an orange-yellow solid, 221.5 mg, with a yield of 61.7%. 1 H NMR (400 MHz, DMSO-d6) δ 8.28(d, J =8.8 Hz,1H),8.23 (d, J =9.0 Hz,1H),7.84(d,J=2.1Hz,1H),7.65(dd,J=8.8,2.1Hz,1H),6.95(dd, J =9.1, 2.5 Hz,1H),6.81(d, J=2.4Hz,1H). 13 CNMR (101MHz, DMSO-d6) δ 164.77, 157.41, 155.78, 155.31, 155.07, 152.29, 131.48, 131.32, 128.36, 128.22, 120.81, 115.15, 114.76, 107.53, 103.46, 103.38. 3) Synthesis of DCF-Hg, DCCl-Hg, and DCBr-Hg: DCou-F (149.1 mg, 0.5 mmol), DCou-Cl (315 mg, 0.5 mmol), and DCou-Br (359 mg, 0.5 mmol) were weighed into reaction flasks, respectively. 20 mL of ultra-dry dichloromethane was added, and the mixture was stirred. Then, 20 μL of N,N-diisopropylethylamine was added, and the mixture was stirred for 15 min. Next, phenyl thiochloroformate (103.8 μL, 0.75 mmol) was added, and the mixture was reacted at room temperature for 30 min. After the reaction was complete, the solvent was removed by rotary evaporation, and 5 mL of DCM and 20 mL of MeOH were added. The mixture was sonicated for 5 min, filtered, washed with copious amounts of methanol, dried, and weighed.

[0051] DCF-Hg is a white solid, 175.1 mg, with a yield of 80.6%. 1 H NMR (600 MHz, Chloroform-d) δ 8.27(dd,J=9.2,4.2Hz,1H),7.45–7.41(m,1H),7.33–7.28(m,2H),7.20–7.13(m, 3H). 19 FNMR (565MHz, Chloroform-d) δ -98.67. 13 CNMR (151MHz, Chloroform-d) δ 191.95, 156.53, 155.08, 153.82, 152.33, 150.94, 129.71, 129.64, 128.86, 128.62, 126.23, 120.55, 118.51, 112.72, 112.51, 112.36, 111.47, 110.88, 104.97, 104.81. DCCl-Hg is a white solid, 177.1 mg, yield 78.7%. 1 H NMR (400 MHz, Chloroform-d) δ 8.37(d,J=8.7Hz,1H),8.29(d,J=8.7Hz,1H),7.56–7.46(m,4H),7.41(d,J=8.6Hz,3H),7.29(d,J=2.3Hz,2H). 13 C NMR (101MHz, Chloroform-d) δ192.99, 157.66, 156.11, 155.42, 154.91, 154.83, 153.38, 151.95, 141.82, 129.93, 129.68, 129.46, 127.30, 125.73, 121.61, 119.69, 118.72, 113.73, 112.55, 106.33. DCBr-Hg is a white solid, 201.3 mg, yield 81.3%. 1 H NMR (400 MHz, Chloroform-d) δ 8.37(d,J =8.7 Hz,1H),8.20(d,J=8.8 Hz,1H),7.71(s,1H),7.63(d,J =8.7Hz,1H),7.53(t,J=7.9Hz,2H),7.44 –7.37(m,3H),7.27(s,2H). 13 C NMR (101MHz, Chloroform-d) δ 193.05, 157.62, 156.10, 155.19, 154.88, 154.74, 153.36, 130.12, 129.95, 129.67, 129.42, 128.57, 127.34, 121.74, 121.63, 119.69, 114.09, 113.71, 112.56, 106.49. Figure 2 For compound DCF-Hg 1 H NMR (600 MHz). Figure 3 For compound DCF-Hg 13 C NMR (600MHz). Figure 4 For compound DCF-Hg 19 F NMR (600 MHz). Figure 5 HRMS of compound DCF-Hg. Figure 6 For the compound DCCl-Hg 1 H NMR (400 MHz). Figure 7 For the compound DCCl-Hg 13 C NMR (400 MHz). Figure 8 For the compound DCBr-Hg 1 H NMR (400 MHz). Figure 9 For the compound DCBr-Hg 13 C NMR (400 MHz).

[0052] Example 2: Optical property testing of probes DCF-Hg, DCCl-Hg, and DCBr-Hg (1) Preparation of probe and various analyte solutions: Weigh DCF-Hg (3.48 mg, 4 mmol), DCCl-Hg (3.6 mg, 4 mmol), and DCBr-Hg (3.96 mg, 4 mmol) into 2 mL of acetonitrile to prepare a 2 mmol / L probe stock solution. Dilute 200 mmol / L standard mercuric nitrate solution with ultrapure water to 4 mmol / L for later use. Select VPBS:VDMSO=1:1, pH = 7.4 as the test system. Accurately weigh appropriate amounts of the following analytes Ag2SO4, CoCl2, BaCl2, CuCl2, MnCl2, KCl, CrCl3, Pb(CH3COO)2, CdSO4, FeCl3, ZnCl2, MgCl2, GSH, and Cys using an analytical balance to prepare a 20 mmol / L analyte solution. H2O2 solution: Dilute 30% H2O2 stock solution with ultrapure water to 20 mmol / L.

[0053] (2) The effect of probes DCF-Hg, DCCl-Hg, and DCBr-Hg on Hg under different pH conditions 2+ response Equal volumes of PBS buffer at different pH values ​​(3-11) were mixed with DMSO to prepare the test system. Then, probe stock solution was added to bring the final concentration to 10 μmol / L. Finally, 12 µL of 4 mmol / L Hg was added to each system. 2+ After reacting the solution for 20 min, the fluorescence spectra before and after the response were scanned at 25 °C.

[0054] To investigate the effect of pH on probe stability and its effect on Hg 2+ To assess the impact of Hg on identification performance and determine the optimal applicable pH range, this experiment measured the effects of three probes (DCF-Hg, DCCl-Hg, and DCBr-Hg) on ​​Hg in buffer systems ranging from pH 3 to 11. 2+ Changes in fluorescence intensity before and after. For example... Figure 10 As shown, without the addition of Hg 2+ At that time, all three probes maintained low fluorescence background throughout the entire test pH range, indicating good pH stability. (Adding Hg...) 2+ Subsequently, the fluorescence intensity of DCF-Hg remained at a high and stable level within the pH range of 6-9, demonstrating its efficacy against Hg. 2+ The three probes showed a stable response; while DCCl-Hg and DCBr-Hg exhibited relatively stable fluorescence enhancement within the pH range of 6-8. Overall, all three probes effectively recognized Hg under weakly acidic to weakly alkaline conditions (pH 6-9).2+ Among them, DCF-Hg has a slightly wider applicable pH range, making it suitable for the detection of a wider range of physiological and environmental samples.

[0055] (3) Ultraviolet spectral testing of probes DCF-Hg, DCCl-Hg, and DCBr-Hg Add 10 µL of 2 mmol / L probe stock solution to 2 mL of test system (pH=7.4), then add 12 µL of 4 mmol / L Hg. 2+ The solution was tested, and the changes in the ultraviolet spectrum were recorded.

[0056] Depend on Figure 11 As can be seen, the probes DCF-Hg, DCCl-Hg, and DCBr-Hg, when Hg is added to the system... 2+ New ultraviolet absorption peaks then appeared at 450 nm, 458 nm, and 460 nm, respectively, indicating the formation of new compounds.

[0057] (4) Probes DCF-Hg, DCCl-Hg, DCBr-Hg and Hg 2+ kinetics of the reaction Add 10 μL of probe solution to the test system (pH=7.4) to make the probe concentration 10 μmol / L, mix thoroughly, and immediately monitor the fluorescence intensity change within 30 min at the maximum emission wavelength; then add 10 μL of Hg. 2+ Solution, test system Hg 2+ The concentration was 24 μmol / L, and the fluorescence intensity at this wavelength was recorded. After the test, the data was saved.

[0058] The optimal excitation wavelength and the reaction of the fluorescent probe can be determined by ultraviolet absorption spectroscopy. Under the condition of 450 nm excitation, Hg is added. 2+ Subsequently, the fluorescence intensity of all three probe systems increased rapidly. Among them, the fluorescence intensity of probe DCF-Hg was significantly higher than that of probes DCCl-Hg and DCBr-Hg, with fluorescence enhancement factors of 33-fold, 14-fold, and 13-fold, respectively. Figure 12 It is speculated that the probe DCF-Hg may exhibit a greater fluorescence enhancement factor because the strong electron-withdrawing effect of the F atom allows for a more complete reaction within a similar time frame.

[0059] (5) Fluorescence titration spectroscopy experiments and detection limit tests of probes DCF-Hg, DCCl-Hg, and DCBr-Hg Add 10 μL of probe solution to a 2 mL PBS / DMSO test system (pH=7.4), mix thoroughly, and then gradually add 12 μL of Hg. 2+ (4 mmol / L) solution. Record the amount of Hg added at different equivalents.2+ The change in fluorescence intensity. Then, with Hg 2+ Concentration was plotted on the x-axis, and fluorescence intensity on the y-axis. Points exhibiting a linear relationship were selected for fluorescence titration curve fitting. The limit of detection (LOD) was calculated using the formula LOD = 3σ / k, where σ represents the value without Hg. 2+ The standard deviation of the fluorescence values ​​obtained from 10 measurements of the fluorescent probe solution, where k is the fluorescence probe's response to Hg. 2+ The slope of the concentration linear fitting curve.

[0060] To study the effects of probes DCF-Hg, DCCl-Hg, and DCBr-Hg on Hg 2+ The response characteristics were determined using a fluorescence spectrophotometer, and fluorescence titration spectroscopy was performed on a probe with a concentration of 10 µmol / L. Figure 13 As shown, the initial fluorescence intensities of probes DCF-Hg, DCCl-Hg, and DCBr-Hg were all low, increasing with the fluorescence intensity of Hg. 2+ As the molecules were gradually added, the fluorescence intensity of the three probes increased, but the emission wavelengths were slightly different. The emission wavelength of probe DCF-Hg was 550 nm, while the emission wavelengths of DCCl-Hg and DCBr-Hg were 560 nm.

[0061] The titration results show that within the range of 0-20 μmol / L ( Figure 14 ), Hg 2+ The concentration of the catalyst showed a linear relationship with the fluorescence of the reaction system. The linear fitting equations for DCF-Hg, DCCl-Hg, and DCBr-Hg were y = 13616x + 3204 (R²). 2 =0.99089), y = 36910x+1424 (R 2 = 0.98445), y = 30780x+2515 (R 2 = 0.98645). The calculated LODs were 43.7 nmol / L, 140 nmol / L, and 163 nmol / L, respectively. Among them, the probe DCF-Hg showed better sensitivity.

[0062] (6) Probes DCF-Hg, DCCl-Hg, and DCBr-Hg are used to identify Hg. 2+ Anti-interference capability test Prepare several 2 mL PBS / DMSO test systems (pH=7.4), add probe solution to make the concentration 10 μmol / L, and then add interfering substances and Hg respectively. 2+ The concentration of the interfering substance in the reaction system was set to 100 μmol / L. Fluorescence changes were recorded after 30 min of reaction.

[0063] To evaluate the probe's effect on Hg2+ To assess the specificity of the response, some interfering substances were selected for specificity testing, and the results were as follows: Figure 15 As shown, only when Hg is added 2+ The reaction system showed a significant increase in fluorescence intensity, while the reaction system with added interfering substances showed almost no change in fluorescence intensity and did not interfere with the probe's response to Hg. 2+ The above results indicate that the probe can identify Hg. 2+ It has relatively high specificity.

[0064] Based on the above spectral experimental results, it can be seen that probes DCF-Hg, DCCl-Hg, and DCBr-Hg are effective in recognizing Hg. 2+ The time-selectivity and stability under different pH conditions were similar. However, within a similar reaction time, the probe DCF-Hg exhibited a greater fluorescence enhancement factor and also higher sensitivity. Therefore, the probe DCF-Hg was chosen for subsequent experiments in the following study.

[0065] Example 3: Probe DCF-Hg against Hg 2+ Validation of the recognition mechanism Add probe solution to chromatographically pure acetonitrile solution to achieve a final concentration of 200 μmol / L. Then add Hg. 2+ The solution was prepared to a final concentration of 100 μmol / L, and the reaction was carried out for 20 min before analysis by high-resolution mass spectrometry (HRMS).

[0066] In this experiment, Hg was added to an acetonitrile solution containing the probe DCF-Hg. 2+ Then, analysis was performed using HRMS, and the results are as follows: Figure 16 As shown in (a), add Hg 2+ Later, due to Hg 2+ The fluorophore, being chalcophilic, coordinates with the sulfur atom, thereby reactivating the CO bond, leading to hydrolysis, the release of the fluorophore, and fluorescence recovery. HRMS analysis further validated the above recognition mechanism, such as... Figure 16 As shown in (b), Hg is added to the probe. 2+ Subsequently, in addition to detecting fragment ion peaks (C23H11FO6S, [M+Na]) of the probe itself, + (calcd m / z = 457.0152), and a fragment ion peak (C16H7FO5, [M+Na]) belonging to the newly formed fluorophore DCou-F was also observed. + (calcd m / z = 321.0169). This result confirms the interaction between the probe and Hg. 2+ A specific reaction occurred, and the expected fluorescent product was generated.

[0067] Example 4: Detection of DCF-Hg probe-loaded test paper and environmental samples (1) Hg 2+ Preparation and detection of test strips Filter paper of uniform size was immersed in DCM solution (containing 100 μmol / L probe) for 10 min. After that, the filter paper was removed and allowed to dry naturally to obtain Hg. 2+ Test strips. Then, place the test strips into Hg solutions of different concentrations. 2+ Immerse in (0, 5, 10, 20, 30, 40, 50 μmol / L) solutions for 1 min, then remove, air dry, and observe under UV light.

[0068] Using fluorescent probes in test strip detection greatly facilitates the visual assessment of whether various samples are contaminated. To explore the potential practical applications of the probes, they were loaded onto test strips and tested with different concentrations of Hg. 2+ The detection. For example... Figure 17 As shown, the blank test strip itself and the test strip loaded with only the probe are similar in color, both being blue-purple, indicating that the fluorescence of the blank test strip has little interference with the experimental results. However, immersing the test strip in 5 μmol / L Hg... 2+ After being placed in the solution, the fluorescence of the test paper turned pale yellow-white, and as Hg... 2+ As the concentration increases, the yellow color on the test paper deepens, turning bright yellow when immersed in a 50 μmol / L solution. This indicates that the probe can be used not only for Hg... 2+ The detection can also distinguish between different concentrations of Hg. 2+ The sample has great practical application value.

[0069] (2) The probe detects Hg in the environment 2+ Detection Water sample collection and processing: Water samples were collected from Donghu Lake in Baoding City. After centrifugation (to remove large suspended particles), the supernatant was filtered and the filtrate was collected for later use.

[0070] Soil sample collection and processing: Soil samples were collected from 10 cm below the surface near the wastewater treatment plant of Hebei University, at three sampling points. After natural air drying and grinding, 10 g of each soil sample was accurately weighed and placed in a beaker, 50 mL of ultrapure water was added, and the sample was sonicated for 30 min, then magnetically stirred for 2 h. The filtrate was then collected and analyzed.

[0071] Hg 2+ Preparation of standard solutions and establishment of standard curves: The detection system consisted of sample solution, PBS buffer, and DMSO in a volume ratio of 2:1:1 (pH=7.4). After adding the probe to the test system, the standard curve was established based on the obtained spectral data. Figure 14a) Calculate the Hg in the sample 2+ Content. Then, a series of solutions containing Hg at different concentrations were prepared. 2+ For test systems with solutions (0, 5, 10, 15, 20 μmol / L), add 10 μL of probe solution to the reaction system, mix well, and then measure the fluorescence intensity. Using Hg... 2+ A standard curve for environmental samples was obtained by linearly fitting the concentration on the x-axis and the fluorescence intensity on the y-axis. The recovery rate was calculated by subtracting the concentration of the unspiked sample from the concentration of the spiked sample and then dividing by the spiked concentration.

[0072] To further explore the practical application value of the DCF-Hg probe in the environment, samples of water from East Lake and soil samples from the ground below 10 cm near the wastewater treatment plant on the Hebei University campus were selected, with tap water used as a control. After treatment, the Hg content was measured using the probe. 2+ The tests were conducted. The results are shown in Table 2; Hg was not detected in any of the three samples. 2+ Furthermore, the error value was within a reasonable range. A certain concentration of Hg was further added to the environmental sample. 2+ After adjusting the concentrations (5 μmol / L, 10 μmol / L, 15 μmol / L, 20 μmol / L), the fluorescence intensity of the system was tested, and a good linear relationship was found between the spiked concentration and the concentration. Figure 18 In summary, this demonstrates that probes have high application potential in real-world environments.

[0073] Table 2. The effect of probe DCF-Hg on Hg in real-world environments. 2+ Detection

[0074] Note: "-" indicates that the test was not successful (in three tests).

[0075] Example 5 Cytotoxicity test (1) Cell culture and passage HeLa, HepG2, and A549 cells were cultured in culture flasks containing DMEM medium (supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin solution) and routinely cultured in a cell culture incubator at 37°C and 5% CO2. When the cells reached 80%–90% confluence, the old medium was discarded, the cells were washed three times with PBS, and then adherent cells were digested with 0.25% trypsin solution before passage.

[0076] (2) Cytotoxicity assay of probe DCF-Hg HeLa, HepG2, and A549 cells were seeded into 96-well plates, with 100 μL of cell suspension added to each well, at a cell density of 3 × 10⁶ cells / well. 4Cells / mL were collected, and a blank control group containing only culture medium and no cells was set up. After 24 h of cell adhesion and growth, the supernatant was discarded, and 90 μL of fresh culture medium containing different concentrations of probe were added to make final concentrations of 0, 1, 5, 10, 15, and 20 μmol / L, with 6 replicates for each group. A control group with a concentration of 0 μmol / L was used. After incubating the cells in an incubator for another 24 h, 10 μL of CCK-8 solution was added to each well, and the cells were incubated for 2 h. The absorbance at 450 nm was then measured using a microplate reader, with each well measured 3 times. Cell viability was calculated using the following formula.

[0077] Cell viability % = (OD) 实验组 -OD 空白组 ) / (OD 对照组 -OD 空白组 ).

[0078] To evaluate the cytotoxicity of the probe DCF-Hg, three cell types—HeLa, HepG2, and A549—were selected, and the CCK-8 assay was used for detection. Figure 19 As shown, when the probe concentration in the culture medium was 20 μmol / L, after co-incubation with the cells for 24 h, the survival rate of the three cell types was still above 85%, indicating that the probe has low cytotoxicity and is suitable for subsequent cell experiments.

[0079] (3) Hg 2+ The half-inhibitory concentration (IC50 value) The method is the same as step (2), except that Hg is added. 2+ The final concentrations were set to 0, 5, 10, 15, 20, 25, and 30 μmol / L. Hg was analyzed using GraphPad Prism software. 2+ The concentration logarithm was nonlinearly fitted to the inhibition rate, and the half-maximal inhibitory concentration (IC50 value) was calculated using an S-shaped dose-response curve model.

[0080] To determine Hg 2+ The cytotoxicity of Hg at different concentrations was tested using the CCK-8 assay in HeLa, HepG2, and A549 cells. 2+ Cell viability in the presence of (0 μmol / L, 5 μmol / L, 10 μmol / L, 15 μmol / L, 20 μmol / L, 25 μmol / L, 30 μmol / L). Figure 20 As shown in the figure, the IC50 values ​​for the three cell types were calculated to be 19.4 μmol / L, 20.6 μmol / L, and 22.6 μmol / L, respectively.

[0081] Example 6: The effect of probe DCF-Hg on Hg in plant roots 2+Detection (1) Seed treatment and germination The test plant was *Cabbage de Chine*, and the seeds were purchased from a local farmers' market. Plump and uniform seeds were selected, surface-sterilized with 70% ethanol for 30 seconds, and immediately rinsed three times with sterile water. The sterilized seeds were then placed at a constant temperature of 37℃ for 40 minutes to germinate, and subsequently wrapped in moist gauze and germinated at room temperature in the dark.

[0082] (2) Seedling culture and Hg 2+ deal with After the seeds sprout, select healthy seedlings with uniform growth and transfer them to the opening of centrifuge tubes containing Stanley plant nutrient solution. Cover the opening with a single layer of gauze so that the seedling roots extend vertically into the nutrient solution. Spray with sterile water regularly to keep the soil moist until the roots are fully submerged. The cultivation temperature is 23℃, and incandescent lamps are used for illumination. [The last sentence appears to be incomplete and unrelated to the preceding text. It likely refers to a specific nutrient solution and its dosage, but without further context, a precise translation is impossible.] 2+ The seedlings were divided into 5 groups with final concentrations of 0, 10, 20, 50, and 100 μmol / L, respectively. Each group had 3 biological replicates, and each replicate contained 3 seedlings. Seedling growth parameters were recorded starting the day after the seeds showed signs of germination, and seedlings were exposed to different concentrations of Hg. 2+ Continue culturing in the nutrient solution.

[0083] (3) Root staining and fluorescence imaging Journey to the West (Hg) 2+ Four days after treatment, the root tips of Chinese cabbage seedlings were carefully harvested. The root tip tissue was placed in PBS buffer containing 10 μmol / L DCF-Hg probe and incubated at 37°C in the dark for 40 min. After incubation, the tissue was gently rinsed three times with ultrapure water to remove unbound probe. The root tip tissue was transferred to a confocal dish and observed under an inverted fluorescence microscope. Fluorescence images were acquired using a GFP filter (excitation wavelength 488 nm, emission wavelength 510-530 nm).

[0084] Hg 2+ As a typical heavy metal pollutant, it can be absorbed and accumulated by plants, thereby affecting their normal growth and development. The four-season butter bok choy has a short growth cycle and is widely consumed, making it an ideal model crop for studying the toxicity of heavy metals in plants.

[0085] This experiment used different concentrations of Hg 2+ Treatment of Chinese cabbage seedlings with (0, 10, 20, 50, 100 μmol / L) to investigate Hg levels 2+ The effects on its growth and the plant's response to Hg 2+ Tolerance levels and visualization of Hg using a DCF-Hg probe. 2+ Distribution in the root region. As shown in Table 3, with Hg 2+With increasing treatment concentration, the seedling height, taproot length, and number of lateral roots of Chinese cabbage seedlings all gradually decreased, exhibiting a significant concentration-dependent inhibitory effect. Even at 100 μmol / L Hg... 2+ The seedlings survived and maintained slow growth even after treatment, indicating that Chinese cabbage is resistant to Hg. 2+ It has a certain enrichment capacity and tolerance.

[0086] Table 3. At different concentrations of Hg, cabbage seeds begin to sprout. 2+ Seedling height, taproot and lateral root growth in the culture medium

[0087] Note: Nine seeds are used for each concentration. "-" indicates that the concentration cannot be measured.

[0088] To further observe Hg 2+ The distribution of the root system and its damage to the root tip structure were investigated using fluorescent staining with a probe. The results are as follows: Figure 21 As shown. Control group (0 μmol / L Hg) 2+ The root tip fluorescence signal was extremely low, almost undetectable, but the root tip structure was clearly visible under bright field: the root cap was intact, the cells in the meristematic and elongation zones were tightly arranged and clearly layered, and the epidermal cells were smooth and intact. (20 μmol / L Hg) 2+ After treatment, a distinct green fluorescence signal appeared at the root tip, indicating that Hg... 2+ It has been enriched in the root tissue; at the same time, the root tip morphology shows slight damage, the cells in the meristematic and elongation zones are slightly swollen and loosely arranged, the root cap outline is blurred, and the overall root length is thinner than the control group. 100 μmol / L Hg 2+ After treatment, the fluorescence signal was significantly enhanced, and the root tip structure was severely damaged: the root cap almost disappeared, epidermal cells ruptured and sloughed off, cells were arranged in a disordered manner, and the root tip was twisted, deformed, and darkened in color, indicating a high concentration of Hg. 2+ It causes severe damage to the root tip tissue.

[0089] The above results indicate that the probe DCF-Hg can effectively trace Hg. 2+ Distribution of Hg in plant roots, and 2+ The toxic damage to the root tips of Chinese cabbage is concentration-dependent, providing a visualization tool for monitoring heavy metal pollution in plants.

[0090] Example 7: The effect of probe DCF-Hg on Hg in cells 2+ Imaging The following cells were imaged using laser confocal microscopy at λex = 488 nm and λem = 490-590 nm.

[0091] (1) The probe's effect on Hg in HeLa, A549, and HepG2 cells2+ Imaging Add Hg to the small dishes inoculated with HeLa, A549, and HepG2 cells respectively. 2+ After incubating in a cell culture incubator for 50 min with 20 μmol / L medium, the medium was removed, the cells were washed three times with PBS buffer, and then medium containing the probe (20 μmol / L) was added. After incubation for 40 min, the cells were washed three times with PBS buffer, and then 1 mL of medium was added before imaging was performed.

[0092] Hg 2+ Once inside the cell, Hg binds to it, thereby reducing its harmful effects on the cell. Therefore, using Hg... 2+ Imaging was performed after incubating HeLa, A549, and HepG2 cells, respectively. Figure 22 As shown, the probe exhibits the highest fluorescence intensity on HeLa cells. This may be because HepG2 cells, as liver cancer cells, express GSH and metallothionein more strongly, enabling them to bind more Hg. 2+ This effectively quenched the fluorescence of the probe, resulting in the lowest fluorescence intensity. A549 cells showed the second highest intensity, while HeLa cells exhibited the lowest expression level of the relevant protein, thus showing the strongest fluorescence signal.

[0093] (2) The probe DCF-Hg affects intracellular Hg 2+ Sensitivity experiment To further investigate the detection of Hg by the probe in cells 2+ Two sets of experiments were conducted to assess the sensitivity.

[0094] (a) Add Hg of different concentrations to the inoculated HeLa cells. 2+ The culture media (0, 5, 10, 15, 20 μmol / L) were incubated in an incubator for 50 min. After incubation, the culture media were removed, the sample was washed three times with PBS buffer, and then culture media containing the probe (20 μmol / L) was added. After incubation for 40 min, the culture media without the probe was replaced, and then imaging was performed.

[0095] Depend on Figure 23 It can be seen that cells exposed to a low concentration (5 μmol / L) of Hg... 2+ After incubation and the addition of the fluorescent probe, a significant fluorescence signal was still generated, and this was observed even after incubation with different concentrations of Hg. 2+ The incubated cells can be distinguished by the probe.

[0096] (b) Using the method described in (a) above, with Hg-containing 2+After incubating in a medium of 20 μmol / L for 50 min, the medium was replaced with medium containing different concentrations of probe (0, 1, 5, 10, 15, 20 μmol / L), and imaging was performed after incubation for 40 min.

[0097] Figure 24 As shown, in Hg 2+ At a constant concentration, no fluorescence was produced in cells when the probe concentration was 0. However, incubation of HeLa cells with 1 μmol / L DCF-Hg resulted in weak fluorescence intensity, which increased with increasing probe concentration. This indicates that the probe possesses high sensitivity and is effective in detecting Hg in vivo. 2+ Its potential.

[0098] (3) Long-term imaging of probe DCF-Hg in cells HeLa cells were seeded in confocal culture dishes and cultured at 37°C in a 5% CO2 incubator until the cells adhered well and reached approximately 80%-90% confluence. The old culture medium was discarded, and a solution containing 20 μmol / L Hg was added. 2+ The culture was incubated in fresh culture medium for 50 min. Then, the probe was added to the culture system to a final concentration of 20 μmol / L, gently mixed, and immediately the dish was placed on the stage of a laser confocal microscope. Time-series imaging was performed within 36 min after probe addition, with fluorescence images acquired every 4 min for a total of 10 time points (0, 4, 8, 12, 16, 20, 24, 28, 32, and 36 min). The changes in intracellular fluorescence intensity at each time point were analyzed to assess Hg levels. 2+ The dynamic distribution and accumulation process within cells.

[0099] To investigate the effect of the probe on Hg in living cells 2+ The real-time response process and photostability of HeLa cells were recorded upon the addition of Hg. 2+ Fluorescence changes over the next 36 minutes. (Example:) Figure 25 As shown, almost no fluorescence was initially observed in the cells; a weak fluorescence signal appeared at 4 min; the fluorescence intensity gradually increased with prolonged incubation time, reaching its maximum intensity at approximately 24 min; and remained stable for the subsequent 12 min. This result indicates that the probe interacts with Hg... 2+ The fluorophores generated by the reaction exhibit good photostability within the cells.

[0100] (4) The probe DCF-Hg on mercury poisoned cells Hg 2+ In situ imaging HeLa cells were seeded in confocal culture dishes and cultured at 37°C in a 5% CO2 incubator until the cells adhered well and reached approximately 80%-90% confluence. Four experimental groups were set up: a blank control group, a model group, a DMSA intervention group, and a GSH intervention group. The blank control group received no treatment; the other three groups were first treated with a solution containing 20 μmol / L Hg. 2+ Cells were incubated with culture medium for 50 min to establish a mercury poisoning cell model. Subsequently, the model group was incubated with a probe at a final concentration of 20 μmol / L for another 40 min. The DMSA intervention group and GSH intervention group were incubated with DMSA at a final concentration of 50 μmol / L or GSH at a final concentration of 1 mmol / L, and a probe at a final concentration of 20 μmol / L, respectively, for a total of 40 min. After treatment, the culture medium in each well was discarded, and the cells were gently washed three times with PBS buffer. 1 mL of fresh culture medium was added to each dish. Immediately, the cells were imaged under a laser confocal microscope to observe and compare the differences in intracellular fluorescence intensity among the groups, evaluating the effects of DMSA and GSH on Hg. 2+ The cleaning effect.

[0101] To investigate the use of probes to detect the clearance of Hg from cells by different analytes 2+ The ability to detoxify poisoned cells was assessed using DMSA and GSH. DMSA is a commonly used hemoglobin in clinical practice. 2+ Chelating agents, while GSH can be produced endogenously by cells and has low biotoxicity, thus both have certain research value. Results are as follows... Figure 26 As shown, DMSA exhibits lower fluorescence intensity and the best detoxification effect. DMSA is Hg. 2+ Classic specific thiol chelating agents for detoxification contain two highly active thiol groups in their molecules, which can bind with Hg. 2+ It forms stable, water-soluble chelates and is expelled from the cell, while GSH and Hg... 2+ Its binding ability is weaker than that of DMSA, therefore its detoxification ability is worse than that of DMSA.

[0102] Example 8 Imaging experiment of probe DCF-Hg in zebrafish Zebrafish, as a model organism, are widely used in in vivo imaging research due to their transparent embryos and larvae, short reproductive cycle, and high genetic homology with humans. (The last sentence appears to be incomplete and unrelated to the preceding text. It likely refers to Hg in water.) 2+ Hg can easily accumulate through the food chain, and fish, as a daily food source for humans, have high levels of Hg in their bodies, especially in heavily polluted areas. 2+ The enrichment problem is particularly prominent. Therefore, this experiment used 4-day-old zebrafish juveniles as a model to investigate Hg. 2+ Distribution within organisms and the effects of drug intervention.

[0103] Four-day-old zebrafish juveniles were randomly divided into four groups: a blank control group, a model group, a DMSA intervention group, and a GSH intervention group. All groups were cultured and treated with E3 medium. The blank control group received no treatment; the other three groups were initially treated with a medium containing 20 µmol / L Hg. 2+ The zebrafish were incubated in E3 culture medium for 40 min to induce mercury exposure. Subsequently, the model group was incubated with a probe at a final concentration of 20 µmol / L for 40 min; the DMSA intervention group and GSH intervention group were incubated with E3 culture medium containing 40 µmol / L DMSA or 50 µmol / L GSH, respectively, along with a 20 µmol / L probe, and incubated together for 40 min. After treatment, the zebrafish in each group were gently rinsed three times with fresh E3 culture medium to remove residual probe and drug.

[0104] After cleaning, zebrafish were placed under an inverted fluorescence microscope and imaged using a GFP filter channel (excitation wavelength 488 nm, emission wavelength 510-530 nm). The differences in fluorescence intensity among the juvenile fish in each group were observed and compared to evaluate the effects of DMSA and GSH on Hg. 2+ The cleaning effect.

[0105] To better investigate the detoxification effects of different drugs at the individual level, 4-day-old zebrafish were used with 20 µmol / L Hg. 2+ After incubation, it was detoxified with DMSA and GSH, and the results were as follows. Figure 27 As can be seen, DMSA still has a good detoxification ability in zebrafish, while GSH also has a certain detoxification ability. GSH can be produced endogenously by cells, so its toxicity is relatively low, and it can be used as a good auxiliary detoxification agent.

[0106] The above description of the disclosed embodiments enables those skilled in the art to make or use the invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A Hg based on dicoumarin 2+ The detection fluorescent probe is characterized by, Its structural formula is as follows: 。 2. The Hg based on dicoumarin as described in claim 1 2+ A method for preparing a fluorescent probe for detection, characterized in that, Includes the following steps: (1) Synthesis of Cou-F, Cou-Cl, and Cou-Br: 569 mg of 4-fluoro-2-hydroxybenzaldehyde, 634 mg of 4-chloro-2-hydroxybenzaldehyde, and 812 mg of 4-bromo-2-hydroxybenzaldehyde were weighed and placed in a reaction flask. 20 mL of anhydrous ethanol was added, and 961 mg of diethyl malonate was added while stirring. Then, 40 μL of acetic acid and 140 μL of piperidine were added, and the mixture was refluxed at 80 °C for 5 h. After the reaction was completed, the solvent was removed by rotary evaporation, and the compounds were purified by rapid column chromatography to obtain Cou-F, Cou-Cl, and Cou-Br. (2) Synthesis of DCou-F, DCou-Cl, and DCou-Br: 236.2 mg Cou-F, 253 mg Cou-Cl, 297 mg Cou-Br, 220.2 mg resorcinol, and 6.1 mg 4-dimethylaminopyridine were weighed and added to a reaction flask. After stirring at 140 °C for 3 h, 20 mL of methanol was added, and the mixture was sonicated for 15 min. The mixture was then filtered, washed with dichloromethane and methanol, and dried to obtain DCou-F, DCou-Cl, and DCou-Br. (3) Synthesis of DCF-Hg, DCCl-Hg, and DCBr-Hg: 149.1 mg DCou-F, 315 mg DCou-Cl, and 359 mg DCou-Br were weighed into a reaction flask, 20 mL of ultra-dry dichloromethane was added, and the mixture was stirred. Then, 20 μL of N,N-diisopropylethylamine was added, and the mixture was stirred for 15 min. Then, 103.8 μL of phenyl thiochloroformate was added, and the mixture was reacted at room temperature for 30 min. After the reaction was completed, the solvent was removed by vortexing, and 5 mL of DCM and 20 mL of MeOH were added. The mixture was sonicated for 5 min, filtered, and washed with methanol to obtain DCF-Hg, DCCl-Hg, and DCBr-Hg.

3. The Hg based on dicoumarin as described in claim 1 2+ Detection of fluorescent probes in the detection of Hg 2+ Applications in [the field].

4. The Hg based on dicoumarin as described in claim 1 2+ Detection of fluorescent probes in the visual detection of Hg 2+ Applications in [the field].

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