Stable fluorescent probe for detecting hydrogen peroxide as well as preparation method and application of stable fluorescent probe

Abstract

The invention discloses a stable fluorescent probe for detecting hydrogen peroxide as well as a preparation method and application thereof, and belongs to the technical field of fluorescent probe materials. The probe is a compound with a structure shown in a general formula (I). According to the fluorescent probe for detecting hydrogen peroxide provided by the invention, the probe is based on a 1, 8-naphthalimide fluorophore intermediate, and the water solubility of the whole molecule is improved by introducing a hydrophilic group of a 2-(2-aminoethoxy) ethanol group; phenylboronic acid ester is used as a recognition group, so that good specific recognition of hydrogen peroxide and improvement of probe stability can be realized. The probe A reacts with other ROS, and the probe A has high specificity on H2O2. In biological imaging, the probe can be successfully applied to HeLa cells and zebrafish of targeted liver tissues, and endogenous and exogenous H2O2 in the zebrafish can be sensitively recognized at the same time.

Description

Technical Field

[0001] This invention belongs to the field of fluorescent probe materials technology, specifically relating to a stable fluorescent probe for detecting hydrogen peroxide, its preparation method, and its application. Background Technology

[0002] In biological systems, oxygen-containing free radicals with unpaired electrons and their derivatives associated with oxygen metabolism are collectively referred to as reactive oxygen species (ROS). These highly reactive molecules are chemically active oxygen-containing derivatives produced during the oxidative metabolism of aerobic organisms. The ROS family mainly includes superoxide anion (O2⁻), hydrogen peroxide (H2O2), hydroxyl radicals (OH), and singlet oxygen (¹O2). Among them, H2O2 is one of the most important ROS members, playing a central role in oxidative stress-related signal transduction, cellular homeostasis regulation, and pathophysiological processes. The balance of ROS is not only affected by the organism's own metabolism, but changes in the external environment can also disrupt this balance. This is the case in aquatic environments, where eutrophication is a major cause of rampant algal growth. Fish living in such environments for extended periods experience an abnormal increase in ROS due to MC-LR stimulation in the lake water, leading to impaired cardiopulmonary function. In fact, the harm caused by abnormal reactive oxygen species also extends to biological cells. For example, abnormal levels of H2O2 in cells can lead to various inflammations and diseases, including cancer, Alzheimer's disease, and cardiovascular diseases. Therefore, developing an efficient method for detecting hydrogen peroxide has significant academic and practical value.

[0003] The concentration of hydrogen peroxide in cells is generally around 10. -5 The range is from mM to 0.1mM. Currently, methods for detecting hydrogen peroxide include chemiluminescence, spectrophotometry, chromatography, fluorescent probe methods, and sensor analysis. For example, Fang's research group reported a ratiometric fluorescent probe, which uses gold nanoclusters synthesized with starch as a template to detect reactive oxygen species, but also responds to other reactive oxygen species; Zhao...

[36] An electrochemical sensor reported by Hufeng et al. (Hufeng, F., Huan, Y., Qi, L., Xun, F., Qunlin, Z., Junting, Z., Lili, Z., Quanbao, M., 2020. A new ratiometric fluorescent probe for specific monitoring of hROS under physiological conditions using boric acid-protected L -DOPA goldnanoclusters. Anal. Chem .(Fujia, Z., Shan, Z., Yingjie, Z., 2021. Ultrasensitive detection of hydrogen peroxide using Bi2Te3 electrochemical sensors. ACS Appl. Mater. Interfaces. 13 (3), 4761-476.) This is a novel electrochemical sensor using Bi2Te3 nanoparticles, a topological insulator, to detect hydrogen peroxide, but its preparation process is complex. Ahmed's research group reported a doped and modified semiconductor carbon-based nanomaterial for colorimetric detection of hydrogen peroxide. It uses PEG-1500 as a soft template to dope zinc ions in thin nanosheets in mesoporous g-C3N4, but its sensitivity is low (Xiangdong, B., Wei, D., Xueying, W., Shaojie, D., Shulin, Z., Dajuan, Z., Miao, W. 2019. Microcystins distribution, bioaccumulation, and microcystisgenotype succession in A fish culture pond. Sci. Total Environ. 688, 380–388.); Siddiqui reported a synthesis of nitrogen-doped graphene oxide using the Hummers method, which served as a catalyst for the oxidation of Rhoda mine B by hydrogen peroxide. However, it exhibited poor selectivity for reactive oxygen species and readily reacted with organisms or other substances in the reaction system, such as ferric ions and hypochlorous acid (Ayesha, SS, Muhammad, AA, Mian, HN, Akhtar, H., Muhammad, N., 2020. Nitrogen-doped graphene oxide as a catalyst for the oxidation of Rhoda mine B by hydrogen peroxide: application to a sensitive fluorometric assay for hydrogen peroxide. 187: 47.); Hossain et al. reported a colorimetric sensor that uses the interaction of Ti(IV) with H2O2 to form a Ti(IV)-hydrogen peroxide coordination complex to detect hydrogen peroxide. However, improper storage conditions can affect the accuracy of the results (Rayhan, H.)., Allen, A., Nicholas, FM, 2025. Designing tunable paper-based colorimetric sensor for precise detection of hydrogen peroxide vapor. ACS Omega 10 (31), 34276 – 34283.). .

[0004] In recent years, fluorescent probes using 1,8-naphthalimide as the fluorescent parent compound have become important tools for detecting various targets in analytical chemistry and biomedicine due to their excellent photochemical stability, tunable fluorescence emission wavelength, and easily functionalizable molecular structure. The synergistic effect between its rigid naphthalene ring framework and imide group not only provides good fluorescence quantum yield but also allows for the introduction of specific recognition groups at active sites such as the N- and 4-positions, providing a structural basis for highly selective and sensitive detection. However, current fluorescent probes using 1,8-naphthalimide as the fluorescent parent compound suffer from poor water solubility, high detection limits, insensitive reactions, and low stability, limiting their practical application in the complex environments of living organisms. Summary of the Invention

[0005] This invention addresses the problems of existing technologies by providing a novel fluorescent probe A for detecting hydrogen peroxide. This probe uses a phenylboronic ester as a recognition group, achieving good specificity for hydrogen peroxide and improved probe stability. Probe A reacts with other ROS, exhibiting high specificity for H2O2. In bioimaging, this probe has been successfully applied to HeLa cells and zebrafish targeting liver tissue, while sensitively identifying both endogenous and exogenous H2O2 in zebrafish.

[0006] To achieve the above-mentioned technical objectives, the technical solution adopted by the present invention is as follows: A stable fluorescent probe for detecting hydrogen peroxide, comprising a compound having the following general formula (I): .

[0007] A method for preparing a stable fluorescent probe for detecting hydrogen peroxide includes the following steps: (1) Synthesis of compound A-1: ​​7.9 mmol of 4-bromo-1,8-naphthoic anhydride was placed in a 100 mL three-necked flask, 40 mL of ethanol was added, and 4 mL of diethylene glycolamine was added dropwise. The mixture was refluxed at 75 °C for 2.5 h with stirring. After cooling to room temperature, a small amount of ethanol was added to the reaction solution, and the solvent was removed by rotary evaporation to obtain product A-1. (2) Synthesis of compound A-2: 1.36 g of compound A-1, 5.59 mmol of pinacol diborate, 1.1 g of potassium acetate, and 1.87 mmol of Pd(dppf)Cl2 were added to a 100 mL round-bottom flask; the mixture was evacuated and purged with nitrogen three times; 25 mL of 1,4-dioxane was added; the mixture was refluxed under stirring for 12 h; after the reaction was completed, the mixture was cooled to room temperature; the reaction solution was filtered; the solid was washed three times with dichloromethane; the solvent was removed from the filtrate under reduced pressure; the obtained solid was purified by column chromatography (eluent composition: DCM:EA = 5:1) to obtain solid A-2; (3) Synthesis of compound A-3: 0.353 g of compound A-2 was added to a 50 mL round bottom flask, 10 mL of ethanol and 2 mL of LDMF were added, and 5 mL of 30% hydrogen peroxide solution was added dropwise. The mixture was stirred at room temperature for 2 h, extracted with ethyl acetate, washed with saturated sodium chloride aqueous solution, dried the organic layer with anhydrous sodium sulfate, removed the solvent by rotary evaporation, and purified by column chromatography to obtain solid A-3. (4) Synthesis of target probe compound A: 0.211 g of compound A-3, 1.394 g of potassium carbonate, and 0.25 g of pinacol ester of 4-bromomethylphenylboronic acid were added to a 50 mL single-necked flask, followed by 20 mL of acetonitrile. The mixture was refluxed for 10 h with stirring. After cooling to room temperature, 20 mL of dichloromethane was added to the reaction solution. The mixture was filtered, and the residue was washed three times with 10 mL of dichloromethane. The solvent was removed from the filtrate under reduced pressure, and the crude product obtained was purified by column chromatography. The eluent composition was EA:DCM = 4.5:1.5.

[0008] The application of the fluorescent probe in the detection of hydrogen peroxide for purposes other than disease diagnosis and treatment.

[0009] Beneficial effects: This invention provides a novel fluorescent probe A for detecting hydrogen peroxide. The probe is based on a 1,8-naphthalimide fluorophore intermediate, and its overall water solubility is improved by introducing a hydrophilic group, 2-(2-aminoethoxy)ethanol. A phenylboronic acid ester is used as a recognition group, enabling good specificity for hydrogen peroxide and enhancing probe stability. Probe A reacts with other ROS, exhibiting high specificity for H2O2. Its fluorescence intensity shows a good linear relationship with H2O2 concentration (0-90 μM), with a detection limit (LOD) of 53.23 nM. In bioimaging, this probe has been successfully used in HeLa cells and zebrafish targeting liver tissue, and can sensitively identify both endogenous and exogenous H2O2 in zebrafish, specifically manifested as enhanced fluorescence signal. Attached Figure Description

[0010] Figure 1 This is the mass spectrum of compound A-1; Figure 2 This is the mass spectrum of compound A-2; Figure 3 This is the mass spectrum of compound A-3; Figure 4 This is the mass spectrum of probe compound A; Figure 5 The 1H NMR spectrum of probe compound A; Figure 6 The image shows the carbon NMR spectrum of probe compound A. Figure 7 This is a diagram illustrating the reaction mechanism between probe A and hydrogen peroxide. Figure 8 The fluorescence spectra of probe A (10 µM) and probe A with 90 µM H2O2 in PBS (pH 7.4, containing 20% ​​DMSO) buffer solution are shown. Figure 9 The reaction time variation spectrum of probe A (10 μM) and hydrogen peroxide (90 μM); Figure 10 The graph shows the fluorescence changes of probe A in the pH range of 5-9. Figure 11 The fluorescence spectrum changes of probe A (10 μM) after treatment with different concentrations of hydrogen peroxide (0-90 μM); Figure 12 Linear fitting plot of probe A with different concentrations of hydrogen peroxide (0-90 μM); Figure 13 Probe A (10 μM) was present with hydrogen peroxide (20 μM) and other reactive oxygen species (20 μM) (1:Cl). - Ions, 2: H2PO4 - Ions, 3:SO4 2- Ions, 4:HCO3 - 5: Black ion, 6: -OH ion, 7: NO2 ion - Ions, 8: 1 O2 ions, 9: NaCl - A bar chart of selective determination of ions (10:H2O2); Figure 14 Fluorescence spectra of probe A (10 μM) and other reactive oxygen species (20 μM) in PBS (pH = 7.4) containing 20% ​​DMSO. Figure 15Confocal fluorescence microscopy images of HeLa cells; Probe A (10 μM) incubated with HeLa cells for 30 min (a1–c1), Probe A (10 μM) incubated with 0.05 mM hydrogen peroxide for 30 min (a2–c2), Probe A (10 μM) incubated with 0.35 mM hydrogen peroxide for 30 min (a3–c3), Probe A (10 μM) incubated with 0.5 mM hydrogen peroxide for 30 min (a4–c4), Probe A (10 μM) incubated with 3.0 mM hydrogen peroxide for 30 min (a5–c5); Scale bar: 10 μm, blue channel (λem = 425–475 nm, λex = 402.9 nm); Figure 16 Fluorescence confocal imaging of fish liver slices; treated with 0.05 mM H2O2 (a1-c1), 0.35 mM H2O2 (a2-c2), and 3 mM H2O2 (a3-c3) for 30 min; scale bar: 300 μm, blue channel (λem = 425 – 475 nm, λex = 402.9 nm); Figure 17 The images are confocal fluorescence micrographs of zebrafish. Probe A (10 μM) was incubated with zebrafish for 30 min (a1–c1). The probe was then incubated with 1.5 μg / L MC-LR for 20 min (a2–c2), 40 min (a3–c3), 60 min (a4–c4), and 80 min (a5–c5). Scale bar: 250 μm, blue channel (λem = 425–475 nm, λex = 402.9 nm). Figure 18 The image shows a confocal fluorescence image of zebrafish. Probe A (10 μM) was incubated with zebrafish for 30 min (a1–c1), and then incubated with 0.15 μg / L MC-LR (a2–c2), 1.5 μg / L MC-LR (a3–c3), and 3.0 μg / L MC-LR (a4–c4) for 1 h. Scale bar: 250 μm, blue channel (λem = 425–475 nm, λex = 402.9 nm). Detailed Implementation

[0011] The technical solution of the present invention will be further described below with reference to specific embodiments, but it is not limited thereto.

[0012] Example 1 A method for preparing a stable fluorescent probe for detecting hydrogen peroxide includes the following steps: (1) Synthesis of compound A-1: ​​7.9 mmol of 4-bromo-1,8-naphthoic anhydride was placed in a 100 mL three-necked flask, 40 mL of ethanol was added, and 4 mL of diethylene glycolamine was added dropwise. The mixture was refluxed at 75 °C with stirring for 2.5 h. After cooling to room temperature, a small amount of ethanol was added to the reaction solution, and the solvent was removed by rotary evaporation to obtain product A-1 with a yield of 75.98% (mass spectrum shown). Figure 1 ); (2) Synthesis of compound A-2: 1.36 g of compound A-1, 5.59 mmol of pinacol diboronate, 1.1 g of potassium acetate, and 1.87 mmol of Pd(dppf)Cl2 were added to a 100 mL round-bottom flask; the mixture was evacuated and purged with nitrogen three times, and 25 mL of 1,4-dioxane was added. The mixture was refluxed for 12 h with stirring. After the reaction was completed, the mixture was cooled to room temperature. The reaction solution was filtered, and the solid was washed three times with dichloromethane. The solvent was removed from the filtrate under reduced pressure. The obtained solid was purified by column chromatography (DCM:EA = 5:1) to obtain solid A-2 (mass spectrum as shown in Figure 1). Figure 2 ), 0.6312g, yield 88%; (3) Synthesis of compound A-3: 0.353 g of compound A-2 was added to a 50 mL round-bottom flask, along with 10 mL of ethanol and 2 mL of DMF. 5 mL of 30% hydrogen peroxide solution was added dropwise. The mixture was stirred at room temperature for 2 h, extracted with ethyl acetate, washed with saturated sodium chloride aqueous solution, dried over anhydrous sodium sulfate in the organic layer, and the solvent was removed by rotary evaporation. The resulting solid was purified by column chromatography to obtain solid A-3 (mass spectrum shown in figure). Figure 3 The yield was 38.63%. (4) Synthesis of target probe compound A: 0.211 g of compound A-3, 1.394 g of potassium carbonate, and 0.25 g of pinacol 4-bromomethylphenylboronic acid were added to a 50 mL single-necked flask, followed by 20 mL of acetonitrile. The mixture was refluxed for 10 h with stirring. After cooling to room temperature, 20 mL of dichloromethane was added to the reaction solution. The mixture was filtered, and the residue was washed three times with 10 mL of dichloromethane. The solvent was removed from the filtrate under reduced pressure, and the crude product was purified by column chromatography to obtain 0.125 g of probe compound A, with a yield of 34.51% (mass spectrum shown). Figure 4 The eluent composition is EA:DCM = 4.5:1.5.

[0013] The response mechanism of probe A to hydrogen peroxide: When the probe undergoes an oxidation reaction with hydrogen peroxide, the phenylboronic ester moiety is oxidized to a phenolic hydroxyl group. This product retains the basic structure of naphthalimide, and one of its sites is oxidized and carries a negative charge. To verify the above-mentioned detection mechanism of probe A for hydrogen peroxide, the spectral characteristics of probe A before and after the addition of hydrogen peroxide were analyzed.

[0014] The test method is as follows: Probe A was dissolved in DMSO to prepare a stock solution with a concentration of 3 mmol / L, which was diluted before use. A 30% hydrogen peroxide (H₂O₂) stock solution was diluted with deionized water to prepare hydrogen peroxide solutions of 3 mM, 2 mM, 1 mM, 0.5 mM, 0.35 mM, 0.25 mM, 0.15 mM, 0.05 mM, and 0 mM. Fluorescence spectroscopy was performed in a PBS (pH = 7.4) solution containing 20% ​​DMSO. λex = 430 nm, λem = 450–700 nm, slit width 5.5 mm.

[0015] Experimental results are as follows Figure 8 In PBS (pH = 7.4, containing 20% ​​DMSO) buffer solution, probe A showed a weak fluorescence intensity at 550 nm, but after reacting with hydrogen peroxide, probe A showed a strong fluorescence intensity at 550 nm.

[0016] Example 2 Probe response time and pH adaptability The test method is as follows: Response time method: Probe A was added to a cuvette and reacted with 90 μM H2O2. Three sets of data were measured every 5 minutes using a fluorescence instrument. The fluorescence intensity tended to stabilize after about 90 minutes.

[0017] pH adaptation method: The pH values ​​were adjusted to 5.0, 6.0, 7.4, 8.0, and 9.0 using a pH meter with disodium hydrogen phosphate and sodium dihydrogen phosphate. In a PBS buffer solution system, the same concentration of probe A and 90 μM H2O2 were added respectively, and the solution was incubated for a period of time before detection.

[0018] Experimental results are as follows Figure 9-10 , Figure 9 The probe reacts with hydrogen peroxide, and as time increases, the fluorescence of the probe at 550 nm gradually increases, stabilizing at around 90 minutes.

[0019] Figure 10 This indicates that probe A exhibits the best fluorescence intensity at a weakly alkaline pH, demonstrating its suitability for in vivo detection.

[0020] Example 3 Sensitivity of the probe to hydrogen peroxide (H2O2) The test method is as follows: Different concentrations of H2O2 solution (1.5 μM, 4.5 μM, 7.5 μM, 10.5 μM, 15 μM, 30 μM, 60 μM, 90 μM, etc.) were prepared using distilled water to make a 30% H2O2 solution. The probe was prepared using 20% ​​DMSO solution. In a PBS buffer solution system, probe A and different concentrations of H2O2 were added and incubated for a period of time in the dark. The results were then detected using a fluorescence instrument.

[0021] Experimental results are as follows Figure 11-12 , Figure 11 The results showed that the fluorescence intensity of probe A at 550 nm increased with increasing hydrogen peroxide concentration. Figure 12 A good linear relationship was shown between fluorescence intensity and hydrogen peroxide concentration in the range of 0–90 μM (R² = 0.97286). The limit of detection (LOD) was calculated to be 53.23 nM using a signal-to-noise ratio of 3. The LOD is lower than that of other detection methods using hydrogen peroxide concentrations.

[0022] Example 5 Selectivity and interference resistance are key factors in evaluating probe quality and potential applications. Here, we investigate the selectivity using optical spectroscopy probe A.

[0023] The test method is as follows: Prepare solutions of 10 mM ferrous sulfate, sodium molybdate, anhydrous sodium sulfate, sodium bicarbonate, sodium hypochlorite, sodium chloride, sodium dihydrogen phosphate, sodium hydroxide, and 30% H₂O₂. Add probe A and the different solutions of the same concentration to cuvettes for detection.

[0024] like Figure 13 As shown, in multiple analytes (Cl) - H2PO4 - SO4 2- HCO3 - Black, -OH, NO2 - , 1 O2, NaCl - In the study, only hydrogen peroxide caused a significant change in the spectrum. Subsequently, the fluorescence response of probe A to hydrogen peroxide was investigated in the presence of other reactive oxygen species. Figure 14 As shown, when probe A reacts with other reactive oxygen species (20 μM), probe A has the best response to hydrogen peroxide. This result confirms that the probe has good selectivity and strong anti-interference ability.

[0025] Example 6 Confocal fluorescence imaging analysis of actual samples Cell imaging: HeLa cells were commonly cultured in DMEM medium containing 10% placenta at 37°C in a 5% CO2 incubator. The medium was changed 2-3 times daily, and cells were passaged at a 1:2 ratio and trypsinized at room temperature for 2-3 minutes. First, HeLa cells were incubated with probe A (10 μM) for 30 min, washed 2-3 times with PBS, and then divided into four groups: incubated with 0.05 μM, 0.35 μM, 0.5 μM, and 3 μM hydrogen peroxide, respectively, for 30 min. Cell fluorescence images were acquired using confocal microscopy. (Blue channel: λem = 425 – 475 nm, λex = 402.9 nm)

[0026] Hydrogen peroxide (H2O2), as a stable member of reactive oxygen species (ROS), plays a central role in oxidative stress-related signal transduction, cellular homeostasis regulation, and pathophysiological processes. Therefore, fluorescence imaging of hydrogen peroxide in living organisms is important. Probe A is used for live-cell imaging to track hydrogen peroxide levels. Figure 15 As shown, after incubation with probe A (10 μM) for 30 min, HeLa cells exhibited weak blue fluorescence, indicating that probe A has strong cell membrane penetration ability and good cytoplasmic staining effect. When further incubated with different concentrations of hydrogen peroxide (0.05, 0.35, 0.5, and 3 mM, respectively) for 30 min, HeLa cells showed gradually brightening blue fluorescence. These results demonstrate that probe A, as a highly efficient ratiometric fluorescent probe, can sensitively visualize the H2O2 level in HeLa cells.

[0027] Example 7 Fish liver slice imaging The liver was dissected from the fish and soaked in 4% paraformaldehyde for 24-48 hours, then soaked in 15% sucrose solution until it sank, and finally soaked in 30% sucrose solution overnight. First, the fish liver was incubated with probe A for 1 hour, then treated with 0.05 μM H2O2 (a2-c2), 0.35 μM H2O2 (a3-c3), and 3 μM H2O2 (a4-c4) for 30 minutes each. Fluorescence images of the fish liver sections were obtained using confocal microscopy. Scale bar 100 μm, blue channel (λem = 425 – 475 nm, λex = 402.9 nm).

[0028] Experimental results are as follows Figure 16As shown, fish liver was incubated in 10 μM probe A for 1 hour, and then reacted with different concentrations of hydrogen peroxide (0.05, 0.35 and 3 mM) for 1 hour. The results showed that the fluorescence signal in the blue channel gradually increased with the increase of hydrogen peroxide concentration, indicating that probe A can be used to visualize the hydrogen peroxide concentration in fish liver through fluorescence imaging.

[0029] Example 8 Zebrafish Imaging To study the ability of probe A to target endogenous hydrogen peroxide in zebrafish, we chose zebrafish around 6 days old as an animal model for fluorescence imaging because their genes are highly homologous to those of humans, and they are low in cost and easy to observe.

[0030] Five-day-old zebrafish were used in bioimaging experiments. The zebrafish were cultured in E3 medium in a standardized incubator at 28 °C. First, the zebrafish were incubated with probe A for 30 min as a control group. In the experimental group, after incubation with 10 μL of probe A for 30 min, the zebrafish were divided into three groups: incubated with 1.5 μg / L MC-LR for different time periods (20 min, 40 min, and 60 min), followed by washing three times with PBS solution. Glycerol was used to fix the zebrafish for imaging. Confocal fluorescence imaging (Nikon inverted research Ti2-E) was used for capture, λem = 425 – 475 nm, λex = 402.9 nm (blue channel).

[0031] The results are as follows Figure 17 As shown, Figure 17 As shown, zebrafish were first incubated with probe A for 30 minutes, and then treated with 1.5 μg / L MC-LR for different durations. The results showed that the fluorescence effect remained essentially unchanged after 1 hour of MC-LR incubation. Figure 18 As shown, after the probe was treated with zebrafish for 30 minutes, it was then treated with different concentrations of MC-LR (0.15 μg / L, 1.5 μg / L, 3.0 μg / L) for 1 hour. The results showed that the fluorescence intensity increased significantly with the increase of MC-LR concentration.

[0032] Comparative Example 1 Prepare probe compound B, with the structure shown in II: , The detection stability of probe B and probe A was compared using the following experimental method: Add probe A or probe compound B to a cuvette and react with 90 μM H2O2, respectively. Measure the data using a fluorescence instrument.

[0033] Probe A or probe compound B reacted with hydrogen peroxide under the same experimental conditions, and the changes in fluorescence intensity were detected at different time points. The results showed that the fluorescence signal of probe B increased rapidly within 40 minutes, but then showed a significant decay, with the intensity decreasing by about 30% by 120 minutes. In contrast, the fluorescence intensity of probe A remained stable under the same conditions, without significant decay. This indicates that probe A has superior detection stability and is suitable for long-term dynamic monitoring of changes in hydrogen peroxide.

[0034] It should be noted that the above embodiments are only some preferred embodiments of the present invention, and not all embodiments. Obviously, based on the above embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort should fall within the scope of protection of the present invention.

Claims

1. A stable fluorescent probe for detecting hydrogen peroxide, characterized by, It is a compound with the following general formula (I).

2. A method for preparing the stable fluorescent probe for detecting hydrogen peroxide according to claim 1, characterized by, It comprises the following steps: (1) Synthesis of compound A-1: 7.9 mmol 4-bromo-1,8-naphthalene anhydride is placed in a 100 mL three-necked flask, 40 mL of ethanol is added, 4 mL of diethylene glycol amine is added dropwise, and stirring is carried out at 75°C for 2.5 h; after cooling to room temperature, a small amount of ethanol is added to the reaction solution, and the solvent is removed by rotary evaporation to obtain the product A-1; (2) Synthesis of compound A-2: 1.36 g of compound A-1, 5.59 mmol of pinacol diboronic acid, 1.1 g of potassium acetate, and 1.87 mmol of Pd(dppf)Cl2 are added to a 100 mL round-bottom flask; vacuum and nitrogen are repeated three times; 25 mL of 1,4-dioxane is added, and stirring is carried out under reflux for 12 h; after the reaction is completed, the reaction solution is cooled to room temperature; the reaction solution is filtered, the solid is washed with dichloromethane three times, the filtrate is concentrated under reduced pressure, and the obtained solid is purified by column chromatography to obtain solid A-2; (3) Synthesis of compound A-3: 0.353 g of compound A-2 is added to a 50 mL round-bottom flask, 10 mL of ethanol and 2 mL of DMF are added, 5 mL of 30% hydrogen peroxide solution is added dropwise, stirring is carried out at room temperature for 2 h, ethyl acetate extraction is carried out, saturated sodium chloride aqueous solution washing is carried out, the organic layer is dried with anhydrous sodium sulfate, the solvent is removed by rotary evaporation, and the obtained solid is purified by column chromatography to obtain solid A-3; (4) Synthesis of target probe compound A: 0.211 g of compound A-3, 1.394 g of potassium carbonate, and 0.25 g of 4-bromomethylphenylboronic acid pinacol ester are added to a 50 mL single-necked flask, 20 mL of acetonitrile is added, and stirring is carried out under reflux for 10 h; after cooling to room temperature, 20 mL of dichloromethane is added to the reaction solution; filtration is carried out, the filter residue is washed with 10 mL of dichloromethane three times; the solvent is removed from the filtrate under reduced pressure, and the obtained crude product is purified by column chromatography to obtain probe compound A.

3. Use of the compound of formula (I) in claim 1 as a fluorescent probe for detecting hydrogen peroxide for the purpose of non-disease diagnosis and treatment.

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