Fluorescent probe for detecting hydroxyl radicals as well as preparation method and application of fluorescent probe

Abstract

The invention provides a fluorescent probe for detecting hydroxyl radicals as well as a preparation method and application thereof, and the preparation method comprises the following steps: (S10) synthesizing a first intermediate compound by taking 2, 3, 3-trimethyl-3H-indoline and methyl iodide as raw materials; (S20) synthesizing a second intermediate compound by taking phosphorus trichloride, cyclohexanone and anhydrous dimethylformamide as raw materials; (S30) mixing and reacting the first intermediate compound and the second intermediate compound to synthesize a compound IR780; and (S40) synthesizing OH-probe on the basis of the IR780. The prepared OH-probe has extremely high sensitivity and excellent selectivity on hydroxyl radicals, can be effectively used for monitoring and mapping the level of the hydroxyl radicals, and can be applied to detection of atherosclerosis, obesity, cancers, neurodegenerative diseases, inflammatory diseases and other free radical related diseases. Wide development prospects and practical application values are realized.

Description

Technical Field

[0001] The invention relates to the technical field of biomedical detection, and in particular to a fluorescent probe for detecting hydroxyl free radicals and a preparation method and application thereof. Background Art

[0002] Atherosclerosis and obesity are major health problems worldwide, which not only affect the quality of life, but are also closely related to the development of a variety of chronic diseases. Through the study of the pathological mechanisms of these diseases, it is found that the pathological processes of these diseases are complex, involving a variety of biochemical and molecular mechanisms, among which free radicals, especially hydroxyl free radicals, play a key role.

[0003] For example, the pathological mechanism of atherosclerosis is as follows: Atherosclerosis is a disease that affects arterial blood vessels, characterized by the accumulation of lipids and fibrous tissue in the vessel wall, leading to vascular hardening, stenosis, and even occlusion. In this process, hydroxyl radicals promote the formation and development of plaques by oxidizing low-density lipoprotein (LDL). In addition, hydroxyl radicals can activate vascular endothelial cells, induce inflammatory responses, and aggravate vascular damage. The pathological mechanism of obesity is as follows: Obesity is the result of the combined effects of genetic and environmental factors. Its core feature is the abnormal increase of adipose tissue in the body. Obesity is associated with a variety of metabolic disorders, such as type 2 diabetes, heart disease, and hypertension. In the pathological process of obesity, hydroxyl radicals aggravate cell dysfunction and insulin resistance by promoting inflammatory responses and oxidative stress.

[0004] Free radicals are highly reactive molecules with unpaired electrons that can damage cellular components such as lipids, proteins, and DNA. Hydroxyl radicals, as one type of free radical, are extremely reactive and are produced by metabolic processes in the body or by external environmental factors (such as tobacco smoke, pollutants, etc.). Excessive production and accumulation of hydroxyl radicals are considered to be important driving factors in the development of many diseases.

[0005] Although the role of hydroxyl radicals in disease has been widely recognized, it is challenging to accurately detect and quantify hydroxyl radicals in vivo due to their high reactivity and extremely short life span. Although current detection methods such as electron spin resonance (ESR) and various chemical probes provide certain detection means, they are often limited by the complexity of the equipment, high cost or strict requirements on environmental conditions, making the detection of hydroxyl radicals not widely used. Therefore, a more efficient and easy-to-use fluorescent probe is urgently needed to overcome the shortcomings of existing technologies and better serve clinical services. Summary of the invention

[0006] One advantage of the present invention is that it provides a fluorescent probe for detecting hydroxyl radicals, and a preparation method and application thereof. Through a simple, sensitive and highly biocompatible new fluorescent probe, the problem of insufficient sensitivity and specificity in detecting hydroxyl radicals in the prior art can be overcome, the technical level of hydroxyl radical detection is improved, and a more effective tool is provided for research and clinical application, which has high application value and broad application prospects.

[0007] Another advantage of the present invention is that it provides a fluorescent probe for detecting hydroxyl radicals and its preparation method and application. The fluorescent probe OH-probe can monitor and detect hydroxyl radicals in vivo in real time, accurately, quantitatively, efficiently and specifically, which is of great significance for improving the diagnosis and treatment of diseases such as atherosclerosis and obesity.

[0008] Another advantage of the present invention is that it provides a fluorescent probe for detecting hydroxyl radicals, and a preparation method and application thereof. The OH-probe probe is based on the IR780 molecular structure and introduces specific functional groups through chemical modification so that it can specifically react with hydroxyl radicals, which not only improves the selectivity of the probe, but also enhances its stability and penetration ability in the body.

[0009] Another advantage of the present invention is to provide a fluorescent probe for detecting hydroxyl radicals, and a preparation method and application thereof. The preparation method of the fluorescent probe is simple, has high synthesis efficiency, good repeatability and operability, and the purity of the fluorescent probe prepared by the method is high.

[0010] Another advantage of the present invention is that it provides a fluorescent probe for detecting hydroxyl radicals, and a preparation method and application thereof, wherein the preparation method of the fluorescent probe introduces a hydroxyl radical-sensitive structure by reacting IR780 with a specific precursor compound, and then through a series of optimized reaction conditions, the synthesis efficiency and product purity can be ensured, and the product is purified by high performance liquid chromatography (HPLC), which can improve the quality of the fluorescent probe.

[0011] Another advantage of the present invention is that it provides a fluorescent probe for detecting hydroxyl radicals, and a preparation method and application thereof. Since the OH-probe has extremely high sensitivity and excellent selectivity to hydroxyl radicals, it can specifically react with hydroxyl radicals at low concentrations, so that its fluorescence intensity is significantly enhanced, thereby achieving quantitative detection of hydroxyl radicals, which is helpful for early diagnosis and pathological research.

[0012] Another advantage of the present invention is that it provides a fluorescent probe for detecting hydroxyl radicals and its preparation method and application. The OH-probe probe can be used to study other free radical-related diseases such as atherosclerosis and obesity, and provides an important tool for studying the pathological mechanisms of these diseases by monitoring and mapping the level of hydroxyl radicals.

[0013] Another advantage of the present invention is that it provides a fluorescent probe for detecting hydroxyl radicals, and a preparation method and application thereof. The OH-probe has good biocompatibility within the concentration range used in biology and has no toxic side effects, which provides a guarantee for its safety in clinical applications.

[0014] According to one aspect of the present invention, the present invention provides a method for preparing a fluorescent probe for detecting hydroxyl radicals, comprising the following steps: (S10) using 2,3,3-trimethyl-3H-indoline and iodomethane as raw materials to synthesize a first intermediate compound; (S20) using phosphorus trichloride, cyclohexanone and anhydrous dimethylformamide as raw materials to synthesize a second intermediate compound; (S30) mixing the first intermediate compound and the second intermediate compound to react to synthesize compound IR780; and (S40) Synthesis of OH-probe based on the IR780.

[0015] The step (S30) comprises the following steps: (S301) dissolving the first intermediate compound and the second intermediate compound in a mixed solvent of n-butanol and benzene; (S302) the mixture is refluxed in a round-bottom flask for reaction; (S303) after reflux, it is dried under vacuum to obtain a green solid; and (S304) purified using silica gel column chromatography to obtain compound IR780.

[0016] Wherein in the step (S301), the mass ratio of the first intermediate compound to the second intermediate compound is 2:1-4:1, and the mixing volume ratio of n-butanol to benzene is 7:3-9:1.

[0017] The step (S40) comprises the following steps: (S401) dissolving IR780 in methanol; (S402) controlling the temperature of the reaction system to be maintained at 0°C in an ice bath; (S403) gradually adding sodium borohydride and stirring the reaction at 0°C; (S404) monitoring the reaction progress using thin layer chromatography; (S405) after the reaction is completed, extracting the mixture using dichloromethane and saturated brine, and collecting the organic phase; and (S406) purifying using ethyl acetate / petroleum ether as an eluent to finally obtain a yellow solid OH-probe.

[0018] The step (S10) comprises the following steps: (S101) mixing 2,3,3-trimethyl-3H-indoline and methyl iodide in a round-bottom flask; (S102) adding anhydrous acetonitrile, anhydrous toluene or anhydrous dimethylformamide as a solvent into the flask; (S103) heating the mixture under reflux conditions for reaction; and (S104) separating the formed precipitate using a Buchner funnel and washing the residual solid with ether to obtain a first intermediate compound.

[0019] Wherein in the step (S101), the molar ratio of 2,3,3-trimethyl-3H-indoline to methyl iodide is 1:1.

[0020] The step (S20) comprises the following steps: (S201) adding anhydrous dimethylformamide and anhydrous dichloromethane or anhydrous chloroform into a round-bottom flask, and cooling and stirring the mixture; (S202) gradually adding phosphorus trichloride in anhydrous dimethylformamide or anhydrous chloroform through a constant pressure addition funnel; (S203) slowly adding cyclohexanone, and the color of the mixture changes from transparent to yellow; (S204) gradually raising the temperature of the mixture to 45-55°C and maintaining it for 3-5 hours; and (S205) after cooling, pouring the mixture into a large amount of ice water, and obtaining a yellow second intermediate compound after overnight.

[0021] The step (S40) comprises the following steps: (S401) dissolving 50 mg of IR780 in 10 mL of methanol; (S402) controlling the temperature of the reaction system to be maintained at 0°C in an ice bath; (S403) gradually adding sodium borohydride and stirring at 0°C for 2 hours; (S404) monitoring the progress of the reaction using thin layer chromatography; (S405) after the reaction is completed, extracting the mixture using dichloromethane and saturated brine, and collecting the organic phase; and (S406) purifying using ethyl acetate / petroleum ether as an eluent to finally obtain a yellow solid OH-probe.

[0022] In the step (S405), 2 to 4 equivalents of sodium borohydride are added, and in the step (S406), the volume ratio of ethyl acetate to petroleum ether is 1:1 to 9:1.

[0023] According to another aspect of the present invention, the present invention provides a fluorescent probe for detecting hydroxyl radicals, wherein the fluorescent probe is an OH-probe probe, and the chemical structure of the OH-probe probe is based on the IR780 molecule, wherein the IR780 molecule contains a functional group that is sensitive to hydroxyl radicals.

[0024] According to one aspect of the present invention, the present invention provides an application of a fluorescent probe for detecting hydroxyl radicals, wherein the fluorescent probe is an OH-probe probe, and the OH-probe probe is suitable for specific reaction with hydroxyl radicals, and is suitable for application in detecting atherosclerosis, obesity, cancer, neurodegenerative diseases, inflammatory diseases and other diseases associated with free radicals.

[0025] The fluorescent probe is an OH-probe probe, and the chemical structure of the OH-probe probe is based on the IR780 molecule, wherein the IR780 molecule contains a functional group that is sensitive to hydroxyl free radicals.

[0026] The OH-probe probe is used to detect hydroxyl free radicals, including the following detection steps: (A) Two PBS buffer solutions were prepared, one of which was a PBS buffer solution in a low viscosity environment, and the other was a PBS buffer solution in a high viscosity environment with glycerol added; (B) OH-probe solution was added to two PBS buffer solutions with different viscosities; (C) Baseline data recording: Use a near-infrared (NIR) absorption spectrometer to record the initial absorption of the OH-probe in the two solutions and the absorption of the probe in the absence of hydroxyl radicals; (D) Adding hydroxyl free radicals: adding a certain amount of hydroxyl free radicals to the two solutions respectively and mixing them thoroughly; (E) Using the NIR absorption spectrometer again to record the absorption spectrum after adding hydroxyl radicals, and observing the change of the absorption peak; and (F) Fluorescence spectrum test: Use a fluorescence spectrometer to test and record the changes in the fluorescence intensity of OH-probe under two environments.

[0027] According to another aspect of the present invention, the present invention provides a method for preparing a fluorescent probe for detecting hydroxyl radicals, comprising the following steps: (S10) synthesizing a first intermediate compound; (S20) synthesizing a second intermediate compound; (S30) synthesizing compound IR780 through the first intermediate compound and the second intermediate compound, wherein the IR780 molecule contains a functional group that is sensitive to hydroxyl radicals; and (S40) Synthesis of OH-probe based on the IR780. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 It is a schematic diagram of the synthesis route of the OH-probe prepared by the present invention.

[0029] Figure 2 It is a response schematic diagram of the OH-probe prepared in the present invention.

[0030] Figure 3 It is a schematic diagram of the absorption spectrum of the OH-probe prepared in the present invention in response to hydroxyl radicals and viscosity.

[0031] Figure 4 It is a schematic diagram of the fluorescence spectrum of the OH-probe prepared in the present invention in response to hydroxyl radicals and viscosity.

[0032] Figure 5 It is a schematic diagram of the fluorescence intensity of the selectivity of the OH-probe prepared by the present invention to hydroxyl radicals.

[0033] Figure 6 It is a schematic diagram of the selective fluorescence intensity quantification of hydroxyl radicals by the OH-probe prepared by the present invention.

[0034] Figure 7 It is a schematic diagram of the cytotoxicity test of the OH-probe prepared by the present invention on macrophages.

[0035] Figure 8 It is a schematic diagram of the cytotoxicity test of the OH-probe prepared by the present invention on HEK293T cells.

[0036] Fig. 9 It is a schematic diagram of the changes in liver signals in the control group and the atherosclerosis group after injection of the OH-probe prepared by the present invention.

[0037] Fig.10 It is a schematic diagram of the changes in liver signals of the control group and the obese group after injection of OH-probe prepared by the present invention. DETAILED DESCRIPTION

[0038] The following description is used to disclose the present invention so that those skilled in the art can implement the present invention. The preferred embodiments described below are only examples, and those skilled in the art can think of other obvious variations. The basic principles of the present invention defined in the following description can be applied to other embodiments, variations, improvements, equivalents, and other technical solutions that do not deviate from the spirit and scope of the present invention.

[0039] Reference Figure 1 To Attachment Fig.10 As shown, the OH-probe probe according to the present invention, its preparation method and corresponding functional verification detection are illustrated. The OH-probe probe is specially designed for detecting and imaging hydroxyl radical activity and has high specificity and sensitivity. Embodiment 1

[0040] The preparation method of the OH-probe provided by the present invention comprises the following steps: (S10) synthesizing a first intermediate compound; (S20) synthesizing a second intermediate compound; (S30) synthesizing compound IR780; and (S40) Synthesis of OH-probe.

[0041] The step (S10) comprises the following steps: (S101) in a 500 mL round-bottom flask, 2,3,3-trimethyl-3H-indoline (8.0 g, 50 mmol) and iodomethane (7.1 g, 50 mmol) are mixed; (S102) 30 mL of anhydrous acetonitrile is added to the flask as a solvent; (S103) the mixture is heated under reflux conditions for 4 to 12 hours; and (S104) the formed precipitate is separated using a Buchner funnel, and the residual solid is washed with ether to obtain a first intermediate compound.

[0042] The step (S20) comprises the following steps: (S201) adding anhydrous dimethylformamide (anhydrous DMF) and anhydrous dichloromethane (anhydrous DCM) into a 250 mL round-bottom flask, and the mixture is cooled and stirred for 20 minutes; (S202) gradually adding phosphorus trichloride (POCl3) in anhydrous DCM through a constant pressure addition funnel; (S203) slowly adding cyclohexanone (2.9 g, 30 mmol), and the color of the mixture changes from transparent to yellow; (S204) gradually raising the temperature of the mixture to 45°C and maintaining it for 3 hours; and (S205) after cooling, pouring the mixture into a large amount of ice water, and obtaining a yellow second intermediate compound overnight.

[0043] The step (S30) comprises the following steps: (S301) dissolving the first intermediate compound (1.74 g, 10 mmol) and the second intermediate compound (0.86 g, 5 mmol) in 150 mL of a mixed solvent of n-butanol and benzene (volume ratio 7:3); (S302) refluxing the mixture in a 250 mL round-bottom flask for 3 hours; (S303) drying under vacuum after reflux to obtain a green solid; and (S304) purifying the raw material using silica gel column chromatography to obtain compound IR780.

[0044] The step (S40) comprises the following steps: (S401) dissolving 50 mg of compound IR780 in 10 mL of methanol; (S402) controlling the temperature of the reaction system to be maintained at 0°C in an ice bath; (S403) gradually adding 2 equivalents of sodium borohydride and stirring at 0°C for 2 hours; (S404) monitoring the progress of the reaction using thin layer chromatography (TLC); (S405) after the reaction is completed, extracting the mixture using dichloromethane and saturated brine, and collecting the organic phase; and (S406) purifying using ethyl acetate / petroleum ether (volume ratio 1:1) as an eluent to finally obtain a yellow solid OH-probe. Embodiment 2

[0045] The synthetic route of the OH-probe probe provided by the present invention is as follows Figure 1 As shown, the OH-probe preparation method comprises the following steps: (S10) synthesizing a first intermediate compound; (S20) synthesizing a second intermediate compound; (S30) synthesizing compound IR780; and (S40) Synthesis of OH-probe.

[0046] The step (S10) comprises the following steps: (S101) in a 500 mL round-bottom flask, 2,3,3-trimethyl-3H-indoline (9.6 g, 60 mmol) and iodomethane (8.52 g, 60 mmol) are mixed; (S102) 40 mL of anhydrous toluene is added to the flask as a solvent; (S103) the mixture is heated under reflux conditions for 8 hours; and (S104) the formed precipitate is separated using a Buchner funnel, and the residual solid is washed with ether to obtain a first intermediate compound.

[0047] The step (S20) comprises the following steps: (S201) adding anhydrous dimethylformamide (anhydrous DMF) and aqueous chloroform (CH3Cl) into a 250 mL round-bottom flask, and the mixture is cooled and stirred for 20 minutes; (S202) gradually adding anhydrous chloroform (CH3Cl) of phosphorus trichloride (POCl3) through a constant pressure addition funnel; (S203) slowly adding cyclohexanone (3.9 g, 40 mmol), and the color of the mixture changes from transparent to yellow; (S204) gradually raising the temperature of the mixture to 55°C and maintaining it for 5 hours; and (S205) after cooling, pouring the mixture into a large amount of ice water, and obtaining a yellow second intermediate compound overnight.

[0048] The step (S30) comprises the following steps: (S301) dissolving the first intermediate compound (3.48 g, 20 mmol) and the second intermediate compound (0.86 g, 5 mmol) in 150 mL of a mixed solvent of n-butanol and benzene (volume ratio 9:1); (S302) refluxing the mixture in a 250 mL round-bottom flask for 3 hours; (S303) drying under vacuum after reflux to obtain a green solid; and (S304) purifying the raw material using silica gel column chromatography to obtain compound IR780.

[0049] The step (S40) comprises the following steps: (S401) dissolving 60 mg of compound IR780 in 15 mL of methanol; (S402) controlling the temperature of the reaction system to be maintained at 0°C in an ice bath; (S403) gradually adding 4 equivalents of sodium borohydride and stirring at 0°C for 3 hours; (S404) monitoring the reaction progress using thin layer chromatography (TLC); (S405) after the reaction is completed, extracting the mixture using dichloromethane and saturated brine, and collecting the organic phase; and (S406) purifying using ethyl acetate / petroleum ether (volume ratio 9:1) as an eluent to finally obtain a yellow solid OH-probe. Embodiment 3

[0050] The synthetic route of the OH-probe probe provided by the present invention is as follows Figure 1 As shown, the OH-probe preparation method comprises the following steps: (S10) synthesizing a first intermediate compound; (S20) synthesizing a second intermediate compound; (S30) synthesizing compound IR780; and (S40) Synthesis of OH-probe.

[0051] The step (S10) comprises the following steps: (S101) in a 500 mL round-bottom flask, 2,3,3-trimethyl-3H-indoline (8.0 g, 50 mmol) and iodomethane (7.1 g, 50 mmol) are mixed; (S102) 30 mL of anhydrous dimethylformamide (anhydrous DMF) is added to the flask as a solvent; (S103) the mixture is heated under reflux for 10 hours; and (S104) the formed precipitate is separated using a Buchner funnel, and the residual solid is washed with ether to obtain a first intermediate compound.

[0052] The step (S20) comprises the following steps: (S201) adding anhydrous dimethylformamide (anhydrous DMF) and anhydrous dichloromethane (anhydrous DCM) into a 250 mL round-bottom flask, and the mixture is cooled and stirred for 20 minutes; (S202) gradually adding phosphorus trichloride (POCl3) in anhydrous DCM through a constant pressure addition funnel; (S203) slowly adding cyclohexanone (2.9 g, 30 mmol), and the color of the mixture changes from transparent to yellow; (S204) gradually raising the temperature of the mixture to 50°C and maintaining it for 4 hours; and (S205) after cooling, pouring the mixture into a large amount of ice water, and obtaining a yellow second intermediate compound overnight.

[0053] The step (S30) comprises the following steps: (S301) dissolving the first intermediate compound (3.13 g, 18 mmol) and the second intermediate compound (1.03 g, 6 mmol) in 150 mL of a mixed solvent of n-butanol and benzene (volume ratio 9:2); (S302) refluxing the mixture in a 250 mL round-bottom flask for 3 hours; (S303) drying under vacuum after reflux to obtain a green solid; and (S304) purifying the raw material using silica gel column chromatography to obtain compound IR780.

[0054] The step (S40) comprises the following steps: (S401) dissolving 50 mg of compound IR780 in 10 mL of methanol; (S402) controlling the temperature of the reaction system to be maintained at 0°C in an ice bath; (S403) gradually adding 3 equivalents of sodium borohydride and stirring at 0°C for 2 hours; (S404) monitoring the reaction progress using thin layer chromatography (TLC); (S405) after the reaction is completed, extracting the mixture using dichloromethane and saturated brine, and collecting the organic phase; and (S406) purifying using ethyl acetate / petroleum ether (volume ratio 4:1) as an eluent to finally obtain a yellow solid OH-probe. Embodiment 4

[0055] The preparation method of the OH-probe provided by the present invention comprises the following steps: (S10) synthesizing a first intermediate compound; (S20) synthesizing a second intermediate compound; (S30) synthesizing compound IR780; and (S40) Synthesis of OH-probe.

[0056] The step (S10) comprises the following steps: (S101) in a 500 mL round-bottom flask, 2,3,3-trimethyl-3H-indoline (8.0 g, 50 mmol) and iodomethane (7.1 g, 50 mmol) are mixed; (S102) 30 mL of anhydrous toluene is added to the flask as a solvent; (S103) the mixture is heated under reflux for 6 hours; and (S104) the formed precipitate is separated using a Buchner funnel, and the residual solid is washed with ether to obtain a first intermediate compound.

[0057] The step (S20) comprises the following steps: (S201) adding anhydrous dimethylformamide (anhydrous DMF) and anhydrous chloroform (CH3Cl) into a 250 mL round-bottom flask, and the mixture is cooled and stirred for 20 minutes; (S202) gradually adding anhydrous chloroform (CH3Cl) of phosphorus trichloride (POCl3) through a constant pressure addition funnel; (S203) slowly adding cyclohexanone (2.9 g, 30 mmol), and the color of the mixture changes from transparent to yellow; (S204) gradually raising the temperature of the mixture to 50°C and maintaining it for 3.5 hours; and (S205) after cooling, pouring the mixture into a large amount of ice water, and obtaining a yellow second intermediate compound overnight.

[0058] The step (S30) comprises the following steps: (S301) dissolving the first intermediate compound (1.74 g, 10 mmol) and the second intermediate compound (0.86 g, 5 mmol) in 150 mL of a mixed solvent of n-butanol and benzene (volume ratio 7:2); (S302) refluxing the mixture in a 250 mL round-bottom flask for 3 hours; (S303) drying under vacuum after reflux to obtain a green solid; and (S304) purifying the raw material using silica gel column chromatography to obtain compound IR780.

[0059] The step (S40) comprises the following steps: (S401) dissolving 55 mg of compound IR780 in 12 mL of methanol; (S402) controlling the temperature of the reaction system to be maintained at 0°C in an ice bath; (S403) gradually adding 4 equivalents of sodium borohydride and stirring at 0°C for 3 hours; (S404) monitoring the progress of the reaction using thin layer chromatography (TLC); (S405) after the reaction is completed, extracting the mixture using dichloromethane and saturated brine, and collecting the organic phase; and (S406) purifying using ethyl acetate / petroleum ether (volume ratio 7:1) as an eluent to finally obtain a yellow solid OH-probe.

[0060] The functional verification of the OH-probe includes the following steps: (A) Prepare PBS buffer solution and OH-probe solution; (B) OH-probe solution was added to two PBS buffer solutions with different viscosities; (C) Baseline data recording: recording the absorption of the probe in the absence of hydroxyl radicals; (D) Addition of hydroxyl radicals; (E) recording an absorption spectrum: recording an absorption spectrum after adding hydroxyl radicals; and (F) Fluorescence spectrum test: record the changes in the fluorescence intensity of OH-probe under two environments.

[0061] The step (A) comprises the following steps: (A1) preparing PBS buffer solution: preparing two phosphate buffer solutions (PBS), one for a low viscosity environment and the other adding glycerol to simulate a high viscosity environment; (A2) OH-probe solution: dissolving the OH-probe probe in an appropriate amount of PBS buffer solution to ensure that the concentration of the probe is appropriate for subsequent experimental observations.

[0062] In the step (C), a near infrared (NIR) absorption spectrometer is used to record the initial absorption of the OH-probe in the two solutions to confirm the absorption of the probe in the absence of hydroxyl radicals.

[0063] In the step (D), a certain amount of hydroxyl radicals are added to the two solutions respectively and mixed thoroughly.

[0064] In the step (E), the absorption spectrum after adding hydroxyl radicals is recorded again using the NIR absorption spectrometer to observe the change of the absorption peak.

[0065] In the step (F), a fluorescence spectrometer is used to test the fluorescence response of the OH-probe under two environments, and the change of fluorescence intensity is recorded.

[0066] Analysis of the results of the functional verification of the OH-probe probe: Reference Figure 2 Since OH-probe has a corresponding hydroxyl radical response site (attached Figure 2 When hydroxyl radicals are added and the viscosity is high, the hydroxyl radicals can oxidize the indole of the OH-probe (attached Figure 2The circled part in the figure shows the product IR780. Since the conjugated system of IR780 is significantly increased, the OH-probe and hydroxyl radicals show enhanced absorption after reaction. At the same time, the rotation of the OH-probe and the hydroxyl radical oxidation product (IR780) is restricted in a high viscosity environment, so the absorption and fluorescence intensity are further enhanced. In summary, OH-probe shows a dual response to hydroxyl radicals and viscosity.

[0067] Absorption spectrum analysis: Analyze the changes in the NIR absorption spectrum of OH-probe in two environments before and after the addition of hydroxyl radicals. Figure 3 As shown, the results show that in a high viscosity environment, the addition of hydroxyl radicals significantly enhances the NIR absorption of OH-probe.

[0068] Fluorescence spectroscopy analysis: Compare the fluorescence response of OH-probe in high viscosity and low viscosity environments. Figure 4 As shown, the results showed that only in a high viscosity environment could hydroxyl radicals significantly activate the near-infrared fluorescence of OH-probe, while in a low viscosity environment, the change of the fluorescence signal was not obvious.

[0069] To further demonstrate the selectivity of the probe, OH-probe was incubated with several other reactive oxygen species with similar activity under the same conditions. For selectivity testing, various reactive species were incubated with OH-probe for 30 minutes, such as nitric oxide (NO), singlet oxygen ( 1 O2), peroxynitrite anion (ONOO - ), hypochlorous acid (HClO), hyponitrous acid (HNO), hydrogen peroxide (H2O2), superoxide anion (O2 ·- ) and hydroxyl radicals (·OH), where the concentration of all reactive oxygen species was 50 μM. Figure 5 and attached Figure 6 As shown, Figure 5 The changes in fluorescence signal intensity after incubation of OH-probe with different analytes are shown. Figure 6 The results show that the fluorescence signal of the probe does not show obvious changes when the OH-probe reacts with other active substances, but the fluorescence intensity of the OH-probe after the reaction with the hydroxyl radical reaches 15000 (such as the attached Figure 6 as shown).

[0070] The biocompatibility of OH-probe was verified by cytotoxicity experiments In the present invention, in order to evaluate the biosafety of the novel probe OH-probe, we first systematically tested its cytotoxicity. The cytotoxicity test was performed using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) method, which is a standard method widely used to evaluate cell activity. The specific experimental steps and results are as follows: Cell culture: Two cell lines were selected for cytotoxicity testing, including mouse macrophage cell line RAW264.7 and human embryonic kidney cell line HEK293T. These two cells were cultured at 37°C and 5% CO2 until the cells grew to a suitable level for the experiment.

[0071] Probe treatment: OH-probe was prepared at different concentrations from 0 to 10 μM and added to the cultured cells. Three replicates were performed for each concentration to ensure the reliability of the results.

[0072] MTT assay: After probe treatment, add MTT reagent to the cells and continue incubating in the incubator for a certain period of time. MTT reagent is reduced to a purple formazan compound by mitochondrial enzymes of living cells.

[0073] Dissolve the formazan compound: After incubation, use DMSO or other solvents to dissolve the purple formazan compound in the cells for subsequent absorbance measurement.

[0074] Absorbance determination: Use an enzyme-labeled instrument to measure the absorbance of each sample at a wavelength of 570 nm. The absorbance reflects the survival rate of the cells.

[0075] According to the experimental results (see Appendix Figure 7 and attached Figure 8 As shown in Figure 3, even when the concentration of OH-probe increased from 0 to 10 μM, the survival rate of both cell types remained above 90%. This result shows that OH-probe has low cytotoxicity and little effect on cells, showing good biosafety.

[0076] The above experimental results provide important safety evidence for the future clinical application and further biomedical research of OH-probe. In addition, these data also support the possibility of using this probe for a wider range of biomedical detection and treatment.

[0077] OH-probe probe detects the activity of hydroxyl radicals in atherosclerosis mouse model by fluorescence imaging Experimental materials and animal models: Animal model selection: Select genetically modified ApoE knockout mice (ApoE - / -), these mice lack the apolipoprotein E gene involved in fat metabolism. By feeding a high-fat diet, these mice developed atherosclerosis and served as the experimental group. The control group was C57BL / 6 mice fed a regular diet. The OH-probe probe was then used for experiments.

[0078] Probe injection and imaging: After injecting OH-probe into two groups of mice, fluorescence imaging was performed to monitor the response of the probe. The results are shown in the attached figure. Fig. 9 and attached Fig.10 As shown in the figure, there was no significant fluorescence signal in the liver area of ​​the control group mice after injection, indicating that the probe did not respond to other factors. However, the fluorescence signal in the liver area of ​​the experimental group mice was significantly enhanced after the probe was injected, indicating that OH-probe effectively detected the changes in the level of hydroxyl radicals in the mouse model of atherosclerosis.

[0079] OH-probe probe detects the activity of hydroxyl radicals in obese mouse models by fluorescence imaging Experimental materials and animal models: Animal model selection: C57BL / 6 mice were used, a mouse strain commonly used in obesity research because it is easy to induce obesity through diet. The experimental group of mice was fed a high-fat diet for 16 weeks to induce obesity (obese group, OB group), while the control group of mice was fed a regular diet.

[0080] Experimental steps: After the OH-probe probe was injected into the two groups of mice, fluorescence imaging was performed after injection to monitor the response of the probe. It was observed that the fluorescence signal in the liver area of ​​the obese group mice was significantly enhanced after the probe was injected, which was similar to the previous observations in the atherosclerosis model, while there was no obvious signal in the liver of the control group mice. This shows that OH-probe is also suitable for detecting liver hydroxyl radicals in the obesity model.

[0081] This example demonstrates the effectiveness of the OH-probe probe in detecting changes in liver hydroxyl radicals in an obesity model. Therefore, this study provides valuable insights into the role of oxidative stress in obese mice. These findings will help develop new diagnostic and therapeutic strategies to address obesity and its related complications.

[0082] Through the above experiments, it can be found that the OH-probe probe provided by the present invention is not only simple in preparation method and high in synthesis efficiency, but also the chemical structure of the prepared OH-probe probe is based on the IR780 molecule, and the selectivity and reaction sensitivity to hydroxyl radicals are enhanced by introducing a functional group sensitive to hydroxyl radicals, which is due to the extremely high sensitivity and excellent selectivity of OH-probe to hydroxyl radicals, and can distinguish other types of highly reactive reactive oxygen species, such as superoxide anions and hydrogen peroxide. Therefore, in animal models of atherosclerosis and obesity, OH-probe can be effectively used to monitor and map the level of hydroxyl radicals, providing an important tool for the study of the pathological mechanisms of these diseases. Through this method, the oxidative stress state of related diseases can be effectively evaluated and studied, which is of great clinical significance for the development of new diagnostic and therapeutic strategies, especially in the early diagnosis of atherosclerosis and obesity, pathological research and application in the evaluation of therapeutic effects.

[0083] This application is based on the high sensitivity and specificity of the fluorescent probe as well as its excellent biocompatibility and fluorescence properties, which can provide important information about the role of hydroxyl radicals in disease progression. Therefore, OH-probe has the potential to be used in disease diagnosis and evaluation of therapeutic effects in future preclinical studies.

[0084] The OH-probe probe provided by the present invention is not only suitable for studying atherosclerosis and obesity, but can also be extended to other free radical-related diseases, such as cancer, neurodegenerative diseases and inflammatory diseases. In short, the development of the OH-probe probe not only improves the technical level of hydroxyl radical detection, but also provides new perspectives and tools for the diagnosis, treatment and research of diseases. It has broad development prospects and practical application value and is worthy of promotion and application.

[0085] It should be understood by those skilled in the art that the embodiments of the present invention described above and shown in the accompanying drawings are only examples and do not limit the present invention. The purpose of the present invention has been fully and effectively achieved. The functional and structural principles of the present invention have been demonstrated and explained in the embodiments, and the embodiments of the present invention may be deformed or modified in any way without departing from the principles.

Claims

1. A method for preparing a fluorescent probe for detecting hydroxyl radicals, characterized in that: The following steps are involved: (S10) using 2,3,3-trimethyl-3H-indoline and iodomethane as raw materials to synthesize a first intermediate compound; (S20) using phosphorus trichloride, cyclohexanone and anhydrous dimethylformamide as raw materials to synthesize a second intermediate compound; (S30) mixing the first intermediate compound and the second intermediate compound to react to synthesize compound IR780; as well as (S40) Synthesis of OH-probe based on the IR780.

2. The method for preparing a fluorescent probe for detecting hydroxyl radicals according to claim 1, wherein the step (S30) comprises the following steps: (S301) dissolving the first intermediate compound and the second intermediate compound in a mixed solvent of n-butanol and benzene; (S302) the mixture is refluxed in a round-bottom flask for reaction; (S303) after reflux, it is dried under vacuum to obtain a green solid; and (S304) it is purified using silica gel column chromatography to obtain compound IR780.

3. The method for preparing a fluorescent probe for detecting hydroxyl radicals according to claim 2, wherein in the step (S301), the molar ratio of the first intermediate compound to the second intermediate compound is 2:1~4:1, and the mixing volume ratio of n-butanol and benzene is 7:3~9:

1.

4. The method for preparing a fluorescent probe for detecting hydroxyl radicals according to claim 2, wherein the step (S40) comprises the following steps: (S401) dissolving IR780 in methanol; (S402) controlling the temperature of the reaction system to be maintained at 0°C in an ice bath; (S403) gradually adding sodium borohydride and stirring the reaction at 0°C; (S404) monitoring the progress of the reaction using thin layer chromatography; (S405) after the reaction, extracting the mixture using dichloromethane and saturated brine, and collecting the organic phase; and (S406) purifying using ethyl acetate / petroleum ether as an eluent to finally obtain a yellow solid OH-probe.

5. The method for preparing a fluorescent probe for detecting hydroxyl radicals according to claim 4, wherein the step (S10) comprises the following steps: (S101) 2,3,3-trimethyl-3H-indoline and iodomethane are mixed in a round-bottom flask, (S102) anhydrous acetonitrile, anhydrous toluene or anhydrous dimethylformamide is added to the flask as a solvent; (S103) the mixture is heated under reflux conditions for reaction; and (S104) a formed precipitate is separated using a Buchner funnel, and the residual solid is washed with ether to obtain a first intermediate compound. 6 . The method for preparing a fluorescent probe for detecting hydroxyl radicals according to claim 5 , wherein in the step ( S101 ), the molar ratio of 2,3,3-trimethyl-3H-indoline to iodomethane is 1:

1.

7. The method for preparing a fluorescent probe for detecting hydroxyl radicals according to claim 5, wherein the step (S20) comprises the following steps: (S201) In a round-bottom flask, anhydrous dimethylformamide and anhydrous dichloromethane or anhydrous chloroform are added, and the mixture is cooled and stirred; (S202) gradually adding phosphorus trichloride in anhydrous dimethylformamide or anhydrous chloroform through a constant pressure addition funnel; (S203) slowly adding cyclohexanone, and the color of the mixture changes from transparent to yellow; (S204) gradually raising the temperature of the mixture to 45-55°C and maintaining it for 3-5 hours; and (S205) after cooling, pouring the mixture into a large amount of ice water, and obtaining a yellow second intermediate compound after overnight.

8. The method for preparing a fluorescent probe for detecting hydroxyl radicals according to any one of claims 1 to 7, wherein the step (S40) comprises the following steps: (S401) Dissolve 50 mg of IR780 in 10 mL of methanol; (S402) controlling the temperature of the reaction system to be maintained at 0°C in an ice bath; (S403) gradually adding sodium borohydride and stirring at 0°C for 2 hours; (S404) monitoring the progress of the reaction using thin layer chromatography; (S405) after the reaction, extracting the mixture using dichloromethane and saturated brine, and collecting the organic phase; and (S406) purifying using ethyl acetate / petroleum ether as eluent to finally obtain a yellow solid OH-probe.

9. The method for preparing a fluorescent probe for detecting hydroxyl radicals according to claim 8, wherein in the step (S405), 2 to 4 equivalents of sodium borohydride are added, and in the step (S406), the volume ratio of ethyl acetate to petroleum ether is 1:1 to 9:

1.

10. The fluorescent probe prepared according to the method for preparing a fluorescent probe for detecting hydroxyl radicals according to any one of claims 1 to 9, characterized in that: The fluorescent probe is an OH-probe probe, and the chemical structure of the OH-probe probe is based on the IR780 molecule, wherein the IR780 molecule contains a functional group that is sensitive to hydroxyl free radicals.

11. Use of the fluorescent probe prepared by the method for preparing a fluorescent probe for detecting hydroxyl radicals according to any one of claims 1 to 9, characterized in that: The fluorescent probe is an OH-probe probe, which is suitable for specific reaction with hydroxyl free radicals and is suitable for detecting atherosclerosis, obesity, cancer, neurodegenerative diseases, inflammatory diseases and other diseases related to free radicals.

12. The use according to claim 11, wherein the fluorescent probe is an OH-probe probe, the chemical structure of the OH-probe probe is based on the IR780 molecule, wherein the IR780 molecule contains a functional group that is sensitive to hydroxyl radicals.

13. The use according to claim 12, wherein the OH-probe probe is used to detect hydroxyl free radicals, comprising the following detection steps: (A) Two PBS buffer solutions were prepared, one of which was a PBS buffer solution in a low viscosity environment, and the other was a PBS buffer solution in a high viscosity environment with glycerol added; (B) OH-probe solution was added to two PBS buffer solutions with different viscosities; (C) Baseline data recording: Use a near-infrared absorption spectrometer to record the initial absorption of the OH-probe in the two solutions, and record the absorption of the probe in the absence of hydroxyl radicals; (D) Adding hydroxyl free radicals: adding a certain amount of hydroxyl free radicals to the two solutions respectively and mixing them thoroughly; (E) Using a near-infrared absorption spectrometer again to record the absorption spectrum after adding hydroxyl radicals, and observing the change in the absorption peak; and (F) Fluorescence spectrum test: Use a fluorescence spectrometer to test and record the changes in the fluorescence intensity of OH-probe under two environments.

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