A Selective Fluorescent Probe for Hydrogen Peroxide, Preparation Method, Kit and Application

Abstract

The present invention belongs to the technical field of analytical detection, and particularly relates to a selective hydrogen peroxide fluorescent probe, a preparation method, a kit and an application thereof. After reacting with hydrogen peroxide, the main absorption peak of this fluorescent probe redshifts from 560 nm to 605 nm, and a new fluorescence emission peak is formed at 648 nm. The linear range for detecting hydrogen peroxide can reach 0 µM - 460 µM, and the detection limit can be as low as 80 nM. After investigation, this fluorescent probe has advantages such as strong specificity or selectivity, high sensitivity, wide linear range, and fast response speed in the detection of hydrogen peroxide in milk samples, cell samples and plant samples. Therefore, it has a broad application prospect and can be combined with means such as mobile phone software or laser confocal microscope for fluorescence analysis, improving the convenience of application and having a good application prospect.

Description

Technical Field

[0001] The present invention belongs to the technical field of analytical detection, and particularly relates to a hydrogen peroxide fluorescent probe compound, a preparation method, a kit and an application thereof. Background Art

[0002] Hydrogen peroxide (H 2 O 2 ) is a common reactive oxygen species (ROS), which exists in environmental water bodies and animals and plants, and can cause oxidative stress reactions, gradually accumulating to induce or exacerbate cell and tissue organ damage. For example, plants are continuously affected by various stress factors in the environment throughout their life cycle, including metals, drought, low or high temperature, floods, etc. These stresses will seriously damage plant tissues and hinder plant growth. Hydrogen peroxide, as an important reactive oxygen species (ROS), participates in a variety of biological processes and has become a key signaling molecule regulating stress responses and is considered an indicator of acute stress. In addition, hydrogen peroxide is also used as an antifungal agent and is widely used in food disinfection and sterilization. Excessive hydrogen peroxide residues in food can cause various diseases in humans, such as stroke, depression, air embolism, etc. The U.S. Food and Drug Administration stipulates that the residual amount of hydrogen peroxide in dairy products cannot exceed 0.5 mg / L. In the milk industry, excessive hydrogen peroxide is also harmful to the nutritional value of milk. For example, too much hydrogen peroxide will destroy the nutritional component folic acid in milk, and its degradation will reduce the nutritional value of milk. The residual hydrogen peroxide will accelerate the deterioration process of milk, resulting in a worse taste of milk and shortening the shelf life of milk. Therefore, it is necessary to develop a simple, efficient and intuitive method for monitoring hydrogen peroxide.

[0003] In response to this, various hydrogen peroxide analysis methods have been developed in this field, such as high performance liquid chromatography, colorimetry, mass spectrometry, spectrophotometry, electron spin resonance method, electrochemistry method, etc. However, these methods all have certain limitations, such as long response time, poor specificity, high cost, inability to achieve on-site detection, etc. In contrast, fluorescence probe monitoring has the advantages of high selectivity, simple operation, low cost, real-time in-situ imaging, etc., and is considered a promising method for determining hydrogen peroxide. However, for animals and plants, due to the different tissue penetration abilities of light with different wavelengths and the background light interference of animals and plants themselves, the application of hydrogen peroxide fluorescence detection in animals and plants is restricted. Moreover, in animals and plants, especially in plants, various metal ions, reactive oxygen species, reactive sulfur species, fluorescent substances (such as anthocyanins, lignin, etc.) contained in themselves under stress conditions or themselves will interfere with fluorescence detection. Therefore, it poses a severe challenge to the development of fluorescence probes for hydrogen peroxide fluorescence detection in animals and plants (Long Yi. Construction of hydrogen peroxide-responsive cyanine fluorescent probes and their application in detection and imaging in plants. Dissertation of South China University of Technology, 2023).

[0004] Although a variety of hydrogen peroxide fluorescent probes have been developed in recent years, such as the hydrogen peroxide fluorescent probes disclosed in CN106632436A and CN118955544A, etc., there are few reports on fluorescent probes with high selectivity / high sensitivity and wide linear range that are applicable to animals and plants, especially for the detection of hydrogen peroxide fluorescence in plants with extremely many interfering factors. Therefore, there is an urgent need to develop a hydrogen peroxide fluorescent probe with a wide application range, high selectivity, wide linear range and fast response speed. Summary of the Invention

[0005] In view of the deficiencies of the prior art, the present invention provides a selective hydrogen peroxide fluorescent probe, a preparation method, a kit and an application thereof, which are used for the precise recognition and fluorescence imaging of hydrogen peroxide in foods, isolated cells and plants. The probe is simple to synthesize and easy to operate, has high detection sensitivity, good selectivity, wide linear range and fast response speed for hydrogen peroxide, and has higher practical value for visual detection of hydrogen peroxide in foods, cells and plants; and can be combined with mobile phone software or a laser confocal microscope, etc. for fluorescence analysis, improving the convenience of application.

[0006] In the first aspect of the present invention, a selective hydrogen peroxide fluorescent probe is provided, and its structural formula is shown as formula (I) (the naming code is NBP, and hereinafter the selective hydrogen peroxide fluorescent probe of formula (I) is referred to as NBP):

[0007]

[0008] (I).

[0009] In the second aspect of the present invention, a preparation method of NBP is provided: the method includes the following steps: in the presence of a base and an organic solvent, reacting the compound shown in formula (Ia) with the compound shown in formula (Ib) to obtain NBP. The reaction formula is as follows:

[0010]

[0011] Formula (Ia) Formula (Ib) Formula (I);

[0012] Wherein, X is a halogen, preferably chlorine.

[0013] Preferably, in the foregoing reaction, the molar ratio of the compound shown in formula (Ia) to the compound shown in formula (Ib) is 1:1 to 1:3; more preferably, the molar ratio of the compound shown in formula (Ia) to the compound shown in formula (Ib) is 1:2.

[0014] Preferably, in the aforementioned reaction, the base is any one of triethylamine, potassium carbonate, and cesium carbonate; the organic solvent is any one of dichloromethane, anhydrous acetonitrile, anhydrous methanol, and N,N-dimethylformamide. Further preferably, the base is triethylamine; the organic solvent is dichloromethane.

[0015] Preferably, in the aforementioned reaction, the reaction condition is to react at room temperature for 0.5 h to 1.5 h. Further preferably, the reaction condition is to react at room temperature for 1 h. The term "room temperature" is 10°C to 30°C as defined in the Chinese Pharmacopoeia.

[0016] In the third aspect of the present invention, a kit for detecting hydrogen peroxide containing NBP is provided.

[0017] The present invention further provides the application of NBP in the detection of hydrogen peroxide, including:

[0018] (1) Application in the detection of hydrogen peroxide in food samples, and the food is dairy products. Preferably, the dairy product is any one of whole milk, skim milk, and organic milk.

[0019] (2) Application in the detection of hydrogen peroxide in isolated cells for non-disease diagnosis or treatment purposes. Preferably, the isolated cells are liver cancer cells; further preferably, the liver cancer cells are HepG2 liver cancer cells.

[0020] (3) Application in the detection of hydrogen peroxide in plants. Preferably, the plant is Platycodon grandiflorum, and further preferably, the detection part of Platycodon grandiflorum is the root of Platycodon grandiflorum.

[0021] The present invention further provides a method for detecting hydrogen peroxide in plant roots, including the following steps: washing the plant roots with water, immersing them in a probe solution containing NBP for incubation, washing with water, and then performing root fluorescence imaging analysis.

[0022] The beneficial effects of the present invention are as follows:

[0023] 1. The fluorescent probe NBP provided by the present invention uses benzopyrylium salt as the fluorophore and aryl boronic ester as the specific deprotection group mediated by hydrogen peroxide, and 4-(hydroxymethyl)phenylboronic acid pinacol ester is covalently conjugated to one end of the double condensation part. In spectral studies, the probe shows good selectivity and high sensitivity to hydrogen peroxide, has a single selective recognition of hydrogen peroxide in the acetonitrile / PBS (pH = 7.4, v / v = 4:6) system, and the detection limit is as low as 80 nM. The fluorescence intensity has a good linear relationship with the hydrogen peroxide concentration in the range of 0 μM to 460 μM. The raw materials for preparing this probe are easily available, the synthesis is simple, and the operation is convenient.

[0024] 2. The present invention applies the fluorescence probe imaging technology to the detection of hydrogen peroxide in HepG2 living cells, and studies the imaging detection ability of the probe for the levels of endogenous and exogenous hydrogen peroxide in living cells. The results show that after induction with phorbol 12-myristate 13-acetate (abbreviated as PMA) and then treatment with the probe, clear red fluorescence can be seen. After pretreatment with N-acetylcysteine (abbreviated as NAC, a hydrogen peroxide inhibitor), the fluorescence inside the cells is significantly weakened and can be ignored. In addition, by externally adding hydrogen peroxide at different concentrations (50 and 100 μM), it can be observed that the fluorescence signal exhibited by the cells gradually increases with the increase in the concentration of hydrogen peroxide. The results indicate that this fluorescence probe can be used for the fluorescence imaging of endogenous and exogenous hydrogen peroxide in living cells.

[0025] 3. The present invention combines the fluorescence probe with a smartphone software platform to complete the on-site detection of hydrogen peroxide in milk samples, and the color change can be visually observed without other additional equipment. Compared with using large instruments for data analysis, this method can visualize the content of hydrogen peroxide in milk, making the results more intuitive and significantly reducing the detection cost at the same time. In the range of 0 μM to 100 μM of hydrogen peroxide, this detection method has a good linear relationship, and the detection limits are 0.46 μM to 0.54 μM respectively.

[0026] 4. The present invention applies the fluorescence probe NBP technology to detect the accumulation of hydrogen peroxide in Platycodon grandiflorum under drought stress and the change in fluorescence intensity of Platycodon grandiflorum after externally adding hydrogen peroxide. The results show that as the treatment time of simulated drought stress prolongs, the fluorescence signal in the red channel gradually increases. The research indicates that this fluorescence probe has good anti-interference ability in the detection of hydrogen peroxide in plants, with a low detection limit (2.03 μM), a wide linear range (0 μM to 150 μM), a fast response speed (20 min), and does not need to be injected into the plant body (only soaking and incubation are convenient and practical). The linear range is greater than the range of plant physiological hydrogen peroxide concentration, and it is applicable to detect endogenous or externally added (such as artificial hydrogen peroxide treatment) hydrogen peroxide under physiological or pathological conditions of plants. Description of the Drawings

[0027] Figure 1 is the synthetic route of the fluorescence probe NBP;

[0028] Figure 2 is the 1H NMR spectrum of the fluorescence probe NBP;

[0029] Figure 3 is the 13C NMR spectrum of the fluorescence probe NBP;

[0030] Figure 4 is the mass spectrum of the fluorescence probe NBP;

[0031] Figure 5UV absorption spectra changes of 10 µM fluorescent probe NBP after adding 0 µM - 460 µM hydrogen peroxide and photos of 10 µM fluorescent probe NBP under daylight before and after adding hydrogen peroxide;

[0032] Figure 6 Fluorescence spectra changes of 10 µM fluorescent probe NBP after adding 0 µM - 460 µM hydrogen peroxide and photos of 10 µM fluorescent probe NBP under 365 nm UV lamp before and after adding hydrogen peroxide;

[0033] Figure 7 Linear relationship diagram between the fluorescence intensity of 10 µM fluorescent probe NBP at 648 nm wavelength and hydrogen peroxide concentration;

[0034] Figure 8 Fluorescence intensity change diagram of 10 µM fluorescent probe NBP after adding 460 µM hydrogen peroxide over time;

[0035] Figure 9 Fluorescence spectra changes of 10 µM fluorescent probe NBP after adding 460 µM hydrogen peroxide or 500 µM other analytes; In the figure: 1 is Blank; 2 is Na + ; 3 is K + ; 4 is Li + ; 5 is NH 4 + ; 6 is Mg 2+ ; 7 is Ca 2+ ; 8 is Fe 2+ ; 9 is Zn 2+ ; 10 is Ba 2+ ; 11 is Mn 2+ ; 12 is Co 2+ ; 13 is Al 3+ ; 14 is Cl - ; 15 is ClO - ; 16 is Br - ; 17 is SO 4 2- ; 18 is HCO 3 - ; 19 is CO 3 2- ; 20 is H 2 PO 4 - ; 21 is HPO 4 2- ; 22 is F - ; 23 is I - ; 24 is NO 2 - ; 25 is NO 3- ; 26 is Na 2 S; 27 is GSH (glutathione); 28 is CyS (cysteine); 29 is Hcy (homocysteine); 30 is H 2 O 2 ;

[0036] Figure 10 is the fluorescence spectrum change diagram of 10 µM fluorescent probe NBP after adding 460 µM hydrogen peroxide in the presence of 500 µM different other analytes; In the figure: 1 is Blank (blank); 2 is Na + ; 3 is K + ; 4 is Li + ; 5 is NH 4 + ; 6 is Mg 2+ ; 7 is Ca 2+ ; 8 is Fe 2+ ; 9 is Zn 2+ ; 10 is Ba 2+ ; 11 is Mn 2+ ; 12 is Co 2+ ; 13 is Al 3+ ; 14 is Cl - ; 15 is ClO - ; 16 is Br - ; 17 is SO 4 2- ; 18 is HCO 3 - ; 19 is CO 3 2- ; 20 is H 2 PO 4 - ; 21 is HPO 4 2- ; 22 is F - ; 23 is I - ; 24 is NO 2 - ; 25 is NO 3 - ; 26 is Na 2 S; 27 is GSH (glutathione); 28 is CyS (cysteine); 29 is Hcy (homocysteine);

[0037] Figure 11 is the fluorescence spectrum change diagram of 10 µM fluorescent probe NBP before and after reacting with 460 µM hydrogen peroxide under different pH conditions;

[0038] Figure 12Intracellular endogenous hydrogen peroxide fluorescence imaging of HepG2 cells treated with PMA or PMA + NAC for 30 min and then incubated with the fluorescent probe NBP for 30 min, and extracellular exogenous hydrogen peroxide fluorescence imaging of cells incubated with 50 μM and 100 μM hydrogen peroxide for 30 min and then treated with the probe for 30 min;

[0039] Figure 13 Fluorescence photographs (13a) of three milk samples added with 10 μM fluorescent probe NBP and 0, 10, 50, and 100 μM hydrogen peroxide under 365 nm ultraviolet light irradiation, the RGB data analysis display interface (13b) of each group of milk collected by the smartphone software ColorAssist, the R values (13c) of three milk samples added with different concentrations of hydrogen peroxide, and the linear relationship between the R value and the hydrogen peroxide concentration (13d);

[0040] Figure 14 Fluorescence imaging of Platycodon grandiflorum roots incubated with 5% polyethylene glycol for different times (0, 6, 12, 18, 24 h) and then incubated with 10 μM fluorescent probe NBP for 30 min;

[0041] Figure 15 Fluorescence imaging of Platycodon grandiflorum added with different concentrations of hydrogen peroxide (0, 50, 100, 150 μM) and then incubated with 10 μM fluorescent probe NBP for 30 min. Specific implementation manners

[0042] The present invention will be further explained and described below in conjunction with specific embodiments and the accompanying drawings, so that the technical solutions of the present invention are clearer and more complete. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the scope of protection of the present invention.

[0043] Example 1 Synthesis of fluorescent probe NBP

[0044] The synthesis route is as Figure 1 shown. The following are Figure 1 the preparation processes of NBP-OH and NBP in

[0045] (1) Synthesis of NBP-OH: 9-Formyl-8-hydroxynaphthyridine (1.09 g, 5 mmol), 6-hydroxy-1-tetralone (0.81 g, 5 mmol) and glacial acetic acid were added to a round-bottom flask. The mixture was stirred for 2 min at 250 rpm on a magnetic stirrer, and 2 mL of 70% perchloric acid was slowly added with a syringe. The mixture was refluxed at 90 °C for 2 h, then cooled to room temperature (25 °C). 15 mL of a petroleum ether / ethyl acetate = 1 / 1 mixture was added for washing, and the mixture was stirred for 30 min, filtered by suction, washed with a small amount of petroleum ether / ethyl acetate (v / v) = 1 / 1 mixture, and then washed with absolute ethanol. Then the product was purified by a silica gel chromatography column (dichloromethane / methanol (v / v) = 100 / 1) to obtain a blackish-green solid powder NBP-OH (1 g) with a yield of 46%.

[0046] of NBP-OH 1 H NMR (600 MHz, DMSO-d 6 ): δ 8.35 (s, 1H), 7.99 (d, J = 8.6Hz, 1H), 7.49 (s, 1H), 6.90 (d, J = 8.6 Hz, 1H), 6.81 (s, 1H), 3.55 (d, J =5.8 Hz, 4H), 3.01 (t, J = 6.4 Hz, 2H), 2.96 (s, 4H), 2.87 (t, J = 5.9 Hz,2H), 2.00 (s, 2H), 1.93 (s, 2H).

[0047] (2) Synthesis of NBP: NBP-OH (20 mg, 0.045 mmol) and triethylamine (9 mg, 0.09 mmol) were dissolved in analytical grade dichloromethane. After stirring at 25 °C for 10 min, 4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzyl chloroformate (26 mg, 0.09 mmol) was added to the system, and the reaction was carried out at 25 °C for 1 h. After the reaction was completed, the solvent was evaporated to dryness with a rotary evaporator, and then the product was purified by a silica gel chromatography column (dichloromethane / methanol (v / v) = 50 / 1) to obtain a black solid powder NBP (18 mg) with a yield of 57%. The 1H NMR spectrum, 13C NMR spectrum and mass spectrum of NBP are shown as Figures 2 to 4 shown.

[0048] of NBP 1 H NMR (600 MHz, DMSO-d 6):δ 8.46 (s, 1H), 8.14 (d, J = 8.4 Hz,1H), 7.73 (d, J = 8.1 Hz, 2H), 7.57 (s, 1H), 7.48 (d, J = 8.1 Hz, 2H), 7.43(d, J = 8.4 Hz, 2H), 5.33 (s, 2H), 3.61 (d, J = 5.7 Hz, 4H), 3.06 (d, J = 7.3Hz, 2H), 3.03 (s, 4H), 2.90 (s, 2H), 2.02 (s, 2H), 1.96 (s, 2H), 1.30 (s,12H).

[0049] of NBP 13 C NMR (151 MHz, DMSO-d 6 ):δ 158.49, 153.91, 152.75, 152.25,145.92, 142.92, 138.06, 134.67, 129.16, 127.52, 126.96, 126.51, 124.67,121.66, 120.88, 119.81, 104.53, 83.79, 69.93, 50.77, 50.33, 41.31, 31.28,29.02, 27.00, 26.17, 24.68, 24.12, 22.09, 19.74, 18.87, 18.70, 11.01.

[0050] HRMS (ESI-TOF) of NBP: m / z: [M] + Calculated for C 37 H 39 BNO 6 + 604.2869, found 604.2865.

[0051] Example 2 Spectral Response of Fluorescent Probe NBP to Hydrogen Peroxide

[0052] The detection method steps are as follows:

[0053] 1. UV / Fluorescence Spectral Changes of Fluorescent Probe NBP to H 2 O 2 Adopt the fluorescent probe NBP prepared in Example 1, and prepare a probe stock solution with a concentration of 1 mmol / L with dimethyl sulfoxide (DMSO); use deionized water to prepare 30% H

[0054] O 2 O2 The solution was prepared into a hydrogen peroxide stock solution with a concentration of 10 mmol / L; a spectral solution of acetonitrile / PBS (pH = 7.4, v / v = 4:6) (PBS is phosphate buffer solution) was prepared.

[0055] Accurately pipette 3 mL of the spectral solution into a quartz cuvette. After adding 30 μL of the probe stock solution to the cuvette, different volumes of the hydrogen peroxide stock solution were added in sequence. After reacting for 20 min, the spectral diagrams of the probe and the probe plus hydrogen peroxide solution were measured using a UV-2401PC ultraviolet-visible absorption spectrometer (zeroed with a blank control) and an RF-5301PC fluorescence absorption spectrometer (excitation wavelength was 560 nm, slit width was 3 nm).

[0056] From Figure 5 and Figure 6 it can be seen that after adding different concentrations of H 2 O 2 (0 μM~460 μM), the main absorption peak of the probe NBP (10 μM) red-shifted from 560 nm to 605 nm, and the color of the solution changed from purple to blue ( Figure 5 upper right inset). At the same time, it was recorded with a fluorescence spectrophotometer that a new fluorescence emission peak appeared at 648 nm, and the fluorescence gradually increased with the increase of the H 2 O 2 concentration, and the fluorescence changed from light red to bright red ( Figure 6 upper right inset).

[0057] From Figure 7 and Figure 8 it can be seen that in the concentration range of 0 μM~460 μM H 2 O 2 , there was a good linear relationship between the H 2 O 2 concentration and the fluorescence intensity (R 2 = 0.9824). The detection limit of the probe NBP for H 2 O 2 was calculated to be 80 nM through the formula for the detection limit LOD = 3δ / k. At the same time, with the increase of the reaction time (0~20 min), the fluorescence at 648 nm gradually increased.

[0058] 2. Selectivity and anti-interference experiments

[0059] Accurately pipette 3 mL of the spectral solution into a quartz cuvette. After adding 30 μL of the probe stock solution to the cuvette, H 2 O 2 (460 μM) and other different analytes (500 μM) were added in sequence: Na + , K + , Li+ , NH 4 + , Mg 2+ , Ca 2+ , Fe 2+ , Zn 2+ , Ba 2+ , Mn 2+ , Co 2+ , Al 3+ , Cl - , ClO - , Br - , SO 4 2- , HCO 3 - , CO 3 2- , H 2 , PO 4 - , HPO 4 2- , F - , I - , NO 2 - , NO 3 - , Na 2 S, GSH, CyS, Hcy, and then spectroscopic tests were performed at 648 nm using an RF-5301PC fluorescence absorption spectrometer.

[0060] As Figure 9 shown, the fluorescence intensity of other analytes added was very weak, and only when H 2 O 2 was added did it exhibit strong red fluorescence, indicating that the probe has strong specificity and selectivity for identifying H 2 O 2 .

[0061] Accurately pipette 3 mL of the spectroscopic solution into a cuvette. After taking 30 μL of the probe mother liquor into a quartz cuvette, other analytes (500 μM) were added in sequence: Na + , K + , Li + , NH 4 + , Mg 2+ , Ca 2+ , Fe 2+ , Zn 2+ , Ba 2+ , Mn 2+ , Co 2+ , Al 3+ , Cl - , ClO -, Br - , SO 4 2- , HCO 3 - , CO 3 2- , H 2 , HPO 4 - , HPO 4 2- , F - , I - , NO 2 - , NO 3 - , Na 2 S, GSH, CyS, HCy, and then H 2 O 2 (460 µM) was added and spectral tests were performed at 648 nm using an RF-5301PC fluorescence absorption spectrometer.

[0062] As Figure 10 shown, the addition of other analytes had a minimal interference on the fluorescence signal of the probe for recognizing H 2 O 2 , indicating that the probe for detecting H 2 O 2 had good anti-interference ability.

[0063] 3. Effect of pH on the fluorescence probe

[0064] Solutions with different pH values (2, 3, 4, 5, 6, 7, 7.4, 8, 9, 10, 11) were prepared respectively. A spectral test system was prepared according to the ratio of acetonitrile / each pH solution = 4 / 6. After adding NBP (10 µM), H 2 O 2 (460 µM) was added. After reacting at room temperature (25 °C) for 20 min, spectral tests were performed at 648 nm using an RF-5301PC fluorescence absorption spectrometer, and the fluorescence intensity was recorded.

[0065] As Figure 11 shown, when the pH was 2 - 6, the fluorescence intensity before and after the addition of H 2 O 2 could be ignored. In the presence of H 2 O 2 , when the pH value was 6 - 8, the fluorescence intensity of the NBP solution gradually increased and reached the peak at pH = 8. When the pH was 8 - 11, the fluorescence intensity decreased rapidly. While for the probe itself, when the pH value was 6 - 11, the increase in fluorescence intensity was relatively slow. It indicated that the probe NBP was suitable for detection applications under physiological pH and alkaline conditions.

[0066] Example 3 Detection of Hydrogen Peroxide in Living Cells Using the Fluorescent Probe NBP

[0067] Detection was carried out using the fluorescent probe NBP prepared in Example 1, and the detection object was HepG2 cells. The detection imaging method is as follows:

[0068] (1)Detection of endogenous hydrogen peroxide in HepG2 cells

[0069] After the HepG2 cells were resuscitated, the cell concentration was adjusted and divided into three groups with equal cell concentrations (all seeded into 24-well plates at a density of 2×10 4 cells / well). The first group was the control group without adding PMA and NAC; the second group was incubated with PMA (1 μg / mL) for 30 min; the third group was treated with PMA (1 μg / mL) for 30 min and then with NAC (1 mM) for 30 min. Finally, all cells were treated with NBP (10 μM) for 30 min.

[0070] (2)Detection of exogenously added hydrogen peroxide in HepG2 cells

[0071] After the HepG2 cells were resuscitated, the cell concentration was adjusted and grouped. Each group of cells was added with different concentrations of H 4 O 2 O 2 (50 and 100 μM) at the same cell concentration (all seeded into 24-well plates at a density of 2×10 2 cells / well) and treated for 30 min. Finally, all cells were treated with NBP (10 μM) for 30 min.

[0072] The above detections were all subjected to fluorescence imaging analysis of each group of cells under a laser confocal microscope Olympus FV3000 (excitation wavelength λex = 561 nm, emission wavelength λem = 570 nm - 670 nm).

[0073] It can be seen that the fluorescence of the probe itself is weak ( Figure 12 a1 - a3), red fluorescence can be seen after induction with PMA ( Figure 12 b1 - b3), and the fluorescence is significantly weakened after treatment with NAC ( Figure 12 c1 - c3). As the concentration of exogenous H Figure 12 O 2 O 2 increases, the fluorescence gradually increases ( Figure 12 d1 - d3 and 12e1 - e3). Through gradient investigation and analysis, in the fluorescence imaging of extracellular hydrogen peroxide in cells, the probe and hydrogen peroxide concentration show a good linear relationship in the range of 0 μM - 100 μM (R 2= 0.9979), the detection limit was calculated to be 3.2 μM according to the formula LOD = 3δ / k.

[0074] Example 4 Detection of hydrogen peroxide in food using the fluorescent probe NBP

[0075] Detection was carried out using the fluorescent probe NBP prepared in Example 1, and the detection objects were milk (whole milk, skim milk, and organic milk). The detection method was as follows:

[0076] Put 25 mL of each of the three milk samples (whole milk, skim milk, and organic milk) into a 100 mL volumetric flask, add zinc acetate solution and potassium ferrocyanide solution, and then dilute to 100 mL with deionized water. The mixture was centrifuged at 1000 rpm for 10 min, and the supernatant of each sample was mixed with acetonitrile at a volume ratio of 6 / 4 as the test sample.

[0077] Add NBP to each sample and treat for 30 min and treat with different concentrations of H 2 O 2 for 30 min, and after ultraviolet light irradiation, fluorescence analysis was carried out using the mobile phone software ColorAssist in the iphone13.

[0078] As Figure 13 shown in a, when 10 μM NBP was added to the milk, under the irradiation of 365 nm ultraviolet light, the red fluorescence of the solution was very weak. As the concentration of exogenous H 2 O 2 increased, the red fluorescence of the solution gradually enhanced.

[0079] The fluorescence RGB values of each group of milk samples were analyzed using the mobile phone software ColorAssist in the iphone13 (as Figure 13 shown in b-13c), and it was found that there was a good linear relationship between the R value of each group of milk and the concentration of H 2 O 2 in the concentration range of 0 μM to 100 μM (R 2 = 0.9848, 0.9246, 0.9693) ( Figure 13 d), and the detection limits of the probe NBP for detecting hydrogen peroxide in milk were calculated to be 0.46 μM, 0.52 μM, and 0.54 μM respectively according to the detection limit formula LOD = 3δ / k. The above results indicate that the NBP probe can visually and quantitatively visualize the detection of residual hydrogen peroxide in various milk samples, and flexible and convenient on-site detection can be carried out using mobile phone software. And the irradiation of 365 nm ultraviolet light is a common device that is easily available in the market.

[0080] Example 5 Detection of hydrogen peroxide in plants using the fluorescent probe NBP

[0081] Platycodon grandiflorum belongs to the family Campsis grandiflora and is an important medicinal and edible plant in East Asia, with potential therapeutic effects. And H 2 O 2 As an important signaling molecule, it can regulate the growth of Platycodon grandiflorum. Measuring the H 2 O 2 level in Platycodon grandiflorum under stress by fluorescence imaging is of great significance for evaluating the risk of oxidative stress, improving the stress resistance and productivity of Platycodon grandiflorum. Before studying the H 2 O 2 fluorescence imaging performance of NBP in plants, the inventors analyzed the toxicity of NBP to Platycodon grandiflorum by observing the overall morphological changes of Platycodon grandiflorum seedlings. The morphology of the seedlings treated with 0 μM - 30 μM NBP was similar to that of the normal control group. When the concentration of the probe NBP was no higher than 20 μM, the average root length of the seedlings incubated with the probe NBP was not different from that of the normal control group, indicating that the probe NBP can be safely used for staining the roots of living plants and performing fluorescence imaging, with low toxicity to the whole plant, and thus can be used for H 2 O 2 fluorescence imaging detection of living plants.

[0082] Inspired by the above experimental results, the inventors further used NBP to perform fluorescence imaging on endogenous H 2 O 2 and exogenously added H 2 O 2 in the roots of Platycodon grandiflorum. All the following Platycodon grandiflorum were subjected to fluorescence imaging analysis under a laser confocal microscope Olympus FV1000.

[0083] (1) Detection of endogenous hydrogen peroxide in the roots of Platycodon grandiflorum using NBP

[0084] After incubating the roots of Platycodon grandiflorum in a 5% polyethylene glycol solution for different times (0, 6, 12, 18, 24 h), the roots were washed three times with deionized water, immersed in deionized water containing the probe (10 μM) for incubation for 20 - 30 min (either 20 - 30 min is acceptable, 30 min was used in this study), and then washed three times with deionized water before performing root fluorescence imaging (excitation wavelength λex = 559 nm, emission wavelength λem = 570 nm - 670 nm; scale bar = 100 μm).

[0085] The results are shown in Figure 14 the brightfield imaging diagram (Brightfield), the red channel imaging diagram (Red channel) and the merged imaging diagram (Merge). As the stress treatment time extended, the red channel fluorescence signal gradually increased, and the fluorescence intensity had a good linear relationship with time within 0 - 24 h (R 2= 0.9143).

[0086] (2)NBP was used for the detection of exogenous hydrogen peroxide added to the roots of Platycodon grandiflorum

[0087] Platycodon grandiflorum seedlings were incubated with different concentrations of H 2 O 2 (0, 50, 100, 150 μM; prepared by diluting 30% hydrogen peroxide with deionized water) for 30 min, and then incubated with deionized water containing the probe NBP (10 μM) for 20 - 30 min (either 20 - 30 min is acceptable, 30 min was used in this study). After washing three times with deionized water, root fluorescence imaging was performed (excitation wavelength λex = 559 nm, emission wavelength λem = 570 nm - 670 nm; scale bar = 100 μm).

[0088] The results are shown in the bright - field imaging diagram (Brightfield), red - channel imaging diagram (Red channel), and merged imaging diagram (Merge) as follows: incubated with 0 (a1 - a3), 50 (b1 - b3), 100 (c1 - c3), 150 (d1 - d3) μM of H Figure 15 for 30 min, and then incubated with 10 μM of NBP for 30 min. As the concentration of H 2 O 2 increased, the red - channel fluorescence signal gradually enhanced, and the fluorescence intensity had a good linear relationship with the concentration within the range of 0 μM - 150 μM (R 2 O 2 = 0.9758). According to the detection limit formula LOD = 3δ / k, the detection limit of the probe for detecting hydrogen peroxide in Platycodon grandiflorum was calculated to be 2.03 μM. 2 = 0.9758), and according to the detection limit formula LOD = 3δ / k, the detection limit of the probe for detecting hydrogen peroxide in Platycodon grandiflorum was calculated to be 2.03 μM.

[0089] The physiological concentration range of H 2 O 2 in plants is 10 μM - 100 μM, and the concentration of H 2 O 2 increases under stress or pathological conditions. Since the NBP of the present invention has a good linear relationship within the range of 0 μM - 150 μM and a detection limit of 2.03 μM, it is suitable for the detection of endogenous or exogenous addition of H 2 O 2 in plants such as Platycodon grandiflorum under physiological or pathological conditions. This detection limit and linear range are significantly better than other reported plant H 2 O 2 fluorescent probes. Moreover, this NBP fluorescent probe does not need to be injected into the plant body, but can be detected by soaking and incubating for 20 - 30 min, having the advantages of easy use, low destructiveness, and fast response speed.

[0090] In summary, the fluorescence imaging of exogenous and endogenous H in living cells was studied using the fluorescent probe NBP. 2 O 2 Combining the fluorescent probe NBP with a smartphone enables the visual detection of residual H in milk. 2 O 2 Using this probe, the accumulation of H in the roots of Platycodon grandiflorum under drought stress and the fluorescence signal intensity after adding exogenous H can be detected. 2 O 2 Similarly, the fluorescent probe NBP provided by the present invention has good selectivity, high sensitivity, a low detection limit (80 nM), and a wide linear range for H. 2 O 2 It is applicable to the detection of H in dairy products, ex vivo living cells, and plants. 2 O 2 2 2 O 2

[0091] The preferred embodiments of the present invention have been described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the scope of the technical concept of the present invention, various simple transformations can be made to the technical solutions of the present invention, and these simple transformations all fall within the protection scope of the present invention.​

Claims

1. A selective hydrogen peroxide fluorescent probe, characterized in that: The structural formula of the hydrogen peroxide fluorescent probe is shown in formula (I): ； （Ⅰ）。 2. A method for preparing the selective hydrogen peroxide fluorescent probe according to claim 1, characterized in that: The preparation method comprises the following steps: reacting the compound represented by formula (Ia) with the compound represented by formula (Ib) in the presence of a base and an organic solvent to obtain a hydrogen peroxide fluorescent probe represented by formula (I), and the reaction formula is as follows: ； Formula (Ia) Formula (Ib) Formula (I); Wherein, X is a halogen.

3. The method for preparing the selective hydrogen peroxide fluorescent probe according to claim 2, characterized in that: In the reaction, the molar ratio of the compound represented by formula (Ia) to the compound represented by formula (Ib) is 1:1 to 1:

3.

4. The method for preparing the selective hydrogen peroxide fluorescent probe according to claim 2, characterized in that: The base is any one of triethylamine, potassium carbonate and cesium carbonate; the organic solvent is any one of dichloromethane, anhydrous acetonitrile, anhydrous methanol and N,N-dimethylformamide.

5. The method for preparing the selective hydrogen peroxide fluorescent probe according to claim 2, characterized in that: The reaction conditions are as follows: reaction at room temperature for 0.5 h to 1.5 h.

6. A kit for detecting hydrogen peroxide, characterized in that: The kit contains the hydrogen peroxide fluorescent probe according to claim 1.

7. Use of the selective hydrogen peroxide fluorescent probe according to claim 1 in the detection of hydrogen peroxide in food samples, characterized in that: The food is a dairy product.

8. Use of the selective hydrogen peroxide fluorescent probe according to claim 1 in detecting hydrogen peroxide in ex vivo cells for purposes other than disease diagnosis or treatment.

9. Use of the selective hydrogen peroxide fluorescent probe according to claim 1 in the detection of hydrogen peroxide in plants.

10. A method for detecting hydrogen peroxide in plant roots, characterized in that The method comprises the following steps: washing plant roots with water, immersing them in a probe solution containing the hydrogen peroxide fluorescent probe according to claim 1 for incubation, and performing root system fluorescence imaging analysis after washing with water.

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