Selective hydrogen peroxide fluorescent probe, preparation method, kit and application

Abstract

The invention belongs to the technical field of analysis and detection, and particularly relates to a selective hydrogen peroxide fluorescent probe, a preparation method, a kit and application. After the fluorescent probe reacts with hydrogen peroxide, the main absorption peak is subjected to red shift from 560 nm to 605 nm, and a new fluorescence emission peak is formed at 648 nm. The linear range of hydrogen peroxide detection can reach 0M-460M, and the detection limit can be as low as 80nM. Through investigation, the fluorescent probe has the advantages of high specificity or selectivity, high sensitivity, wide linear range, high response speed and the like in detection of hydrogen peroxide in a milk sample, a cell sample and a plant sample, so that the fluorescent probe has a wide application prospect and can be combined with means such as mobile phone end software or a laser confocal microscope and the like for fluorescence analysis; and the application convenience is improved, and the method has a good application prospect.

Description

Technical Field

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

[0002] Hydrogen peroxide (H2O2) is a common reactive oxygen species (ROS) that exists in environmental water bodies and in plants and animals. It can cause oxidative stress reactions, gradually accumulate, and induce or aggravate damage to cells, tissues, and organs. For example, plants are constantly affected by various stress factors in the environment throughout their life cycle, including metals, drought, low or high temperatures, floods, etc. These stresses can seriously damage plant tissues and hinder plant growth. As an important reactive oxygen species (ROS), hydrogen peroxide participates in a variety of biological processes and has become a key signal molecule for regulating stress responses. It is considered an indicator of acute stress. In addition, hydrogen peroxide is also widely used in food disinfection and sterilization as an antifungal agent. Excessive hydrogen peroxide residues in food can cause various diseases in the human body, such as cerebral stroke, depression, and gas embolism. 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 dairy industry, excessive hydrogen peroxide is also harmful to the nutritional value of milk. For example, excessive hydrogen peroxide will destroy the nutrient folic acid in milk, and its degradation will reduce the nutritional value of milk. Hydrogen peroxide residues will accelerate the deterioration of milk, causing the milk to taste bad and shortening its shelf life. Therefore, it is necessary to develop a simple, efficient and intuitive method for hydrogen peroxide monitoring.

[0003] In this regard, a variety of hydrogen peroxide analysis methods have been developed in this field, such as high performance liquid chromatography, colorimetry, mass spectrometry, spectrophotometry, electron spin resonance, electrochemical method, etc. However, these methods all have certain limitations, such as long response time, poor specificity, high cost, and inability to achieve on-site detection. In contrast, fluorescent probe monitoring has the advantages of high selectivity, simple operation, low cost, and real-time in situ imaging, and is considered to be a very promising method for measuring hydrogen peroxide. However, in terms of plants and animals, the application of hydrogen peroxide fluorescence detection in animals and plants is limited due to the different tissue penetration capabilities of light of different wavelengths and the background light interference of the plants and animals themselves. Moreover, various metal ions, reactive oxygen species, reactive sulfides, fluorescent substances (such as anthocyanins, lignin, etc.) in animals and plants, especially in plants themselves under stress or other conditions, will interfere with fluorescence detection. Therefore, it poses a severe challenge to the development of fluorescent 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. Master's thesis 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, there are few reports of fluorescent probes with high selectivity / high sensitivity and wide linear range that are suitable for the fluorescence detection of hydrogen peroxide in animals and plants, especially in plants with many interfering factors. Therefore, it is urgent to develop a hydrogen peroxide fluorescent probe with a wide range of applications, 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 and preparation method, kit and application for accurate identification and fluorescence imaging of hydrogen peroxide in food, in vitro cells and plants. The probe is simple to synthesize and easy to operate, has high sensitivity, good selectivity, wide linear range and fast response speed for hydrogen peroxide detection, and has higher practical value for visual detection of hydrogen peroxide in food, cells and plants; and can be used with mobile phone software or laser confocal microscope for fluorescence analysis, which improves the convenience of application.

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

[0007] (I).

[0008] The second aspect of the present invention provides a method for preparing NBP, comprising the following steps: reacting a compound represented by formula (Ia) with a compound represented by formula (Ib) in the presence of a base and an organic solvent to obtain NBP. The reaction formula is as follows:

[0009] Formula (Ia) Formula (Ib) Formula (I) ; Wherein, X is a halogen, preferably chlorine.

[0010] Preferably, in the above reaction, the molar ratio of the compound represented by formula (Ia) to the compound represented by formula (Ib) is 1:1 to 1:3; further preferably, the molar ratio of the compound represented by formula (Ia) to the compound represented by formula (Ib) is 1:2.

[0011] Preferably, in the above 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.

[0012] Preferably, in the above reaction, the reaction conditions are 0.5h to 1.5h at room temperature. More preferably, the reaction conditions are 1h at room temperature. The term "room temperature" is defined as 10°C to 30°C according to the Chinese Pharmacopoeia.

[0013] The third aspect of the present invention provides a kit for detecting hydrogen peroxide containing NBP.

[0014] The present invention further provides an application of NBP in hydrogen peroxide detection, comprising: (1) Application in the detection of hydrogen peroxide in food samples, wherein the food is a dairy product. Preferably, the dairy product is any one of whole milk, skim milk and organic milk.

[0015] (2) Application in the detection of hydrogen peroxide in ex vivo cells for purposes other than disease diagnosis or treatment. Preferably, the ex vivo cells are liver cancer cells; more preferably, the liver cancer cells are HepG2 liver cancer cells.

[0016] (3) Application in the detection of hydrogen peroxide in plants. Preferably, the plant is Platycodon grandiflorum, and more preferably, the detection site of Platycodon grandiflorum is the root of Platycodon grandiflorum.

[0017] The present invention further provides a method for detecting hydrogen peroxide in plant roots, comprising the following steps: washing the plant roots with water, immersing them in a probe solution containing NBP for incubation, and performing root system fluorescence imaging analysis after washing with water.

[0018] The beneficial effects of the present invention are: 1. The fluorescent probe NBP provided by the present invention uses benzopyran salt as a fluorophore and aryl borate as a specific deprotection group mediated by hydrogen peroxide, wherein 4-(hydroxymethyl)phenylboronic acid pinacol ester is covalently conjugated to one end of the double condensed part. In spectral studies, the probe exhibits 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, and its fluorescence intensity has a good linear relationship with the hydrogen peroxide concentration in the range of 0µM~460µM. The raw materials for the preparation of the probe are easily available, the synthesis is simple, and the operation is simple.

[0019] 2. The present invention applies fluorescent probe imaging technology to the detection of hydrogen peroxide in HepG2 living cells, and studies the imaging detection ability of the probe for the level of endogenous / exogenous hydrogen peroxide in living cells. The results show that after induction by phorbol 12-myristate 13-acetate (PMA for short) and then treatment with the probe, clear red fluorescence can be seen. After pretreatment with N-acetylcysteine ​​(NAC for short, a hydrogen peroxide inhibitor), the intracellular fluorescence was significantly weakened and negligible. In addition, by exogenously adding different concentrations (50 and 100 μM) of hydrogen peroxide, it can be observed that the fluorescence signal exhibited by the cells gradually increases with the increase in hydrogen peroxide concentration. The results show that this fluorescent probe can be used for fluorescent imaging of endogenous / exogenous hydrogen peroxide in living cells.

[0020] 3. The present invention uses a fluorescent probe combined with a smartphone software platform to complete the on-site detection of hydrogen peroxide in milk samples, and the color change can be observed intuitively without any additional equipment. Compared with the use of large instruments for data analysis, this method can visualize the content of hydrogen peroxide in milk, making the results more intuitive while greatly reducing the detection cost. In the range of 0μM~100μM hydrogen peroxide, the detection method has a good linear relationship, and the detection limit is 0.46μM~0.54μM.

[0021] 4. The present invention applies the fluorescent 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 exogenous addition of hydrogen peroxide. The results show that as the simulated drought stress treatment time increases, the red channel fluorescence signal gradually increases. Studies have shown that this fluorescent probe has good anti-interference ability in the detection of hydrogen peroxide in plants, with a low detection limit (2.03μM), a wide linear range (0μM~150μM), a fast response speed (20min), and no need to be injected into the plant body (only soaking and incubation is convenient and practical). The linear range is greater than the physiological hydrogen peroxide concentration range of the plant, and is suitable for detecting endogenous or exogenously added (such as artificial hydrogen peroxide treatment) hydrogen peroxide under plant physiological or pathological conditions. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 This is the synthetic route of the fluorescent probe NBP; Figure 2 This is the nuclear magnetic resonance hydrogen spectrum of the fluorescent probe NBP; Figure 3 This is the carbon NMR spectrum of the fluorescent probe NBP; Figure 4 is the mass spectrum of the fluorescent probe NBP; Figure 5The UV absorption spectrum of 10µM fluorescent probe NBP after adding 0µM to 460µM hydrogen peroxide and the photos of 10µM fluorescent probe NBP under fluorescent light before and after adding hydrogen peroxide reaction; Figure 6 The fluorescence spectrum change diagram of 10µM fluorescent probe NBP after adding 0µM~460µM hydrogen peroxide and the photos of 10µM fluorescent probe NBP before and after adding hydrogen peroxide under 365nm ultraviolet light irradiation; Figure 7 This is the linear relationship between the fluorescence intensity of 10µM fluorescent probe NBP at 648nm and the concentration of hydrogen peroxide; Figure 8 This is a graph showing the change in fluorescence intensity over time after adding 460µM hydrogen peroxide to 10µM fluorescent probe NBP; Fig. 9 The fluorescence spectrum changes after adding 460µM hydrogen peroxide or 500µM other analytes to 10µM fluorescent probe NBP; in the figure: 1 is Blank; 2 is Na + ; 3 is K + ; 4 is Li + ; 5 is NH4 + ; 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 SO4 2- ; 18 is HCO3 - ; 19 is CO3 2- ; 20 is H2PO4 - ; 21 is HPO4 2- ; 22 is F - ; 23 I - ; 24 is NO2 - ; 25 for NO3 - ; 26 is Na2S; 27 is GSH (glutathione); 28 is CyS (cysteine); 29 is Hcy (homocysteine); 30 is H2O2; Fig.10The fluorescence spectrum of 10µM fluorescent probe NBP after adding 460µM hydrogen peroxide in the presence of 500µM other analytes; in the figure: 1 is Blank; 2 is Na + ; 3 is K + ; 4 is Li + ; 5 is NH4 + ; 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 SO4 2- ; 18 is HCO3 - ; 19 is CO3 2- ; 20 is H2PO4 - ; 21 is HPO4 2- ; 22 is F - ; 23 I - ; 24 is NO2 - ; 25 for NO3 - ; 26 is Na2S; 27 is GSH (glutathione); 28 is CyS (cysteine); 29 is Hcy (homocysteine); Fig.11 The fluorescence spectrum change diagram of 10µM fluorescent probe NBP before and after the reaction with 460µM hydrogen peroxide under different pH conditions; Fig.12 The fluorescence images of endogenous hydrogen peroxide in HepG2 cells treated with PMA or PMA+NAC for 30 minutes, then incubated with the fluorescent probe NBP for 30 minutes, and the fluorescence images of exogenous hydrogen peroxide in HepG2 cells treated with 50μM and 100μM hydrogen peroxide for 30 minutes, then treated with the probe for 30 minutes. Fig.13 Fluorescence photos of three milk samples after adding 10 µM fluorescent probe NBP and 0, 10, 50 and 100 μM hydrogen peroxide under 365 nm ultraviolet light (13a), the RGB data analysis display interface of each group of milk collected by the smartphone software ColorAssist (13b), the R values ​​of three milk samples after adding different concentrations of hydrogen peroxide (13c) and the linear relationship between the R value and the hydrogen peroxide concentration (13d); Fig.14The fluorescence imaging image is shown after the roots of Platycodon grandiflorum were incubated with 5% polyethylene glycol for different time periods (0, 6, 12, 18, 24 h) and then incubated with 10 µM fluorescent probe NBP for 30 min. Fig.15 This is the fluorescence imaging after adding different concentrations of hydrogen peroxide (0, 50, 100, 150μM) to Platycodon grandiflorum and then incubating with 10μM fluorescent probe NBP for 30 minutes. DETAILED DESCRIPTION

[0023] The present invention is further explained and illustrated below in conjunction with specific embodiments and drawings to make the technical solution of the present invention clearer and more complete. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

[0024] Example 1 Synthesis of fluorescent probe NBP Synthesis route such as Figure 1 As shown, the following Figure 1 The preparation process of NBP-OH and NBP, the reagents used are all commercially available reagents: (1) Synthesis of NBP-OH: 9-formaldehyde-8-hydroxyjulolidine (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 and stirred at 250 rpm for 2 min. 2 mL of 70% perchloric acid was slowly added with a syringe and refluxed at 90°C for 2 h. After cooling to room temperature (25°C), 15 mL of a 1 / 1 mixture of petroleum ether and ethyl acetate was added for washing. The mixture was stirred for 30 min, filtered, washed with a small amount of a 1 / 1 mixture of petroleum ether and ethyl acetate (v / v), and washed with anhydrous ethanol. The product was then purified by silica gel column chromatography (dichloromethane / methanol (v / v) = 100 / 1) to obtain a dark blue solid powder NBP-OH (1 g) with a yield of 46%.

[0025] NBP-OH 1H NMR (600 MHz, DMSO-d6): 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).

[0026] (2) Synthesis of NBP: NBP-OH (20 mg, 0.045 mmol) and triethylamine (9 mg, 0.09 mmol) were dissolved in analytical grade dichloromethane, stirred at 25°C for 10 min, and then 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 reacted at 25°C for 1 h. After the reaction, the solvent was dried using a rotary evaporator, and the product was purified using a silica gel column (dichloromethane / methanol (v / v) = 50 / 1) to obtain a black solid powder NBP (18 mg) with a yield of 57%. The hydrogen NMR spectrum, carbon NMR spectrum, and mass spectrum of NBP are shown in Figure 1. Figure 2~Figure 4 shown.

[0027] NBP 1 H NMR (600 MHz, DMSO-d6): 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).

[0028] NBP 13C NMR (151 MHz, DMSO-d6): 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.

[0029] NBP’s HRMS (ESI-TOF): m / z:[M] + Calculated as C 37 H 39 BNO6 + 604.2869, found to be 604.2865.

[0030] Example 2 Spectral response of fluorescent probe NBP to hydrogen peroxide The detection method steps are as follows: 1. Changes in UV / fluorescence spectra of fluorescent probe NBP to H2O2 The fluorescent probe NBP prepared in Example 1 was used to prepare a probe stock solution with a concentration of 1 mmol / L using dimethyl sulfoxide (DMSO); a 30% H2O2 solution was prepared into a hydrogen peroxide stock solution with a concentration of 10 mmol / L using deionized water; and a spectral solution of acetonitrile / PBS (pH = 7.4, v / v = 4:6) (PBS is phosphate buffered saline) was prepared.

[0031] Accurately transfer 3 mL of spectral solution into a quartz cuvette, take 30 μL of probe stock solution into the cuvette, and then add different volumes of hydrogen peroxide stock solution in sequence. After reacting for 20 minutes, use a UV-2401PC ultraviolet-visible absorption spectrometer (using a blank control for zero adjustment) and a RF-5301PC fluorescence absorption spectrometer (excitation wavelength of 560 nm, slit width of 3 nm) to test the spectra of the probe and the probe plus hydrogen peroxide solution.

[0032] from Figure 5 and Figure 6 It can be seen that after adding different concentrations of H2O2 (0µM~460µM), the main absorption peak of the probe NBP (10 µM) red-shifted from 560nm to 605nm, and the color of the solution changed from purple to blue ( Figure 5At the same time, a new fluorescence emission peak was recorded at 648 nm using a fluorescence spectrophotometer, and the fluorescence gradually increased with the increase of H2O2 concentration, and the fluorescence changed from light red to bright red ( Figure 6 upper right illustration).

[0033] from Figure 7 and Figure 8 It can be seen that in the range of 0µM~460µM H2O2 concentration, there is a good linear relationship between H2O2 concentration and fluorescence intensity (R 2 = 0.9824). The detection limit of probe NBP for H2O2 was calculated to be 80 nM using the detection limit formula LOD = 3δ / k. At the same time, as the reaction time (0-20 min) increased, the fluorescence at 648 nm gradually increased.

[0034] 2. Selectivity and Anti-interference Experiments Accurately pipette 3 mL of the spectral solution into a quartz cuvette, take 30 μL of the probe stock solution into the cuvette, and then add H2O2 (460 μM) and other different analytes (500 μM): Na + , K + , Li + NH4 + Mg 2+ , Ca 2+ , Fe 2+ 、Zn 2+ , Ba 2+ , Mn 2+ 、Co 2+ 、Al 3+ , Cl - , ClO - Br - 、SO4 2- 、HCO3 - 、CO3 2- 、H2PO4 - 、HPO4 2- 、F - ,I - 、NO2 - 、NO3 - , Na2S, GSH, CyS, Hcy, and then the spectrum was tested at 648nm using RF-5301PC fluorescence absorption spectrometer.

[0035] like Fig. 9 As shown, the fluorescence intensity of other analytes is very weak, and only when H2O2 is added will it show strong red fluorescence, which indicates that the probe has strong specificity and selectivity for recognizing H2O2.

[0036] Accurately pipette 3 mL of the spectral solution into the cuvette, take 30 μL of the probe stock solution into the quartz cuvette, and then add other analytes (500 μM): Na + , K + , Li + NH4 + Mg 2+ , Ca 2+ , Fe 2+ 、Zn 2+ , Ba 2+ , Mn 2+ 、Co 2+ 、Al 3+ , Cl - , ClO - Br - 、SO4 2- 、HCO3 - 、CO3 2- 、H2PO4 - 、HPO4 2- 、F - ,I - 、NO2 - 、NO3 - , Na2S, GSH, CyS, and HCy, and then H2O2 (460 µM) was added respectively, and the spectrum was tested at 648 nm using RF-5301PC fluorescence absorption spectrometer.

[0037] like Fig.10 As shown, the addition of other analytes has minimal interference on the fluorescence signal of the probe recognizing H2O2, indicating that the probe has good anti-interference ability in detecting H2O2.

[0038] 3. Effect of pH on fluorescent probes Solutions with different pH values ​​(2, 3, 4, 5, 6, 7, 7.4, 8, 9, 10, and 11) were prepared respectively, and the spectral test system was prepared according to the ratio of acetonitrile / each pH solution = 4 / 6. After adding NBP (10 µM), H2O2 (460 µM) was added. After reacting for 20 min at room temperature (25°C), the spectral test was performed at 648 nm using an RF-5301PC fluorescence absorption spectrometer, and the fluorescence intensity was recorded.

[0039] like Fig.11As shown in the figure, when the pH is 2-6, the fluorescence intensity before and after the addition of H2O2 is negligible. In the presence of H2O2, when the pH is 6-8, the fluorescence intensity of the NBP solution gradually increases, reaches a peak when the pH reaches 8, and decreases rapidly when the pH is 8-11. The fluorescence intensity of the probe itself increases slowly when the pH is 6-11. This shows that the probe NBP is suitable for detection applications under physiological pH and alkaline conditions.

[0040] Example 3: Application of fluorescent probe NBP in the detection of hydrogen peroxide in living cells The fluorescent probe NBP prepared in Example 1 was used for detection, and the detection object was HepG2 cells. The detection imaging method was as follows: (1) Detection of endogenous hydrogen peroxide in HepG2 cells After HepG2 cells were revived, the cell concentration was adjusted and divided into three groups. The cell concentrations of the three groups were equal (all 2×10 4 The cells were inoculated into 24-well plates at a density of 10 cells / well. The first group was the control group, without 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, followed by NAC (1 mM) for 30 min. Finally, all cells were treated with NBP (10µM) for 30 min.

[0041] (2) Detection of exogenously added hydrogen peroxide in HepG2 cells After HepG2 cells were revived, the cell concentration was adjusted and grouped. Cells in each group were cultured at the same cell concentration (2×10 4 The cells were seeded in 24-well plates at a density of 10 cells / well and treated with different concentrations of H2O2 (50 and 100 μM) for 30 min. Finally, all cells were treated with NBP (10 μM) for 30 min.

[0042] The above tests were performed by fluorescence imaging analysis of each group of cells under a laser confocal microscope Olympus FV3000 (excitation wavelength λex = 561 nm, emission wavelength λem = 570nm~670nm).

[0043] Depend on Fig.12 It can be seen that the fluorescence of the probe itself is weak ( Fig.12 a1-a3), red fluorescence was observed after PMA induction ( Fig.12 b1-b3), and then the fluorescence was significantly weakened after NAC treatment ( Fig.12 c1-c3). As the concentration of exogenous H2O2 increases, the fluorescence gradually increases ( Fig.12d1-d3 and 12e1-e3). Gradient investigation and analysis showed that in the fluorescence imaging of exogenous hydrogen peroxide in cells, the probe and hydrogen peroxide concentration showed a good linear relationship in the range of 0μM~100μM (R 2 = 0.9979), the detection limit was calculated to be 3.2 μM according to the formula LOD = 3δ / k.

[0044] Example 4: Detection of hydrogen peroxide in food using fluorescent probe NBP The fluorescent probe NBP prepared in Example 1 was used for detection, and the detection object was milk (whole milk, skim milk and organic milk). The detection method is as follows: 25 mL of each of the three milk samples (whole milk, skim milk, and organic milk) were placed in a 100 mL volumetric flask, zinc acetate solution and potassium ferrocyanide solution were added, and then diluted 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.

[0045] NBP was added to each sample for 30 minutes and different concentrations of H2O2 were treated for 30 minutes. After ultraviolet light irradiation, fluorescence analysis was performed using the mobile phone software ColorAssist in iPhone13.

[0046] like Fig.13 As shown in a, when 10 μM NBP was added to milk, the red fluorescence of the solution was very weak under 365 nm ultraviolet light. As the concentration of exogenous H2O2 increased, the red fluorescence of the solution gradually increased.

[0047] The fluorescence RGB values ​​of each group of milk samples were analyzed using the mobile phone software ColorAssist in iPhone 13 (such as Fig.13 b-13c), it was found that the R value of each group of milk had a good linear relationship with the H2O2 concentration in the concentration range of 0μM~100μM (R 2 = 0.9848, 0.9246, 0.9693)( Fig.13 d), according to the detection limit formula LOD = 3δ / k, the detection limits of probe NBP for hydrogen peroxide in milk were calculated to be 0.46 μM, 0.52 μM and 0.54 μM respectively. The above results show that the NBP probe can visually and quantitatively detect residual hydrogen peroxide in various milk samples, and mobile phone software can be used for flexible and convenient on-site detection. 365 nm ultraviolet light irradiation is a common device that is easily available in the market.

[0048] Example 5: Fluorescent probe NBP for detection of hydrogen peroxide in plants Platycodon grandiflorum belongs to the Campestaceae family and is an important medicinal and edible plant in East Asia with potential therapeutic effects. H2O2, as an important signal molecule, can regulate the growth of Platycodon grandiflorum. Determining the H2O2 level of Platycodon grandiflorum under stress by fluorescence imaging is of great significance for evaluating the risk of oxidative stress and improving the stress resistance and productivity of Platycodon grandiflorum. Before studying the H2O2 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 seedlings treated with 0μM~30μM NBP is similar to that of the normal control group. When the concentration of probe NBP is not higher than 20μM, the average root length of seedlings incubated with probe NBP is no different from that of the normal control group, indicating that the probe NBP can be safely used for root staining and fluorescence imaging of living plants, and has low toxicity to the entire plant, so it can be used for H2O2 fluorescence imaging detection of living plants.

[0049] Inspired by the above experimental results, the inventors further used NBP to perform fluorescence imaging of endogenous H2O2 and exogenously added H2O2 in the roots of Platycodon grandiflorum. All the following Platycodon grandiflorum were analyzed by fluorescence imaging under a laser confocal microscope Olympus FV1000.

[0050] (1) Application of NBP in the detection of endogenous hydrogen peroxide in Platycodon grandiflorum roots The roots of Platycodon grandiflorum were placed in a 5% polyethylene glycol solution and incubated for different periods of time (0, 6, 12, 18, and 24 h). The roots were then washed three times in deionized water and immersed in deionized water containing the probe (10 μM) for incubation for 20 to 30 min (20 to 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 to 670 nm; scale bar = 100 μm).

[0051] The results are as follows Fig.14 As shown in the brightfield, red channel and merged images, the red channel fluorescence signal gradually increased with the extension of stress treatment time, and the fluorescence intensity showed a good linear relationship with time within 0-24h (R 2 = 0.9143).

[0052] (2) NBP is used to detect exogenous addition of hydrogen peroxide to Platycodon grandiflorum roots The roots of Platycodon grandiflorum seedlings were incubated with different concentrations of H2O2 (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 (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 = 570nm-670nm; scale bar = 100 μm).

[0053] The results are as follows Fig.15 The brightfield image (Brightfield), red channel image (Red channel) and merged image (Merge) are shown: 0 (a1-a3), 50 (b1-b3), 100 (c1-c3), 150 (d1-d3) μM H2O2 were used for incubation for 30 min, and then 10 μM NBP was used for incubation for 30 min. With the increase of H2O2 concentration, the red channel fluorescence signal gradually increased, and the fluorescence intensity showed a good linear relationship with the concentration in the range of 0μM~150μM (R 2 =0.9758), and according to the detection limit formula LOD = 3δ / k, the detection limit of the probe pair for detecting hydrogen peroxide in Platycodon grandiflorum was calculated to be 2.03 μM.

[0054] The physiological H2O2 concentration range of plants is 10μM~100μM, and the H2O2 concentration increases under stress or pathological conditions. Since the NBP of the present invention has a good linear relationship within 0μM~150μM and a detection limit of 2.03μM, it is suitable for the detection of endogenous or exogenous addition of H2O2 in physiological or pathological states of plants such as Platycodon grandiflorum. The detection limit and linear range are significantly better than other disclosed plant H2O2 fluorescent probes. Moreover, the NBP fluorescent probe does not need to be injected into the plant body, but can be detected by soaking and incubating for 20~30min, which has the advantages of ease of use, low destructiveness and fast response speed.

[0055] In summary, the fluorescent probe NBP was used to study the fluorescence imaging of exogenous and endogenous H2O2 in living cells; the fluorescent probe NBP was combined with a smart phone to achieve visual detection of residual H2O2 in milk; the probe was used to detect the accumulation of H2O2 in the roots of Platycodon grandiflorum under drought stress and the intensity of the fluorescence signal after adding exogenous H2O2. Similar to the research, the fluorescent probe NBP provided by the present invention has good selectivity for H2O2, high sensitivity, low detection limit (80 nM), and wide linear range, and is suitable for the detection of H2O2 in dairy products, isolated living cells, and plants.

[0056] The preferred embodiments of the present invention are described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the technical concept of the present invention, a variety of simple changes can be made to the technical solution of the present invention, and these simple changes all belong to 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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