Fluorescent probe as well as preparation method and application thereof

Abstract

The invention discloses a fluorescent probe and a preparation method and application thereof, and belongs to the technical field of biological detection.2-formyl phenyl boronic acid pinacol ester is grafted to a polysiloxane main chain, due to the fact that a boric acid ester structure in the 2-formyl phenyl boronic acid pinacol ester is prone to being oxidized by HCIO, the 2-formyl phenyl boronic acid pinacol ester is synthesized through an intramolecular charge transfer mechanism of the fluorescent probe, and the fluorescence intensity of the 2-formyl phenyl boronic acid pinacol ester is improved. The electron donating ability of the initial borate structure is weak, so that the fluorescence of the fluorescent probe is in a closed state, and after the HCIO is added, the borate structure is oxidized into a hydroxyl structure with strong electron donating ability to promote electron transfer, so that the fluorescence of the fluorescent probe is turned on along with charge transfer. The 2-formyl phenylboronic acid pinacol ester has an electron withdrawing group, namely a boron atom adjacent to an aldehyde group, and polysiloxane is modified by the electron withdrawing group, so that the ICT effect and the response sensitivity of the fluorescent probe to polarity are remarkably improved.

Description

Technical Field

[0001] The present invention relates to the field of biological detection technology, and in particular to a fluorescent probe and a preparation method and application thereof. Background Art

[0002] Reactive oxygen is closely related to the pathogenesis of many diseases. HCIO is an important representative of reactive oxygen. In the immune process, HCIO is the nemesis of microorganisms and can eliminate invading bacteria. However, excessive HCIO can lead to cardiovascular disease, arteriosclerosis, etc. In addition, polarity, as an indispensable microenvironmental parameter in biological systems, can activate functional proteins and trigger signal transduction and membrane system processes. Abnormal polarity changes are also involved in the occurrence and development of pathological physiological processes such as inflammation and cancer.

[0003] So far, there are many methods to detect changes in HCIO content, such as reduction titration, liquid chromatography, etc. These methods are often complicated, and the detection environment in organic media limits the study of biological systems. Compared with the above methods, the fluorescent probe method has advantages in selectivity, detection limit and operability. However, the existing fluorescent probes can respond to reactive oxygen, ions, pH, viscosity and polarity respectively, but cannot achieve dual response to reactive oxygen and polarity. Summary of the invention

[0004] The present invention provides a fluorescent probe and a preparation method and application thereof. The fluorescent probe prepared by the present invention is a dual-emission dual-target ratio fluorescent probe, which has good response ability to HCIO and polarity. When irradiated with an excitation wavelength of 417nm, HCIO is added to start the response, and when irradiated with an excitation wavelength of 550nm, HCIO is added to quench. In cell imaging, the probe targets lipid droplets and mitochondria, the green channel targets lipid droplets, and the red and deep red channels target mitochondria.

[0005] The first object of the present invention is to provide a fluorescent probe, characterized in that its general structural formula is: , The molecular weight of the fluorescent probe is 4132-4621, and the molar ratio of m to n is 1:0.9-1.1.

[0006] The second object of the present invention is to provide a method for preparing the above fluorescent probe, comprising the following steps: Take 2,3,3-trimethyl-3H-benzidindole of formula I and iodoethane as raw materials, add ethanol to dissolve, condense and reflux under a protective atmosphere of 80°C to 88°C, and a nucleophilic substitution reaction occurs to obtain a solid product of formula II.

[0007] Cyclohexanone and phosphorus oxychloride are used as raw materials, added into a mixed solvent of dichloromethane and N,N-dimethylformamide, mixed, and heated to 60°C to 70°C to cause a Vilsmeier-Hake reaction to obtain a compound of formula III.

[0008] The solid product of formula II and the compound of formula III are used as raw materials, added into a mixed solvent of n-butanol and cyclohexane, and subjected to affinity addition reaction at 110° C. to 120° C. to obtain a compound of formula IV.

[0009] The compound of formula IV and the silicone oil of formula V are used as raw materials, an ethanol solution is added, and a substitution reaction occurs at 60° C. to 70° C. to obtain a compound of formula VI.

[0010] The compound of formula VI and 2-formylphenylboronic acid pinacol ester of formula VII are used as raw materials, ethanol solution is added, and a nucleophilic addition reaction occurs at 55°C to 65°C to obtain a compound of formula VIII, i.e., a fluorescent probe. The synthesis route is as follows: , wherein the molecular weight of the silicone oil of formula V is 1600~2667, a is the percentage of amino group in the silicone oil of formula V, which is 15%~25%, and the molar ratio of m to n is 1:0.9~1.1.

[0011] As a preferred embodiment, the molar ratio of 2,3,3-trimethyl-3H-benzidindole of formula I to ethyl iodide is 1:1.75~2.

[0012] As a preferred embodiment, the molar ratio of cyclohexanone to phosphorus oxychloride is 1:1.5-2.

[0013] As a preferred embodiment, the molar ratio of the solid product of formula II to the compound of formula III is 2-2.5:1.

[0014] As a preferred embodiment, the mass ratio of the compound of formula IV to the silicone oil of formula V is 1:5.5-6.

[0015] As a preferred embodiment, the mass ratio of the compound of formula VI to 2-formylphenylboronic acid pinacol ester of formula VII is 1:0.517~0.8.

[0016] The third object of the present invention is to provide an application of the above fluorescent probe in detecting hypochlorous acid and polar organic polymers.

[0017] Compared with the prior art, the present invention has the following beneficial effects: The present invention provides a fluorescent probe, wherein 2-formylphenylboronic acid pinacol ester is grafted onto a main chain of polysiloxane, and an N atom with a lone electron pair on the polysiloxane attacks a C atom with a positive charge on a carbonyl group on the 2-formylphenylboronic acid pinacol ester to complete a nucleophilic addition reaction, and the boron atom in the 2-formylphenylboronic acid pinacol ester is an electron-withdrawing group, which makes the carbon atom carry more positive charge, thereby promoting the reaction. Since the borate ester structure in the 2-formylphenylboronic acid pinacol ester is easily oxidized by HCIO, through the intramolecular charge transfer mechanism of the fluorescent probe, i.e., the ICT mechanism, the electron-donating ability of the initial borate ester structure is relatively weak, so that the fluorescence of the fluorescent probe is in an off state, and after the addition of HCIO, the borate ester structure is oxidized to a hydroxyl structure with a strong electron-donating ability, which promotes electron transfer, so the fluorescence of the fluorescent probe is turned on as the charge transfer occurs.

[0018] The fluorescent probe provided by the present invention is composed of an electron-donating group and an electron-withdrawing group. The push-pull electronic structure of the donor-acceptor causes the excited-state electron to be transferred from the electron-donating group to the electron-withdrawing group accompanied by fluorescence emission. At the moment when the fluorescent probe molecule is excited, the excited-state electron is in an energy state higher than the equilibrium state. At this time, the high-polarity solvent can stabilize the excited-state molecule. In this process, the greater the solvent polarity, the stronger the ability to stabilize the fluorescent molecule, and the emission wavelength of the probe produces a red shift phenomenon. At the same time, in the luminescence process of the fluorescent molecule, the energy is lost due to the vibration relaxation phenomenon, so that the fluorescence intensity decreases. The solvation effect can affect the vibration relaxation of the molecule. In the present invention, in a medium polar solvent, the solvation effect is the strongest, the relaxation is suppressed, the energy loss is reduced, and the fluorescence intensity is enhanced. Since there is an electron-withdrawing group of the boron atom adjacent to the aldehyde group in 2-formylphenylboronic acid pinacol ester, the electron-withdrawing group is used to modify the polysiloxane, thereby significantly improving the ICT effect and the response sensitivity of the fluorescent probe to polarity. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 To gradually add ClO - The fluorescence change diagram of the fluorescent probe of the present invention at an excitation wavelength of 417 nm.

[0020] Figure 2 To gradually add ClO - The fluorescence change diagram of the fluorescent probe of the present invention at an excitation wavelength of 550nm.

[0021] Figure 3 The fluorescence change diagram of the fluorescent probe of the present invention in different solvents at an excitation wavelength of 417 nm.

[0022] Figure 4The cellular localization diagram of the fluorescent probe of the present invention, wherein Figure (a) is a composite field imaging diagram, Figure (b) is a green channel imaging diagram of the fluorescent probe, Figure (c) is a Nile red red channel imaging diagram of the fluorescent probe, Figure (d) is a bright field imaging diagram of the fluorescent probe, Figure (e) is a fluorescence intensity scatter diagram of the FITC green channel and the TRITC channel, and Figure (f) is an intensity change matching diagram of the FITC channel and the TRITC channel.

[0023] Figure 5 It is a mitochondrial targeting localization diagram of the fluorescent probe of the present invention, wherein Figure (a) is a composite field imaging diagram, Figure (b) is a green channel imaging diagram of Mito Tracker Green, Figure (c) is a red channel imaging diagram of the fluorescent probe, Figure (d) is a Cy5 channel imaging diagram of the fluorescent probe, Figure (e) is a fluorescence intensity scatter diagram of the FITC green channel and the Cy5 channel, and Figure (f) is an intensity change matching diagram of the FITC channel and the Cy5 channel.

[0024] Figure 6 Different concentrations of HClO were added to the present invention, and confocal imaging of Hela cells and exogenous HClO was performed. DETAILED DESCRIPTION

[0025] In order to enable those skilled in the art to better understand the technical solution of the present invention and implement it, the present invention is further described below in conjunction with specific embodiments and drawings, but the embodiments are not intended to limit the present invention. The following test methods and detection methods, unless otherwise specified, are conventional methods; the reagents and raw materials, unless otherwise specified, are commercially available.

[0026] In view of the fact mentioned in the background technology of this application that existing fluorescent probes can respond to active oxygen, ions, pH, viscosity and polarity respectively, but cannot achieve dual response to active oxygen and polarity, the present invention provides a fluorescent probe and a preparation method and application thereof.

[0027] The technical solution of the present invention is described in detail below.

[0028] The present invention first provides a fluorescent probe, the general structural formula of which is: , The molecular weight of the fluorescent probe is 4132-4621, and the molar ratio of m to n is 1:0.9-1.1.

[0029] In the fluorescent probe of the above structural formula, the boron atom in 2-formylphenylboronic acid pinacol ester is an electron-withdrawing group, which makes the carbon atom carry more positive charge. Since the borate structure in 2-formylphenylboronic acid pinacol ester is easily oxidized by HCIO, through the charge transfer mechanism within the fluorescent probe molecule, after adding HCIO, the borate structure is oxidized to a hydroxyl structure with strong electron-donating ability, which promotes electron transfer, so the fluorescence of the fluorescent probe turns on with the occurrence of charge transfer. In medium polar solvents, the solvation effect is the strongest, relaxation will be suppressed, the energy loss is reduced, and the fluorescence intensity is enhanced. Since there is an electron-withdrawing group of the boron atom adjacent to the aldehyde group in 2-formylphenylboronic acid pinacol ester, the electron-withdrawing group is used to modify the polysiloxane, thereby significantly improving the ICT effect and the response sensitivity of the fluorescent probe to polarity.

[0030] The second object of the present invention is to provide a method for preparing the above fluorescent probe, comprising the following steps: Take 2,3,3-trimethyl-3H-benzidindole of formula I and iodoethane as raw materials, add ethanol to dissolve, condense and reflux under a protective atmosphere of 80°C~88°C, and a nucleophilic substitution reaction occurs. After the reaction is completed, rotary evaporation is performed, and after being spin-dried, ethanol is added to completely dissolve the dried solid product again, and then a large amount of petroleum ether is added to precipitate, and stirred for 2 minutes. After the stirring is completed, suction filtration is performed, and the cycle is repeated three times, and air-dried at room temperature to obtain a solid product of formula II.

[0031] Cyclohexanone and phosphorus oxychloride are used as raw materials, added to a mixed solvent of dichloromethane and N,N-dimethylformamide, mixed, and heated to 60°C to 70°C to cause a Vilsmeier-Hack reaction. After the reaction is completed, the solution is cooled to room temperature, ice is added to the solution, and then the solution is aged overnight and filtered to obtain a compound of formula III.

[0032] The solid product of formula II and the compound of formula III are used as raw materials, added to a mixed solvent of n-butanol and cyclohexane, and affinity addition reaction occurs at 110° C. to 120° C. After the reaction is completed, the solvent is evaporated under reduced pressure, and the product is separated by wet column chromatography, and dichloromethane / methanol with a volume ratio of 25 to 100:1 is used as an elution reagent, and rotary evaporation is performed to obtain a compound of formula IV.

[0033] The compound of formula IV and the silicone oil of formula V are used as raw materials, ethanol solution is added, and a substitution reaction occurs at 60° C. to 70° C. After the reaction is completed, the mixture is cooled to room temperature, and the solvent is removed by rotary evaporation to obtain a compound of formula VI.

[0034] The compound of formula VI and 2-formylphenylboronic acid pinacol ester of formula VII are used as raw materials, and an ethanol solution is added to react at 55°C to 65°C to cause a nucleophilic addition reaction. After the reaction is completed, anhydrous MgSO4 is added, stirred, and filtered to obtain a compound of formula VIII, i.e., a fluorescent probe. The synthesis route is as follows: , wherein the molecular weight of the silicone oil of formula V is 1600~2667, a is the percentage of amino group in the silicone oil of formula V, which is 15%~25%, and the molar ratio of m to n is 1:0.9~1.1.

[0035] The reaction can proceed smoothly within the temperature values ​​specified in the above reaction steps. If the reaction temperature of each step is too high, the solvent will volatilize and some side reactions will occur; if the reaction temperature is too low, the reaction will not be complete or cannot proceed.

[0036] In the above preparation process, the molar ratio of 2,3,3-trimethyl-3H-benzoindole of formula I to ethyl iodide is 1:1.75~2. When the molar ratio of 2,3,3-trimethyl-3H-benzoindole of formula I to ethyl iodide is 1:1.75, a solid product of formula II with optimal yield and purity is obtained. If the amount of ethyl iodide added is too little, the yield of the product is affected. If the amount of ethyl iodide added is too much, side reactions and the generation of by-products will occur, which will seriously affect the purity of the solid product of formula II.

[0037] It should be noted that the molar ratio of cyclohexanone to phosphorus oxychloride is 1:1.5 to 2. If the amount of phosphorus oxychloride added is too little, the yield of the compound of formula III is affected, and if the amount of phosphorus oxychloride added is too much, side reactions and the generation of by-products are caused, which seriously affects the purity of the compound of formula III.

[0038] It should be emphasized that the molar ratio of the solid product of formula II to the compound of formula III is 2 to 2.5: 1. When the molar ratio of the solid product of formula II to the compound of formula III is 2: 1, the compound of formula IV with the best yield and purity can be obtained.

[0039] In order to obtain a compound of formula VI with higher yield and better purity, the mass ratio of the compound of formula IV to the silicone oil of formula V is 1:5.5-6.

[0040] In order to obtain a compound of formula VIII with a higher yield and better purity, that is, to obtain a fluorescent probe with excellent performance, the mass ratio of the compound of formula VI to 2-formylphenylboronic acid pinacol ester of formula VII is 1:0.517~0.8.

[0041] It should be noted that when the percentage of amino groups in the silicone oil is 20%, the silicone oil of formula V has good fluidity.

[0042] The technical effects of the present invention are described below through specific embodiments.

[0043] Example 1 A method for preparing a fluorescent probe comprises the following steps: Take 1.045g, i.e. 0.005mol, of 2,3,3-trimethyl-3H-benzidindole of formula I and 1.36g, i.e. 0.00875mol of iodoethane, add 7mL of ethanol, dissolve, and react under condensation reflux at 85°C in a nitrogen atmosphere for 10h. Rotary evaporate, and after being spin-dried, add ethanol to completely dissolve the dried solid product again, then add a large amount of petroleum ether to precipitate, stir for 2min, and after stirring, filter with suction, repeat three times, and air-dry at room temperature to obtain a solid product of formula II.

[0044] A mixture of 5 mL of dichloromethane and 5 mL of DMF was added to a round-bottom flask, which was placed in an ice bath and allowed to stand for 30 min; 7 mL of phosphorus oxychloride was dissolved in 7 mL of dichloromethane and slowly added dropwise to the round-bottom flask; 5 g of cyclohexanone was dissolved in 5 mL of dichloromethane and slowly added dropwise to the round-bottom flask; mixed under ice water for 30 min; the reaction solution was gradually heated to 65° C. and refluxed for 3 h; after the solution was cooled to room temperature, 50 g of ice was added to the solution; the solution was then aged overnight and filtered to obtain a compound of formula III.

[0045] 2.38 g, i.e., 10.0 mmol, of the solid product of formula II, 0.86 g, i.e., 5.0 mmol, of the compound of formula III, 105 mL of n-butanol and 45 mL of cyclohexane were added into a round-bottom flask, and affinity addition reaction was carried out at 117° C. for 4 h. After completion of the reaction, the solvent was evaporated under reduced pressure, and the product was separated by wet column chromatography, and dichloromethane / methanol in a volume ratio of 25:1 was used as an elution reagent, and rotary evaporation was performed to obtain a compound of formula IV.

[0046] 0.22 g of the compound of formula IV and 1 g of silicone oil of formula V were dissolved in 100 mL of ethanol solution, and the substitution reaction was carried out at 65° C. for 9 hours. After the reaction was completed, the mixture was cooled to room temperature and the solvent was removed by rotary evaporation to obtain a compound of formula VI.

[0047] 1000 mg of the compound of formula VI and 517 mg of 2-formylphenylboronic acid pinacol ester of formula VII were dissolved in 20 mL of ethanol solution and subjected to nucleophilic addition reaction at 60°C for 6 h. After the reaction, anhydrous MgSO4 was added, stirred, and filtered to obtain a compound of formula VIII, i.e., a fluorescent probe.

[0048] Example 2 A method for preparing a fluorescent probe comprises the following steps: Take 1.045g, i.e. 0.005mol, of 2,3,3-trimethyl-3H-benzidindole of formula I and 1.36g, i.e. 0.00875mol of iodoethane, add 7mL of ethanol, dissolve, and react under condensation reflux at 80°C in a nitrogen atmosphere for 10h. Rotary evaporate, and after being spin-dried, add ethanol to completely dissolve the dried solid product again, then add a large amount of petroleum ether to precipitate, stir for 2min, and after stirring, filter with suction, repeat three times, and air-dry at room temperature to obtain a solid product of formula II.

[0049] A mixture of 5 mL of dichloromethane and 5 mL of DMF was added to a round-bottom flask, which was placed in an ice bath and allowed to stand for 30 min; 7 mL of phosphorus oxychloride was dissolved in 7 mL of dichloromethane and slowly added dropwise to the round-bottom flask; 5 g of cyclohexanone was dissolved in 5 mL of dichloromethane and slowly added dropwise to the round-bottom flask, mixed under ice water for 30 min, the reaction solution was gradually heated to 60° C. and refluxed for 3 h. After the solution was cooled to room temperature, 50 g of ice was added to the solution, and then the solution was aged overnight and filtered to obtain a compound of formula III.

[0050] 2.38 g, i.e., 10.0 mmol, of the solid product of formula II, 0.86 g, i.e., 5.0 mmol, of the compound of formula III, 105 mL of n-butanol and 45 mL of cyclohexane were added into a round-bottom flask, and affinity addition reaction was carried out at 110° C. for 4 h. After completion of the reaction, the solvent was evaporated under reduced pressure, and the product was separated by wet column chromatography, and dichloromethane / methanol with a volume ratio of 25:1 was used as an elution reagent, and rotary evaporation was carried out to obtain a compound of formula IV.

[0051] 0.22 g of the compound of formula IV and 1 g of silicone oil of formula V were dissolved in 100 mL of ethanol solution, and the substitution reaction was carried out at 60° C. for 9 hours. After the reaction was completed, the mixture was cooled to room temperature and the solvent was removed by rotary evaporation to obtain a compound of formula VI.

[0052] 1000 mg of the compound of formula VI and 517 mg of 2-formylphenylboronic acid pinacol ester of formula VII were dissolved in 20 mL of ethanol solution and subjected to nucleophilic addition reaction at 55°C for 6 hours. After the reaction, anhydrous MgSO4 was added, stirred, and filtered to obtain a compound of formula VIII, i.e., a fluorescent probe.

[0053] Example 3 A method for preparing a fluorescent probe comprises the following steps: Take 1.045g, i.e. 0.005mol, of 2,3,3-trimethyl-3H-benzidindole of formula I and 1.36g, i.e. 0.00875mol of iodoethane, add 7mL of ethanol, dissolve, and react under condensation reflux at 88°C in a nitrogen atmosphere for 10h. Rotary evaporate, and after being spin-dried, add ethanol to completely dissolve the dried solid product again, then add a large amount of petroleum ether to precipitate, stir for 2min, and after stirring, filter with suction, repeat three times, and air-dry at room temperature to obtain a solid product of formula II.

[0054] A mixture of 5 mL of dichloromethane and 5 mL of DMF was added to a round-bottom flask, which was placed in an ice bath and allowed to stand for 30 min; 7 mL of phosphorus oxychloride was dissolved in 7 mL of dichloromethane and slowly added dropwise to the round-bottom flask; 5 g of cyclohexanone was dissolved in 5 mL of dichloromethane and slowly added dropwise to the round-bottom flask, mixed under ice water for 30 min, the reaction solution was gradually heated to 70° C. and refluxed for 3 h. After the solution was cooled to room temperature, 50 g of ice cubes were added to the solution, and then the solution was aged overnight and filtered to obtain a compound of formula III.

[0055] 2.38 g, i.e., 10.0 mmol, of the solid product of formula II, 0.86 g, i.e., 5.0 mmol, of the compound of formula III, 105 mL of n-butanol and 45 mL of cyclohexane were added into a round-bottom flask, and affinity addition reaction was carried out at 120° C. for 4 h. After completion of the reaction, the solvent was evaporated under reduced pressure, and the product was separated by wet column chromatography, and dichloromethane / methanol in a volume ratio of 100:1 was used as an elution reagent, and rotary evaporation was performed to obtain a compound of formula IV.

[0056] 0.22 g of the compound of formula IV and 1 g of the silicone oil of formula V were dissolved in 100 mL of ethanol solution, and the substitution reaction was carried out at 70° C. for 9 h. After the reaction was completed, the solution was cooled to room temperature and the solvent was removed by rotary evaporation to obtain a compound of formula VI; 1000 mg of the compound of formula VI and 517 mg of 2-formylphenylboronic acid pinacol ester of formula VII were dissolved in 20 mL of ethanol solution and subjected to nucleophilic addition reaction at 65°C for 6 hours. After the reaction, anhydrous MgSO4 was added, stirred, and filtered to obtain a compound of formula VIII, i.e., a fluorescent probe.

[0057] Example 4 A method for preparing a fluorescent probe comprises the following steps: Take 1.045g, i.e. 0.005mol, of 2,3,3-trimethyl-3H-benzidindole of formula I and 1.36g, i.e. 0.00875mol of iodoethane, add 7mL of ethanol, dissolve, and react under condensation reflux at 82°C in a nitrogen atmosphere for 10h. Rotary evaporate, and after being spin-dried, add ethanol to completely dissolve the dried solid product again, then add a large amount of petroleum ether to precipitate, stir for 2min, and after stirring, filter with suction, repeat three times, and air-dry at room temperature to obtain a solid product of formula II.

[0058] A mixture of 5 mL of dichloromethane and 5 mL of DMF was added to a round-bottom flask, which was placed in an ice bath and allowed to stand for 30 min; 7 mL of phosphorus oxychloride was dissolved in 7 mL of dichloromethane and slowly added dropwise to the round-bottom flask; 5 g of cyclohexanone was dissolved in 5 mL of dichloromethane and slowly added dropwise to the round-bottom flask, mixed under ice water for 30 min, the reaction solution was gradually heated to 68° C. and refluxed for 3 h. After the solution was cooled to room temperature, 50 g of ice was added to the solution, and then the solution was aged overnight and filtered to obtain a compound of formula III.

[0059] 2.38 g, i.e., 10.0 mmol, of the solid product of formula II, 0.86 g, i.e., 5.0 mmol, of the compound of formula III, 105 mL of n-butanol and 45 mL of cyclohexane were added into a round-bottom flask, and affinity addition reaction was carried out at 115° C. for 4 h. After completion of the reaction, the solvent was evaporated under reduced pressure, and the product was separated by wet column chromatography, and dichloromethane / methanol with a volume ratio of 50:1 was used as an elution reagent, and rotary evaporation was carried out to obtain a compound of formula IV.

[0060] 0.22 g of the compound of formula IV and 1 g of silicone oil of formula V were dissolved in 100 mL of ethanol solution, and the substitution reaction was carried out at 62° C. for 9 hours. After the reaction was completed, the solution was cooled to room temperature and the solvent was removed by rotary evaporation to obtain a compound of formula VI.

[0061] 1000 mg of the compound of formula VI and 517 mg of 2-formylphenylboronic acid pinacol ester of formula VII were dissolved in 20 mL of ethanol solution and subjected to nucleophilic addition reaction at 58°C for 6 hours. After the reaction, anhydrous MgSO4 was added, stirred, and filtered to obtain a compound of formula VIII, i.e., a fluorescent probe.

[0062] The performance of the fluorescent probes prepared in Examples 1 to 4 of the present invention was tested, and the specific process and results are as follows: 1. HClO response test: Preparation: 1 mg of the fluorescent probe prepared in Example 1 was dissolved in 1 mL of dimethyl sulfoxide to prepare a stock solution. The PBS buffer solution was prepared using PBS phosphate buffer, which was purchased from Beijing Dingguo Changsheng Biotechnology Co., Ltd. After preparation, the concentration of the PBS buffer solution was 0.01 mol / L and the pH value was 7.2-7.4.

[0063] 417nm excitation: Use a pipette to take 1mL PBS buffer into a fluorescent dish, add 1µL of the mother solution, irradiate at 417nm excitation wavelength, test, then add 2µL HClO at a time. Figure 1 It can be seen that the fluorescence intensity increases with the increase of HClO concentration.

[0064] 550nm excitation: Use a pipette to take 1mL PBS buffer into a fluorescent dish, add 1µL of the mother solution, irradiate at 550nm excitation wavelength, test, then add 1µL HClO at a time. Figure 2 It can be seen that the fluorescence intensity decreases with the increase of HClO concentration.

[0065] 2. Polarity response Use a pipette to take 1 mL of dichloromethane, 1 mL of tetrahydrofuran, 1 mL of N,N-dimethylformamide, 1 mL of ethanol, 1 mL of methanol, and 1 mL of H2O into a fluorescent dish, add 10 µL of the mother solution, and irradiate at an excitation wavelength of 417 nm. Figure 3 It can be seen that as the polarity increases, the fluorescence intensity first increases and then decreases, and the emission red-shifts.

[0066] 3. Cell experiments (1) Cell localization step: Culture the cells and incubate them in a medium containing 10% serum for 24 hours. Add 10 µL of the 1 mg / mL stock solution and incubate with the cells for 20 minutes.

[0067] Locate the green channel and add 10 µL of 1 mg / mL Nile Red, a commercial dye that stains lipid droplets. Figure 4 It can be seen that the Pearson codomain coefficient of the red channel stained with the commercial dye Nile Red and the green channel stained with the fluorescent probe is 0.985, indicating that the fluorescent probe has stained the lipid droplets and determined the location of the lipid droplets.

[0068] Change another plate of cells and incubate the cells with the same medium containing 10% serum for 24 hours. Add 10 µL of the 1 mg / mL stock solution and incubate with the cells for 20 minutes.

[0069] Add 10µL of 1mg / mL commercial dye Mito Tracker Green for co-localization experiment. Red channel: 0.904, dark red channel: 0.889, confirming the location of mitochondria. Figure 5 It can be seen that the overlap coefficient between the green channel stained with the commercial dye Mito Tracker Green and the deep red channel stained with the fluorescent probe is 0.889, indicating that the fluorescent probe of the present invention can locate mitochondria.

[0070] (2) HClO exogenous cell imaging Four plates of cells were cultured and incubated with medium containing 10% serum for 24 h. 10 µL of the 1 mg / mL stock solution was added and incubated with the cells for 20 min.

[0071] Four plates of cells were added with 0uM, 3uM, 6uM, and 9uM HClO and incubated for 5 min. Figure 6 It can be observed that the green fluorescence becomes brighter and the red fluorescence becomes weaker.

[0072] In summary, the fluorescent probe prepared by the present invention is easy to be oxidized by HCIO because the borate structure in 2-formylphenylboronic acid pinacol ester is easily oxidized by HCIO. After adding HCIO, the borate structure is oxidized to a hydroxyl structure with strong electron-donating ability, which promotes electron transfer. Therefore, the fluorescence of the fluorescent probe is turned on as the charge transfer occurs. In a medium polar solvent, the solvation effect is the strongest, relaxation is suppressed, the energy loss is reduced, and the fluorescence intensity is enhanced. Since there is an electron-withdrawing group of a boron atom adjacent to the aldehyde group in 2-formylphenylboronic acid pinacol ester, the electron-withdrawing group is used to modify the polysiloxane, thereby significantly improving the ICT effect and the response sensitivity of the fluorescent probe to polarity.

[0073] Obviously, those skilled in the art can make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include these modifications and variations.

Claims

1. A fluorescent probe, characterized in that Its general structural formula is: The molecular weight of the fluorescent probe is 4132~4621, and the molar ratio of m to n is 1:0.9~1.

1.

2. A method for preparing the fluorescent probe according to claim 1, characterized in that: The following steps are involved: Using 2,3,3-trimethyl-3H-benzidindole of formula I and iodoethane as raw materials, adding ethanol to dissolve, condensing and refluxing at 80°C to 88°C under a protective atmosphere, a nucleophilic substitution reaction occurs to obtain a solid product of formula II; Cyclohexanone and phosphorus oxychloride are used as raw materials, added into a mixed solvent of dichloromethane and N,N-dimethylformamide, mixed, heated to 60°C to 70°C, and a Vilsmeier-Hake reaction occurs to obtain a compound of formula III; The solid product of formula II and the compound of formula III are used as raw materials, added into a mixed solvent of n-butanol and cyclohexane, and reacted at 110° C. to produce an affinity addition reaction to obtain a compound of formula IV; The compound of formula IV and the silicone oil of formula V are used as raw materials, an ethanol solution is added, and a substitution reaction occurs at 60° C. to 70° C. to obtain a compound of formula VI; The compound of formula VI and 2-formylphenylboronic acid pinacol ester of formula VII are used as raw materials, ethanol solution is added, and a nucleophilic addition reaction occurs at 55°C to 65°C to obtain a compound of formula VIII, i.e., a fluorescent probe. The synthesis route is as follows: ， Wherein, the molecular weight of the silicone oil of formula V is 1600-2667, a is the percentage of amino groups in the silicone oil of formula V, which is 15%-25%, and the molar ratio of m to n is 1:0.9-1.

1.

3. The method for preparing a fluorescent probe according to claim 2, characterized in that: The molar ratio of 2,3,3-trimethyl-3H-benzidindole of formula I to ethyl iodide is 1:1.75-2.

4. The method for preparing a fluorescent probe according to claim 2, characterized in that: The molar ratio of cyclohexanone to phosphorus oxychloride is 1:1.5-2.

5. The method for preparing a fluorescent probe according to claim 2, characterized in that: The molar ratio of the solid product of formula II to the compound of formula III is 2-2.5:

1.

6. The method for preparing a fluorescent probe according to claim 2, characterized in that: The mass ratio of the compound of formula IV to the silicone oil of formula V is 1:5.5-6.

7. The method for preparing a fluorescent probe according to claim 2, characterized in that: The mass ratio of the compound of formula VI to the 2-formylphenylboronic acid pinacol ester of formula VII is 1:0.517-0.

8.

8. Use of the fluorescent probe according to claim 1 in detecting hypochlorous acid and polar organic polymers.

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