A fluorescent probe and its preparation method and application

Abstract

The present invention provides a fluorescent probe and its preparation method and application. The structural formula of the fluorescent probe is as follows: The preparation method is: 7-N,N-diethylamino-2H-chromene-3-aldehyde, 4-methyl-N-(4'-boronic acid pinacol benzyl) pyridine bromide, a catalyst and a solvent are stirred, mixed and heated under reflux for reaction, and then filtered and recrystallized to obtain a fluorescent probe product. The probe has good water solubility, biocompatibility and is positively charged. The solution color is purple-red, the absorption peak is at 540nm, and the fluorescence peak is at 720nm (near-infrared region), which can be used for near-infrared biological fluorescence imaging. The probe has the property of realizing ratiometric colorimetry and ratiometric fluorescence response to peroxynitrite. After reacting with peroxynitrite in a CH3CN-pbs (v / v=1:1, v / v, pH=7.4) solution, the color of the probe solution changes from purple-red to slightly yellow, A 410nm / A 540nm The probe has a linear relationship with the concentration of peroxynitrite in the range of 0 to 10 μM, with a detection limit of 95 nM. The red fluorescence of the probe gradually weakens at 720 nm and the bright yellow fluorescence gradually increases at 568 nm. 568nm / I 720nm The probe has a linear relationship with peroxynitrite concentration in the range of 0 to 16 μM, with a detection limit of 10 nM. The probe has the advantages of high sensitivity, low detection limit, and low background fluorescence interference.

Description

Technical Field

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

[0002] Reactive oxygen is an important substance in the life system and plays an important regulatory role in physiological and pathological processes. - ) is a common reactive oxygen species in cells, which is formed by the reaction of nitric oxide and superoxide anion free radicals. - It has extremely high reactivity and participates in important physiological activities such as balancing the redox process and signal transmission in cells. - Abnormal concentration of ONOO can cause loss of function of some important protein molecules in the body, DNA chain breakage, lipid oxidation, and disrupt the cell environment, thereby inducing cancer, cardiovascular and cerebrovascular diseases, etc. - Often leads to cell apoptosis or necrosis. Therefore, real-time and non-destructive detection of ONOO in biological systems is possible. - It is of great significance.

[0003] Viscosity is a crucial parameter of the cellular microenvironment, influencing processes such as interactions between biomacromolecules, signal transduction, and metabolite diffusion. Abnormal viscosity levels in vivo are closely associated with a variety of diseases, such as diabetes, atherosclerosis, and Alzheimer's disease. Therefore, accurately measuring changes in intracellular viscosity is crucial.

[0004] Fluorescent probes offer advantages such as simple pretreatment, convenient operation, high sensitivity, rapid response, low cost, non-destructive testing of biological samples, and in situ detection. They are important analytical methods for monitoring target analytes in vivo. Intracellular fluorescence imaging has attracted considerable attention as a highly effective detection method in biological and pharmaceutical sciences. Fluorescent small molecules are being used for intracellular imaging due to their excellent biocompatibility and superior photophysical properties. Summary of the Invention

[0005] The present invention provides a fluorescent probe and its preparation method and application, which solves the problems of the existing probe technology, such as short fluorescence wavelength, low sensitivity, interference from background fluorescence in biological detection, and poor water solubility.

[0006] In order to achieve the above object, the technical solution of the present invention is achieved as follows:

[0007] A fluorescent probe, the structural formula of the fluorescent probe is shown in formula (I):

[0008]

[0009] The fluorescent probe is named 7-N,N-diethylamino-3E-(2'-(4"-(N-4"'-boronic acid pinacol ester benzyl)pyridylvinyl))-2H-chromene bromide, hereinafter referred to as PBVC.

[0010] The method for preparing the fluorescent probe comprises the following steps:

[0011] 7-N,N-diethylamino-2H-chromene-3-aldehyde, 4-methyl-N-(4'-boronic acid pinacol ester benzyl)pyridine bromide, a catalyst, and a solvent are mixed, stirred, and refluxed, and then filtered, washed, and recrystallized to purify the fluorescent probe product.

[0012] The synthetic route is:

[0013]

[0014] Preferably, the molar ratio of 7-N,N-diethylamino-2H-chromene-3-al, 4-methyl-N-(4'-boronic acid pinacolyl benzyl)pyridine bromide and the catalyst is 1:(0.8-1.5):(0.05-0.5).

[0015] Preferably, the reaction temperature is 60-140°C and the reaction time is 6-18h;

[0016] Preferably, the molar ratio of 7-N,N-diethylamino-2H-chromene-3-al, 4-methyl-N-(4'-boronic acid pinacolyl benzyl)pyridine bromide and the catalyst is preferably 1:1.2:0.3; the reaction temperature is preferably 100° C., and the reaction time is preferably 12 h.

[0017] Preferably, the solvent is any one of methanol, ethanol, propanol, tetrahydrofuran, dichloromethane, acetonitrile, acetone, 1,4-dioxane, ethyl acetate and N,N-dimethylformamide.

[0018] Preferably, the solvent is ethanol.

[0019] Preferably, the solvent used for washing and recrystallization is methanol, ethanol, n-propanol, isopropanol, tetrahydrofuran, dichloromethane, acetonitrile, acetone, 1,4-dioxane, or ethyl acetate.

[0020] Preferably, the catalyst is triethylamine, pyridine, piperidine or ammonium acetate, and the yield of the fluorescent probe is 40-80%.

[0021] Preferably, the fluorescent probe is used in the preparation of products for detecting peroxynitrite concentration, viscosity, and near-infrared mitochondria in biological samples.

[0022] Preferably, the fluorescent probe realizes ratiometric colorimetric and ratiometric fluorescence response to peroxynitrite; the linear equation of the absorbance ratio of the fluorescent probe and the peroxynitrite concentration C in the range of 0-10 μM is A 410nm / A 540nm =0.3318+31892.0569C, R 2 =0.9928 The detection limit is 95nM; the linear equation of the fluorescence ratio of the fluorescent probe and the peroxynitrite concentration in the range of 0-16μM is I 568nm / I 720 nm =1.8503+441502.3682C,R 2 =0.9902, detection limit 10nM.

[0023] A 410nm / A 540nm is the ratio of the absorbance of the fluorescent probe at 410 nm to the absorbance at 540 nm, I 568nm / I 720 nm is the ratio of the fluorescence intensity of the fluorescent probe at 568 nm to the fluorescence intensity at 720 nm; C is ONOO - The concentration of the substance is expressed in mol / L.

[0024] Preferably, the logarithm of the fluorescence intensity (I) of the fluorescent probe is linearly related to the logarithm of the viscosity (η), and the linear equation is logI=0.12253+1.53602logη, R 2 =0.9937. I is the fluorescence intensity of the fluorescent probe at 710 nm, η is the viscosity of the system, and the unit of viscosity is cp.

[0025] In the present invention, the method of ONOO based on phenylboronic acid pinacol ester is described. - The redox recognition effect of pyridinium salt, the water solubility of pyridinium salt, and the red shift of fluorescence spectrum by the electron-pushing and pulling groups in the molecule through conjugated double bonds were used to design a fluorescent probe. The probe reacted with ONOO in a CH3CN-PBS (v / v=1:1, v / v, pH=7.4) buffer solution. - After the reaction, ratio colorimetry and ratio fluorescence identification of ONOO can be realized. - .

[0026] Due to the strong electron-withdrawing ability of the pyridinium salt in the probe, a strong intramolecular charge transfer effect (ICT) is generated, and the probe emits red fluorescence at 720nm, which is in the near-infrared region. - In the environment, its recognition group phenylboronic acid pinacol ester is ONOO -Nucleophilic attack, followed by 1,2-migration of the aromatic group from the boron atom to the adjacent oxygen atom to form a borate ester, which is hydrolyzed to form boronic acid and the corresponding phenol intermediate. The carbon-nitrogen bond in the phenol intermediate spontaneously cleaves through a 1,6-elimination reaction, and finally the recognition group is removed to form a new compound 7-N,N-diethylamino-3E-(2'-(4"-pyridinylvinyl))-2H-chromene, which emits fluorescence. As a result, the fluorescence of the probe at 720nm is gradually quenched, and a new fluorescence peak appears at 568nm, that is, bright yellow fluorescence is emitted, thereby realizing the probe's detection of ONOO - Ratiometric colorimetric and ratiometric fluorescence detection.

[0027] Based on the fact that viscosity can restrict the rotation of double bonds and carbon-carbon single bonds between pyridinium and chromene groups, the probe exhibits a light-up type fluorescence response at 720 nm in high viscosity glycerol, so the probe can be used for dual-channel detection of ONOO - and viscosity.

[0028] The present invention has the following beneficial effects:

[0029] 1. The fluorescent probe PBVC of the present invention is prepared by the condensation reaction of 7-N,N-diethylamino-2H-chromene-3-al and 4-methyl-N-(4'-boronic acid pinacolyl benzyl)pyridine bromide. The reaction of the present invention has the advantages of simple synthesis steps, mild reaction conditions, and simple post-processing operations.

[0030] 2. The fluorescent probe PBVC of the present invention has good water solubility and biocompatibility. The probe is positively charged and can be used for mitochondrial localization. Its fluorescence peak is at 720nm (near-infrared region), making it suitable for near-infrared biological imaging.

[0031] 3. The fluorescent probe PBVC of the present invention can realize dual detection (ONOO - and viscosity), has the effect of peroxynitrite ONOO - The ratiometric colorimetric and ratiometric fluorescence response properties can be achieved, thereby reducing the interference caused by the background fluorescence of the organism. - It has the advantages of high sensitivity and low detection limit. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0033] Figure 1 PBVC is a fluorescent probe1 H NMR spectrum (solvent: deuterated dimethyl sulfoxide CD3SOCD3).

[0034] Figure 2 PBVC is a fluorescent probe 13 C NMR spectrum (solvent: CD3SOCD3).

[0035] Figure 3 This is the mass spectrum of the fluorescent probe PBVC.

[0036] Figure 4 Absorption and fluorescence spectra of probe PBVC (10 μM) in water (excitation wavelength 540 nm).

[0037] Figure 5 The CH3CN-PBS (v / v=1:1, v / v, pH=7.4) solution of fluorescent probe PBVC (10μM) and its anion, active oxygen (including blank, ClO3 - ,NO3 - ,AcO - ,ClO - ,F - ,Cl - ,Br - ,I - ,SO3 2- ,S2O3 2- ,S 2- ,SO4 2- ,ONOO - ,t-BuO·,·OH, 1 O2, NO, H2O2, O2 -· ,CO3 2- ,PO4 3- ,P2O7 4- ) in the presence of .

[0038] Figure 6 The CH3CN-PBS (v / v=1:1, v / v, pH=7.4) solution of fluorescent probe PBVC (10μM) and its anion, active oxygen (including blank, ClO3 - ,NO3 - ,AcO - ,ClO - ,F - ,Cl - ,Br - ,I - ,SO3 2- ,S2O3 2- ,S 2- ,SO4 2- ,ONOO- ,t-BuO·,·OH, 1 O2,NO,H2O2,O2 -· ,CO3 2- ,PO4 3- ,P2O7 4- ) in the presence of (excitation wavelength 410 nm).

[0039] Figure 7 The CH3CN-PBS (v / v=1:1, v / v, pH=7.4) solution of fluorescent probe PBVC (10μM) was added to 10 times the analyte (blank, ClO3 - ,NO3 - ,AcO - ,ClO - ,F - ,Cl - ,Br - ,I - ,SO3 2- ,S2O3 2- ,S 2- ,SO4 2- ,ONOO - ,t-BuO·,·OH, 1 O2,NO,H2O2,O2 -· ,CO3 2- ,PO4 3- ,P2O7 4- ) in the presence of the probe solution at 410 nm and 540 nm (A 410 / A 540 , orange); continue to add 10 times of ONOO to the above coexistence solution - The ratio of the probe solution at 410 nm and 540 nm (A 410 / A 540 ,green).

[0040] Figure 8 The CH3CN-PBS (v / v=1:1, v / v, pH=7.4) solution of fluorescent probe PBVC (10μM) was added to 10 times the analyte (blank, ClO3 - ,NO3 - ,AcO - ,ClO - ,F - ,Cl - ,Br - ,I - ,SO3 2- ,S2O3 2- ,S 2- ,SO42- ,ONOO - ,t-BuO·,·OH, 1 O2, NO, H2O2, O2 -· ,CO3 2- ,PO4 3- ,P2O7 4- ) in the presence of the probe solution at 568 nm (orange); 10 times the amount of ONOO was added to the coexisting solution. - Fluorescence intensity of the probe solution at 568 nm (green, excitation wavelength 410 nm).

[0041] Figure 9 9a is a fluorescent probe PBVC (10 μM) in CH3CN-pbs (v / v=1:1, v / v, pH=7.4) solution under different ONOO - Absorption spectra in concentrations; Figure 9 b is the ratio of the absorbance of the probe (10 μM) at 410 nm to the absorbance at 540 nm (A 410 / A 540 ) and ONOO - concentration relationship; Figure 9 c is the ratio of the absorbance at 410 nm to the absorbance at 540 nm (A 410 / A 540 ) and ONOO - The linear relationship between the concentration (0 ~ 10μM).

[0042] Figure 10 middle Figure 10 a is the CH3CN-pbs (v / v=1:1, v / v, pH=7.4) solution of fluorescent probe PBVC (10μM) in different ONOO - Fluorescence spectrum in concentration, excitation wavelength is 450nm; Figure 10 b is the ratio of the fluorescence intensity of the probe (10 μM) at 568 nm to the fluorescence intensity at 720 nm (I 568 / I 720 ) and ONOO - concentration relationship; Figure 10 c is the ratio of the fluorescence intensity of the probe (10 μM) at 568 nm to the fluorescence intensity at 720 nm (I 568 / I 720 ) and ONOO - The linear relationship between the concentration (0 ~ 16μM).

[0043] Figure 11The fluorescence intensity of the CH3CN-PBS (v / v=1:1, v / v, pH=7.4) solution containing the fluorescent probe PBVC (10 μM) at 568 nm changes with time in the presence of 10 times ONOO-.

[0044] Figure 12 The fluorescent probe PBVC prepared in Example 1 and 10 times ONOO - High-resolution mass spectrometry after the reaction.

[0045] Figure 13 Photographs of the solution of the fluorescent probe PBVC (10 μM) prepared in Example 1 in CH3CN-pbs (v / v=1:1, v / v, pH=7.4) reacted with different concentrations of ONOO- under natural light and 365 nm illumination conditions, respectively.

[0046] Figure 14 middle Figure 14 a is the fluorescence spectrum (excitation wavelength 568 nm) of the fluorescent probe PBVC (10 μM) prepared in Example 1 in glycerol-methanol solutions with different glycerol contents, Figure 14 b is the relationship between the logarithm of the fluorescence intensity at 720 nm (logI) and the logarithm of the viscosity (logη). DETAILED DESCRIPTION

[0047] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. All other embodiments obtained by ordinary technicians in this field based on the embodiments of the present invention without creative work are within the scope of protection of the present invention.

[0048] Example 1

[0049] A fluorescent probe, the preparation steps are as follows:

[0050] 7-N,N-diethylamino-2H-chromene-3-al (231 mg, 1 mmol), 4-methyl-N-(4'-boronic acid benzylpinacolyl)pyridine bromide (468 mg, 1.2 mmol), anhydrous ethanol (20 ml) as solvent, and pyridine (24 μl, 0.3 mmol) as catalyst were added to a 100 ml round-bottom flask. The mixture was heated to 100°C under magnetic stirring and refluxed for 14 h. After the reaction was completed, the reaction solution was cooled, the precipitate was separated, and filtered. The solid was washed twice with 0.5 ml of ethanol and recrystallized from anhydrous ethanol to obtain 290 mg of a purple-red solid with a yield of 48%.

[0051] The prepared fluorescent probe PBVC was analyzed by nuclear magnetic resonance using a nuclear magnetic resonance instrument, and the results are as follows:

[0052] 1 H NMR (400 MHz, DMSO) δ 1 H NMR (400 MHz, DMSO) δ8.86 (d, J = 7.0 Hz, 2H), 8.01 (d, J = 6.9 Hz, 2H), 7.78 (d, J = 8.0 Hz, 2H), 7.69 (d, J = 15.8 Hz, 1H), 7.39 (d, J = 7.9 Hz, 2H), 7.05 (d, J = 8.7 Hz, 1H), 6.96 (s, 1H), 6.60 (d, J = 16.0 Hz, 1H), 6.31 (d, J = 11.0 Hz, 1H), 6.13 (s, 1H), 5.64 (s, 2H), 5.02 (s, 2H), 3.44–3.38 (m, 4H), 1.23 (s, 6H), 1.10 (q, J = 7.2 Hz, 12H). Figure 1 shown.

[0053] 13 C NMR (101 MHz, DMSO) δ 156.87, 153.90, 150.98, 144.13, 140.14, 136.14, 135.21, 133.71, 130.45, 127.69, 124.37, 123.28, 119.07, 110.88, 106.13, 97.91, 65.06, 62.50, 49.07, 46.11, 44.43, 23.88, 13.03, 12.05, C NMR spectrum is shown in Figure 2. Figure 2 shown.

[0054] High-resolution mass spectrometry HR-MS: C 33 H 40 BN2O3 + Calculated value: 523.4938, measured value 441.2359, the fragment peak may have been lost in the mass spectrometry test, and the analysis found that it was consistent with C 33 H 40 BN2O3 + After removing the fragments -2C(CH3)2 and adding 2H, that is, C 27 H 30 The calculated value of BN2O3+ is 441.2344. The mass spectrum data is as follows Figure 3 shown.

[0055] After characterization, it was determined to be the fluorescent probe PBVC.

[0056] Example 2

[0057] A fluorescent probe, the preparation steps are as follows:

[0058] 7-N,N-diethylamino-2H-chromen-3-al (231 mg, 1 mmol), 4-methyl-N-(4'-boronic acid benzylpinacolyl)pyridine bromide (390 mg, 1 mmol), acetonitrile (20 ml) as solvent, and triethylamine (55 μl, 0.4 mmol) as catalyst were added to a 100 ml round-bottom flask. The mixture was heated to 100°C with magnetic stirring and refluxed for 12 hours. After the reaction was completed, the reaction solution was cooled, the precipitate was separated, and filtered. The solid was washed twice with 0.5 ml of acetonitrile and recrystallized from anhydrous ethanol to obtain 338 mg of a purple-red solid with a yield of 56%. Characterization confirmed that it was the fluorescent probe PBVC.

[0059] Example 3

[0060] A fluorescent probe, the preparation steps are as follows:

[0061] 7-N,N-diethylamino-2H-chromen-3-al (231 mg, 1 mmol), 4-methyl-N-(4'-boronic acid benzylpinacolyl)pyridine bromide (468 mg, 1.2 mmol), anhydrous ethanol (20 ml) as solvent, and piperidine (39 μl, 0.4 mmol) as catalyst were added to a 100 ml round-bottom flask. The mixture was heated to 100°C with magnetic stirring and refluxed for 12 hours. After the reaction was completed, the reaction solution was cooled, the precipitate was separated, filtered, and the solid was washed twice with 0.5 ml of ethanol. It was then recrystallized from anhydrous ethanol to obtain 482 mg of a purple-red solid with an 80% yield. Characterization confirmed that it was the fluorescent probe PBVC.

[0062] Example 4

[0063] A fluorescent probe, the preparation steps are as follows:

[0064] 7-N,N-diethylamino-2H-chromene-3-al (231 mg, 1 mmol), 4-methyl-N-(4'-boronic acid benzylpinacolyl)pyridine bromide (390 mg, 1 mmol), anhydrous ethanol (20 ml) as solvent, and ammonium acetate (32 mg, 0.4 mmol) as catalyst were added to a 100 ml round-bottom flask. The mixture was heated to 60°C under magnetic stirring and reacted for 15 h. After the reaction was completed, the reaction solution was cooled, the precipitate was precipitated, filtered, washed twice with 0.5 ml of ethanol, and recrystallized from acetonitrile to obtain 250 mg of a purple-red solid with a yield of 40%. After characterization, it was confirmed to be the fluorescent probe PBVC.

[0065] Example 5

[0066] A fluorescent probe, the preparation steps are as follows:

[0067] 7-N,N-diethylamino-2H-chromen-3-al (231 mg, 1 mmol), 4-methyl-N-(4'-boronic acid benzylpinacolyl)pyridine bromide (312 mg, 0.8 mmol), acetonitrile (20 ml), and ammonium acetate (4 mg, 0.05 mmol) as catalyst were added to a 100 ml round-bottom flask and heated to 140°C under magnetic stirring for 6 h. After the reaction was completed, the reaction solution was cooled, the precipitate was precipitated, filtered, washed twice with 0.5 ml of ethanol, and recrystallized from 1,4-dioxane to obtain 250 mg of a purple-red solid with a yield of 40%. After characterization, it was determined to be the fluorescent probe PBVC.

[0068] Example 6

[0069] A fluorescent probe, the preparation steps are as follows:

[0070] 7-N,N-diethylamino-2H-chromene-3-al (231 mg, 1 mmol), 4-methyl-N-(4'-boronic acid benzylpinacolyl)pyridine bromide (585 mg, 1.5 mmol), tetrahydrofuran (40 ml), and catalyst pyridine (40 μl, 0.5 mmol) were added to a 100 ml round-bottom flask and heated to 60°C under magnetic stirring for 18 hours. After the reaction was completed, the reaction solution was cooled, the precipitate was precipitated, filtered, washed twice with 0.5 ml of ethanol, and recrystallized from acetone to obtain 250 mg of a purple-red solid, which was identified as the fluorescent probe PBVC after characterization.

[0071] Implementation Effect

[0072] Preparation of 1 mM probe solution: Accurately weigh the probe PBVC prepared in Example 1 and dissolve it in dimethyl sulfoxide to prepare a 1 mM solution for later use.

[0073] Separately add anion sodium salt or potassium salt: ClO3 - ,NO3 - ,AcO - ,ClO - ,F - ,Cl - ,Br - ,I - ,SO3 2- ,S2O3 2- ,S 2- ,SO4 2- ,CO3 2- ,PO4 3- ,P2O7 4- , active oxygen species include t-BuO·,·OH, 1 O2,ClO - , ONOO - ,NO,H2O2,O2-· Prepared in distilled water with a concentration of 10 mM, stored in the refrigerator for future use.

[0074] Water Solubility and Spectral Properties of Probe PBVC

[0075] The aqueous solution of the probe PBVC of Example 1 can be directly prepared (concentration is 10 μM). The aqueous solution is purple-red. The absorption spectrum of the probe PBVC in aqueous solution is tested. The results are shown in the attached figure. Figure 4 The absorption peak is at 540 nm. When the excitation wavelength is 540 nm, the fluorescence spectrum is measured at 720 nm, which is in the near-infrared region. The probe also has a large Stokes shift (180 nm), which effectively avoids fluorescence background interference and inner filter effects during detection.

[0076] Probe PBVC for ONOO - Spectral response selectivity

[0077] To the solution of the probe (10 μM) in CH3CN-PBS (v / v=1:1, v / v, pH=7.4) of Example 1, 10 times the amount of anionic sodium salt or potassium salt and active oxygen were added. The probe was investigated for ONOO by absorption spectroscopy and fluorescence spectroscopy. - Selectivity. Figure 5 As shown in the figure, it can be seen that the probe (blank) has an obvious absorption peak at 540nm. After adding the above anions and active oxygen, in addition to adding ONOO - In addition to causing the probe solution to have a significant decrease in absorbance at 540nm and a significant absorption peak at 410nm, the addition of other anions ClO3 - ,NO3 - ,AcO - ,F - ,Cl - ,Br - ,I - ,SO3 2- ,S2O3 2- ,S 2- ,SO4 2- ,CO3 2- ,PO4 3- ,P2O7 4- 、Active oxygen (t-BuO·,·OH, 1 O2,ClO - , NO, H2O2, O2 -· ) did not cause significant changes in the absorbance of the probe at 540 nm and 410 nm; under 410 nm light excitation, the probe had a very weak fluorescence peak at 568 nm, as shown in Figure 6 After adding the above anions and active oxygen, in addition to adding ONOO- In addition to causing the probe solution to show a significantly enhanced fluorescence peak at 568 nm, the addition of other anions ClO3 - ,NO3 - ,AcO - ,F - ,Cl - ,Br - ,I - ,SO3 2- ,S2O3 2- ,S 2- ,SO4 2- ,CO3 2- ,PO4 3- ,P2O7 4- 、Active oxygen (t-BuO·,·OH, 1 O2,ClO - , NO, H2O2, ClO - , O2 -· ) did not cause obvious changes in probe fluorescence, such as Figure 6 The results showed that the probe was sensitive to ONOO - It has excellent absorption selectivity and fluorescence selectivity.

[0078] Probe PBVC for ONOO - Absorption and fluorescence response anti-interference

[0079] In order to verify the selectivity of the probe, an anti-interference experiment was conducted when anions, reactive oxygen species and ONOO- coexisted. To the acetonitrile-PBS (v / v=1:1, v / v, pH=7.4) buffer solution of the probe (10 μM) of Example 1, 10 times the amount of anions and reactive oxygen species were added, and then 10 times the amount of ONOO- was added. The absorbance ratio of each solution at 410 nm and 540 nm was measured to test the probe's recognition of ONOO - The ability to resist absorption and interference of common anions and active oxygen, such as Figure 7 As shown, the probe (10 μM) was detected in blank sample and 100 μM analyte ClO3 - ,NO3 - ,AcO - ,F - ,Cl - ,Br - ,I - ,SO3 2- ,S2O3 2- ,S 2- ,SO4 2- ,CO3 2- ,PO4 3- ,P2O7 4-、Active oxygen (t-BuO·,·OH, 1 O2,ClO - ,NO,H2O2,O2 -· ) in the presence of, the absorbance ratio of the resulting solution at 410nm and 540nm, respectively, as Figure 7 As shown in orange, the intensity is basically zero, which means that the above anions and reactive oxygen species do not react with the probe. When 10 times the amount of ONOO- (i.e., 100 μM ONOO) is added to the above analyte, the - ), and the addition of 100 μM ONOO to the probe alone - The probe solution A 410 nm / A 540 nm Basically the same, such as Figure 7 Shown in green.

[0080] At the same time, by the Figure 8 It can be seen that the blank solution and 100 μM ClO3 were added to the acetonitrile-PBS (v / v=1:1, v / v, pH=7.4) buffer solution of the probe (10 μM) of Example 1. - ,NO3 - ,AcO - ,F - ,Cl - ,Br - ,I - ,SO3 2- ,S2O3 2- ,S 2- ,SO4 2- ,CO3 2- ,PO4 3- ,P2O7 4- 、Active oxygen (t-BuO·,·OH, 1 O2,ClO - ,NO,H2O2,O2 -· ), the fluorescence intensity of the above solutions at 568nm (such as Figure 8 100 μM ONOO was added to the above analytes. - , compared with the addition of 100 μM ONOO - The fluorescence intensity of the probe solution obtained at 568 nm is basically the same (such as Figure 8 (shown in green in the middle), indicating that the probe is sensitive to ONOO - The detection has strong anti-interference ability and is expected to be applicable to complex biological environments.

[0081] Probe PBVC for ONOO - Ratio colorimetric response, linear range and detection limit

[0082] The width of the linear response range and the sensitivity are important criteria for judging whether a probe molecule has application value. - The probe (10 μM) of Example 1 was titrated in acetonitrile-PBS (v / v=1:1, v / v, pH=7.4) buffer solution to determine the effect of the probe on different concentrations of ONOO - The absorption spectrum of the response. Figure 9 a It can be seen that the absorbance of the probe at 540 nm increases with the increase of ONOO - The absorbance gradually decreases with the increase of concentration, and a new peak appears at 410nm. The absorbance gradually increases, and an isosbestic point appears at 450nm. This shows that ONOO - The presence of causes the probe structure to change, generating a new structure. Figure 9 b It can be seen that adding 5 times the amount of ONOO - The ratio of the absorbance at 410 nm to that at 540 nm (A 410 / A 540 ) reaches 5. And A 410 / A 540 with ONOO - The concentration of α-glucose was linear between 0 and 10 μM ( Figure 9 c) The linear equation is A 410 / A 540 =0.3318+31892.0569C(A 410 / A 540 is the ratio of the absorbance at 410 nm to the absorbance at 540 nm, and C is ONOO - The concentration unit is mol / L), the linear correlation coefficient R 2 =0.9928, according to the formula LOD = 3σ / k, σ is the standard deviation of the ratio of the absorbance at 410nm to the absorbance at 540nm of the probe measured 10 times, and k is the slope of the linear relationship curve. - The limit of detection (LOD) was 95 nM.

[0083] Probe PBVC for ONOO - Ratio fluorescence response, linear range and detection limit

[0084] With different concentrations of ONOO - The probe (10 μM) was titrated in acetonitrile-PBS (v / v=1:1, v / v, pH=7.4) buffer solution to determine the effect of the probe on different concentrations of ONOO - The fluorescence spectrum of the response, from Figure 10 aIt can be seen that with ONOO -With the continuous addition of , under 450nm excitation, the fluorescence peak of the probe at 720nm gradually weakened, the fluorescence peak at 568nm gradually increased, and an isofluorescence point appeared at 680nm, indicating that ONOO - The change in the probe structure caused by the change in the fluorescence spectrum. Figure 10 b It can be seen that ONOO - When the concentration reaches 50 μM, the ratio of fluorescence intensity (I 568 / I 720 ) reached 50. The ratio of fluorescence intensity (I 568 / I 720 ) and ONOO - The concentration showed a linear relationship between 0 and 16 μM ( Figure 10 c), the linear equation is I 568 / I 720 =1.8503+441502.3682C,R 2 =0.9902(I 568 / I 720 is the ratio of the fluorescence intensity at 568 nm to the fluorescence intensity at 720 nm, C is ONOO - concentration), linear correlation coefficient R 2 =0.9902, according to the formula LOD = 3σ / k, σ is the I of the probe measured 10 times 568 / I 720 The standard deviation of the probe molecule is , and k is the slope of the linear relationship curve. - The detection limit LOD was 10 nM, indicating that the probe was sensitive to ONOO - The fluorescence response enables ratiometric detection with high sensitivity.

[0085] Probe PBVC for ONOO - Speed ​​of fluorescence response

[0086] The response speed is also an important basis for judging whether a probe molecule has potential application value. Figure 11 It can be seen that when ONOO was added to the probe (10 μM), - After 100 μM, the fluorescence intensity of the probe solution at 568 nm almost reached its maximum at 20 min. The fluorescence intensity hardly changed significantly after the time was prolonged. - The response speed is fast, which is beneficial to the environment or biological system - Real-time and online detection.

[0087] Probe PBVC for ONOO - Mechanism of fluorescence response

[0088] The probe PBVC of Example 1 was mixed with ONOO - (10eq) was subjected to mass spectrometry analysis and ONOO - Afterwards, with Figure 3 In comparison, a new molecular ion peak appeared at m / z 307.1802 (see attached Figure 12 This peak is attributed to the (M+1) peak of the new compound 7-N,N-diethylamino-3E-(2'-(4"-pyridinylvinyl))-2H-chromene.

[0089] When the probe is exposed to ONOO - In the environment, the electrophilic boron atom in the molecule is firstly - The attack forms a boronic acid tetrahedral intermediate. The aromatic group then undergoes a 1,2-migration from the boron atom to the adjacent oxygen atom, forming a boronate ester. Subsequently, the boronate ester hydrolyzes to produce the boronic acid / ester and the corresponding phenol intermediate. Finally, the C-N bond in the phenol intermediate spontaneously cleaves via a 1,6-elimination reaction to form the oxidized product, 7-N,N-diethylamino-3E-(2'-(4"-pyridinylvinyl))-2H-chromene (the process is shown in the figure below). This product emits bright yellow fluorescence at 568 nm.

[0090]

[0091] Probe PBVC for ONOO - Fluorescent naked eye identification

[0092] The probe PBVC solution is purple-red under natural light. - With the increase of concentration, the purple-red color gradually becomes lighter until it is slightly yellow. Under 365nm light, the probe PBVC emits red fluorescence. - With the increase of concentration, the fluorescence gradually changes from red to orange to yellow, and finally turns into bright yellow fluorescence. Figure 13 As shown in the figure, under natural light or 365nm illumination, ONOO can be identified by naked eyes using the probe PBVC. - .

[0093] Fluorescence response of probe PBVC to viscosity

[0094] In order to test the responsiveness of the probe PBVC to the system viscosity, the viscosity of the system was changed by adjusting the mass ratio of glycerol in the glycerol-methanol system (0% to 100%), and the fluorescence spectrum of the probe PBVC was tested to see how it was affected by the viscosity. Figure 14 It can be seen from a that as the proportion of glycerol in the glycerol-methanol system increases, that is, the viscosity increases, the fluorescence intensity of the probe at 710 nm gradually increases. The logarithm of the fluorescence intensity (logI) is linearly related to the logarithm of the corresponding viscosity (logη) ( Figure 14 b), the linear equation is logI=0.12253+1.53602logη, R 2 =0.9937. The results show that the probe PBVC has excellent responsiveness to viscosity and is expected to become an ideal probe for detecting viscosity changes in microenvironments.

[0095] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A fluorescent probe, characterized in that: The structural formula of the fluorescent probe is shown in formula (I):

2. The method for preparing the fluorescent probe according to claim 1, wherein The steps include: 7-N,N-diethylamino-2H-chromene-3-aldehyde, 4-methyl-N-(4'-boronic acid pinacol ester benzyl)pyridine bromide, a catalyst, and a solvent are mixed, stirred, and refluxed, and then filtered, washed, and recrystallized to purify the fluorescent probe product.

3. The method for preparing a fluorescent probe according to claim 2, wherein: The molar ratio of the 7-N,N-diethylamino-2H-chromene-3-al, 4-methyl-N-(4'-boronic acid pinacolyl benzyl)pyridine bromide and the catalyst is 1:(0.8-1.5):(0.05-0.5).

4. The method for preparing a fluorescent probe according to claim 3, wherein: The molar ratio of the 7-N,N-diethylamino-2H-chromene-3-aldehyde, 4-methyl-N-(4'-boronic acid pinacolyl benzyl)pyridine bromide and the catalyst is 1:1.2:0.

3.

5. The method for preparing a fluorescent probe according to claim 2, wherein: The reaction temperature is 60-140° C., and the reaction time is 6-18 hours.

6. The method for preparing a fluorescent probe according to claim 5, wherein: The reaction temperature is 100° C. and the reaction time is 12 h.

7. The method for preparing a fluorescent probe according to claim 2, wherein: The solvent is any one of methanol, ethanol, n-propanol, isopropanol, tetrahydrofuran, dichloromethane, acetonitrile, acetone, 1,4-dioxane, ethyl acetate and N,N-dimethylformamide.

8. The method for preparing a fluorescent probe according to claim 2, wherein: The solvent used for the washing and recrystallization is methanol, ethanol, n-propanol, isopropanol, tetrahydrofuran, dichloromethane, acetonitrile, acetone, 1,4-dioxane or ethyl acetate.

9. The method for preparing a fluorescent probe according to claim 2, wherein: The catalyst is triethylamine, pyridine, piperidine or ammonium acetate.

10. Use of the fluorescent probe according to claim 1 in preparing a product for detecting peroxynitrite concentration, viscosity, and near-infrared mitochondria in biological samples.

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