High-sensitivity polarity and ONOO <-> dual-response fluorescent probe and preparation method thereof

Abstract

The preparation method comprises the following steps: (1) adding 1, 3-dicyclohexyl barbituric acid into acetic anhydride, then adding boron trifluoride diethyl etherate, and carrying out a reaction, so as to obtain an intermediate product; (2) adding 7-(diethylamino) coumarin-3-formaldehyde, the intermediate product and piperidine into a first organic solvent, and carrying out heating reflux, extraction, filtration and recrystallization to obtain a fluorescent dye LD-YB; (3) preparing a metal organic framework material UiO from hafnium tetrachloride and an organic ligand; and (4) adding the LD-YB and UiO into a second organic solvent, and reacting to obtain the polarity and ONOO <-> dual-response fluorescent probe. The polarity and ONOO <-> dual-response fluorescent probe is obtained by loading LD-YB on UiO, so that the detection accuracy is improved, and the hydrophilicity is good.

Description

Technical Field

[0001] The present invention relates to the field of fluorescent probes, in particular to a highly sensitive polar and ONOO - Dual-response fluorescent probe and preparation method thereof. Background Art

[0002] Fluorescent molecular probe imaging has the advantages of sensitivity and intelligent response, and has great application potential in the biomedical field. For example, the incidence of non-alcoholic fatty liver disease (NAFLD) has been increasing year by year in recent years. The development of a highly sensitive and specific fluorescent probe is of great significance for the early diagnosis of NAFLD.

[0003] Due to the influence of lipid accumulation, lipotoxicity and other factors, there are some studies on NAFLD based on polarity, viscosity, ONOO - Such fluorescent probes have the problems of low detection accuracy and specificity. Later, researchers developed dual-response probes based on polarity-viscosity, viscosity-ONOO - The dual-response probes such as ONOO have improved the detection accuracy, but most of these fluorescent probes are single molecules with two responses, which are prone to false positive results and poor hydrophilicity of the probes, which is not conducive to biomedical applications. - Dual-responsive, highly sensitive, and hydrophilic fluorescent probes have particularly important practical application significance. Summary of the invention

[0004] The purpose of the present invention is to overcome the above technical deficiencies and provide a highly sensitive polar and ONOO - Dual-response fluorescent probe and preparation method to solve the problem of polar response or ONOO in the prior art - The detection accuracy of the responsive fluorescent probe is not high and the hydrophilicity is poor.

[0005] In order to achieve the above technical purpose, the technical solution provided by the present invention is: In a first aspect, the present invention provides a highly sensitive polar and ONOO - The preparation method of a dual-response fluorescent probe comprises the following steps: (1) adding 1,3-dicyclohexylbarbituric acid to acetic anhydride, and then adding boron trifluoride ether to react to obtain an intermediate product; (2) adding 7-(diethylamino)coumarin-3-carboxaldehyde, the intermediate product and piperidine to a first organic solvent, and obtaining a fluorescent dye LD-YB after heating and refluxing, extraction, filtration and recrystallization; (3) preparing a metal organic framework material UiO with hafnium tetrachloride and an organic ligand; (4) adding the fluorescent dye LD-YB and the metal organic framework material UiO to a second organic solvent, and obtaining polar and ONOO - Dual-response fluorescent probe.

[0006] In a second aspect, the present invention provides a highly sensitive polar and ONOO prepared by the above preparation method. - Dual-response fluorescent probe.

[0007] Compared with the prior art, the present invention has the following beneficial effects: The present invention prepares ONOO - The responsive fluorescent UiO and the polar responsive fluorescent dye LD-YB were used as the main raw materials, and LD-YB was loaded on UiO to obtain polar and ONOO - The dual-response fluorescent probe avoids the problem of low accuracy of detection results that is prone to occur when a single molecule performs multiple responses in conventional material design. The fluorescent probe obtained by the present invention improves detection accuracy and has good hydrophilicity, which is conducive to subsequent biological applications, and the particle size of the obtained material is uniform and stable. BRIEF DESCRIPTION OF THE DRAWINGS

[0008] Figure 1 is a particle size distribution diagram of UiO and Uio-YB prepared in Example 1 of the present invention; Figure 2 is a TEM image of Uio-YB prepared in Example 1 of the present invention; Figure 3 is the excitation emission spectrum of UiO prepared in Example 1 of the present invention; Figure 4 is the excitation emission spectrum of LD-YB prepared in Example 1 of the present invention; Figure 5 is a polarity response relationship diagram of LD-YB prepared in Example 1 of the present invention; Figure 6 The UiO prepared in Example 1 of the present invention is subjected to ONOO - Fluorescence response linear relationship; Figure 7 It is the hydrophilicity (contact angle) characterization of LD-YB, UiO and Uio-YB prepared in Example 1 of the present invention. DETAILED DESCRIPTION

[0009] In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention.

[0010] Regarding the polarity response or ONOO in the existing technology -In the responsive fluorescent probe, a single molecule is prone to multiple responses, which may result in low detection specificity and poor hydrophilicity. The present invention provides a highly sensitive polar and ONOO - A dual-response fluorescent probe and preparation method, wherein the probe is a material of UiO loaded with a polarity-responsive fluorescent dye; wherein the metal organic framework material (abbreviated as MOF or MOFs) is composed of metal ions and organic ligands, and has the characteristics of diverse components, simple synthesis, easy surface functionalization, large specific surface area, adjustable porosity, and controllable biocompatibility. The UiO used in the present invention is a kind of MOFs material. Since the UiO skeleton can respond to ONOO - , and is not affected by environmental factors, showing excellent fluorescence effects; at the same time, UiO can be used as a carrier of fluorescent dyes to enhance the hydrophilicity and stability of fluorescent dyes, showing potential application value. In addition, YB is a fluorescent dye designed based on lipophilic coumarin, which has a sensitive response to changes in polarity.

[0011] In a first aspect, the present invention provides a highly sensitive polar and ONOO - The preparation method of the dual-response fluorescent probe comprises the following steps: (1) Add 1,3-dicyclohexylbarbituric acid to acetic anhydride, and then add boron trifluoride etherate to react to obtain an intermediate product; (2) adding 7-(diethylamino)coumarin-3-carboxaldehyde (CAS No.: 57597-64-5), the intermediate product and piperidine to a first organic solvent, heating and refluxing, extracting, filtering and recrystallizing to obtain a fluorescent dye LD-YB; (3) Preparation of metal organic framework material UiO using hafnium tetrachloride and organic ligands; (4) Adding fluorescent dye LD-YB and metal organic framework material UiO into the second organic solvent, and reacting to obtain polar and ONOO - Dual-response fluorescent probe.

[0012] Preferably, in step (1), the ratio of 1,3-dicyclohexylbarbituric acid, acetic anhydride and boron trifluoride etherate is 1 g: (8-12) mL: (500-550) μL.

[0013] Preferably, in step (1), the reaction conditions include: temperature of 90-100°C, time of 30-40 min, and stirring speed of 500-800 rpm.

[0014] Preferably, in step (2), the mass ratio of 7-(diethylamino)coumarin-3-carboxaldehyde to the intermediate product is (0.5-1):1, and the ratio of the intermediate product to piperidine is (0.1-0.5) g:0.1 mL.

[0015] Preferably, in step (2), the first organic solvent comprises acetonitrile, and the ratio between the intermediate product and the first organic solvent is (0.1-0.5) g:10 mL.

[0016] Preferably, in step (2), the heating under reflux is carried out at 60-70° C. for 4-6 hours.

[0017] Preferably, in step (2), the recrystallization is carried out using a mixture of dichloromethane and anhydrous ethanol in a volume ratio of 1:3.

[0018] Preferably, in step (3), the step of preparing the metal organic framework material UiO comprises: adding hafnium tetrachloride and an organic ligand into a third organic solvent for heating reaction, and then performing post-treatment to obtain UiO.

[0019] Further preferably, the organic ligand includes 2-hydroxyterephthalic acid or 2,5-dihydroxyterephthalic acid; the molar ratio of hafnium tetrachloride to the organic ligand is 1: (1-1.2) (the metal organic framework material UiO prepared by the present invention has the same structure as UiO-66, and the ratio needs to preferably maintain the same stoichiometric ratio as UiO-66); the third organic solvent includes a mixed solution of DMF and formic acid in a volume ratio of 9: (1-3); the heating reaction is carried out at 130-150 ° C for 36-48 h; the post-treatment includes centrifugal separation, washing and drying, wherein the centrifugal speed is 8000-10000 rpm, the time is 15-25 min, and the washing solvent is acetone.

[0020] Preferably, in step (4), the mass ratio of the fluorescent dye LD-YB to the metal organic framework material UiO is (1-3):10.

[0021] Preferably, in step (4), the reaction is stirred at a speed of 500 to 800 rpm for 16 to 24 hours.

[0022] Preferably, in step (4), after the reaction is completed, the reaction mixture is centrifuged at a speed of 9000 to 11000 rpm for 15 to 25 min to obtain polar and ONOO - Dual-response fluorescent probe.

[0023] Preferably, in step (4), the second organic solvent comprises DMF ( N,N -dimethylformamide) and DMSO.

[0024] In a second aspect, the present invention provides a highly sensitive polar and ONOO prepared by the above preparation method. - Dual-response fluorescent probe.

[0025] In a third aspect, the present invention provides a polar and ONOO- Dual-responsive fluorescent probes for either polarity and ONOO - Application in microenvironment-sensitive fluorescence imaging or content detection.

[0026] Preferred, polar and ONOO - Microenvironment-sensitive fluorescence imaging includes, but is not limited to, early-stage imaging of non-alcoholic fatty liver disease.

[0027] The main mechanism of action and advantages of the present invention are: (1) In the preparation method of the present invention, first, the methylene group in the malonylurea structure of 1,3-dicyclohexylbarbituric acid is deprotonated, and the formed carbon anion undergoes addition elimination with acetic anhydride to obtain the acetylated structure 1,3-dicyclohexylbarbituric acid, and the newly introduced acetyl group undergoes enolization, and the oxygen anion and the carbonyl oxygen on the original structure attack the boron atom in boron trifluoride ether together, and the ethoxy group leaves to finally obtain a structural intermediate product; in the second step, the methyl group is deprotonated under the catalysis of piperidine, and the generated carbon anion attacks the aldehyde carbon in the structural fluorescent dye precursor 7-(diethylamino)coumarin-3-carboxaldehyde, and an aldol condensation reaction occurs and dehydration is performed to obtain the product LD-YB; hafnium tetrachloride and 2-hydroxyterephthalic acid are added to a mixed solution of DMF / formic acid, heated, filtered, washed, and dried to obtain UiO; LD-YB is added to a DMF solution of UiO, and after the reaction, centrifuged to obtain polar and ONOO - Dual-response fluorescent probe Uio-YB.

[0028] (2) The present invention prepares a fluorescent probe with dual response function. The fluorescent probe is a fluorescent dye loaded on a metal organic framework. The present invention avoids the problems of low specificity, low sensitivity and poor hydrophilicity of the traditional single molecule dual response. The Uio-YB prepared by this method has excellent hydrophilicity and polarity and ONOO - Dual response capability, in polarity and ONOO - The field of microenvironment-sensitive response fluorescence imaging has great application prospects.

[0029] The present invention is further described in detail below through specific embodiments.

[0030] Example 1 The specific steps for preparing Uio-YB in this embodiment are as follows: (1) Take 1.0 g of 1,3-dicyclohexylbarbituric acid and add it to 10 mL of acetic anhydride. Then add 520 μL of boron trifluoride ether dropwise. After reacting at 95 °C for 30 min, the solution becomes uniform and light yellow. The solvent is evaporated by rotary evaporation, methanol is added and the white solid is filtered to obtain LD-YB intermediate a.

[0031] (2) 0.21 g of 7-(diethylamino)coumarin-3-carboxaldehyde, 0.30 g of LD-YB intermediate a and 0.1 mL of piperidine were added to 10 mL of acetonitrile, heated under reflux at 60 °C for 4 h, cooled to room temperature, extracted and filtered, and recrystallized by adding 5 mL of dichloromethane and 15 mL of anhydrous ethanol to obtain the fluorescent dye LD-YB.

[0032] (3) 100 mg of hafnium tetrachloride (0.3 mmol) and 65 mg of 2-hydroxyterephthalic acid (0.36 mmol) were added to a mixed solution of 10 mL of DMF / formic acid (9:1), heated at 130 °C for 48 h in a Teflon-lined autoclave, cooled to room temperature, and centrifuged at 10,000 rpm for 25 min. The white precipitate was repeatedly washed with acetone and dried to obtain UiO. (4) Take 54 mg of UiO and 5.4 mg of LD-YB and dissolve them in 10 mL of DMF. Stir them thoroughly at 600 rpm for 24 h. After the reaction is completed, centrifuge at 10,000 rpm for 25 min and dry at 80 °C overnight to obtain Uio-YB sample 1.

[0033] The particle sizes of UiO and Uio-YB prepared in Example 1 were tested, wherein the average particle size of UiO was in the range of 80 to 95 nm, and the average particle size of Uio-YB was in the range of 85 to 100 nm. Specifically, the particle size distribution statistics of UiO and Uio-YB prepared in Example 1 were selected, and the results were as follows: Figure 1 As shown, the morphology of Uio-YB was characterized, and the results are shown Figure 2 shown.

[0034] At the same time, the excitation and emission spectra of UiO and Uio-YB were characterized. The results are as follows: Figure 3 , Figure 4 As shown, the emission wavelength of UiO is around 440 nm, and the emission wavelength of LD-YB is around 600 nm.

[0035] Subsequently, the prepared stable Uio-YB was subjected to polar and ONOO - Fluorescence response test. Uio-YB was added to a set of solutions with polarity from low to high, which were obtained by mixing high-polarity water and low-polarity solvent 1,4-dioxane in different proportions. The fluorescence intensity of Uio-YB in solutions of different polarity was tested using a fluorescence spectrometer. The results are shown in the figure. Figure 5 As shown, Uio-YB can exhibit good polarity response characteristics, and the fluorescence intensity is significantly enhanced with the increase of polarity.

[0036] Subsequently, Uio-YB was added to the mixture containing different concentrations of ONOO- Fluorescence spectrometer was used to test the activity of Uio-YB in solutions containing different concentrations of ONOO - The fluorescence intensity in the solution, such as Figure 6 As shown, with ONOO - With the increase of content, the fluorescence intensity of Uio-YB increased, and ONOO - The content and fluorescence intensity maintained a good linear relationship within a certain range, and the detection limit was as low as 51.7 nM, showing high sensitivity.

[0037] Finally, the effect of the introduction of Uio on the hydrophilicity of the water-insoluble dye LD-YB was verified. Figure 7 As shown in the figure, the hydrophilicity of the material was characterized by a contact angle meter. The contact angle of LD-YB was 79.44°, the contact angle of UiO was 50.22°, and the contact angle of Uio-YB was 48.3°. The hydrophilicity of UiO decreased slightly after loading LD-YB. This is because the UiO skeleton has good hydrophilicity, which significantly improves the hydrophilicity of LD-YB. Therefore, the above results prove that this polarity and ONOO - Dual-responsive fluorescent probe, showing good hydrophilicity and polarity and ONOO - Dual-response capability enables applications such as fatty liver, polar and ONOO - Specific testing location.

[0038] Example 2 The only difference from Example 1 is that the centrifugal speed of UiO in step (3) is adjusted to 7000 rpm, 9000 rpm, 10000 rpm and 11000 rpm, and the other steps and conditions are the same as in Example 1.

[0039] The results show that as the centrifugal speed increases, the particle size of the obtained UiO is more uniform and stable. Therefore, the centrifugal speed of UiO is preferably 9000-11000 rpm, and more preferably 10000 rpm.

[0040] Example 3 The only difference from Example 1 is that the mass ratio of LD-YB to UiO in step (4) is adjusted to 0.5:10, 3:10 and 5:10 respectively. The other steps and conditions are the same as those in Example 1.

[0041] The results show that with the increase of LD-YB dosage, the fluorescence intensity of the response increases, but the excessive increase of fluorescence intensity is not significant and causes reagent loss. In Example 1, the fluorescence intensity basically reaches the highest when the mass ratio of 1:10 is used. Therefore, the preferred mass ratio of LD-YB to UiO in the present invention is (1-3):10, and more preferably 1:10.

[0042] Comparative Example 1 The only difference from Example 1 is that the fluorescent dye LD-YB is directly used as the comparison probe 1, and the specific preparation steps and conditions are the same as those in Example 1.

[0043] The results showed that only LD-YB can be used as a fluorescent probe with a single polarity response. As the polarity of the test environment decreases, the fluorescence increases. However, if it is used for in vivo testing or imaging, fluorescence changes will also occur at other sites in the body where polarity changes occur, which does not have selective specificity. Uio-YB also has ONOO - Fluorescence response and polarity response, with high specificity. In addition, LD-YB is a fat-soluble fluorescent probe, which is subject to certain limitations in practical use.

[0044] Comparative Example 2 The only difference from Example 1 is that UiO is used as the comparison probe 2, and the specific preparation steps and conditions are the same as those in Example 1.

[0045] The results showed that only using UiO as a ligand can only play a single role in ONOO - Fluorescence response, along with the test environment ONOO - As the concentration increases, the fluorescence response of UiO increases. However, the emission light of Uio is around 450 nm, which does not have good tissue penetration ability, so it is greatly limited in deep tissue imaging. - The dual response functions of fluorescence response concentration and polar environment location response, in addition to the 450 nm emission light of Uio, also have emission light of about 600 nm, with strong tissue penetration ability, in actual use, with better imaging effect.

[0046] Different from the prior art, the present invention provides a highly sensitive polarity and ONOO-dual-response fluorescent probe and a preparation method thereof, by loading polarity-response fluorescent dye on ONOO - The dual-responsive probe Uio-YB was prepared by a responsive metal-organic framework method, avoiding the situation in which a single compound is used to respond to two markers in conventional preparation, resulting in inaccurate detection results; Uio-YB prepared by this method improves the accuracy of detection, while improving the poor hydrophilicity problem that fluorescent dyes usually have, which is beneficial for subsequent biological applications; at the same time, the particle size of the obtained product is uniform and stable.

[0047] The specific implementation of the present invention described above does not constitute a limitation on the protection scope of the present invention. Any other corresponding changes and modifications made based on the technical concept of the present invention should be included in the protection scope of the claims of the present invention.

Claims

1. A highly sensitive polar and ONOO - The preparation method of the dual-response fluorescent probe is characterized in that The following steps are involved: (1) Add 1,3-dicyclohexylbarbituric acid to acetic anhydride, and then add boron trifluoride etherate to react to obtain an intermediate product; (2) adding 7-(diethylamino)coumarin-3-carboxaldehyde, the intermediate product and piperidine to a first organic solvent, heating and refluxing, extracting, filtering and recrystallizing to obtain a fluorescent dye LD-YB; (3) Preparation of metal organic framework material UiO using hafnium tetrachloride and organic ligands; (4) Adding fluorescent dye LD-YB and metal organic framework material UiO into the second organic solvent, and reacting to obtain polar and ONOO - Dual-response fluorescent probe.

2. The highly sensitive polar and ONOO according to claim 1 - A method for preparing a dual-response fluorescent probe, characterized in that: In step (1), the ratio of 1,3-dicyclohexylbarbituric acid, acetic anhydride and boron trifluoride etherate is 1 g: (8-12) mL: (500-550) μL.

3. The highly sensitive polar and ONOO according to claim 1 - A method for preparing a dual-response fluorescent probe, characterized in that: In step (1), the reaction conditions include: temperature of 90-100°C, time of 30-40 min, and stirring speed of 500-800 rpm.

4. The highly sensitive polar and ONOO according to claim 1 - A method for preparing a dual-response fluorescent probe, characterized in that: In step (2), the mass ratio of 7-(diethylamino)coumarin-3-carboxaldehyde to the intermediate product is (0.5-1):1, and the ratio of the intermediate product to piperidine is (0.1-0.5) g:0.1 mL; The first organic solvent includes acetonitrile, and the ratio between the intermediate product and the first organic solvent is (0.1-0.5) g:10 mL.

5. The highly sensitive polar and ONOO according to claim 1 - A method for preparing a dual-response fluorescent probe, characterized in that: In step (2), the heating reflux reaction is carried out at 60-70° C. for 4-6 hours.

6. The highly sensitive polar and ONOO according to claim 1 - A method for preparing a dual-response fluorescent probe, characterized in that: In step (3), the preparation step of the metal organic framework material UiO comprises: adding hafnium tetrachloride and an organic ligand into a third organic solvent for heating reaction, and then performing post-treatment to obtain UiO.

7. The highly sensitive polar and ONOO according to claim 6 - A method for preparing a dual-response fluorescent probe, characterized in that: The organic ligand includes 2-hydroxyterephthalic acid or 2,5-dihydroxyterephthalic acid; the molar ratio of the hafnium tetrachloride to the organic ligand is 1: (1-1.2); The third organic solvent includes a mixed solution of DMF and formic acid in a volume ratio of 9:(1-3); The heating reaction is carried out at 130-150°C for 36-48 hours; The post-treatment includes centrifugal separation, washing and drying, wherein the centrifugal speed is 8000-10000 rpm and the time is 15-25 min.

8. The highly sensitive polar and ONOO according to claim 1 - A method for preparing a dual-response fluorescent probe, characterized in that: In step (4), the mass ratio of the fluorescent dye LD-YB to the metal organic framework material UiO is (1-3):

10.

9. The highly sensitive polar and ONOO according to claim 1 - A method for preparing a dual-response fluorescent probe, characterized in that: The reaction in step (4) is stirred at a speed of 500 to 800 rpm for 16 to 24 hours. After the reaction is completed, the reaction is centrifuged at a speed of 9000 to 11000 rpm for 15 to 25 minutes to obtain polar and ONOO - Dual-response fluorescent probe; The second organic solvent includes one or more of DMF and DMSO.

10. A highly sensitive polar and ONOO prepared by the method according to any one of claims 1 to 9. - Dual-response fluorescent probe.

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