A highly sensitive probe for polarity and onoo - Dual-responsive fluorescent probe and preparation method

Abstract

This invention relates to a highly sensitive polarity and ONOO ‑ The dual-response fluorescent probe and its preparation method include the following steps: (1) adding 1,3-dicyclohexylbarbituric acid to acetic anhydride, and then adding boron trifluoride diethyl ether to react and obtain an intermediate product; (2) adding 7-(diethylamino)coumarin-3-carboxaldehyde, the intermediate product and piperidine to a first organic solvent, and after heating and reflux, extraction, filtration and recrystallization, obtaining the fluorescent dye LD-YB; (3) preparing the metal-organic framework material UiO with hafnium tetrachloride and organic ligands; (4) adding LD-YB and UiO to a second organic solvent, and reacting to obtain polar and ONOO. ‑ Dual-response fluorescent probe. This invention obtains polar and ONOO fluorescent probes by loading LD-YB onto UiO. ‑ The dual-response fluorescent probe improves detection accuracy and has good hydrophilicity.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of fluorescent probes, and particularly relates to a high-sensitivity polar and ONOO - Dual-response fluorescent probe and preparation method thereof. BACKGROUND

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

[0003] Due to the influence of factors such as lipid accumulation, lipid toxicity, etc., there are currently some single-response probes based on polarity, viscosity, ONOO - , etc. Such fluorescent probes have problems of low detection accuracy and specificity. Later, researchers developed dual-response probes based on polarity-viscosity, viscosity-ONOO - , etc., which improved the detection accuracy. However, such fluorescent probes are mostly single molecules responding to two responses, which can easily produce false positive results, and also have problems of poor hydrophilicity, which is not conducive to biomedical applications. Therefore, it is of great practical significance to design a fluorescent probe with high sensitivity, good hydrophilicity, and dual response to polarity and ONOO - . SUMMARY

[0004] The present application aims to overcome the above technical deficiencies and provide a high-sensitivity polar and ONOO - dual-response fluorescent probe and a preparation method thereof, which solves the technical problems of low detection accuracy and poor hydrophilicity of the existing polar response or ONOO - response fluorescent probes.

[0005] To achieve the above technical purpose, the technical solution provided by the present application is as follows:

[0006] In a first aspect, the present application provides a preparation method of a high-sensitivity polar and ONOO - dual-response fluorescent probe, comprising the following steps: (1) adding 1,3-biscyclohexylbarbituric acid to acetic anhydride, and then adding boron trifluoride ether to react to obtain an intermediate product; (2) adding 7-(diethylamino) coumarin-3-formaldehyde, the intermediate product and piperidine to a first organic solvent, and after heating and refluxing, extracting and filtering, and recrystallizing, a fluorescent dye LD-YB is obtained; (3) preparing a metal-organic framework material UiO with hafnium tetrachloride and an organic ligand; and (4) adding the fluorescent dye LD-YB and the metal-organic framework material UiO to a second organic solvent, and reacting to obtain a polar and ONOO -Dual-responsive fluorescent probe

[0007] In a second aspect, the present application provides a high-sensitivity polar and ONOO - Dual-responsive fluorescent probe

[0008] Compared with the prior art, the present application has the following beneficial effects:

[0009] The present application uses the fluorescent UiO with ONOO - response and the fluorescent dye LD-YB with polar response as main raw materials, and then loads the LD-YB on the UiO to obtain the polar and ONOO - Dual-responsive fluorescent probe, which avoids the problem of low detection accuracy of single molecule in conventional material design. The fluorescent probe obtained by the present application improves the detection accuracy, has good hydrophilicity, is beneficial to subsequent biological application, and has uniform and stable particle size. BRIEF DESCRIPTION OF DRAWINGS

[0010] Figure 1 is a particle size distribution diagram of the UiO and Uio-YB prepared in Example 1 of the present application;

[0011] Figure 2 is a TEM diagram of the Uio-YB prepared in Example 1 of the present application;

[0012] Figure 3 is an excitation emission spectrum diagram of the UiO prepared in Example 1 of the present application;

[0013] Figure 4 is an excitation emission spectrum diagram of the LD-YB prepared in Example 1 of the present application;

[0014] Figure 5 is a polar response relationship diagram of the LD-YB prepared in Example 1 of the present application;

[0015] Figure 6 is a ONOO - fluorescent response linear relationship of the UiO prepared in Example 1 of the present application;

[0016] Figure 7 is a hydrophilicity (contact angle) characterization of the LD-YB, UiO and Uio-YB prepared in Example 1 of the present application. DETAILED DESCRIPTION

[0017] In order to make the purpose, technical scheme and advantages of the present application clearer, the present application will be further described in detail below in combination with the drawings and examples. It should be understood that the specific examples described herein are only used to explain the present application, and are not used to limit the present application.

[0018] The present application is directed to a polar response or ONOO - In the prior art, a single molecule in a multi-response fluorescent probe has defects of low detection specificity and poor hydrophilicity, and the present application provides a high-sensitivity polar and ONOO - The present application provides a polar and ONOO - response fluorescent probe and a preparation method thereof. The probe is a material of UiO loaded with a polar response fluorescent dye. The metal organic framework material (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, controllable biocompatibility, etc. The UiO used in the present application is a kind of MOF material, and the UiO framework can respond to ONOO - , and is not disturbed by environmental factors, showing excellent fluorescence effect. At the same time, UiO can be used as a carrier of fluorescent dye to enhance the hydrophilicity and stability of fluorescent dye, showing potential application value. In addition, YB is a fluorescent dye designed based on lipophilic coumarin, which has a sensitive response to polarity change.

[0019] In a first aspect, the present application provides a high-sensitivity polar and ONOO - response fluorescent probe and a preparation method thereof. The probe is a material of UiO loaded with a polar response fluorescent dye. The metal organic framework material (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, controllable biocompatibility, etc. The UiO used in the present application is a kind of MOF material, and the UiO framework can respond to ONOO - .

[0020] (1) 1,3-dicyclohexylbarbituric acid is added to acetic anhydride, and then boron trifluoride ether is added for reaction to obtain an intermediate product;

[0021] (2) 7-(diethylamino) coumarin-3-formaldehyde (CAS No.: 57597-64-5), the intermediate product and piperidine are added to a first organic solvent, and after heating reflux, extraction filtration and recrystallization, a fluorescent dye LD-YB is obtained;

[0022] (3) Metal organic framework material UiO is prepared from hafnium tetrachloride and organic ligand;

[0023] (4) The fluorescent dye LD-YB and the metal organic framework material UiO are added to a second organic solvent, and a polar and ONOO - response fluorescent probe is prepared by reaction.

[0024] Preferably, in step (1), the ratio among 1,3-dicyclohexylbarbituric acid, acetic anhydride and boron trifluoride ether is 1g: (8-12) mL: (500-550) μL.

[0025] Preferably, in step (1), the reaction conditions include: temperature of 90-100 ℃, time of 30-40 min, and stirring speed of 500-800 rpm.

[0026] 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 between the intermediate product and piperidine is (0.1-0.5) g:0.1 mL.

[0027] 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.

[0028] Preferably, in step (2), the heating reflux is refluxed at 60-70 °C for 4-6 h.

[0029] Preferably, in step (2), the recrystallization is performed using a mixture of dichloromethane and anhydrous ethanol in a volume ratio of 1:3.

[0030] Preferably, in step (3), the preparation of the metal-organic framework material UiO includes: adding hafnium tetrachloride and an organic ligand into a third organic solvent for heating reaction, and then performing post-treatment to obtain UiO.

[0031] Further preferably, the organic ligand comprises 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 application is consistent with the structure of UiO-66, and the ratio needs to be preferably maintained at the same stoichiometric ratio as UiO-66); the third organic solvent comprises a mixed solution of DMF and formic acid in a volume ratio of 9:(1-3); the heating reaction is performed 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, and the time is 15-25 min, and the washing solvent is acetone.

[0032] Preferably, in step (4), the mass ratio of the fluorescent dye LD-YB to the metal-organic framework material UiO is (1-3):10.

[0033] Preferably, in step (4), the reaction is stirred at a speed of 500-800 rpm for 16-24 h.

[0034] Preferably, in step (4), after the reaction is completed, centrifugal separation is performed at a speed of 9000-11000 rpm and for a time of 15-25 min to obtain the polar and ONOO - dual-response fluorescent probe.

[0035] Preferably, in step (4), the second organic solvent comprises one or more of DMF (dimethylformamide) and DMSO. N,N ​

[0036] In a second aspect, the present application provides a high-sensitivity polar and ONOO - Dual-responsive fluorescent probe.

[0037] In a third aspect, the present application provides a polar and ONOO - Dual-responsive fluorescent probe for use in any one of polar and ONOO - Microenvironment-sensitive fluorescent imaging or content detection.

[0038] Preferably, the polar and ONOO - The microenvironment-sensitive fluorescent imaging includes but is not limited to early imaging of non-alcoholic fatty liver.

[0039] The main mechanism and advantages of the present application are:

[0040] (1) In the preparation method of the present application, first, the methylene in the malonylurea structure of 1,3-biscyclohexylbarbituric acid is deprotonated to form a carbon negative ion, which undergoes addition-elimination with acetic anhydride to obtain acetylated 1,3-biscyclohexylbarbituric acid. The newly introduced acetyl group undergoes enol tautomerism, and the oxygen negative ion together with the carbonyl oxygen on the original structure attacks the boron atom in boron trifluoride ether, and the ethoxy group is removed to finally obtain the intermediate product in the structure; in the second step reaction, the methyl group is deprotonated under the catalysis of piperidine to produce a carbon negative ion which attacks the aldehyde carbon in 7-(diethylamino) coumarin-3-formaldehyde, a fluorescent dye precursor, to undergo aldol condensation and dehydration to obtain the product LD-YB; the hafnium tetrachloride and 2-hydroxyterephthalic acid are added to the mixed solution of DMF / formic acid, heated, filtered, washed, and dried to obtain UiO; LD-YB is added to the DMF solution of UiO, and after reaction and centrifugation, the polar and ONOO - Dual-responsive fluorescent probe Uio-YB.

[0041] (2) The present application prepares a fluorescent probe with dual response function. The fluorescent probe is a fluorescent dye loaded on a metal-organic framework. The present application avoids the problems of low specificity, low sensitivity and poor hydrophilicity of traditional single molecules in dual response. The Uio-YB prepared by the method has excellent hydrophilicity and polar and ONOO - Dual response capability, in polar and ONOO - Microenvironment-sensitive response fluorescent imaging field has great application prospect.

[0042] The present application will be further described in detail through specific embodiments.

[0043] Example 1

[0044] The specific steps for preparing Uio-YB in this embodiment are as follows:

[0045] (1) Take 1.0 g of 1,3-biscyclohexylbarbituric acid into 10 mL of acetic anhydride, then add 520 μL of boron trifluoride ether dropwise, after 30 min of reaction at 95℃, the solution is uniform and light yellow, evaporate the solvent by rotary evaporation, add methanol to filter the white solid, to obtain the LD-YB intermediate a.

[0046] (2) Add 0.21 g of 7-(diethylamino)coumarin-3-formaldehyde, 0.30 g of LD-YB intermediate a and 0.1 mL of piperidine into 10 mL of acetonitrile, heat to reflux at 60℃ for 4 h, cool to room temperature, extract and filter, add 5 mL of dichloromethane and 15 mL of anhydrous ethanol to recrystallize, to obtain the fluorescent dye LD-YB.

[0047] (3) Add 100 mg of hafnium tetrachloride (0.3 mmol) and 65 mg of 2-hydroxyterephthalic acid (0.36 mmol) into 10 mL of a mixed solution of DMF / formic acid (9:1), heat in a Teflon-lined autoclave at 130℃ for 48 h, cool to room temperature, centrifuge at 10000 rpm for 25 min, wash the white precipitate with acetone repeatedly, dry to obtain UiO.

[0048] (4) Take 54 mg of UiO and 5.4 mg of LD-YB, dissolve in 10 mL of DMF, stir at 600 rpm for 24 h, centrifuge at 10000 rpm for 25 min after the reaction is completed, dry at 80℃ overnight to obtain Uio-YB sample 1.

[0049] The particle size of the UiO and Uio-YB prepared in the above example 1 was tested, wherein the average particle size of the UiO was in the range of 80-95 nm, and the average particle size of the Uio-YB was in the range of 85-100 nm. Specifically, the particle size distribution of the UiO and Uio-YB prepared in example 1 was counted, and the results are shown in Figure 1 , and the morphology of Uio-YB was characterized, and the results are shown in Figure 2 .

[0050] At the same time, the excitation and emission spectra of the UiO and Uio-YB were also characterized, and the results are shown in Figure 3 , Figure 4 , it can be seen that the emission wavelength of the UiO is near 440 nm, and the emission wavelength of the LD-YB is near 600 nm.

[0051] Subsequently, the polarity and ONOO -Test of fluorescence response. Uio-YB was added to a group of solutions with different polarities obtained by mixing high-polarity water and low-polarity solvent 1,4-dioxane in different proportions, and the fluorescence intensity of Uio-YB in different polarity solutions was tested by a fluorescence spectrometer, and the results are shown in Figure 5 Fig. 1. It is shown that Uio-YB can exhibit good polarity response characteristics, and the fluorescence intensity is significantly enhanced with the increase of polarity.

[0052] Subsequently, Uio-YB was added to solutions containing different concentrations of ONOO - solution, and the fluorescence intensity of Uio-YB in the solution containing different concentrations of ONOO - solution was tested by a fluorescence spectrometer, and the results are shown in Figure 6 Fig. 2. It is shown that the response fluorescence intensity of Uio-YB increases with the increase of ONOO - content, and the ONOO - content and the fluorescence intensity maintain a good linear relationship within a certain range, and the detection limit is as low as 51.7 nM, showing high sensitivity.

[0053] Finally, the influence of Uio on the hydrophilicity of non-water-soluble dye LD-YB was verified, and the results are shown in Figure 7 Fig. 3. The hydrophilicity of the material was characterized by a contact angle measuring instrument, 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 slightly decreased after loading LD-YB, because the UiO skeleton has good hydrophilicity, which significantly improves the hydrophilicity of LD-YB. Therefore, the above results prove that the fluorescence probe with the above-mentioned polarity and ONOO - double response exhibits good hydrophilicity and the ability of polarity and ONOO - response, and can be applied to specific detection sites such as fatty liver and polarity and ONOO - .

[0054] Example 2

[0055] The 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 other steps and conditions are the same as those in Example 1.

[0056] It is found that with the increase of centrifugal speed, the particle size of the obtained UiO is more uniform and stable, therefore, the centrifugal speed of UiO in the present application is preferably 9000-11000 rpm, and more preferably 10000 rpm.

[0057] Example 3

[0058] The difference from Example 1 is only that the mass ratio of LD-YB to UiO in step (4) is 0.5:10, 3:10 and 5:10 respectively, and other steps and conditions are the same as those in Example 1.

[0059] It is found that the fluorescence intensity increases with the increase of the amount of LD-YB, but too much fluorescence intensity increase is not significant and causes reagent loss, wherein the fluorescence intensity basically reaches the highest when the mass ratio of 1:10 is used in Example 1, therefore, the mass ratio of LD-YB to UiO is preferably (1-3):10, and more preferably 1:10.

[0060] Comparative Example 1

[0061] The difference from Example 1 is only that the fluorescent dye LD-YB is directly used as the contrast probe 1, and the specific preparation steps and conditions are the same as those in Example 1.

[0062] It is found that only LD-YB is used as a fluorescent probe, only a single polarity response can be played; with the decrease of the polarity of the test environment, the fluorescence is enhanced, but if it is used for in vivo test or imaging, fluorescence changes will also occur at other sites with polarity changes in vivo, and it does not have selectivity; while Uio-YB has ONOO - fluorescence response and polarity response, and has high specificity. In addition, LD-YB is a fat-soluble fluorescent probe, which is actually limited in use.

[0063] Comparative Example 2

[0064] The difference from Example 1 is only that UiO is used as the contrast probe 2, and the specific preparation steps and conditions are the same as those in Example 1.

[0065] It is found that only UiO is used as a ligand, only a single ONOO - fluorescence response can be played; with the increase of the ONOO - concentration of the test environment, the fluorescence response of UiO is enhanced, however, the emission light of Uio is about 450 nm, which does not have good tissue penetration ability, and therefore is greatly limited in deep tissue imaging; while Uio-YB has dual response functions of ONOO - fluorescence response concentration and polarity environment site response, in addition to the 450 nm emission light of Uio, it also has an emission light of about 600 nm, which has strong tissue penetration ability, and has better imaging effect in actual use.

[0066] Different from the prior art, the present application provides a high-sensitivity polarity and ONOO- dual-response fluorescent probe and a preparation method, which loads a polarity response fluorescent dye on an ONOO -The dual-responsive probe Uio-YB is prepared in response to the metal organic framework, avoiding the inaccurate detection results caused by using a single compound to respond to two markers in conventional preparation; the Uio-YB prepared by the method improves the detection accuracy, improves the poor hydrophilicity problem of the fluorescent dye, and is conducive to subsequent biological level application; meanwhile, the particle size of the obtained product is uniform and stable.

[0067] The specific embodiments of the application described above do not constitute a limitation on the protection scope of the application. Any various other corresponding changes and modifications made according to the technical concept of the application should be included in the protection scope of the claims of the application.

Claims

1. A highly sensitive polarity and ONOO - A method for preparing a dual-response fluorescent probe, characterized in that... Includes the following steps: (1) 1,3-Bicyclohexylbarbituric acid was added to acetic anhydride, and then boron trifluoride diethyl ether was added to react and the intermediate product was obtained. (2) 7-(diethylamino)coumarin-3-carboxaldehyde, intermediate product and piperidine were added to the first organic solvent, and after heating and reflux, extraction, filtration and recrystallization, the fluorescent dye LD-YB was obtained. (3) Preparation of metal-organic framework material UiO using hafnium tetrachloride and organic ligands; (4) Fluorescent dye LD-YB and metal-organic framework material UiO are added to a second organic solvent, and polar and ONOO are prepared by reaction. - Dual-response fluorescent probe; In step (1), the ratio of 1,3-dicyclohexylbarbituric acid, acetic anhydride and boron trifluoride diethyl ether is 1 g: (8-12) mL: (500-550) μL; In step (2), the mass ratio of 7-(diethylamino)coumarin-3-carboxaldehyde to the intermediate product is (0.5-1):1, and the ratio between the intermediate product and 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; In step (4), the mass ratio of the fluorescent dye LD-YB to the metal-organic framework material UiO is (1-3):10; The reaction in step (4) is carried out by stirring at 500–800 rpm for 16–24 h; after the reaction is completed, the product is separated by centrifugation at 9000–11000 rpm for 15–25 min to obtain polar and ONOO. - Dual-response fluorescent probe; The second organic solvent includes one or more of DMF and DMSO.

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

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

4. The highly sensitive polarity and ONOO as described in claim 1 - A method for preparing a dual-response fluorescent probe, characterized in that, In step (3), the preparation steps of the metal-organic framework material UiO include: adding hafnium tetrachloride and organic ligands to a third organic solvent for heating reaction, and then obtaining UiO through post-treatment.

5. The highly sensitive polarity and ONOO as described in claim 4 - A method for preparing a dual-response fluorescent probe, characterized in that, The organic ligand comprises 2-hydroxyterephthalic acid or 2,5-dihydroxyterephthalic acid; the molar ratio of 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 was carried out at 130–150 °C for 36–48 h; The post-processing includes centrifugation, washing, and drying, wherein the centrifugation speed is 8000-10000 rpm and the time is 15-25 min.

6. The highly sensitive polar and ONOO prepared by any one of the preparation methods of claims 1-5 - Dual-response fluorescent probe.

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