Rhodamine-based pH fluorescent probe as well as preparation method and application thereof

Abstract

The invention discloses a rhodamine-based pH fluorescent probe as well as a preparation method and application thereof. According to the invention, a trans-1, 4-cyclohexanediamine group is introduced on a rhodamine 6G molecule through an amine ester condensation reaction, so that regulation and control on the acid response performance of the rhodamine-based pH fluorescent probe are realized. Researches show that pKa of the probe is greatly improved through modification of trans-1, 4-cyclohexanediamine, detection of the probe on a weak acid solution (pH is 5-7) is achieved, the acid response speed is increased, and the weak acid solution can be rapidly detected through color and fluorescence double channels. On the basis, coupling probe degreasing cotton is prepared through oxidation of carriers such as degreasing cotton and an aldehyde amine condensation reaction, and the coupling probe degreasing cotton can be used for monitoring and imaging gastric juice leakage in an operation to prevent occurrence of postoperative gastric fistula.

Description

Technical Field

[0001] The invention relates to the field of biological analysis and detection, and in particular to a rhodamine-based pH fluorescent probe and a preparation method and application thereof. Background Art

[0002] Rhodamine is an important class of organic fluorescent dyes with the advantages of high quantum yield, high sensitivity, good stability, and low molecular weight. It has high application value in the biomedical field, such as cell staining, biological imaging, tumor diagnosis and treatment, especially specific fluorescence imaging and detection.

[0003] pH fluorescent probes based on rhodamine dyes have become an important tool in fluorescent bioimaging technology. The most common construction method is to modify rhodamine with organic amines of different structures through amine ester condensation reaction. However, after modification with organic amines, the pK value of the probe is a For example, after R6G is modified with 2-hydroxyethylamine, the pK a Only 2.8, pK after modification of 4-hydroxyaniline a is 3.2, and the pK after modification with 2-hydroxyaniline a is 3.6 (Org. Biomol. Chem., 2011, 9, 1723-1726). Patent CN113072559A reports that after modification with organic amines, the pK a In the range of 3.51 to 4.68, patent CN108997363B modified a complex ethylenediamine derivative on rhodamine, and the pK a Still only 4.32. Based on the lower pK a , these probes only exhibit fluorescence under strongly acidic conditions, which greatly hinders their application in the biomedical field.

[0004] In addition, although the prior art also includes pK a Improved rhodamine probes, such as patent document CN112574227A discloses that the pK of rhodamine derivatives can be regulated by introducing spirolactone to connect morpholine structure. a , pH detection range is 7-10. Other researchers introduced benzimidazole groups into the rhodamine molecule to increase its pK a The pH value was increased to between 6 and 7, and the new pH fluorescent probe thus constructed can be used for pH monitoring in mitochondria and lysosomes of living cells (New J. Chem., 2018, 42, 11102-11108). However, the synthesis of these probes is complex, and dangerous reducing agents such as catalysts and lithium aluminum hydride are required, and high-purity probes need to be obtained through post-processing of complex processes such as column chromatography, which limits the production and application of the probes. In addition, these probes no longer have highly active modification sites, and their application range is greatly limited. SUMMARY OF THE INVENTION

[0005] In order to overcome the deficiencies of the prior art, the present invention provides a rhodamine-based pH fluorescent probe with a wide acid response range, high detection sensitivity, and a simple preparation process, as well as a preparation method and application thereof. By improving the amine-ester condensation reaction process of rhodamine, the trans-1,4-cyclohexanediamine (CDA) group is efficiently modified on rhodamine, significantly increasing the pK a and acid response rate of the probe, and realizing the detection of weakly acidic solutions by the probe. In addition, the modified probe still has a primary amine group as a site for further modification. Based on this modification site, the probe is further coupled with a carrier to realize the imaging of gastric juice leakage through fluorescence and apparent color, and can be used to prevent the occurrence of postoperative gastric fistula.

[0006] In the first aspect of the present invention, a rhodamine-based pH fluorescent probe is provided. The rhodamine-based pH fluorescent probe (R6G-CDA) is obtained by modifying the trans-1,4-cyclohexanediamine (CDA) group on a rhodamine 6G (R6G) molecule, and its structural formula is

[0007] In the second aspect of the present invention, a preparation method of a rhodamine-based pH fluorescent probe is provided. The method includes the following steps: 1) Dissolve R6G in a polar organic solvent and add CDA for reaction; 2) Separate and purify the product obtained in step 1) to obtain a rhodamine-based pH fluorescent probe (R6G-CDA).

[0008] Furthermore, in step 1), the polar organic solvent is selected from one or more of N,N-dimethylformamide (DMF), methanol, ethanol, isopropanol, and N,N-dimethylacetamide (DMAC), and preferably DMF.

[0009] The inventors found through experiments that when preparing R6G-CDA in a polar organic solvent, the reaction efficiency is greatly improved. This is mainly because there are a large number of polar groups such as carbonyl and amino groups on the reactant molecules of R6G and CDA. Based on the principle of solvation, the solvent molecules of the polar solvent can generate multiple interactions such as hydrogen bonds and electrostatics with these groups, making the reactant molecules better dispersed in the solvent system, thus improving the reaction efficiency. If the reaction is carried out in a low-polar organic solvent such as dichloromethane (DCM), although the reaction can also proceed, due to the large polarity difference between the two, the dispersibility decreases, resulting in a significant decrease in the reaction rate, indicating that polar solvents such as DMF are more suitable for this reaction.

[0010] Furthermore, the mass ratio of R6G to CDA is 0.2-4:1 (such as 0.2:1, 0.25:1, 0.5:1, 0.75:1, 1:1, 2:1, 3:1, 4:1), and preferably 0.2-2:1.

[0011] Further, the temperature of the reaction is room temperature.

[0012] Further, the reaction time is 0.5 - 4 hours (such as 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4 hours), preferably 1 - 3 hours.

[0013] Further, the reaction is carried out under stirring.

[0014] In a specific embodiment of the present invention, step 1) includes: dissolving R6G in DMF, adding CDA, and stirring and reacting at room temperature.

[0015] Further, the separation and purification in step 2) includes the following steps: subjecting the product obtained in step 1) to extraction, rotary evaporation and recrystallization, and solid-liquid separation in sequence.

[0016] Further, the extraction includes the following steps: adding DCM and water to the reaction system obtained, placing it in a separating funnel and mixing well, and collecting the DCM phase after standing and separating layers.

[0017] Further, the volume ratio of the DCM to water is 1:1 - 5 (such as 1:1, 1:2, 1:3, 1:4, 1:5), preferably 1:2.

[0018] Further, the extraction can be carried out once or multiple times, preferably 3 times.

[0019] Further, the extraction also includes the following steps: adding a desiccant to the collected DCM phase to obtain an anhydrous DCM solution.

[0020] Further, the desiccant is selected from one or more of anhydrous sodium sulfate, anhydrous magnesium sulfate, and molecular sieve, preferably anhydrous sodium sulfate.

[0021] Further, the drying time is 5 - 30 min (such as 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 min), preferably 10 min.

[0022] Further, the rotary evaporation and recrystallization includes the following steps: adding a non-polar organic solvent to the DCM solution, and obtaining a solid-liquid mixture through rotary evaporation and recrystallization.

[0023] Further, the non-polar organic solvent is selected from one or more of petroleum ether, n-pentane, isopentane, n-hexane, n-heptane, cyclohexane, and toluene, preferably n-hexane.

[0024] Further, the volume ratio of the DCM solution to the nonpolar organic solvent is 0.5 - 2:1 (such as 0.5:1, 1:1, 1.5:1, 2:1), preferably 1:1.

[0025] Further, the sign for stopping rotary evaporation and recrystallization is that there are 20 - 80 mL of solid-liquid mixture remaining, preferably 50 mL remaining.

[0026] Further, the solid-liquid separation includes the following steps: centrifuging and / or filtering the solid-liquid mixture to obtain solid powder.

[0027] Further, the separation and purification also include the following steps: washing and drying the solid powder with a nonpolar organic solvent.

[0028] Further, the nonpolar organic solvent is one or more of petroleum ether, n-pentane, isopentane, n-hexane, n-heptane, cyclohexane, toluene, preferably n-hexane.

[0029] Further, the washing can be carried out once or multiple times, preferably 2 times.

[0030] Further, the drying method is vacuum drying.

[0031] After the reaction of the present invention is completed, by first using water and DCM for extraction to remove the organic solvent with relatively high polarity and the reaction by-products with relatively high polarity, and then removing other low-polarity impurities by rotary evaporation and recrystallization, a product with high purity is obtained, without the need to adopt complex processes such as column chromatography, greatly simplifying the post-treatment process and reducing the reaction cost.

[0032] In some embodiments of the present invention, the preparation method of the rhodamine-based pH fluorescent probe includes the following steps: (1) Dissolve R6G in DMF, add CDA, and stir and react at room temperature to obtain a reaction solution; (2) Mix the reaction solution with DCM and water for extraction, collect the DCM phase, and add a drying agent (such as anhydrous sodium sulfate) to the DCM phase; (3) Add a nonpolar organic solvent (such as n-hexane, n-heptane) to the dried DCM phase, carry out rotary evaporation and recrystallization to obtain a solid-liquid mixture; (4) Centrifuge the solid-liquid mixture to obtain solid powder; (5) Wash the solid powder with a nonpolar organic solvent (such as n-hexane, n-heptane), and vacuum dry at room temperature to obtain pure R6G-CDA.

[0033] The R6G-CDA prepared by the present invention still has a primary amine group as a reaction site and can be further modified, thus having a wide range of applications. For example, by Schiff base reaction, amidation reaction, etc., R6G-CDA can be modified onto substrates such as cellulose, hyaluronic acid, chitosan, alginate, starch, gelatin, and sponges, hydrogels, swabs, papers, absorbent cotton, etc. composed of them or with them as the main components to prepare acid-responsive fluorescence detection materials; or R6G-CDA can be covalently coupled with drug molecules to monitor the blood circulation cycle and metabolic pathway of drugs; or R6G-CDA can be coupled with specific targeting groups for fluorescence imaging at the tissue, cell, and subcellular levels.

[0034] As an example of the application, in the present invention, oxidized absorbent cotton is covalently linked with R6G-CDA through Schiff base reaction to obtain absorbent cotton conjugated with R6G-CDA.

[0035] In the third aspect of the present invention, there is provided a carrier conjugated with R6G-CDA, which is obtained by oxidizing the carrier and then conjugating it with the R6G-CDA of the first aspect or the R6G-CDA prepared by the preparation method of the second aspect.

[0036] Furthermore, the carrier is selected from: cellulose, hyaluronic acid, chitosan, alginate, starch, gelatin, and sponges, hydrogels, swabs, papers, absorbent cotton, etc. composed of them or with them as the main components.

[0037] In the fourth aspect of the present invention, there is provided a preparation method of the carrier conjugated with R6G-CDA described in the third aspect, and the preparation method includes the following steps:

[0038] (1) Carrier oxidation: Mix the carrier, an oxidant, and water, add an acidic reagent to adjust the pH of the solution to obtain an oxidized carrier;

[0039] (2) Coupling of R6G-CDA with the carrier: Dissolve the R6G-CDA of the first aspect or obtained by the preparation method of the second aspect in an organic solvent, add the oxidized carrier described in step (1) for reaction to obtain a carrier conjugated with R6G-CDA.

[0040] Furthermore, in step (1), the oxidant is selected from one or more of sodium periodate, hydrogen peroxide, chromium trioxide, potassium permanganate, 2-iodoxybenzoic acid (IBX), phenyliodine diacetate (PIDA), iodobenzene (PhIO), sodium chlorite, and preferably sodium periodate.

[0041] Further, in step (1), the mass ratio of the carrier to the oxidant is 1:3 - 5 (such as 1:1, 1:1.2, 1:1.3, 1:1.4, 1:1.5, 1:1.6, 1:1.7, 1:1.8, 1:1.9, 1:2, 1:2.1, 1:2.2, 1:2.3, 1:2.4, 1:2.5, 1:2.6, 1:2.7, 1:2.8, 1:2.9, 1:3, 1:3.1, 1:3.2, 1:3.3, 1:3.4, 1:3.5, 1:3.6, 1:3.7, 1:3.8, 1:3.9, 1:4, 1:4.1, 1:4.2, 1:4.3, 1:4.4, 1:4.5, 1:4.6, 1:4.7, 1:4.8, 1:4.9, 1:5), preferably 1:3 - 4, and more preferably 1:3.3.

[0042] Further, in step (1), the acidic reagent is sulfuric acid, hydrochloric acid, nitric acid, phosphoric acid, perchloric acid, acetic acid, trifluoromethanesulfonic acid or their aqueous solutions, preferably a sulfuric acid solution, and more preferably a 10 - 30% sulfuric acid solution (such as 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30%), and even more preferably a 20% sulfuric acid solution.

[0043] Further, in step (1), adding the acidic reagent to adjust the pH of the solution to 2.0 - 6.0 (such as 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0), preferably 2.5 - 3.5, and more preferably 3.0.

[0044] Further, in step (1), the temperature of the reaction is room temperature.

[0045] Further, in step (1), the reaction time is 6 - 24 hours (such as 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 hours).

[0046] Further, in step (1), the reaction is carried out under stirring.

[0047] Further, in step (2), the organic solvent is selected from one or more of DMF, DMAC, N-methylpyrrolidone (NMP), dimethyl sulfoxide (DMSO), N,N-diethylformamide (DEF), tetrahydrofuran (THF), and cyclohexanone, preferably DMF.

[0048] Further, in step (2), the mass ratio of R6G-CDA to the carrier is 1:5 - 20 (such as 1:5, 1:5.5, 1:6, 1:6.5, 1:7, 1:7.5, 1:8, 1:8.5, 1:9, 1:9.5, 1:10, 1:11.5, 1:12, 1:12.5, 1:13, 1:13.5, 1:14, 1:14.5, 1:15, 1:15.5, 1:16, 1:16.5, 1:17, 1:17.5, 1:18, 1:18.5, 1:19, 1:19.5, 1:20).

[0049] Further, in step (2), the reaction temperature is 30 - 60 °C (such as 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60 °C), preferably 35 - 45 °C.

[0050] Further, in step (2), the reaction time is 1 - 10 hours (such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 hours), preferably 3 - 5 hours.

[0051] Further, in step (2), the reaction is carried out under shaking.

[0052] Preferably, in step (1): The carrier, sodium periodate, and water are mixed, and the pH of the solution is adjusted to 2.5 - 3.5 with sulfuric acid solution, and the reaction is stirred at room temperature to obtain an oxidized carrier.

[0053] Preferably, in step (2): R6G-CDA is dissolved in DMF, and the oxidized carrier obtained in step (1) is added, and the reaction is carried out under shaking at 35 - 45 °C to obtain a carrier conjugated with R6G-CDA.

[0054] Further, the steps of washing and drying are also included in step (1).

[0055] Further, the steps of washing and drying are also included in step (2).

[0056] In some embodiments of the present invention, the method for preparing the degreased cotton conjugated with R6G-CDA includes the following steps:

[0057] (1) Mix absorbent cotton, sodium periodate and water, adjust the pH of the solution to 2.5 - 3.5 with sulfuric acid solution, stir and react at room temperature for 12 hours. After the reaction, wash the absorbent cotton with water and dry it at 30 - 40 °C to obtain oxidized absorbent cotton;

[0058] (2) Dissolve R6G-CDA in DMF, add the oxidized absorbent cotton described in step (1), and oscillate and react at 35 - 45 °C for 4 hours. After the reaction, wash the oxidized absorbent cotton with DMF and absolute ethanol in sequence, and dry it under vacuum to obtain absorbent cotton coupled with R6G-CDA.

[0059] In the fifth aspect of the present invention, there is provided an application of R6G-CDA of the first aspect or R6G-CDA prepared by the preparation method of the second aspect as a pH fluorescent probe.

[0060] Furthermore, the pH fluorescent probe is used for detecting the pH of different systems.

[0061] Furthermore, the system is selected from: aqueous system, organic system or biological system, such as gastric juice, ribozyme, lysosome, etc.

[0062] Furthermore, the pH range detected by the pH fluorescent probe is pH ≤ 7.0.

[0063] Furthermore, the detection includes: contacting the R6G-CDA with the sample to be detected (for example, dissolving R6G-CDA in a solvent (such as DMSO) and adding it to the sample to be detected), observing the color change, and / or fluorescence under 365 nm ultraviolet excitation.

[0064] It is known in the art that the opening and closing ring conversion of rhodamine derivatives with the structure shown in formula (1) depends on acidity and alkalinity: when the acidity is strong enough, the lactam ring opens, and the xanthene part of rhodamine forms an electron push-pull structure, thereby emitting yellow or green fluorescence, and at the same time the apparent color presents red.

[0065] In the present invention, by selecting CDA and modifying CDA on the R6G molecule, effective regulation of the acid response performance of the probe can be achieved. On the one hand, due to the large six-membered ring structure of CDA, when it is introduced into the rhodamine structure as the R group, the lactam ring closure of rhodamine is hindered by the steric hindrance effect of its ring structure, so that the pK of the probe a is significantly improved. Compared with organic amines such as amantadine commonly used in the prior art, the steric hindrance effect of CDA can effectively delay the lactam ring closure of rhodamine without hindering the amine-ester condensation reaction of rhodamine. Therefore, the reaction conditions are mild, R6G-CDA can be synthesized at room temperature, the reaction efficiency is high, and the post-treatment process is simple; while the ring structure of amantadine has a large steric hindrance, although it is beneficial to further improve the pK of the rhodamine pH fluorescent probe a, but the excessive steric hindrance hinders the aminolysis reaction of rhodamine, making the reaction conditions harsh. Auxiliaries such as catalysts and reducing agents need to be added, and the post-treatment process is complex.

[0066] On the other hand, the large steric hindrance, rigidity, and trans configuration characteristics of CDA change the planarity of the rhodamine molecule, inhibit the π-π stacking effect of rhodamine in aqueous solution, and make the probe molecules in a relatively dispersed state, which kinetically promotes the ring-opening reaction and thus speeds up the response rate.

[0067]

[0068] In some embodiments of the present invention, the pH fluorescent probe can be used for detecting gastric juice leakage during surgery to help prevent the occurrence of postoperative gastric fistula.

[0069] In the sixth aspect of the present invention, there is provided an application of the carrier conjugated with R6G-CDA as described in the third aspect or the carrier conjugated with R6G-CDA prepared by the preparation method as described in the fourth aspect in the preparation of products for detecting the pH of different systems.

[0070] Further, the product is a medical device with a visual detection function.

[0071] Further, the medical device is a kit or a dressing.

[0072] Further, the system is selected from: an aqueous system, an organic system, or a biological system, such as gastric juice, ribozyme, lysosome, etc.

[0073] Further, the pH range to be detected is pH ≤ 7.0.

[0074] Further, the detection includes: contacting the carrier conjugated with R6G-CDA with the sample to be tested (for example, dropping the sample to be tested onto the carrier, or dipping the carrier into the sample to be tested), observing the color change of the carrier, and / or the fluorescence under 365 nm ultraviolet excitation.

[0075] Further, the application is an application of the carrier conjugated with R6G-CDA in the preparation of products for detecting gastric juice leakage during surgery and preventing the occurrence of postoperative gastric fistula.

[0076] In some embodiments of the present invention, the product can be used for detecting gastric juice leakage during surgery and preventing the occurrence of postoperative gastric fistula.

[0077] Compared with the prior art, the present invention has the following advantages:

[0078] (1) By selecting CDA as the modifying molecule of R6G, the acid-responsive performance of the probe can be effectively regulated. On the one hand, due to the large six-membered ring structure of CDA, after introducing it as the R group into the rhodamine structure, the ring closure of rhodamine is hindered by the steric hindrance effect of the ring structure. Therefore, the pK a of the probe is significantly increased. Moreover, the steric hindrance of CDA can effectively delay the ring closure of rhodamine without hindering the amine-ester condensation reaction of rhodamine. Therefore, the reaction conditions are mild, and R6G-CDA can be synthesized at room temperature with high reaction efficiency. On the other hand, the large steric hindrance, rigidity, and trans configuration characteristics of CDA change the planarity of the rhodamine molecule, inhibit the π-π stacking effect of rhodamine in aqueous solution, and make the probe molecules in a relatively dispersed state, which kinetically promotes the ring-opening reaction and thus accelerates the response speed.

[0079] (2) The present invention improves the process for preparing rhodamine-based pH fluorescent probes through amine-ester condensation reaction: First, react at room temperature in a polar organic solvent to complete the coupling of rhodamine and an amine (such as CDA); Subsequently, remove the polar solvent and higher-polarity by-products through extraction; Finally, remove the lower-polarity impurities through rotary evaporation, recrystallization, and centrifugation to obtain the target product with high purity. Compared with the existing technology for preparing rhodamine derivatives through amine-ester condensation reaction, which usually requires complicated purification processes such as column chromatography and high-performance liquid chromatography separation, the preparation process of the present invention is milder and the process is simplified, greatly reducing the preparation cost.

[0080] (3) The present invention modifies CDA on the R6G molecule through the above preparation process, realizing the regulation of the acid-responsive performance of the probe. And through comparative experiments, it is found that compared with the R6G-HDA control probe modified with 1,6-hexanediamine (HDA), R6G-CDA significantly increases the pK a of the probe and realizes the detection of weakly acidic solutions (pH 5-7).

[0081] (4) The R6G-CDA of the present invention can rapidly respond to weakly acidic solutions through dual channels of apparent color and fluorescence, while R6G-HDA can only respond to strongly acidic solutions; The inventors also found that compared with the introduction of HDA, after introducing CDA, the acid response speed of the probe is increased by about 17 times, indicating a significant improvement in the detection sensitivity of the probe.

[0082] (5) The R6G-CDA of the present invention still has a primary amine group as a modification site. Based on this modification site, R6G-CDA is further subjected to an aldehyde-amine condensation reaction with carriers such as oxidized absorbent cotton to obtain absorbent cotton coupled with R6G-CDA, which immediately turns red and exhibits bright yellow fluorescence after absorbing acidic gastric juice, and can be used to detect intraoperative gastric juice leakage and prevent the occurrence of postoperative gastric fistula. Brief Description of the Drawings

[0083] Figure 1 Shown is the 1 1H-NMR spectrum of the fluorescent probe R6G-CDA of Example 1.

[0084] Figure 2 Shown is the 1 1H-NMR spectrum of the fluorescent probe R6G-HDA of Comparative Example 1.

[0085] Figure 3 Shown are the apparent photos of the fluorescent probe R6G-CDA of Example 1 in phosphate buffered saline (PBS) at different pH values and the fluorescence photos under 365 nm ultraviolet light excitation (pH values from left to right are 5.0, 5.8, 6.5, 7.0, 7.5, 8.0).

[0086] Figure 4 Shown are the apparent photos of the fluorescent probe R6G-HDA of Comparative Example 1 in PBS at different pH values and the fluorescence photos under 365 nm ultraviolet light excitation (pH values from left to right are 5.0, 5.8, 6.5, 7.0, 7.5, 8.0).

[0087] Figure 5 Shown is the fluorescence spectrum of the fluorescent probe R6G-CDA of Example 1 in PBS at different pH values (excitation wavelength: 530 nm).

[0088] Figure 6 Shown is the fluorescence spectrum of the fluorescent probe R6G-HDA of Comparative Example 1 in PBS at different pH values (excitation wavelength: 530 nm).

[0089] Figure 7 Shown is the response time-fluorescence intensity relationship curve of the fluorescent probe R6G-CDA of Example 1 at pH 5.63 and the fluorescent probe R6G-HDA of Comparative Example 1 at pH 3.49 (excitation: 530 nm, reception: 555 nm).

[0090] Figure 8 Shown is the appearance photo of the conjugated probe absorbent cotton of Example 5.

[0091] Figure 9 Shown are the apparent photos of the conjugated probe absorbent cotton of Example 5 after absorbing PBS at different pH values (pH values from left to right are 6.1, 6.4, 7.2, 8.0).

[0092] Figure 10 Shown are the fluorescence photos of the conjugated probe absorbent cotton of Example 5 after absorbing PBS at different pH values (excited by 365 nm ultraviolet light, pH values from left to right are 6.1, 6.4, 7.2, 8.0).

[0093] Figure 11 The coupling probe of Example 5 is shown as degreased cotton for imaging of gastric juice leakage simulation. Detailed implementation mode

[0094] Unless otherwise defined, all scientific and technical terms used in the present invention have the same meaning as commonly understood by those skilled in the technical field to which the present invention pertains.

[0095] The disclosures of various publications, patents, and published patent specifications cited herein are incorporated herein by reference in their entirety.

[0096] The term "polar organic solvent" refers to an organic solvent with a relative dielectric constant greater than 10.

[0097] The term "non-polar organic solvent" refers to an organic solvent with a relative dielectric constant less than 3.

[0098] The term "room temperature" means that the temperature of an article is close to or the same as the temperature of the space (such as the place of the fume hood where the article is located). Generally, room temperature is about 20 °C to about 30 °C, or about 22 °C to 27 °C, or about 25 °C.

[0099] Next, in combination with the embodiments of the present invention, the technical solutions of the present invention will be clearly and completely described. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without making creative efforts belong to the scope of protection of the present invention.

[0100] Example 1: Synthesis of R6G-CDA by CDA modification of R6G

[0101] Weigh 100 mg of R6G and add it to a reaction flask. Add 4 mL of DMF and stir for 5 min to fully dissolve R6G. Weigh 200 mg of CDA and add it to the reaction flask. Stir at room temperature for 2 h. During this period, the reaction solution gradually changes from deep purple-red to orange-red. After the reaction is completed, pour the reaction solution into a separatory funnel, add 100 mL of DCM and mix well. Then add 200 mL of purified water for extraction, and repeat the extraction 3 times by replacing the aqueous phase. Take the DCM phase, add an excessive amount of anhydrous sodium sulfate and stir for 10 min. Then perform rotary evaporation and recrystallization: Pour the supernatant into a rotary evaporation flask, add 100 mL of n-hexane, rotary evaporate and concentrate until 50 mL of solution remains and a large amount of solid precipitates. Centrifuge the solid-liquid mixture to obtain solid powder. Then wash the solid powder with n-hexane 2 times, and then perform vacuum drying treatment at room temperature to obtain the probe R6G-CDA. The reaction route is shown in formula (2). Through 1 1H NMR characterization confirmed the structure of the fluorescent probe R6G-CDA ( Figure 1)。

[0102]

[0103] The NMR characterization data of the product are as follows: 1 1H NMR (400 MHz, DMSO-d6): δ (ppm): 7.70 (m, 1H), 7.45 (m, 2H), 6.89 (m, 1H), 6.24 (s, 2H), 6.09 (s, 2H), 5.03 (s, 2H), 3.42 (q, 2H), 3.11 (m, 4H), 2.70 (m, 1H), 2.32 (m, 1H), 2.06 (q, 2H), 1.85 (s, 6H), 1.53 (d, 2H), 1.21 (s, 6H), 1.04 (m, 4H). The NMR spectrum and NMR data are consistent with the theoretical data of R6G-CDA, and it can be determined that the synthesized compound is R6G-CDA. From the few impurity peaks in the spectrum, it can be judged that the high-purity product can be obtained by the relatively simple post-treatment process in the present invention.

[0104] Example 2: Synthesis of R6G-CDA by modifying R6G with CDA

[0105] Weigh 100 mg of R6G and add it to a reaction flask. Add 4 mL of DMF and stir for 5 min to fully dissolve R6G. Weigh 100 mg of CDA and add it to the reaction flask, and stir at room temperature for 4 h. During this period, the reaction solution gradually changes from dark purple-red to orange-red. After the reaction is completed, pour the reaction solution into a separatory funnel, add 100 mL of DCM and mix well. Then add 200 mL of purified water for extraction, and repeat the extraction 3 times by changing the aqueous phase. Take the DCM phase, add an excessive amount of anhydrous sodium sulfate and stir for 10 min. Then carry out rotary evaporation and recrystallization: Pour the supernatant into a rotary evaporation flask, add 100 mL of n-hexane, rotary evaporate and concentrate until 50 mL of solution remains and a large amount of solid precipitates. Centrifuge the solid-liquid mixture to obtain solid powder. Then wash the solid powder with n-hexane twice, and then carry out vacuum drying treatment at room temperature to obtain the probe R6G-CDA. The product is 1 characterized by 1H NMR, and the results show that R6G-CDA has been successfully obtained.

[0106] Example 3: Synthesis of R6G-CDA by modifying R6G with CDA

[0107] Weigh 100 mg of R6G and add it to a reaction flask. Add 4 mL of DMF and stir for 5 min to fully dissolve R6G. Weigh 400 mg of CDA and add it to the reaction flask. Stir at room temperature for 1 h. During this period, the reaction solution gradually changes from deep purple-red to orange-red. After the reaction is completed, pour the reaction solution into a separatory funnel, add 100 mL of DCM and mix evenly. Then add 200 mL of purified water for extraction, and repeat the extraction 3 times by changing the aqueous phase. Take the DCM phase, add an excessive amount of anhydrous sodium sulfate and stir for 10 min. Then perform rotary evaporation and recrystallization: Pour the supernatant into a rotary evaporation flask, add 100 mL of n-hexane, rotary evaporate and concentrate until 50 mL of solution remains and a large amount of solid precipitates. Centrifuge the solid-liquid mixture to obtain a solid powder. Then wash the solid powder with n-hexane 2 times, and then perform vacuum drying at room temperature to obtain the probe R6G-CDA. The product is subjected to 1 HNMR characterization, and the results show that R6G-CDA has been successfully obtained.

[0108] Example 4: Synthesis of R6G-CDA by modifying R6G with CDA

[0109] Weigh 100 mg of R6G and add it to a reaction flask. Add 4 mL of DMF and stir for 5 min to fully dissolve R6G. Weigh 200 mg of CDA and add it to the reaction flask. Stir at room temperature for 2 h. During this period, the reaction solution gradually changes from deep purple-red to orange-red. After the reaction is completed, pour the reaction solution into a separatory funnel, add 100 mL of DCM and mix evenly. Then add 200 mL of purified water for extraction, and repeat the extraction 3 times by changing the aqueous phase. Take the DCM phase, add an excessive amount of anhydrous sodium sulfate and stir for 10 min. Then perform rotary evaporation and recrystallization: Pour the supernatant into a rotary evaporation flask, add 100 mL of n-heptane, rotary evaporate and concentrate until 50 mL of solution remains and a large amount of solid precipitates. Centrifuge the solid-liquid mixture to obtain a solid powder. Then wash the solid powder with n-heptane 2 times, and then perform vacuum drying at room temperature to obtain the probe R6G-CDA. The product is subjected to 1 HNMR characterization, and the results show that R6G-CDA has been successfully obtained.

[0110] Example 5: Coupling of the probe with absorbent cotton

[0111] Put 1 g of absorbent cotton into a reaction flask, add 3.3 g of sodium periodate and 150 mL of purified water, and adjust the pH of the solution to 3.0 with 20% concentrated sulfuric acid. Then stir at room temperature for 12 h for the oxidation of absorbent cotton. After the reaction is completed, pour out the reaction solution, and wash the absorbent cotton with excessive purified water 3 times. Finally, put the absorbent cotton into a blast drying oven and dry it at 35 °C to obtain oxidized absorbent cotton.

[0112] Take 50 mg of the R6G-CDA probe obtained in Example 1 and dissolve it in 15 mL of DMF to obtain an R6G-CDA solution. Add 0.5 g of oxidized absorbent cotton to the reaction system, and then add the above R6G-CDA solution. Oscillate at 40 °C for 4 h. After the reaction is completed, take out the absorbent cotton, wash the absorbent cotton 3 times with DMF, and then wash it 3 times with absolute ethanol. After vacuum drying, the absorbent cotton coupled with the R6G-CDA fluorescent probe is obtained.

[0113] Comparative Example 1: Synthesis of R6G-HDA by modifying R6G with HDA

[0114]

[0115] Weigh 100 mg of R6G and add it to a reaction flask. Add 4 mL of DMF and stir for 5 min to fully dissolve R6G. Weigh 200 mg of HDA and add it to the reaction flask, and stir at room temperature for 2 h. During this period, the reaction solution gradually changes from dark purple-red to orange-red. After the reaction is completed, pour the reaction solution into a separatory funnel, add 100 mL of DCM and mix well. Then add 200 mL of purified water for extraction, and repeat the extraction 3 times by changing the aqueous phase. Take the DCM phase, add an excessive amount of anhydrous sodium sulfate and stir for 10 min. Then perform rotary evaporation and recrystallization: Pour the supernatant into a rotary evaporation flask, add 100 mL of n-hexane, rotary evaporate and concentrate until 50 mL of solution remains and a large amount of solid precipitates. Centrifuge the solid-liquid mixture to obtain a solid powder. Then wash the solid powder 2 times with n-hexane, and then perform vacuum drying at room temperature to obtain the probe R6G-HDA. The structure of the fluorescent probe R6G-HDA was confirmed by 1 1H NMR characterization ( Figure 2 ). Judging from the 1 1H NMR spectrum, high-purity R6G-HDA can be obtained through the above purification process.

[0116] The NMR characterization data of the product are as follows: 1 1H NMR (400 MHz, DMSO-d6): δ (ppm): 7.75 (m, 1H), 7.48 (m, 2H), 6.97 (m, 1H), 6.24 (s, 2H), 6.03 (s, 2H), 5.04 (t, 2H), 4.43 (m, 2H), 3.12 (m, 4H), 2.91 (m, 2H), 2.36 (t, 2H), 1.84 (s, 6H), 1.20 (t, 6H), 1.09 (m, 2H), 0.95 (m, 6H).

[0117] Comparative Example 2: Synthesis of R6G-CDA using DCM as a solvent

[0118] Weigh 100 mg of R6G and add it to the reaction flask. Then add 4 ml of DCM and stir for 5 min to fully dissolve R6G. Weigh 200 mg of CDA and add it to the reaction flask, and stir at room temperature for 24 h. During this period, the reaction solution slowly changes from purplish red to orange red. After the reaction is completed, pour the reaction solution into a separatory funnel, add 100 mL of DCM and mix well. Then add 200 mL of purified water for extraction, and repeat the extraction 3 times by changing the aqueous phase. Take the DCM phase, add an excessive amount of anhydrous sodium sulfate and stir for 10 min. Subsequently, perform rotary evaporation and recrystallization: Pour the supernatant into the rotary evaporation flask, add 100 mL of n-hexane, and rotary evaporate and concentrate until 50 mL of solution remains and a large amount of solid precipitates. Centrifuge the solid-liquid mixture to obtain solid powder. Then wash the solid powder with n-hexane for many times, and then perform vacuum drying treatment at room temperature to obtain the probe R6G-CDA. Perform 1 1H NMR characterization on the product, and the results show that R6G-CDA has been successfully obtained.

[0119] Compared with Example 1, when the reaction solvent is changed to DCM, the reaction rate becomes significantly slower and it takes 24 h to complete the reaction. The decrease in the reaction rate in the DCM system is mainly due to the relatively low polarity of the solvent. Due to the insufficient stability of R6G, the extension of the reaction time may increase the reaction by-products. The above results indicate that a solvent system with a relatively large polarity (such as the DMF system) is more suitable for the amine-ester condensation reaction of R6G and CDA.

[0120] Comparative Example 3: Amine-ester condensation reaction of adamantylamine and R6G

[0121] Weigh 100 mg of R6G and add it to the reaction flask. Then add 4 mL of DMF and stir for 5 min to fully dissolve R6G. Weigh 300 mg of adamantylamine and add it to the reaction flask, and stir at room temperature for 24 h. During this period, no obvious color change is observed in the reaction solution. Monitor the reaction by thin-layer chromatography, and no product is found to be generated. This indicates that adamantylamine cannot be coupled with R6G under these conditions, probably because the steric hindrance of adamantylamine is too large, which hinders the progress of the reaction.

[0122] Performance detection:

[0123] Detection Example 1, probe pK a Test

[0124] Prepare a series of phosphate buffer solutions with pH values ranging from 1 to 9. Dissolve the rhodamine-based pH fluorescent probes obtained in Examples 1-4 and Comparative Example 1 in DMSO respectively to prepare a stock solution with a concentration of 1 mg / mL. Dilute the probe stock solution 1:100 into the phosphate buffer solution to prepare the test solution, let it stand for 12 h, and measure the fluorescence intensity value of the test solution at 555 nm under excitation at 530 nm. Then, calculate the pK of the probe based on the fluorescence intensity - pH relationship and the Henderson Hasselbalch equationa value

[0125] After calculation, the pK of R6G-HDA a is 3.49, while the pK of R6G-CDA obtained in Examples 1-4 a is 5.63. The results show that introducing CDA into the R6G structure significantly increases the pK of the probe a .

[0126] Detection Example 2, Probe pH Responsiveness Test

[0127] The rhodamine-based pH fluorescent probes obtained in Example 1 and Comparative Example 1 were respectively dissolved in DMSO to prepare a stock solution of 1 mg / mL. For each probe, 100 μL of the stock solution was diluted to 1.9 mL of phosphate buffer with pH values of 5.0, 5.8, 6.5, 7.0, 7.5, and 8.0, and allowed to stand for 12 h. Then, the color differences of each probe at different pH values and the fluorescence differences under 365 nm ultraviolet light excitation were observed and compared.

[0128] As Figure 3 shown, the probe R6G-CDA appears orange in weakly acidic conditions. As the pH of the solution increases, the color gradually disappears; under 365 nm ultraviolet light excitation, R6G-CDA exhibits yellow-green fluorescence in weakly acidic conditions. As the pH of the solution increases, the fluorescence gradually weakens and disappears at pH = 8. This indicates that R6G-CDA can determine the pH of weakly acidic aqueous solutions through two channels: apparent color and fluorescence, specifically for the effective determination of acidity and alkalinity in the pH range of 5-7.

[0129] As Figure 4 shown, the probe R6G-HDA shows no significant color and fluorescence changes in the pH range of 5.0 - 8.0, indicating that it cannot be used to detect the pH of weakly acidic aqueous solutions. This is because R6G-HDA is in a closed-loop state under weakly acidic conditions and cannot exhibit fluorescence and color.

[0130] The fluorescence spectra of the probes R6G-CDA and R6G-HDA in different pH buffers were further tested ( Figure 5 and Figure 6 ), and it was found that R6G-HDA only has a significant fluorescence response below pH 4, while R6G-CDA can achieve a fluorescence response under weakly acidic conditions with a pH of 5-7. Since the pH of the physiological environment is generally around neutral, the above results indicate that modifying CDA can expand the application of rhodamine-based pH fluorescent probes in the biomedical field.

[0131] The experiment also found that the acid response rate of R6G-CDA is significantly faster than that of R6G-HDA. During the experiment, at pH values of 5.63 and 3.49 (the pK of the probe a)The acid-responsive kinetics tests of R6G-CDA and R6G-HDA were carried out as follows. As Figure 7 shown, the response rate of R6G-HDA is extremely slow, and the fluorescence intensity only stabilizes after 180 min, that is, it takes 180 min to fully respond; while for R6G-CDA, the fluorescence intensity has stabilized within 10 min, that is, full response is achieved within 10 min. The response rate is 17 times higher than that of R6G-HDA. This is mainly because CDA on R6G-CDA has the characteristics of large steric hindrance, rigidity and trans configuration, which changes the planarity of the rhodamine molecule, thereby inhibiting the π-π stacking effect of rhodamine in aqueous solution and making the probe molecules in a relatively dispersed state. Therefore, the response rate is greatly accelerated. After R6G is modified with HDA, it is still a planar molecule, which is prone to form aggregates through π-π stacking in aqueous solution, hindering the kinetic process of acid-responsive ring opening, so the response rate is slow.

[0132] Detection Example 3. Response study of the coupled probe absorbent cotton

[0133] The acid-base response test was carried out on the absorbent cotton sample of the coupled probe R6G-CDA in Example 5. Figure 8 is the appearance of the absorbent cotton sample of the coupled probe R6G-CDA dried in vacuum. In the experiment, 0.1 M PBS with pH values of 6.1, 6.4, 7.2, and 8.0 was first prepared as the aqueous solution to be tested.

[0134] After the absorbent cotton sample of the coupled probe R6G-CDA absorbs the aqueous solution to be tested, it can indicate the acidity and alkalinity of the aqueous solution through apparent color change ( Figure 9 ) and fluorescence change ( Figure 10 ). As can be seen from Figure 9-10 , as the pH drops from 8.0 to 6.1, the absorbent cotton of the coupled probe R6G-CDA gradually changes from colorless to red. At the same time, it changes from non-fluorescent to emitting yellow fluorescence under the excitation of 365 nm ultraviolet light.

[0135] Furthermore, the absorbent cotton of the coupled probe R6G-CDA was made into thin slices for gastric juice leakage imaging experiments. Dilute hydrochloric acid with pH = 3 was prepared to simulate gastric juice. A small amount of simulated gastric juice was dropped on some areas of the thin slice of the coupled probe absorbent cotton. After 1 min, photos were taken without ultraviolet light excitation and under the excitation of 365 nm ultraviolet light respectively. The results are as Figure 11 shown.

[0136] As can be seen from Figure 11It can be seen that the area of absorbent cotton absorbing simulated gastric juice shows dark red, while the areas of other absorbent saline are close to colorless. Under the excitation of 365 nm ultraviolet light, the area absorbing gastric juice shows significant yellow fluorescence, and the area not absorbing gastric juice shows basically no fluorescence. The results confirm that the absorbent cotton conjugated with the probe can be used for imaging gastric juice leakage during gastric surgery, checking the closure of the surgical incision, and preventing the occurrence of postoperative gastric fistula.

[0137] The foregoing are only the preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent substitutions, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

[0138] The foregoing embodiments and methods described in the present invention may vary based on the capabilities, experience, and preferences of those skilled in the art.

[0139] The steps of the method listed only in a certain order in the present invention do not constitute any limitation on the order of the method steps.

Claims

1. A rhodamine-based pH fluorescent probe, characterized in that, The rhodamine-based pH fluorescent probe (R6G-CDA) is obtained by modifying a rhodamine 6G (R6G) molecule with a trans-1,4-cyclohexanediamine (CDA) group, and the structural formula of the rhodamine-based pH fluorescent probe is 2. The preparation method of the rhodamine-based pH fluorescent probe according to claim 1, characterized in that, The method includes the following steps: 1) Dissolve R6G in a polar organic solvent and add CDA for reaction; 2) Separate and purify the product obtained in step 1) to obtain R6G-CDA.

3. The method according to claim 1, wherein In step 1), the polar organic solvent is selected from one or more of N,N-dimethylformamide (DMF), methanol, ethanol, isopropanol, or N,N-dimethylacetamide (DMAC), preferably DMF; Preferably, the mass ratio of R6G to CDA is 0.2-4:1, preferably 0.2-2:1; Preferably, the reaction temperature is room temperature and the reaction time is 0.5-4 hours.

4. The method according to any one of claims 2-3, characterized in that, The separation and purification in step 2) includes the following steps: subjecting the product obtained in step 1) to extraction, rotary evaporation and recrystallization, and solid-liquid separation in sequence.

5. The method according to claim 4, characterized in that The extraction includes the following steps: adding dichloromethane (DCM) and water to the reaction system, placing it in a separatory funnel and mixing well, and collecting the DCM phase after standing and separating layers; Preferably, the rotary evaporation and recrystallization includes the following steps: adding a non-polar organic solvent to the DCM solution and obtaining a solid-liquid mixture by rotary evaporation and recrystallization; Preferably, the non-polar organic solvent is selected from one or more of petroleum ether, n-pentane, isopentane, n-hexane, n-heptane, cyclohexane, toluene, preferably n-hexane; Preferably, the solid-liquid separation includes the following steps: centrifuging and / or filtering the solid-liquid mixture to obtain a solid powder; Preferably, the separation and purification further includes the following steps: washing and drying the solid powder with a non-polar organic solvent.

6. A carrier conjugated with R6G-CDA, characterized in that, It is obtained by oxidizing the carrier and then coupling it with the R6G-CDA as described in claim 1 or the R6G-CDA obtained by the method according to any one of claims 2-5.

7. The carrier conjugated with R6G-CDA according to claim 6, characterized in that, The carrier is selected from cellulose, hyaluronic acid, chitosan, alginate, starch, gelatin, and sponges, hydrogels, swabs, papers, absorbent cotton composed of them or mainly composed of them.

8. A method for preparing a carrier conjugated with R6G-CDA as described in claim 6 or 7, characterized in that, The preparation method includes the following steps: (1) Carrier oxidation: Mix the carrier, an oxidant and water, add an acidic reagent to adjust the pH of the solution for reaction to obtain an oxidized carrier; (2) Coupling of R6G-CDA with the carrier: Dissolve R6G-CDA in an organic solvent, add the oxidized carrier described in step (1) for reaction to obtain a carrier coupled with R6G-CDA.

9. The preparation method according to claim 8, characterized in that, In step (1), the oxidant is selected from one or more of sodium periodate, hydrogen peroxide, chromium trioxide, potassium permanganate, 2-iodoxybenzoic acid (IBX), phenyliodine diacetate (PIDA), iodobenzene (PhIO), or sodium chlorite, preferably sodium periodate; Preferably, the mass ratio of the carrier to the oxidant is 1:3-5; Preferably, the acidic reagent is sulfuric acid, hydrochloric acid, nitric acid, phosphoric acid, perchloric acid, acetic acid, trifluoromethanesulfonic acid or their aqueous solutions; Preferably, the pH of the solution is adjusted to 2.0 - 6.0 by adding an acidic reagent; Preferably, the temperature of the reaction is room temperature.

10. The preparation method according to claim 8 or 9, characterized in that, In step (2), the organic solvent is selected from one or more of DMF, DMAC, N-methylpyrrolidone (NMP), dimethyl sulfoxide (DMSO), N,N-diethylformamide (DEF), tetrahydrofuran (THF), and cyclohexanone, preferably DMF; Preferably, the mass ratio of R6G-CDA to the carrier is 1:5 - 20; Preferably, the reaction temperature is 30 - 60 °C.

11. Use of R6G-CDA as claimed in claim 1 or R6G-CDA obtained by the method as claimed in any one of claims 2 - 5 as a pH fluorescent probe; Preferably, the pH fluorescent probe is applied to the detection of gastric juice leakage during surgery to help prevent the occurrence of postoperative gastric fistula.

12. Use of the carrier conjugated with R6G-CDA as claimed in any one of claims 6 - 7 or the carrier conjugated with R6G-CDA prepared by the preparation method as claimed in any one of claims 8 - 10 in the preparation of a product for detecting the pH of different systems; Preferably, the product is a medical device with a visual detection function; More preferably, the medical device is a kit or a dressing.

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