In-situ anchored fluorescent probe as well as preparation method and application thereof
By designing an in-situ anchored fluorescent probe, the problem of easy diffusion of traditional probes was solved, achieving high selectivity, rapid response and long-term dynamic tracking of hypochlorous acid. It is suitable for fluorescence imaging of live cells and zebrafish and has commercial potential.
Patent Information
- Application Number
- CN202511819603.9
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-12-04
- Publication Date
- 2026-03-17
AI Technical Summary
Traditional fluorescent probes are prone to diffusion and displacement within cells, resulting in low signal-to-noise ratios and ambiguous localization, making it impossible to achieve long-term, stable tracking of hypochlorous acid and limiting their application in studying the mechanisms of oxidative stress-related diseases.
A novel in-situ anchored fluorescent probe based on an active quinone structure was developed. By covalently bonding to specific biomolecules and cellular structures, the probe can achieve long-term residence and high-fidelity imaging. It is simple to synthesize, highly sensitive, selective, and can respond rapidly to hypochlorous acid.
It achieves precise localization and long-term dynamic tracking of hypochlorous acid, with high selectivity and rapid response capabilities. It is suitable for detection at physiological levels and for fluorescence imaging of live cells and zebrafish. It is simple to synthesize and easy to commercialize.
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Figure CN121673223A_ABST
Abstract
Description
TECHNICAL FIELD
[0001] The present application belongs to the field of fluorescent probes, and particularly relates to an in-situ anchoring type fluorescent probe and application thereof. BACKGROUND
[0002] Hypochlorous acid (HOCl) is a specific reaction product catalyzed by myeloperoxidase (MPO), which plays an important role in immune defense and redox balance. The accurate detection of its physiological concentration level is of great significance for understanding various pathological processes such as neurodegenerative diseases and cancer. Therefore, it is crucial to develop a method for accurately and long-term detection of HOCl.
[0003] Currently, fluorescent probe technology provides an effective method for in-situ detection of HOCl in biological systems. However, traditional fluorescent probes are prone to displacement or leakage due to diffusion in cells, resulting in low signal-to-noise ratio and ambiguous localization, which cannot achieve long-term and stable tracking of their generation sites, severely limiting their application in studying the mechanisms of oxidative stress-related diseases.
[0004] The in-situ anchoring type fluorescent probe constructed by using active quinone structure provides an effective means to solve the above problems. This kind of probe can be covalently bonded to specific biological macromolecules and cell structures, overcoming the shortcomings of traditional fluorescent probes, effectively preventing probe leakage, achieving accurate positioning and long-term tracking, and thus realizing long-term residence and high-fidelity imaging of fluorescent probes in living organisms. However, existing probes of this type usually have limitations such as complex structure, multiple synthesis steps, and hydrolysis. Therefore, it is of great significance to develop a simple structure of in-situ anchoring type fluorescent probe activated by hypochlorous acid. SUMMARY
[0005] In view of this, the present application aims to provide a more universally applicable in-situ anchoring type fluorescent probe, as well as its preparation method and use, for the detection of HOCl at physiological levels. The probe has the characteristics of simple synthesis, high sensitivity, good selectivity and rapid response to HOCl, and can be anchored on proteins to achieve long-term in-situ dynamic tracking of HOCl.
[0006] Specifically, the present application provides an in-situ anchoring type hypochlorous acid fluorescent probe, characterized in that the chemical structure of the fluorescent probe is shown as (I): wherein R1, R2, R3, R6 and R7 are independently selected from the group consisting of hydrogen atom, straight-chain or branched alkyl group, straight-chain or branched alkoxy group, sulfonic acid group, ester group and carboxyl group; and wherein R1, R2, R3, R6 and R7 can be the same or different; R4and R5are independently selected from the group consisting of hydrogen atom, straight or branched alkyl, straight or branched alkoxy, sulfonic acid group, ester group and carboxyl group; and wherein R4and R5may be the same or different; or, R4and R5together with the N atom to which they are attached form a carbocyclic ring, an aromatic ring or a heterocyclic ring.
[0007] In some embodiments of the present application, the fluorescent probe is of the following structural formula: The present application also provides a preparation method of the fluorescent probe of formula (I) or formula (II), comprising the following synthesis route and method: wherein, R1, R2, R3, R6and R7are independently selected from the group consisting of hydrogen atom, straight or branched alkyl, straight or branched alkoxy, sulfonic acid group, ester group and carboxyl group; and wherein R1, R2, R3, R6and R7may be the same or different; R4and R5are independently selected from the group consisting of hydrogen atom, straight or branched alkyl, straight or branched alkoxy, sulfonic acid group, ester group and carboxyl group; and wherein R4and R5may be the same or different; or, R4and R5together with the N atom to which they are attached form a carbocyclic ring, an aromatic ring or a heterocyclic ring.
[0008] In some embodiments of the present application, the specific preparation method is: dissolving the compound of formula (III) and formula (IV), (V) in anhydrous ethanol, and heating the mixture to reflux for 5-10 hours; after the reaction is completed, the product is obtained by suction filtration at room temperature from the reaction solution; after the reaction is completed and cooled, the product is obtained by suction filtration; then, the product is further purified by recrystallization method.
[0009] In some embodiments of the present application, the molar ratio of the compound of formula (III), formula (IV) and formula (V) is 1: (1.2-3.0): (1.2-5.0).
[0010] The present application also provides a fluorescent probe composition for measuring, identifying or detecting HOCl, which comprises the fluorescent probe of formula (I) or formula (II).
[0011] In some embodiments of the present application, the fluorescent probe composition further comprises a pharmaceutically acceptable carrier or buffer system.
[0012] The present application also provides the use of the fluorescent probe of formula (I) or formula (II) in the preparation of a reagent for measuring, identifying or detecting hypochlorous acid.
[0013] The present invention also provides a kit for detecting the presence of hypochlorous acid in a sample or determining the content of hypochlorous acid in a sample, comprising the fluorescent probe of formula (I) or formula (II).
[0014] The present invention also provides a method for detecting the presence of hypochlorous acid in a sample or determining the hypochlorous acid content in a sample, comprising the following steps: a) Contact the fluorescent probe of formula (I) or formula (II) with the sample to generate a product of fluorescence quenching; b) Measure the change in fluorescence intensity before and after the reaction.
[0015] In some specific embodiments of the present invention, the sample is a chemical sample or a biological sample.
[0016] In some specific embodiments of the present invention, the sample is a biological sample.
[0017] In some specific embodiments of the present invention, the sample is an aquatic environment sample.
[0018] In some specific embodiments of the present invention, the sample is a food sample.
[0019] The present invention also provides the application of fluorescent probes of formula (I) or formula (II) in cell fluorescence imaging.
[0020] The present invention also provides the application of fluorescent probes of formula (I) or formula (II) in zebrafish fluorescence imaging.
[0021] The present invention also provides the application of the fluorescent probe of formula (I) or formula (II) in the long-term dynamic tracking of intracellular hypochlorous acid; the fluorescent probe is induced to anchor in situ under the activation of hypochlorous acid, thereby realizing the long-term dynamic tracking of intracellular hypochlorous acid.
[0022] The present invention has the following significant advantages and effects compared with the prior art: 1. Quantitative detection of hypochlorous acid: The hypochlorous acid fluorescent probe provided by this invention can react with hypochlorous acid and produce obvious linear changes in fluorescence spectrum, thereby realizing accurate quantitative detection of hypochlorous acid.
[0023] 2. High selectivity recognition: The hypochlorous acid fluorescent probe provided by this invention can not only react with hypochlorous acid, but also specifically recognize hypochlorous acid in a variety of ions and compounds, ensuring the accuracy and reliability of the detection results.
[0024] 3. High sensitivity and rapid response: The hypochlorous acid fluorescent probe provided by this invention has a fast reaction rate with hypochlorous acid and high sensitivity, which can quickly and sensitively respond to hypochlorous acid, facilitating real-time detection and analysis.
[0025] 4. High fluorescence quantum yield: The hypochlorous acid fluorescent probe provided by this invention has a large molar absorptivity and a high fluorescence quantum yield.
[0026] 5. Long-term tracking imaging: The fluorescent probe of this invention utilizes the properties of o-hydroxybenzylamine, which can covalently bind to proteins after activation by hypochlorous acid, thereby realizing the detection of HOCl and long-term dynamic tracking.
[0027] 6. Applicable under physiological conditions: The fluorescent probe of the present invention can be applied under physiological conditions and can be used for fluorescence imaging of live cells and zebrafish.
[0028] 7. Simple synthesis and easy commercialization: The hypochlorous acid fluorescent probe provided by this invention has a simple synthesis process, is easy to mass-produce and commercialize, reduces production costs and improves market competitiveness.
[0029] In summary, the hypochlorous acid fluorescent probe provided by this invention has multiple beneficial technical effects, such as quantitative detection, high selective recognition, rapid and sensitive reaction, high fluorescence quantum yield, simple synthesis, and long-term dynamic tracking, providing an effective tool and method for the detection and analysis of hypochlorous acid. Attached Figure Description
[0030] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.
[0031] Figure 1 The response time spectrum of probe (II) (5 μM) before and after the addition of hypochlorous acid (10 μM) is shown.
[0032] Figure 2 The fluorescence spectra of the (II) probe (5 µM) before and after the addition of hypochlorous acid (0-10 μM) are shown.
[0033] Figure 3 The graph shows the linear relationship between the fluorescence intensity of the formula (II) probe (5 µM) at 537 nm and the concentration of hypochlorous acid (0-1 µM).
[0034] Figure 4This study describes the effect of common substances in the human body on the fluorescence intensity of probe (II) (5 µM). The analytes include: magnesium ions, sodium ions, potassium ions, copper ions, calcium ions, ferrous ions, ferric ions, zinc ions, nitrate ions, sulfate ions, carbonate ions, bromide ions, chloride ions, nitrite ions, nitric oxide, superoxide ions, singlet oxygen, hydroxide ions, tert-butylhydrogen peroxide, tert-butyloxygen radical, hydrogen peroxide, and HOCl (unless otherwise specified, all analytes are at a concentration of 10 μM). The bar chart represents the fluorescence intensity at 537 nm of the probe in the presence of different analytes.
[0035] Figure 5 The fluorescence imaging of HeLa cells by the fluorescent probe (5 µM) of formula (II) is shown.
[0036] Figure 6 Fluorescence imaging of exogenous HOCl in HeLa cells using the fluorescent probe of formula (II) (5 µM).
[0037] Figure 7 The fluorescence imaging of exogenous and endogenous HOCl in zebrafish using the formula (II) fluorescent probe (5 µM).
[0038] Figure 8 This is the long-term dynamic tracking of intracellular HOCl by the fluorescent probe of formula (II) (5 µM). Detailed Implementation
[0039] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only a part of the embodiments of the present invention and should not be used to limit the scope of protection of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.
[0040] Example 1: The synthetic route of the fluorescent probe of formula (II) is as follows: The specific steps are as follows: Compound (VI) (300 mg, 1.12 mmol) was uniformly dispersed in anhydrous ethanol (10 mL). Then, thiomorpholine (129 mg, 1.25 mmol) and paraformaldehyde (38 mg, 1.25 mmol) were added sequentially to the solution, and the mixture was heated under reflux for 7 hours. After the reaction was complete and cooled, the crude product was obtained by filtration. Subsequently, recrystallization with anhydrous ethanol yielded the pure product (yield: 42%).
[0041] Example 2: Testing the reaction kinetics of fluorescent probes A 10 mL test system with a 5 μM concentration of the fluorescent probe of formula (II) was prepared. Then, 10 µM HOCl was added to the test system, and the mixture was quickly shaken until homogeneous. The fluorescence intensity change was immediately measured using a fluorescence spectrometer. The above determination was performed in a system of ethanol:water at a ratio of 2:8 (10 mM PBS, pH 7.4). The probe used was the probe prepared in Example 1, and the fluorescence spectrum was measured at 25 °C. Unless otherwise specified, all spectral data are expressed in λ. ex Data was collected at 430 nm, slit: W ex =W em =3 nm.
[0042] Test results are as follows Figure 1 As shown, it can be clearly seen that when HOCl is added, the fluorescence intensity instantly reaches its lowest value and remains basically unchanged. This indicates that the probe reacts rapidly with HOCl and can provide a rapid analytical method for the determination of HOCl.
[0043] Example 3: Quantitative analysis of the fluorescence spectrum of hypochlorous acid using fluorescent probes Multiple parallel samples with a probe concentration of 5 µM were prepared in 10 mL colorimetric tubes. Then, hypochlorous acid of different concentrations (0-10 µM) was added to the test system. After shaking and allowing to stand for 10 minutes, the fluorescence intensity changes were measured using a fluorescence spectrometer. The above measurements were performed in an ethanol:water ratio of 2:8 (10 mM PBS, pH 7.4). The probe used was the one prepared in Example 1, and all spectral measurements were taken at 25 °C. The test results are as follows: Figure 2 and 3 As shown.
[0044] from Figure 2 It can be clearly seen that the fluorescence intensity at 537 nm gradually decreases with increasing hypochlorous acid concentration. Furthermore, from... Figure 3 It can be seen that after the probe (5 µM) is added to hypochlorous acid (0-10 µM), there is a good linear relationship between the fluorescence intensity at 537 nm and the concentration of hypochlorous acid, which proves that this fluorescent probe can be used to perform quantitative analysis of hypochlorous acid.
[0045] Example 4: Selectivity test of fluorescent probe for hypochlorous acid The analytes were: blank, magnesium ion, sodium ion, potassium ion, copper ion, calcium ion, ferrous ion, ferric ion, zinc ion, nitrate ion, sulfate ion, carbonate ion, bromide ion, chloride ion, nitrite ion, nitric oxide, superoxide ion, singlet oxygen, hydroxyl ion, tert-butyl hydroperoxide, tert-butyloxy radical, hydrogen peroxide, and HOCl (unless otherwise specified, the concentration of all other analytes was 10 μM). The bar chart represents the fluorescence intensity value at 537 nm of the probe in the presence of different analytes. The above measurements were performed in a system of ethanol:water at a ratio of 2:8 (10 mM PBS, pH 7.4), using the probe prepared in Example 1, and all spectral measurements were taken at 25 °C. Specifically, multiple parallel samples with a probe concentration of 5 µM were prepared in 10 mL colorimetric tubes, then a certain amount of analyte was added, shaken well, and allowed to stand for 5 minutes before measuring the fluorescence intensity value. The results are as follows: Figure 4 As shown.
[0046] from Figure 4 It can be seen that the probe of the present invention has high selectivity for hypochlorous acid and can selectively react with hypochlorous acid.
[0047] Example 5: Detection limit test and calculation of hypochlorous acid using a probe The limit of detection (LOD) was calculated using fluorescence titration. The formula for calculating the LOD is as follows: Detection limit = 3σ / k σ is the standard deviation of the fluorescence intensity of the blank probe, and k is... Figure 3 The slope of the linear relationship graph. Based on this, the detection limit of the probe for hypochlorous acid calculated by formula (II) is 29.8 nM.
[0048] Example 6: Fluorescence microscopy imaging of endogenous and exogenous HOCl in HeLa cells using fluorescent probes Fluorescence microscopy analysis of endogenous HOCl in cells: HeLa cells were divided into four groups: Group 1 consisted of HeLa cells under normal conditions; Group 2 was incubated with probe (10 µM) for 30 min; Group 3 was pretreated with LPS (1 μg / mL) for 1 h and then incubated with probe (10 µM) for 30 min; Group 4 was pretreated with NAC (200 μM) for 1 h and then incubated with probe (10 µM) for 30 min. Fluorescence microscopy analysis of exogenous HOCl in cells: HeLa cells were divided into five groups: Group 1 consisted of HeLa cells in normal condition; Group 2 was incubated with a probe (10 µM) for 30 min; Group 3 was incubated with a probe (10 µM) for 30 min followed by incubation with HOCl (10 μM) for 20 min; Group 4 was incubated with a probe (10 µM) for 30 min followed by incubation with HOCl (20 μM) for 20 min; and Group 5 was incubated with a probe (10 µM) for 30 min followed by incubation with HOCl (30 μM) for 20 min. Finally, confocal microscopy was performed on each group of cells, and the results are as follows: Figure 5 and 6 As shown.
[0049] from Figure 5 and Figure 6 It can be seen that this probe can detect endogenous and exogenous HOCl in HeLa cells; experiments have shown that this probe can be applied to the detection of HOCl in biological samples.
[0050] Example 7: Fluorescence microscopic imaging of endogenous and exogenous HOCl in zebrafish using fluorescent probes Zebrafish were divided into eight groups: Group 1 consisted of zebrafish in normal condition; Group 2 was incubated with a probe (10 µM) for 30 min; Group 3 was pretreated with LPS (1 μg / mL) for 1 h, then incubated with a probe (10 µM) for 30 min; Group 4 was pretreated with NAC (200 μM) for 1 h, then incubated with a probe (10 µM) for 30 min; Group 5 was incubated with a probe (10 µM) for 30 min, then incubated with HOCl (10 μM) for 20 min; Group 6 was incubated with a probe (10 µM) for 30 min, then incubated with HOCl (20 μM) for 20 min; Group 7 was incubated with a probe (10 µM) for 30 min, then incubated with HOCl (30 μM) for 20 min; and Group 8 was incubated with a probe (10 µM) for 10 min, then incubated with HOCl (50 μM) for 20 min. min; finally, confocal microscopy was performed on each group of cells, and the test results are as follows. Figure 7 As shown.
[0051] from Figure 7It can be seen that the probe can detect endogenous and exogenous HOCl in zebrafish; further experiments have demonstrated that the probe can be applied to the detection of HOCl in live samples.
[0052] Example 8: Long-term dynamic tracking of intracellular HOCl by fluorescent probes Cells pretreated with LPS (1 μg / mL) were incubated with the formula (II) probe (10 μM) and imaged over 0–4 hours. Figure 8 As shown, the fluorescent probe exhibits higher fluorescence intensity before the response. After the response, the fluorescence intensity of the cells incubated with the fluorescent probe remains essentially unchanged over time. This demonstrates that the probe of formula (II) can be anchored in situ within the cell after activation by HOCl, and can be used to dynamically monitor changes in intracellular HOCl levels.
[0053] Although the present invention has been described with reference to the above embodiments, it should be understood that the present invention may be further modified and varied without departing from the spirit of the present invention, and all such modifications and variations are within the protection scope of the present invention.
Claims
1. An in situ anchoring type hypochlorous acid fluorescent probe, characterized by The chemical structure of the fluorescent probe is shown in the following formula, and the chemical structure is shown in formula (I): R1, R2, R3, R6 and R7 are independently selected from the group consisting of hydrogen atom, straight-chain or branched alkyl, straight-chain or branched alkoxy, sulfonic acid group, ester group and carboxyl group; and R1, R2, R3, R6 and R7 can be the same or different; R4 and R5 are independently selected from the group consisting of hydrogen atom, straight-chain or branched alkyl, straight-chain or branched alkoxy, sulfonic acid group, ester group and carboxyl group; and R4 and R5 can be the same or different; or R4 and R5 together with the N atom to which they are connected form a carbon ring, an aromatic ring or a heterocyclic ring.
2. The in situ anchoring hypochlorous acid fluorescent probe according to claim 1, characterized in that The following structural formula (II): 。 3. A method for preparing the fluorescent probe according to claim 1 or 2, characterized by, The compound of formula (I) is prepared by reacting the compound of formula (III), the compound of formula (IV) and the compound of formula (V), and the reaction formula is as follows: R1, R2, R3, R6 and R7 are independently selected from the group consisting of hydrogen atom, straight-chain or branched alkyl, straight-chain or branched alkoxy, sulfonic acid group, ester group and carboxyl group; and R1, R2, R3, R6 and R7 can be the same or different; R4 and R5 are independently selected from the group consisting of hydrogen atom, straight-chain or branched alkyl, straight-chain or branched alkoxy, sulfonic acid group, ester group and carboxyl group; and R4 and R5 can be the same or different; or R4 and R5 together with the N atom to which they are connected form a carbon ring, an aromatic ring or a heterocyclic ring.
4. The present application also provides a fluorescent probe composition for measuring, discriminating or detecting hypochlorous acid, characterized in that, The fluorescent probe of any one of claims 1 or 2.
5. The fluorescent probe composition of claim 4, wherein, The fluorescent probe composition further comprises a pharmaceutically acceptable carrier or buffer system.
6. The present application also provides the use of the fluorescent probe of any one of claims 1 or 2 in the preparation of a reagent for measuring, identifying or detecting hypochlorous acid.
7. The present application also provides a kit for detecting the presence of hypochlorous acid in a sample or measuring the content of hypochlorous acid in a sample, comprising the fluorescent probe of any one of claims 1 or 2.
8. The present application also provides a method for detecting the presence of hypochlorous acid in a sample or measuring the content of hypochlorous acid in a sample, comprising the following steps: a) contacting the fluorescent probe of any one of claims 1 or 2 with the sample to generate a product with quenched fluorescence; b) measuring the change in fluorescence intensity before and after the reaction; Preferably, the sample is a chemical sample or a biological sample.
9. The use of the fluorescent probe of any one of claims 1 or 2 in cell fluorescence imaging.
10. Use of the fluorescent probe according to any one of claims 1 or 2 for long-term dynamic tracing of hypochlorous acid in cells, characterized in that, The fluorescent probe induces in-situ anchoring under the activation of hypochlorous acid, achieving long-term dynamic tracing of intracellular hypochlorous acid.