A water-soluble low-background cy5 probe, a construction method thereof and cross-instrument hypochlorous acid consistent detection application

Abstract

The application belongs to the field of biomedical detection and molecular imaging technology, and specifically discloses a water-soluble low-background Cy5 probe, a construction method thereof and application of the probe to consistent hypochlorous acid detection across instruments. The probe Cy5Q1-HClO of the application takes Cy5 cyanine dye as a skeleton, realizes near-zero intrinsic fluorescence by means of a double photo-induced electron transfer (PET) mechanism, triggers a self-elimination cascade reaction after specific recognition of hypochlorous acid, and restores strong near-infrared fluorescence. The double-cation structure of the probe endows it with good water solubility, facilitating its use in biological systems. The spectral shift of the probe is extremely small before and after response, only the absorption and fluorescence intensity change, so similar working curves can be obtained on instruments with different excitation and detection channels, the probe has cross-platform and instrument-independent signal quantitative capability, thereby realizing high-sensitivity, zero-background interference and in-vivo precise imaging of HClO. The probe has the characteristics of water solubility, low background and high sensitivity, can not only visualize tumors with high contrast, but also guide precise resection of less than 2mm resection margin, and for the first time realizes longitudinal monitoring of dynamic changes of HClO in the postoperative wound healing process. The application also provides a preparation method and application of the Cy5Q1-HClO probe.

Description

Technical Field

[0001] This invention relates to the fields of biomedical detection and molecular imaging technology, and in particular to a water-soluble low-background Cy5 probe, its construction method, and its cross-instrument consistent detection application for hypochlorous acid. Background Technology

[0002] Hypochlorous acid (HClO) is an important reactive oxygen species (ROS) in the body, reacting with H2O2 and Cl- catalyzed by myeloperoxidase (MPO). - HClO plays a dual crucial role in both the tumor inflammatory microenvironment and wound repair. In solid tumors, HClO is mainly secreted in large quantities by tumor-associated macrophages and infiltrative neutrophils, participating in tumor progression, immunosuppression, and matrix remodeling. In postoperative wounds, HClO released by neutrophils exerts bactericidal, anti-inflammatory, and tissue regeneration-initiating functions, making it an ideal biomarker connecting tumor diagnosis and treatment with postoperative repair.

[0003] Although HClO is widely recognized as a key biomarker for tumor diagnosis, surgical navigation, and wound healing monitoring, quantitative detection and accurate imaging of HClO still face technical challenges. Current detection technologies for HClO suffer from the following bottlenecks: 1) High background fluorescence and limited signal amplification: Traditional small molecule probes often employ a single photoinduced electron transfer (PET) or intramolecular charge transfer (ICT) quenching mechanism, achieving only a fluorescence enhancement of tens of times (typically 10-30 times). Incomplete suppression of background fluorescence makes it difficult to achieve highly sensitive and accurate detection of low concentrations and short-lived HClO at physiological levels, failing to meet the required standards. 1) It meets the clinical need for high-contrast imaging; 2) It is highly dependent on instruments and the quantification is inaccurate: The fluorescence response mechanism of most probes is strongly correlated with the excitation / emission wavelength. The signal-to-background ratio fluctuates with the change of equipment parameters. The quantitative results vary greatly between different detection platforms (fluorescence spectrometer, in vivo imaging instrument) and the repeatability is poor, which seriously limits its clinical translation and promotion; 3) It has a single function and cannot achieve full-process integration: Existing probes can only be used for tumor imaging or wound monitoring. They cannot cover the complete clinical diagnosis and treatment path of "tumor detection - precise surgical navigation - postoperative healing dynamic monitoring". Moreover, the tissue imaging contrast is low and it is difficult to meet the needs of integrated diagnosis and treatment.

[0004] Given the aforementioned shortcomings of current HClO detection technologies, there is an urgent need to develop novel HClO detection technologies that combine zero intrinsic fluorescence, high signal-to-background ratio, instrument-independent quantification, and full-process imaging capabilities. Summary of the Invention

[0005] This invention aims to address at least one of the technical problems existing in the prior art. To this end, this invention provides a water-soluble, low-background Cy5 probe, its construction method, and its cross-instrumental consistent detection application for hypochlorous acid. The Cy5Ql-HClO probe of this invention utilizes a synergistic mechanism of complete background elimination through dual photoinduced electron transfer (PET) quenching, recovery of strong fluorescence through hypochlorous acid (HClO) specific recognition triggering a self-elimination cascade reaction, and cross-platform instrument-independent signal quantification. This probe is characterized by water solubility, low background, and high sensitivity, achieving HClO detection technology with high sensitivity, zero background interference, and accurate imaging capabilities. Furthermore, this probe also possesses integrated diagnostic and therapeutic capabilities, including high-contrast visualization of tumors, precise surgical navigation with resection margins smaller than 2 mm, and dynamic monitoring of postoperative wound healing, effectively expanding the application value of fluorescent probes in the entire process of tumor diagnosis and treatment.

[0006] This invention also provides a method for preparing a Cy5Ql-HClO probe.

[0007] This invention also provides applications of the Cy5Ql-HClO probe.

[0008] This invention also proposes an integrated kit for tumor detection and postoperative wound monitoring.

[0009] In a first aspect, the present invention provides a fluorescent probe Cy5Ql-HClO for detecting hypochlorous acid, with the following structural formula:

[0010] .

[0011] According to specific embodiments of the present invention, the dual PET-quenched zero intrinsic fluorescent probe (Cy5Ql-HClO) based on Cy5 cyanine dye achieves a technological breakthrough through the following synergistic mechanism:

[0012] Achieving Zero Intrinsic Fluorescence through Dual PET Quenching: The probe Cy5Ql-HClO of this invention uses Cy5 cyanine dye as the fluorescent core. Quinolineon is directly introduced at the meso site of the Cy5 core as the first PET quenching unit, and simultaneously, dimethyl thiocarbamate is covalently linked via a self-eliminating linker as the second PET quenching unit, constructing a dual photoinduced electron transfer (PET) synergistic quenching system. First PET quenching pathway: Quinolineon at the meso site forms the shortest electron transport channel with the Cy5 core, with an energy level difference ΔE = 0.12 eV, satisfying the effective PET quenching condition (ΔE < 0.6 eV), achieving primary fluorescence quenching. Second PET quenching pathway: Dimethyl thiocarbamate acts as an independent quenching unit, with an energy level difference ΔE = 0.52 eV between it and the Cy5 core, also satisfying the effective PET quenching condition, achieving secondary fluorescence quenching. Two quenching units work independently yet collaboratively, simultaneously suppressing fluorescence emission from the Cy5 parent nucleus through two electron transport pathways. This results in a fluorescence quantum yield of less than 0.001 in the unactivated state, achieving true zero intrinsic fluorescence. Traditional HClO fluorescent probes employ only a single PET quenching mechanism. The energy level difference ΔE between the quenching unit and the fluorescent parent nucleus is greater than 0.6 eV, exceeding the effective quenching threshold. This only achieves partial fluorescence suppression, with a background fluorescence quantum yield of approximately 0.1, and still exhibits significant background interference.

[0013] This invention's dual PET quenching offers significantly superior technical advantages: First, quenching is more thorough; the dual-path synergistic quenching reduces the background quantum yield by more than 100 times compared to a single PET probe, eliminating background interference at its source. Second, signal amplification is stronger; after specific oxidative cleavage with HClO, the dual PET quenching effect is simultaneously relieved, resulting in a fluorescence enhancement factor of more than 20 times and a signal-to-background ratio (SBR) far exceeding that of a single PET probe. Third, sensitivity is higher; zero background combined with high signal amplification lowers the probe's detection limit to as low as 26 nM, enabling precise detection of low concentrations and short-lived HClO at physiological levels. Cross-platform, instrument-independent signal quantification: The probe's PET response mechanism is completely decoupled from the excitation / emission wavelength, and the SBR remains unchanged regardless of instrument parameters. Quantitative results are highly consistent across different platforms such as fluorescence spectrometers and in vivo imaging systems, exhibiting excellent repeatability and overcoming the instrument dependence of traditional probes.

[0014] Integrated high-contrast imaging of tumors and wounds: The Cy5Ql-HClO probe of this invention specifically responds to the endogenously highly expressed HClO in the tumor microenvironment and postoperative wound, achieving high-contrast visualization of tumor tissue and precisely guiding tumor resection surgery with margins smaller than 2 mm. Simultaneously, it can longitudinally monitor the dynamic changes of HClO during postoperative wound healing, completing the entire imaging process of tumor diagnosis and wound repair. In vitro detection method: Mix 10 μM Cy5Ql-HClO working solution (PBS:DMF=99:1, v / v) with the sample to be tested, incubate at 37 ℃ for 10 min, and analyze using a fluorescence spectrometer (λ). ex / λ em The signal can be directly read from 580 / 657 nm without complex preprocessing, enabling high-sensitivity, zero-background-interference detection of HClO. In vivo imaging method: The Cy5Ql-HClO probe is applied locally to the tumor or wound site. Fluorescence is activated within 1 minute, and the signal peak is reached within 30 minutes. A near-infrared in vivo imaging system (λ...) is then used... ex =605 nm, λ em High-contrast imaging (640-660 nm) can be obtained, enabling real-time surgical guidance and dynamic monitoring of wound healing.

[0015] A second aspect of the present invention provides a method for preparing the fluorescent probe Cy5Ql-HClO as described in the first aspect of the present invention, comprising the following steps:

[0016] S1. 4-hydroxybenzaldehyde and dimethylthiocarbamoyl chloride are mixed in a solvent and reacted. After the reaction is completed, the solvent is removed, a reducing agent is added for reduction, and then the mixture is extracted and concentrated. The resulting mixture is mixed with phosphorus tribromide in a solvent and the reaction is continued. After the reaction is completed, the purified compound is: O-(4-bromomethylphenyl)N,N-dimethylthiocarbamate.

[0017] S2. 4-Methylquinoline was added to a mixture of phosphorus oxychloride and N,N-dimethylformamide and heated to react, yielding the intermediate (E)-3-hydroxy-2-(quinoline-4-yl)propenal. The intermediate was mixed with 1-ethyl-2,3,3-trimethyl-3H-indole-1-onium in a solvent and the reaction was continued under heating. After the reaction was completed, the product was purified to obtain the dye Cy5Ql.

[0018] S3. Mix Cy5Ql and O-(4-bromomethylphenyl)N,N-dimethylthiocarbamate in a solvent and heat to react. After the reaction is complete, purify to obtain the fluorescent probe Cy5Ql-HClO.

[0019] According to some embodiments of the present invention, in step S1, the molar ratio of 4-hydroxybenzaldehyde and dimethylthiocarbamoyl chloride is (1~1.5):1; preferably, the molar ratio of the two is (1.1~1.4):1.

[0020] According to some embodiments of the present invention, in step S1, the concentration of 4-hydroxybenzaldehyde in the reaction system is 1~5 mmol / mL.

[0021] According to some embodiments of the present invention, in step S1, the reaction conditions for the mixture of 4-hydroxybenzaldehyde and dimethylthiocarbamoyl chloride are: reaction at 0-4 °C for 3-6 h under an inert atmosphere.

[0022] According to some embodiments of the present invention, in step S1, the reaction conditions for the mixture with phosphorus tribromide are: reaction at 0~4℃ for 10~16 h under an inert atmosphere.

[0023] According to some embodiments of the present invention, in step S2, the volume ratio of 4-methylquinoline, phosphorus oxychloride and N,N-dimethylformamide is 1:(1.5~2.5):(2~3).

[0024] According to some embodiments of the present invention, in step S2, the molar ratio of 4-methylquinoline and 1-ethyl-2,3,3-trimethyl-3H-indole-1-onium is (1.5~4):1.

[0025] According to some embodiments of the present invention, in step S2, the heating reaction conditions for the two consecutive heating reactions are as follows: heating and refluxing at 80~95 °C for 5~8 h, and heating and refluxing at 60~70 °C for 10~16 h.

[0026] According to some embodiments of the present invention, in step S2, the purification is carried out by silica gel column separation and purification, using a mixed solution of dichloromethane and methanol as the eluent, wherein the volume ratio of dichloromethane to methanol in the eluent is (20~100):1.

[0027] According to some embodiments of the present invention, in step S3, the molar ratio of Cy5Ql and O-(4-bromomethylphenyl)N,N-dimethylthiocarbamate is 1:(1.2~2); preferably, the molar ratio of the two is 1:(1.3~1.7).

[0028] According to some embodiments of the present invention, in step S3, the solvent includes anhydrous acetonitrile, and the concentration of Cy5Ql in the reaction system is 0.05~0.1 mmol / mL.

[0029] According to some embodiments of the present invention, in step S3, the heating reaction is carried out under reflux at 80-95 °C for 5-8 h.

[0030] According to some embodiments of the present invention, in step S3, the purification is carried out by silica gel column separation and purification, using a mixed solution of dichloromethane and methanol as the eluent, wherein the volume ratio of dichloromethane to methanol in the eluent is (10~100):1.

[0031] A third aspect of the present invention provides the application of the fluorescent probe Cy5Ql-HClO as described in the first aspect of the present invention in the detection of hypochlorous acid (HClO).

[0032] According to some embodiments of the present invention, the fluorescent probe Cy5Ql-HClO is used for the quantitative or qualitative detection of hypochlorous acid.

[0033] According to some embodiments of the present invention, the fluorescent probe Cy5Ql-HClO is used for the detection of endogenous hypochlorous acid in the tumor microenvironment and postoperative wounds.

[0034] A fourth aspect of the present invention provides the use of the fluorescent probe Cy5Ql-HClO as described in the first aspect of the present invention in the preparation of tumor detection reagents.

[0035] A fifth aspect of the present invention provides the use of the fluorescent probe Cy5Ql-HClO as described in the first aspect of the present invention in the preparation of a postoperative wound healing monitoring reagent.

[0036] According to some embodiments of the present invention, the fluorescent probe Cy5Ql-HClO is used for tumor surgical navigation, dynamic monitoring of postoperative wound healing, in vivo inflammation imaging, and tumor tissue fluorescence imaging.

[0037] In a sixth aspect, the present invention provides an integrated kit for tumor detection and postoperative wound monitoring, the kit comprising the fluorescent probe Cy5Ql-HClO as described in the first aspect of the present invention.

[0038] The beneficial effects of this invention are:

[0039] Compared with existing hypochlorite fluorescent probes and tumor diagnostic imaging technologies, the dual PET-quenched zero intrinsic fluorescence Cy5 probe Cy5Ql-HClO of this invention has outstanding features and significant advantages in four dimensions: structural design, photophysical properties, detection mechanism, and clinical application, as detailed below:

[0040] 1) A pioneering dual-PET synergistic quenching structure achieves true zero intrinsic fluorescence. Existing probes mostly employ a single PET or ICT quenching mechanism, resulting in incomplete quenching, high background fluorescence, and difficulty in detecting low concentrations and short lifetimes of HClO. This application uses Cy5 as the fluorescent core, introduces quinolineonium at the meso site as the first PET quenching unit, and introduces dimethyl thiocarbamate as the second PET quenching unit through a self-eliminating linker. The synergistic effect of the dual pathways reduces the quantum yield of the unactivated state of the probe to below 0.001, and the background fluorescence is reduced by more than 100 times compared to traditional single PET probes, eliminating background interference at its source and achieving true zero intrinsic fluorescence.

[0041] 2) Significantly enhanced HClO specificity and signal amplification. The Cy5Ql-HClO probe exhibits a specific response only to HClO, resisting interference from various active substances such as H2O2, ROS, and RNS, demonstrating selectivity far superior to existing broad-spectrum probes. Upon reaction with HClO, the recognition group is oxidized and cleaved, triggering a self-elimination cascade reaction, simultaneously eliminating the dual PET quenching effect. Fluorescence enhancement exceeds 20-fold within 15 minutes, with a detection limit as low as 26 nM and a linear range covering 0–10 μM. It can accurately capture endogenous HClO at physiological and pathological levels, meeting the requirements for high-sensitivity in vivo detection.

[0042] 3) Cross-platform, instrument-independent quantification, overcoming the bottlenecks of traditional probe detection. Existing probes exhibit strong correlation between fluorescence signals and excitation / emission wavelengths, with signal-to-background ratios fluctuating with instrument parameters, resulting in poor repeatability of quantitative results. The probe in this application is based on the PET quenching mechanism, with the response process completely decoupled from wavelength. Detection results are highly consistent across different platforms such as fluorescence spectrometers, in vivo imaging systems, and microplate detectors, with a high linear correlation coefficient R0. 2 >0.997, achieving stable quantification independent of instrument, significantly improving the reliability of clinical translation and multi-center applications.

[0043] 4) Excellent near-infrared optical performance, making it more suitable for in vivo deep imaging. The probe uses a near-infrared Cy5 dye as a backbone, with an excitation wavelength of 580 nm and an emission wavelength of 657 nm, resulting in deep tissue penetration and low interference from biological autofluorescence. At the same time, the probe has a dual-cationic structure, excellent water solubility, no obvious aggregation at a concentration of 1 mM, strong photostability, no spontaneous hydrolysis in cell culture medium, and can be stored at low temperatures for a long time, making it suitable for non-invasive in vivo imaging and long-term monitoring.

[0044] 5) Integration of tumor diagnosis and treatment with postoperative wound monitoring, covering the entire clinical pathway. Existing probes have limited functionality, only enabling tumor imaging or wound monitoring, and cannot connect to the entire clinical process. The probe in this application can specifically target HClO, which is highly expressed in the tumor microenvironment, achieving high-contrast visualization of the tumor with a tumor / normal tissue fluorescence intensity ratio of 4:1 or higher, accurately guiding radical resection of tumors with margins <2 mm; simultaneously, it can monitor the dynamic changes of HClO during postoperative wound healing for the first time longitudinally, with fluorescence signals highly correlated with neutrophil infiltration and MPO expression, providing an integrated solution for intraoperative navigation and postoperative assessment.

[0045] 6) Highly efficient and convenient detection, meeting the needs of rapid clinical diagnosis. In vitro detection requires no complex pretreatment; readings can be obtained after incubation at 37 ℃ for 10 minutes, enabling high-throughput rapid screening in 96-well plates. Fluorescence is activated within 1 minute after local administration of the drug to the living organism, reaching peak signal in 30 minutes. The imaging speed is fast, the window is sufficient, and the efficiency of real-time intraoperative interpretation is 90% higher than that of traditional pathological slides. The detection cycle is shortened from 3 days to 30 minutes, significantly improving the efficiency of clinical diagnosis and treatment.

[0046] Other features and advantages of the invention will be set forth in the description which follows, and will be apparent in part from the description, or may be learned by practicing the invention. Attached Figure Description

[0047] The present invention will be further described below with reference to the accompanying drawings and embodiments, wherein:

[0048] Figure 1 This is a schematic diagram of the synthesis process of the probe Cy5Ql-HClO in Example 1 of the present invention;

[0049] Figure 2 This is a diagram illustrating the design strategy and response mechanism of the fluorescent probe in Example 2 of the present invention; wherein, (a) is a schematic diagram of the probe's response mechanism to HClO; (b) and (c) are density functional theory (DFT) level calculation comparison diagrams of the probe's dual PET quenching mechanism and the traditional single PET quenching mechanism; (d) is a table of spectral and photophysical performance data of the probe and intermediates; and (e) is a fluorescence enhancement response diagram of the probe and control probe under stimulation by sodium hypochlorite (NaClO) and hydrogen peroxide (H2O2).

[0050] Figure 3The following are spectral characterization diagrams of the fluorescent probe Cy5Ql-HClO in Example 3 of this invention: (a) real-time fluorescence kinetic curves of the probe under stimulation with different concentrations of NaClO; (b) absorption spectra of the probe at different concentrations of NaClO; (c) fluorescence spectra of the probe at different concentrations of NaClO; (d) fluorescence imaging of the probe in response to hypochlorous acid in a 96-well plate; (e) and (f) linear relationships between the probe fluorescence intensity ratio and NaClO concentration; (g) quantum yield characterization diagrams of the probe and intermediates; (h) dynamic light scattering (DLS) spectra of the probe before and after activation; and (i) high-performance liquid chromatography (HPLC) tracking diagram of the reaction between the probe and NaClO.

[0051] Figure 4 The images show cell imaging and activity verification of the fluorescent probe Cy5Ql-HClO in Example 4 of this invention; (a) real-time confocal fluorescence imaging of the probe in Hep3B cells; (b) cell colocalization imaging of the probe; (c) cell fluorescence imaging of the NAC inhibition experiment; (d) cell fluorescence imaging under different treatment conditions; (e) statistical graph of intracellular fluorescence intensity changes over time; (f) quantitative statistical graph of fluorescence intensity in the NAC inhibition experiment; and (g) quantitative statistical graph of fluorescence intensity in different treatment groups.

[0052] Figure 5 The following are in vivo experimental results of the fluorescent probe Cy5Ql-HClO used in Example 5 of this invention for real-time monitoring of hypochlorous acid (HClO) during mouse wound healing: (a) Schematic diagram of mouse wound model construction and experimental process; (b) Time-dependent fluorescence imaging of wound sites at different time points; (c) Quantitative statistical graph of fluorescence intensity at wound sites; (d) Time-dependent change of wound fluorescence intensity ratio (F / F0); (e) Immunohistochemistry and H&E staining of wound tissue; (f) Western blot detection of MPO protein expression in wound tissue; (g) Quantitative statistical graph of relative MPO protein expression level.

[0053] Figure 6The images show the fluorescence imaging results of the fluorescent probe Cy5Ql-HClO used in the subcutaneous Hep3B tumor model and postoperative wound in Example 6 of this invention. (a) Schematic diagram of the tumor model construction and imaging experiment; (b) Time-dependent near-infrared fluorescence imaging of the tumor site; (c) Quantitative statistical graph of fluorescence intensity between tumor and normal tissue; (d) Time-dependent fluorescence imaging of the postoperative wound site; (e) Quantitative statistical graph of fluorescence intensity at the wound site; (f) Time-dependent change graph of the wound fluorescence intensity ratio (F / F0); (g) and (h) Western blot detection of MPO protein expression in tumor tissue and postoperative wound tissue; (i) and (j) Quantitative statistical graphs of the corresponding relative MPO protein expression levels; (k) Immunohistochemistry and H&E staining of tumor tissue; (l) Immunohistochemistry and H&E staining of postoperative wound tissue.

[0054] Figure 7 This diagram illustrates the dual PET quenching mechanism, core performance advantages, and near-infrared guided tumor resection and postoperative wound monitoring applications of the fluorescent probe Cy5Ql-HClO of this invention. Detailed Implementation

[0055] The following will describe the concept and technical effects of the present invention clearly and completely with reference to embodiments, so as to fully understand the purpose, features and effects of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are all within the scope of protection of the present invention.

[0056] Unless otherwise specified in the examples, the procedures should be performed under standard conditions or conditions recommended by the manufacturer. Reagents or instruments whose manufacturers are not specified are all commercially available products.

[0057] Ethical Permissions: All animal experiments strictly comply with the Hunan Province Regulations on the Management of Laboratory Animals (License No.: SYXK (Xiang) 2020-0012), and all animal experiment protocols have been approved by the Animal Ethics Committee of Hunan Normal University (No.: 2024-185).

[0058] Example 1

[0059] This embodiment provides a method for synthesizing the dual PET-quenched hypochlorous acid-responsive fluorescent probe Cy5Ql-HClO, and the specific steps are as follows:

[0060] 1) Add 10 mL of N,N-dimethylformamide (DMF) to a round-bottom flask, and slowly add 8 mL of phosphorus oxychloride (POCl3) dropwise under ice bath conditions. After removing the ice bath, stir the mixture at room temperature for 30 min. Then add 4 mL of 4-methylquinoline (30.25 mmol), and reflux at 90 °C for 6 h. Pour the reaction mixture into ice water, adjust to alkalinity with sodium hydroxide solution, and extract with dichloromethane (DCM). After drying, concentrate the organic phase using a rotary evaporator to obtain a pale yellow solid compound (3.68 g) (E)-3-hydroxy-2-(quinoline-4-yl)propenal. The crude product does not require further purification and can be used directly in the next reaction.

[0061] 2) Take the pale yellow solid obtained in step 1) (1.00 g, 5.02 mmol), 1-ethyl-2,3,3-trimethyl-3H-indole-1-onium (2.19 g, 12.55 mmol), and a small amount of sodium acetate (AcONa), mix them, dissolve them in 20 mL of acetic anhydride (Ac2O), and reflux at 65 °C for 12 h. Remove the solvent by rotary evaporation, and purify the product by silica gel column chromatography (elution gradient: DCM / MeOH = (20~100):1) to obtain the blue solid dye Cy5Ql (468 mg, yield 20.8%).

[0062] The structural formula of dye Cy5Ql: ;

[0063] NMR and mass spectrometry characterization of Cy5Ql:

[0064] 1 H NMR (700 MHz, DMSO) δ 9.09 (s, 1H), 8.74 (m, 2H), 8.18 (d, J = 8.5Hz, 1H), 7.81 (d, J = 7.5 Hz, 1H), 7.68 (d, J = 7.7 Hz, 3H), 7.59 (d, J = 7.3Hz, 1H), 7.50 (s, 1H), 7.38 (m, 2H), 7.34 (s, 2H), 7.26 (d, J = 6.9 Hz, 2H), 5.29 (m, 2H), 3.60 (m, 4H), 1.79 (s, 12H), 0.77 (t, J = 6.4 Hz, 6H).

[0065] 13C NMR (176 MHz, DMSO) δ 173.19, 153.10, 151.23, 148.75, 142.58,141.76, 130.44, 130.26, 130.11, 129.35, 128.96, 127.64, 126.51, 125.74,125.65, 123.77, 123.09, 111.58, 101.07, 55.44, 49.65, 49.07, 40.47, 38.91,34.13, 31.77, 29.55, 29.51, 29.19, 29.05, 27.42, 27.35, 27.02, 25.59, 24.96, 22.58, 14.46, 11.85.

[0066] MS (MALDI-TOF): calc. for C 38 H 40 N3 + 538.76, found 538.466.

[0067] 3) 4-Hydroxymethylbenzaldehyde (5 g, 40.28 mmol) was mixed with dimethylthiocarbamoyl chloride (3.98 g, 32.22 mmol) and dissolved in anhydrous tetrahydrofuran (THF, 15 mL), with an appropriate amount of cesium carbonate (Cs₂CO₃) added. The mixture was stirred at 0 °C under nitrogen protection for 4 h. After filtration, the solvent was removed by rotary evaporation under reduced pressure. Sodium borohydride (NaBH₄) was added to the residue for reduction, followed by extraction and concentration by rotary evaporation. The resulting mixture was dissolved in dichloromethane (DCM, 15 mL), and phosphorus tribromide (PBr₃) was added at 0 °C, with stirring continued for 12 h. After filtration and concentration under reduced pressure, the crude product was purified by rapid silica gel column chromatography (elution gradient: DCM / MeOH = 20:1, v / v) to give a white solid O-(4-bromomethylphenyl)N,N-dimethylthiocarbamate (820 mg, yield 36%).

[0068] 4) Cy5Ql (200 mg, 0.371 mmol) and O-(4-bromomethylphenyl)N,N-dimethylthiocarbamate (152.6 mg, 0.557 mmol) were dissolved in 15 mL of anhydrous acetonitrile (ACN), and a catalytic amount of sodium iodide was added. The mixture was refluxed at 90 °C for 6 h. After the reaction was completed, the solvent was removed by rotary evaporation, and the product was purified by silica gel column chromatography (elution gradient: DCM / MeOH = (10~100):1) to obtain the target probe Cy5Ql-HClO (108 mg, yield 51%).

[0069] A schematic diagram of the synthesis process of the probe Cy5Ql-HClO is shown below. Figure 1 As shown.

[0070] Structure of probe Cy5Ql-HClO: ;

[0071] Mass spectrometry and NMR characterization of probe Cy5Ql-HClO:

[0072] 1 H NMR (700 MHz, DMSO) δ 9.94 (d, J = 5.9 Hz, 1H), 8.78 (m, 2H), 8.63 (d, J = 9.0 Hz, 1H), 8.36 (d, J = 5.9 Hz, 1H), 8.23 ​​(t, J = 7.3 Hz, 1H), 8.10(dd, J = 8.5, 1.4 Hz, 1H), 7.98 – 7.92 (m, 1H), 7.71 (dd, J = 7.4, 1.2 Hz,2H), 7.53 – 7.50 (m, 2H), 7.41 (t, J = 7.0 Hz, 2H), 7.37 (d, J = 8.0 Hz, 2H),7.33 – 7.28 (m, 2H), 7.15 (d, J = 8.6 Hz, 2H), 6.57 (s, 2H), 5.36 (m, 2H), 3.86 – 3.73 (m, 4H), 3.37 (s, 3H), 3.32 (s, 3H), 1.80 (s, 12H), 0.74 (t, J =7.3 Hz, 6H).

[0073] 13C NMR (176 MHz, DMSO) δ 186.52, 173.97, 155.78, 154.32, 152.39,151.00, 141.98, 141.67, 138.65, 136.04, 132.04, 130.79, 130.14, 129.31,129.04, 128.56, 128.49, 126.01, 125.75, 123.93, 123.41, 123.14, 122.64,120.82, 111.87, 101.27, 59.85, 49.83, 43.33, 43.29, 40.49, 40.36, 39.64, 39.02, 38.96, 31.76, 30.86, 30.31, 29.46, 29.17, 27.35, 27.29, 22.99, 22.57, 21.55, 14.43, 12.13, 11.83.

[0074] MS (MALDI-TOF): calc. for C 48 H 52 N4OS 2+ 733.03, found 731.635; HRMS,calc. for C 48 H 52 N4OS 2+ , m / z = 366.19, found 366.1929.

[0075] Example 2

[0076] This embodiment provides verification of the low background fluorescence and strong specificity of the dual PET-quenched hypochlorous acid-responsive fluorescent probe Cy5Ql-HClO.

[0077] The fluorescent probe Cy5Ql-HClO is based on the principles of synergistic optimization of dual PET quenching, HClO specific response, and bandgap and transfer gating, such as... Figure 2 As shown in Figure (a), a cationic quenching framework was constructed by using Cy5 cyanine dye, which has excellent biocompatibility and is suitable for bioimaging, as the parent core. Quinolinium was introduced at the meso site of the Cy5 parent core as the first PET quenching unit. Then, dimethyl thiocarbamate was introduced as the second PET quenching unit and HClO specific recognition group through a self-eliminating linker to design and synthesize a fluorescent probe Cy5Ql-HClO targeting hypochlorous acid (HClO).

[0078] To verify the HClO detection specificity of probe Cy5Ql-HClO and the superiority of the dual PET quenching mechanism, this embodiment simultaneously designed and synthesized a control probe Cy5Ql-H2O2. This control probe uses borate ester as the H2O2 response group, retains only the meso-position quinolineonium as a single PET quenching unit, and does not have a second PET quenching unit, thus constituting the traditional single PET quenching system described in this application.

[0079] This embodiment provides a method for synthesizing the control probe Cy5Ql-H2O2, and the specific steps are as follows:

[0080] Cy5Ql (200 mg, 0.371 mmol) and 4-bromomethylphenylboronic acid (220 mg, 0.742 mmol) were dissolved in 15 mL of anhydrous acetonitrile (ACN), and a catalytic amount of sodium iodide was added. The mixture was refluxed at 90 °C for 6 h. After the reaction was complete, the solvent was removed by rotary evaporation, and the product was purified by silica gel column chromatography (elution gradient: DCM / MeOH = 100:1 to 10:1) to obtain the control probe Cy5Ql-H2O2 (125 mg, yield 53%).

[0081] The structure of the reference probe Cy5Ql-H2O2: ;

[0082] Mass spectrometry and NMR characterization of the control probe Cy5Ql-H2O2:

[0083] 1 H NMR (700 MHz, DMSO) δ 9.94 (d, J = 5.8 Hz, 1H), 8.79 (m, 2H), 8.57(d, J = 9.1 Hz, 1H), 8.35 (t, J = 5.9 Hz, 1H), 8.19 (d, J = 9.3 Hz, 1H), 8.16(s, 1H), 8.09 (d, J = 8.4 Hz, 1H), 7.94 (t, J = 7.7 Hz, 1H), 7.84 (d, J = 8.1Hz, 1H), 7.71 (d, J = 7.3 Hz, 2H), 7.49 (d, J = 8.1 Hz, 1H), 7.41 (d, J = 7.9Hz, 4H), 7.39 (d, J = 7.6 Hz, 1H), 7.31 (t, J = 7.3 Hz, 2H), 6.58 (m, 2H), 5.37 (s, 2H), 3.82 (s, 4H), 1.81 (s, 12H), 1.30 (s, 6H), 0.76 (s, 6H).

[0084] 13 C NMR (176 MHz, DMSO) δ 173.99, 155.72, 152.43, 151.02, 141.98,141.68, 138.70, 138.03, 136.51, 136.01, 135.44, 135.17, 130.77, 129.24,129.20, 129.03, 128.50, 127.00, 126.40, 126.01, 125.76, 123.13, 120.84,111.89, 101.23, 84.37, 73.99, 60.46, 49.84, 44.57, 40.49, 40.35, 38.97, 35.58, 32.01, 31.76, 30.29, 29.44, 29.29, 29.16, 29.05, 27.35, 27.29, 25.58, 25.44, 25.13, 22.56, 19.24, 14.44, 12.15, 12.13.

[0085] MS (MALDI-TOF): calc. for C 51 H 58 BN3O2 2+ 755.85, found 754.684.

[0086] The quenching mechanism of the probe was verified by density functional theory (DFT) calculations, and the results are as follows: Figure 2 As shown in Figures (b) and (c), Cy5Ql-HClO employs a dual-PET synergistic quenching mechanism. The energy level difference ΔE between the first PET quenching unit (quinolineon) and the Cy5 parent nucleus is 0.12 eV, and the energy level difference ΔE between the second PET quenching unit (dimethylthiocarbamate) and the Cy5 parent nucleus is 0.52 eV. Both meet the energy conditions for effective PET quenching (ΔE < 0.6 eV). The synergistic effect of the dual pathways can completely suppress background fluorescence. In contrast, the traditional single-PET quenching system only has a single quenching unit, with an energy level difference ΔE = 0.85 eV, which exceeds the effective quenching threshold and cannot achieve zero background fluorescence. This directly demonstrates the technical advantages of the dual-PET mechanism of this invention.

[0087] The photophysical properties of the dye and probe were characterized, and the results are as follows: Figure 2As shown in Figure (d), the absolute fluorescence quantum yield of the Cy5 parent nucleus was 14.90%, while the quantum yield of the dye Cy5Ql obtained by quinoline ondenylation modification decreased to 8.69%. The finally constructed Cy5Ql-HClO probe had an extremely low quantum yield (not detected, below 0.001) in the unactivated state, achieving true zero intrinsic fluorescence. In contrast, the control probe Cy5Ql-H2O2 had a quantum yield of 0.73% in the unactivated state, and its background fluorescence suppression effect was significantly worse than that of Cy5Ql-HClO.

[0088] like Figure 2 As shown in Figure (e), after stimulation with 10 μM sodium hypochlorite (NaClO, HClO donor), the thiocarbamate group of Cy5Ql-HClO was oxidized and cleaved, triggering a self-elimination cascade reaction. The double PET quenching effect was completely relieved, and the fluorescence was significantly restored, with a total fluorescence enhancement of about 22 times. In contrast, the control probe Cy5Ql-H2O2 only produced about 9 times fluorescence enhancement to H2O2, verifying the high selectivity of the probe to HClO. This further demonstrates that the double PET quenching design can bring better quenching effect and response performance.

[0089] Example 3

[0090] This embodiment verifies the spectroscopic properties and reaction mechanism of the Cy5Ql-HClO probe.

[0091] 3.67 mg of probe Cy5Ql-HClO prepared in Example 1 was dissolved in 5 mL of DMF to prepare a 1 mM stock solution, which was stored at -20 °C. The detection system was PBS buffer solution (10 mM, pH 7.4, containing 1% DMF). The reaction system of probe Cy5Ql-HClO and NaClO was incubated at room temperature for 10 min, and its UV-Vis absorption and fluorescence emission spectra were measured. The excitation wavelength of the fluorometer was set to 580 nm, the emission wavelength receiving range was 600–800 nm, the slit width was 10 nm / 10 nm, and the absorption spectrum measurement range was set to 400–800 nm. The test results are as follows. Figure 3 As shown.

[0092] in, Figure 3 Figure (a) shows the real-time fluorescence kinetic curves, which demonstrate the change of fluorescence intensity of Cy5Ql-HClO (10 μM) at 657 nm over time (0~1800 s) under stimulation with different concentrations of NaClO (0, 1, 4, 7, 10 μM). The results show that the probe can reach the fluorescence plateau within 15 min under stimulation with 10 μM NaClO, and the total fluorescence enhancement is more than 20 times, proving its rapid response characteristics.

[0093] Figure 3Figures (b) and (c) show the absorption spectra and fluorescence titration curves of different concentrations of NaClO (0–10 μM), respectively. The results show that the fluorescence intensity significantly increases at 657 nm under stimulation with 10 μM NaClO, proving the probe's specific recognition response to hypochlorous acid. Figure (d) shows the fluorescence imaging of the probe in response to different concentrations of NaClO in a 96-well plate, enabling naked-eye visualization detection. Figures (e) and (f) show the dose-response curves of the activated state fluorescence intensity ratio (F / F0 at 657 nm) versus NaClO concentration (0–10 μM), corresponding to two detection platforms: a fluorescence spectrophotometer and a multimodal imaging instrument, respectively. The linear correlation coefficient R0 is shown. 2 All values ​​were greater than 0.997, demonstrating its linear quantitative ability for hypochlorous acid and its instrument-independent detection characteristics; Figure (g) shows the absolute fluorescence quantum yield (QY) of Cy5Ql dye, Cy5Ql-HClO probe, and probe + NaClO. QY was undetectable in the unactivated state, but recovered to 1.43% after activation, further demonstrating its responsiveness; Figure (h) shows the hydration particle size (DLS) spectrum of Cy5Ql-HClO (5~1000 μM), showing no significant probe aggregation and excellent water solubility; Figure (i) shows the high-performance liquid chromatography (HPLC) tracer curves of Cy5Ql-HClO (10 μM) and NaClO (10 μM) co-incubated for different times, verifying the oxidative cleavage-self-elimination reaction mechanism of the probe and hypochlorous acid. The limit of detection (LOD) was 26 nM (n=3) calculated based on 3σ / S, demonstrating that the probe not only has a rapid response but also extremely high sensitivity.

[0094] The combined results of the above tests indicate that the probe Cy5Ql-HClO has excellent specific qualitative and quantitative analytical capabilities for hypochlorous acid.

[0095] Example 4

[0096] This embodiment provides fluorescence imaging and specificity verification experiments of the Cy5Ql-HClO probe for hypochlorous acid in Hep3B cells.

[0097] 3.67 mg of the probe Cy5Ql-HClO prepared in Example 1 was dissolved in 5 mL of DMF to prepare a 1 mM stock solution, which was stored at -20 °C. The cell experiment system was DMEM complete medium containing 10% fetal bovine serum, and the working concentration of the probe was 4 μM. The Hep3B cells incubated with the Cy5Ql-HClO probe were placed under a laser confocal microscope with an excitation wavelength of 640 nm and an emission wavelength receiving range of 663–738 nm. Fluorescence images were acquired, and the test results are as follows. Figure 4 As shown

[0098] in, Figure 4 Figure (a) shows a real-time confocal fluorescence imaging image, illustrating the endogenous ClO₂ levels in Hep3B cells after incubation with 4 μM Cy5Ql-HClO for 5, 15, 30, and 60 min. - The results showed that the red fluorescence signal in the cells gradually increased with the extension of incubation time, proving that the probe can effectively enter the cells and respond to endogenous hypochlorous acid; (b) The figure shows the colocalization fluorescence imaging, which shows the cell imaging results and colocalization scatter plot of Cy5Ql-HClO co-incubated with Mito-Tracker Green and Lyso-Tracker Green, respectively. The Pearson correlation coefficient PCC with the mitochondrial probe is 0.945, and the PCC with the lysosomal probe is 0.596, which proves that the probe is mainly localized in mitochondria; (c) The figure shows the fluorescence imaging of the NAC inhibition experiment, which shows the Hep3B cells pretreated with 0, 200, 500, and 1000 μM NAC for 1 hour. Imaging results after incubation with Cy5Ql-HClO for h h showed that the cell fluorescence intensity decreased significantly with increasing NAC concentration, proving that the probe fluorescence signal is hypochlorous acid specific (N-acetylcysteine ​​(NAC) is a thiol-containing antioxidant that can undergo a specific redox reaction with hypochlorous acid (HClO) through its thiol group, efficiently clearing endogenous HClO from cells. After NAC pretreatment, the intracellular HClO level decreased significantly, and the probe Cy5Ql-HClO could not be activated, resulting in a significantly weakened fluorescence signal, thus verifying the probe's specificity in response to intracellular HClO); (d) Figure shows cell fluorescence imaging under different treatment conditions. The first column is the blank control group, the second column is the APAP (1000 μM) treatment group, the third column is the NaClO (5 μM) treatment group, and the fourth column is the NAC (1000 μM) pretreatment group. The results showed that both APAP and exogenous NaClO could significantly enhance cell fluorescence, while NAC could effectively inhibit fluorescence recovery, further verifying the probe's specificity in response to ClO. -The specific response of acetaminophen (APAP) to cellular oxidative stress, glutathione (GSH) consumption, and myeloperoxidase (MPO) activation significantly promotes the production of endogenous hypochlorous acid. After APAP pretreatment, the intracellular HClO level increased, and the probe Cy5Ql-HClO was specifically activated, with a significantly enhanced fluorescence signal, further verifying the probe's specific response to endogenous HClO. (e) Figure is the statistical graph of the average fluorescence intensity of cells in Figure (a), showing that the fluorescence intensity increases in a time-dependent manner with incubation time. (f) Figure is the statistical graph of the relative fluorescence intensity (F / F0) of cells in Figure (c), and Figure (g) Figure is the statistical graph of the relative fluorescence intensity (F / F0) of cells in Figure (d). The statistical results show that the differences between the groups are significant (*P < 0.1, **P < 0.01, ***P < 0.001, ****P < 0.0001, ns indicates no significant difference), proving that the probe can accurately quantify the intracellular hypochlorous acid level.

[0099] The combined results of the above cell experiments indicate that the probe Cy5Ql-HClO has excellent specific imaging ability for intracellular hypochlorous acid and can be used for real-time and quantitative detection of endogenous hypochlorous acid in living cells.

[0100] Example 5

[0101] This embodiment provides a method for monitoring the real-time in vivo fluorescence of hypochlorous acid using a Cy5Ql-HClO probe in a mouse wound healing model.

[0102] The Cy5Ql-HClO probe was dissolved in PBS at pH 7.4 to prepare a 50 μM working solution for fluorescence imaging studies in a mouse wound healing model. SPF-grade BALB / c mice were used as experimental animals. A full-thickness dorsal skin wound model was constructed for in vivo hypochlorous acid (ClO) imaging. - Real-time monitoring of ).

[0103] Mice were anesthetized with isoflurane, and a uniform full-thickness skin wound was created on their backs. They were then fed as usual. On Day 1, Day 2, Day 2 + NAC treatment group, and Day 3, 50 μM Cy5Ql-HClO probe was sprayed onto the wound site. In vivo fluorescence imaging (λ) was used to visualize the wound. ex =605 nm, λ em Fluorescence images were acquired at different time points (1 min, 5 min, 10 min, 30 min, 60 min, 90 min) using a fluorescence sensor (nm = 640-660 nm). The test results are as follows: Figure 5 As shown.

[0104] in, Figure 5 Figure (a) shows a schematic diagram of the mouse wound model construction and experimental process, illustrating the complete experimental procedure of modeling, probe administration, NAC control, in vivo imaging, and tissue sampling; Figure (b) shows in vivo fluorescence imaging at different time points. The results show that obvious fluorescence appeared at the wound site on Day 1, and the fluorescence intensity gradually increased with the healing process (Day 2, Day 3), while the fluorescence of the NAC-treated group was significantly weakened, proving that the fluorescence signal originated from the highly expressed ClO at the wound site. - (c) The figure shows a quantitative statistical graph of the fluorescence radiation intensity at the wound site, which intuitively presents the trend of fluorescence intensity changes at different days and time points. The fluorescence intensity on Day 2 and Day 3 is significantly higher than that on Day 1, and the fluorescence intensity of the NAC group is significantly lower than that of the control group at the same time point. (d) The figure shows the time-dependent change curve of the wound fluorescence intensity ratio (F / F0), which further verifies the presence of ClO at the wound site. - The dynamic increasing trend of MPO levels during the healing process; (e) shows the immunohistochemistry and H&E staining of wound tissue, which shows that Ly6G-positive neutrophil infiltration is obvious in the wound site and MPO expression level is significantly increased, which is highly consistent with the trend of fluorescence signal; (f) shows the Western blot detection of MPO protein expression in wound tissue; (g) shows the quantitative statistical graph of MPO relative protein expression level, which shows that MPO protein expression in wound tissue is significantly upregulated on Day 2, which is consistent with the results of in vivo fluorescence imaging, further proving the specificity and reliability of probe imaging.

[0105] The above mouse wound model results indicate that the probe Cy5Ql-HClO can induce ClO during wound healing at the in vivo level. - It provides non-invasive, real-time, and specific fluorescence monitoring, with fluorescence signals highly correlated with the degree of wound inflammation, demonstrating excellent potential for in vivo applications.

[0106] Example 6

[0107] This embodiment provides fluorescence imaging experiments using the Cy5Ql-HClO probe in a subcutaneous Hep3B tumor model and postoperative wound.

[0108] The Cy5Ql-HClO probe was dissolved in PBS at pH 7.4 to prepare a 50 μM working solution, which was used for fluorescence imaging studies of subcutaneous Hep3B tumor models and postoperative wounds.

[0109] A subcutaneous Hep3B tumor model was constructed in BALB / c nude mice. After the tumor grew to a suitable size, fluorescence imaging of the tumor site and postoperative wound monitoring were performed. Following intratumoral injection of 50 μL Cy5Ql-HClO (50 μM), in vivo near-infrared fluorescence imaging (λ) was used. ex =605 nm, λem Time-series fluorescence images were acquired from 1 to 120 minutes at a wavelength of 640-660 nm. After tumor resection, Cy5Ql-HClO was applied topically on postoperative days 1, 2, and 3, and fluorescence images of the wound site were acquired. The test results are as follows: Figure 6 As shown.

[0110] in, Figure 6 Figure (a) shows a schematic diagram of the experimental procedure for tumor model construction, intratumoral injection, in vivo imaging, and postoperative wound monitoring; Figure (b) shows a time-dependent fluorescence imaging map of the tumor site, showing that the fluorescence intensity of the tumor area (red) increases rapidly over time after probe injection, while the fluorescence signal of normal tissue (blue) is extremely low, clearly defining the tumor boundary; Figure (c) shows a quantitative statistical graph of the fluorescence radiation intensity of tumor and normal tissue, showing that the fluorescence intensity of the tumor site is significantly higher than that of normal tissue, demonstrating high-contrast imaging capability; Figure (d) shows a time-dependent fluorescence imaging map of the postoperative wound site, showing that the fluorescence intensity of the postoperative wound site gradually increases over time, reflecting postoperative inflammation and hypochlorous acid (ClO₂). - (e) Figure shows the quantitative statistical graph of fluorescence radiation intensity at the wound site, which intuitively presents the dynamic changes in fluorescence intensity at the wound site from day 1 to day 3 after surgery; (f) Figure shows the time-dependent change curve of the fluorescence intensity ratio (F / F0) between the tumor and the wound site, which further verifies the response characteristics of the probe; (g) and (h) Figures are Western blot detection diagrams of MPO protein expression in tumor tissue and postoperative wound tissue, respectively; (i) and (j) Figures are quantitative statistical graphs of the relative protein expression level of MPO, which show that MPO protein expression in both tumor tissue and postoperative wound tissue is significantly upregulated, which is consistent with the fluorescence imaging results; (k) Figure shows the immunohistochemistry and H&E staining of tumor tissue, and (l) Figure shows the immunohistochemistry and H&E staining of postoperative wound tissue, which show that both tumor and wound tissue have obvious neutrophil infiltration and high MPO expression, which further proves the specificity and reliability of probe imaging.

[0111] The results of the above tumor model and postoperative wound experiments show that the probe Cy5Ql-HClO can achieve high-contrast near-infrared fluorescence imaging of subcutaneous Hep3B tumors, and can also be used for real-time monitoring of postoperative wound inflammation. It integrates precise tumor imaging and postoperative wound assessment, and has important application value in precision tumor surgery and postoperative management.

[0112] In summary, such as Figure 7The diagram illustrates the dual PET quenching response mechanism, core performance advantages, and near-infrared guided tumor resection and postoperative wound monitoring application mode of the Cy5Ql-HClO fluorescent probe of this invention. This invention discloses a novel probe, Cy5Ql-HClO. This hypochlorous acid (HClO)-based fluorescent probe achieves highly sensitive quantitative detection of inflammation-related hypochlorous acid and endogenous HClO. - This probe enables integrated and precise fluorescence monitoring of imaging and tumors / wounds. Through its dual PET quenching structure design, hypochlorous acid-specific oxidative cleavage-self-elimination cascade response, and excellent quantitative detection performance, it overcomes the key limitations of traditional hypochlorous acid probes—high inherent background fluorescence, poor signal enhancement, and poor consistency across multiple platforms.

[0113] The innovative design of the Cy5Ql-HClO fluorescent probe of this invention is reflected in the following aspects: (1) Through double PET quenching and charge conversion, extremely low intrinsic fluorescence is achieved in the unactivated state, reducing background interference from the source; (2) Under the action of hypochlorous acid, specific oxidation cleavage occurs, triggering a self-elimination cascade reaction, relieving the PET quenching process, significantly restoring fluorescence, and greatly improving the detection signal-to-noise ratio; (3) It shows good quantitative relationship under multiple platforms such as fluorescence spectrophotometer and in vivo imaging system, and the detection results are highly consistent, which can be applied to various scenarios such as in vitro high-throughput detection and in vivo in vivo imaging.

[0114] The aforementioned structural and response characteristics resulted in a 22-fold increase in fluorescence of Cy5Ql-HClO within 15 minutes under the action of 10 μM sodium hypochlorite (NaClO), significantly improving the sensitivity and specificity of detection. The Cy5Ql-HClO fluorescent probe of this invention can also detect endogenous hypochlorous acid in cells with high specificity, achieving real-time fluorescence imaging in Hep3B cells, with the fluorescence signal highly correlated with the level of cellular oxidative stress. At the in vivo level, the Cy5Ql-HClO probe of this invention can achieve non-invasive real-time monitoring of hypochlorous acid during wound healing in mice, with the fluorescence signal highly consistent with the degree of wound inflammation (neutrophil infiltration, MPO expression); simultaneously, it can be used for high-contrast near-infrared fluorescence imaging of subcutaneous Hep3B tumors, clearly defining tumor boundaries, achieving precise tumor resection, and further used for dynamic monitoring of postoperative wound inflammation, integrating tumor navigation and postoperative assessment.

[0115] Based on the advantages of the Cy5Ql-HClO fluorescent probe, this invention provides a reliable tool for integrated diagnosis and treatment of inflammation-related diseases, precision oncology surgery, and postoperative management. It can monitor the dynamic changes of hypochlorous acid in real time to guide personalized treatment. By combining molecular engineering with clinical application, this Cy5Ql-HClO probe opens up new avenues for non-invasive inflammation monitoring, precision oncology diagnosis and treatment, and postoperative healing assessment.

[0116] The embodiments of the present invention have been described in detail above. However, the present invention is not limited to the above embodiments. Within the scope of knowledge possessed by those skilled in the art, various changes can be made without departing from the spirit of the present invention. Furthermore, the embodiments of the present invention and the features thereof can be combined with each other unless otherwise specified.

Claims

1. A fluorescent probe Cy5Ql-HCIO for detecting hypochlorous acid, characterized in that, The structural formula is as follows: 。 2. The method for preparing fluorescent probe Cy5Ql-HClO according to claim 1, characterized in that, Includes the following steps: S1. 4-hydroxybenzaldehyde and dimethylthiocarbamoyl chloride are mixed in a solvent and reacted. After the reaction is completed, the solvent is removed, a reducing agent is added for reduction, and then the mixture is extracted and concentrated. The resulting mixture is mixed with phosphorus tribromide in a solvent and the reaction is continued. After the reaction is completed, the purified compound is: O-(4-bromomethylphenyl)N,N-dimethylthiocarbamate. S2. 4-Methylquinoline was added to a mixture of phosphorus oxychloride and N,N-dimethylformamide and heated to react, yielding the intermediate (E)-3-hydroxy-2-(quinoline-4-yl)propenal. The intermediate was mixed with 1-ethyl-2,3,3-trimethyl-3H-indole-1-onium in a solvent and the reaction was continued under heating. After the reaction was completed, the product was purified to obtain the dye Cy5Ql. S3. Mix Cy5Ql and O-(4-bromomethylphenyl)N,N-dimethylthiocarbamate in a solvent and heat to react. After the reaction is complete, purify to obtain the fluorescent probe Cy5Ql-HClO.

3. The production method according to claim 2, characterized by, In step S1, the molar ratio of 4-hydroxybenzaldehyde to dimethylthiocarbamoyl chloride is (1~1.5):

1.

4. The preparation method according to claim 2, characterized in that, In step S2, the volume ratio of 4-methylquinoline, phosphorus oxychloride and N,N-dimethylformamide is 1:(1.5~2.5):(2~3); the molar ratio of 4-methylquinoline and 1-ethyl-2,3,3-trimethyl-3H-indole-1-onium is (2~3):

1.

5. The preparation method according to claim 2, characterized in that, In step S3, the molar ratio of Cy5Ql to O-(4-bromomethylphenyl)N,N-dimethylthiocarbamate is 1:(1.2~2).

6. The application of the fluorescent probe Cy5Ql-HClO as described in claim 1 in the detection of hypochlorous acid (HClO).

7. Use according to claim 6, characterized in that, The fluorescent probe Cy5Ql-HClO is used to detect endogenous hypochlorous acid in the tumor microenvironment and postoperative wounds.

8. The application of the fluorescent probe Cy5Ql-HClO as described in claim 1 in the preparation of tumor detection reagents.

9. The application of the fluorescent probe Cy5Ql-HClO as described in claim 1 in the preparation of a postoperative wound healing monitoring reagent.

10. An integrated kit for tumor detection and postoperative wound monitoring, characterized in that, The kit contains the fluorescent probe Cy5Ql-HClO as described in claim 1.

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