PH-viscosity dual-response fluorescent probe, preparation method and application

Abstract

The invention provides a PH-viscosity dual-response fluorescent probe, a preparation method and application, belongs to the technical field of fluorescent probes, and is technically characterized in that the structural formula of a probe molecule is shown in the specification. By optimizing a fluorescent skeleton and hydrophilic modification and calibrating pH / viscosity response mapping, the application can be used for rapidly screening a blood coagulation function, evaluating an anticoagulation treatment effect or researching and developing a portable thrombus detection device.

Description

Technical Field

[0001] This invention belongs to the technical field of fluorescent probes, specifically relating to a pH-viscosity dual-response fluorescent probe, its preparation method, and its application. Background Technology

[0002] Thrombosis detection mainly employs methods such as ultrasound imaging, CT angiography, magnetic resonance angiography, and angiography. However, these imaging methods cannot detect early-stage microthrombi.

[0003] In recent years, real-time fluorescence / optical detection methods based on molecular probes have attracted attention (e.g., CN114874142B, “Xu Huiting, Ni Jianming, Han Cuiping, et al. Preparation and cell imaging of fluorescence-magnetic resonance dual-modal nanoprobes targeting ovarian cancer cells [J]. Chinese Journal of Nuclear Medicine and Molecular Imaging, 2019, 39(4):5”).

[0004] However, fluorescent molecular probes for thrombosis detection have not yet been studied.

[0005] When fluorescent molecular probes were used for real-time detection of thrombosis and coagulation processes, the research team discovered that two significant changes simultaneously occur in the local microenvironment during thrombosis: first, the pH value typically shifts towards acidity (due to metabolic and coagulation-related enzyme activity); second, local rheological properties change significantly, with clot formation causing a sharp increase in local micro-region viscosity. Therefore, designing a dual-response probe that simultaneously responds to pH and viscosity to detect thrombosis time has become a novel research and development approach.

[0006] The existing dual-response probes for pH and viscosity are summarized in Table 1 below.

[0007] In summary, there is a lack of fluorescent molecular probes for thrombosis detection in the current technology. Summary of the Invention

[0008] The purpose of this invention is to solve the problems existing in the prior art and provide a pH-viscosity dual-response fluorescent probe.

[0009] Another objective of this application is to provide a method for preparing a pH-viscosity dual-responsive fluorescent probe.

[0010] Another object of this application is to provide an application.

[0011] A pH-viscosity dual-responsive fluorescent probe for detecting thrombus formation time, the structural formula of the probe molecule is as follows: .

[0012] Furthermore, the excitation wavelength of the pH-viscosity dual-response fluorescent probe is 630 nm, and the fluorescence emission wavelength of the pH-viscosity dual-response fluorescent probe is 670 nm.

[0013] A method for preparing a pH-viscosity dual-responsive fluorescent probe for detecting thrombus formation time, comprising the following steps: Step A: Synthesize compound I: The solution of DMF and DCM was placed in an ice-water bath, and phosphorus oxychloride was added dropwise with stirring, followed by cyclohexanone. The mixture was refluxed for 6 hours, cooled, poured into ice water, stirred, filtered, and dried under vacuum to obtain compound I. The reaction formula is as follows: Step B: Synthesize compound II: 2,3,3-Trimethyl-3H-indole and 1,4-butanesulfonate lactone were dissolved in toluene and reacted at 80 °C for 24 h under N2 atmosphere. After cooling, the solvent was removed by rotary evaporation. The residue was dissolved in methanol and added dropwise to ethyl acetate with stirring. The mixture was filtered and dried under vacuum to obtain compound II. The reaction formula is as follows: Step C: Synthesize compound III: Compounds I and II were dissolved in anhydrous n-butanol and toluene, and refluxed at 130 °C for 4 h under N2 protection. After purification, compound III was obtained. The reaction formula is as follows: Step D: Synthesis of the pH-viscosity dual-responsive fluorescent probe Cy: Compound III and NaOAc were dissolved in acetic anhydride and refluxed at 100°C for 6 h under N2 protection. After cooling to room temperature, the solution was diluted with dichloromethane and washed successively with water and physiological saline. Then, the solution was dried over anhydrous sodium sulfate, filtered, and concentrated by rotary evaporation to obtain a crude product. The crude product was purified by silica gel column chromatography to obtain solid powder Cy. The reaction formula is as follows: .

[0014] Based on the aforementioned application of a pH-viscosity dual-response fluorescent probe in the preparation of thrombosis detection reagents.

[0015] Based on the aforementioned application of a pH-viscosity dual-response fluorescent probe in screening and / or studying anticoagulants.

[0016] The advantages of the technical solution of this invention are mainly reflected in: First, this application provides a pH-viscosity dual-responsive fluorescent probe. Its research and development needs and solutions are as follows: a. Solving the pH response design problem. Upper limit of pH response: Without thrombus formation in the blood, the pH value will not exceed 7.45. Lower limit of pH response: When a thrombus forms, the pH can drop significantly to 6.8. Therefore, the pH response range determined in this application should be between 6.8 and 7.45. Thus, using the baseline (7.4) as the starting point of the response, any enhancement of the signal clearly points to "acidification." The solution to the above research and development needs is to introduce a proton-receptible weak base group (pyridine) into one end of the fluorescent backbone. The nitrogen atom on the pyridine ring possesses a lone pair of electrons, capable of binding protons. The balance between protonation and deprotonation determines the pH response range of the probe. Its molecular center, cyclohexanone, contains a carbonyl group (C=O) bridging structure. Under alkaline conditions, the carbonyl group can be partially converted into an enol group (–C(OH)=CH–), increasing the degree of conjugation. Under acidic conditions, it exists in the form of a carbonyl group, and the degree of conjugation is weakened, which is manifested by a significant shift in absorption peak position and color change (e.g., acidic blue-green → basic pink).

[0017] b. Addressing the issue of maintaining a linear response in the low viscosity range. When the overall viscosity change is small (i.e., in the low viscosity range), the probe's brightness change during thrombus formation is proportional to the thrombus density / maturity (i.e., local viscosity) (linear relationship).

[0018] If a molecular probe lacks linear response capability, it can only perform qualitative analysis on the presence or absence of a thrombus. However, it cannot determine the "quality" of the thrombus formation.

[0019] A linear response, in in vitro experiments, yields the key parameter of the "fluorescence intensity-time curve" slope. This reflects the rate of thrombus formation and plays an important role in evaluating the efficacy of anticoagulants.

[0020] Linear response can detect early microthrombi. In the early stages of thrombus formation, viscosity changes are small and gradual. A probe with a sensitive linear response in the low viscosity range is needed.

[0021] The solution to this problem in this application is as follows: b.1 Design of the conjugate chain and cyanine rotor skeleton.

[0022] When environmental constraints are slight, the rotational motion of the conjugated chain is significantly affected, leading to a substantial change in the fluorescence quantum yield. This design enables significant signal variations even at low viscosity levels.

[0023] Meanwhile, the indole and pyridine rings at both ends of the cyanine rotor framework form a powerful push-pull electron system. This structure makes the excited-state energy of the molecule highly dependent on the rotor's twist angle. A tiny rotational confinement can cause a huge change in the excited-state energy, thus drastically altering the fluorescence efficiency.

[0024] b.2 The introduction of sulfonic acid groups solves the water solubility problem and also provides targeted localization. That is, it can increase the effective local concentration: at the site of the thrombus, the probe concentration is much higher than the average concentration in the blood. It senses the "real" microenvironment: the probe no longer senses the viscosity of the overall blood, but directly senses the extremely high local viscosity within the fibrin network. It can also generate a synergistic response: a large number of probe molecules are simultaneously confined within the clot, producing a collective, amplified fluorescence signal. This allows the system to convert minute physical changes (a slight increase in macroscopic viscosity) into a powerful, detectable optical signal.

[0025] c. A balance is achieved between water solubility, stability, and responsiveness. Cyanide base dyes have large π-conjugated backbones and strong hydrophobicity, making them prone to self-aggregation in aqueous environments (forming H / J aggregates), leading to fluorescence quenching or spectral drift.

[0026] To address this issue, sulfonic acid groups were introduced into the design of the molecular probe to improve its dispersibility and prevent it from self-aggregating in an aqueous environment.

[0027] d. The spectral characteristics should match the biological detection window. Ideally, the excitation light of a fluorescent probe for thrombosis detection should be in the near-infrared region, i.e., a wavelength of 700–850 nm. However, elongating the conjugated chain of the dye can lead to decreased photostability and increased background signal.

[0028] Solving this problem requires a systematic approach: d.1, Introducing a pyridine ring to construct a "strong donor-π-acceptor" system. Near-infrared emission is achieved with a shorter conjugated chain, avoiding excessive chain length extension.

[0029] d.2, Introducing a rigid ring structure into the conjugated chain. This suppresses the dissipation of excited-state energy as heat, significantly enhancing the resistance to photobleaching.

[0030] d.3, strong hydrophilic sulfonic acid groups are linked via flexible alkyl chains. This prevents aggregation quenching, reduces non-specific binding, and achieves a high signal-to-noise ratio.

[0031] e. Improve the stability and signal specificity of molecular probes. Traditional cyanine probe dyes often carry a positive charge, which makes them prone to non-specific binding to plasma proteins, cell membranes, or the negatively charged platelet surface, resulting in high signal background and poor response reproducibility.

[0032] To address this issue, a design that combines "introducing sulfonic acid groups to form electrically neutral internal salts" with "rigidifying the core framework and regulating electronic effects to ensure both photostability and chemical stability" is employed to solve the problem.

[0033] f. Thrombosis involves fibrin aggregation, erythrocyte capture, and plasma protein cross-linking. Macroscopically, this is accompanied by changes in microviscosity and polarity, but these changes do not necessarily correspond linearly to a single spectral signal. Probe signal changes can be affected by multiple factors (pH, ionic strength, protein binding, redox state), leading to complex analysis and poor reproducibility.

[0034] Second, this application proposes a reproducible synthesis of a pH-viscosity dual-response fluorescent probe and a complete operational protocol for its use in in vitro thrombosis time detection. By optimizing the fluorescent backbone and hydrophilic modification, and calibrating the pH / viscosity response mapping, this method can be used for rapid screening of coagulation function, evaluation of anticoagulation therapy efficacy, or development of portable thrombosis detection devices. Attached Figure Description

[0035] The present invention will be further described in detail below with reference to the embodiments shown in the accompanying drawings, but this does not constitute any limitation on the present invention.

[0036] Figure 1 The diagram illustrates the verification of the probe's acidic response and the reaction mechanism.

[0037] Figure 2 The UV absorption of the probe under different pH conditions is illustrated.

[0038] Figure 3 The fluorescence intensity of the probe under different pH conditions is illustrated.

[0039] Figure 4 The fluorescence intensity of the probe under different viscosity conditions is shown (the percentage represents the glycerol doping ratio). Detailed Implementation

[0040] The objectives, advantages, and features of this invention will be explained through the following non-limiting description of preferred embodiments. These embodiments are merely typical examples of applying the technical solutions of this invention, and all technical solutions formed by equivalent substitutions or equivalent transformations fall within the scope of protection claimed by this invention.

[0041] Analysis of R&D needs and challenges (1) pH response design is difficult The pH of the human blood system is normally maintained between 7.35 and 7.45; a pH below 7.35 indicates acidosis. Lower limit of pH response: Although the overall pH of human blood is stable, the microenvironment of thrombus formation, due to thrombin activation, platelet activation, and metabolic bursts (which consume large amounts of oxygen and produce lactic acid), creates an acidic microenvironment. Within or immediately adjacent to a clot, the pH can drop significantly to 6.8. Upper limit of pH response: Without a thrombus, the pH of the blood will not exceed 7.45. When there is no thrombus in the blood, the probe needs to output a baseline signal.

[0042] Therefore, the pH response range determined in this application should be between 6.8 and 7.45. Thus, taking the baseline (7.4) as the starting point of the response, any enhancement of the signal clearly points to "acidification". However, the acid-base responses of existing pH-responsive probes are often observed in the pH range of 4–6. There is a lack of existing technological references on how to respond precisely within this pH range and how to adapt to changes in pH.

[0043] (2) The contradiction between viscosity response and the complexity of the blood system Existing molecular probes, when responding to viscosity, rely on changes in intramolecular rotational (TICT) or aggregation state. Specifically, they only produce significant fluorescence changes when the apparent viscosity of the solution is high. The apparent viscosity of blood plasma is approximately 1.2–1.5 mPa·s; even with the presence of microthrombi, as long as the bulk blood remains fluid, the overall viscosity value does not change significantly. In such cases, existing fluorescence enhancement is minimal, resulting in a lack of sensitivity.

[0044] The viscosity response requirement is that the probe needs to "light up" during thrombus formation, and its brightness change should be proportional (linearly related) to the density / maturity of the thrombus (i.e., local viscosity). This necessitates that the probe also exhibits a smooth and significant response gradient in the low viscosity range. In other words, maintaining a linear response in the low viscosity range is another major challenge of this application.

[0045] (3) It is difficult to balance water solubility and aggregation.

[0046] Molecular structure requirements: In order to have excellent fluorescence and viscosity response (molecular rotor) properties, a large-area, rigid π-conjugated framework is required.

[0047] Working environment requirements: In order to work effectively in the blood, molecules must be hydrophilic, soluble, and able to be uniformly dispersed.

[0048] However, cyanine base dyes have large π-conjugated backbones and strong hydrophobicity, making them prone to self-aggregation in aqueous environments (forming H / J aggregates), leading to fluorescence quenching or spectral drift.

[0049] Achieving a balance between water solubility, stability, and responsiveness is a key challenge in molecular structure optimization.

[0050] (4) Matching spectral characteristics with biological detection window Hemoglobin in the blood terminal layer exhibits very strong absorption of light with wavelengths of 400–600 nm. If the probe operates in this region, the excitation light cannot effectively reach the probe, resulting in no detectable signal. Furthermore, many biomolecules produce background fluorescence under ultraviolet and visible light excitation, which can mask the probe's signal. Therefore, the ideal excitation light for a fluorescent probe for thrombosis detection should be in the near-infrared region, specifically with wavelengths of 700–850 nm.

[0051] However, the most common way to achieve a shift of the spectrum to the near-infrared region is to lengthen the conjugated chain of the dye. However, the lengthening of the conjugated chain leads to: decreased photostability and increased background signal (the lengthening of the conjugated chain leads to a significant increase in the hydrophobicity of the molecule, which in turn leads to molecular aggregation).

[0052] Therefore, a trade-off needs to be struck between "redshifting the emission wavelength" and "maintaining optical stability" in the design.

[0053] (5) Selectivity and stability in complex biological systems Traditional cyanine probe dyes often carry a positive charge, making them prone to non-specific binding to plasma proteins, cell membranes, or the negatively charged surface of platelets, resulting in high background signals and poor response reproducibility. Furthermore, they may be degraded or bleached under oxidative, esterase, or thiol conditions, making it difficult to maintain stable detection over long periods.

[0054] Therefore, improving the stability and signal specificity of molecular probes is also a key issue.

[0055] (6) It is difficult to verify the correspondence between the response mechanism and physiological events. Thrombosis involves fibrin aggregation, erythrocyte capture, and plasma protein cross-linking. Macroscopically, this is accompanied by changes in microviscosity and polarity, but these changes do not necessarily correspond linearly to a single spectral signal. Probe signal variations can be affected by multiple factors (pH, ionic strength, protein binding, redox state), leading to complex analysis and poor reproducibility.

[0056] Example 1: A pH-viscosity dual-response fluorescent probe A pH-viscosity dual-responsive fluorescent probe, the structure of which is shown below: .

[0057] The pH-viscosity dual-responsive fluorescent probe comprises three parts: (1) Fluorescent backbone: Dyes with molecular rotor characteristics [rich in polymethylene chains (-CH=CH-)] are selected as viscosity-responsive units; their fluorescence is weak at low viscosity and significantly enhanced at high viscosity; (2) pH-responsive site: A proton-receptible weak base group (pyridine and cyclohexanone) is introduced at one end of the fluorescent backbone. This site causes the molecule to undergo spectral / quantum yield changes in an acidic environment. (3) Water solubility / localization modification: Introduce hydrophilic chains (sulfonic acid groups) to improve dispersibility and local enrichment in blood. Ensure probe solubility in blood and local enrichment at thrombus sites.

[0058] The mechanism of action of the pH-viscosity dual-response fluorescent probe in Example 1: The probe exhibits low fluorescence output at low viscosity and weak alkalinity (or physiological pH); when the local pH decreases (acidification) and / or the viscosity increases (increased micro-region viscosity caused by clot formation), the probe fluorescence is significantly enhanced.

[0059] like Figure 1 As shown, under neutral to slightly alkaline conditions (pH=7.4), its color is purplish-red. With the addition of acid to the system, the color also turns light green, thus proving the pH response and indication ability of the probe synthesized in this application.

[0060] like Figure 2 As shown, during the pH range from 8.0 to 5.5, the UV absorption peak at 520 nm gradually decreases, while the intensity of the absorption peak at 720 nm gradually increases. Such a large Stokes shift reduces tissue self-absorption, improves signal resolution, enhances tissue penetration, and minimizes background noise during probe monitoring of thrombus formation, ultimately improving monitoring effectiveness.

[0061] like Figure 3 As shown in the fluorescence emission spectra under different pH conditions, the fluorescence emission intensity at 750 nm under 720 nm laser irradiation also increases with decreasing pH value, increasing by 25 times within the tested pH range, demonstrating the probe's excellent acid-base indication performance.

[0062] like Figure 4 As shown in the fluorescence emission spectra under different viscosity conditions, as the glycerol ratio increases from 50% to 100%, the fluorescence intensity also increases by 1.4 times, demonstrating that Cy has good viscosity response and indication performance.

[0063] Example 2: A method for preparing a pH-viscosity dual-responsive fluorescent probe Reagents and materials required for synthesis (1) Cyclohexanone, phosphorus oxychloride (POCl3), 2,3,3-trimethyl-3H-indole, 1,4-butanesulfonate lactone, sodium acetate (NaOAc), n-butanol and toluene (2) Consumables: No RNA / DNA enzyme-grade solvents (DMF, DMSO, MeOH), bases (triethylamine, TEA), coupling reagents (EDC / NHS), etc.; (3) Purification: silica gel column chromatography, gel dialysis (MWCO selects accordingly); (4) Characterization: UV-Vis, fluorescence spectrometer.

[0064] The synthetic route of a pH-viscosity dual-responsive fluorescent probe is as follows: .

[0065] The preparation method of the pH-viscosity dual-responsive fluorescent probe in Example 2 includes the following steps: Step A: Synthesize the fluorescent backbone – rotor module; A solution of N,N-dimethylformamide (DMF, 40 mL) and dichloromethane (DCM, 40 mL) was placed in an ice-water bath. Phosphorus oxychloride (38 mL, 0.407 mol) was added dropwise with stirring, followed by cyclohexanone (10.00 g, 0.102 mol). The mixture was refluxed for 6 hours. After cooling, the solution was poured into 200 mL of ice water and stirred overnight. The mixture was filtered and dried under vacuum to obtain a yellow solid, compound I.

[0066] Step B: Introduce pH-responsive side chain blocks; 30 mmol of 2,3,3-trimethyl-3H-indole and 45 mmol of 1,4-butanesulfonate lactone were dissolved in 40 mL of toluene and reacted at 80 °C for 24 h under N2 atmosphere. After cooling, the solvent was removed by rotary evaporation. The residue was dissolved in methanol and added dropwise to ethyl acetate, stirred for 1.5 h, and sonicated for 3 h. The mixture was filtered and dried under vacuum to give a purple precipitate, compound II.

[0067] Step C: Hydrophilization modification; 1 mmol of compound 1 and 2 mmol of compound 2 were dissolved in anhydrous n-butanol (60 mL) and toluene (25 mL), and refluxed at 130 °C for 4 h under N2 protection. After purification, a purple-red solid compound III was obtained.

[0068] Step D: Final purification and characterization; 0.3 mmol of compound III and 1.2 mmol of NaOAc were dissolved in 10 mL of acetic anhydride and refluxed at 100 °C for 6 h under N2 protection. After cooling to room temperature, the solution was diluted with 40 mL of dichloromethane and washed successively with water and physiological saline. The solution was dried over anhydrous sodium sulfate, filtered, and concentrated by rotary evaporation.

[0069] The crude product was purified by silica gel column chromatography (DCM / MeOH=20:1, v / v) to obtain a dark red solid powder Cy.

[0070] Stock solution (for long-term stable storage): Dissolve the probe in DMSO to prepare a 10 mM stock solution, aliquot and store at -20°C protected from light.

[0071] Working solution (use immediately): Dilute the stock solution to 2-0 μM with PBS (pH 7.4); final DMSO concentration ≤1% (volume fraction).

[0072] Example 3, Application Instructions The method in Example 3 can be used to study anticoagulants, which can be studied using comparative experiments.

[0073] (1) Blood collection: Venous blood is collected from healthy volunteers or laboratory animals. The blood is collected into a vacuum blood collection tube containing an anticoagulant. The anticoagulant is 3.2% or 3.8% sodium citrate, which prevents the initiation of the clotting process by binding with calcium ions in the blood.

[0074] (2) Preparation of reagents and instruments 2.1 Preparation of probe working solution: Remove the DMSO stock solution of the probe (stored at -20°C) and thaw at room temperature. Serially dilute the stock solution with phosphate-buffered saline (PBS, pH 7.4) to prepare working solutions of the desired concentrations. Ensure the final concentration of DMSO in the working solution is ≤1% to avoid cytotoxicity.

[0075] 2.2 Preparation of coagulation triggering agents: A calcium chloride solution of a specific concentration is prepared to reverse anticoagulation and restart the coagulation process. The final concentration of the calcium chloride solution is 10-25 mM.

[0076] 2.3, Drug treatment: Pre-incubate plasma with different candidate drug solutions (or different concentrations of the same drug) (e.g., incubate at 37°C for 5–10 minutes). Set up a blank control group (with buffer added only) and a positive control group (e.g., heparin at a known concentration).

[0077] 2.4 Setting up the instrument Turn on the fluorescence microplate reader and preheat it to 37°C to simulate the core temperature of the human body.

[0078] Set the fluorescence detection parameters: excitation wavelength Ex and emission wavelength Em are 630 / 670nm respectively; read interval: set to 15-30 seconds to capture rapid changes in coagulation dynamics; total monitoring time: set to 30-60 minutes to ensure coverage of the entire coagulation process.

[0079] A 96-well black microplate is used because the black plate wall effectively reduces cross-interference of optical signals between the holes.

[0080] (3) Experimental operation and data acquisition 3.1 Add the following to each well of the microplate: 180 μL blood sample + 20 μL probe working solution; then add 20 μL CaCl2 solution to the same well to trigger coagulation. 3.2 Real-time monitoring: Place the microplate in the ELISA reader and immediately start running the preset program. The instrument will automatically and continuously record the fluorescence intensity value of each well at the set time point.

[0081] (4) Test results Record the fluorescence intensity versus time curve (kinetic curve).

[0082] This curve provides the TFT (time to thrombosis), the time it takes for fluorescence intensity to exceed a certain threshold from the baseline (e.g., a 20% increase from the baseline). This is the most direct indicator for assessing drug efficacy. The longer the TFT, the stronger the anticoagulant effect of the drug.

[0083] Maximum response rate (slope): Calculate the slope of the steepest part of the rising segment of the curve. The smaller the slope, the more the intensity of the burst is weakened by the drug, even if coagulation is initiated.

[0084] Maximum fluorescence intensity: reflects the size / density of the final clot. The lower the intensity, the more likely the drug has inhibited fibrin polymerization or network compaction.

[0085] Constructing a dose-response curve: Plot a curve with the logarithm of drug concentration on the X-axis and the TFT (or slope / percentage of inhibition at maximum intensity) on the Y-axis. By fitting the curve, the half-maximal effective concentration (IC50) of the drug can be calculated, which is the drug concentration required to double the TFT (or inhibit the clotting rate by 50%). IC50 is the gold standard for quantifying drug efficacy.

[0086] The above-described embodiments are preferred embodiments of the present invention and are only used to facilitate the illustration of the present invention. They are not intended to limit the present invention in any way. Any person skilled in the art who makes local modifications or alterations to the technical content disclosed in the present invention without departing from the scope of the technical features of the present invention shall still fall within the scope of the technical features of the present invention.

Claims

1. A pH-viscosity dual-responsive fluorescent probe, characterized in that, The structural formula of the probe molecule is as follows: 。 2. The pH-viscosity dual-response fluorescent probe as described in claim 1, characterized in that, The excitation wavelength of the pH-viscosity dual-response fluorescent probe is 630 nm, and the fluorescence emission wavelength of the pH-viscosity dual-response fluorescent probe is 670 nm.

3. A method for preparing a pH-viscosity dual-responsive fluorescent probe, characterized in that, Includes the following steps: Step A: Synthesize compound I: The solution of DMF and DCM was placed in an ice-water bath, and phosphorus oxychloride was added dropwise with stirring, followed by cyclohexanone. The mixture was refluxed for 6 hours, cooled, poured into ice water, stirred, filtered, and dried under vacuum to obtain compound I. The reaction formula is as follows: ， Step B: Synthesize compound II: 2,3,3-trimethyl-3H-indole and 1,4-butanesulfonate lactone were dissolved in toluene and reacted at 80 °C for 24 h under N2 atmosphere. After cooling, the solvent was removed by rotary evaporation. The residue was dissolved in methanol and added dropwise to ethyl acetate with stirring. After filtration and vacuum drying, compound II was obtained. The reaction formula is as follows: ， Step C: Synthesize compound III: Compounds I and II were dissolved in anhydrous n-butanol and toluene, and refluxed at 130 °C for 4 h under N2 protection. After purification, compound III was obtained. The reaction formula is as follows: ， Step D: Synthesis of the pH-viscosity dual-responsive fluorescent probe Cy: Compound III and NaOAc were dissolved in acetic anhydride and refluxed at 100°C for 6 h under N2 protection. After cooling to room temperature, the solution was diluted with dichloromethane and washed successively with water and physiological saline. The solution was then dried over anhydrous sodium sulfate, filtered, and concentrated by rotary evaporation to obtain a crude product. The crude product was purified by silica gel column chromatography to obtain a solid powder Cy. The reaction formula is as follows: 。 4. The application of the pH-viscosity dual-response fluorescent probe according to claim 1 in the preparation of thrombosis detection reagents.

5. The application of the pH-viscosity dual-response fluorescent probe according to claim 1 in screening and / or studying anticoagulants.

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