A Carboxylesterase Fluorescent Probe, Preparation Method Thereof and Application Thereof

Abstract

The present invention belongs to the technical field of the preparation of biological compounds, and discloses a carboxylesterase fluorescent probe, a preparation method thereof and an application thereof. The preparation method includes: adding BDP, compound 1 and toluene into a Schlenk flask, and obtaining a mixed solution after complete dissolution; heating the mixed solution at 80-120 °C for 10-16 hours; after the reaction is completed, cooling the mixed solution to room temperature, adding dichloromethane to the mixed solution, filtering the precipitated solid under reduced pressure, and drying it in vacuum, thus obtaining the carboxylesterase fluorescent probe. The preparation method of the carboxylesterase fluorescent probe of the present invention is simple, has a high yield and low cost, and is conducive to industrial production; the carboxylesterase fluorescent probe prepared by the present invention has a 656-fold enhancement in fluorescence after reacting with carboxylesterase, and has an extremely high signal-to-noise ratio; the carboxylesterase fluorescent probe prepared by the present invention changes the solution color from red to yellow after reacting with carboxylesterase, and the increase in the concentration of carboxylesterase can be detected with the naked eye.

Description

Technical Field

[0001] The present invention belongs to the technical field of the preparation of biological compounds, and particularly relates to a carboxylesterase fluorescent probe, a preparation method thereof, and an application thereof. Background Art

[0002] As an important hydrolase, carboxylesterase can catalyze the hydrolysis metabolism of esters, amides, carbamates, and thioesters into corresponding alcohols and acids. Research shows that abnormal carboxylesterase metabolic function is closely related to diseases such as high blood lipid, arteriosclerosis, and obesity. At present, the detection of carboxylesterase includes immunoassay, chemiluminescence method, real-time fluorescence quantitative PCR, and Western blotting (WB), etc. However, these technical means have disadvantages such as low detection sensitivity, cumbersome detection steps, and the need for sample pretreatment. It is not only difficult to achieve real-time detection of carboxylesterase, but also cannot be applied to complex biological systems.

[0003] Therefore, developing a method that is accurate, sensitive, simple, specific and can detect carboxylesterase in real-time in situ at the level of living cells and organisms is one of the research hotspots of scientific researchers. Fluorescent probe technology has the advantages of simple operation, high sensitivity, and real-time rapid analysis, and is widely used in the fields of biological detection, optical imaging, etc. Therefore, designing a fluorescent probe that specifically recognizes carboxylesterase and realizing the detection of the concentration of intracellular carboxylesterase under drug stimulation has important clinical value.

[0004] Through the above analysis, the problems and defects existing in the prior art are as follows: In recent years, more and more carboxylesterases have been reported, but the reported carboxylesterase probes generally have challenges such as long detection time (for example, the detection time of the probe reported by Liu et al. in Biosens. Bioelectron. 2022, 211, 114392 is 5 hours), low detection sensitivity (the signal enhancement before and after carboxylesterase recognition is less than 10 times, Patent CN108164450A), cumbersome synthesis steps, and inability to be used for live cell detection. At the same time, the study of the configurational relationship of carboxylesterase probes has important reference significance for the development and design of carboxylesterase probes with superior performance. Summary of the Invention

[0005] In order to overcome the problems existing in the related art, the disclosed embodiments of the present invention provide a carboxylesterase fluorescent probe, a preparation method thereof, and an application thereof.

[0006] The technical solution is as follows: A carboxylesterase fluorescent probe selects boron dipyrromethene with excellent luminescence performance as the parent body, and through the screening of the position of the recognition group, a carboxylesterase probe with ultra-high response signal-to-noise ratio is prepared, and the structural general formula is:

[0007]

[0008] The structural general formula indicates that the fluorophore in the lower half can be connected to the ortho-position, meta-position, and para-position of the N atom.

[0009] In one embodiment, the carboxylesterase fluorescent probe is:

[0010]

[0011] In one embodiment, the carboxylesterase fluorescent probe is:

[0012]

[0013] In one embodiment, the carboxylesterase fluorescent probe is:

[0014]

[0015] Another object of the present invention is to provide a preparation method of the carboxylesterase fluorescent probe, including:

[0016] S101, adding BDP, 4-bromomethylphenyl acetate (Compound 1), and toluene into a Schlenk flask, and obtaining a mixed solution after fully dissolving;

[0017] S102, heating the mixed solution at 80 - 120 °C for 10 - 16 hours;

[0018] S103, after the reaction ends, cooling the mixed solution to room temperature, adding one of dichloromethane, ethyl acetate, acetonitrile, and acetone to the mixed solution, filtering the precipitated solid under reduced pressure, and drying it in vacuum to obtain the carboxylesterase fluorescent probe.

[0019] In one embodiment, in step S101, the molar ratio of BDP to 4-bromomethylphenyl acetate is 1:8 - 12.

[0020] In one embodiment, in step S101, BDP is one of BPD-CE2, BPD-CE3, and BPD-CE4.

[0021] In one embodiment, the chemical reaction formula of the preparation method of the carboxylesterase fluorescent probe is:

[0022]

[0023] Another object of the present invention is to provide an application of a sensor for detecting abnormal diseases of carboxylesterase concentration in vitro in cancer live cells, using the carboxylesterase fluorescent probe in imaging detection of carboxylesterase concentration changes.

[0024] Another object of the present invention is to provide a sensor for detecting abnormal concentration of solution carboxylesterase, and its application in imaging detection of the change in carboxylesterase concentration by using the carboxylesterase fluorescent probe.

[0025] Combining all the above technical solutions, the advantages and positive effects of the present invention are as follows:

[0026] First, aiming at the technical problems existing in the above-mentioned prior art and the difficulty of solving this problem, closely combining the technical solution to be protected by the present invention and the results and data in the research and development process, etc., analyze in detail and deeply how the technical solution of the present invention solves the technical problems, and some creative technical effects brought after solving the problems, which are specifically described as follows:

[0027] Based on the problems existing in the existing technology, the present invention provides an organic small molecule fluorescent probe with good detection ability for carboxylesterase. By comparing the detection performances of three compounds, a fluorescent probe that can rapidly, sensitively and specifically recognize carboxylesterase is screened out, providing a reference idea for the construction of such probes.

[0028] The present invention also provides a preparation method of a carboxylesterase fluorescent probe. This method uses simple and easily available raw materials, and the synthesis steps are simple and the cost is low.

[0029] Compared with the prior art, the beneficial effects of the present invention also include: the preparation method of the carboxylesterase fluorescent probe of the present invention is simple, with high yield and low cost, which is conducive to industrial production;

[0030] The carboxylesterase fluorescent probe BDP-CE2 prepared by the present invention has a fluorescence enhancement of 656 times in the presence of 0.5 U / mL carboxylesterase, with an extremely high signal-to-noise ratio; the solution color of the carboxylesterase fluorescent probe prepared by the present invention changes from red to yellow after reacting with carboxylesterase, and the increase in carboxylesterase concentration can be detected with the naked eye; the carboxylesterase fluorescent probe prepared by the present invention can work in a buffer solution without adding extra organic solvents, has good biocompatibility, and can track the change in carboxylesterase concentration at the live cell level.

[0031] Second, regarding the technical solution as a whole or from the perspective of the product, the technical effects and advantages of the technical solution to be protected by the present invention are specifically described as follows:

[0032] The present invention provides a carboxylesterase fluorescent probe, its preparation method and application. The carboxylesterase fluorescent probe provided by the present invention has the advantages of rapid detection, high selectivity, good sensitivity, simple synthesis, and can be used for imaging tracking of the change in carboxylesterase concentration in living cells. The present invention also studies the configurational relationship of this series of carboxylesterase probes. Description of the Drawings

[0033] The accompanying drawings here are incorporated into the specification and form a part of this specification, showing embodiments consistent with the present disclosure, and are used together with the specification to explain the principles of the present disclosure;

[0034] Figure 1 It is a flowchart of the preparation method of the carboxylesterase fluorescent probe provided by an embodiment of the present invention;

[0035] Figure 2 It is a graph showing the change of the fluorescence spectrum of BDP-CE2 after incubation for different times in the presence of 0.5 U / mL carboxylesterase provided by an embodiment of the present invention;

[0036] Figure 3 It is a graph showing the change of the fluorescence intensity at 520 nm of BDP-CE2, BDP-CE3 and BDP-CE4 respectively after incubation for different times in the presence of 0.5 U / mL carboxylesterase provided by an embodiment of the present invention;

[0037] Figure 4 It is the change of the absorption spectrum of the mixture of BDP-CE2 and carboxylesterase with time provided by an embodiment of the present invention. As time increases, the maximum absorption peak of the probe blue-shifts from 510 nm to 502 nm, and it is a graph showing the color change of the solution;

[0038] Figure 5 It is a fluorescence spectrum graph after incubation with different concentrations of carboxylesterase added to a 10 μM solution provided by an embodiment of the present invention;

[0039] Figure 6 It is a graph and linear curve showing the relationship between the fluorescence intensity of the probe at 520 nm and the concentration of carboxylesterase provided by an embodiment of the present invention;

[0040] Figure 7 It is a graph of the fluorescence intensity after adding different species to the BDP-CE2 solution provided by an embodiment of the present invention;

[0041] Figure 8 It is a graph of the change at 520 nm of BDP-CE2 and BDP-CE2 + carboxylesterase in PBS buffer solutions with different pH values provided by an embodiment of the present invention;

[0042] Figure 9 It is a confocal fluorescence effect diagram provided by an embodiment of the present invention. Detailed implementation manners

[0043] To make the above objects, features, and advantages of the present invention more apparent and understandable, the following detailed description of the specific embodiments of the present invention will be provided in conjunction with the accompanying drawings. Many specific details are set forth in the following description to facilitate a full understanding of the present invention. However, the present invention can be implemented in many other ways different from those described herein, and those skilled in the art can make similar improvements without departing from the spirit of the present invention. Therefore, the present invention is not limited by the specific embodiments disclosed below.

[0044] I. Explanation of the embodiments:

[0045] In the embodiments of the present invention, through the study of the compound configuration relationship, a fluorescent probe with high sensitivity, high selectivity, rapid detection, etc. for carboxylesterase is screened out, and the synthesis method is simple and the raw materials are easily available.

[0046] Using boron dipyrromethene with excellent luminescence performance as the parent, a carboxylesterase probe with ultra-high response signal-to-noise ratio is prepared through the screening of the recognition group position. Among them, the general structural formula of the carboxylesterase fluorescent probe is:

[0047]

[0048] As Figure 1 shown, the embodiments of the present invention provide a preparation method of a carboxylesterase fluorescent probe, including:

[0049] S101, Add BDP, 4-bromomethylphenyl acetate (Compound 1), and toluene into a Schlenk flask, and obtain a mixed solution after fully dissolving;

[0050] S102, Heat the mixed solution at 80 - 120 °C for 10 - 16 hours;

[0051] S103, After the reaction ends, cool the mixed solution to room temperature, add one of dichloromethane, ethyl acetate, acetonitrile, and acetone to the mixed solution, filter the precipitated solid under reduced pressure, and dry it in vacuum to obtain the carboxylesterase fluorescent probe.

[0052] In the embodiments of the present invention, in step S101, the molar ratio of BDP to 4-bromomethylphenyl acetate (Compound 1) is 1:8 - 12.

[0053] Example 1

[0054] The embodiments of the present invention provide a preparation method of a carboxylesterase fluorescent probe with boron dipyrromethene (BODIPY) as the fluorescent parent, and the chemical reaction formula is as follows:

[0055]

[0056] Example 2

[0057] The embodiment of the present invention provides a carboxylic esterase fluorescent probe (BPD-CE2) with the molecular formula:

[0058]

[0059] Synthesis of probe BPD-CE2:

[0060]

[0061] Weigh 4-bromomethylphenyl acetate (compound 1 (710.2 mg, 3.1 mmol)) and BDP-2 (100 mg, 0.31 mmol) respectively into a Schlenk flask, add toluene (6 mL), and dissolve. Heat the mixture solution under reflux for 12 hours. After cooling to room temperature, slowly add dichloromethane to the system. The precipitated solid is filtered under reduced pressure and dried in vacuo. 54.4 mg of brick-red solid compound BDP-CE2 is obtained, and the calculated yield is 37.0%. The 1H NMR characterization of compound BDP-CE2 is as follows: 1 H NMR (500 MHz, DMSO-d6, δ): δ 9.66 (s, 1H), 9.57 (d, J = 10.0 Hz, 1H), 8.91 (d, J = 10.0 Hz, 2H), 8.43 (s, 1H), 7.72 (d, J = 10.0 Hz, 2H), 7.24 (d, J = 10.0 Hz, 2H), 6.28 (s, 2H), 5.96 (s, 2H), 2.50 (s, 6H), 2.29 (s, 3H), 1.23 (s, 6H).

[0062] Example 3

[0063] The embodiment of the present invention provides a carboxylic esterase fluorescent probe (BPD-CE3) with the molecular formula:

[0064]

[0065] Synthesis of probe BPD-CE3:

[0066]

[0067] Weigh 4-bromomethylphenyl acetate (compound 1 (710.2 mg, 3.1 mmol)) and BDP-3 (100 mg, 0.31 mmol) respectively into a Schlenk flask, add toluene (6 mL), and dissolve. Heat the mixture solution under reflux for 12 hours. After cooling to room temperature, slowly add dichloromethane to the system. The precipitated solid is filtered under reduced pressure and dried in vacuo. 58.2 mg of brick-red solid compound BDP-CE3 is obtained, and the calculated yield is 39.6%. The 1H NMR characterization of compound BDP-CE3 is as follows: 11H NMR (500 MHz, DMSO-d6, δ): δ 9.66 (s, 1H), 9.58 (d, J = 10.0 Hz, 1H), 8.90 (d, J = 10.0 Hz, 1H), 8.43 (t, J = 14.32 Hz, 1H), 7.72 (d, J = 10.0 Hz, 2H), 7.24 (d, J = 10.0 Hz, 2H), 6.28 (s, 2H), 5.96 (s, 2H), 2.50 (s, 6H), 2.28 (s, 3H), 1.22 (s, 6H).

[0068] Example 4

[0069] The carboxylic esterase fluorescent probe (BPD-CE4) provided by the embodiment of the present invention has the molecular formula:

[0070]

[0071] Synthesis of probe BPD-CE4:

[0072]

[0073] Weigh 4-bromomethylphenyl acetate (Compound 1 (710.2 mg, 3.1 mmol)) and BDP-4 (100 mg, 0.31 mmol) into a Schlenk flask, add toluene (6 mL) and dissolve. Heat the mixture solution under reflux for 12 hours. After cooling to room temperature, slowly add dichloromethane to the system. The precipitated solid is filtered by suction under reduced pressure and dried in vacuo. 67.2 mg of brick-red solid compound BDP-CE4 is obtained, and the calculated yield is 45.7%. The 1H NMR characterization of compound BDP-CE4 is as follows: 1 1H NMR (500 MHz, DMSO-d6, δ): δ 9.48 (d, J = 5.0 Hz, 2H), 8.51 (d, J = 5.0 Hz, 2H), 7.62 (d, J = 10.0 Hz, 2H), 7.26 (d, J = 10.0 Hz, 2H), 6.30 (s, 2H), 6.04 (s, 2H), 2.50 (s, 6H), 2.29 (s, 3H), 1.39 (s, 6H). 13C NMR (125 MHz, DMSO-d6, δ) 169.56, 157.38, 151.72, 151.67, 146.73, 142.86, 135.09, 132.30, 130.47, 129.87, 129.45, 129.30, 123.21, 122.97, 63.44, 21.36, 15.20, 14.85.

[0074] In the above embodiments, the descriptions of the respective embodiments have their own focuses. For parts not detailed or recorded in a certain embodiment, reference may be made to the relevant descriptions of other embodiments.

[0075] II. Application embodiments:

[0076] Application example

[0077] An embodiment of the present invention provides an application of a carboxylesterase fluorescent probe. Using it as a sensor for imaging detection of changes in the concentration of carboxylesterase in solutions and living cells, it can be used for in vitro detection of diseases with abnormal carboxylesterase concentrations such as tumors.

[0078] III. Evidence of related effects of the embodiments:

[0079] Experimental analysis of the spectral performance of the probe:

[0080] Weigh 4.7 mg of the probe into a 10 mL volumetric flask, add DMSO to make up the volume to prepare a 1.0 mM concentrated storage solution of the probe. After storing the concentrated solution away from light, it is stored in a refrigerator at 4 °C.

[0081] (1) Determination of the response time and detection signal-to-noise ratio of the carboxylesterase probe: Add 30 μL of the DMSO solution (1.0 mM) of the probe to 2970 μL of PBS buffer solution (pH value 7.4) to obtain a 10 μM probe solution, and then add carboxylesterase so that the working concentration of the enzyme is 0.5 U / mL. The resulting mixed solution is incubated at 37 °C for different times (0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 min), and the fluorescence spectrometer is used to test the change of the solution fluorescence spectrum with time. Figure 2 is the change diagram of the fluorescence spectrum of BDP-CE2 after incubation for different times in the presence of 0.5 U / mL carboxylesterase. From Figure 3 is the change diagram of the fluorescence intensity at 520 nm of BDP-CE2, BDP-CE3, and BDP-CE4 respectively after incubation for different times in the presence of 0.5 U / mL carboxylesterase. Figure 3 It shows that BDP-CE2 and BDP-CE3 have a shorter response time, about 10 minutes. At the same time, the fluorescence signal of BDP-CE2 increases by 656 times before and after the response with carboxylesterase, while the fluorescence of BDP-CE3 only increases by 463 times before and after the response with carboxylesterase. To sum up, BDP-CE2 has better detection performance than BDP-CE3 and BDP-CE4. Figure 4 is the change situation of the absorption spectrum of the mixture of BDP-CE2 and carboxylesterase with time. As time increases, the maximum absorption peak of the probe blue-shifts from 510 nm to 502 nm, and the color of the solution changes from light red to yellow, indicating that the probe BDP-CE2 can be used for the naked-eye detection of carboxylesterase.

[0082] (2) Detection limit test of BDP-CE2 for carboxylesterase: Add 30 μL of the DMSO solution (1.0 mM) of the probe to 2970 μL of PBS buffer solution (pH = 7.4) to obtain a 10 μM probe solution. Then add solutions of carboxylesterase at different concentrations. After incubating at 37 °C for 10 minutes, collect the spectrogram with a fluorescence spectrometer. Establish a linear curve for the detection of carboxylesterase by BDP-CE2 with the fluorescence intensity at 520 nm as the ordinate and the concentration of carboxylesterase as the abscissa. Figure 5 Fluorescence spectrogram after incubating different concentrations of carboxylesterase in a 10 μM solution; Figure 6 Relationship and linear curve diagram between the fluorescence intensity of the probe at 520 nm and the concentration of carboxylesterase. The specific linear curve is y = 3737.49x - 8.99, and the linear response range is 0 - 0.15 U / mL (R 2 = 99.1%), and the detection limit is 3.39×10 -5 U / mL, indicating that the probe BDP-CE2 is sensitive to carboxylesterase and can be used for the detection of low-concentration carboxylesterase. [[ID=|11]]

[0083] (3) Specificity test of BDP-CE2 for carboxylesterase response: Add 30 μL of the DMSO solution (1.0 mM) of the probe to 2970 μL of PBS buffer solution (pH = 7.4) to obtain a 10 μM probe solution. Add common species in the biological system respectively, incubate at 37 °C for 10 minutes, and then measure the fluorescence spectrogram of the solution. Figure 7 Fluorescence intensity diagrams after adding different species to the BDP-CE2 solution, where they are respectively 1. BDP-CE2; 2. BDP-CE2 + carboxylesterase; 3. BDP-CE2 + trypsin; 4. BDP-CE2 + cysteine; 5. BDP-CE2 + glutathione; 6. BDP-CE2 + homocysteine; 7. BDP-CE2 + serine; 8. BDP-CE2 + arginine; 9. BDP-CE2 + histidine; 10. BDP-CE2 + lysine; 11. BDP-CE2 + dithiothreitol; 12. BDP-CE2 + hydrazine; 13. BDP-CE2 + hydrogen peroxide; 14. BDP-CE2 + sodium hypochlorite; 15. BDP-CE2 + aluminum chloride; 16. BDP-CE2 + magnesium chloride; 17. BDP-CE2 + sodium sulfide; 18. BDP-CE2 + zinc nitrate; 19. BDP-CE2 + sodium chloride; 20. BDP-CE2 + iron chloride; 21. BDP-CE2 + nickel chloride; 21. BDP-CE2 + potassium chloride. It can be seen that Figure 7 the detection of carboxylesterase by BDP-CE2 is not interfered by common species and has very good specificity.

[0084] (4) Determination of the pH sensitivity of BDP-CE2: Add 30 μL of the DMSO solution (1.0 mM) of the probe to 2970 μL of PBS buffer solution to obtain a 10 μM probe solution. Adjust the pH of the solution to 4, 5, 6, 7, 8, and 9 respectively, and then collect the fluorescence spectra. Add 0.05 U / mL of carboxylesterase to the BDP-CE2 solution at different pH values, and collect the fluorescence spectra after incubating the resulting mixed solution at 37 °C for 10 minutes. Figure 8 Plots of the changes of BDP-CE2 and BDP-CE2 + carboxylesterase at 520 nm in PBS buffer solutions at different pH values, from Figure 8 which it can be seen that BDP-CE2 can detect carboxylesterase within the physiological pH range.

[0085] (5) Testing the change of carboxylesterase concentration in living cells by BDP-CE2: Co-incubate 5 μM BDP-CE2 with 4T1 (breast cancer) cells or 4T1 cells treated with the carboxylesterase inhibitor bis(4-nitrophenyl) phosphate for 20 minutes, and then observe under a laser confocal fluorescence microscope, using 488 nm excitation as the excitation light source, Figure 9 Confocal fluorescence photographs. (A) is the bright-field photograph of BDP-CE2 co-incubated with 4T1 cells at 25 °C for 20 minutes, (B) is the fluorescence confocal imaging photograph of BDP-CE2 co-incubated with 4T1 cells at 25 °C for 20 minutes, (C) is the bright-field photograph of the inhibitor bis(4-nitrophenyl) phosphate co-incubated with 4T1 cells at 25 °C for 20 minutes and then co-incubated with BDP-CE2 for 20 minutes, and (D) is the fluorescence confocal imaging photograph of the inhibitor bis(4-nitrophenyl) phosphate co-incubated with 4T1 cells at 25 °C for 20 minutes and then co-incubated with BDP-CE2 for 20 minutes. From Figure 9 which it can be seen that after treatment with the carboxylesterase inhibitor bis(4-nitrophenyl) phosphate, the concentration of carboxylesterase in 4T1 cells decreases and the fluorescence intensity weakens, indicating that BDP-CE2 can trace the fluctuations of carboxylesterase concentration in living cells. The probe of the present invention has the advantages of a large signal increase multiple before and after response, and can be detected simultaneously by fluorescence and the naked eye. The present invention also screened out the probe BDP-CE2 with the best performance by changing the position of the recognition group. The reason for the optimal performance of BDP-CE2 may be that it is more compatible with the spatial structure of carboxylesterase.

[0086] The above is only a relatively preferred specific embodiment of the present invention, but the protection scope of the present invention is not limited thereto. Any person skilled in the art within the technical scope disclosed by the present invention, any modification, equivalent replacement, and improvement made within the spirit and principle of the present invention shall be covered by the protection scope of the present invention.

Claims

1. A carboxylic esterase fluorescent probe, characterized in that, Using fluoroboron dipyrrole with luminescence properties as the parent body, a carboxylesterase probe with a response signal-to-noise ratio was prepared by screening the position of the recognition group. The carboxylesterase fluorescence probe is as follows:

2. The carboxylic esterase fluorescent probe according to claim 1, wherein The carboxylesterase fluorescence probe is as follows:

3. The carboxylic esterase fluorescent probe according to claim 1, wherein The carboxylesterase fluorescence probe is as follows:

4. A method for preparing the carboxylic esterase fluorescent probe according to claim 1, characterized in that, The preparation method includes: S101, Add BDP, 4-bromomethylphenyl acetate and toluene into a Schlenk flask, and obtain a mixed solution after complete dissolution; S102, Heat the mixed solution at 80-120 °C for 10-16 hours; S103, After the reaction is completed, cool the mixed solution to room temperature, add one of dichloromethane, ethyl acetate, acetonitrile, and acetone to the mixed solution, filter the precipitated solid under reduced pressure, and dry it in vacuo to obtain the carboxylesterase fluorescence probe.

5. The preparation method according to claim 4, wherein In step S101, the molar ratio of BDP to 4-bromomethylphenyl acetate is 1:8-12.

6. The preparation method according to claim 4, characterized in that, In step S101, BDP is one of BPD-CE2, BPD-CE3, and BPD-CE4.

7. Use of the carboxylesterase fluorescent probe according to any one of claims 1-3 in the preparation of a sensor for detecting diseases with abnormal carboxylesterase concentration in living cancer cells in vitro, characterized in that, This sensor is applied to the imaging detection of changes in carboxylesterase concentration.

8. Use of the carboxylesterase fluorescent probe according to any one of claims 1-3 in the preparation of a sensor for detecting abnormal concentration of carboxylesterase in a solution, characterized in that, This sensor is applied to the imaging detection of changes in carboxylesterase concentration.

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