Preparation method and application of a bio-fluorescent probe molecule based on AIE effect in response to cathepsin B

Abstract

The application belongs to the field of biological medicine, and provides a fluorescent probe which is specifically responsive to cathepsin B. The probe is a biological fluorescent probe with a fluorescent 'on' response which is developed based on an aggregation-induced emission effect, and the amino acid sequence is Ac-rrrr-AEEA-GIVRAK(Gl-TPE)-COOH. The probe is synthesized by a solid-phase synthesis method, cathepsin B specifically cuts the recognition site GIVRAK, after cutting, the part containing tetraphenyl ethylene is aggregated, the aggregation-induced emission effect is triggered, a strong fluorescent signal is generated, and the specific recognition and monitoring of the probe to cathepsin B are realized. Meanwhile, the probe introduces four D-arginines to help the probe enter cells, and the monitoring of cathepsin B at the cell level is realized. The fluorescent probe has high practical application value, and has the advantages of strong monitoring specificity of cathepsin B, simple method and mild conditions.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of Aggregation-Induced Emission (AIE) technology and biological fluorescent probes, in particular, it is a preparation method of a cathepsin B responsive biological fluorescent probe molecule based on AIE effect and its application in tumor diagnosis. The technology belongs to the field of biological detection and polypeptide probes. BACKGROUND

[0002] Cancer is one of the leading causes of premature death worldwide and has become one of the major diseases threatening the health of Chinese residents. It is expected that in the coming decades, with the aging of the population and changes in lifestyle, the incidence and mortality of cancer will further rise. Therefore, early diagnosis and precise treatment of tumors are of great significance to improve the survival rate and quality of life of patients.

[0003] Fluorescent probes, as a key tool in the field of biological analysis, play an important role in detecting and monitoring biologically active molecules. In recent years, the application of fluorescent probes in tumor marker detection has attracted much attention. Cathepsin B (CatB) as an important tumor marker is involved in various pathological processes and carcinogenic mechanisms. Studies have shown that cathepsin B is overexpressed in various malignant tumors, including brain cancer, lung cancer, prostate cancer, breast cancer, and colorectal cancer. Cathepsin B plays an important role in tumor cell invasion, metastasis, and drug resistance by degrading extracellular matrix (ECM) and regulating the function of cell surface receptors.

[0004] Currently, the commonly used methods for detecting cathepsin B in clinical practice include enzyme-linked immunosorbent assay, Western blotting, and mass spectrometry. However, these methods usually require complex sample preparation processes or expensive instruments and equipment, making it difficult to achieve rapid, sensitive, and real-time detection. Therefore, developing fluorescent probes that can sensitively and specifically detect cathepsin B is of great significance for early diagnosis and treatment monitoring of tumors. However, traditional fluorescent probes are prone to Aggregation-Caused Quenching (ACQ) phenomenon at high concentrations, leading to decreased detection sensitivity. In addition, existing cathepsin B responsive fluorescent probes are mostly limited to in vitro detection, making it difficult to achieve real-time imaging and monitoring of intracellular cathepsin B.

[0005] In view of the above problems, the application provides an aggregation-induced emission (AIE) effect-based biological fluorescent probe molecule. The probe comprises a polypeptide sequence specifically recognizing cathepsin B and a tetraphenylethylene (TPE) connected to the polypeptide sequence. The TPE molecule is a typical AIE molecule, which almost does not emit light in a monomer form, and in an aggregated state, due to restricted intramolecular rotation (RIR), energy loss of non-radiative transition is reduced, so that the fluorescence is significantly enhanced. When the probe specifically reacts with the cathepsin B, the polypeptide chain is broken, and the released TPE group is partially aggregated, so that the fluorescence signal is turned on, and the ACQ phenomenon is avoided. At present, there is no related report on the TPE fluorescent probe based on the polypeptide as a substrate for detecting the cathepsin B in vivo and in vitro. SUMMARY

[0006] In view of the above problems, the application provides an AIE effect-based biological fluorescent probe ZSY-3. The probe introduces TPE as an AIE fluorescent molecule, and combines a protease-specific recognition polypeptide sequence and a functional modification unit, so as to realize specific fluorescence response to the cathepsin, and overcome the deficiencies of the traditional probe in sensitivity, specificity and application range. The probe molecule is composed of the following parts: an AIE group capable of significantly enhancing the fluorescence signal in an aggregated state, preferably TPE; a polypeptide sequence capable of specifically recognizing and being cut by the cathepsin B, preferably GIVRAK; a polypeptide fragment capable of enhancing the water solubility and cell penetration capacity of the probe, preferably a plurality of D-arginines; a linker between the enzymolytic polypeptide sequence and the penetration sequence, preferably an AEEA fragment with high hydrophilicity and biocompatibility; and a flexible linker between the AIE group and the polypeptide, preferably Gl. The probe realizes the ability of the AIE effect-based biological fluorescent probe to detect the cathepsin B at the molecular level and the cell level, and has the characteristics of high specificity and high sensitivity.

[0007] Specifically, the application realizes the technical solutions as follows.

[0008] A first aspect of the application provides an AIE effect-based cathepsin B-responsive biological fluorescent probe ZSY-3. The chemical structural formula and the primary amino acid sequence thereof are as follows:

[0009]

[0010] The above-mentioned biological fluorescent probe has a fluorescent "opening" ability in response to cathepsin B. In a solution without cathepsin B, the probe molecule exists in a monomer form, and the TPE group does not emit light. When the probe is enzymatically degraded by cathepsin B, the polypeptide chain is cut, and the part with the TPE group is released to aggregate, and the TPE group in the aggregated state releases a fluorescent signal due to the restriction of intramolecular rotation.

[0011] In a second aspect of the present application, a preparation method of the above-mentioned biological fluorescent probe is provided. Specifically, the target polypeptide is synthesized by a 9-fluorenylmethoxycarbonyl (Fmoc)-based solid-phase polypeptide synthesis method (Fmoc-SPPS). Preferably, Wang Resin resin is used for solid-phase synthesis, Fmoc is used as a protective group, and the target polypeptide amino acid is coupled step by step. After the synthesis of the polypeptide chain is completed, deprotection and purification treatment are performed, and finally the biological fluorescent probe ZSY-3 with a cathepsin B response function is obtained.

[0012] In a third aspect of the present application, an application of the biological fluorescent probe based on the AIE effect is provided, and specifically relates to an application of the biological fluorescent probe ZSY-3 for detecting cathepsin B at a molecular level. At a molecular level, ZSY-3 can sensitively and specifically detect cathepsin B. Through the design of a specific polypeptide sequence (GIVRAK), the probe can be specifically cut by cathepsin B. After cutting, the part containing the TPE group aggregates, triggering the aggregation-induced emission effect, thereby generating a strong fluorescent signal. This fluorescent "opening" mechanism makes ZSY-3 have extremely high sensitivity and specificity in detecting cathepsin B.

[0013] In a fourth aspect of the present application, an application of the biological fluorescent probe based on the AIE effect in detecting cathepsin B in a cell lysate is provided. By incubating the cell lysate with high expression of cathepsin B with ZSY-3, the experimental results show that the fluorescent signal gradually increases with time. In order to verify the specificity of the probe, the cathepsin B specific inhibitor CA-074Me is added in the experiment, and the results show that the fluorescent signal of the inhibitor group is significantly reduced. It is shown that ZSY-3 can specifically detect the presence of cathepsin B in the cell lysate, and provides a powerful tool for studying the expression and function of cathepsin B in tumor cells.

[0014] The fifth aspect of the present application is to provide a biological fluorescent probe based on AIE effect for detecting and imaging Cathepsin B in living cells. The polypeptide chain of ZSY-3 is introduced with D-arginine which has cell penetration effect, so that it can effectively penetrate the cell membrane and monitor the expression level of Cathepsin B in living cells in real time. Through confocal microscope observation, it is found that the fluorescence intensity is significantly enhanced with the increase of incubation time of the probe. This real-time imaging capability makes ZSY-3 have important value in studying the dynamic expression and functional regulation of Cathepsin B in living cells.

[0015] The beneficial technical effects of the above technical solution are as follows:

[0016] 1. The above technical solution can provide a high-sensitivity biological fluorescent probe. ZSY-3 significantly enhances the fluorescence signal through AIE effect, overcoming the problem of insufficient sensitivity of traditional fluorescent probes due to ACQ phenomenon.

[0017] 2. The above technical solution can provide a high-specificity biological fluorescent probe. The specific polypeptide sequence (GIVRAK) is introduced in the design of ZSY-3, which can be specifically recognized and cut by Cathepsin B. The cleavage site of ZSY-3 is verified by reverse phase high performance liquid chromatography (RP-HPLC) and mass spectrometry analysis, further proving its high specificity for Cathepsin B. In addition, in cell level detection, after adding the specific inhibitor CA-074Me of Cathepsin B, the fluorescence signal is significantly reduced, further verifying the specific response ability of ZSY-3 to Cathepsin B.

[0018] 3. The above technical solution can provide an excellent biological fluorescent probe with cell penetration ability. ZSY-3 is introduced with D-arginine which has cell penetration effect in the polypeptide chain, which can effectively penetrate the cell membrane and monitor the expression level of Cathepsin B in living cells in real time. Through confocal microscope observation, it is found that ZSY-3 can quickly enter the cells and accumulate in the cells, and the fluorescence signal in the cells gradually increases with the extension of incubation time, proving the specific response ability of ZSY-3 to Cathepsin B in cells.

[0019] The present application synthesizes biological fluorescent probes based on AIE effect, which have simple preparation process and high repeatability. The probes can exhibit excellent detection performance at the molecular level and cell level, providing new tools and technical means for tumor marker detection, disease diagnosis and treatment monitoring. By monitoring the expression level of Cathepsin B in real time, the probe provides important support for early diagnosis and precise treatment of tumors. BRIEF DESCRIPTION OF DRAWINGS

[0020] The accompanying drawings, which constitute a part of this specification, are included to provide a further understanding of the present application, and are incorporated in and constitute a part of this specification, illustrate embodiments of the present application and together with the description serve to explain the present application. Embodiments of the present application alone are not to be unduly limited as only specified in the claims

[0021] Figure 1 The schematic diagram of the solid-phase polypeptide synthesis method of the biological fluorescent probe of the present application;

[0022] Figure 2 The chemical structural formula, primary amino acid sequence, and analytical reverse-phase high performance liquid chromatogram and mass spectrum of ZSY-3;

[0023] Figure 3 The fluorescence intensity change of the biological fluorescent probe ZSY-3 before and after being incubated with cathepsin B and the contrast diagram under 365nm ultraviolet;

[0024] Figure 4 The fluorescence spectrum of the biological fluorescent probe ZSY-3 incubated with cathepsin B for 0 to 60min;

[0025] Figure 5 The high performance liquid chromatogram of ZSY-3 before and after being incubated with cathepsin B;

[0026] Figure 6 The mass spectrum of ZSY-3 after being incubated with cathepsin B;

[0027] Figure 7 The curve of the fluorescence of the probe treated by the lysate of cathepsin B high-expression cell MDA-MB-231 and the inhibitor CA-074Me with time;

[0028] Figure 8 The confocal imaging of 20μM ZSY-3 acting on the cathepsin B high-expression cell MDA-MB-231 with time increase. Specific embodiments

[0029] The specific embodiments of the present application will be further described in detail below in combination with the drawings and specific embodiments. In the specific embodiments, each original reagent and raw material can be purchased. It should be pointed out that the following detailed description is exemplary and is intended to provide further description of the present application. Unless otherwise specified, all technical and scientific terms used in the present application have the same meaning as generally understood by those skilled in the art to which the present application belongs.

[0030] In the specific embodiments of the present application, the specific synthesis, separation and purification method of the above biological fluorescent probe ZSY-3 is provided, and the structure thereof is verified.

[0031] In another specific embodiment of the present application, there is provided an application of the biological fluorescent probe ZSY-3 in detecting cathepsin B at a molecular level and in a cell lysate.

[0032] In another specific embodiment of the present application, there is provided an application of the biological fluorescent probe ZSY-3 in detecting cathepsin B in MDA-MB-231 cells, i.e., detecting cathepsin B at a cellular level.

[0033] The preparation of the biological fluorescent probe ZSY-3 and its application will be further described in detail below through specific embodiments. The present application will be further explained and described below through examples, but it does not constitute a limitation on the present application. It should be understood that these examples are only used to illustrate the present application and not to limit the scope of the present application.

[0034] Example 1: Synthesis method of ZSY-3

[0035] In a specific embodiment of the present application, there is provided a synthesis method of the above biological fluorescent probe, and the synthesis method is as follows.

[0036] Solid-phase synthesis method based on 9-fluorenylmethyloxycarbonyl Figure 1 ) :

[0037] According to the amount of the reaction, a certain amount of Wang Resin is weighed, and standard washing is performed once, i.e., using DMF to wash twice, DCM to wash twice, DMF to wash once, DCM to wash once, and DMF to wash three times. Then, the resin is soaked in 3-4 mL of analytical pure DMF solvent for 2 h at room temperature for swelling, and the resin is pre-activated. Then, the resin is soaked in 3-4 mL of analytical pure DMF / DCM mixed solvent (4:1, v:v) at 28°C in a constant temperature shaker for 1 h to completely activate the resin. After activation, the first amino acid is connected. Then, the Fmoc protecting group is removed, and the amino acid sequence is connected. After all the reactions are completed, standard washing is performed.

[0038] Method for connecting the first amino acid: analytical pure DMF is used to completely dissolve the amino acid and the condensation reagent and add them to the resin, and condensation is performed twice at 28°C, with 8 h for the first reaction and 12 h for the second reaction. When the amino acid is condensed, the ratio of the reactants is amino acid: DIC: Oxyma: DAMP = 3 equivalents: 6 equivalents: 3 equivalents: 0.4 equivalents (molar ratio). After two reactions, standard washing is performed once, and then the residual solvent is pumped dry with water.

[0039] Method for removing Fmoc protecting group: using 2-3 mL of 20% piperidine in DMF, remove Fmoc protecting group twice at 28°C, the removal time is 5 min and 10 min respectively. Other amino acid linking method: using analytical pure DMF to completely dissolve amino acid and condensation reagent, add to the resin, and shake condensation twice at 28°C. To ensure full reaction, shake condensation twice at 28°C, the first reaction time is 30 min, and the second reaction time is 40 min. When the amino acid is condensed, the ratio of reactants is amino acid: HCTU: DIEA = 3 equivalents: 2.8 equivalents: 6 equivalents (molar ratio). After two reactions, to completely remove impurities in the resin, standard wash once, and then use a water pump to dry the residual solvent.

[0040] Method for linking AEEA: using analytical pure DMF to completely dissolve amino acid and condensation reagent, add to the resin, and shake condensation twice at 28°C. To ensure full reaction, shake condensation twice at 28°C, the first reaction time is 40 min, and the second reaction time is 50 min. When the amino acid is condensed, the ratio of reactants is Fmoc-AEEA-COOH: HATU: HOAt: DIEA = 3 equivalents: 2.8 equivalents: 3 equivalents: 6 equivalents (molar ratio). After two reactions, to completely remove impurities in the resin, standard wash once, i.e. wash twice with DMF, twice with DCM, once with DMF, once with DCM, and three times with DMF, and then use a water pump to dry the residual solvent.

[0041] Method for acetylating the end of the peptide chain: first remove the end Fmoc using 20% piperidine in DMF, use acetic anhydride: DIEA: DMF = 1 equivalent: 1 equivalent: 8 equivalents (volume ratio) to prepare 4 mL of solution, and the reaction time is 5 min and 10 min. After two reactions, standard wash once.

[0042] The connection method of TPE: first, remove the Alloc protecting group on the lysine side chain, specifically, use 72 mg Pd(PPh3)4, 300 μL PhSiH3 to remove Alloc for every 0.10 mmol resin. Take Pd(PPh3)472 mg in 2 mL DCM, shake to completely dissolve, then add 2 mL DMF, finally add an appropriate amount (300 μL) of PhSiH3, avoid light, shake and react at 28°C for 1.5 h and 2 h. Dry, wash twice with DCM, twice with DMF, once with DMF, twice with DCM, and then standard wash once, and finally use a water pump to dry the residual solvent. When the lysine side chain is condensed with amino acids, the reaction ratio of the reactants is amino acid: HCTU: DIEA = 3 equivalents: 2.8 equivalents: 6 equivalents (molar ratio), and the reaction time is 50 min and 60 min. The synthesized polypeptide is condensed with a fluorescent group. The reaction ratio of the reactants is TPE: HATU: HOAt: DIEA = 3 equivalents: 2.8 equivalents: 3 equivalents: 6 equivalents (molar ratio), and the reaction time is 1.5 h and 2 h. The method for cutting the peptide chain from the resin: standard wash once before cutting the peptide, wash five times with analytical DCM, pump for 5 min, and pump for 4 min. Add the prepared polypeptide to the peptide cleavage reagent with a ratio of TFA: water: TIPS = 95: 2.5: 2.5 (v: v: v), add the pre-cooled peptide cleavage reagent to the synthesis tube, and react in a shaking bed at 28-30°C for 2.5 h. After the peptide is cut, the solution is flowed into a three-necked bottle through the synthesis tube, 0.5 mL of TFA is used to wash the resin twice, and the peptide cleavage reagent and the washing liquid are combined. Blow with N2 to 2-3 mL, then precipitate with pre-cooled anhydrous ether, centrifuge for 3 min, discard the supernatant, and obtain the crude peptide after ether precipitation. Repeat the precipitation and centrifugation three times, and dry the crude product in a fume hood.

[0043] Separate and purify the obtained crude product: dissolve the crude peptide product in a mixed solution of acetonitrile and water containing 1‰ TFA, analyze the obtained crude peptide using analytical RP-HPLC, and the experimental conditions are as follows: use a C18 reversed-phase chromatographic column; the mobile phase is 1‰ TFA in water (A phase) and 1‰ TFA in acetonitrile (B phase); use 40%-80% acetonitrile in water to perform a linear gradient over 30 min (20%-40% acetonitrile for 2 min, then 40%-80% for 30 min); the flow rate is 1 mL / min; the detection wavelength is 214 nm; and the injection volume is 20 μL. Identify the obtained crude peptide using ESI-MS. Freeze-dry the identified crude peptide using a freeze-dryer, dissolve it, separate and purify it using semi-preparative reversed-phase high-performance liquid chromatography, and obtain the target pure peptide by freeze-drying.

[0044] Figure 2is the chemical structure of the probe, and the reverse phase high performance liquid chromatogram and ESI-MS mass spectrum of the synthesis of the probe. The theoretical molecular weight is consistent with the actual molecular weight, which proves that the target product is successfully synthesized.

[0045] Application of the biological fluorescence probe based on AIE effect in Example 2

[0046] Detection of Cathepsin B by ZSY-3 probe at the molecular level

[0047] The enzymatic reaction of the present application is carried out at 37℃ in an acetic acid buffer solution (pH 5.0, 1mM EDTA, 4mM DTT). First, 40nM Cathepsin B is incubated in the buffer solution at 37℃ for 15min to activate Cathepsin B. Then 50μL of 80μM probe solution, 50μL of acetic acid buffer solution and 100μL of activated Cathepsin B are added to a 96-well plate, and fluorescence detection is carried out using a fluorescence microplate reader (λ ex =335nm, λ em =470nm).

[0048] Through full-spectrum scanning analysis, it can be clearly observed that the fluorescence intensity of ZSY-3 is significantly enhanced after the addition of Cathepsin B Figure 3 , 4). Specifically, when detecting the fluorescence intensity at 470nm, ZSY-3 without the addition of Cathepsin B hardly emits light. However, after the addition of Cathepsin B, the fluorescence intensity of ZSY-3 gradually increases with the extension of incubation time, and reaches a maximum value at 60min, which is about 64 times higher than that of the initial state Figure 3 , Figure 4 ). Under the 365nm ultraviolet lamp, ZSY-3 without the addition of Cathepsin B hardly emits light. However, after the addition of Cathepsin B, ZSY-3 rapidly aggregates and triggers the aggregation-induced emission effect, emitting strong blue fluorescence Figure 3 . This phenomenon further verifies the specific response ability of ZSY-3 to Cathepsin B. This significant fluorescence enhancement phenomenon indicates that ZSY-3 has a high sensitivity of “fluorescence opening” response to Cathepsin B. The biological fluorescence probe ZSY-3 exhibits excellent performance when detecting Cathepsin B at the molecular level.

[0049] Dynamic analysis of the interaction between the biological fluorescence probe ZSY-3 and Cathepsin B and structural identification

[0050] Dynamic analysis was performed to verify the interaction between the probe and the enzyme using analytical RP-HPLC coupled with ESI-MS. A single-component solution of ZSY-3 and a co-incubation system of ZSY-3 and cathepsin B were prepared and incubated for 1 hour under standard reaction conditions (37℃, pH 5.0, acetate buffer). Samples were then taken at different time points (0h and 1h) for analysis of the probe and evaluation of the enzymatic hydrolytic effect of cathepsin B on the ZSY-3 probe using analytical RP-HPLC. Experimental conditions were as follows: a C4 reversed-phase column was used; the mobile phase consisted of an aqueous solution containing 1‰ trifluoroacetic acid (phase A) and an acetonitrile solution containing 1‰ trifluoroacetic acid (phase B); the flow rate was 1 mL / min; the detection wavelength was 214 nm; and the injection volume was 20 μL. Elution was performed with 5% acetonitrile for 2 min, followed by linear elution with 5%-90% for 30 min. Furthermore, mass spectrometry was used to detect newly emerging peaks and deduce possible lysis products.

[0051] like Figure 5 , 6 As shown, the interaction between the biofluorescent probe ZSY-3 and cathepsin B was analyzed in detail using high-performance liquid chromatography (HPLC) and mass spectrometry (MS). RP-HPLC analysis revealed a new peak in the incubated sample. Figure 5 Comparing the chromatograms before and after incubation, it was found that the original peak a (retention time approximately 23.8 min) of ZSY-3 significantly decreased after incubation, while a new peak b appeared at a retention time of approximately 26.4 min. This result indicates that the ZSY-3 polypeptide chain underwent specific cleavage during co-incubation with cathepsin B, producing new degradation products. Mass spectrometry analysis determined the molecular weight and cleavage site of the new peaks. The mass spectra showed that the molecular weights of the new peaks were 744.94 and 673.86, corresponding to the products resulting from the cleavage of the GIVRAK sequence in the ZSY-3 polypeptide chain, with the cleavage site located at the RA and AK bonds. Figure 6 This result is consistent with the specific cleavage site of cathepsin B reported in previous literature, further verifying the specific cleavage of ZSY-3 by cathepsin B. The experimental results provide crucial experimental evidence for studying the enzyme's response mechanism and further validate the probe's specific response to the target enzyme.

[0052] Response of the biofluorescent probe ZSY-3 in cancer cell lysates

[0053] The cells used in the experiment were MDA-MB-231 human breast cancer cells, which were selected as the research object because they highly expressed cathepsin B. The MDA-MB-231 cells were prepared into cell lysates by repeated freeze-thaw method. Specifically, 1640 culture medium containing 10% serum was used, and the culture conditions were 37°C and 5% CO2. When the MDA-MB-231 cells were cultured to 80%-90% confluence, the culture medium was removed, 1 mL of sodium acetate buffer solution was added, and the cells were scraped off and collected using a cell scraper and placed in a cryotube. The cryotube was placed in liquid nitrogen for 15 s and then placed in a 37°C water bath for 3 min, repeated three times. Then centrifuged at 14000 rmp for 3 min, and the supernatant was taken as the cell lysate.

[0054] When using the cell lysate, first preheat the cell lysate at 37°C for 15 min. Then add 80 μM probe 50 μL, acetic acid buffer solution 50 μL and cell lysate 100 μL into a 96-well plate for fluorescence detection. In the inhibitor control group, another 50 μL of cathepsin B specific inhibitor CA-074Me containing 40 μM was added. The fluorescence signal was detected over time using a fluorescence microplate reader (excitation λ ex = 335 nm, λ em = 470 nm).

[0055] As shown in Figure 7 , in this experiment, the bioluminescent probe ZSY-3 was incubated with MDA-MB-231 cell lysate which highly expressed cathepsin. At the beginning of incubation (0 min), the fluorescence intensity of probe ZSY-3 was low, which was consistent with its low fluorescence characteristics in the inactive state. With the increase of incubation time, the fluorescence intensity gradually increased, and reached a plateau at 30 min, and then tended to be stable. This time-dependent fluorescence enhancement phenomenon indicates that the probe ZSY-3 can effectively respond to the activity of cathepsin B, and its response speed is fast, and it can reach the maximum fluorescence intensity in a short time.

[0056] Under the same incubation conditions, no fluorescence signal was found in the control group with only ZSY-3 or only cathepsin B, indicating that the probe would not produce false positive signals due to non-specific factors, further proving the specificity of the probe.

[0057] In the inhibitor control group, the probe ZSY-3 was incubated with the cell lysate while adding the cathepsin B specific inhibitor CA-074Me. The experimental results show that the fluorescence intensity of the inhibitor control group remained at a low level throughout the incubation process. After the addition of CA-074Me, the activity of cathepsin B was effectively inhibited, and the probe molecule could not be cut, so it could not trigger the AIE effect, resulting in the fluorescence intensity remaining at a low level. The results of the inhibitor control group show that the activity of cathepsin B is a key factor for the fluorescence enhancement of probe ZSY-3, which proves its specific response to cathepsin B.

[0058] Imaging and specific response of bio-fluorescent probe ZSY-3 in cancer cells

[0059] Logarithmic growth phase MDA-MB-231 cells were selected, and the cells were collected and inoculated on a 24-well plate at a density of 2.5×10 4 / well under the culture conditions of 37℃ and 5% CO2 overnight. Bio-fluorescent probe ZSY-3 was added, and the final concentration was 20μM. Confocal laser scanning microscope (CLSM) was used to observe the fluorescence changes in cells at different times, and the fluorescence intensity was quantified.

[0060] As shown in Figure 8 , the probe ZSY-3 was incubated with MDA-MB-231 cells, and the fluorescence intensity gradually increased with the increase of incubation time, reaching a maximum at 4h, and uniformly distributed in the cells. It is proved that the probe can effectively enter the cells and realize real-time monitoring of cathepsin B at the level of living cells.

[0061] Finally, it should be noted that the above description is only a preferred embodiment of the present application and is not intended to limit the present application. Although the present application has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent replacements to some of them. Any modification, equivalent replacement, improvement, etc. made within the spirit and principles of the present application shall be included in the protection scope of the present application. Although the specific embodiments of the present application have been described above, they are not intended to limit the protection scope of the present application, and those skilled in the art should understand that various modifications or variations made by those skilled in the art on the basis of the technical solutions of the present application without creative labor are still within the protection scope of the present application.

Claims

1. A fluorescent probe molecule for cathepsin B detection, characterized in that, The probe is a polypeptide-based aggregation-induced emission response fluorescent molecule, and its amino acid sequence is: Ac-rrrr-AEEA-GIVRAK(Gl-TPE)-COOH.

2. A process for the preparation of a fluorescent probe molecule for cathepsin B detection according to claim 1, characterized by The preparation method comprises: 9-fluorenylmethyloxycarbonyl-based solid-phase synthesis through Wang Resin resin; 4 D-type arginines are connected to the N end of the probe; 2-(2-(2-aminoethoxy)ethoxy)acetic acid is connected between the arginine and the enzyme cleavage polypeptide sequence; the enzyme cleavage polypeptide sequence is GIVRAK; and the enzyme cleavage polypeptide sequence is connected with the aggregation-induced emission group tetraphenyl ethylene through Gl.

3. The fluorescent probe of claim 1 is applied to the preparation of a cathepsin B detection reagent.

4. Use of the fluorescent probe according to claim 3 for the preparation of a cathepsin B detection reagent. By using a fluorescence detection method, the fluorescent probe is hydrolyzed by cathepsin B, and the fluorescence intensity is significantly enhanced with the increase of the concentration of cathepsin B and the incubation time.

Images