Preparation method and application of biological fluorescent probe molecule responding to cathepsin B based on AIE effect

Abstract

The invention belongs to the field of biological medicine, and provides a fluorescent probe for specific response to cathepsin B. The probe is a biological fluorescent probe which is developed on the basis of an aggregation-induced emission effect and has fluorescence'opening 'response, and the amino acid sequence of the probe is Ac-rrrr-AEA-GIVRAK (Gl-TPE)-COOH. The probe is synthesized through a solid-phase synthesis method, a cathepsin B specific cleavage recognition site GIVRAK is cleaved, after cleavage, a part containing tetraphenylethylene is aggregated, an aggregation-induced emission effect is triggered, a strong fluorescence signal is generated, and specific recognition and monitoring of the probe on cathepsin B are achieved. Meanwhile, four D-type arginine is introduced into the probe to help the probe enter cells, so that the cathepsin B is monitored on the cellular level. The fluorescent probe is high in cathepsin B monitoring specificity, the method is simple, the condition is mild, and the high practical application value is achieved.

Description

Technical Field

[0001] The present invention relates to aggregation-induced emission (AIE) technology and the field of bioluminescent probes. Specifically, it relates to a method for preparing a cathepsin B-responsive bioluminescent probe molecule based on the AIE effect and its application in tumor diagnosis. This technology belongs to the field of biological detection and polypeptide probes. Background Art

[0002] Cancer is a leading cause of premature death worldwide and has become a major health threat to Chinese residents. Cancer incidence and mortality are projected to rise further in the coming decades as the population ages and lifestyles change. Therefore, early diagnosis and targeted treatment of tumors are crucial for improving patient survival and quality of life.

[0003] As a key tool in the field of bioanalysis, fluorescent probes play an important role in the detection and monitoring of bioactive 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 a variety of pathological processes and carcinogenic mechanisms. Studies have shown that cathepsin B is overexpressed in a variety of malignant tumors, including brain cancer, lung cancer, prostate cancer, breast cancer, and colorectal cancer. Cathepsin B plays an important role in the invasion, metastasis, and drug resistance of tumor cells by degrading the extracellular matrix (ECM) and regulating the function of cell surface receptors.

[0004] Currently, commonly used methods for detecting cathepsin B in clinical practice include enzyme-linked immunosorbent assay, protein immunoblotting, 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, the development of 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-induced fluorescence quenching (ACQ) at high concentrations, resulting in a decrease in detection sensitivity. In addition, most existing cathepsin B-responsive fluorescent probes are limited to in vitro detection, making it difficult to achieve real-time imaging and monitoring of cathepsin B in cells.

[0005] In response to the above problems, the present invention proposes a biological fluorescent probe molecule based on the aggregation-induced emission (AIE) effect. The probe comprises a polypeptide sequence that specifically recognizes cathepsin B and is connected to tetraphenylethylene (TPE). The TPE molecule is a typical AIE molecule. It hardly emits light when it exists in monomeric form. In the aggregated state, due to restricted intramolecular rotation (RIR), the energy loss of non-radiative transitions is reduced, resulting in a significant enhancement of fluorescence. When the probe specifically interacts with cathepsin B, the polypeptide chain breaks and the released TPE group partially aggregates, thereby "turning on" the fluorescent signal and avoiding the ACQ phenomenon. At present, there are no reports on the use of TPE fluorescent probes based on polypeptides as substrates for the detection of cathepsin B in vivo and in vitro. Summary of the Invention

[0006] In response to the above problems, the present invention proposes a bioluminescent probe ZSY-3 based on the AIE effect. The probe introduces TPE as an AIE fluorescent molecule and combines a protease-specifically recognized polypeptide sequence and a functional modification unit to achieve a specific fluorescent response to cathepsins, overcoming the shortcomings of traditional probes in terms of sensitivity, specificity and application range. The probe molecule is composed of the following parts: an AIE group that can significantly enhance the fluorescence signal in an aggregated state, preferably TPE; a polypeptide sequence that can specifically recognize and be cleaved by cathepsin B, preferably GIVRAK; a polypeptide fragment that can enhance the water solubility and cell penetration ability of the probe, preferably multiple D-arginines; a linker between the enzymatic polypeptide sequence and the penetrating 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 bioluminescent probe to detect cathepsin B at the molecular and cellular levels, and has the characteristics of strong specificity and high sensitivity.

[0007] Specifically, the present invention is achieved through the following technical solutions:

[0008] The first aspect of the present invention provides a cathepsin B-responsive bioluminescent probe ZSY-3 based on the AIE effect. Its chemical structure and primary amino acid sequence are as follows:

[0009]

[0010] The bioluminescent probe has a fluorescent "on" capability that responds to cathepsin B. In a solution without cathepsin B, the probe molecule exists as a monomer, and the TPE group does not emit light. When the probe is digested by cathepsin B, the polypeptide chain is cleaved, releasing the TPE group-bearing segments that aggregate. The aggregated TPE groups emit a fluorescent signal due to restricted intramolecular rotation.

[0011] A second aspect of the present invention provides a method for preparing the aforementioned bioluminescent probe. Specifically, the target polypeptide is synthesized using 9-fluorenylmethoxycarbonyl (Fmoc)-based solid-phase peptide synthesis (Fmoc-SPPS). Wang Resin is preferably used for solid-phase synthesis, with Fmoc as the protecting group. The amino acids of the target polypeptide are coupled stepwise. After the polypeptide chain is synthesized, deprotection and purification are performed to obtain the cathepsin B-responsive bioluminescent probe ZSY-3.

[0012] A third aspect of the present invention provides an application of a bioluminescent probe based on the AIE effect, specifically the application of the ZSY-3 bioluminescent probe for detecting cathepsin B at the molecular level. At the molecular level, ZSY-3 enables sensitive and specific fluorescent detection of cathepsin B. By designing a specific polypeptide sequence (GIVRAK), the probe is specifically cleaved by cathepsin B. After cleavage, the portion containing the TPE group aggregates, triggering the aggregation-induced emission effect, thereby generating a strong fluorescent signal. This fluorescence "on" mechanism gives ZSY-3 extremely high sensitivity and specificity in detecting cathepsin B.

[0013] A fourth aspect of the present invention is to provide an application of an AIE-based bioluminescent probe for detecting cathepsin B in cell lysates. By incubating cell lysates high in cathepsin B with ZSY-3, experimental results showed that the fluorescence signal gradually increased over time. To verify the specificity of the probe, the cathepsin B-specific inhibitor CA-074Me was added, and the results showed that the fluorescence signal in the inhibitor group was significantly reduced. This demonstrates that ZSY-3 can specifically detect the presence of cathepsin B in cell lysates, providing a powerful tool for studying the expression and function of cathepsin B in tumor cells.

[0014] A fifth aspect of the present invention is to provide a fluorescent probe based on the AIE effect for detecting and imaging cathepsin B in living cells. The cell-penetrating D-arginine is introduced into the polypeptide chain of ZSY-3, enabling it to effectively penetrate the cell membrane and monitor the expression level of cathepsin B in real time within living cells. Confocal microscopy revealed that the fluorescence intensity significantly increased with increasing probe incubation time. This real-time imaging capability makes ZSY-3 valuable for 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 highly sensitive biological fluorescent probe. ZSY-3 significantly enhances the fluorescence signal through the AIE effect, overcoming the lack of sensitivity of traditional fluorescent probes caused by the ACQ phenomenon.

[0017] 2. The above technical solution can provide a highly specific bioluminescent probe. A specific polypeptide sequence (GIVRAK) is introduced into the design of ZSY-3, which can be specifically recognized and cleaved by cathepsin B. The cleavage site of ZSY-3 enzymatic hydrolysis was verified by reversed-phase high-performance liquid chromatography (RP-HPLC) and mass spectrometry analysis, further demonstrating its high specificity for cathepsin B. In addition, in cellular level detection, the fluorescence signal was significantly reduced after the addition of CA-074Me, a specific inhibitor of cathepsin B, further verifying the specific response ability of ZSY-3 to cathepsin B.

[0018] 3. The above technical solution can provide an excellent bioluminescent probe with cell-penetrating ability. The cell-penetrating D-arginine is introduced into the polypeptide chain of ZSY-3, which can effectively penetrate the cell membrane and monitor the expression level of cathepsin B in real time within living cells. Confocal microscopy revealed that ZSY-3 can rapidly enter cells and accumulate within them. With the extension of incubation time, the intracellular fluorescence signal gradually increases, demonstrating the specific responsiveness of ZSY-3 to cathepsin B in cells.

[0019] This study synthesized a bioluminescent probe based on the AIE effect. The preparation process is simple and highly reproducible. The probe exhibits excellent detection performance at both the molecular and cellular levels, providing a novel tool and technology for tumor marker detection, disease diagnosis, and treatment monitoring. By monitoring cathepsin B expression in real time, the probe provides important support for early diagnosis and precision treatment of tumors. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] The accompanying drawings, which constitute part of the present invention, are intended to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are intended to explain the present invention and do not constitute an improper limitation of the present invention.

[0021] Figure 1 Schematic diagram of the solid-phase polypeptide synthesis method of the bioluminescent probe of the present invention;

[0022] Figure 2 The chemical structure, primary amino acid sequence, and analytical reverse-phase high-performance liquid chromatography and mass spectrometry of ZSY-3;

[0023] Figure 3 The fluorescence intensity changes of bioluminescent probe ZSY-3 before and after co-incubation with cathepsin B and the comparison chart under 365nm ultraviolet light;

[0024] Figure 4 Fluorescence spectra of bioluminescent probe ZSY-3 incubated with cathepsin B from 0 to 60 min;

[0025] Figure 5 HPLC chromatograms of ZSY-3 before and after incubation with cathepsin B;

[0026] Figure 6 Mass spectrum of ZSY-3 after incubation with cathepsin B;

[0027] Figure 7 Curves of probe fluorescence changes over time after treatment with cathepsin B-high-expressing cell MDA-MB-231 lysate and inhibitor CA-074Me;

[0028] Figure 8 Confocal imaging of high-expressing MDA-MB-231 cells treated with 20 μM ZSY-3 over time. Specific implementation plan

[0029] Below in conjunction with accompanying drawing and specific embodiment, specific embodiment of the present invention is described in further detail.In specific embodiment, each original reagent and raw material all can be purchased.It should be pointed out that following detailed description is all exemplary, is intended to provide further explanation of the present invention.Unless otherwise indicated, all technical and scientific terms used in the present invention have the same meaning that those of ordinary skill in the art to which the present invention belongs are usually understood.

[0030] In a specific embodiment of the present invention, a specific method for synthesizing, separating and purifying the above-mentioned bioluminescent probe ZSY-3 is provided, and its structure is verified.

[0031] In another embodiment of the present invention, there is provided the use of the bioluminescent probe ZSY-3 for detecting cathepsin B at the molecular level and in cell lysates.

[0032] In another specific embodiment of the present invention, a method for detecting cathepsin B in MDA-MB-231 cells using the bioluminescent probe ZSY-3 is provided, namely, the application of detecting cathepsin B at the cellular level.

[0033] The preparation and application of the bioluminescent probe ZSY-3 of the present invention are further described in detail below through specific implementations. The present invention is further explained by examples below, but is not intended to limit the present invention. It should be understood that these examples are intended only to illustrate the present invention and are not intended to limit the scope of the present invention.

[0034] Example 1 Synthesis of ZSY-3

[0035] In a specific embodiment of the present invention, a method for synthesizing the above-mentioned biological fluorescent probe is provided, and the synthesis method is as follows.

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

[0037] Depending on the reaction volume, weigh a certain amount of Wang Resin and perform a standard wash, i.e., wash twice with DMF, twice with DCM, once with DMF, once with DCM, and three times with DMF. Then, soak the resin in 3-4 mL of analytical grade DMF solvent and swell it at room temperature for 2 hours to pre-activate the resin. Then, soak the resin in 3-4 mL of an analytical grade DMF / DCM mixture (4:1, v:v) and shake it in a 28°C constant temperature shaker for 1 hour to fully activate the resin. After activation, connect the first amino acid. Then, remove the Fmoc protecting group and connect according to the amino acid sequence. After all reactions are complete, perform a standard wash.

[0038] Method for attaching the first amino acid: Dissolve the amino acid and condensation reagent completely in analytical-grade DMF and add to the resin. Shake and condense the resin twice at 28°C, with the first reaction taking 8 hours and the second 12 hours. For the amino acid condensation, the reactant ratio is: amino acid: DIC: Oxyma: DAMP = 3 equivalents: 6 equivalents: 3 equivalents: 0.4 equivalents (molar ratio). After the two reactions, perform a standard wash, then remove any residual solvent with a pump.

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

[0040] AEEA ligation method: Completely dissolve the amino acid and condensation reagent in analytical-grade DMF and add them to the resin. Shake and condense twice at 28°C. To ensure complete reaction, shake and condense twice at 28°C, with the first reaction time of 40 minutes and the second reaction time of 50 minutes. For amino acid condensation, the reactant ratio is Fmoc-AEEA-COOH: HATU: HOAt: DIEA = 3 equivalents: 2.8 equivalents: 3 equivalents: 6 equivalents (molar ratio). After the two reactions, to completely remove impurities from the resin, perform a standard wash (two washes with DMF, two washes with DCM, one wash with DMF, one wash with DCM, and three washes with DMF), then remove any residual solvent with a water pump. D-amino acid ligation method: Condense the amino acid sequence synthesized in step 2 with D-arginine: Completely dissolve the amino acid and condensation reagent in analytical-grade DMF and add them to the resin. To ensure complete reaction, shake and condense twice at 28°C, with the first reaction time of 40 minutes and the second reaction time of 50 minutes. When amino acids are condensed, the ratio of reactants is amino acid: HCTU: DIEA = 3 equivalents: 2.8 equivalents: 6 equivalents (molar ratio). After two reactions, standard washing is performed once, and then the residual solvent is pumped out with a water pump.

[0041] The peptide end acetylation method was as follows: first, the terminal Fmoc residue was removed using 20% piperidine in DMF. A 4 mL solution of acetic anhydride: DIEA: DMF (1 equivalent: 1 equivalent: 8 equivalents, by volume) was prepared and the reaction times were 5 and 10 minutes. After the two reactions, standard washes were performed.

[0042] TPE connection method: First, remove the Alloc protecting group on the lysine side chain. Specifically, remove the Alloc protecting group using 72 mg of Pd(PPh3)4 and 300 μL of PhSiH3 per 0.10 mmol of resin. Dissolve 472 mg of Pd(PPh3)3 in 2 mL of DCM and shake until completely dissolved. Then add 2 mL of DMF and, finally, an appropriate amount of PhSiH3 (300 μL). Incubate at 28°C with shaking in the dark for 1.5 and 2 hours. Drain the solution, wash twice with DCM, twice with DMF, once with DMF, twice with DCM, and then perform a standard wash. Finally, remove any remaining solvent using a pump. For the condensation of the lysine side chain with the amino acid, the reactant ratio is amino acid: HCTU: DIEA = 3 equivalents: 2.8 equivalents: 6 equivalents (molar ratio), and the reaction time is 50 and 60 minutes. The synthesized peptide is then condensed with the fluorescent group. The reactant ratio was TPE: HATU: HOAt: DIEA = 3 equivalents: 2.8 equivalents: 3 equivalents: 6 equivalents (molar ratio), and the reaction time was 1.5 h and 2 h. The attached peptide chain was cleaved from the resin: before cleavage, a standard wash was performed once, followed by five washes with analytical-grade DCM, followed by 5 min of water pumping and 4 min of oil pumping. A cleavage reagent was added to the prepared peptide in a ratio of TFA: water: TIPS = 95:2.5:2.5 (v:v:v). The pre-chilled cleavage reagent was added to a synthesis tube and allowed to react on a shaker at 28-30°C for 2.5 h. The cleavage solution was poured through the synthesis tube into a three-necked flask. The resin was washed twice with 0.5 mL of TFA, and the cleavage reagent and wash solution were combined. The resin was purged with N2 to 2-3 mL, then precipitated with pre-chilled anhydrous ether. Centrifuged for 3 min, the supernatant discarded, and the crude peptide was obtained after ether precipitation. Repeat the precipitation and centrifugation three times, and place the crude product in a fume hood to dry.

[0043] The crude product was separated and purified: the crude peptide product was dissolved in a mixed solution of acetonitrile and water containing 1‰ TFA, and the crude peptide was analyzed using analytical RP-HPLC. The experimental conditions were as follows: a C18 reversed-phase column was used; the mobile phase was an aqueous solution of 1‰ TFA (phase A), and acetonitrile of 1‰ TFA (phase B); a linear gradient of 40%-80% acetonitrile in water was performed over 30 minutes (20%-40% acetonitrile for 2 minutes, then 40%-80% for 30 minutes); the flow rate was 1 mL / min; the detection wavelength was 214 nm; the injection volume was 20 μL. The crude peptide was identified by ESI-MS. The identified crude peptide was freeze-dried in a freeze dryer, dissolved, and separated and purified using semi-preparative reversed-phase high-performance liquid chromatography, and the target pure peptide was obtained by freeze-drying.

[0044] Figure 2The chemical structure of the probe is shown below, along with its reversed-phase HPLC and ESI-MS spectra. The theoretical molecular weight is consistent with the actual molecular weight, demonstrating successful synthesis of the target product.

[0045] Example 2 Application of bioluminescent probe based on AIE effect

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

[0047] The enzymatic hydrolysis reaction of the present invention is carried out at 37°C in an acetate buffer solution (pH 5.0, 1mM EDTA, 4mM DTT). First, 40nM cathepsin B is added to the buffer solution and incubated at 37°C for 15 minutes to activate cathepsin B. Then, 50μL of 80μM probe solution, 50μL of acetate buffer solution, and 100μL of activated cathepsin B are added to a 96-well plate, and fluorescence detection is performed using a fluorescence microplate reader (excitation 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 ( Figure 3 Specifically, when the fluorescence intensity was detected at 470 nm, ZSY-3 without cathepsin B showed almost no luminescence. However, after the addition of cathepsin B, the fluorescence intensity of ZSY-3 gradually increased with the extension of the incubation time and reached a maximum at 60 min. Compared with the initial state, the fluorescence intensity increased by about 64 times ( Figure 3 , Figure 4 ). Under 365nm ultraviolet light, ZSY-3 without cathepsin B hardly emits light. However, when cathepsin B is added, ZSY-3 rapidly aggregates and triggers the aggregation-induced emission effect, emitting strong blue fluorescence ( Figure 3 This phenomenon further validates ZSY-3's specific response to cathepsin B. This significant fluorescence enhancement demonstrates ZSY-3's highly sensitive "fluorescence on" response to cathepsin B. The bioluminescent probe ZSY-3 demonstrates excellent performance in detecting cathepsin B at the molecular level.

[0049] Dynamic Analysis and Structural Elucidation of the Interaction between Bioluminescent Probe ZSY-3 and Cathepsin B

[0050] Analytical reversed-phase HPLC coupled with electrophoretic resonance imaging (ESI-MS) was used to validate the interaction between the probe and the enzyme and to conduct dynamic analysis. A single-component solution of ZSY-3 and a co-incubation system of ZSY-3 and cathepsin B were prepared. The solutions were incubated for 1 hour under standard reaction conditions (37°C, pH 5.0 acetate buffer). Samples were then taken at different time points (0 and 1 hour) for analysis by analytical reversed-phase HPLC to assess the enzymatic degradation of the ZSY-3 probe by cathepsin B. The experimental conditions were as follows: a C4 reversed-phase column was used, with the mobile phase consisting of 1‰ trifluoroacetic acid in water (phase A) and 1‰ trifluoroacetic acid in acetonitrile (phase B), at a flow rate of 1 mL / min, a detection wavelength of 214 nm, and a 20 μL injection volume. A 5% acetonitrile solution was used for 2 minutes, followed by a 5% to 90% linear elution for 30 minutes. Newly emerged peaks were further analyzed by mass spectrometry to identify potential cleavage products.

[0051] like Figure 5 、 6 As shown in Figure 2, the interaction between bioluminescent probe ZSY-3 and cathepsin B was deeply analyzed by high performance liquid chromatography and mass spectrometry. RP-HPLC analysis revealed that a new peak ( Figure 5 ). By comparing the chromatograms before and after incubation, it can be found that the original peak a of ZSY-3 (retention time is about 23.8min) is significantly weakened after incubation, and a new peak b appears at a retention time of about 26.4min. This result shows that the polypeptide chain of ZSY-3 undergoes specific cleavage during the co-incubation with cathepsin B, resulting in new degradation products. The molecular weight corresponding to the new peak and its cleavage site were determined by mass spectrometry analysis. The mass spectrum shows that the molecular weight of the new peak is 744.94 and 673.86, corresponding to the product after the GIVRAK sequence in the ZSY-3 polypeptide chain is cleaved, and the cleavage site is 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 validating the specific cleavage of ZSY-3 by cathepsin B. The experimental results provide key experimental evidence for studying the enzyme's response mechanism and further verify the probe's specific response to the target enzyme.

[0052] Response of the bioluminescent 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 of their high expression of cathepsin B. MDA-MB-231 cells were subjected to repeated freeze-thaw method to prepare cell lysates. 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 aspirated, 1 mL of sodium acetate buffer solution was added, and the cells were scraped and collected using a cell scraper and placed in a cryovial. The cryovial was placed in liquid nitrogen for 15 seconds, then placed in a 37°C water bath to thaw for 3 minutes, and repeated three times. After centrifugation at 14,000 rpm for 3 minutes, the supernatant was taken as the cell lysate.

[0054] When using cell lysate, preheat the cell lysate at 37°C for 15 minutes. Then, add 50 μL of 80 μM probe, 50 μL of acetate buffer, and 100 μL of cell lysate to a 96-well plate for fluorescence detection. In the inhibitor control group, add 50 μL of a solution containing 40 μM of the cathepsin B-specific inhibitor CA-074Me. Fluorescence signals (excitation λ) are measured over time using a fluorescence microplate reader. ex =335nm,λ em =470nm).

[0055] like Figure 7 As shown in the figure, this experiment incubated the bioluminescent probe ZSY-3 with lysate from MDA-MB-231 cells, which overexpress cathepsin B. At the beginning of the incubation (0 min), the fluorescence intensity of the ZSY-3 probe was low, consistent with its low fluorescence characteristic in its inactive state. With increasing incubation time, the fluorescence intensity gradually increased, reaching a plateau at 30 min and then stabilizing. This time-dependent fluorescence enhancement indicates that the ZSY-3 probe effectively responds to cathepsin B activity and does so rapidly, reaching maximum fluorescence intensity within a short period of time.

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

[0057] In the inhibitor control group, probe ZSY-3 was incubated with cell lysate and the cathepsin B-specific inhibitor CA-074Me was added. The experimental results showed that the fluorescence intensity in the inhibitor control group remained low throughout the incubation process. The addition of CA-074Me effectively inhibited cathepsin B activity, preventing the probe from being cleaved and thus failing to trigger the AIE effect, resulting in a low fluorescence intensity. The results from the inhibitor control group indicate that cathepsin B activity is a key factor in the fluorescence enhancement of probe ZSY-3, demonstrating its specific response to cathepsin B.

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

[0059] MDA-MB-231 cells in the logarithmic growth phase were selected and collected and plated at 2.5×10 4 Cells were seeded at a density of 100 μg / well in a 24-well plate and cultured overnight at 37°C in 5% CO2. The bioluminescent probe ZSY-3 was added at a final concentration of 20 μM. Fluorescence changes in the cells were observed and quantified using a confocal laser scanning microscope (CLSM) with an excitation wavelength of 405 nm and an emission wavelength of 470 nm at different times.

[0060] like Figure 8 As shown, when the ZSY-3 probe was co-incubated with MDA-MB-231 cells, the fluorescence intensity gradually increased with increasing incubation time, reaching a maximum at 4 hours and being evenly distributed within the cells. This demonstrates that the probe can effectively enter cells and achieve real-time monitoring of cathepsin B at the living cell level.

[0061] Finally, it should be noted that the above is only a preferred embodiment of the present invention and is not intended to limit the present invention. Although the present invention has been described in detail with reference to the aforementioned embodiment, those skilled in the art can still modify the technical solutions described in the aforementioned embodiment or make equivalent replacements for parts thereof. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention. Although the above describes the specific embodiments of the present invention, it is not intended to limit the scope of protection of the present invention. Those skilled in the art should understand that, based on the technical solution of the present invention, various modifications or variations that can be made by those skilled in the art without creative work are still within the scope of protection of the present invention.

Claims

1. A method for preparing a bioluminescent probe molecule that responds to cathepsin B based on the aggregation-induced emission (AIE) effect and its application, characterized in that: The amino acid sequence of the biological probe is: Ac-rrrr-AEEA-GIVRAK(Gl-TPE)-COOH. The molecule can specifically respond to cathepsin B at the molecular and cellular levels and monitor it in real time.

2. The method for preparing a bioluminescent probe based on the AIE effect according to claim 1, characterized in that The preparation method includes: selecting Wang Resin resin for solid-phase synthesis based on 9-fluorenylmethyloxycarbonyl to prepare a probe with a carboxyl group at the N-terminus; selecting a hydrophilic penetrating peptide in the probe to be connected to the N-terminus of the probe, preferably, four D-arginines are used to improve the penetration ability of the probe into cells; connecting the arginine to the enzyme cleavage site through a water-soluble linker, preferably AEEA, and the enzyme cleavage site is preferably GIVRAK; connecting the enzyme cleavage site to the AIE group through a flexible linker, wherein the AIE probe is preferably tetraphenylethylene (TPE), and the soluble linker is preferably Gl, to ensure the generation of the AIE effect.

3. The use of a bioluminescent probe based on the AIE effect for monitoring cathepsin B at the molecular level according to claim 1, characterized in that: The bioluminescent probe was tested at 37°C in a sodium acetate buffer solution (pH 5.0, 1 mM EDTA, 4 mM DTT) containing cathepsin B. HPLC and mass spectrometry results demonstrated that cathepsin B could specifically cleave the recognition site of cathepsin B in ZSY-3. After cleavage, the portion containing the TPE group aggregated, triggering aggregation-induced emission, thereby generating a strong fluorescence signal. The fluorescence intensity significantly increased over time, making it suitable for the detection of cathepsin B.

4. Use of a novel AIE-responsive bioluminescent probe according to claim 1 for detecting cathepsin B at the cellular level, characterized in that: Using MDA-MB-231 cells with high expression of cathepsin B, cell lysates were obtained by repeated freeze-thaw cycles. ZSY-3 was used to detect cathepsin B in tumor cell lysates. The fluorescence signal increased significantly over time and decreased significantly in the presence of the cathepsin-specific inhibitor CA-074Me.

5. The use of a novel AIE-based bioluminescent probe for detecting cathepsin B in living cells according to claim 1, characterized in that: MDA-MB-231 cells with high expression of cathepsin B were co-incubated with ZSY-3. As the incubation time increased, the fluorescence intensity within the cells significantly increased, demonstrating that the probe has cell penetrability, can be effectively taken up by cells, and can achieve the monitoring of intracellular cathepsin B.

Images