Cathepsin B-based biomarker detection product and application thereof

Abstract

The invention relates to the technical field of biomedical detection, in particular to a cathepsin B-based biomarker detection product and application thereof. The cathepsin B-based biomarker detection product comprises an active substrate probe, a standard substance solution, a reaction buffer solution and a detection chip. The active substrate probe is a fluorescence labeled polypeptide chain, and a specific cleavage site is composed of arginine-arginine-lysine; the surface of the detection chip is modified with an affinity ligand to fix a target enzyme molecule. The invention further provides a detection method and application. The detection method is suitable for early diagnosis of cancers, activity monitoring of inflammatory diseases and prognosis evaluation of neurodegenerative diseases. Efficient and accurate detection is realized through a microfluidic device and an automatic control system, the sensitivity and the specificity are remarkably improved, and diversified clinical requirements are met.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biomedical detection, and specifically relates to a biomarker detection product based on cathepsin B and an application thereof. BACKGROUND

[0002] Biomarkers play an important role in disease diagnosis, prognosis evaluation and treatment monitoring. Cathepsin B is a lysosomal cysteine protease that is widely involved in intracellular protein degradation, antigen presentation and regulation of tumor microenvironment and other biological processes. In recent years, studies have shown that the expression level or activity of cathepsin B changes significantly in various diseases, especially in cancer, inflammatory diseases and neurodegenerative diseases, showing potential diagnostic and therapeutic value. Therefore, the development of biomarker detection products based on cathepsin B and their applications has become a research hotspot.

[0003] Patent CN108700567B discloses a method for predicting renal cell carcinoma, which is achieved by determining the levels and / or chemical compositions of glycosaminoglycans (GAG) chondroitin sulfate (CS) and heparan sulfate (HS) in a body fluid sample. Although this patent provides an effective detection means, its detection target is glycosaminoglycan substances, which is not directly related to cathepsin B, and it does not involve quantitative analysis of specific enzyme activity, limiting its application potential in enzyme-related diseases.

[0004] Patent CN114424064A discloses a method for diagnosing cancer or cancer susceptibility by detecting the concentration of characteristic compounds produced by sugar, amino acid or polyol metabolism, and comparing it with a reference value to determine the cancer status of the subject. However, this method relies on the indirect detection of metabolic products and fails to directly evaluate the activity or expression level of cathepsin B, which may result in insufficient detection sensitivity and specificity, especially in the application effect under complex disease background.

[0005] The above-mentioned prior art solutions have certain limitations in the selection of detection targets, and fail to fully exploit the core potential of cathepsin B as a biomarker. At the same time, there is a lack of development of efficient tools for activity detection of cathepsin B, which provides room for improvement for the innovation of biomarker detection products based on cathepsin B. SUMMARY

[0006] The present application provides a biomarker detection product based on cathepsin B and an application thereof, aiming to develop an efficient, specific and sensitive detection tool to directly evaluate the activity or expression level of cathepsin B, solving the problem of insufficient detection sensitivity and specificity of the prior art under complex disease background.

[0007] In a first aspect, the present application provides a kit for preparing a cathepsin B-based biomarker detection product, comprising the following components: an active substrate probe, which is a fluorescently labeled polypeptide chain with an amino acid sequence comprising a cathepsin B-specific cleavage site; a standard solution containing a known concentration of recombinant cathepsin B; a reaction buffer with a pH value ranging from 6 to 8, containing sodium chloride, dithiothreitol, and ethylenediaminetetraacetic acid; a detection chip, the surface of which is modified with an affinity ligand capable of binding to cathepsin B and immobilizing the target enzyme molecule.

[0008] According to the present application, the design of the active substrate probe is based on the selective cleavage ability of cathepsin B on specific polypeptide sequences. When cathepsin B acts on the active substrate probe, the fluorescent group is released from the polypeptide chain, thereby generating a detectable fluorescent signal. This design makes the detection process highly specific, avoiding interference from other proteases. In addition, the introduction of the standard solution provides a reliable quantitative basis for the detection results, while the reaction buffer optimizes the environmental conditions for enzyme activity determination. The use of the detection chip further improves the sample processing efficiency and enhances the sensitivity of the detection.

[0009] In some embodiments, the polypeptide chain of the active substrate probe is 8 to 15 amino acid residues in length, and the specific cleavage site consists of an arginine-arginine-lysine triplet. This triplet structure has been experimentally verified to be highly resistant to other proteases while being efficiently cleaved by cathepsin B.

[0010] In some embodiments, the fluorescent label is selected from at least one of carboxyfluorescein, rhodamine B, or Cy5. These fluorescent labels have high quantum yield and good light stability, enabling high-sensitivity detection at low concentrations.

[0011] In some embodiments, the concentration of sodium chloride in the reaction buffer ranges from 100 millimoles per liter to 200 millimoles per liter, the concentration of dithiothreitol ranges from 1 millimole per liter to 5 millimoles per liter, and the concentration of ethylenediaminetetraacetic acid ranges from 0.5 millimoles per liter to 2 millimoles per liter. The combination of the above components not only maintains the optimal state of enzyme activity but also effectively suppresses the interference of non-specific proteases.

[0012] In some embodiments, the affinity ligand of the detection chip is an antibody fragment or an aptamer. The antibody fragment is prepared by genetic engineering technology, which retains the highly specific binding ability to cathepsin B while reducing the non-specific adsorption problems that may be caused by the intact antibody. The aptamer is obtained by SELEX technology, which has higher thermal stability and chemical resistance, and is suitable for various complex detection environments.

[0013] In some embodiments, the preparation method of the detection chip comprises the following steps: S1: immerse the glass substrate in an ethanol solution containing a silane coupling agent, the reaction temperature is 50 to 70 degrees Celsius, and the reaction time is 2 to 4 hours; S2: immerse the silanized glass substrate in a phosphate buffer containing an affinity ligand, the reaction temperature is 4 to 25 degrees Celsius, and the reaction time is 12 to 24 hours; S3: rinse the surface of the glass substrate with deionized water and dry it under a nitrogen stream to obtain a modified detection chip.

[0014] According to the present application, the use of silane coupling agent enhances the binding strength between the affinity ligand and the glass substrate, ensuring that the detection chip can maintain stable performance after multiple uses. In addition, the low-temperature reaction condition effectively avoids the denaturation or inactivation of the affinity ligand during immobilization.

[0015] In a second aspect, the present application provides a cathepsin B-based biomarker detection method, comprising the following steps: T1: mix the sample to be tested with an active substrate probe, add a reaction buffer, and incubate at 37 degrees Celsius for 30 to 60 minutes; T2: drop the incubated mixture onto the surface of the detection chip, and rinse with buffer after standing for 5 to 10 minutes; T3: measure the fluorescence intensity on the surface of the detection chip using a fluorescence microscope or a fluorescence spectrophotometer; T4: compare the measured fluorescence intensity with the standard curve generated by the standard solution, and calculate the activity or concentration of cathepsin B in the sample to be tested.

[0016] According to the present application, the sample to be tested can be derived from blood, urine or tissue extract, and has a wide range of applications. The optimization of incubation time and temperature ensures the full action of cathepsin B on the active substrate probe, while avoiding the occurrence of non-specific reactions. The use of the detection chip significantly improves the automation level of sample processing and reduces the operation error.

[0017] In some embodiments, the sample to be tested needs to be pretreated before mixing with the active substrate probe, including centrifugal separation and filtration. The centrifugal speed is 3000 to 5000 revolutions per minute, and the centrifugal time is 5 to 10 minutes; the filtration uses a filter membrane with a pore size of 0.22 microns to remove particulate impurities in the sample.

[0018] In some embodiments, the method for drawing the standard curve comprises the following steps: U1: dilute the standard solution into a series of different concentrations ranging from 0.1 nanomole per liter to 100 nanomole per liter; U2: mix each concentration of the standard solution with the active substrate probe, and measure the fluorescence intensity according to steps T1 to T3; U3: draw the standard curve with the concentration of the standard solution as the abscissa and the corresponding fluorescence intensity as the ordinate.

[0019] According to the present application, the linear range of the standard curve covers the common concentration interval of cathepsin B under physiological and pathological conditions, providing a guarantee for the accuracy of the detection results.

[0020] In a third aspect, the present application provides a use of a biomarker detection product based on cathepsin B, including but not limited to the following applications: For early diagnosis of cancer, especially screening of breast cancer, lung cancer and colorectal cancer; For activity monitoring of inflammatory diseases, such as rheumatoid arthritis and Crohn's disease; For prognosis evaluation of neurodegenerative diseases, such as Alzheimer's disease and Parkinson's disease.

[0021] According to the present application, the changes in the expression level or activity of cathepsin B in the above diseases have been confirmed by multiple studies, so the application of the present product has a clear scientific basis. In addition, the present product overcomes the limitations of the prior art by directly detecting the activity or concentration of cathepsin B, significantly improving the sensitivity and specificity of the detection.

[0022] In some embodiments, the specific operation steps for early diagnosis of cancer include: V1: collect the serum sample of the patient, and measure the activity of cathepsin B according to steps T1 to T4; V2: compare the measured activity value with the reference value of healthy people, if the activity value is more than twice the reference value, it suggests that there may be a risk of cancer; V3: further confirm the type and stage of cancer in combination with imaging examination or other diagnostic methods.

[0023] In some embodiments, the specific operation steps for activity monitoring of inflammatory diseases include: W1: periodically collect blood or joint fluid samples from the patient, and determine the activity of Cathepsin B according to steps T1 to T4; W2: record the activity value of each determination, and plot a time-activity curve; W3: if the activity value continues to rise or fluctuates by more than 50%, it indicates that the disease activity is increasing, and the treatment plan needs to be adjusted.

[0024] In some embodiments, the specific operation steps of the prognosis evaluation of the neurodegenerative disease include: X1: collect cerebrospinal fluid samples from the patient, and determine the activity of Cathepsin B according to steps T1 to T4; X2: correlate the measured activity value with different stages of disease progression; X3: if the activity value decreases significantly, it indicates that the treatment is effective, otherwise the intervention measures need to be adjusted.

[0025] According to the present application, the implementation of the above-mentioned uses relies on the accurate detection of Cathepsin B activity or concentration by the product, providing an important basis for decision-making for clinicians. In addition, the modular design of the product enables it to be flexibly applied to different detection scenarios, meeting diverse clinical needs.

[0026] In a fourth aspect, the present application provides a construction method of a Cathepsin B-based biomarker detection system, including the following steps: Y1: integrate the detection chip into the microfluidic device to form an integrated detection platform; Y2: set multiple independent reaction chambers in the microfluidic device, each chamber loaded with active substrate probes, reaction buffer and standard solution; Y3: realize the whole-process operation of sample loading, reaction incubation and fluorescence signal reading through an automatic control system.

[0027] According to the present application, the introduction of the microfluidic device greatly improves the detection efficiency, while reducing the consumption of samples and reagents. The application of the automatic control system further reduces the complexity of manual operation, making the product more easily used in clinical laboratories.

[0028] In some embodiments, the material of the microfluidic device is polydimethylsiloxane, and the surface thereof is subjected to plasma treatment to improve hydrophobicity and chemical stability.

[0029] In some embodiments, the automatic control system includes a stepper motor, a photoelectric sensor and a data acquisition module. The stepper motor is used to accurately control the flow of liquid, the photoelectric sensor is used to monitor the reaction process in real time, and the data acquisition module is used to record and analyze the fluorescence signal.

[0030] According to the present application, the comprehensive application of the above technical means makes the cathepsin B-based biomarker detection system efficient, accurate and reliable, providing strong support for the diagnosis and treatment of related diseases. DETAILED DESCRIPTION

[0031] In the description of the present specification, the description of the terms "one embodiment", "some embodiments", "exemplary embodiment", "example", "specific example", or "some examples" means that the specific features, structures, materials or characteristics described in connection with the embodiment or example are included in at least one embodiment or example of the present application. In the description of the present specification, the exemplary description of the above terms does not necessarily mean the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described can be combined in any one or more embodiments or examples as appropriate.

[0032] In addition, the terms "first", "second" are only for descriptive purposes, and cannot be understood as indicating or implying relative importance or implicitly indicating the number of the indicated technical features. Therefore, the features defined with "first", "second" can explicitly or implicitly include at least one of the features. In the description of the present application, the meaning of "multiple" is at least two, for example, two, three, etc., unless otherwise explicitly specified.

[0033] As described in the background section above, the current detection method for cathepsin B mainly relies on the analysis of indirect metabolites, which has the problem of insufficient sensitivity and specificity. Based on this, the present application provides a cathepsin B-based biomarker detection product and its use, aiming to develop an efficient, specific and sensitive detection tool to directly evaluate the activity or expression level of cathepsin B, and solve the problem of insufficient detection sensitivity and specificity in the prior art under complex disease background.

[0034] In a first aspect, the present application provides a kit for preparing a cathepsin B-based biomarker detection product, comprising the following components: active substrate probe, standard solution, reaction buffer and detection chip.

[0035] According to the present application, the design of the active substrate probe is based on the selective cleavage ability of cathepsin B on specific polypeptide sequences. The active substrate probe is a fluorescently labeled polypeptide chain, and its amino acid sequence contains a cathepsin B-specific cleavage site. When cathepsin B acts on the active substrate probe, the fluorescent group is released from the polypeptide chain, thereby generating a detectable fluorescent signal. This design makes the detection process highly specific and avoids interference from other proteases.

[0036] In some embodiments, the polypeptide chain of the active substrate probe has a length of 8 to 15 amino acid residues, and the specific cleavage site is composed of an arginine-arginine-lysine triplet. This triplet structure has been experimentally verified to be highly efficient in being cleaved by cathepsin B while maintaining high resistance to other proteases.

[0037] In some embodiments, the fluorescent label is selected from at least one of carboxyfluorescein, rhodamine B, or Cy5. These fluorescent labels have high quantum yield and good light stability, and can achieve high-sensitivity detection at low concentrations.

[0038] According to the present application, the standard solution contains a known concentration of recombinant cathepsin B. The introduction of the standard solution provides a reliable quantitative basis for the detection results. In some embodiments, the concentration of the standard solution ranges from 0.1 nanomole per liter to 100 nanomoles per liter.

[0039] The reaction buffer has a pH range of 6 to 8 and contains sodium chloride, dithiothreitol, and ethylenediaminetetraacetic acid. In some embodiments, the concentration of sodium chloride in the reaction buffer ranges from 100 millimoles per liter to 200 millimoles per liter, the concentration of dithiothreitol ranges from 1 millimole per liter to 5 millimoles per liter, and the concentration of ethylenediaminetetraacetic acid ranges from 0.5 millimoles per liter to 2 millimoles per liter. The combination of the above components not only maintains the optimal state of enzyme activity, but also effectively suppresses the interference of non-specific proteases.

[0040] The detection chip is modified with affinity ligands that can bind to cathepsin B and immobilize the target enzyme molecules. In some embodiments, the affinity ligand is an antibody fragment or a nucleic acid aptamer. Antibody fragments are prepared by genetic engineering techniques, retaining the ability to specifically bind to cathepsin B while reducing the non-specific adsorption problems that may be caused by intact antibodies. Nucleic acid aptamers are obtained by SELEX technology and have higher thermal stability and chemical resistance, making them suitable for a variety of complex detection environments.

[0041] The preparation method of the detection chip includes the following steps: S1: soaking the glass substrate in an ethanol solution containing a silane coupling agent, with a reaction temperature of 50 to 70 degrees Celsius and a reaction time of 2 to 4 hours; S2: immersing the silanized glass substrate in a phosphate buffer solution containing affinity ligands, with a reaction temperature of 4 to 25 degrees Celsius and a reaction time of 12 to 24 hours; S3: rinsing the surface of the glass substrate with deionized water and drying it under a nitrogen stream to obtain the modified detection chip.

[0042] According to the present application, the use of silane coupling agent enhances the binding strength between the affinity ligand and the glass substrate, ensuring that the detection chip can maintain stable performance after multiple uses. In addition, the low-temperature reaction conditions effectively avoid denaturation or inactivation of the affinity ligand during immobilization.

[0043] In a second aspect, the present application provides a cathepsin B-based biomarker detection method, comprising the following steps: T1: mixing the sample to be tested with the active substrate probe, adding the reaction buffer, and incubating at 37°C for 30 to 60 minutes; T2: dropping the incubated mixture onto the surface of the detection chip, and after standing for 5 to 10 minutes, washing with buffer; T3: determining the fluorescence intensity on the surface of the detection chip using a fluorescence microscope or a fluorescence spectrophotometer; T4: comparing the measured fluorescence intensity with the standard curve generated by the standard solution, and calculating the activity or concentration of cathepsin B in the sample to be tested.

[0044] According to the present application, the sample to be tested can be derived from blood, urine or tissue extract, with a wide range of applications. The optimization of incubation time and temperature ensures the full action of cathepsin B on the active substrate probe, while avoiding non-specific reactions. The use of detection chips significantly improves the automation of sample processing and reduces operational errors.

[0045] In some embodiments, the sample to be tested needs to undergo a pretreatment step before mixing with the active substrate probe, including centrifugal separation and filtration. The centrifugal speed is 3000 to 5000 revolutions per minute, and the centrifugal time is 5 to 10 minutes; filtration uses a filter membrane with a pore size of 0.22 microns to remove particulate impurities in the sample.

[0046] The method for drawing the standard curve comprises the following steps: U1: diluting the standard solution into a series of different concentrations, ranging from 0.1 nanomole per liter to 100 nanomole per liter; U2: mixing each concentration of the standard solution with the active substrate probe, and determining the fluorescence intensity according to steps T1 to T3; U3: taking the concentration of the standard solution as the abscissa and the corresponding fluorescence intensity as the ordinate, drawing the standard curve.

[0047] In a third aspect, the present application provides the use of a cathepsin B-based biomarker detection product, including but not limited to the following applications: for early diagnosis of cancer, especially screening for breast cancer, lung cancer and colorectal cancer; for monitoring of inflammatory diseases, such as rheumatoid arthritis and Crohn's disease; for prognosis evaluation of neurodegenerative diseases, such as Alzheimer's disease and Parkinson's disease.

[0048] In some embodiments, the specific operation steps for early diagnosis of cancer include: V1: collecting serum samples from patients, and determining the activity of cathepsin B according to steps T1 to T4; V2: comparing the measured activity value with the reference value of healthy people, and if the activity value is more than twice the reference value, it indicates that there may be a risk of cancer; V3: combining imaging examination or other diagnostic methods to further confirm the type and stage of cancer.

[0049] In some embodiments, the specific operation steps for monitoring the activity of inflammatory diseases include: W1: periodically collecting blood or joint fluid samples from patients, and determining the activity of cathepsin B according to steps T1 to T4; W2: recording the activity value of each determination and drawing a time-activity curve; W3: if the activity value continues to rise or fluctuates more than 50%, it indicates that the disease activity is increasing, and the treatment plan needs to be adjusted.

[0050] In some embodiments, the specific operation steps for prognosis evaluation of neurodegenerative diseases include: X1: collecting cerebrospinal fluid samples from patients, and determining the activity of cathepsin B according to steps T1 to T4; X2: correlational analysis of the measured activity value with different stages of disease progression; X3: if the activity value decreases significantly, it indicates that the treatment effect is good, otherwise the intervention measures need to be adjusted.

[0051] In a fourth aspect, the present application provides a construction method of a cathepsin B-based biomarker detection system, including the following steps: Y1: integrating the detection chip into the microfluidic device to form an integrated detection platform; Y2: setting multiple independent reaction chambers in the microfluidic device, each chamber is loaded with active substrate probe, reaction buffer and standard solution; Y3: realizing the whole process operation of sample loading, reaction incubation and fluorescence signal reading through an automatic control system.

[0052] In some embodiments, the material of the microfluidic device is polydimethylsiloxane, and the surface thereof is treated by plasma to improve hydrophobicity and chemical stability.

[0053] In some embodiments, the automatic control system includes a stepper motor, a photoelectric sensor and a data acquisition module. The stepper motor is used to accurately control the flow of liquid, the photoelectric sensor is used to monitor the reaction process in real time, and the data acquisition module is used to record and analyze the fluorescence signal.

[0054] Hereinafter, the embodiments of the present application will be described. The embodiments described below are exemplary and are only used to explain the present application, and cannot be understood as a limitation of the present application. If the specific technology or condition is not specified in the embodiments, it is carried out according to the technology or condition described in the literature in the art or according to the product instruction. If the reagent or instrument used is not specified by the manufacturer, it is a conventional product that can be obtained by purchase.

[0055] Example 1 Preparation of active substrate probe: a polypeptide chain containing arginine-arginine-lysine triplet is covalently connected with carboxyfluorescein to prepare an active substrate probe. The length of the polypeptide chain is 10 amino acid residues.

[0056] Preparation of standard solution: the recombinant cathepsin B is dissolved in reaction buffer to prepare a standard solution with a concentration of 1 nanomole per liter.

[0057] Preparation of reaction buffer: sodium chloride, dithiothreitol and ethylenediaminetetraacetic acid are dissolved in deionized water, and the pH value is adjusted to 7.4 to prepare the reaction buffer. The concentration of sodium chloride is 150 millimoles per liter, the concentration of dithiothreitol is 3 millimoles per liter, and the concentration of ethylenediaminetetraacetic acid is 1 millimole per liter.

[0058] Preparation of detection chip: the glass substrate is soaked in an ethanol solution containing a silane coupling agent, and reacted at 60 degrees Celsius for 3 hours; the silanized glass substrate is immersed in a phosphate buffer containing antibody fragments, and reacted at 20 degrees Celsius for 18 hours; the surface of the glass substrate is washed with deionized water and dried under a nitrogen stream to obtain a modified detection chip.

[0059] Implementation of detection method: the serum sample to be tested is mixed with the active substrate probe, and the reaction buffer is added, and incubated at 37 degrees Celsius for 45 minutes; the mixed solution after incubation is added dropwise to the surface of the detection chip, and after standing for 8 minutes, it is washed with buffer; the fluorescence intensity on the surface of the detection chip is measured by fluorescence microscope; the measured fluorescence intensity is compared with the standard curve generated by the standard solution, and the activity of cathepsin B in the sample to be tested is calculated.

[0060] Example 2 Preparation of active substrate probe: a polypeptide chain containing arginine-arginine-lysine triplet is covalently connected with carboxyfluorescein to prepare an active substrate probe. The length of the polypeptide chain is 10 amino acid residues.

[0061] Preparation of standard solution: the recombinant cathepsin B is dissolved in reaction buffer to prepare a standard solution with a concentration of 10 nanomoles per liter.

[0062] Preparation of reaction buffer: the preparation method of the reaction buffer is the same as that of Example 1.

[0063] Preparation of detection chip: the glass substrate is soaked in an ethanol solution containing a silane coupling agent, and reacted at 60 degrees Celsius for 3 hours; the silanized glass substrate is immersed in a phosphate buffer containing antibody fragments, and reacted at 20 degrees Celsius for 18 hours; the surface of the glass substrate is washed with deionized water and dried under a nitrogen stream to obtain a modified detection chip.

[0064] Implementation of detection method: the serum sample to be tested is mixed with the active substrate probe, and the reaction buffer is added, and incubated at 37 degrees Celsius for 45 minutes; the mixed solution after incubation is added dropwise to the surface of the detection chip, and after standing for 8 minutes, it is washed with buffer; the fluorescence intensity on the surface of the detection chip is measured by fluorescence microscope; the measured fluorescence intensity is compared with the standard curve generated by the standard solution, and the activity of cathepsin B in the sample to be tested is calculated.

[0065] Comparative Example 1 Preparation of active substrate probe: An active substrate probe was prepared by covalently linking a polypeptide chain without the arginine-arginine-lysine triplet to carboxyfluorescein. The polypeptide chain was 10 amino acid residues in length.

[0066] The preparation of the standard solution is the same as in Example 1.

[0067] The reaction buffer was prepared in the same manner as in Example 1.

[0068] The fabrication of the detection chip is the same as in Example 1.

[0069] The detection method is implemented in the same way as in Example 1.

[0070] Comparative Example 2 The preparation of the active substrate probe is the same as in Example 1.

[0071] The preparation of the standard solution is the same as in Example 1.

[0072] Preparation of the reaction buffer: Sodium chloride, dithiothreitol, and ethylenediaminetetraacetic acid (EDTA) were dissolved in deionized water, and the pH was adjusted to 9.0 to prepare the reaction buffer. The concentration of sodium chloride was 150 mmol / L, the concentration of dithiothreitol was 3 mmol / L, and the concentration of EDTA was 1 mmol / L.

[0073] The fabrication of the detection chip is the same as in Example 1.

[0074] The detection method is implemented in the same way as in Example 1.

[0075] The detection results obtained from the above embodiments and comparative examples are compared, and the results are shown in the table below:

[0076] The results in the table show that the detection product obtained in this application has higher detection sensitivity and specificity than the comparative example, while also exhibiting higher fluorescence signal intensity and shorter sample processing time. The active substrate probe used in Comparative Example 1 lacked the arginine-arginine-lysine triplet, resulting in a significant decrease in detection sensitivity and specificity; the pH value of the reaction buffer in Comparative Example 2 exceeded the optimal range, leading to a decrease in fluorescence signal intensity and detection sensitivity.

[0077] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of this application, and are not intended to limit them. Although this application has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features therein. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of this application.

Claims

1. A kit for preparing biomarker detection products based on cathepsin B, characterized in that, Includes the following components: An active substrate probe, wherein the active substrate probe is a fluorescently labeled polypeptide chain whose amino acid sequence contains a cathepsin B-specific cleavage site; A standard solution containing a known concentration of recombinant cathepsin B; The reaction buffer has a pH range of 6 to 8 and contains sodium chloride, dithiothreitol, and ethylenediaminetetraacetic acid; The detection chip has its surface modified with affinity ligands that can bind to cathepsin B and immobilize the target enzyme molecule.

2. The reagent kit according to claim 1, characterized in that, The active substrate probe has a polypeptide chain length of 8 to 15 amino acid residues, and its specific cleavage site is composed of an arginine-arginine-lysine triplet.

3. The reagent kit according to claim 1, characterized in that, The fluorescent label is selected from at least one of carboxyfluorescein, rhodamine B, or Cy5.

4. The reagent kit according to claim 1, characterized in that, The concentration range of sodium chloride in the reaction buffer is 100 mmol / L to 200 mmol / L, the concentration range of dithiothreitol is 1 mmol / L to 5 mmol / L, and the concentration range of ethylenediaminetetraacetic acid is 0.5 mmol / L to 2 mmol / L.

5. The reagent kit according to claim 1, characterized in that, The affinity ligand of the detection chip is an antibody fragment or a nucleic acid aptamer, wherein the antibody fragment is prepared by genetic engineering technology and the nucleic acid aptamer is obtained by screening using SELEX technology.

6. A method for detecting biomarkers based on cathepsin B, characterized in that, Includes the following steps: T1: Mix the sample to be tested with the active substrate probe, add the reaction buffer, and incubate at 37 degrees Celsius for 30 to 60 minutes; T2: Add the incubated mixture to the surface of the detection chip, let it stand for 5 to 10 minutes, and then rinse with buffer solution; T3: Measure the fluorescence intensity on the surface of the detection chip using a fluorescence microscope or fluorescence spectrophotometer; T4: Compare the measured fluorescence intensity with the standard curve generated by the standard solution to calculate the activity or concentration of cathepsin B in the sample to be tested.

7. The method according to claim 6, characterized in that, The sample to be tested needs to undergo a pretreatment step before being mixed with the active substrate probe, including centrifugation and filtration. The centrifugation speed is 3000 to 5000 rpm and the centrifugation time is 5 to 10 minutes. The filtration uses a filter membrane with a pore size of 0.22 micrometers.

8. The method according to claim 6, characterized in that, The method for plotting the standard curve includes the following steps: U1: The standard solution is diluted to a series of different concentrations, ranging from 0.1 nanomoles per liter to 100 nanomoles per liter; U2: Mix the standard solutions of each concentration with the active substrate probe, and measure the fluorescence intensity according to steps T1 to T3; U3: Plot a standard curve with the concentration of the standard solution on the x-axis and the corresponding fluorescence intensity on the y-axis.

9. A method for constructing a biomarker detection system based on cathepsin B, characterized in that, Includes the following steps: Y1: Integrate the detection chip into the microfluidic device to form an integrated detection platform; Y2: Multiple independent reaction chambers are set up in the microfluidic device, and each chamber is loaded with an active substrate probe, a reaction buffer and a standard solution respectively; Y3: The entire process of sample loading, reaction incubation, and fluorescence signal reading is realized through an automated control system.

10. The method according to claim 9, characterized in that, The microfluidic device is made of polydimethylsiloxane, and its surface is treated with plasma to improve its hydrophobicity and chemical stability.

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