A method for detecting the activity of membrane type I matrix metalloproteinase

Abstract

The present invention discloses a method for detecting the activity of membrane type I matrix metalloproteinase. First, a Raman-fluorescent dual-mode probe particle is constructed using a polypeptide sequence. Then, the probe is added to a cell sample, and the specific cleavage of the polypeptide sequence by membrane type I matrix metalloproteinase causes a switch between Raman signals and fluorescent signals, realizing the visualization of the activity of membrane type I matrix metalloproteinase in cells. Finally, the Raman spectrum and fluorescent spectrum in the cells are collected, and a corresponding relationship between the signal intensity and the activity of membrane type I matrix metalloproteinase is established to achieve the purpose of semi-quantitative analysis. The Raman-fluorescent switch-type detection method adopted by the present invention can effectively reduce the interference of background signals and has the advantages of high sensitivity, good specificity, and strong reliability.

Description

Technical Field

[0001] The present invention relates to the field of preparation of nanomaterials and detection of biomolecules, and particularly relates to a method for detecting the activity of membrane type-I matrix metalloproteinase. Background Art

[0002] Membrane type-I matrix metalloproteinase (MT1-MMP) is one of the most important proteases for degrading components of the extracellular matrix. As an endopeptidase, MT1-MMP degrades the extracellular matrix and basement membrane, and at the same time remodels the intercellular adhesion force, promoting the growth of tumor cells along the matrix gaps and basement membrane defects to the surrounding. Therefore, accurate detection of the activity of membrane type-I matrix degrading enzyme is of extremely important significance.

[0003] Currently, conventional detection methods for matrix degrading enzymes include immunoblotting experiments, enzyme-linked immunosorbent assay, immunohistochemistry, etc. These methods usually use proteases as molecular targets and fluorescent or isotope-labeled monoclonal antibodies as molecular probes to quantitatively detect the concentration of matrix degrading enzymes through specific binding of antibody-antigen. However, these methods are also restricted by some antibody properties in actual use, such as cumbersome preparation steps, high cost, batch differences, etc., which affect the accuracy of detection. In addition, the immunological method based on antibody-antigen can only detect the concentration of the target protease and is difficult to effectively determine its hydrolysis activity.

[0004] As a new type of protein recognition molecular probe, polypeptides are expected to make up for the above deficiencies of antibodies. A polypeptide is an amino acid sequence formed by multiple amino acids connected by peptide bonds, and a specific polypeptide sequence can be specifically cleaved by the corresponding matrix degrading enzyme. By using the structural change of this polypeptide, a switch-type biosensing probe can be constructed to detect the activity of the target protease. Fluorescence method is a commonly used optical labeling method and is widely used in the detection of various biomolecules. However, the stability of the fluorescence spectrum is poor and the phenomenon of photobleaching is likely to occur. At the same time, the components of the cells to be detected are complex, and many components themselves also have fluorescence characteristics, so the detection results are easily interfered. Surface enhanced Raman scattering (SERS) spectroscopy is a newly emerging biomarking means with extremely high sensitivity and has shown unique advantages in the fields of biological detection, sensing and imaging. At the same time, the SERS spectrum has high optical stability and is not easily photobleached, enabling people to track the target in a complex biological environment. Therefore, combining Raman and fluorescence technologies to construct a dual-mode probe and developing a new method for detecting the activity of membrane type-I matrix metalloproteinase to achieve highly sensitive and highly reliable detection is an urgent problem to be solved at present. Summary of the Invention

[0005] Objective of the Invention: Aiming at the deficiencies and defects of the prior art, the present invention provides a method for detecting the activity of membrane type I matrix metalloproteinase. The Raman-fluorescence switch-type detection method can effectively reduce the interference of background signals and has the advantages of high sensitivity, good specificity and strong reliability.

[0006] Technical Solution: A method for detecting the activity of membrane type I matrix metalloproteinase according to the present invention is characterized by comprising the following steps:

[0007] 1) Prepare metal nanoparticles as a surface-enhanced Raman scattering substrate;

[0008] 2) Modify a functional polypeptide sequence on the surface of the particles to obtain a Raman-fluorescence dual-mode probe;

[0009] 3) Add the probe to the cells to be tested and react with membrane type I matrix metalloproteinase;

[0010] 4) Use a confocal Raman microscope to analyze the Raman and fluorescence signals on the cells to be tested, collect spectra and images, and realize semi-quantitative detection of the activity of membrane type I matrix metalloproteinase according to the changes in the intensities of Raman and fluorescence signals.

[0011] Wherein, in the step 1), the metal nanoparticles are gold nanospheres or silver nanospheres.

[0012] Wherein, in the step 2), the functional polypeptide sequence is CGPLGLGK, and this polypeptide sequence is covalently connected to the surface of the metal nanoparticles through the sulfhydryl group of the N-terminal cysteine.

[0013] Wherein, in the step 2), a fluorescent Raman molecule is modified at the C-terminus of the functional polypeptide sequence, and the fluorescent Raman molecule is one of rhodamine 6G, rhodamine B, carboxyfluorescein, methylfluorescein or cyanine 5.

[0014] Wherein, in the step 3), the polypeptide sequence of the probe will be specifically cleaved by membrane type I matrix metalloproteinase to release the fluorescent Raman molecule.

[0015] Wherein, in the step 4), the decrease ΔI of the surface-enhanced Raman signal intensity S and the increase ΔI of the fluorescence signal intensity F are used to characterize and analyze the activity of membrane type I matrix metalloproteinase in the cells to be tested. The larger ΔI S and ΔI F are, the higher the activity of membrane type I matrix metalloproteinase in the cells to be tested is.

[0016] The method of the present invention first constructs Raman-fluorescence dual-mode probe particles using a polypeptide sequence, then adds the probe to a cell sample, and realizes the visualization of the activity of membrane type I matrix metalloproteinase in cells through the specific cleavage effect of membrane type I matrix metalloproteinase on the polypeptide sequence, resulting in the switching between Raman signals and fluorescence signals. Finally, the Raman spectrum and fluorescence spectrum in the cells are collected to establish the corresponding relationship between the signal intensity and the activity of membrane type I matrix metalloproteinase, achieving the purpose of semi-quantitative analysis.

[0017] Utilizing the specific cleavage effect of membrane type I matrix metalloproteinase and the corresponding polypeptide sequence, membrane type I matrix metalloproteinases with different activities will have different degrees of influence on the desorption of fluorescent Raman molecules on the surface of metal nanoparticles, thereby generating surface-enhanced Raman scattering signals and fluorescence signals with different intensities. Combining the changes in signal intensity, ΔI S (ΔI F ) and the activity of membrane type I matrix metalloproteinase are established to realize the semi-quantitative detection and analysis of the activity of membrane type I matrix metalloproteinase in cells.

[0018] Advantages: Compared with the prior art, the present invention has the following remarkable advantages:

[0019] 1. The present invention uses an enzyme-cleavable polypeptide substrate as the recognition molecule for membrane type I matrix metalloproteinase, which is beneficial for detecting the hydrolysis activity of membrane type I matrix metalloproteinase.

[0020] 2. The present invention uses a Raman-fluorescence dual-mode probe, which can specifically switch signals in the cells to be detected, has dual-mode imaging performance, reduces the interference of background signals, and improves the stability of signals.

[0021] 3. The present invention realizes the semi-quantitative analysis of the activity of membrane type I matrix metalloproteinase in the cells to be detected by using the change in signal intensity, and has the advantages of high sensitivity, good reliability, and high specificity. Description of the Drawings

[0022] Figure 1 It is a schematic diagram of the detection method of the present invention;

[0023] Figure 2 It is a schematic diagram of the structure of the Raman-fluorescence dual-mode probe of the present invention;

[0024] Figure 3 It is a schematic diagram of the principle of the detection method of the present invention. Detailed Embodiments

[0025] The technical solution of the present invention will be further described below in conjunction with the drawings and specific embodiments.

[0026] Example 1: In this example, silver nanospheres were used as the SERS enhancement substrate, rhodamine 6G was used as the fluorescent Raman molecule, and breast cancer cells (MDA-MB-231) were used as the cells to be tested. The method of the present invention was used to detect the activity of membrane type I matrix metalloproteinase.

[0027] 1. Preparation of silver nanosphere colloid: 50 mL of silver nitrate solution (molar concentration 10 mM) was added to 450 mL of deionized water, and it was vigorously stirred and heated to boiling. Subsequently, 10 mL of sodium citrate solution (mass fraction 1%) was added, and heating and stirring were continued for 60 minutes. Heating was stopped, and it was stirred and cooled to room temperature to obtain a silver nanosphere colloid solution.

[0028] 2. Preparation of the probe: 5 μL of the polypeptide sequence (10 mM, CGPLGLGK-R6G) dissolved in DMSO was added to 5 mL of the silver nanosphere colloid solution. After the mixed solution was stirred at room temperature for 12 hours, it was centrifuged and washed twice at 6000 rpm for 12 minutes, and the precipitate was dispersed in 5 mL of deionized water to obtain a Raman-fluorescent dual-mode probe, as Figure 2 shown.

[0029] 3. Preparation of cell samples: MDA-MB-231 cells were inoculated into a 35 mm glass-bottom culture dish and cultured for 24 hours. They were gently rinsed twice with PBS buffer.

[0030] 4. Probe recognition of membrane type I matrix metalloproteinase: 200 μL of the dual-mode probe and 1.8 mL of PBS buffer were mixed evenly and added to the glass-bottom culture dish to uniformly cover the glass bottom. The culture dish was incubated at 37 °C for 2 hours and gently rinsed twice with PBS buffer to remove excess probe particles.

[0031] 5. Detection of membrane type I matrix metalloproteinase activity: The SERS image and fluorescence image of the probe in the cells were collected using a confocal Raman microscope to visualize the activity of membrane type I matrix metalloproteinase in breast cancer cells. The SERS spectrum and fluorescence spectrum in the cells were taken, the intensities of the SERS signal and fluorescence signal were analyzed, and the decrease ΔI S of the SERS signal intensity and the increase ΔI F of the fluorescence signal intensity were used to achieve semi-quantitative detection of the activity of membrane type I matrix metalloproteinase.

[0032] Effect analysis: The detection method proposed by the present invention can perform qualitative and semi-quantitative detection of the activity of membrane type I matrix metalloproteinase through Raman and fluorescence dual-mode signals; at the same time, it can visualize the activity of membrane type I matrix metalloproteinase in the cells to be tested, so as to achieve the purpose of auxiliary detection. Compared with the prior art, it has unique advantages such as strong anti-interference ability, high reliability, and high repeatability.

[0033] Example 2: In this example, gold nanospheres were used as the SERS enhancement substrate, carboxyfluorescein FAM was used as the fluorescent Raman molecule, and human fibrosarcoma cells (HT-1080) were used as the cells to be detected. The method of the present invention was used to detect the activity of membrane type I matrix metalloproteinase.

[0034] 1. Preparation of gold nanosphere colloid: 100 μL of chloroauric acid solution (mass fraction 10%) was added to 100 mL of deionized water, and it was vigorously stirred and heated to boiling. Subsequently, 4 mL of sodium citrate solution (mass fraction 1%) was added, and heating and stirring were continued for 20 minutes. The heating was stopped, and it was stirred and cooled to room temperature to obtain a gold nanosphere colloid solution.

[0035] 2. Preparation of the probe: 5 μL of the polypeptide sequence (10 mM, CGPLGLGK-FAM) dissolved in DMSO was added to 5 mL of the gold nanosphere colloid solution. After the mixed solution was stirred at room temperature for 12 hours, it was centrifuged and washed twice at 6000 rpm for 12 minutes, and the precipitate was dispersed in 5 mL of deionized water to obtain a Raman-fluorescence dual-mode probe.

[0036] 3. Preparation of cell samples: HT-1080 cells were inoculated into a 35 mm glass-bottom culture dish and cultured for 24 hours. They were gently rinsed twice with PBS buffer.

[0037] 4. Probe recognition of membrane type I matrix metalloproteinase: 200 μL of the dual-mode probe and 1.8 mL of PBS buffer were mixed evenly and added to the glass-bottom culture dish to uniformly cover the glass bottom. The culture dish was incubated at 37°C for 2 hours, and it was gently rinsed twice with PBS buffer to remove excess probe particles.

[0038] 5. Detection of membrane type I matrix metalloproteinase activity: A confocal Raman microscope was used to collect the SERS image and fluorescence image of the probe in the cells to visualize the activity of membrane type I matrix metalloproteinase in breast cancer cells. The SERS spectrum and fluorescence spectrum in the cells were taken to analyze the intensities of the SERS signal and fluorescence signal, and the decrease ΔI S of the SERS signal intensity and the increase ΔI F of the fluorescence signal intensity were used to achieve semi-quantitative detection of the activity of membrane type I matrix metalloproteinase.

[0039] Effect analysis: The detection method proposed by the present invention can perform qualitative and semi-quantitative detection of the activity of membrane type I matrix metalloproteinase through Raman and fluorescence dual-mode signals; at the same time, it can visualize the activity of membrane type I matrix metalloproteinase in the cells to be detected, so as to achieve the purpose of auxiliary detection. Compared with the prior art, it has unique advantages such as strong anti-interference ability, high reliability, and high repeatability.

Claims

1. A method for detecting the activity of membrane type I matrix metalloproteinase, characterized in that: Comprising the following steps: 1) Preparing metal nanoparticles as a surface-enhanced Raman scattering substrate; 2) Modifying a functional polypeptide sequence on the surface of the particles to obtain a Raman-fluorescent dual-mode probe; the functional polypeptide sequence is CGPLGLGK, and this polypeptide sequence is covalently linked to the surface of the metal nanoparticles through the thiol group of the N-terminal cysteine; the C-terminus of the functional polypeptide sequence is modified with a fluorescent Raman molecule, and the fluorescent Raman molecule is one of rhodamine 6G, rhodamine B, carboxyfluorescein, methylfluorescein or cyanine 5; 3) Adding the probe to the cells to be tested and reacting with membrane type I matrix metalloproteinase; 4) Using a confocal Raman microscope to analyze the Raman and fluorescent signals on the cells to be tested, collecting spectra and images, and realizing semi-quantitative detection of the activity of membrane type I matrix metalloproteinase according to the changes in the intensities of the Raman and fluorescent signals.

2. The method for detecting the activity of membrane type I matrix metalloproteinase according to claim 1, wherein: In step 3) described above, the polypeptide sequence of the probe will be specifically cleaved by membrane type I matrix metalloproteinase to release the fluorescent Raman molecule.

3. The method for detecting the activity of membrane type I matrix metalloproteinase according to claim 1, wherein: In step 4), the decrease in surface-enhanced Raman signal intensity, ΔI S and the increase in fluorescence signal intensity, ΔI F are used to characterize the activity of membrane type I matrix metalloproteinase in the cells to be tested. The larger ΔI S and ΔI F are, the higher the activity of membrane type I matrix metalloproteinase in the cells to be tested is.

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