System for detecting PTK7 molecular diffusion coefficient on cell surface based on single molecule tracing technology and application thereof

Abstract

The invention relates to a system for detecting a cell surface PTK7 molecular diffusion coefficient based on a single molecule tracing technology and application thereof, and belongs to the technical field of cancer cell detection. The system comprises a single-molecule tracing fluorescence labeling probe, a single-molecule total internal reflection fluorescence microscope, an image acquisition module and an image processing module. The single-molecule tracing fluorescence labeling probe comprises a labeling unit targeting PTK7 and a single-molecule tracing fluorescence group. The probe has small interference on the structure and movement of PTK7 protein molecules, can realize excellent in-situ, interference-free, real-time and dynamic tracking of the movement of PTK7 protein in various tumor cells, and accurately displays the movement track of the target protein. The system can track and detect motion data of PTK7 proteins of normal cells and tumor cells, and further performs statistics, arrangement and analysis to obtain a dynamic motion rule of PTK7 molecules. The rule can be used as a novel biophysical index for evaluating the invasion and metastasis potential of tumor cells, and is used for early diagnosis and targeted therapy of tumor metastasis.

Description

Technical Field

[0001] The present invention relates to the technical field of cancer cell detection, and in particular to a system for detecting the diffusion coefficient of PTK7 molecules on the cell surface based on single molecule tracing technology and its application. Background Art

[0002] The information disclosed in the background of the invention is only intended to enhance the understanding of the overall background of the invention and should not be necessarily regarded as an acknowledgment or any form of suggestion that the information constitutes the prior art already known to a person skilled in the art.

[0003] Cancer is currently one of the diseases with the highest morbidity and mortality rates in the world. It ranks alongside cardiovascular disease as one of the three major diseases that threaten human health. The main treatments for cancer currently include surgery, chemotherapy, immunotherapy, and targeted therapy. Surgery is the only curative method, but a large proportion of patients have already developed metastasis when the disease is discovered, completely losing the opportunity for surgery. At present, the clinical assessment of tumor metastasis mainly relies on B-ultrasound examinations, CT scans, and magnetic resonance imaging, which can usually only be discovered after a more obvious tumor appears, and often the best time for treatment has been missed when it is discovered. Therefore, it is necessary to develop a new means and method for evaluating tumor metastasis. When the early tumor has not metastasized, it is possible to assess the probability of subsequent metastasis in the patient and take early intervention treatment measures. This is of great significance for improving the cure rate of patients and prolonging their survival.

[0004] Single-molecule tracing technology refers to the use of fluorescent proteins, fluorescent dyes or nanoparticles to specifically label target molecules, and then dynamically track fluorescence or scattered light signals in real time, supplemented by data analysis algorithms, and quantitatively characterize the dynamic movement of molecules using diffusion coefficients, etc. It has been widely used in research in the fields of materials, biology, and medicine. Although single-molecule tracing technology has been widely used, choosing a suitable method to label cell surface molecules is a problem facing this technology. Fluorescent protein labeling is the most commonly used labeling method in single-molecule tracing. Fluorescent proteins are fused with target protein genes to express membrane proteins with fluorescent labels when expressed. Although this method has little effect on cell activity, the experimental operation steps are complicated and can only be constructed on specific cell lines, making it difficult to easily compare the differences between different cell lines. Antibody-coupled fluorescent dyes or nanoparticles are another method for labeling target proteins, which uses the specific binding of antibodies to target proteins to achieve labeling. This method can easily label target proteins on different cell lines, but due to the large size of antibodies, it will cause great steric hindrance, interfere with the structure and function of target proteins, and antibody labeling has certain nonspecific binding, which makes the fluorescence background interference larger. Therefore, traditional single-molecule tracing technology is difficult to directly apply to the comparison of tumor tissues at different stages to evaluate their invasion and metastasis potential.

[0005] Therefore, a new technical solution is needed to enable earlier, faster and more effective assessment of the risk of tumor metastasis after the diagnosis of tumor tissue, improve the patient's treatment effect and prolong his life cycle. Summary of the invention

[0006] In order to quickly and effectively evaluate the metastasis risk of tumors, the present invention provides a system for detecting the diffusion coefficient of PTK7 molecules on the cell surface based on single molecule tracing technology. The system includes: a single molecule tracing fluorescent labeling probe, a fluorescence microscope, an image acquisition module and an image processing module.

[0007] The single-molecule tracer fluorescent labeling probe comprises a labeling unit targeting PTK7 and a single-molecule tracer fluorescent group. In some embodiments, the labeling unit is a nucleic acid aptamer targeting PTK7. In some embodiments, the labeling unit is a polypeptide targeting PTK7. In some embodiments, the labeling unit is a peptide nucleic acid targeting PTK7. In some embodiments, the labeling unit is a nanobody targeting PTK7.

[0008] Preferably, the labeling unit is a Sgc8 nucleic acid aptamer, which can specifically recognize and bind to the PTK7 molecule on the surface of the sample cell. PTK7 (Protein Tyrosine Kinase 7) is a receptor tyrosine kinase that lacks catalytic activity. It was initially discovered that its gene expression was upregulated in colorectal cancer cells, so it is also called colon cancer kinase-4 (CCK-4). PTK7 is mainly distributed at cell junctions and on cell membranes, and plays an important role in physiological processes such as cell polarization, proliferation, and migration. In addition to colorectal cancer, PTK7 has also been found to be abnormally expressed in many other tumors, such as breast cancer, lung cancer, pancreatic cancer, esophageal cancer, and liver cancer, and is closely related to tumor cell proliferation, invasion, metastasis, and multiple tumor pathological indicators. The nucleic acid aptamer is an oligonucleotide with a length of approximately 25-80 bases that can bind to various specific targets. The Sgc8 nucleic acid aptamer used in the present invention (sequence and structure such as Figure 1 As shown in the figure, it can specifically recognize and bind to PTK7. It was screened by the inventor's research group through the systematic evolution of ligands by exponential enrichment (SELEX) technology in the early stage. The nucleic acid aptamer has excellent targeting and specificity for the membrane protein PTK7.

[0009] The present invention uses the Sgc8 nucleic acid aptamer with small molecular weight and simple synthesis as a targeting unit, and couples it with a single-molecule tracing fluorescent group to construct a single-molecule tracing probe. The probe has good targeting and little interference with the structure and movement of the PTK7 protein molecule, so it can achieve excellent in-situ, non-interference, and real-time dynamic tracking of the movement of the PTK7 protein.

[0010] The single molecule tracer fluorescent group is a fluorescent dye, quantum dot and fluorescent protein. Common fluorescent dyes include organic dyes, such as Alexa Flour, Cyanine, ATTO series and other dyes with different wavelengths.

[0011] Preferably, the single-molecule tracer fluorescent group is Atto647N, and the Sgc8 nucleic acid aptamer is coupled with the fluorescent dye Atto647N with high quantum efficiency and good photostability as a labeling probe, making the experimental results more reliable and accurate.

[0012] Furthermore, the single-molecule tracing fluorescent labeling probe is prepared by the following preparation method: the 5' end of the Sgc8 nucleic acid aptamer is coupled to the fluorescent dye Atto647N through N-hydroxysuccinimide reaction.

[0013] The imaging system used in this patent is a single-molecule total internal reflection fluorescence microscope, including an electric TIRF module that accurately realizes the total internal reflection conditions, and a laser that can be turned on at fixed intervals to excite fluorescence. The image acquisition module is arranged at the front camera outlet of the fluorescence microscope, and is used to collect and record single-molecule fluorescence signals to obtain the motion trajectory of the PTK7 molecule. After the sample cells are incubated with the probe, the laser is used to excite fluorescence at fixed intervals under a fluorescence microscope, and single-molecule fluorescence signals are continuously collected and recorded to obtain the motion trajectory of the PTK7 molecule. Furthermore, the fixed interval time is 100-300ms, and the image acquisition module continuously records more than 300 frames of images. The smaller the interval time, the higher the number of frames, and the more accurate the calculated data of the diffusion coefficient obtained.

[0014] The visual processing module is used to draw an MSD-t curve (a curve of the relationship between mean square displacement and time t) for each PTK7 molecule of the sample cell and perform statistical processing. The diffusion coefficient of each PTK7 molecule is calculated through the relationship between the logarithm of the mean square displacement (MSD) and the logarithm of the interval time (t). By counting the diffusion coefficients of all PTK7 molecules, the statistical distribution of the diffusion coefficients of PTK7 molecules on the surface of the sample cells is obtained, and the median is taken as the diffusion coefficient of the PTK7 molecules on the surface of the sample cells.

[0015] The present invention also provides an application of the system for detecting the diffusion coefficient of PTK7 molecules on the cell surface based on the single molecule tracing technology in the preparation of a product for detecting the invasion and metastasis ability of cancer cells. The product can be used to predict the metastasis ability of tumors and optimize the treatment plan of tumors. The process of detecting the invasion and metastasis ability of cancer cells by the product includes the following steps:

[0016] S1. Take one or more primary cancer cells and one or more metastatic cancer cells from the patient's tumor tissue, use the detection system as described above to measure the diffusion coefficient distribution of each group of PTK7 molecules, and take the median as the diffusion coefficient (D value) of the PTK7 molecules on the surface of the sample cells. The linear range of each D value reflects different invasion and metastasis capabilities;

[0017] S2, obtaining the patient's tumor tissue, isolating and culturing primary cells, and using the detection system as described above to obtain the diffusion coefficient distribution and diffusion coefficient (D value);

[0018] S3. Compare the D value measured in step S2 with the D value range of each cell line in step S1 to determine the invasion and metastasis capabilities of the patient's cancer cells.

[0019] In some embodiments, the tumor site is pancreas, and the diffusion coefficient measured for primary cells is greater than or equal to 0.17 μm 2 / s, it is judged that the cancer cell has a strong invasion and metastasis ability.

[0020] In some embodiments, the tumor site is colorectal, and the diffusion coefficient measured for primary cells is greater than or equal to 0.13 μm 2 / s, it is judged that the cancer cell has a strong invasion and metastasis ability.

[0021] In some embodiments, the tumor site is breast, and the diffusion coefficient measured for primary cells is greater than or equal to 0.15 μm 2 / s, it is judged that the cancer cell has a strong invasion and metastasis ability.

[0022] The present invention verifies that the diffusion coefficient of each tumor development stage of each specific tissue is positively correlated with its invasion and metastasis ability. By detecting primary cells in the patient's tumor site and comparing its D value with the D value range of cell lines with known metastasis ability, the present invention preliminarily explores a feasible method for detecting the invasion and metastasis ability of cancer cells based on the dynamic movement law of PTK7 molecules.

[0023] The beneficial effects of the present invention are:

[0024] The present invention provides a system for detecting the diffusion coefficient of PTK7 molecules on the cell surface based on single-molecule tracing technology. The system uses the nucleic acid aptamer Sgc8, which is simple to synthesize, highly specific and small in size, as a new single-molecule tracing carrier, and couples it with an organic fluorescent dye or a quantum dot as a labeling probe for single-molecule tracing. The probe has little interference with the structure and movement of the PTK7 protein molecule, and can achieve excellent in-situ, non-interference, and real-time dynamic tracking of the movement of the PTK7 protein, and can accurately display the movement trajectory of the target protein on the surface of multiple types of cancer cells; then, by real-time tracking, collecting, and recording the movement of PTK7 on the surface of different cells, the movement trajectory of the PTK7 single molecule is obtained, and the distribution of the diffusion coefficient of its movement is calculated and counted. The median is taken as an indicator to measure the diffusion ability of the PTK7 molecule on the surface of the sample cell, and the measured value of the tumor cell sample to be tested is compared with the D value interval obtained by the cell line with known invasion and metastasis ability, so that the cell invasion and metastasis ability can be quickly and accurately evaluated, and the early prediction of the metastasis of tumor patients can be achieved, and subsequent treatment can be guided. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] The accompanying drawings in the specification, which constitute a part of the present invention, are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute improper limitations on the present invention.

[0026] Figure 1 : Example 1 Schematic diagram of constructing the Sgc8-Atto647N single-molecule imaging probe.

[0027] Figure 2 : Fluorescence image of the position and movement of single PTK7 molecules on the surface of Patu-8988t cells in Example 2.

[0028] Figure 3 : In Example 2, the PTK7 molecular motion trajectory shown in the Patu-8988t cell fluorescence image was statistically analyzed to obtain a diffusion coefficient distribution histogram.

[0029] Figure 4 : Comparison of diffusion coefficient distribution and D value of four types of cells in Example 2: pancreatic ductal cells hTERT-HPNE, pancreatic cancer cells Miapaca-2, Panc-1 and Patu-8988t.

[0030] Figure 5 : In Example 2, the Transwell assay was used to detect the cell invasion ability of four pancreatic cancer cell lines.

[0031] Figure 6 : In Example 2, a correlation analysis was performed between the diffusion coefficient (D value) and the number of cell migration.

[0032] Figure 7 : Single-molecule fluorescence image and movement trajectory of PTK7 protein on the surface of SW620 cells in Example 3.

[0033] Figure 8 : The distribution diagram of the diffusion coefficient of PKT7 molecules was quantitatively measured by the MSD-t curve of SW620 cells in Example 3.

[0034] Fig. 9 : Comparison of the diffusion coefficient distribution and D value of PTK7 protein on the surface of three cell lines NCM460, HCT116 and SW620 in Example 3.

[0035] Fig.10 : Single molecule fluorescence image and movement trajectory of PTK7 protein on the cell surface of MDA-MB-231, a highly metastatic malignant breast cancer cell line, in Example 4.

[0036] Fig.11 : The distribution diagram of the diffusion coefficient of PKT7 molecules was quantitatively measured by the MSD-t curve of MDA-MB-231 cells in Example 4.

[0037] Fig.12 : Comparison of the diffusion coefficient distribution and D value of PTK7 protein on the surface of two cell lines MCF-7 and MDA-MB-231 in Example 4. DETAILED DESCRIPTION

[0038] The following detailed descriptions are all illustrative and are intended to provide further explanation of the present application. Unless otherwise specified, all technical and scientific terms used herein have the same meanings as those generally understood by those of ordinary skill in the art to which the present application belongs. The following examples are intended only to explain the present invention and are not intended to limit their contents. If the specific experimental conditions not specified in the examples are generally in accordance with conventional conditions or in accordance with the conditions recommended by the reagent company; the reagents, consumables, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial sources.

[0039] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

[0040] Example 1: Construction of single-molecule tracing fluorescent labeling probe

[0041] In this example, the nucleic acid aptamer Sgc8 was synthesized by solid phase DNA synthesizer, and the fluorescent dye Atto647N was coupled to the 5' end by N-hydroxysuccinimide (NHS) reaction to construct a probe labeled PTK7 ( Figure 1As shown). Atto647N is an organic fluorescent dye with strong absorption, high fluorescence quantum yield and photostability, suitable for single molecule imaging. Other suitable fluorescent dyes in the prior art, such as Alexa Flour, Cyanine, ATTO series and other organic dyes with different wavelengths, can also be used.

[0042] The fluorescent labeled probe was separated and purified by HPLC and identified by LC-MS, confirming the successful synthesis of Sgc8-Atto647N.

[0043] Example 2: Detecting the invasion and metastasis ability of pancreatic cancer cells based on the movement of PTK7 single molecules

[0044] (1) Detection of single-molecule motion trajectory of PTK7 in Patu-8988t cells

[0045] In this example, the nucleic acid aptamer single-molecule tracer probe Sgc8-atto647N synthesized in Example 1 was used to obtain a single-molecule fluorescence image and movement trajectory of the PTK7 protein on the cell surface of the pancreatic cancer cell line Patu-8988t through single-molecule tracer technology.

[0046] The experimental steps of single molecule tracing technology are as follows:

[0047] Step 1. Cell sample preparation: Inoculate cells into a confocal culture dish. After culturing the cells for 24 hours, until the cell density reaches about 80%, add 0.02 nM Sgc8-Atto647N and incubate for 30 minutes, then wash to remove unbound probes.

[0048] Step 2. Set up single-molecule imaging conditions: Use an Olympus IX83 inverted TIRF fluorescence microscope equipped with a live cell culture device, adjust the temperature to 37°C, and the gas atmosphere to 5% CO2. Use a 100× oil objective (NA=1.49) and an ultra-sensitive sCMOS camera Prime95B (1024×1024 pixels, pixel size: 11μm) to collect and record single-molecule fluorescence signals. Use a Coherent OBIS 647nm laser to excite the fluorescence of Atto647N. Use a larger excitation power (20% or 40%) to ensure that a single-molecule fluorescence signal is obtained. The exposure time is 30ms, and the laser is turned on every 100ms. Usually 300 frames of images are recorded continuously.

[0049] The position and movement of PTK7 single molecules obtained by single-molecule tracing are shown in Figure 2 .

[0050] Figure 2In the figure, the position of PTK7 molecules on the surface of Patu-8988t cells is shown in the left figure (a), and each PTK7 molecule appears as a single fluorescent bright spot. The right figure (b) shows the movement trajectory of PTK7 molecules. One color represents a movement trajectory of a PTK7 in a fixed time. Different colors are used in the figure to show the movement trajectory and movement of different PTK7 in this time period.

[0051] (2) Calculate the diffusion coefficient (D) of PTK7 molecules based on the MSD-t curve

[0052] In this section, the MSD-t curve (mean square displacement, the relationship curve with t) of Patu-8988t cells was analyzed to quantitatively obtain the parameter reflecting the speed of PKT7 molecular movement, namely the diffusion coefficient (D).

[0053] The principle of obtaining the diffusion coefficient (D) through the relationship curve between mean square displacement (MSD) and t is briefly introduced as follows:

[0054] When describing molecular motion, the mean square displacement (MSD) is often used to measure the displacement of molecules relative to a reference position over time. The following is the calculation formula for MSD:

[0055]

[0056] In addition to the mean square displacement, the diffusion coefficient (D) is another very important physical quantity used to measure the diffusion speed of a substance. There is a relationship between the diffusion coefficient and the mean square displacement that is related to the time parameter, as shown below:

[0057] MSD=4Dt α

[0058] The diffusion coefficient D can be obtained by plotting the mean square displacement (MSD) against the interval time (t) in double logarithmic form.

[0059] After the present invention obtains the distribution of diffusion coefficients of all PTK molecules by statistics, the representative value (such as mean or median) of the distribution of diffusion coefficients of all trajectories can be taken as the diffusion coefficient representing the speed of PTK7 molecule movement on the surface of a certain cell line. Because the D value distribution span itself is relatively wide, this embodiment uses the median value, which is relatively more representative.

[0060] By statistically analyzing the 2783 molecular motion trajectories of PTK7 in experiment (1), we can obtain the distribution histogram of the diffusion coefficient on Patu-8988t cells and take the median to obtain the diffusion coefficient D value of 0.17 μm2 / s(reference Figure 3 Where lg(D) = -0.769, we can calculate D = 0.17 μm 2 / s), as the diffusion coefficient of PTK molecules in this cell line.

[0061] (3) Correlation between diffusion coefficient D and cell metastasis ability

[0062] In this example, four different cell lines were selected, pancreatic ductal cells tTERT-HPNE, pancreatic cancer cells Miapaca-2, Panc-1 and Patu-8988t, to explore the quantitative relationship between the diffusion coefficient D and its cell metastasis ability. Miapaca-2 is a human pancreatic ductal adenocarcinoma cell; Panc-1 is an epithelial cell carcinoma of the pancreatic duct; Patu-8988t is a pancreatic cancer cell metastasis to the liver. Generally speaking, the invasion and metastasis ability of the metastatic lesion is higher than that of the primary lesion.

[0063] Referring to the single-molecule tracing experiment (1), the movement trajectory of PTK7 protein on the surface of four cell lines was detected and the calculation method in (2) was used to statistically obtain the diffusion coefficient D corresponding to each cell line. The results are shown in the attached figure. Figure 4 It is not difficult to see that there are obvious differences in the diffusion coefficient distribution histograms and diffusion coefficient D of these four types of cells.

[0064] At the same time, we used Transwell assay to detect the cell invasion ability of the four cell lines and quantitatively counted the number of cells that metastasized in each cell line (see Appendix Figure 5 ), it can be seen that there are significant differences in the metastatic ability of each cell line.

[0065] Furthermore, we conducted a quantitative correlation analysis on the quantitative relationship between the diffusion coefficient (D value) and the number of cell migration. Figure 6 The preliminary results show that the two show a significant linear positive correlation in quantity, which indicates that the D value selected by the present invention as an indicator of the invasion and metastasis ability of pancreatic cancer cells is more accurate.

[0066] (4) Early prediction of pancreatic cancer invasion and metastasis

[0067] The distribution diagrams and medians of the diffusion coefficients of PTK7 proteins of the above-mentioned pancreatic cancer cell lines were collated, and the D value ranges indicated different invasion and metastasis capabilities, forming a set of databases for reference.

[0068] After obtaining the patient's tissue, primary cells were isolated and cultured, and the above experiment (single molecule experiment operation process) was performed on the patient's primary cells to obtain the D value distribution and median value, and the data were compared with the above D value intervals. When it is greater than or equal to 0.17 μm 2 / s, the patient's pancreatic cancer tissue can be judged to have "strong potential for invasion".

[0069] Example 3: Detection of the invasion and metastasis ability of colorectal cancer cells based on the movement of PTK7 single molecules

[0070] In this embodiment, except that the cell samples used for detection are three cell lines, NCM460, HCT116 and SW620, the single molecule tracing experiment of PTK7 molecules and the statistical calculation method of the diffusion coefficient are the same as those in Example 2. Among them, NCM460 is a normal colon epithelial cell, HCT116 is a colorectal cancer cell of the primary lesion, and SW620 is a colorectal cancer cell of the metastatic lesion. It is generally believed that the invasion and metastasis ability of cancer cells in the metastatic lesion is greater than that of the primary lesion, so these three types of cells were selected for verification.

[0071] The single-molecule fluorescence image and movement trajectory of PTK7 protein on the surface of colorectal cancer cell SW620 in metastatic lesions can be clearly acquired by single-molecule tracing technology (see Appendix Figure 7 The MSD-t curve of SW620 cells was analyzed and the diffusion coefficient (D) of PKT7 molecules was quantitatively measured. The results are shown in the attached Figure 8 , the diffusion coefficient (D) of SW620 is 0.13 μm 2 / s.

[0072] In this example, the diffusion coefficient distribution and median value of PTK7 protein on the surface of three cell lines, NCM460, HCT116 and SW620, were measured respectively. The three increased in sequence and there was a clear distinction.

[0073] It can be found that for colorectal cancer cells, the stronger the invasion and metastasis ability of the cell tissue, the greater the D value (reference Fig. 9 ), the D value is a relatively accurate indicator of the invasion and metastasis ability of colorectal cancer cells. Based on this, those skilled in the art can detect and count the diffusion coefficient distribution of patient sample cell tissues and the D value, and compare the data obtained in this embodiment. When it is greater than or equal to 0.13 μm 2 / s, it can be estimated that the patient's colorectal cancer cells have strong invasion and metastasis capabilities.

[0074] Example 4: Detecting the invasion and metastasis ability of breast cancer cells based on the movement of PTK7 single molecules

[0075] The single molecule tracing experiment of PTK7 molecules and the statistical calculation method of the diffusion coefficient in this example are the same as those in Example 2, except that the cell samples used for detection are MCF-7 and MDA-MB-231 cell lines.

[0076] The single-molecule fluorescence image and movement trajectory of PTK7 protein on the surface of MDA-MB-231, a highly metastatic malignant breast cancer cell line, can be clearly acquired by single-molecule tracking technology (see Appendix Fig.10 The MSD-t curve of MDA-MB-231 cells was analyzed and the diffusion coefficient (D value) of PKT7 molecules was quantitatively measured. The results are shown in the attached Fig.11 The diffusion coefficient (D) of this cell line is 0.15 μm 2 / s.

[0077] In this example, the diffusion coefficient distribution and D value (median) of PTK7 protein on the surface of two cell lines, MCF-7 (less malignant) and MDA-MB-231, were measured respectively. There was a clear difference in the D values ​​of the two cell lines (refer to Fig.12 ).

[0078] Therefore, for breast cancer cells, the stronger the invasion and metastasis ability of the cells, the larger the D value. The D value is a more accurate indicator of the invasion and metastasis ability of breast cancer cells. Based on this, those skilled in the art can detect and count the diffusion coefficient distribution of the patient's cancer cell tissue and the D value, and compare the data obtained in this embodiment. When it is greater than or equal to 0.15 μm 2 / s, it can be estimated that the patient's breast cancer cells have a strong ability to invade and metastasize.

[0079] Although the technical solutions of the present invention have been described and listed in detail, it should be understood that it is obvious to those skilled in the art to make modifications to the above-mentioned embodiments or adopt equivalent alternatives. These modifications or improvements made without departing from the spirit of the present invention are within the scope of protection claimed by the present invention.

Claims

1. A system for detecting the diffusion coefficient of PTK7 molecules on the cell surface based on single molecule tracing technology, characterized in that: include: A single-molecule tracing fluorescent labeling probe, wherein the single-molecule tracing fluorescent labeling probe comprises a labeling unit targeting PTK7 and a single-molecule tracing fluorescent group; A single-molecule total internal reflection fluorescence microscope, comprising a motorized TIRF module and a laser that can be turned on at fixed intervals; An image acquisition module, which is disposed at the front camera outlet of the fluorescence microscope and is used to acquire and record single-molecule fluorescence signals to obtain the motion trajectory of the PTK7 molecule; The image processing module is connected to the image acquisition module and is used to draw a logarithmic relationship curve between the mean square displacement and the interval time for each PTK7 molecule of the sample cell and perform statistical processing, calculate the diffusion coefficient of each PTK7 molecule through the relationship between the logarithm of the mean square displacement and the logarithm of the interval time, obtain the statistical distribution of the diffusion coefficient of the PTK7 molecules on the surface of the sample cell by counting the diffusion coefficients of all PTK7 molecules, and take the median as the diffusion coefficient of the PTK7 molecules on the surface of the sample cell.

2. The system for detecting the diffusion coefficient of PTK7 molecules on the cell surface based on single molecule tracing technology according to claim 1, characterized in that: The labeling unit is a nucleic acid aptamer targeting PTK7.

3. The system for detecting the diffusion coefficient of PTK7 molecules on the cell surface based on single molecule tracing technology according to claim 1, characterized in that: The labeling unit is a polypeptide targeting PTK7.

4. The system for detecting the diffusion coefficient of PTK7 molecules on the cell surface based on single molecule tracing technology according to claim 1, characterized in that: The labeling unit is a peptide nucleic acid targeting PTK7.

5. The system for detecting the diffusion coefficient of PTK7 molecules on the cell surface based on single molecule tracing technology according to claim 1, characterized in that: The labeling unit is a nano antibody targeting PTK7.

6. The system for detecting the diffusion coefficient of PTK7 molecules on the cell surface based on single molecule tracing technology according to claim 1, characterized in that: The single molecule tracer fluorescent group is a fluorescent dye, a quantum dot and a fluorescent protein.

7. The system for detecting the diffusion coefficient of PTK7 molecules on the cell surface based on single molecule tracing technology according to claim 6, characterized in that: The single-molecule tracing fluorescent group is selected from any one of ATTO, Alexa Fluor and Cyanine series.

8. Use of a system for detecting the diffusion coefficient of PTK7 molecules on the cell surface based on single molecule tracing technology as claimed in any one of claims 1 to 7 in the preparation of a product for detecting the invasion and metastasis ability of tumor cells.

9. The use according to claim 8, characterized in that The product can be used to predict the metastatic ability of a tumor.

10. The use according to claim 8, characterized in that The product can be used to optimize the treatment plan for tumors.

Images