Magnetic polypeptide nanoparticles, methods for their production and use thereof

Description

TECHINICAL FIELD

[0001] The invention belongs to the field of medical detection, and specifically relates to a magnetic nanoparticle bearing a polypeptide, preparation method therefor and use thereof.BACKGROUND OF THE INVENTION

[0002] Cancer has become one of diseases that seriously endanger human health and life, and more than 8 million people die of cancer each year in the world. Conventional tumor detection methods in clinic are mostly imaging and tissue biopsy, which are limited by the resolution of imaging, and it is difficult to find tumors less than 5mm. While tissue biopsy is difficult to achieve multiple sampling, and brings pain and risks to patients. Circulating tumor cells (CTC) shed from the primary tumor focus and enter the blood circulation, carry almost all the genetic and protein information of the tumor tissue in situ. CTC detection, as a form of liquid biopsy at present, can dynamically reflect the progress of the tumor, and provide basis for predicting the curative effect, evaluating the prognosis and monitoring the recurrence of tumor.

[0003] Tumors are highly heterogeneous. Even tumors with the same histology and morphology have different molecular biological changes. Different biological changes have different biological behaviors and sensitivity to treatment. At present, the traditional pathological classification, such as staging and grading, which is commonly used in clinic, has limited ability to predict tumors. In recent years, with the development of science and technology, targeted therapy and immunotherapy for tumors have attracted more and more attention. In order to achieve the maximum efficacy and minimum toxicity, molecular diagnosis and precise typing of tumor types are the key to treatment. Therefore, molecular typing of tumors is an inevitable requirement for individualized tumor treatment. Breast cancer can be taken as an example to illustrate the molecular diagnosis and precise typing of tumor-targeting drugs as well as the companion diagnostic of targeted drugs. Breast cancer is one of the common malignant tumors in women, and its incidence rate increases year by year, which seriously threatens physical and mental health of women. At present, more than 40 genes related to the occurrence and development of breast cancer have been found, the most important of which are human epidermal growth factor receptor 2 (HER2), ER (estrogen receptor), PR (progesterone receptor) and androgen receptor (AR), etc., and various targeted therapies and hormone therapies have been researched and developed. In 2002, the US Food and Drug Administration

[0004] (FDA) approved the drug targeting HER2 positive breast cancer-the monoclonal antibody Herceptin for the treatment of breast cancer. Clinical efficacy shows that the drug can improve the treatment efficiency of patients and prolong the survival time of patients in pre-surgery and post-surgery adjuvant treatment of advanced breast cancer. Therefore, molecular typing of breast cancer is of great significance in tumor therapy, especially in targeted drug therapy. Since CTC in peripheral blood carries almost all of the genetic and protein information of in situ tumor tissue, molecular typing of CTC in peripheral blood of the detected patients such as HER2, ER, and PR is of great significance for guiding the clinical treatment of the patients. Similarly, for almost all other malignant tumors, molecular typing and companion diagnostic of targeted drugs have important clinical value.

[0005] In addition to targeted therapy for tumor, immunotherapy for tumor has also made a series of progress in recent years, which has changed the treatment pattern of many cancers. PD-1 / PD-L1 antibody drug for immune checkpoints is currently the most anticipated and fastest-developing tumor immunotherapy, so the PD-L1 expression of tumor cells is crucial for the pre-evaluation of the immunotherapy effect. Therefore, the companion diagnostic of PD-L1 expression at the CTC level has important clinical guiding significance for the immunotherapy of PD-1 / PD-L1 antibody drug.

[0006] Bai, Linling et al. (Journal of materials chemistry. B vol. 2,26 (2014): 4080-4088)report a new CTC isolation method with high efficiency by using the EpCAM recognition peptide functionalized iron oxide magnetic nanoparticles (MNPs) (Pep@MNPs). The article discloses peptide-based isolation ofcirculating tumor cells by magnetic nanoparticles, wherein the targeting peptide is VRRDAPRFSMQGLDA-CGGNNCNN.SUMMARY OF THE INVENTION

[0007] Therefore, the invention aims to overcome the defects in the prior art and provides a magnetic nanoparticles bearing polypeptides for detecting circulating tumor cells and molecular typing of tumor markers as well as the preparation method and use thereof.

[0008] Before explaining the present invention, the terms used herein are defined as follows: The term "PBS" refer to: phosphate buffer solution. The term "HEPES" refer to: 4- hydroxyethylpiperazine ethanesulfonic acid buffer. The term "PD-L1" refer to: programmed death receptor ligand-1. The term "HER2" refer to: human epidermal growth factor receptor 2. The term "ER" refer to: estrogen receptor. The term "PR" refer to: progesterone receptor. The term "AR" refer to: androgen receptor. The term "EGFR" refer to: epidermal growth factor receptor. The term "CXCR4" refer to: chemokine receptor 4. The term "VEGFR" refer to: vascular endothelial growth factor receptor.

[0009] The invention is set out in the appended set of claims.

[0010] In order to achieve the above purpose, the technical scheme of the present invention is as follows: The first aspect of the present invention provides a magnetic nanoparticle bearing a polypeptide, and the magnetic nanoparticle bearing a polypeptide comprises: a specific targeting polypeptide and magnetic nanoparticle; wherein the amino acid sequence of the specific targeting polypeptide is SEQ ID NO: 1.

[0011] The magnetic nanoparticle bearing a polypeptide according to the first aspect of the present invention, wherein the magnetic nanoparticle is magnetic nanoparticle with streptavidin; preferably, the particle size of the magnetic nanoparticle is 100 to 900 nm; more preferably, the particle size of the magnetic nanoparticle is 300 nm to 800 nm.

[0012] The second aspect of the present invention provides a production method of a magnetic nanoparticle bearing a polypeptide according to the first aspect, and the method includes the following steps: (1) preparing polypeptide and magnetic nanoparticle solutions; (2) mixing and reacting the polypeptide and the magnetic nanoparticle solutions prepared in step (1) to obtain the magnetic nanoparticles bearing polypeptides.

[0013] According to the method of the second aspect of the present invention, wherein, in the step (1), the solvent for preparing the polypeptide solution is one or more selected from the following: water, physiological saline, PBS, HEPES; and / or the solvent for preparing the magnetic nanoparticle solution is one or more selected from the following: water, PBS, HEPES.

[0014] According to the method of the second aspect of the present invention, wherein, in the step (1), the final concentration of the polypeptide solution is 1-1000 µg / mL, preferably 100-500 µg / mL; and / or the final concentration of the magnetic nanoparticle solution is 1-10000 µg / mL, preferably 1000-5000 µg / mL.

[0015] According to the method of the second aspect of the present invention, wherein, in the step (2), the mass ratio of the polypeptide and the magnetic nanoparticles is 1:10-5:1, preferably 2:5.

[0016] The third aspect of the present invention provides use of a magnetic particle according to the first aspect of the present invention for the diagnosis of cancer, and / or detection and / or molecular typing of circulating tumor cells, in clinical blood samples.

[0017] According to the use of the third aspect of the present invention, wherein the biomarker for circulating tumor cell detection and / or molecular typing is one or more selected from the following: Programmed death receptor ligand-1(PD-L1), Human epidermal growth factor receptor 2 (HER2), Estrogen receptor (ER), Progesterone receptor (PR), Androgen receptor (AR), Epidermal growth factor receptor (EGFR), Chemokine receptor 4( CXCR4), Vascular endothelial growth factor receptor (VEGF).

[0018] The present invention provides a magnetic nanoparticle bearing a polypeptide for CTC detection, comprising: 1) A specific recognition polypeptide targeting epithelial cell adhesion molecule (EpCAM), with the corresponding preferred sequence is VRRDAPRFSMQGLDACGGNNCNNNNN and its possible mutants. 2)The magnetic nanoparticles with streptavidin having a particle size of 100-900 nm, preferably, the particle size is 300-800 nm.) combining the above 1) and 2).

[0019] The method includes the following steps: a) Dissolve the peptide powder in a certain amount of solvent to obtain a peptide solution with a concentration of 1-1000 µg / mL; preferably, the solvent is selected from the good solvent for polypeptide, which is water, physiological saline, PBS, HEPES. b) Dilute the magnetic nanoparticles with a certain amount of solvent to obtain a magnetic nanoparticle solution with a concentration of 1-10000 µg / mL; Preferably, the solvent is magnetic bead dispersant, which is water, PBS, HEPES. c) Mix the polypeptide solution and the magnetic nanoparticle solution according to a certain proportion, place the mixture on a shaking table react for 0.5-2h at the temperature of 25-37°C and the rotating speed of 100-160rpm, and centrifuge and wash the obtained magnetic nanoparticle bearing a polypeptide assembly, preferably, the centrifugal rotating speed is 5000-10000 rpm, and the obtained magnetic nanoparticle bearing a polypeptide suspension is stored at 4°C.

[0020] The invention also provides the detection of CTC by the polypeptide nano detection device.

[0021] Preferably, the CTC is SK-BR-3, MCF-7, MDA-MB-231, H1975, H1650 and A549 tumor cells.

[0022] The present invention also provides the polypeptide nano detection device for detecting CTCs in peripheral blood of tumor patients and performing molecular typing of tumor markers on the detected CTC. Preferably, it is suitable for the detection and molecular typing of CTC in the peripheral blood of cancer patients such as breast cancer, esophageal cancer, gastric cancer, liver cancer, lung cancer, colorectal cancer, cervical cancer and prostate cancer. The steps of detecting CTC by polypeptide nanotechnology include incubation, washing, centrifugation, fixation, sealing, immunofluorescence staining, CTC identification.

[0023] Polypeptide nano magnetic bead technology can be used for almost all solid tumors except brain tumors, osteosarcoma, and lymphoma, including esophageal cancer, liver cancer, lung cancer, stomach cancer, breast cancer, colorectal cancer, cervical cancer, thyroid cancer, prostate cancer, pancreatic cancer, renal cancer, bladder cancer, skin cancer, melanoma.

[0024] One object of the present invention is to provide a magnetic nanoparticle bearing a polypeptide for circulating tumor cell detection and tumor marker molecular typing and its use. This magnetic nanoparticle bearing a polypeptide can realize in vitro diagnosis and molecular typing of breast cancer. It is simple and convenient to operate, low in cost, rapid in detection process, and non-invasive in nature, which avoids the pain caused by conventional pathological detection to patients. In addition, it is expected to track the condition in real time and adjust the treatment plan in time according to the development of the condition, which provides guidance for the realization of personalized medicine.

[0025] The references to the methods of treatment by therapy or surgery or in vivo diagnosis methods in examples 1-17 of this description are to be interpreted as references to compounds, pharmaceutical compositions and medicaments of the present invention for use in those methods. Such subject-matter is not claimed as such, but is useful for understanding the invention

[0026] The magnetic nanoparticles bearing polypeptides for detection and molecular typing of circulating tumor cells of the present invention may have the following beneficial effects: The polypeptide nanotechnology of the present invention has high sensitivity and specificity for detecting CTC, which can detect CTC in the peripheral blood of patients with various clinical tumors, including breast cancer, liver cancer, lung cancer, gastric cancer, esophageal cancer, colorectal cancer, prostate cancer, cervical cancer and other tumors.BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Hereinafter, the embodiments of the present invention will be described in detail with reference to the drawings, in which: Figure 1 shows the results of NO:1 polypeptide nanomagnetic beads of test example 1enriching breast cancer cells and performing HER2 molecular typing, wherein Figure 1A shows the capture of breast cancer cells SK-BR-3, MCF-7, and MDA-MB-231 by SEQ ID NO:1 polypeptide nanomagnetic beads of test example 1. Figure 1B shows typical breast cancer cells with different HER2 expression levels detected by the SEQ ID NO:1 polypeptide nanomagnetic beads of test example 1. Figure 2 shows the results of SEQ NO:1 polypeptide nanomagnetic beads of test example 2 enriching lung cancer cells and performing PD-L1 molecular typing, wherein Figure 2A shows the capture of lung cancer cells H1975, H1650 and A549 by SEQ ID NO:1 polypeptide nanomagnetic beads of test example 2. Figure 2B shows typical lung cancer cells with different PD-L1 expression levels detected by the SEQ ID NO:1 polypeptide nanomagnetic beads of test example 2. Figure 3 shows the typical CTCs in the peripheral blood of breast cancer patients with different HER2 expression levels detected in test example 3. Figure 4 shows typical CTCs in the peripheral blood of breast cancer patients with different ER expression levels detected in test example 4. Figure 5 shows the CTCs in the peripheral blood of breast cancer patients with different expression levels of typical PR molecules detected in test example 5. Figure 6 shows the CTCs in the peripheral blood of breast cancer patients with different expression levels of typical AR molecules detected in test example 6. Figure 7 shows the CTCs in the peripheral blood of esophageal cancer patients with different expression levels of typical PD-L1 molecules detected in test example 7. Figure 8 shows the CTCs in the peripheral blood of lung cancer patients with different expression levels of typical PD-L1 molecules detected in test example 8. Figure 9 shows the CTCs in the peripheral blood of lung cancer patients with different expression levels of typical EGFR molecules detected in test example 9. Figure 10 shows the CTCs in the peripheral blood of liver cancer patients with different expression levels of typical PD-L1 molecules detected in test example 10. Figure 11 shows the CTCs in the peripheral blood of cervical cancer patients with different expression levels of typical PD-L1 molecules detected in test example 11. Figure 12 shows the typical CTCs in the peripheral blood of gastric cancer patients with different expression levels of PD-L1 molecules detected in test example 12. Figure 13 shows the CTCs in the peripheral blood of breast cancer patients with different expression levels of typical CXCR4 molecules detected in test example 13. Figure 14 shows the CTCs in the peripheral blood of gastric cancer patients with different expression levels of typical HER2 molecules detected in test example 14. Figure 15 shows the CTCs in the peripheral blood of colorectal cancer patients with different expression levels of typical HER2 molecules detected in test example 15. Figure 16 shows the CTCs in the peripheral blood of colorectal cancer patients with different expression levels of typical PD-L1 molecules detected in test example 16. Figure 17 shows the CTCs in the peripheral blood of colorectal cancer patients with different expression levels of typical VEGFR molecules detected in test example 17. BEST MODE FOR CARRYING OUT THE INVENTION

[0028] The present invention will be further illustrated by specific examples below. If the specific technology or condition is not indicated in the embodiment, it shall be performed according to the technology or condition described in the literature in the art or according to the product specification. The reagents or instruments that do not indicate the manufacturer are all conventional products that are commercially available from regular channels.

[0029] This section gives a general description of the materials and test methods used in the test of the present invention. Although many materials and operating methods used to achieve the purpose of the present invention are well known in the art, the present invention is still described herein as much detail as possible. It is clear to the person skilled in the art that, in the context, unless otherwise specified, the materials and operating methods used in the present invention are well known in the art.

[0030] Unless otherwise specified, the human tumor cell lines SK-BR-3, MCF-7, MDA-MB-231, H1975, H1650, and A549 used in the following examples were all purchased from the cell bank of the Institute of Basic Research, Chinese Academy of Medical Sciences.

[0031] Unless otherwise specified, the polypeptides used in the following examples have a purity of 98% or more.

[0032] Unless otherwise specified, the solvents of the aqueous solutions used in the following examples are all sterile ultrapure aqueous solutions with a resistivity of 18.2MΩ·cm.

[0033] Unless otherwise specified, the reagents used in the following examples are all analytical reagents.

[0034] Unless otherwise specified, the scanning microscopes used in the following examples are all Olympus microscope IX73.

[0035] The reagents and instruments used in the following examples are as follows:Reagents:

[0036] Magnetic beads, purchased from Thermo Fisher.

[0037] The peptide was synthesized by Beijing Zhongkenatai Biotechnology Co., Ltd., with a purity of 98%.

[0038] PBS, paraformaldehyde, complete medium, DAPI working solution, immunofluorescence staining blocking solution, all purchased from Hyclone.Instruments:

[0039] The magnetic stand is made by Beijing Zhongkenatei Biotechnology Co., Ltd., which can hold 15ml centrifuge tube.

[0040] Fluorescence microscope: Olympus IX73, purchased from Beijing Cold Spring Technology Co., Ltd. ZEISS Axio Vert A1 and ZEISS Z2, purchased from ZEISS Far East Co., Ltd. Thermo Fisher CX5, purchased from Thermo Fisher. Nikon Ti-S, purchased from Beijing Hengsanjiang Instrument Sales Co., Ltd. ZEISS Z2 is recommended firstly, followed by Olympus IX73 and Thermo Fisher CX5. Example 1: Preparation of magnetic nanoparticle bearing a polypeptides assembly

[0041] 1)Took 400µL of 500nm magnetic beads into a 2mL Ep tube, added 1ml PBS to wash, then placed the tube on a magnetic stand to enrich the beads for 10 minutes, and discarded the supernatant. 2)Added 2 mL PBS to wash, then placed the tube on a magnetic stand to enrich the beads for 10 minutes, and discarded the supernatant. 3)Added 1 mL PBS to dissolve the peptide powder, shook and voluted, added the peptide solution into the Ep tube containing the magnetic beads, vortexed for 1 minute with a vortex meter, placed the peptide magnetic bead mixture on a decolorizing shaker, and adjusted the rotating speed to 60rpm , and incubated for 1 hour at room temperature. 4)Placed the Ep tube on the magnetic stand to enrich the peptide beads for 10 minutes, and discarded the supernatant. Add 1.5 mL PBS and wash for 3 times. 5)Added 400µL PBS, vortexed for 1 minute, and stored the prepared polypeptide magnetic beads in a refrigerator at 4°C.

[0042] In the following test examples, the magnetic nanoparticle bearing a polypeptide assembly of SEQ ID NO:1-9 were adopted in test examples 1-2, and the magnetic nanoparticle bearing a polypeptide assembly of SEQ ID NO:1 was adopted in test examples 3-17.Experimental Example 1: magnetic nanoparticles bearing polypeptides enrich breast cancer cells and perform HER2 molecular typing

[0043] Collected SK-BR-3, MCF-7 and MDA-MB-231 cells in logarithmic growth phase, resuspended the cells in their respective complete medium (containing 10% fetal bovine serum, 100U / mL penicillin, 100µg / mL streptomycin), counted the cell concentration, added about 1000 cells of each cell to 2mL of healthy human blood, added 10µL of peptide nanomagnetic beads to mix, and incubated for 1 hour on a shaker at room temperature. Removed the centrifuge tube and add 5mL PBS and mix gently, put it on the magnetic stand, and then placed the magnetic stand on a horizontal shaker for enrichment for 30 min. Removed the magnetic stand, discarded the supernatant, added 5 mL PBS, and placed the magnetic stand on a horizontal shaker for enrichment for 30 min. Removed the magnetic stand, discarded the supernatant, removed the centrifuge tube from the magnetic stand, blew off the magnetic beads on the tube wall with paraformaldehyde, fixed for 30 minutes at room temperature, added 5mL PBS for centrifugal washing; added DAPI working solution dropwise to stain the nucleus, and then added 5mL PBS for centrifugal washing after nuclear staining. Added 200µL immunofluorescence staining blocking solution, blocked for 30 minutes at room temperature, and added 5mL PBS for centrifugal washing. The enriched cells were stained with FITC-CK, PE-CD45 and Alexa Fluor647-HER2 (Abcam) antibodies for 1 hour respectively. Added 5mL PBS for centrifugal washing the mount, observed and found the cell interface under 20 times objective lens, set the corresponding exposure time of each fluorescence channel of DAPI, FITC, PE and Alexa Fluor 647, performed fluorescence scanning on the sample area, and performed CTC identification and HER2 fluorescence intensity analysis on the detected cells. In which DAPI+ / CK+ / CD45- and cells conforming to the cell morphology were referred to as CTC and DAPI+ / CK+ / HER2+ / CD45- cells were referred to as HER2 expressing CTC, and the HER2 expression of CTC was interpreted according to the fluorescence intensity of the HER2 channel. As shown in Figure 1A, the capture rates of SK-BR-3, MCF-7 and MDA-MB-231 by SEQ ID NO:1 polypeptide nanomagnetic beads are stable and all reach over 90%, indicating that the polypeptide nanomagnetic beads have very high enrichment and detection efficiency for breast cancer cells. Figure 1B shows breast cancer cells with different HER2 expression levels enriched by SEQ ID NO:1 polypeptide nanomagnetic beads. Table 1 shows the detection rates of SEQ ID NO:1-9 for SK-BR-3, MCF-7 and MDA-MB-231 three types of breast cancer cells. Table 1. Detection rate for SK-BR-3, MCF-7 and MDA-MB-231 three types of breast cancer cells PolypeptideDetection Rate (%)SK-BR-3MCF-7MDA-MB-231SEQ ID NO:197± 391 ±590 ± 5SEQ ID NO:280 ± 961± 856 ± 12SEQ ID NO:376±1368±749±8SEQ ID NO:461±1470±1158±4SEQ ID NO:563±778±654±7SEQ ID NO:670±551±948±7SEQ ID NO:754±1049±835±14SEQ ID NO:860±747±639±11SEQ ID NO:949±1450±853±13 Experimental Example 2: magnetic nanoparticles bearing polypeptides enrich lung cancer cells and perform PD-L1 molecular typing

[0044] Collected H1975, H1650 and A549 lung cancer cells in logarithmic growth phase, resuspended the cells in their respective complete medium (containing 10% fetal bovine serum, 100U / mL penicillin, 100µg / mL streptomycin), counted the cell concentration, added about 1000 cells of each cell to 2mL of healthy human blood, added 10µL of peptide nanomagnetic beads to mix, and incubated for 1 hour on a shaker at room temperature. Removed the centrifuge tube and add 5mL PBS and mix gently, put it on the magnetic stand, and then placed the magnetic stand on a horizontal shaker for enrichment for 30 min. Removed the magnetic stand, discarded the supernatant, added 5 mL PBS, and placed the magnetic stand on a horizontal shaker for enrichment for 30 min. Removed the magnetic stand, discarded the supernatant, removed the centrifuge tube from the magnetic stand, blew off the magnetic beads on the tube wall with paraformaldehyde, fixed for 30 minutes at room temperature, added 5mL PBS for centrifugal washing; added DAPI working solution dropwise to stain the nucleus, and then added 5mL PBS for centrifugal washing after nuclear staining. Added 200µL immunofluorescence staining blocking solution, blocked for 30 minutes at room temperature, and added 5mL PBS for centrifugal washing. The enriched cells were stained with FITC-CK, PE-CD45 and Alexa Fluor647- PD-L1 (Abcam) antibodies for 1 hour respectively. Added 5mL PBS for centrifugal washing the mount, observed under 20 times objective lens, set the corresponding exposure time of each fluorescence channel of DAPI, FITC, PE and Alexa Fluor 647, performed fluorescence scanning and fluorescence intensity analysis on the sample area. In which DAPI+ / CK+ / CD45- and cells conforming to the cell morphology were referred to as CTC and DAPI+ / CK+ / PD-L1+ / CD45- cells were referred to as PD-L1 expressing CTC, and the PD-L1 expression of CTC was interpreted according to the fluorescence intensity of the PD-L1 channel. As shown in Figure 2A, the polypeptide nanomagnetic beads have a capture rate of over 60% for H1975, H1650 and A549, indicating that the polypeptide nanomagnetic beads have very high enrichment and detection efficiency for breast cancer cells. Figure 2B shows lung cancer cells with different PD-L1 expression levels enriched by SEQ ID NO:1 polypeptide nanomagnetic beads. Table 2 shows the detection rates of SEQ ID NO:1-9 for H1975, H1650 and A549 lung cancer cells. Table 2. Detection rate for H1975, H1650 and A549 lung cancer cells PolypeptideDetection Rate (%)H1975H1650A549SEQ ID NO:165 ± 560 ± 672 ± 7SEQ ID NO:258±949±539±9SEQ ID NO:348±752±440±7SEQ ID NO:438±541±653±8SEQ ID NO:549±1537±842±5SEQ ID NO:655±1148±1347±6SEQ ID NO:739±446±838±7SEQ ID NO:845±1436±749±15SEQ ID NO:942±733±1146±13 Experimental Example 3: magnetic nanoparticles bearing polypeptides detect CTC in peripheral blood of breast cancer patients and perform HER2 molecular typing

[0045] Took 2mL of breast cancer patient's peripheral blood into a 15mL centrifuge tube, added 10µL of peptide nanomagnetic beads and mixed well, and incubated with a shaking table for 1 hour at room temperature. Removed the centrifuge tube, added 5mL PBS and mixed gently, placed it on the magnetic stand, and then the magnetic stand was placed on a horizontal shaker for enrichment for 30 minutes. Removed the magnetic stand, discarded the supernatant, added 5 mL PBS, and placed the magnetic stand on a horizontal shaker for enrichment for 30 min. Removed the magnetic stand, discarded the supernatant, removed the centrifuge tube from the magnetic stand, blew off the magnetic beads on the tube wall with paraformaldehyde, fixed for 30 minutes at room temperature, added 5mL PBS for centrifugal washing. Added DAPI working solution dropwise to stain the nucleus, added 5mL PBS for centrifugal washing after nuclear staining. Added 200µL immunofluorescence staining blocking solution, blocked for 30 minutes at room temperature, added 5mL PBS for centrifugal washing. The enriched cells were respectively stained with FITC-CK, PE-CD45 and AlexaFluor647- HER2 (Abcam) antibodies for 1 hour. Added 5mL PBS centrifuge and wash the mount, observed and found the cell interface under 20 times objective lens, set the corresponding exposure time of each fluorescence channel of DAPI, FITC, PE and Alexa Fluor 647, performed fluorescence scanning on the sample area, and performed CTC identification and HER2 fluorescence intensity analysis on the detected cells. In which DAPI+ / CK+ / CD45- and cells conforming to the cell morphology were referred to as CTCs, and DAPI+ / CK+ / HER2+ / CD45- cells were referred to as HER2 expressing CTCs, and the HER2 expression level of CTCs was interpreted according to the fluorescence intensity of the HER2 channel. Figure 3 shows the detected typical CTCs in the peripheral blood of breast cancer patients with different expression levels of HER2.Experimental Example 4: magnetic nanoparticles bearing polypeptides detect CTC in peripheral blood of breast cancer patients and perform ER molecular typing

[0046] Took 2mL of breast cancer patient's peripheral blood into a 15mL centrifuge tube, added 10µL of peptide nanomagnetic beads and mixed well, and incubated with a shaking table for 1 hour at room temperature. Removed the centrifuge tube, added 5mL PBS and mixed gently, placed it on the magnetic stand, and then the magnetic stand was placed on a horizontal shaker for enrichment for 30 minutes. Removed the magnetic stand, discarded the supernatant, added 5 mL PBS, and placed the magnetic stand on a horizontal shaker for enrichment for 30 min. Removed the magnetic stand, discarded the supernatant, removed the centrifuge tube from the magnetic stand, blew off the magnetic beads on the tube wall with paraformaldehyde, fixed for 30 minutes at room temperature, added 5mL PBS for centrifugal washing. Added DAPI working solution dropwise to stain the nucleus, added 5mL PBS for centrifugal washing after nuclear staining. Added 200µL immunofluorescence staining blocking solution, blocked for 30 minutes at room temperature, added 5mL PBS for centrifugal washing. The enriched cells were respectively stained with FITC-CK, PE-CD45 and AlexaFluor647- ER (Abcam) antibodies for 1 hour. Added 5mL PBS centrifuge and wash the mount, observed and found the cell interface under 20 times objective lens, set the corresponding exposure time of each fluorescence channel of DAPI, FITC, PE and Alexa Fluor 647, performed fluorescence scanning on the sample area, and performed CTC identification and ER fluorescence intensity analysis on the detected cells. In which DAPI+ / CK+ / CD45- and cells conforming to the cell morphology were referred to as CTCs, and DAPI+ / CK+ / ER+ / CD45- cells were referred to as ER expressing CTCs, and the ER expression level of CTCs was interpreted according to the fluorescence intensity of the ER channel. Figure 4 shows the detected typical CTCs in the peripheral blood of breast cancer patients with different expression levels of ER.Experimental Example 5: magnetic nanoparticles bearing polypeptides detect CTC in peripheral blood of breast cancer patients and perform PR molecular typing

[0047] Took 2mL of breast cancer patient's peripheral blood into a 15mL centrifuge tube, added 10µL of peptide nanomagnetic beads and mixed well, and incubated with a shaking table for 1 hour at room temperature. Removed the centrifuge tube, added 5mL PBS and mixed gently, placed it on the magnetic stand, and then the magnetic stand was placed on a horizontal shaker for enrichment for 30 minutes. Removed the magnetic stand, discarded the supernatant, added 5 mL PBS, and placed the magnetic stand on a horizontal shaker for enrichment for 30 min. Removed the magnetic stand, discarded the supernatant, removed the centrifuge tube from the magnetic stand, blew off the magnetic beads on the tube wall with paraformaldehyde, fixed for 30 minutes at room temperature, added 5mL PBS for centrifugal washing. Added DAPI working solution dropwise to stain the nucleus, added 5mL PBS for centrifugal washing after nuclear staining. Added 200µL immunofluorescence staining blocking solution, blocked for 30 minutes at room temperature, added 5mL PBS for centrifugal washing. The enriched cells were respectively stained with FITC-CK, PE-CD45 and AlexaFluor647- PR (Abcam) antibodies for 1 hour. Added 5mL PBS centrifuge and wash the mount, observed and found the cell interface under 20 times objective lens, set the corresponding exposure time of each fluorescence channel of DAPI, FITC, PE and Alexa Fluor 647, performed fluorescence scanning on the sample area, and performed CTC identification and PR fluorescence intensity analysis on the detected cells. In which DAPI+ / CK+ / CD45- and cells conforming to the cell morphology were referred to as CTCs, and DAPI+ / CK+ / PR+ / CD45- cells were referred to as PR expressing CTCs, and the PR expression level of CTCs was interpreted according to the fluorescence intensity of the PR channel. Figure 5 shows the detected typical CTCs in the peripheral blood of breast cancer patients with different expression levels of PR.Experimental Example 6: magnetic nanoparticles bearing polypeptides detect CTC in peripheral blood of breast cancer patients and perform AR molecular typing

[0048] Took 2mL of breast cancer patient's peripheral blood into a 15mL centrifuge tube, added 10µL of peptide nanomagnetic beads and mixed well, and incubated with a shaking table for 1 hour at room temperature. Removed the centrifuge tube, added 5mL PBS and mixed gently, placed it on the magnetic stand, and then the magnetic stand was placed on a horizontal shaker for enrichment for 30 minutes. Removed the magnetic stand, discarded the supernatant, added 5 mL PBS, and placed the magnetic stand on a horizontal shaker for enrichment for 30 min. Removed the magnetic stand, discarded the supernatant, removed the centrifuge tube from the magnetic stand, blew off the magnetic beads on the tube wall with paraformaldehyde, fixed for 30 minutes at room temperature, added 5mL PBS for centrifugal washing. Added DAPI working solution dropwise to stain the nucleus, added 5mL PBS for centrifugal washing after nuclear staining. Added 200µL immunofluorescence staining blocking solution, blocked for 30 minutes at room temperature, added 5mL PBS for centrifugal washing. The enriched cells were respectively stained with FITC-CK, PE-CD45 and AlexaFluor647- AR (Abcam) antibodies for 1 hour. Added 5mL PBS centrifuge and wash the mount, observed and found the cell interface under 20 times objective lens, set the corresponding exposure time of each fluorescence channel of DAPI, FITC, PE and Alexa Fluor 647, performed fluorescence scanning on the sample area, and performed CTC identification and AR fluorescence intensity analysis on the detected cells. In which DAPI+ / CK+ / CD45- and cells conforming to the cell morphology were referred to as CTCs, and DAPI+ / CK+ / AR+ / CD45- cells were referred to as AR expressing CTCs, and the AR expression level of CTCs was interpreted according to the fluorescence intensity of the AR channel. Figure 6 shows the detected typical CTCs in the peripheral blood of breast cancer patients with different expression levels of AR.Experimental Example 7: magnetic nanoparticles bearing polypeptides detect CTC in peripheral blood of esophageal cancer patients and perform PD-L1 molecular typing

[0049] Took 2mL of esophageal cancer patient's peripheral blood into a 15mL centrifuge tube, added 10µL of peptide nanomagnetic beads and mixed well, and incubated with a shaking table for 1 hour at room temperature. Removed the centrifuge tube, added 5mL PBS and mixed gently, placed it on the magnetic stand, and then the magnetic stand was placed on a horizontal shaker for enrichment for 30 minutes. Removed the magnetic stand, discarded the supernatant, added 5 mL PBS, and placed the magnetic stand on a horizontal shaker for enrichment for 30 min. Removed the magnetic stand, discarded the supernatant, removed the centrifuge tube from the magnetic stand, blew off the magnetic beads on the tube wall with paraformaldehyde, fixed for 30 minutes at room temperature, added 5mL PBS for centrifugal washing. Added DAPI working solution dropwise to stain the nucleus, added 5mL PBS for centrifugal washing after nuclear staining. Added 200µL immunofluorescence staining blocking solution, blocked for 30 minutes at room temperature, added 5mL PBS for centrifugal washing. The enriched cells were respectively stained with FITC-CK, PE-CD45 and AlexaFluor647- PD-L1 (Abcam) antibodies for 1 hour. Added 5mL PBS centrifuge and wash the mount, observed and found the cell interface under 20 times objective lens, set the corresponding exposure time of each fluorescence channel of DAPI, FITC, PE and Alexa Fluor 647, performed fluorescence scanning on the sample area, and performed CTC identification and PD-L1 fluorescence intensity analysis on the detected cells. In which DAPI+ / CK+ / CD45- and cells conforming to the cell morphology were referred to as CTCs, and DAPI+ / CK+ / PD-L1+ / CD45- cells were referred to as PD-L1 expressing CTCs, and the PD-L1 expression level of CTCs was interpreted according to the fluorescence intensity of the PD-L1 channel. Figure 7 shows the detected typical CTCs in the peripheral blood of esophageal cancer patients with different expression levels of PD-L1.Experimental Example 8: magnetic nanoparticles bearing polypeptides detect CTC in peripheral blood of lung cancer patients and perform PD-L1 molecular typing

[0050] Took 2mL of lung cancer patient's peripheral blood into a 15mL centrifuge tube, added 10µL of peptide nanomagnetic beads and mixed well, and incubated with a shaking table for 1 hour at room temperature. Removed the centrifuge tube, added 5mL PBS and mixed gently, placed it on the magnetic stand, and then the magnetic stand was placed on a horizontal shaker for enrichment for 30 minutes. Removed the magnetic stand, discarded the supernatant, added 5 mL PBS, and placed the magnetic stand on a horizontal shaker for enrichment for 30 min. Removed the magnetic stand, discarded the supernatant, removed the centrifuge tube from the magnetic stand, blew off the magnetic beads on the tube wall with paraformaldehyde, fixed for 30 minutes at room temperature, added 5mL PBS for centrifugal washing. Added DAPI working solution dropwise to stain the nucleus, added 5mL PBS for centrifugal washing after nuclear staining. Added 200µL immunofluorescence staining blocking solution, blocked for 30 minutes at room temperature, added 5mL PBS for centrifugal washing. The enriched cells were respectively stained with FITC-CK, PE-CD45 and AlexaFluor647- PD-L1 (Abcam) antibodies for 1 hour. Added 5mL PBS centrifuge and wash the mount, observed and found the cell interface under 20 times objective lens, set the corresponding exposure time of each fluorescence channel of DAPI, FITC, PE and Alexa Fluor 647, performed fluorescence scanning on the sample area, and performed CTC identification and PD-L1 fluorescence intensity analysis on the detected cells. In which DAPI+ / CK+ / CD45- and cells conforming to the cell morphology were referred to as CTCs, and DAPI+ / CK+ / PD-L1+ / CD45- cells were referred to as PD-L1 expressing CTCs, and the PD-L1 expression level of CTCs was interpreted according to the fluorescence intensity of the PD-L1 channel. Figure 8 shows the detected typical CTCs in the peripheral blood of lung cancer patients with different expression levels of PD-L1.Experimental Example 9: magnetic nanoparticles bearing polypeptides detect CTC in peripheral blood of lung cancer patients and perform EGFR molecular typing

[0051] Took 2mL of lung cancer patient's peripheral blood into a 15mL centrifuge tube, added 10µL of peptide nanomagnetic beads and mixed well, and incubated with a shaking table for 1 hour at room temperature. Removed the centrifuge tube, added 5mL PBS and mixed gently, placed it on the magnetic stand, and then the magnetic stand was placed on a horizontal shaker for enrichment for 30 minutes. Removed the magnetic stand, discarded the supernatant, added 5 mL PBS, and placed the magnetic stand on a horizontal shaker for enrichment for 30 min. Removed the magnetic stand, discarded the supernatant, removed the centrifuge tube from the magnetic stand, blew off the magnetic beads on the tube wall with paraformaldehyde, fixed for 30 minutes at room temperature, added 5mL PBS for centrifugal washing. Added DAPI working solution dropwise to stain the nucleus, added 5mL PBS for centrifugal washing after nuclear staining. Added 200µL immunofluorescence staining blocking solution, blocked for 30 minutes at room temperature, added 5mL PBS for centrifugal washing. The enriched cells were respectively stained with FITC-CK, PE-CD45 and AlexaFluor647-EGFR (Abcam) antibodies for 1 hour. Added 5mL PBS centrifuge and wash the mount, observed and found the cell interface under 20 times objective lens, set the corresponding exposure time of each fluorescence channel of DAPI, FITC, PE and Alexa Fluor 647, performed fluorescence scanning on the sample area, and performed CTC identification and EGFR fluorescence intensity analysis on the detected cells. In which DAPI+ / CK+ / CD45- and cells conforming to the cell morphology were referred to as CTCs, and DAPI+ / CK+ / EGFR+ / CD45- cells were referred to as EGFR expressing CTCs, and the EGFR expression level of CTCs was interpreted according to the fluorescence intensity of the EGFR channel. Figure 9 shows the detected typical CTCs in the peripheral blood of lung cancer patients with different expression levels of EGFR.Experimental Example 10: magnetic nanoparticles bearing polypeptides detect CTC in peripheral blood of liver cancer patients and perform PD-L1 molecular typing

[0052] Took 2mL of liver cancer patient's peripheral blood into a 15mL centrifuge tube, added 10µL of peptide nanomagnetic beads and mixed well, and incubated with a shaking table for 0.5-1 hour at room temperature. Removed the centrifuge tube, added 5mL PBS and mixed gently, placed it on the magnetic stand, and then the magnetic stand was placed on a horizontal shaker for enrichment for 30 minutes. Removed the magnetic stand, discarded the supernatant, added 5 mL PBS, and placed the magnetic stand on a horizontal shaker for enrichment for 30 min. Removed the magnetic stand, discarded the supernatant, removed the centrifuge tube from the magnetic stand, blew off the magnetic beads on the tube wall with paraformaldehyde, fixed for 30 minutes at room temperature, added 5mL PBS for centrifugal washing. Added DAPI working solution dropwise to stain the nucleus, added 5mL PBS for centrifugal washing after nuclear staining. Added 200µL immunofluorescence staining blocking solution, blocked for 30 minutes at room temperature, added 5mL PBS for centrifugal washing. The enriched cells were respectively stained with FITC-CK, PE-CD45 and AlexaFluor647- PD-L1 (Abcam) antibodies for 1 hour. Added 5mL PBS centrifuge and wash the mount, observed and found the cell interface under 20 times objective lens, set the corresponding exposure time of each fluorescence channel of DAPI, FITC, PE and Alexa Fluor 647, performed fluorescence scanning on the sample area, and performed CTC identification and PD-L1 fluorescence intensity analysis on the detected cells. In which DAPI+ / CK+ / CD45- and cells conforming to the cell morphology were referred to as CTCs, and DAPI+ / CK+ / PD-L1+ / CD45- cells were referred to as PD-L1 expressing CTCs, and the PD-L1 expression level of CTCs was interpreted according to the fluorescence intensity of the PD-L1 channel. Figure 10 shows the detected typical CTCs in the peripheral blood of liver cancer patients with different expression levels of PD-L1.Experimental Example 11: magnetic nanoparticles bearing polypeptides detect CTC in peripheral blood of cervical cancer patients and perform PD-L1 molecular typing

[0053] Took 2mL of cervical cancer patient's peripheral blood into a 15mL centrifuge tube, added 10µL of peptide nanomagnetic beads and mixed well, and incubated with a shaking table for 1 hour at room temperature. Removed the centrifuge tube, added 5mL PBS and mixed gently, placed it on the magnetic stand, and then the magnetic stand was placed on a horizontal shaker for enrichment for 30 minutes. Removed the magnetic stand, discarded the supernatant, added 5 mL PBS, and placed the magnetic stand on a horizontal shaker for enrichment for 30 min. Removed the magnetic stand, discarded the supernatant, removed the centrifuge tube from the magnetic stand, blew off the magnetic beads on the tube wall with paraformaldehyde, fixed for 30 minutes at room temperature, added 5mL PBS for centrifugal washing. Added DAPI working solution dropwise to stain the nucleus, added 5mL PBS for centrifugal washing after nuclear staining. Added 200µL immunofluorescence staining blocking solution, blocked for 30 minutes at room temperature, added 5mL PBS for centrifugal washing. The enriched cells were respectively stained with FITC-CK, PE-CD45 and AlexaFluor647- PD-L1 (Abcam) antibodies for 1 hour. Added 5mL PBS centrifuge and wash the mount, observed and found the cell interface under 20 times objective lens, set the corresponding exposure time of each fluorescence channel of DAPI, FITC, PE and Alexa Fluor 647, performed fluorescence scanning on the sample area, and performed CTC identification and PD-L1 fluorescence intensity analysis on the detected cells. In which DAPI+ / CK+ / CD45- and cells conforming to the cell morphology were referred to as CTCs, and DAPI+ / CK+ / PD-L1+ / CD45- cells were referred to as PD-L1 expressing CTCs, and the PD-L1 expression level of CTCs was interpreted according to the fluorescence intensity of the PD-L1 channel. Figure 11 shows the detected typical CTCs in the peripheral blood of cervical cancer patients with different expression levels of PD-L1.Experimental Example 12: magnetic nanoparticles bearing polypeptides detect CTC in peripheral blood of gastric cancer patients and perform PD-L1 molecular typing

[0054] Took 2mL of gastric cancer patient's peripheral blood into a 15mL centrifuge tube, added 10µL of peptide nanomagnetic beads and mixed well, and incubated with a shaking table for 1 hour at room temperature. Removed the centrifuge tube, added 5mL PBS and mixed gently, placed it on the magnetic stand, and then the magnetic stand was placed on a horizontal shaker for enrichment for 30 minutes. Removed the magnetic stand, discarded the supernatant, added 5 mL PBS, and placed the magnetic stand on a horizontal shaker for enrichment for 30 min. Removed the magnetic stand, discarded the supernatant, removed the centrifuge tube from the magnetic stand, blew off the magnetic beads on the tube wall with paraformaldehyde, fixed for 30 minutes at room temperature, added 5mL PBS for centrifugal washing. Added DAPI working solution dropwise to stain the nucleus, added 5mL PBS for centrifugal washing after nuclear staining. Added 200µL immunofluorescence staining blocking solution, blocked for 30 minutes at room temperature, added 5mL PBS for centrifugal washing. The enriched cells were respectively stained with FITC-CK, PE-CD45 and AlexaFluor647- PD-L1 (Abcam) antibodies for 1 hour. Added 5mL PBS centrifuge and wash the mount, observed and found the cell interface under 20 times objective lens, set the corresponding exposure time of each fluorescence channel of DAPI, FITC, PE and Alexa Fluor 647, performed fluorescence scanning on the sample area, and performed CTC identification and PD-L1 fluorescence intensity analysis on the detected cells. In which DAPI+ / CK+ / CD45- and cells conforming to the cell morphology were referred to as CTCs, and DAPI+ / CK+ / PD-L1+ / CD45- cells were referred to as PD-L1 expressing CTCs, and the PD-L1 expression level of CTCs was interpreted according to the fluorescence intensity of the PD-L1 channel. Figure 12 shows the detected typical CTCs in the peripheral blood of gastric cancer patients with different expression levels of PD-L1.Experimental Example 13: magnetic nanoparticles bearing polypeptides detect CTC in peripheral blood of breast cancer patients and perform CXCR4 molecular typing

[0055] Took 2mL of breast cancer patient's peripheral blood into a 15mL centrifuge tube, added 10µL of peptide nanomagnetic beads and mixed well, and incubated with a shaking table for 1 hour at room temperature. Removed the centrifuge tube, added 5mL PBS and mixed gently, placed it on the magnetic stand, and then the magnetic stand was placed on a horizontal shaker for enrichment for 30 minutes. Removed the magnetic stand, discarded the supernatant, added 5 mL PBS, and placed the magnetic stand on a horizontal shaker for enrichment for 30 min. Removed the magnetic stand, discarded the supernatant, removed the centrifuge tube from the magnetic stand, blew off the magnetic beads on the tube wall with paraformaldehyde, fixed for 30 minutes at room temperature, added 5mL PBS for centrifugal washing. Added DAPI working solution dropwise to stain the nucleus, added 5mL PBS for centrifugal washing after nuclear staining. Added 200µL immunofluorescence staining blocking solution, blocked for 30 minutes at room temperature, added 5mL PBS for centrifugal washing. The enriched cells were respectively stained with FITC-CK, PE-CD45 and AlexaFluor647-CXCR4 (Abcam) antibodies for 1 hour. Added 5mL PBS centrifuge and wash the mount, observed and found the cell interface under 20 times objective lens, set the corresponding exposure time of each fluorescence channel of DAPI, FITC, PE and Alexa Fluor 647, performed fluorescence scanning on the sample area, and performed CTC identification and CXCR4 fluorescence intensity analysis on the detected cells. In which DAPI+ / CK+ / CD45- and cells conforming to the cell morphology were referred to as CTCs, and DAPI+ / CK+ / CXCR4+ / CD45- cells were referred to as CXCR4 expressing CTCs, and the CXCR4 expression level of CTCs was interpreted according to the fluorescence intensity of the PD-L1 channel. Figure 13 shows the detected typical CTCs in the peripheral blood of breast cancer patients with different expression levels of CXCR4.Experimental Example 14: magnetic nanoparticles bearing polypeptides detect CTC in peripheral blood of gastric cancer patients and perform HER2 molecular typing

[0056] Took 2mL of gastric cancer patient's peripheral blood into a 15mL centrifuge tube, added 10µL of peptide nanomagnetic beads and mixed well, and incubated with a shaking table for 1 hour at room temperature. Removed the centrifuge tube, added 5mL PBS and mixed gently, placed it on the magnetic stand, and then the magnetic stand was placed on a horizontal shaker for enrichment for 30 minutes. Removed the magnetic stand, discarded the supernatant, added 5 mL PBS, and placed the magnetic stand on a horizontal shaker for enrichment for 30 min. Removed the magnetic stand, discarded the supernatant, removed the centrifuge tube from the magnetic stand, blew off the magnetic beads on the tube wall with paraformaldehyde, fixed for 30 minutes at room temperature, added 5mL PBS for centrifugal washing. Added DAPI working solution dropwise to stain the nucleus, added 5mL PBS for centrifugal washing after nuclear staining. Added 200µL immunofluorescence staining blocking solution, blocked for 30 minutes at room temperature, added 5mL PBS for centrifugal washing. The enriched cells were respectively stained with FITC-CK, PE-CD45 and AlexaFluor647-HER2 (Abcam) antibodies for 1 hour. Added 5mL PBS centrifuge and wash the mount, observed and found the cell interface under 20 times objective lens, set the corresponding exposure time of each fluorescence channel of DAPI, FITC, PE and Alexa Fluor 647, performed fluorescence scanning on the sample area, and performed CTC identification and HER2 fluorescence intensity analysis on the detected cells. In which DAPI+ / CK+ / CD45- and cells conforming to the cell morphology were referred to as CTCs, and DAPI+ / CK+ / HER2+ / CD45- cells were referred to as HER2 expressing CTCs, and the HER2 expression level of CTCs was interpreted according to the fluorescence intensity of the HER2 channel. Figure 14 shows the detected typical CTCs in the peripheral blood of gastric cancer patients with different expression levels of HER2.Experimental Example 15: magnetic nanoparticles bearing polypeptides detect CTC in peripheral blood of colorectal cancer patients and perform HER2 molecular typing

[0057] Took 2mL of colorectal cancer patient's peripheral blood into a 15mL centrifuge tube, added 10µL of peptide nanomagnetic beads and mixed well, and incubated with a shaking table for 1 hour at room temperature. Removed the centrifuge tube, added 5mL PBS and mixed gently, placed it on the magnetic stand, and then the magnetic stand was placed on a horizontal shaker for enrichment for 30 minutes. Removed the magnetic stand, discarded the supernatant, added 5 mL PBS, and placed the magnetic stand on a horizontal shaker for enrichment for 30 min. Removed the magnetic stand, discarded the supernatant, removed the centrifuge tube from the magnetic stand, blew off the magnetic beads on the tube wall with paraformaldehyde, fixed for 30 minutes at room temperature, added 5mL PBS for centrifugal washing. Added DAPI working solution dropwise to stain the nucleus, added 5mL PBS for centrifugal washing after nuclear staining. Added 200µL immunofluorescence staining blocking solution, blocked for 30 minutes at room temperature, added 5mL PBS for centrifugal washing. The enriched cells were respectively stained with FITC-CK, PE-CD45 and AlexaFluor647-HER2 (Abcam) antibodies for 1 hour. Added 5mL PBS centrifuge and wash the mount, observed and found the cell interface under 20 times objective lens, set the corresponding exposure time of each fluorescence channel of DAPI, FITC, PE and Alexa Fluor 647, performed fluorescence scanning on the sample area, and performed CTC identification and HER2 fluorescence intensity analysis on the detected cells. In which DAPI+ / CK+ / CD45- and cells conforming to the cell morphology were referred to as CTCs, and DAPI+ / CK+ / HER2+ / CD45- cells were referred to as HER2 expressing CTCs, and the HER2 expression level of CTCs was interpreted according to the fluorescence intensity of the HER2 channel. Figure 15 shows the detected typical CTCs in the peripheral blood of colorectal cancer patients with different expression levels of HER2.Experimental Example 16: magnetic nanoparticles bearing polypeptides detect CTC in peripheral blood of colorectal cancer patients and perform PD-L1 molecular typing

[0058] Took 2mL of colorectal cancer patient's peripheral blood into a 15mL centrifuge tube, added 10µL of peptide nanomagnetic beads and mixed well, and incubated with a shaking table for 1 hour at room temperature. Removed the centrifuge tube, added 5mL PBS and mixed gently, placed it on the magnetic stand, and then the magnetic stand was placed on a horizontal shaker for enrichment for 30 minutes. Removed the magnetic stand, discarded the supernatant, added 5 mL PBS, and placed the magnetic stand on a horizontal shaker for enrichment for 30 min. Removed the magnetic stand, discarded the supernatant, removed the centrifuge tube from the magnetic stand, blew off the magnetic beads on the tube wall with paraformaldehyde, fixed for 30 minutes at room temperature, added 5mL PBS for centrifugal washing. Added DAPI working solution dropwise to stain the nucleus, added 5mL PBS for centrifugal washing after nuclear staining. Added 200µL immunofluorescence staining blocking solution, blocked for 30 minutes at room temperature, added 5mL PBS for centrifugal washing. The enriched cells were respectively stained with FITC-CK, PE-CD45 and AlexaFluor647- PD-L1 (Abcam) antibodies for 1 hour. Added 5mL PBS centrifuge and wash the mount, observed and found the cell interface under 20 times objective lens, set the corresponding exposure time of each fluorescence channel of DAPI, FITC, PE and Alexa Fluor 647, performed fluorescence scanning on the sample area, and performed CTC identification and PD-L1 fluorescence intensity analysis on the detected cells. In which DAPI+ / CK+ / CD45- and cells conforming to the cell morphology were referred to as CTCs, and DAPI+ / CK+ / PD-L1+ / CD45- cells were referred to as PD-L1 expressing CTCs, and the PD-L1 expression level of CTCs was interpreted according to the fluorescence intensity of the PD-L1 channel. Figure 16 shows the detected typical CTCs in the peripheral blood of colorectal cancer patients with different expression levels of PD-L1.Experimental Example 17: magnetic nanoparticles bearing polypeptides detect CTC in peripheral blood of colorectal cancer patients and perform VEGFR molecular typing

[0059] Took 2mL of colorectal cancer patient's peripheral blood into a 15mL centrifuge tube, added 10µL of peptide nanomagnetic beads and mixed well, and incubated with a shaking table for 1 hour at room temperature. Removed the centrifuge tube, added 5mL PBS and mixed gently, placed it on the magnetic stand, and then the magnetic stand was placed on a horizontal shaker for enrichment for 30 minutes. Removed the magnetic stand, discarded the supernatant, added 5 mL PBS, and placed the magnetic stand on a horizontal shaker for enrichment for 30 min. Removed the magnetic stand, discarded the supernatant, removed the centrifuge tube from the magnetic stand, blew off the magnetic beads on the tube wall with paraformaldehyde, fixed for 30 minutes at room temperature, added 5mL PBS for centrifugal washing. Added DAPI working solution dropwise to stain the nucleus, added 5mL PBS for centrifugal washing after nuclear staining. Added 200µL immunofluorescence staining blocking solution, blocked for 30 minutes at room temperature, added 5mL PBS for centrifugal washing. The enriched cells were respectively stained with FITC-CK, PE-CD45 and AlexaFluor647-VEGFR (Abcam) antibodies for 1 hour. Added 5mL PBS centrifuge and wash the mount, observed and found the cell interface under 20 times objective lens, set the corresponding exposure time of each fluorescence channel of DAPI, FITC, PE and Alexa Fluor 647, performed fluorescence scanning on the sample area, and performed CTC identification and VEGFR fluorescence intensity analysis on the detected cells. In which DAPI+ / CK+ / CD45- and cells conforming to the cell morphology were referred to as CTCs, and DAPI+ / CK+ / VEGFR + / CD45- cells were referred to as VEGFR expressing CTCs, and the VEGFR expression level of CTCs was interpreted according to the fluorescence intensity of the VEGFR channel. Figure 17 shows the detected typical CTCs in the peripheral blood of colorectal cancer patients with different expression levels of VEGFR.

Claims

1. A magnetic nanoparticle bearing a polypeptide, characterized in that, the magnetic nanoparticles bearing a polypeptide comprises: a specific targeting polypeptide and magnetic nanoparticle; wherein the amino acid sequence of the specific targeting polypeptide is SEQ ID NO: 1.

2. The magnetic nanoparticle bearing a polypeptide according to claim 1, characterized in that, the magnetic nanoparticle is magnetic nanoparticle with streptavidin; preferably, the particle size of the magnetic nanoparticle is 100-900 nm; more preferably, the particle size of the magnetic nanoparticle is 300 nm to 800 nm.

3. A production method of a magnetic nanoparticle bearing a polypeptide as specified in claims 1 or 2, characterized in that, the method includes the following steps: (1) preparing polypeptide and magnetic nanoparticle solutions; (2) mixing and reacting the polypeptide and the magnetic nanoparticle solutions prepared in step (1) to obtain the magnetic nanoparticles bearing a polypeptide.

4. The method according to claim 3, characterized in that, in the step (1), the solvent for preparing the polypeptide solution is one or more selected from the following: water, physiological saline, PBS, HEPES; and / or the solvent for preparing the magnetic nanoparticle solution is one or more selected from the following: water, PBS, HEPES.

5. The method according to claim 3 or 4, characterized in that, in the step (1), the final concentration of the polypeptide solution is 1-1000 µg / mL, preferably 100-500 µg / mL; and / or the final concentration of the magnetic nanoparticle solution is 1-10000 µg / mL, preferably 1000-5000 µg / mL.

6. The method according to any one of claims 3 to 5, characterized in that, in the step (2), the mass ratio of the polypeptide and the magnetic nanoparticles is 1:10-5:1, preferably 2:5.

7. Use of a magnetic particle according to claim 1 or 2 for the diagnosis of cancer, and / or detection and / or molecular typing of circulating tumor cells, in clinical blood samples.

8. The use according to claim 7, characterized in that, the biomarker for circulating tumor cell detection and / or molecular typing is one or more selected from the following: Programmed death receptor ligand-1, Human epidermal growth factor receptor 2, Estrogen receptor, Progesterone receptor, Androgen receptor, Epidermal growth factor receptor, Vascular endothelial growth factor receptor and Chemokine receptor 4.