阅读说明：本技术 一种用于捕获循环肿瘤细胞的免疫磁球组合及制备方法 (Immune magnetic ball composition for capturing circulating tumor cells and preparation method thereof ) 是由 李国雷 于 2021-07-25 设计创作，主要内容包括：本申请提供了一种用于捕获循环肿瘤细胞的免疫磁球组合及制备方法,用于检测已经脱离肺癌实体瘤进入外周血中的循环肿瘤细胞,其特征在于,具有EGFR免疫磁球、Vimentin免疫脂质磁球、FA免疫脂质磁球,能够对血液中的循环肿瘤细胞进行特异性靶向快速分离从而捕获循环肿瘤细胞,具有较高的肺癌细胞亲和能力和比较稳定的捕获能力,能够实现捕获缺乏EpCAM蛋白的循环肿瘤细胞或已经发生上皮间质转化的循环肿瘤细胞,因此通过本申请能够对血液中非常稀少的循环肿瘤细胞进行精确、快速捕获,从而提供肿瘤形成、分化、转移的本质信息。(The application provides an immunomagnetic sphere combination for capturing circulating tumor cells and a preparation method thereof, which are used for detecting circulating tumor cells which are separated from lung cancer solid tumors and enter peripheral blood, and the immunomagnetic sphere is characterized by comprising an EGFR immunomagnetic sphere, a Vimentin immunomagnetic lipid sphere and a FA immunomagnetic lipid sphere, can carry out specific target rapid separation on the circulating tumor cells in blood so as to capture the circulating tumor cells, has higher affinity and more stable capture capability of the lung cancer cells, and can realize capture of circulating tumor cells lacking EpCAM protein or circulating tumor cells which are subjected to epithelial mesenchymal transition, so that the circulating tumor cells which are very rare in blood can be accurately and rapidly captured through the method, thereby providing essential information of tumor formation, differentiation and metastasis.)

1. An immunomagnetic sphere combination for capturing circulating tumor cells, comprising: the anti-tumor vaccine comprises an EGFR immune lipid magnetic sphere, a Vimentin immune lipid magnetic sphere and an FA immune lipid magnetic sphere, wherein the quantity ratio of the EGFR immune lipid magnetic sphere to the Vimentin immune lipid magnetic sphere to the FA immune lipid magnetic sphere is 1:0.6: 0.7-1: 1.5: 1.4.

2. An immunomagnetic sphere composition for capturing circulating tumor cells according to claim 1, wherein: the number ratio of the EGFR immune lipid magnetic spheres, the Vimentin immune lipid magnetic spheres and the FA immune lipid magnetic spheres is 1:1: 1.

3. The method of claim 1, wherein the immunomagnetic beads are used for capturing circulating tumor cells, and the method comprises the following steps: the preparation process of the EGFR immune lipid magnetic ball is as follows:

s1: mixing Fe₃O₄Mixing the magnetic nanoparticles with dichloromethane to obtain a magnetic sphere suspension; said Fe₃O₄The volume ratio of the mass of the magnetic nanoparticles to the dichloromethane is 300mg:3.53 ml;

s2: adding cholesterol, chitosan hexadecyl quaternary ammonium salt, dimethyl octadecyl epoxypropyl ammonium chloride and 1, 2-dioleoyl lecithin into phosphate buffer solution to obtain mixed solution; wherein the mass ratio of the cholesterol, the chitosan hexadecyl quaternary ammonium salt, the dimethyl octadecyl epoxypropyl ammonium chloride and the 1, 2-dioleoyl lecithin is 1:1:1: 1;

s3: mixing the magnetic sphere suspension and the mixed solution, and then carrying out ultrasonic oscillation on the mixture by using a probe type ultrasonic instrument to completely emulsify the mixture to obtain a lipid magnetic sphere solution; wherein the volume ratio of the magnetic ball suspension to the mixed solution is 1: 1-1: 1.3;

s4: dissolving 0.6mg of EGFR polypeptide in 10mL of isopropanol, adding the solution into the lipid magnetic sphere solution obtained in the step S3, and keeping the temperature at 3.9-4.1 ℃ and stirring at a constant speed for 24 hours to obtain an EGFR modified lipid magnetic sphere; wherein the volume ratio of the isopropanol to the lipid magnetic sphere solution is 1: 6-1: 11.

4. The method of claim 1, wherein the immunomagnetic beads are used for capturing circulating tumor cells, and the method comprises the following steps: the preparation process of the Vimentin immune lipid magnetic ball is as follows:

s1: mixing Fe₃O₄Mixing the magnetic nanoparticles with dichloromethane to obtain a magnetic sphere suspension; said Fe₃O₄The volume ratio of the mass of the magnetic nanoparticles to the dichloromethane is 300mg:3.53 ml;

s2: adding cholesterol, chitosan hexadecyl quaternary ammonium salt, dimethyl octadecyl epoxypropyl ammonium chloride and 1, 2-dioleoyl lecithin into phosphate buffer solution to obtain mixed solution; wherein the mass ratio of the cholesterol, the chitosan hexadecyl quaternary ammonium salt, the dimethyl octadecyl epoxypropyl ammonium chloride and the 1, 2-dioleoyl lecithin is 1:1:1: 1;

s3: mixing the magnetic sphere suspension and the mixed solution, and then carrying out ultrasonic oscillation on the mixture by using a probe type ultrasonic instrument to completely emulsify the mixture to obtain a lipid magnetic sphere solution; wherein the volume ratio of the magnetic ball suspension to the mixed solution is 1: 1-1: 1.3;

s4: dissolving 0.6mg of epithelial cell adhesion molecules in 10mL of isopropanol, adding the solution into the lipid magnetic sphere solution obtained in the step S3, and keeping the temperature at 3.9-4.1 ℃ and stirring at a constant speed for 24 hours to obtain Vimentin immune lipid magnetic spheres; wherein the volume ratio of the isopropanol to the lipid magnetic sphere solution is 1: 6-1: 11.

5. The method of claim 1, wherein the immunomagnetic beads are used for capturing circulating tumor cells, and the method comprises the following steps: the preparation process of the FA immune lipid magnetic sphere comprises the following steps:

s1: mixing Fe₃O₄Mixing the magnetic nanoparticles with dichloromethane to obtain a magnetic sphere suspension; said Fe₃O₄The volume ratio of the mass of the magnetic nanoparticles to the dichloromethane is 300mg:3.53 ml;

s2: adding cholesterol, chitosan hexadecyl quaternary ammonium salt, dimethyl octadecyl epoxypropyl ammonium chloride and 1, 2-dioleoyl lecithin into phosphate buffer solution to obtain mixed solution; wherein the mass ratio of the cholesterol, the chitosan hexadecyl quaternary ammonium salt, the dimethyl octadecyl epoxypropyl ammonium chloride and the 1, 2-dioleoyl lecithin is 1:1:1: 1;

s3: mixing the magnetic sphere suspension and the mixed solution, and then carrying out ultrasonic oscillation on the mixture by using a probe type ultrasonic instrument to completely emulsify the mixture to obtain a lipid magnetic sphere solution; wherein the volume ratio of the magnetic ball suspension to the mixed solution is 1: 1-1: 1.3;

s4: dissolving 0.6mg folic acid in 10mL isopropanol, adding the solution into the lipid magnetic sphere solution obtained in the step S3, and keeping the temperature at 3.9-4.1 ℃ and stirring at a constant speed for 24 hours to obtain FA immune lipid magnetic spheres; wherein the volume ratio of the isopropanol to the lipid magnetic sphere solution is 1: 6-1: 11.

6. A method of preparing an immunomagnetic bead composition for capturing circulating tumor cells according to any of claims 3-5, wherein: the phosphate buffered saline solution added in step S2 had a concentration of 0.1mol/L and a pH of 7.4.

7. A method of preparing an immunomagnetic bead composition for capturing circulating tumor cells according to any of claims 3-5, wherein: the ultrasonic oscillation in the step S3 is interval oscillation, and the ultrasonic oscillation is performed for 2S at an interval of 1S at a temperature of 25 ℃, and the total time is 6 min.

Technical Field

The application relates to the fields of biotechnology and biomedicine, in particular to an immunomagnetic ball combination for capturing circulating tumor cells and a preparation method thereof.

Background

Metastasis often occurs in the advanced stages of cancer, and metastasis of tumors causes about 90% of cancer deaths, making it the focus of cancer therapy. Tumor cells can migrate from a primary site to the circulatory system (e.g., blood, lymph, cerebrospinal fluid, etc.) and then spread to other organs. At this time, the cancer cell subpopulation is separated from the primary tumor, migrates through circulation such as blood, and colonizes a new organ tissue to form a new tumor mass. Therefore, early identification of cancer cells that are transmitted through the blood circulation, and timely and accurate detection of rare and rare circulating tumor cells is critical to the success of cancer therapy and to improve patient survival.

However, the abundance of circulating tumor cells in blood is extremely low, detecting circulating tumor cells is a challenge, and identifying and capturing very few tumor cells from a 7.5mL blood sample obtained from a patient presents certain difficulties. The technology commonly used for detecting circulating tumor cells in blood of cancer patients is CellSearch, and other various capture technologies including immunomagnetic beads, functionalized micro-nano structures, living flow cytometry and the like are also under development and application. Among them, the immunochemical-based magnetic nanoparticles are capable of recognizing and capturing circulating tumor cells in whole blood with high efficiency and high selectivity. CellSearch is a technology for capturing circulating tumor cells based on the connection of antibody-coated magnetic nanoparticles and epithelial cell adhesion molecules. In view of the limitations of antibody-based circulating tumor cell capture technologies, for example, the inability to capture circulating tumor cells lacking EpCAM protein or circulating tumor cells that have undergone epithelial-mesenchymal transition.

Therefore, the technical personnel in the field provide an immunomagnetic ball combination for capturing circulating tumor cells and a preparation method thereof, so as to solve the problems in the background technology.

Disclosure of Invention

The present invention has been made to solve the above problems, and an object of the present invention is to provide an immunomagnetic bead assembly for capturing circulating tumor cells, which is modified with a protein antibody to capture circulating tumor cells lacking EpCAM protein or circulating tumor cells that have undergone epithelial-mesenchymal transition, and a method for preparing the same.

The application provides an immune magnetic bead combination for capturing circulating tumor cells, which is used for detecting the circulating tumor cells which are separated from lung cancer solid tumors and enter peripheral blood, and comprises an EGFR immune lipid magnetic bead, a Vimentin immune lipid magnetic bead and an FA immune lipid magnetic bead, wherein the quantity ratio of the EGFR immune lipid magnetic bead to the Vimentin immune lipid magnetic bead to the FA immune lipid magnetic bead is 1:0.6: 0.7-1: 1.5: 1.4.

Further, an immunomagnetic sphere combination for capturing circulating tumor cells, wherein the quantity ratio of the EGFR immunoliposome, the Vimentin immunoliposome and the FA immunoliposome is 1:1: 1.

Further, the preparation method of the immune magnetic sphere combination for capturing the circulating tumor cells comprises the following steps of:

s1: mixing Fe₃O₄Mixing the magnetic nanoparticles with dichloromethane to obtain a magnetic sphere suspension; said Fe₃O₄The volume ratio of the mass of the magnetic nanoparticles to the dichloromethane is 300mg:3.53 ml;

s2: adding cholesterol, chitosan hexadecyl quaternary ammonium salt, dimethyl octadecyl epoxypropyl ammonium chloride and 1, 2-dioleoyl lecithin into phosphate buffer solution to obtain mixed solution; wherein the mass ratio of the cholesterol, the chitosan hexadecyl quaternary ammonium salt, the dimethyl octadecyl epoxypropyl ammonium chloride and the 1, 2-dioleoyl lecithin is 1:1:1: 1;

s3: mixing the magnetic sphere suspension and the mixed solution, and then carrying out ultrasonic oscillation on the mixture by using a probe type ultrasonic instrument to completely emulsify the mixture to obtain a lipid magnetic sphere solution; wherein the volume ratio of the magnetic ball suspension to the mixed solution is 1: 1-1: 1.3;

s4: dissolving 0.6mg of EGFR polypeptide in 10mL of isopropanol, adding the solution into the lipid magnetic sphere solution obtained in the step S3, and keeping the temperature at 3.9-4.1 ℃ and stirring at a constant speed for 24 hours to obtain an EGFR modified lipid magnetic sphere; wherein the volume ratio of the isopropanol to the lipid magnetic sphere solution is 1: 6-1: 11.

Further, a preparation method of the immunomagnetic sphere combination for capturing the circulating tumor cells, which also comprises Vimentin immunoliposome for capturing the circulating tumor cells, and the preparation process comprises the following steps:

s1: mixing Fe₃O₄Mixing the magnetic nanoparticles with dichloromethane to obtain a magnetic sphere suspension; said Fe₃O₄The volume ratio of the mass of the magnetic nanoparticles to the dichloromethane is 300mg:3.53 ml;

s2: adding cholesterol, chitosan hexadecyl quaternary ammonium salt, dimethyl octadecyl epoxypropyl ammonium chloride and 1, 2-dioleoyl lecithin into phosphate buffer solution to obtain mixed solution; wherein the mass ratio of the cholesterol, the chitosan hexadecyl quaternary ammonium salt, the dimethyl octadecyl epoxypropyl ammonium chloride and the 1, 2-dioleoyl lecithin is 1:1:1: 1;

s3: mixing the magnetic sphere suspension and the mixed solution, and then carrying out ultrasonic oscillation on the mixture by using a probe type ultrasonic instrument to completely emulsify the mixture to obtain a lipid magnetic sphere solution; wherein the volume ratio of the magnetic ball suspension to the mixed solution is 1: 1-1: 1.3;

s4: dissolving 0.6mg of epithelial cell adhesion molecules in 10mL of isopropanol, adding the solution into the lipid magnetic sphere solution obtained in the step S3, and keeping the temperature at 3.9-4.1 ℃ and stirring at a constant speed for 24 hours to obtain Vimentin immune lipid magnetic spheres; wherein the volume ratio of the isopropanol to the lipid magnetic sphere solution is 1: 6-1: 11.

Further, a method for preparing an immunomagnetic sphere combination for capturing circulating tumor cells, which further comprises FA immunomagnetic lipid spheres for capturing the circulating tumor cells, the preparation process comprises the following steps:

s1: mixing Fe₃O₄Mixing the magnetic nanoparticles with dichloromethane to obtain a magnetic sphere suspension; said Fe₃O₄The volume ratio of the mass of the magnetic nanoparticles to the dichloromethane is 300mg:3.53 ml;

s2: adding cholesterol, chitosan hexadecyl quaternary ammonium salt, dimethyl octadecyl epoxypropyl ammonium chloride and 1, 2-dioleoyl lecithin into phosphate buffer solution to obtain mixed solution; wherein the mass ratio of the cholesterol, the chitosan hexadecyl quaternary ammonium salt, the dimethyl octadecyl epoxypropyl ammonium chloride and the 1, 2-dioleoyl lecithin is 1:1:1: 1;

s3: mixing the magnetic sphere suspension and the mixed solution, and then carrying out ultrasonic oscillation on the mixture by using a probe type ultrasonic instrument to completely emulsify the mixture to obtain a lipid magnetic sphere solution; wherein the volume ratio of the magnetic ball suspension to the mixed solution is 1: 1-1: 1.3;

s4: dissolving 0.6mg folic acid in 10mL isopropanol, adding the solution into the lipid magnetic sphere solution obtained in the step S3, and keeping the temperature at 3.9-4.1 ℃ and stirring at a constant speed for 24 hours to obtain FA immune lipid magnetic spheres; wherein the volume ratio of the isopropanol to the lipid magnetic sphere solution is 1: 6-1: 11.

Further, a method for preparing an immunomagnetic sphere composition for capturing circulating tumor cells, wherein the phosphate buffered saline solution is added in the step S2, the concentration is 0.1mol/L, and the pH is 7.4.

Further, in the preparation method of the immunomagnetic sphere composition for capturing the circulating tumor cells, the ultrasonic oscillation performed in the step S3 is interval oscillation, and under the condition of the temperature of 25 ℃, the ultrasonic oscillation is performed for 2S, the interval is 1S, and the total time is 6 min.

After the above scheme is adopted, the beneficial effects of the application are as follows:

according to the immune magnetic sphere combination for capturing the circulating tumor cells by the lung cancer and the preparation method thereof, the immune magnetic sphere combination has the EGFR immune magnetic sphere, the Vimentin immune lipid magnetic sphere and the FA immune lipid magnetic sphere, can perform specific targeted rapid separation on the circulating tumor cells in blood so as to capture the circulating tumor cells, and has higher affinity capacity and more stable capturing capacity of the lung cancer cells. In addition, the immunomagnetic beads are magnetic nano microspheres modified by epithelial cell adhesion molecules, and have strong capture capacity on humoral tumor cells and low toxicity on cells.

In addition, in the preparation process of the immunomagnetic beads, the EGFR polypeptide, the epithelial cell adhesion molecules and the folic acid are added finally, so that the problem that the activity of the EGFR polypeptide and the epithelial cell adhesion molecules is reduced due to adverse effects on the activities of the EGFR polypeptide and the epithelial cell adhesion molecules in the subsequent steps after the addition in advance, so that the capturing efficiency of the prepared immunomagnetic beads on the lung cancer circulating tumor cells is influenced is solved, and therefore, the immunomagnetic beads have high activity and high purity, can capture the circulating tumor cells in the peripheral blood of a detected person to the greatest extent with strong specificity and high sensitivity, and the missing of the circulating tumor cells is reduced as much as possible. In addition, the whole preparation process of the immune magnetic ball can be completed at room temperature, the preparation process is simple, and the production cost is low.

Drawings

In order to make the content of the present application easier to understand, the technical solutions described in the present application will be further described below with reference to the accompanying drawings and the detailed description, but the present application is not limited thereto.

FIG. 1 is an atomic force microscope observation view of the present application;

FIG. 2 is a UV spectrum of the present application.

Detailed Description

In order to make the technical means, the creation characteristics, the achievement purposes and the effects of the present application easy to understand, the following embodiments are combined with the accompanying drawings to specifically describe an immunomagnetic ball combination for capturing circulating tumor cells and a preparation method thereof.

Example 1

The embodiment provides an immune magnetic sphere combination for capturing circulating tumor cells, which is used for detecting the circulating tumor cells which are separated from lung cancer solid tumors and enter peripheral blood, and comprises an EGFR immune lipid magnetic sphere, a Vimentin immune lipid magnetic sphere and an FA immune lipid magnetic sphere, wherein the quantity ratio of the EGFR immune lipid magnetic sphere, the Vimentin immune lipid magnetic sphere and the FA immune lipid magnetic sphere is 1:1: 1.

A method for preparing an immune magnetic sphere combination for capturing circulating tumor cells comprises the following steps:

s1: mixing Fe₃O₄Mixing the magnetic nanoparticles with dichloromethane to obtain a magnetic sphere suspension; said Fe₃O₄The volume ratio of the mass of the magnetic nanoparticles to the dichloromethane is 300mg:3.53 ml;

s2: adding cholesterol, chitosan hexadecyl quaternary ammonium salt, dimethyl octadecyl epoxypropyl ammonium chloride and 1, 2-dioleoyl lecithin into phosphate buffer solution with the concentration of 0.1mol/L, pH of 7.4 to obtain mixed solution; wherein the mass ratio of the cholesterol, the chitosan hexadecyl quaternary ammonium salt, the dimethyl octadecyl epoxypropyl ammonium chloride and the 1, 2-dioleoyl lecithin is 1:1:1: 1;

s3: mixing the magnetic sphere suspension and the mixed solution, and then carrying out ultrasonic oscillation on the mixture by using a probe type ultrasonic instrument to completely emulsify the mixture to obtain a lipid magnetic sphere solution; wherein the volume ratio of the magnetic ball suspension to the mixed solution is 1: 1-1: 1.3; the ultrasonic oscillation is interval oscillation, and under the condition of 25 ℃, the ultrasonic treatment is carried out for 2S at intervals of 1S, and the total time is 6 min;

s4: dissolving 0.6mg of EGFR polypeptide in 10mL of isopropanol, adding the solution into the lipid magnetic sphere solution obtained in the step S3, and keeping the temperature at 3.9-4.1 ℃ and stirring at a constant speed for 24 hours to obtain an EGFR modified lipid magnetic sphere; wherein the volume ratio of the isopropanol to the lipid magnetic sphere solution is 1: 6-1: 11.

A method for preparing an immunomagnetic sphere combination for capturing circulating tumor cells, which further comprises Vimentin immunoliposome for capturing the circulating tumor cells, and the preparation process comprises the following steps:

s1: mixing Fe₃O₄Mixing the magnetic nanoparticles with dichloromethane to obtain a magnetic sphere suspension; said Fe₃O₄The volume ratio of the mass of the magnetic nanoparticles to the dichloromethane is 300mg:3.53 ml;

s2: adding cholesterol, chitosan hexadecyl quaternary ammonium salt, dimethyl octadecyl epoxypropyl ammonium chloride and 1, 2-dioleoyl lecithin into phosphate buffer solution with the concentration of 0.1mol/L, pH of 7.4 to obtain mixed solution; wherein the mass ratio of the cholesterol, the chitosan hexadecyl quaternary ammonium salt, the dimethyl octadecyl epoxypropyl ammonium chloride and the 1, 2-dioleoyl lecithin is 1:1:1: 1;

s3: mixing the magnetic sphere suspension and the mixed solution, and then carrying out ultrasonic oscillation on the mixture by using a probe type ultrasonic instrument to completely emulsify the mixture to obtain a lipid magnetic sphere solution; wherein the volume ratio of the magnetic ball suspension to the mixed solution is 1: 1-1: 1.3; the ultrasonic oscillation is interval oscillation, and under the condition of 25 ℃, the ultrasonic treatment is carried out for 2S at intervals of 1S, and the total time is 6 min;

s4: dissolving 0.6mg of epithelial cell adhesion molecules in 10mL of isopropanol, adding the solution into the lipid magnetic sphere solution obtained in the step S3, and keeping the temperature at 3.9-4.1 ℃ and stirring at a constant speed for 24 hours to obtain Vimentin immune lipid magnetic spheres; wherein the volume ratio of the isopropanol to the lipid magnetic sphere solution is 1: 6-1: 11.

A method for preparing an immunomagnetic sphere combination for capturing circulating tumor cells, which further comprises FA immunomagnetic lipid spheres for capturing the circulating tumor cells, and the preparation process comprises the following steps:

s1: mixing Fe₃O₄The magnetic nanoparticles were mixed with methylene chloride,obtaining magnetic ball suspension; said Fe₃O₄The volume ratio of the mass of the magnetic nanoparticles to the dichloromethane is 300mg:3.53 ml;

s2: adding cholesterol, chitosan hexadecyl quaternary ammonium salt, dimethyl octadecyl epoxypropyl ammonium chloride and 1, 2-dioleoyl lecithin into phosphate buffer solution with the concentration of 0.1mol/L, pH of 7.4 to obtain mixed solution; wherein the mass ratio of the cholesterol, the chitosan hexadecyl quaternary ammonium salt, the dimethyl octadecyl epoxypropyl ammonium chloride and the 1, 2-dioleoyl lecithin is 1:1:1: 1;

s3: mixing the magnetic sphere suspension and the mixed solution, and then carrying out ultrasonic oscillation on the mixture by using a probe type ultrasonic instrument to completely emulsify the mixture to obtain a lipid magnetic sphere solution; wherein the volume ratio of the magnetic ball suspension to the mixed solution is 1: 1-1: 1.3; the ultrasonic oscillation is interval oscillation, and under the condition of 25 ℃, the ultrasonic treatment is carried out for 2S at intervals of 1S, and the total time is 6 min;

s4: dissolving 0.6mg folic acid in 10mL isopropanol, adding the solution into the lipid magnetic sphere solution obtained in the step S3, and keeping the temperature at 3.9-4.1 ℃ and stirring at a constant speed for 24 hours to obtain FA immune lipid magnetic spheres; wherein the volume ratio of the isopropanol to the lipid magnetic sphere solution is 1: 6-1: 11.

Application example

1. Material characterization of immunomagnetic spheres

1.1 atomic force microscopy of EGFR, Vimentin and FA immunoliposome magnetic spheres showed the results shown in FIG. 1.

As can be seen from the figure, the three immunomagnetic microspheres are spherical with different sizes, and no agglomeration phenomenon occurs, which indicates that the immunomagnetic microspheres have good stability, regular shapes, sizes of about 200nm, and the vesicular characteristics of liposomes.

1.2 UV spectroscopy test of EGFR, Vimentin and FA immunoliposome, the results are shown in FIG. 2.

As can be seen, a broad absorption peak appears at about 276nm on the immunomagnetic spheres of the three antibodies, which indicates that EGFR, Vimentin and FA are indeed grafted on the surfaces of the immunomagnetic spheres.

2. Capture efficiency experiment of immunomagnetic ball

Blood samples of 26 pathologically confirmed lung cancer patients were selected, with the patients aged 36-75 years and the average age 55.3 years. The sample collection method comprises collecting 7.5mL of peripheral blood of a patient by using a medical anticoagulant blood collection tube, wherein the anticoagulant is EDTA-K₂. All selected cases fulfilled the informed obligation and signed the informed consent.

The medical anticoagulation blood collection tube collects 7.5mL of peripheral blood, and the anticoagulant is EDTA-K₂And storing at 4 ℃, avoiding freezing in the processes of storage, treatment and transportation, and detecting within 48 h. First, blood was centrifuged at 1500r/min for 10min, the supernatant was collected and circulating tumor cells were captured using the immunoliposome magnetic bead assembly prepared in example 1 of the present application, and the captured circulating tumor cells were detected and compared with the results of tissue detection, as shown in table 1:

TABLE 1 comparison of Gene mutations in tissue detection and circulating tumor cell detection

Note: the gene detection result is positive; "-" Gene test result was negative

The result shows that the combined use of the three magnetic spheres has higher detection rate on the circulating tumor cells. The application uses three immune lipid magnetic spheres of EGFR, Vimentin and FA jointly as targets for capturing circulating tumor cells, so that the sensitivity of circulating tumor cell detection is improved, and the method is a novel and practical detection means. By this strategy, more heterogeneous circulating tumor cells can be captured, which can be used for early cancer diagnosis and recurrence assessment thereof.

The above description is only for the purpose of illustrating the preferred embodiments of the present application and is not to be construed as limiting the present application, and all equivalent variations and modifications within the scope of the present application are intended to be covered by the present application. Furthermore, it should be understood that although the present description refers to embodiments, not every embodiment may contain only a single embodiment, and such description is for clarity only, and those skilled in the art should integrate the description, and the embodiments may be combined as appropriate to form other embodiments understood by those skilled in the art.