阅读说明：本技术 用于肺癌循环肿瘤细胞富集分离试剂盒 (Enrichment and separation kit for lung cancer circulating tumor cells ) 是由 邹叶青 申阳 陈志青 滕红 于 2021-09-15 设计创作，主要内容包括：本发明提供了用于肺癌循环肿瘤细胞富集分离试剂盒,采用所述试剂盒进行肺癌循环肿瘤细胞富集分离,包括以下步骤：步骤一：将磁性纳米粒子送入到磁力搅拌器内,然后加入磁性纳米粒子总重量2-3倍的乙醇溶剂,随后再加入磁性纳米粒子总重量20-30％的叶酸,以500r/min的转速搅拌20-30min,随后再加入磁性纳米粒子总重量10％的改性石墨烯,搅拌结束,得到叶酸改性磁性纳米粒子。本发明制备的叶酸修饰的纳米粒子用于癌细胞的分离,分离的肺癌细胞活性可达到92.7％,通过对肺癌细胞的富集分离效率可达到61.3％。(The invention provides a kit for enriching and separating lung cancer circulating tumor cells, which is used for enriching and separating the lung cancer circulating tumor cells and comprises the following steps: the method comprises the following steps: feeding the magnetic nanoparticles into a magnetic stirrer, adding an ethanol solvent 2-3 times of the total weight of the magnetic nanoparticles, then adding folic acid 20-30% of the total weight of the magnetic nanoparticles, stirring at a rotating speed of 500r/min for 20-30min, then adding modified graphene 10% of the total weight of the magnetic nanoparticles, and stirring to obtain the folic acid modified magnetic nanoparticles. The folic acid modified nano particle prepared by the invention is used for separating cancer cells, the activity of the separated lung cancer cells can reach 92.7%, and the separation efficiency can reach 61.3% by enriching the lung cancer cells.)

1. The kit for enriching and separating the lung cancer circulating tumor cells is characterized by comprising the following steps of:

the method comprises the following steps: feeding the magnetic nanoparticles into a magnetic stirrer, adding an ethanol solvent 2-3 times of the total weight of the magnetic nanoparticles, then adding folic acid 20-30% of the total weight of the magnetic nanoparticles, stirring at a rotating speed of 500r/min for 20-30min, then adding modified graphene 10% of the total weight of the magnetic nanoparticles, and stirring to obtain folic acid modified magnetic nanoparticles;

step two: adding the folic acid modified magnetic nanoparticles into lung cancer circulating tumor cells for separation treatment, and finishing the separation;

the separation comprises the following specific steps:

s1: mixing and stirring lung cancer circulating tumor cells and folic acid modified magnetic nanoparticles at a stirring speed of 100-200r/min for 20-30min to obtain a to-be-separated body;

s2: adding phosphoric acid into deionized water, and adjusting the pH value of the solution to 4.0-5.0 to obtain a buffer solution;

s3: and (3) placing the separation column on a magnetic frame, adopting a buffer solution to rinse the separation column, sending the body to be separated to the separation column, and carrying out screening separation treatment by utilizing the principle of gravity.

2. The kit for enriching and separating the circulating tumor cells of the lung cancer according to claim 1, wherein the modified graphene is modified by a method comprising the following steps: feeding graphene into a chitosan solution according to the weight ratio of 1:3, then adding glacial acetic acid, and adjusting the pH value of the solution to 4.5-5.5; and then carrying out ultrasonic dispersion for 10-20min at the ultrasonic power of 300-500W, and finishing the ultrasonic treatment to obtain the modified graphene.

3. The kit for enriching and separating the circulating tumor cells of the lung cancer according to claim 2, wherein the mass fraction of the chitosan solution is 20-40%.

4. The kit for enriching and separating the circulating tumor cells of the lung cancer according to claim 3, wherein the mass fraction of the chitosan solution is 30%.

5. The kit for enriching and separating the circulating tumor cells of the lung cancer as claimed in claim 2, wherein the ultrasound time is 10-20min, and the ultrasound power is 300-500W.

6. The kit for enriching and separating the circulating tumor cells of the lung cancer according to claim 5, wherein the ultrasonic time is 15min and the ultrasonic power is 400W.

7. The kit for enriching and separating lung cancer circulating tumor cells as claimed in claim 1, wherein the rotation speed of the separation treatment in the second step is 500-1000r/min, and the separation time is 20-30 min.

8. The kit for enriching and separating the circulating tumor cells of the lung cancer according to claim 7, wherein the rotation speed of the separation treatment in the second step is 750r/min, and the separation time is 25 min.

Technical Field

The invention relates to the technical field of enrichment and separation, in particular to an enrichment and separation kit for lung cancer circulating tumor cells.

Background

The immunomagnetic separation technology is one of the important components of the peripheral blood cell rapid separation and enrichment technology, and can efficiently capture and concentrate target cells in human peripheral blood samples and improve the detection sensitivity of the target cells. In recent years, an immunomagnetic separation method (IMS) based on magnetic beads is to attach an antibody to magnetic beads, and then put the magnetic beads attached with the antibody into a sample solution to capture, enrich and magnetically separate target cells. The prior separation technology based on micron-sized immunomagnetic beads has the defects of low magnetic bead capture efficiency, longer time for capturing target cells specifically and poor monodispersity of micron-sized magnetic beads.

The existing enrichment and separation method is simple, low in separation efficiency, different in improvement effect of magnetic nanoparticles and different in separation efficiency, and further improvement treatment is needed based on the improvement effect.

Disclosure of Invention

The invention aims to provide a kit for enriching and separating lung cancer circulating tumor cells, which aims to solve the problems in the background technology.

In order to achieve the purpose, the invention provides the following technical scheme:

the kit is used for enriching and separating the lung cancer circulating tumor cells, and the kit is used for enriching and separating the lung cancer circulating tumor cells and comprises the following steps:

the method comprises the following steps: feeding the magnetic nanoparticles into a magnetic stirrer, adding an ethanol solvent 2-3 times of the total weight of the magnetic nanoparticles, then adding folic acid 20-30% of the total weight of the magnetic nanoparticles, stirring at a rotating speed of 500r/min for 20-30min, then adding modified graphene 10% of the total weight of the magnetic nanoparticles, and stirring to obtain folic acid modified magnetic nanoparticles;

step two: adding the folic acid modified magnetic nanoparticles into lung cancer circulating tumor cells for separation treatment, and finishing the separation;

the separation comprises the following specific steps:

s1: mixing and stirring lung cancer circulating tumor cells and folic acid modified magnetic nanoparticles at a stirring speed of 100-200r/min for 20-30min to obtain a to-be-separated body;

s2: adding phosphoric acid into deionized water, and adjusting the pH value of the solution to 4.0-5.0 to obtain a buffer solution;

s3: and (3) placing the separation column on a magnetic frame, adopting a buffer solution to rinse the separation column, sending the body to be separated to the separation column, and carrying out screening separation treatment by utilizing the principle of gravity.

Preferably, the modification method of the modified graphene is as follows: feeding graphene into a chitosan solution according to the weight ratio of 1:3, then adding glacial acetic acid, and adjusting the pH value of the solution to 4.5-5.5; and then carrying out ultrasonic dispersion for 10-20min at the ultrasonic power of 300-500W, and finishing the ultrasonic treatment to obtain the modified graphene.

Preferably, the mass fraction of the chitosan solution is 20-40%.

Preferably, the mass fraction of the chitosan solution is 30%.

Preferably, the ultrasonic time is 10-20min, and the ultrasonic power is 300-.

Preferably, the ultrasonic time is 15min, and the ultrasonic power is 400W.

Preferably, the rotation speed of the separation treatment in the second step is 500-.

Preferably, the rotation speed of the separation treatment in the second step is 750r/min, and the separation time is 25 min.

Compared with the prior art, the invention has the beneficial effects that:

the folic acid modified nano particle prepared by the invention is used for separating cancer cells, the activity of the separated lung cancer cells can reach 92.7%, and the separation efficiency can reach 61.3% by enriching the lung cancer cells.

Drawings

FIG. 1 is a schematic diagram of the separation efficiency of the lung cancer circulating tumor cells enriched and separated by magnetic beads with different particle sizes.

Detailed Description

The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

Example 1.

The kit is used for enriching and separating the lung cancer circulating tumor cells, and the kit is used for enriching and separating the lung cancer circulating tumor cells and comprises the following steps:

the method comprises the following steps: sending the magnetic particles into a magnetic stirrer, adding an ethanol solvent which is 2 times of the total weight of the magnetic particles, then adding folic acid accounting for 20% of the total weight of the magnetic particles, stirring at a rotating speed of 500r/min for 20min, then adding modified graphene accounting for 10% of the total weight of the magnetic particles, and obtaining folic acid modified magnetic particles after stirring;

step two: adding the folic acid modified magnetic particles into lung cancer circulating tumor cells for separation treatment, and finishing the separation;

the separation comprises the following specific steps:

s1: mixing and stirring lung cancer circulating tumor cells and folic acid modified magnetic particles at the stirring speed of 100r/min for 20min to obtain a to-be-separated body;

s2: adding phosphoric acid into deionized water, and adjusting the pH value of the solution to 4.0 to obtain a buffer solution;

s3: and (3) placing the separation column on a magnetic frame, adopting a buffer solution to rinse the separation column, sending the body to be separated to the separation column, and carrying out screening separation treatment by utilizing the principle of gravity.

The modification method of the modified graphene of the embodiment comprises the following steps: feeding graphene into a chitosan solution according to the weight ratio of 1:3, then adding glacial acetic acid, and adjusting the pH value of the solution to 4.5; and then carrying out ultrasonic dispersion for 10min with ultrasonic power of 300W, and finishing the ultrasonic treatment to obtain the modified graphene.

The chitosan solution of this example was 20% by mass.

The ultrasonic time of the present embodiment is 10min, and the ultrasonic power is 300W.

In the second step of this embodiment, the rotation speed of the separation process is 500r/min, and the separation time is 20 min.

In this example, 3 sets of experiments were performed in parallel, which were: the folic acid modified magnetic particles with the particle sizes of 25nm, 150nm and 1um provided by the invention are used for enriching, separating and treating lung cancer circulating tumor cells, and the result is shown in figure 1.

The diagram bcd is a cell state diagram of 10h of the folic acid modified magnetic bead enrichment and separation treatment adopting the particle sizes of 25nm, 150nm and 1um, and the diagram fgh is a cell state diagram of 24h of the folic acid modified magnetic bead enrichment and separation treatment adopting the particle sizes of 25nm, 150nm and 1 um. As is clear from the separation effect chart, folic acid-modified magnetic particles (magnetic beads) having a particle size of 25nm were most effective in separation, and therefore, the subsequent experiments were conducted using magnetic beads having a particle size of 25 nm.

Example 2.

The kit is used for enriching and separating the lung cancer circulating tumor cells, and the kit is used for enriching and separating the lung cancer circulating tumor cells and comprises the following steps:

the method comprises the following steps: sending the magnetic nanoparticles into a magnetic stirrer, adding an ethanol solvent which is 3 times of the total weight of the magnetic nanoparticles, then adding folic acid accounting for 30% of the total weight of the magnetic nanoparticles, stirring at a rotating speed of 500r/min for 30min, then adding modified graphene accounting for 10% of the total weight of the magnetic nanoparticles, and stirring to obtain folic acid modified magnetic nanoparticles;

step two: adding the folic acid modified magnetic nanoparticles into lung cancer circulating tumor cells for separation treatment, and finishing the separation;

the separation comprises the following specific steps:

s1: mixing and stirring lung cancer circulating tumor cells and folic acid modified magnetic nanoparticles at a stirring speed of 200r/min for 30min to obtain a to-be-separated body;

s2: adding phosphoric acid into deionized water, and adjusting the pH value of the solution to 5.0 to obtain a buffer solution;

s3: and (3) placing the separation column on a magnetic frame, adopting a buffer solution to rinse the separation column, sending the body to be separated to the separation column, and carrying out screening separation treatment by utilizing the principle of gravity.

The modification method of the modified graphene of the embodiment comprises the following steps: feeding graphene into a chitosan solution according to the weight ratio of 1:3, then adding glacial acetic acid, and adjusting the pH value of the solution to 5.5; and then carrying out ultrasonic dispersion for 20min with ultrasonic power of 500W, and finishing the ultrasonic treatment to obtain the modified graphene.

The chitosan solution of this example was 40% by mass.

The chitosan solution of this example was 30% by mass.

The ultrasonic time of the present embodiment is 20min, and the ultrasonic power is 500W.

In the second step of this embodiment, the rotation speed of the separation process is 1000r/min, and the separation time is 30 min.

Example 3:

the kit is used for enriching and separating the lung cancer circulating tumor cells, and the kit is used for enriching and separating the lung cancer circulating tumor cells and comprises the following steps:

the method comprises the following steps: sending the magnetic nanoparticles into a magnetic stirrer, adding an ethanol solvent which is 2.5 times of the total weight of the magnetic nanoparticles, then adding folic acid accounting for 25 percent of the total weight of the magnetic nanoparticles, stirring at a rotating speed of 500r/min for 25min, then adding modified graphene accounting for 10 percent of the total weight of the magnetic nanoparticles, and stirring to obtain folic acid modified magnetic nanoparticles;

step two: adding the folic acid modified magnetic nanoparticles into lung cancer circulating tumor cells for separation treatment, and finishing the separation;

the separation comprises the following specific steps:

s1: mixing and stirring lung cancer circulating tumor cells and folic acid modified magnetic nanoparticles at a stirring speed of 150r/min for 25min to obtain a to-be-separated body;

s2: adding phosphoric acid into deionized water, and adjusting the pH value of the solution to 4.5 to obtain a buffer solution;

s3: and (3) placing the separation column on a magnetic frame, adopting a buffer solution to rinse the separation column, sending the body to be separated to the separation column, and carrying out screening separation treatment by utilizing the principle of gravity.

The modification method of the modified graphene of the embodiment comprises the following steps: feeding graphene into a chitosan solution according to the weight ratio of 1:3, then adding glacial acetic acid, and adjusting the pH value of the solution to 5.0; and then carrying out ultrasonic dispersion for 15min, wherein the ultrasonic power is 400W, and finishing the ultrasonic treatment to obtain the modified graphene.

The chitosan solution of this example was 30% by mass.

The ultrasonic time of the present embodiment is 15min, and the ultrasonic power is 400W.

In the second step of this embodiment, the rotation speed of the separation process is 750r/min, and the separation time is 25 min.

Example 4:

the kit is used for enriching and separating the lung cancer circulating tumor cells, and the kit is used for enriching and separating the lung cancer circulating tumor cells and comprises the following steps:

the method comprises the following steps: sending the magnetic nanoparticles into a magnetic stirrer, adding an ethanol solvent which is 2.2 times of the total weight of the magnetic nanoparticles, then adding folic acid accounting for 22 percent of the total weight of the magnetic nanoparticles, stirring at a rotating speed of 500r/min for 23min, then adding modified graphene accounting for 10 percent of the total weight of the magnetic nanoparticles, and stirring to obtain folic acid modified magnetic nanoparticles;

step two: adding the folic acid modified magnetic nanoparticles into lung cancer circulating tumor cells for separation treatment, and finishing the separation;

the separation comprises the following specific steps:

s1: mixing and stirring lung cancer circulating tumor cells and folic acid modified magnetic nanoparticles at a stirring speed of 150r/min for 25min to obtain a to-be-separated body;

s2: adding phosphoric acid into deionized water, and adjusting the pH value of the solution to 4.5 to obtain a buffer solution;

s3: and (3) placing the separation column on a magnetic frame, adopting a buffer solution to rinse the separation column, sending the body to be separated to the separation column, and carrying out screening separation treatment by utilizing the principle of gravity.

The modification method of the modified graphene of the embodiment comprises the following steps: feeding graphene into a chitosan solution according to the weight ratio of 1:3, then adding glacial acetic acid, and adjusting the pH value of the solution to 4.6; and then carrying out ultrasonic dispersion for 12min with ultrasonic power of 320W, and finishing the ultrasonic treatment to obtain the modified graphene.

The chitosan solution of this example was 22% by mass.

The ultrasonic time of the present embodiment is 12min, and the ultrasonic power is 320W.

In the second step of this embodiment, the rotation speed of the separation process is 600r/min, and the separation time is 23 min.

Comparative example 1:

the folic acid modified magnetic nanoparticles are not adopted, and 25nm magnetic nanoparticles are adopted for treatment.

Lung cancer cell activity test criteria:

preparation of mouse lung cancer transplantable tumor:

culturing mouse lung cancer cells by using a DMEM (modified eagle Medium) culture medium containing 8% fetal calf serum, carrying out passage for 2-3 times, taking cells in a logarithmic growth phase, digesting the cells by using 025% trypsin, centrifuging the cells at 1000r/min for 10min, taking a supernatant, adding 0.9% physiological saline into the supernatant, carrying out cell suspension, diluting the suspension to 1.0 x 106/ml, and taking 1ml to inoculate a mouse to be tested;

and (4) measuring the transplanted tumor index. Establishing a model every 3d from the treatment starting day, measuring the maximum diameter (a) and the transverse diameter (b) of the tumor, and calculating the volume; treating 21d in total, taking out the spleen of the mouse after the cervical dislocation method in the 22d row is killed, and measuring the tumor volume;

the tumor cell activity rate was calculated as (control tumor weight-experimental tumor weight)/control tumor weight × 100%.

The efficiency of enriching and separating lung cancer circulating tumor cells by adopting 25nm modified magnetic beads/magnetic beads and the activity test results of the separated cells in the examples 1 to 4 and the comparative example 1 are as follows:

group of	Enrichment and separation efficiency (%)	Cell Activity (%)
			Example 1	60.6	92.3
Example 2	60.9	92.1
			Example 3	61.3	92.7
Example 4	58.6	92.4
			Comparative example 1	45.4	90.1

The magnetic nanoparticles are improved through folic acid, modified graphene added in the improvement is matched with a chitosan solution through graphene, so that the compatibility of the graphene with folic acid and the magnetic nanoparticles is improved, and the connectivity between raw materials is improved; the modified graphene is subjected to improved treatment through graphene, so that the matching effect of raw materials can be improved, the combination effect of folic acid and magnetic nanoparticles is promoted, and the improvement effect of the magnetic nanoparticles is improved; further improving the separation effect of the product on cells, and the highest separation rate can reach 61.3 percent through tests.

The present embodiments are therefore to be considered in all respects as illustrative and not restrictive, the scope of the invention being indicated by the appended claims rather than by the foregoing description, and all changes which come within the meaning and range of equivalency of the claims are therefore intended to be embraced therein. Any reference sign in a claim should not be construed as limiting the claim concerned.

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.