CN110747169B - Method for capturing circulating tumor cells - Google Patents
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- CN110747169B CN110747169B CN201911028894.4A CN201911028894A CN110747169B CN 110747169 B CN110747169 B CN 110747169B CN 201911028894 A CN201911028894 A CN 201911028894A CN 110747169 B CN110747169 B CN 110747169B
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Abstract
The invention relates to a capture cycleA method of tumor cell comprising: (1) Mixing the novel magnetic beads and the anti-tumor cell antibody, adding a mixture of NHS and EDC, and incubating overnight to obtain the immune functional magnetic beads; (2) And (2) mixing and incubating the immune functional magnetic beads in the step (1) with blood to be detected, then moving the mixture into a magnetic field, and discarding waste liquid to obtain the circulating tumor cells. The invention uses carboxyl silicon dioxide microspheres as cores and quantum dots Fe 3 O 4 For the shell, novel magnetic bead wholly is the nucleocapsid structure, has kept magnetism, has reduced holistic molecular weight and has increased whole volume simultaneously to increased the surface area, made novel magnetic bead can combine more tumor cell antibodies, the circulating tumor cell in the contact peripheral blood of more having the chance. Compared with the immunomagnetic beads on the current market, the method increases the number of surface antibodies, improves the capture efficiency of circulating tumor cells, greatly reduces the combination of foreign proteins in peripheral blood and the capture of leukocytes, and has good application prospect.
Description
Technical Field
The invention belongs to the field of biomedicine, relates to a cell capturing technology, and particularly relates to a method for capturing circulating tumor cells.
Background
Malignant tumors have become a major public safety problem over the last 20 years, with nearly millions of people dying from cancer worldwide each year. One of the main causes of cancer mortality is that cancer is susceptible to metastasis, and studies have shown that tumors progress to 1cm 3 Then, the tumor cells are released to enter peripheral blood to form circulating tumor cells, and the 'seed cells' are provided for the metastasis of the tumor. In addition, the tumor mass also secretes a large amount of factors and substances with the aim of improving the environment of the transferred "soil". Circulating Tumor Cells (CTCs) can break through the inner wall of blood vessels to transfer to other tissues to take root and sprout after timely maturation along with the circulation of peripheral blood. Therefore, the monitoring and analysis of the peripheral blood circulation tumor cells are instructive to the early diagnosis of cancer (especially cancer which is extremely difficult to find, such as cervical cancer, breast cancer, etc.) and the personalized treatment of patients.
The concentration of CTCs in peripheral blood is extremely low (about 10 in 1mL of whole blood) 9 Red blood cells and 10 7 Only a few to tens of CTCs), and peripheral blood contains a large amount of proteins and various factors, and the composition is very complicated. At present developVarious methods for enriching and capturing the CTCs, such as density gradient centrifugation, membrane filtration, affinity separation and the like. The method for enriching the immunomagnetic beads is based on antigen-antibody specific binding reaction, and then an external magnetic field is used for separating CTC (CTC), and the method is gradually the most widely applied CTCs separation method due to the advantages of simple operation, high capture efficiency, convenience in combination with other methods and the like. However, the disadvantages of non-uniform size, poor dispersibility, slow magnetic reaction, low functionalization degree and the like exist in the preparation process of the magnetic beads, so that the adsorption efficiency and recovery rate of CTC are low, and the application of modified magnetic beads in the early cancer field is limited.
Disclosure of Invention
The invention aims to overcome the defects of the prior art, provides a method for capturing circulating tumor cells, and particularly provides a preparation method of novel magnetic beads for separating CTCs.
The technical problem to be solved by the invention is realized by adopting the following technical scheme:
a method of capturing circulating tumor cells comprising the steps of:
(1) Mixing the novel magnetic beads and the anti-tumor cell antibody, adding a mixture of NHS and EDC, and incubating overnight to obtain the immune functional magnetic beads;
(2) And (2) adding the immune function magnetic beads in the step (1) and blood to be detected into a centrifugal tube for mixing and incubation, then moving the centrifugal tube into a magnetic field, and discarding waste liquid to obtain the circulating tumor cells.
Furthermore, the anti-tumor cell antibody in step (1) may be one of epithelial cell adhesion molecule (EpCAM), epidermal growth factor receptor (her 2) or Prostate Specific Membrane Antigen (PSMA), and the amount of the antibody is 6ug antibody/mg of the novel magnetic beads.
Furthermore, NHS in step (1) was N-Hydroxysuccinimide (N-Hydroxysuccinimide), EDC in step (1) was 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride having a molecular weight of 191.04
The overnight incubation was carried out at 4 ℃ in a shaker for 14 hours or more.
The incubation time in step (2) is 2.5 to 20min.
Further, the preferable incubation time is 15min.
Moreover, the novel magnetic bead preparation method comprises the following steps:
(1) Adding PEI into the silica microspheres, adding a mixture of NHS and EDC, and incubating overnight;
(2) Centrifuging the overnight product in the step (1), transferring the precipitate to hexanol, and adding quantum dot Fe 3 O 4 Adding PEI for tending, and centrifuging and collecting the precipitate;
(3) Repeating the reaction for 5 times, and adding a PEG aqueous solution into the precipitation product for nurturing to obtain the final water-soluble novel magnetic beads.
Moreover, the silicon dioxide microspheres in the step (1) are modified by carboxyl groups, and the particle size is 500nm;
the PEI in the step (1) is Polyetherimide (polyether imide) with the molecular weight of less than 25kDa;
the molar ratio of the carboxyl silica microspheres to the PEI in the step (1) is 1.
Further, quantum dots Fe in step (2) 3 O 4 The particle size is 30nm, the incubation condition of adding PEI is 37 ℃, shaking table and more than 2 h;
the centrifugation condition in the step (2) is 1000g/min for 3min.
In the step (3), the above reaction is repeated 5 times, that is, the quantum dot Fe 3 O 4 The number of layers of (2) is 6;
in the step (3), the molecular weight of PEG is 3500, and the concentration of the PEG aqueous solution is 10%;
and (4) finally storing the water-soluble novel magnetic beads in the step (3) in 1 xPBS.
The invention has the advantages and positive effects that:
(1) The invention uses carboxyl silicon dioxide microspheres as cores and quantum dots Fe 3 O 4 The novel magnetic beads are of a core-shell structure, the magnetism is reserved, the overall molecular weight is reduced, and the overall volume is increased, so that the surface is increasedThe area of the magnetic beads enables the novel magnetic beads to be combined with more tumor cell antibodies and to be contacted with circulating tumor cells in peripheral blood more opportunistically.
(2) The novel magnetic bead particles prepared by the invention are uniformly dispersed and have regular size and shape, and the novel magnetic bead particles are encapsulated by PEG, so that the novel magnetic bead particles have good storage property in water and can be stored for a long time.
(3) Compared with the immunomagnetic beads on the current market, the immunomagnetic beads prepared by combining the novel magnetic beads with the antibodies increase the number of surface antibodies, improve the capture efficiency of circulating tumor cells, greatly reduce the combination of foreign proteins in peripheral blood and the capture of leukocytes, and have better application prospect.
Drawings
FIG. 1 is a schematic diagram of a process for preparing the novel magnetic beads of the present invention;
FIG. 2 is a diagram showing the characterization results of the novel magnetic beads, wherein A is a Transmission Electron Microscope (TEM) image of the novel magnetic beads, and B is a particle size diagram of the novel magnetic beads;
FIG. 3 is an analysis chart of the optimal conditions for probing the results of experiments with the immuno-functional magnetic beads to capture prostate cells, wherein A is a graph of the capture efficiency of the immuno-functional magnetic beads at different concentrations on PC-3, and B is a graph of the capture efficiency of the immuno-functional magnetic beads on PC-3 cells at different incubation times;
FIG. 4 shows that the pair of immune functional magnetic beads contains a small number of PC-3 cells (PC-3 cell concentration is 5, 50, 100, 500 and 1000/mL, 293T cells are 5X10 5 number/mL) of cancer cells in a mixed system;
fig. 5 is a graph showing the results of the capture of prostate cancer cells by the mixed system PC-3 with the bifunctional magnetic beads, 293t =1, a is a graph showing the results of the sorting of tumor cells by the bifunctional magnetic beads, and B is a graph showing the fluorescence of the sorting of tumor cells by the bifunctional magnetic beads.
Detailed Description
The present invention will be described in further detail with reference to the following embodiments, which are illustrative only and not limiting, and the scope of the present invention is not limited thereby.
Preparation of novel magnetic beads
The novel magnetic bead of the present inventionThe preparation process is schematically shown in figure 1, and the specific novel magnetic bead is formed by assembling quantum dots F on the surface of a carboxyl silicon dioxide microsphere layer by layer through a layer-by-layer assembly method 3 O 4 Thus obtaining the compound. The method comprises the following specific steps: (1) 1mL of silica microspheres with carboxyl on the surface are taken, 1mL of PEI (MW: 25K, concentration: 0.3 g/mL) and a mixture of EDC and NHS are sequentially added, and then the mixture is placed in a shaker at 4 ℃ for reaction overnight, so that a layer of PEI can be assembled on the surfaces of the silica microspheres. (2) And (2) washing the product obtained in the step (1) for several times by using NaCl solution, ultrapure water and absolute ethyl alcohol in sequence, and then uniformly dispersing by using n-hexanol. Adding equal amount of quantum dots F 3 O 4 Mix and shake evenly, put on a shaking bed and react for 2h at 37 ℃. (3) Centrifuging the product obtained in the step (2) to remove the supernatant, and washing with n-hexane, absolute ethyl alcohol and ultrapure water for several times to remove the unassembled quantum dots F 3 O 4 And adding 5mL of PEI solution, uniformly mixing, placing on a shaking table, reacting at room temperature for 2h, and assembling a second layer of PEI. (4) Repeating the steps (2) and (3) to carry out multilayer assembly, and assembling 6 layers of magnetic quantum dots F 3 O 4 The nanospheres of (2) were dispersed in 5ml PEG3500 solution and placed on a shaker at 37 ℃ for 12h reaction. And (3) centrifugally washing the product by using ethanol and ultrapure water in sequence to obtain novel magnetic beads (the particle size is 789 nm) with carboxyl on the surface, and dispersing the novel magnetic beads in 1mL of ultrapure water for storage. (5) About 10. Mu.L of the product obtained in step (4) was diluted in 1mL of ultrapure water, and the morphology and size thereof were measured by a transmission electron microscope and a dynamic light scattering apparatus, and the results are shown in FIG. 2.
Preparation of magnetic beads with immunological function
The immune functional magnetic bead is obtained by reacting carboxyl on the surface of the novel magnetic bead with amino of EpCAM. The method comprises the following specific steps: 0.5mL of novel magnetic beads with carboxyl groups on the surface were added with the antibody EpCAM (120 ug) and a mixture of EDC and NHS in this order, and then they were subjected to shaking reaction at 4 ℃ overnight. The obtained product is dispersed in ultrapure water for storage, and fig. 3 is an analysis chart of the optimal conditions for capturing prostate cells by the immune functional magnetic beads, wherein A is the capture efficiency of the immune functional magnetic beads with different concentrations on PC-3, and B is the capture efficiency of the PC-3 cells under different incubation times. FIG. 4 is a graph showing the results of the capture efficiency of the immuno-functional magnetic beads on cancer cells in a mixed system containing a small number of PC-3 cells (PC-3 cell concentration of 5, 50, 100, 500 and 1000/mL, 293T cells of 5X 105/mL).
Efficiency of capturing circulating tumor cells
The following describes a method for establishing a capture method by taking the mixture of prostate cancer cells (PC-3) and 293T cells as a cell model, wherein the PC-3 cells are transfected with green fluorescent plasmids, and the prostate cancer is reliably captured by using immune functional magnetic beads. The method comprises the following specific steps: 1mL of a mixture of digested PC-3 and 293T cells (total cell mass 5X 10) 4 The concentration of PC-3 cells is about 5, 50, 100, 500 and 1000/mL), adding a certain amount of immune functional magnetic beads, incubating for 15min in a light-shielding manner under the temperature of 37 ℃, carrying out magnetic sorting and washing, respectively collecting clear liquid and turbid liquid in a cell culture dish, counting the PC-3 cells in the clear liquid and calculating the capture efficiency through a fluorescence microscope, and the result is shown in figure 5.
Although the embodiments of the present invention and the accompanying drawings are disclosed for illustrative purposes, those skilled in the art will appreciate that: various substitutions, changes and modifications are possible without departing from the spirit and scope of the invention and the appended claims, and therefore the scope of the invention is not limited to the disclosure of the embodiments and the accompanying drawings.
Claims (6)
1. A method of capturing circulating tumor cells for non-diagnostic purposes, comprising: the method comprises the following steps:
(1) Mixing the magnetic beads and the anti-tumor cell antibody, adding a mixture of NHS and EDC, and incubating overnight to obtain the immune functional magnetic beads;
(2) Adding the immune function magnetic beads and the blood to be detected in the step (1) into a centrifugal tube for mixing and incubation, then moving the centrifugal tube into a magnetic field, discarding waste liquid to obtain circulating tumor cells,
the anti-tumor cell antibody in the step (1) is one of an epithelial cell adhesion molecule EpCAM, an epidermal growth factor receptor her 2 or a prostate specific membrane antigen PSMA, the amount of the antibody is 6 mug of antibody/mg of magnetic bead,
the preparation method of the magnetic bead comprises the following steps:
(1) Adding PEI into the silica microspheres, adding a mixture of NHS and EDC, and incubating overnight;
(2) Centrifuging the overnight product in the step (1), transferring the precipitate to hexanol, and adding quantum dot Fe 3 O 4 Then adding PEI for incubation, centrifuging and collecting the precipitate;
(3) Repeating the reaction for 5 times, adding PEG aqueous solution into the precipitation product, incubating to obtain the final water-soluble magnetic beads,
the silica microspheres in the step (1) of the magnetic bead preparation method are modified by carboxyl, and the particle size is 500nm;
the PEI in the step (1) of the magnetic bead preparation method is Polyetherimide polyethenimide with the molecular weight less than 25kDa;
according to the magnetic bead preparation method, the molar ratio of the silica microspheres to the PEI is 1.
2. A method of capturing circulating tumor cells for non-diagnostic purposes according to claim 1, wherein:
NHS in the step (1) of the magnetic bead preparation method is N-Hydroxysuccinimide with the molecular weight of 115.09
Magnetic bead preparation method EDC in step (1) was 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride having a molecular weight of 191.04.
3. The method of claim 1, wherein the step of capturing the circulating tumor cells comprises: the incubation time in the step (2) of the magnetic bead preparation method is 2.5-20 min.
4. A method of capturing circulating tumor cells for non-diagnostic purposes as claimed in claim 3 wherein: the preferred incubation time is 15min.
5. A method of capturing circulating tumor cells for non-diagnostic purposes according to claim 1, wherein:
quantum dot Fe in step (2) of magnetic bead preparation method 3 O 4 The particle size is 30nm, the incubation condition of adding PEI is 37 ℃, shaking table and more than 2 h;
magnetic bead preparation method the centrifugation condition in step (2) is 1000g/min for 3min.
6. A method of capturing circulating tumor cells for non-diagnostic purposes according to claim 1, wherein:
the step (3) of the magnetic bead preparation method is repeated for 5 times, namely the quantum dot Fe 3 O 4 The number of layers of (2) is 6;
in the step (3) of the magnetic bead preparation method, the molecular weight of PEG is 3500, and the concentration of PEG aqueous solution is 10%;
and (4) finally storing the water-soluble magnetic beads in the step (3) in 1 xPBS.
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| CN111893095A (en) * | 2020-08-05 | 2020-11-06 | 清华大学 | Magnetic bead for capturing circulating tumor cells and preparation method thereof |
| CN117607441B (en) * | 2024-01-23 | 2024-04-12 | 杭州华得森生物技术有限公司 | Circulating tumor cell TROP-2 immune chromogenic detection reagent |
Citations (4)
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| CN103630440A (en) * | 2013-11-28 | 2014-03-12 | 武汉大学 | Enriching method of circulating tumor cells |
| KR20140098334A (en) * | 2013-01-31 | 2014-08-08 | 연세대학교 산학협력단 | Apparatus and method to capture CTCs and detect biomarker |
| CN105807057A (en) * | 2016-03-11 | 2016-07-27 | 武汉大学 | Method for synchronously capturing and identifying circulating tumor cells |
| CN109100511A (en) * | 2018-07-20 | 2018-12-28 | 四川大学 | Capture and the immune magnetic nano particle of release and preparation method thereof are visualized for circulating tumor cell |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| KR20140098334A (en) * | 2013-01-31 | 2014-08-08 | 연세대학교 산학협력단 | Apparatus and method to capture CTCs and detect biomarker |
| CN103630440A (en) * | 2013-11-28 | 2014-03-12 | 武汉大学 | Enriching method of circulating tumor cells |
| CN105807057A (en) * | 2016-03-11 | 2016-07-27 | 武汉大学 | Method for synchronously capturing and identifying circulating tumor cells |
| CN109100511A (en) * | 2018-07-20 | 2018-12-28 | 四川大学 | Capture and the immune magnetic nano particle of release and preparation method thereof are visualized for circulating tumor cell |
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