Separation method of micro circulating tumor cells

Abstract

The invention discloses a method for separating and enriching micro circulating tumor cells (circulating tumor cells, CTCs) in whole blood for further providing a basis for subsequent study of tumor cells, and relates to the biomedical field. The method comprises the following steps of: covalent coupling of dendrimers and matters (special matters) specifically combined with target cells; further coating long-chain biotin molecules by dendrimers modified by the specific matters; capturing CTCs in a whole blood sample by dendrimers co-modified by the special matters and the long-chain biotin; identifying and coupling long-chain biotin dendrimers in the whole blood by streptavidin modified nanomagnetic beads; and separating and resuspending the captured CTCs. The resuspension can be directly used for subsequent analysis. Compared with conventional cell separation methods, the method is more suitable for magnetic separation of CTCs in complex whole blood samples, so that the magnetic separation time is shortened, and the CTCs separation efficiency in the whole blood sample is improved.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a method for separating circulating tumor cells based on nano magnetic beads. Background technique [0002] Circulating tumor cells (CTCs) refer to tumor cells that enter the peripheral blood of the human body. Foreign studies have found that the appearance of CTCs in the peripheral blood of tumor patients is often earlier than that of visible solid tumors. Therefore, the detection of CTCs has been widely used in the early diagnosis of tumor patients in vitro. However, the number of CTCs in the peripheral blood of cancer patients is very rare in the early stage, and the existing methods cannot directly detect trace CTCs in peripheral blood. Therefore, realizing the high-efficiency enrichment of CTCs in human peripheral blood to meet the sensitivity requirements of the detection method is not only for the early detection of tumors, but also for the rapid evaluation of chemotherapy d...

AI Technical Summary

Problems solved by technology

However, the current separation technology based on micron-scale immunomagnetic beads has many limitations: 1) The specific surface area of ​​micron-sized magnetic beads is relatively small, which reduces the capture efficiency of magnetic beads; The combination of cells through a multiphase reaction usually takes longer to specifically capture cells in the whole blood matrix; 3) Micron magnetic beads have poor monodispersity and are prone to self-aggregation in the whole blood matrix Or form a precipitate; 4) The traditional immunomagnetic separation technology often directly couples specific substances to immunomagnetic beads. Changes occur, thereby increasing the steric hindrance effect between specific substances and reducing the capture efficiency of specific substances; 5) The blood viscosity is high and the blood cell concentration of non-CTCs is large, and micron magnetic beads are prone to non-specific adsorption, which is difficult Realize the specific separation of CTCs in blood; 6) Excessive concentration of micron magnetic beads will cause damage to CTCs (the magnetic field causes the magnetic beads on the cell surface to attract each other, causing the cells to be squeezed or even ruptured), resulting in failure of separation; (7) ) When coupling antibodies to magnetic beads, hydrophobic adsorption or chemical coupling is generally used to couple active antibodies to the surface of magnetic beads

The distance between the antibody and the surface of the magnetic bead is too close, the nature of the magnetic bead itself and the residual hydrophobic or strong hydrophilic groups on the surface are likely to cause changes in the spatial conformation of the antibody, resulting in a decrease in the biological activity of the antibody

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