Capture And Release Of Cancer Cells On The Surface Of Site- Specific Immobilized Nanobody

Cancer seriously threatens human health and is one of the most serious lethal diseases in the world.90%of cancer patients die due to cancer metastasis,and the key to cancer metastasis lies in the circulation and transfer of circulating tumor cells（CTCs）.The detection and capture of CTCs in patients’blood are essential for the diagnosis,prognosis,evaluation and treatment of cancer.The non-destructive release of captured cells and further cultivation and analysis are conducive to personalized treatment of cancer.The immunoaffinity method that relies on antibody to specifically recognize cell surface protein markers is the most commonly used method to capture CTCs.However,using a traditional antibody for high-efficiency isolation of CTCs remains a challenge due to the limited loading capacity of the traditional antibody on material surfaces.Using small-size nanobody,we constructed a reversible site-specific immobilized nanobody surface for the capture and release of CTCs.Coordination interaction between Histidine-tagged nanobody and Ni²⁺ions that chelated to Nitrilotriacetic acid（NTA）-modified poly（2-hydroxyethyl methacrylate）（PHEMA）brushes was used to achieve site-specific immobilization of nanobody,using the competitive effect of imidazole to effectively release nanobody.The tumor marker we selected is epidermal growth factor receptor（EGFR）,which is overexpressed in many tumor cells and is closely related to cell proliferation,differentiation,invasion and metastasis.The research contents of this thesis included:（1）Soluble expression of anti-EGFR nanobody was performed in Shuffle T7.The purity of nanobody after purification by GE nickel column system is over 98%.The cell ELISA results proved that nanobody has binding activity.（2）The NTA is modified on the PHEMA polymer brushes.The coordination between the His-tagged nanobody and Ni²⁺chelated to the NTA achieves site-specific immobilization of the nanobody.The competitive effect of imidazole effectively releases nanobody.The reversible immobilization and release of nanobodies were detected by QCM-D.After calculation,350 ng cm^-2 nanobody was immobilized,and 80%of nanobody was released after the treatment of imidazole solution.（3）The specific interaction of antigen-antibody is used to achieve the capture of cancer cells,and 81%of CTCs were captured within 30 min,showing higher capture rate and shorter capture time compared with that achieved by the conventional antibody immobilized on the flat surface.Besides that,the site-specific immobilized anti-EGFR nanobody surface has a lower capture limit,good cell compatibility and sensitivity.After treatment with imidazole,86%of cells can be effectively released.In summary,using nanobody to specifically capture and effectively release circulating tumor cells in this paper provides a new and promising platform for the capture,release and cultivation of circulating tumor cells to aid fundamental and clinical research.