Site-Specific Antibody Modification toward Development Of 2D Immunomagnetic Nano-Net for Isolation of Circulating Tumor Cells in Whole Blood

We focus on site-specifically-modified biotin ligands through N-glycan on Fc domain of anti-EpCAM in improving binding ability for circulating tumor cells (CTCs) through microfluidics. Eight types of biotinylated anti-EpCAMs were synthesized and evaluated for their antigen binding ability by ELISA and QCM assay. One site-specifically-modified anti-EpCAM Ab-2G was further applied in clinical samples in which was shown 1.6 and 2.8 times higher CTCs and CTMs capture abiliby than random modified one. Besides, an immunomagnetic “nano-net” was designed and synthesized for specifically capturing rare cells of interest from mixtures. The nano-net, Ab@Lipo-MNP-GO, consists of conjugated antibody Ab-2G molecules on a lipid coated magnetic nanoparticle-graphene oxide sheet complex. The magnetism, chemical composition, and morphology of the construct and its precursors were characterized by SQUID, FTIR, TGA, DLS and SEM, respectively, to confirm the feasibility of the synthetic steps and the resulting properties suitable for solution phase immuno-recognition for cell capture. When applied to capturing CTCs in clinical patients’ blood samples, the nano-net construct exhibits far superior ability whereas conventional immunomagnetic beads in some cases were unable to capture any CTCs, even by increasing the beads concentration. Confocal images showed that the nano-net wrapped around CTCs while immunomagnetic beads attached them with point contacts. A stable, patch-like multivalent matrix nano-net was demonstrated to tackle the shortcomings of single point contact of immunomagnetic beads to the target cell. This strategy is universal for any cell separation in complex fluids.