Materials Today Advances (Dec 2021)
Detection of a single circulating tumor cell using a genetically engineered antibody-like phage nanofiber probe
Abstract
Detection of circulating tumor cells (CTCs), at the single-cell level, can be of great value for subsequent treatment of tumors but remains a significant challenge. Herein, we report a phage-based detection strategy for the identification of single CTC. The key assay component is the flexible recombinant M13@anti-CEA-scFv probe (M13, CEA, and scFv stand for M13 phage, carcinoembryonic antigen, and single-chain variable region fragment, respectively), which has a high, monoclonal antibody-like binding affinity for tumor cell-expressed CEA antigen, as confirmed by enzyme-linked immunosorbent assay (ELISA). Cell immunofluorescence experiments by confocal microscopy also demonstrate that the M13@anti-CEA-scFv could specifically bind to CEA-positive colon cancer cells such as Caco-2 and HT29, instead of CEA-negative HEK293T cells even when in great excess. Quantitative polymerase chain reaction (qPCR) further proves the ability to pin down single Caco-2 cell in 1 mL of spiked samples by 1,000 copies of M13@anti-CEA-scFv probe, after merely 15 min incubation. Another probe, the rigid magnetic microparticle (MMP), loaded with anti-CEA antibody, is used for the initial screening of the single CTC, which is mixed with numerous lymphocytes. The CEA on the resultant MMPs/CTC complex is subsequently recognized by numerous copies of M13@anti-CEA-scFv to form an MMP/CTC/M13@anti-CEA-scFv sandwich complex. Probing the phage DNA in this complex by qPCR leads to the unambiguous identification of the single Caco-2 cell in 1 mL of spiked blood samples. The phage-based detection strategy holds promise for the single-cell detection of any CTCs since the scFv displayed at the end of phage can be customized to the unique antigen of a specific CTC.
