Detection of bacteria-residing circulating tumor cells (CTCs) using DARMR

Red blood cells were lysed twice by two volumes of ACK lysis buffer (C3702-500mL, Beyotime biotechnology) on ice for 5 min. The pellet was resuspended in PBS, and CTC isolation was performed using a size-dictated immunocapture chip (SDI-Chip) as described previously.The SDI-Chip was fabricated using PDMS through soft lithography, with SU8-3050 photoresist (Alfa Chemistry #SU8-3050) for micropillar mold preparation.

Surface modification involved 4% MPTS (3-mercaptopropyl trimethoxysilane; Sigma-Aldrich #175617-100G), 0.01 μmol/mL GMBS (N-γ-maleimidobutyryl-oxysuccinimide ester; Sigma-Aldrich #442630-100MG), and 20 μg/mL biotinylated anti-EpCAM antibody immobilized via streptavidin (Sigma-Aldrich #S4762). Nonspecific binding was blocked with 3% BSA /0.05% Tween 20 in PBS.Samples were introduced into the chip at 1 mL/h using a syringe pump, and captured cells were released with 0.25% trypsin at 37°C for 10 min.