Microfluidic Immunomagnetic Capture of Circulating Tumor Cells via On-Bead Antibody Conjugation

Microfluidic immunomagnetic platforms have been widely explored for circulating tumor cell (CTC) enrichment; however, operational complexity, unstable flow control, and limited real-time observation remain practical challenges for routine implementation. In this study, we developed an integrated microfluidic chip system for EpCAM-based immunomagnetic enrichment of tumor cells using antibody-functionalized magnetic beads. The platform incorporates a gas-liquid separation structure, pressure-regulated flow control, and a transparent chip design that enables real-time microscopic observation of bead-cell interactions during capture. Biotin loading on streptavidin-coated beads was optimized, with surface saturation observed at 6 nmol. Flow-rate optimization identified 0.4 μL/s as the selected flow rate, balancing capture efficiency and cell-bead interaction time. In whole-blood spike-in experiments using MCF-7 cells, the platform achieved recovery efficiencies ranging from approximately 57% to 91%, depending on the input cell number. Cells with Hoechst⁺/CD326⁺/CD45⁻ phenotypes were also observed in patient-derived blood samples, supporting the feasibility of detecting putative CTCs in clinical specimens. This study positions the system as an engineering-integrated, real-time-observable platform for CTC enrichment. Further validation using larger clinical cohorts, multi-marker capture strategies, quantitative purity analysis, and cell viability assays will be required before translational application.