Magnetically Sculpted Microfluidics for Continuous-Flow Fractionation of Cell Populations by EpCAM Expression Level

Continuous-flow separation of magnetically labeled cells according to surface-marker expression levels is increasingly needed to study phenotypic heterogeneity and support downstream assays. Here, we present a microfluidic platform that uses spatially engineered soft magnetic strips (SMS) to sculpt lateral magnetic deflection fields for quantitative, label-guided cell fractionation. Under a uniform bias field, the SMS generates controllable magnetic gradients within the microchannel, producing distinct lateral velocities among EpCAM-labeled tumor cells that carry different Dynabead loads, which indirectly report membrane protein expression. Multi-outlet collection converts these "race-based" trajectory differences into discrete expression-level-resolved fractions. A COMSOL-MATLAB framework and a force-equivalent metric |(H·∇)H| are used to optimize key structural parameters of the magnetic interface, including strip thickness, width, and vertical spacing from the flow channel. Three journey nodes at 1.5, 3, and 9 mm along the flow path define a three-stage cascade that partitions MDA-MB-231, Caco-2, and A549 cells into four EpCAM-related magnetic subgroups: high (H), medium (M), low (L), and near-negative (N). Experiments show that the sorted fractions follow the expected expression trends reported in the literature, while maintaining high cell recovery (>90%) and viability retention of 98.2 ± 1.3%, indicating compatibility with downstream whole-blood assays and culture. Rather than introducing a new biomarker, this work establishes a quantitative magnetic-field design strategy for continuous microfluidic sorting, in which the spatial configuration of soft magnetic elements is exploited to implement expression-level-dependent fractionation in next-generation magneto-fluidic separation systems.