Inertial−Digital Microfluidics Platform for High-Recovery, High-Purity Circulating Tumor Cell Isolation toward Single-Cell Genomic Analysis

Abstract Isolating circulating tumor cells (CTCs) remains challenging owing to their rarity and the abundant background of white blood cells (WBCs). To overcome the traditional trade-off between the recovery rate and purity, we developed an orthogonal decoupling microfluidic platform consisting of two integrated modules. The integrated modules achieved an overall recovery of ∼87.3% and an overall WBCs depletion of 99.97%, reducing background WBCs contamination to a level that permitted direct microscopic visualization and single-cell isolation. Specifically, the first spiral inertial microfluidic module achieved an approximately 90% recovery rate for MCF-7 and HeLa cells (at a flow speed of 3 mL/min) while depleting 99.4% of WBCs. Second, a downstream digital microfluidic module removed >95% of residual WBCs using immunomagnetic depletion and isolated single MCF-7 cells via electrowetting-on-dielectric actuation combined with hydrophilic microstructures. Subsequent whole-genome sequencing of single MCF-7 cells from spiked samples successfully detected expected copy number variations (1q/8q amplifications) and a PIK3CA mutation, confirming the integrated platform’s reliability. In preliminary clinical testing of blood samples obtained from patients with cancer, the platform successfully identified and isolated EpCAM/PanCK-positive and CD45-negative CTCs. Overall, this integrated approach resolves the CTC recovery−purity bottleneck, enabling precision single-cell genomics from spiked samples and providing a robust foundation for clinical translation.