Improving CTC isolation by controlling orbit engagement in inertial microfluidics.

Inertial microfluidics provides a label-free strategy for isolating circulating tumor cells (CTCs) from blood by exploiting size-dependent lateral migration in microchannels. However, straight-channel devices are often optimized using the final equilibrium position, even though target-cell loss can arise from incomplete or poorly reproducible migration before outlet collection. Here, we show that heteroclinic orbit engagement provides a predictive design criterion for improving CTC scale separation in a rectangular co-flow inertial microchannel. A computational model predicted size-dependent heteroclinic orbits and lateral migration velocities, which were experimentally validated using beads under controlled inlet flow-split conditions. Increasing buffer confinement shifted the sample-buffer interface closer to the sidewall, promoted earlier orbit engagement, improved agreement between measured and simulated velocity profiles, and reduced upstream trajectory variability. Larger CTC scale particles engaged their orbits earlier and migrated more reproducibly, while smaller white blood cell (WBC) scale particles remained laterally displaced from the larger particle focusing region. Biological validation with PANC1 and A549 cells showed that the 1 : 6 : 1 flow-split configuration increased separation efficiency to 99.9 ± 0.2% for PANC1 and maintained high separation efficiency of 99.2 ± 0.9% for A549 cells, although its effect on total recovery was cell line dependent. In an intraductal papillary mucinous neoplasm (IPMN) patient sample, representing a premalignant pancreatic lesion that may progress to invasive pancreatic ductal adenocarcinoma (PDAC), CTC target outlet partitioning was 67% under 1 : 1 : 1 and 100% under 1 : 6 : 1, within the limits of the detected cell numbers. These results establish orbit engagement as a mechanistic design principle for improving clinically relevant CTC enrichment without changing device geometry, total flow rate, or labeling strategy.