Continuous, Label-Free Phenotyping of Single Cells Based on Antibody Interaction Profiling in Microfluidic Channels

Flow cytometry commonly utilizes fluorescence labeling and extensive sample preparation to detect specific cell surface markers, making analysis under native cell conditions impractical. In this work, a label-free flow cytometry technique is presented that spatiotemporally resolves cell-surface interactions in antibody-coated microfluidic channels. Using computational imaging, numerous cells are tracked across a large field of view (12 × 3 mm²) and the resulting motion profiles are used for phenotypic cell characterization. As proof-of-principle, experiments targeting T-cell receptor CD8 are performed directly on cell cultures. Individual T-cells are successfully tracked in 98% cases for flow velocities of 1-3 mm·s^-1. In 14 μm high channels coated with only nonspecific antibodies, both CD8-positive SUP-T1 and CD8-negative Jurkat cells exhibit mostly constant velocities. In contrast, using channels functionalized with CD8-specific antibodies, numerous CD8-positive cells but not CD8-negative cells show temporary delays in motion linked to surface interaction. Cell classification based on the observed interactions results in a clear contrast ratio of 23.9 ± 11.6 (mean ± standard deviation) between SUP-T1 and Jurkat cells at 1 mm·s^-1. The contrast decreases at higher flow velocities as fewer cells interact due to the increased hydrodynamic lift. Our results affirm our method's ability to differentiate cells without prior labeling or sample preparation.