Consecutive Mechano-Immunophenotyping of Single Cells in a Photopatterned Microfluidic Channel

Physical forces act on cells alongside biochemical signals, yet single-cell assays rarely capture both dimensions simultaneously. We present a two-stage microfluidic device that extracts mechanical and molecular phenotypes in one pass. As cells squeeze through dual 6 μm constrictions, their deformability appears as transit time <i>T</i> _<i>D</i> . Immediately downstream, a 350 μm-long hydrogel photopatterned microchannel with antibody transiently captures cells proportionally to the cell surface-antigen density, yielding an additional delay <i>T</i> _<i>A</i> . Using biotinylated anti-CD64 and anti-CD44v9, we profiled five cancer cell lines and uncovered a mechanical-immunochemical correlation up to <i>R</i> ² = 0.41 for HeLa cells and high heterogeneity for HT29 cells. The platform interrogates ∼100 cells min^-1, detects ∼100 membrane proteins μm^-2, and preserves viability for downstream culture or genome sequencing. Because the hydrogel can host multiple ligands, the method enables multiplex, mechanomolecular phenotyping for precision oncology, stem-cell quality control, and mechanotargeted drug screening.