Heteromultivalency Enhances Resolution in Cellular Mechanical Sorting

Cells sense their microenvironment and neighboring cells by exerting finely tuned mechanical forces through receptor–ligand interactions, which shape mechanophenotypes critical to biological development, immunity, and disease. Profiling these mechanophenotypes requires ligands with variable avidities. However, conventional strategies based on discrete valency changes lack the precision for continuous modulation, limiting the resolution of subtle mechanotypic differences. Here, we introduce a bivariate strategy to construct heteromultivalent ligands that independently modulate both unit affinity and valency within a single DNA nanoscaffold. By co‐assembling two aptamers with distinct binding affinities, each targeting different epitopes of the same receptor, we generate ligand architectures with tunable avidities through precise stoichiometric modulation. This strategy produces a continuous avidity spectrum, overcoming the limitations of traditional homovalent systems, which are constrained by the discrete nature of valency adjustments. These heteromultivalent ligands enhance the sensitivity of force measurements, revealing subtle mechanical differences in cells with varying epithelial cell adhesion molecule (EpCAM) expressions, and enabling efficient, sequential sorting of phenotypically similar subpopulations via simple flow rate adjustments. These findings underscore the critical importance of avidity modulation in multivalent interaction engineering and mechanical cell profiling, showcasing the advantages of molecularly assembled heteromultivalent interactions over conventional homovalent strategies.