Probing the Dynamic Strength of Biomolecular Interactions with Single-Cell Centrifugation

Molecular interactions between receptors and ligands govern critical biological processes, from immune surveillance and T-cell activation to tissue development. However, current techniques for studying binding avidity often sacrifice throughput or precision. We introduce a high-throughput method for quantifying molecular and cellular binding kinetics using a centrifuge force microscope (CFM)a compact imaging system integrated into a benchtop centrifuge. The CFM performs real-time force measurements on thousands of single cells in parallel, probing receptor-ligand interactions under controlled mechanical stress. To extend these capabilities, we developed a next-generation CFM with dual-channel fluorescence imaging that enables tracking of individual cell unbinding events. To demonstrate its utility, we profiled the binding mechanics of Bispecific T-cell Engager (BiTE) molecules, immunotherapeutic proteins that facilitate T-cell targeting of cancer cells. In cell-protein assays, we quantified the avidity of T and B cells interacting with BiTE-modified surfaces, revealing receptor-specific correlations between ligand concentration and bond strength. In cell-cell assays, we characterized BiTE-mediated adhesion between Jurkat and Nalm6 cells, demonstrating a time-dependent increase in avidity. By integrating force spectroscopy with fluorescence imaging, the CFM provides a high-throughput approach for investigating the mechanochemical principles underlying receptor-mediated interactions, with broad implications for biophysical chemistry, molecular recognition, and therapeutic development.