Imaging and Calibration of Receptor‐Specific Cellular Forces Using Biotinylated Ligands

ABSTRACT Covalent conjugation of ligand molecules to force sensors provides a powerful approach to image receptor‐transmitted cellular forces. While this strategy is effective for small‐molecule ligands, the conjugation process can damage large protein ligands. To address this challenge, we developed a modular mechano‐fluorescent coating (mMFC) that enables receptor‐specific force imaging using widely available biotinylated ligands. The mMFC construct consists of a poly‐L‐lysine (PLL) backbone for substrate adsorption, polyethylene glycol (PEG) grafts to prevent nonspecific binding, and DNA‐based integrative tension sensors (ITS) as force‐reporting units. Benefiting from copper‐free click chemistry, the mMFC is synthesized via a simple one‐pot reaction. During application, the mMFC is adsorbed onto substrates, and biotinylated ligands are subsequently linked to the mMFC construct via biotin‐avidin interactions, where the ITS reports receptor‐transmitted forces by fluorescence. By testing HeLa cells, platelets, and human T lymphocytes on mMFC platforms, we imaged cellular forces transmitted through RGD peptide, latency‐associated peptide (LAP), ICAM‐1, and anti‐CD3, which target integrins, LFA‐1, and T cell receptor (TCR) complex, respectively. We further demonstrated molecular force calibration and single‐molecule force imaging of these receptors using the mMFC platform. Overall, the mMFC provides a modular, convenient, and robust platform for investigating mechanotransduction across diverse receptors in various cell types.