Multiplexed Mechanochemistry Assay - A Tool For Multiplexed Single Molecule Bond Rupture Force Studies

Studies in mechanochemistry have greatly benefited from advances in single molecule force spectroscopy (SMFS) techniques. Despite success, conventional SMFS techniques are inherently low throughput. For example, Atomic Force Microscopy (AFM) and optical tweezers are serial techniques. In recent years, considerable effort has been made to develop massively parallel force spectroscopy methods such as the centrifugal force microscope (CFM), which applies centrifugal force to tethered beads. The capability to apply centrifugal force on a large number of beads opens new multiplexing possibilities. Here, we report the development of a novel technique — Multiplexed Mechanochemistry Assay (MMA) — enabling large-scale study of single molecule chemical bond rupture events. Briefly, in a bead surface assay configuration, we designed a centrifugal system for the application of pre-calibrated forces on up to 320 samples simultaneously. Our system enables two-dimensional multiplexing, (1) number of beads being studied simultaneously, and (2) number of experimental conditions being probed. The force range afforded by our equipment ranges from a fraction of a pN to ∼30 nN. This unprecedented level of ultraplexing afforded by our approach, hundreds of experimental conditions tested against orders of magnitude variation in force, provides a dramatic improvement in throughput of individual experiments over conventional SMFS techniques. As system validation, we report a case study on studying bond rupture of covalent bonds. We believe this simple tool will greatly increase the rate of progress in probing the relation between force-lifetime and chemistry in single molecular bonds.