On Determination of Force on Bead-Surface Linker Proteins-Comment on a Recent Paper on Small.

In a 2023 Small paper, the authors introduced a microfluidics-based high-throughput force spectroscopy assay to probe protein mechanics by measuring bead displacement under shear flow. Their analysis, however, appears to contain three issues that may have affected the accuracy of the results: (1) setting up the basic force balance equations incorrectly, (2) not considering the torque exerted on the bead by the hydrodynamic forces and not balancing the moments, and (3) assuming the linker anchoring the bead aligns with the bead's radius. Here, we demonstrate that these mistakes lead to erroneous force estimations on the bond. Using established formulations from Goldman, Tissot, Pierres, and others, we present the correct governing equations that balance both forces and moments. Applying this corrected model to the authors' data shows that the forces experienced by single, double, and triple Biotin-NeutrAvidin bonds are 3.17-, 5.54-, and 29.30-fold greater, respectively, than those reported by the authors, while the calculated bond lengths remain consistent. These findings highlight the importance of correctly setting up the basic force balance and incorporating torque in the moment balance in the calculation of force in the microfluidics-based force spectroscopy.