Single-molecule force spectroscopy (SMFS) measurements of protein/DNA co-condensates can identify biophysical mechanisms, such as active force generation or generalized conformational maps. High-throughput is key to fully characterize large phase spaces of condensate formation regimes. Recent flow-based methods have achieved 1,000+ molecule throughput, but high required sample volumes and close surface proximity limit usage for large, “sticky” condensate systems and hard to purify disordered proteins. We have developed a flow-free, surface interaction-free, high-throughput single-molecule force spectroscopy system, herein magnetic arrayed skyscraper tweezers (MAST). MAST combines a 2.5D biopatterning approach with custom magnetic architecture to apply pN-scale forces to tens of thousands of single-molecule systems simultaneously. Using electron beam lithography, chromium lift-off, and reactive ion etching, we create over 10 5 3 μm tall and 0.25–5 μm wide pillars in fused silica coverslips at 1+ μm pitch and high anisotropy over a >1 cm 2 area, validated using scanning electron microscopy, optical profilometry, and bright field imaging. We then coat pillar tops in streptavidin using microcontact printing, passivate the remaining silica surface, and add end-modified DNA to individually bind to pillar tops and superparamagnetic beads. With finite element analysis (COMSOL), we designed an asymmetric permanent magnet architecture to sufficiently achieve the background magnetic field strength and gradient required to controllably apply up to 40 pN of force laterally over the 1 cm 2 working area. MAST can apply forces to protein/DNA systems 3 microns from any device surface without flow, reducing surface interactions and limiting required protein volume to tens of microliters. We anticipate that the MAST platform will support the conformational mapping and force-spectroscopy analysis of protein/DNA systems, particularly for disordered, condensate forming proteins. We will continue to benchmark platform performance against previously performed high-resolution optical tweezer measurements on oncoprotein/DNA co-condensates.
Ghosh et al. (Sun,) studied this question.