Beyond the Single Molecule: Multiplexed Methods in Force Spectroscopy

Single-molecule techniques have transformed biological research by enabling direct observation and manipulation of individual molecules. These methods overcome ensemble averaging inherent in bulk measurements and facilitate studies under physiological stresses and out-of-equilibrium conditions. They have provided valuable insights into diverse biological processes, from stepping mechanisms of molecular motors to mechanical properties of biomolecules to the dynamic strength of intermolecular bonds. Advances in multiplexed and high-throughput single-molecule force spectroscopy methods are improving throughput, capabilities, and accessibility. In this review, we detail the evolution of multiplexed force spectroscopy technologies, highlighting key advances in instrumentation, molecular engineering, and analytical techniques. We discuss diverse applications spanning molecular biophysics, biomolecular sensing, proteomics, and cellular mechanobiology. Finally, we explore ongoing challenges and future opportunities and highlight how the impact of multiplexed single-molecule force spectroscopy can continue to grow through further developments in novel instrumentation, chemical tools, and innovative applications.