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Abstract

Mechanical forces at the molecular scale play critical roles in regulating cellular behavior, yet methods for measuring and applying these forces remain limited. In particular, highly dynamic and fast interactions are a challenge to study. This dissertation addresses these challenges by developing new methodologies for the detection, quantification, and application of molecular forces with high precision and versatility. The research begins with a comprehensive review of molecular force sensors, classifying current designs based on their mechanical properties and fluorescent readout mechanisms. Building on this foundation, an adhesion footprint assay that utilizes irreversible molecular force sensors was developed to quantify the forces experienced by P-selectin:PSGL-1 interactions during cell rolling adhesion – a critical immune process that occurs on blood vessel walls. Complementary statistical modeling of rolling adhesion behavior relates instantaneous rolling velocity distributions to underlying molecular force distributions providing new insights into the mechanical behavior of the rapid adhesion events involved in rolling adhesion. In the latter part of the dissertation, the focus shifts toward tool development for force application. The Flow Force Assay was introduced as a high-throughput single molecule force spectroscopy technique based on hydrodynamic flow-stretching of DNA. By using flow-stretched DNA as a force generator within microfluidic channels, controlled forces were applied to molecular interactions with nanometer and millisecond-scale spatial and temporal resolution, respectively. To enable quantitative force measurements, an empirical model was developed to calibrate the relationship between DNA extension, wall shear stress and piconewton scale forces. Calibration was validated experimentally across multiple DNA constructs, achieving reliable force quantification from 0.5 to 20 pN. Together, the methods developed in this dissertation expand the experimental capabilities for studying mechanical forces at the molecular scale. The combination of irreversible molecular force sensing, flow-based force application, and precise calibration enables the interrogation of molecular adhesion and mechanotransduction processes that were previously inaccessible. Future work will focus on (1) increasing the force capabilities of the Flow Force Assay and (2) using the assay to study a diverse set of biomolecular interactions (e.g., protein-protein, antibody-antigen, protein-DNA). This work contributes to advancing molecular force spectroscopy and provides new tools to probe the mechanical behavior of biomolecular interactions.