In Situ Kinetics of Solution-Phase Biomolecular Reactions and Interactions through Single-Molecule Displacement Statistics

We report a framework for quantifying the in situ reaction kinetics of bio(macro)molecules in the solution phase through single-molecule displacement statistics. Using single-molecule displacement/diffusivity mapping (SMdM), we measure the transient (0.6 ms) displacements of freely diffusing molecules in the wide field at ultrahigh throughput. Fitting the time-dependent displacement distributions of many molecules to a two-component diffusion model quantifies the reactant and product fractions based on molecular-weight differences, thereby enabling kinetic analysis of reaction progress at ∼1 s temporal resolution. Working with ∼100 pM fluorescently tagged reactant while varying the concentration of a second, unlabeled reactant over wide ranges, we determine second-order reaction rate constants for strain-promoted azide–alkyne cycloaddition and N-hydroxysuccinimide ester aminolysis as well as first-order hydrolysis side-reaction rate constants for the latter. For antibody binding kinetics, we quantify association and dissociation rate constants for a monoclonal antibody and unveil the gradual formation of cross-linked complexes/aggregates for a polyclonal antibody. Working with microliter-scale samples, our single-molecule-displacement-based approach provides intuitive readouts linked to the molecular size and is applicable to diverse reaction types and conditions.