Geometric Confinement in Solid-State Nanopores Enables Single-Molecule Discrimination of Ferritin Subunits and Translocation Bias.

Resolving intact (24-mer) ferritin from its heavy and light chain subunits at the single-molecule level has remained a fundamental challenge, owing to their near-identical sizes and complex structural heterogeneity. Here, we quantitatively probe ferritin compositional and dynamic heterogeneity using confinement-modulated solid-state nanopores. Under weak confinement (∼20 nm), intact ferritin is discriminated from individual subunits via volumetric scaling of current blockades and a pronounced free-energy penalty governing its capture kinetics. Furthermore, transitioning to strong confinement (∼10 nm) reveals intrinsic, subunit-specific transport dynamics driven by steric-mechanical coupling. Finally, applying semisupervised learning to mixed samples uncovers a strong accessibility-driven translocation bias, demonstrating that nanopore readouts report capture-accessible molecular populations rather than nominal bulk abundance. This establishes a quantitative, physically grounded framework for decoding complex protein assembly states and transient dynamics in heterogeneous environments.