Electroosmotic Flow-Driven Nanopore Translocation of Large, Conformationally Dynamic Proteins: Overcoming Steric and Electrostatic Barriers.

The translocation of large proteins through nanopores smaller than their native dimensions opens opportunities to probe endogenous biological processes, assess protein stability, and enable nanopore-based linearization for protein sequencing. Here, we demonstrate that electroosmotic flow (EOF) drives the passage of megadalton proteins through geometric constrained nanopores. Using glass nanopipettes with tip diameters up to 2.5-fold smaller than the proteins, we show that EOF actively captures, aligns, and deforms macromolecules─including immunoglobulin M (IgM) and α2-macroglobulin (α2M)─facilitating translocation through extreme confinement. Electrostatic potential analysis and quantitative modeling reveal that dipole enhancement and subsequent protein unfolding reduce the energetic barrier to pore entry and enable structural accommodation during transport. By fine-tuning voltage and ionic strength, a critical EOF-electrophoretic balance governing translocation kinetics is identified. Furthermore, voltage reversals change the EOF direction to achieve >70% recapture efficiency, allowing reversible protein transport and ensemble-level interrogation of conformational dynamics. These findings establish an electrohydrodynamic framework for controlled macromolecular passage and expand nanopore sensing capabilities for precision proteomics and structural biology.