Pressure-retarded DNA translocations of nanopores reveal nanoscale stagnant fluid layers

Although nanopore sequencing and sensing have undergone years of commercial development, accurately interpreting current signals, translating them into molecular length, position, and sequence, depends on knowing the translocation speed, which still lacks a physical model capable of predicting it from first principles. Existing models introduce adjustable parameters that can be tuned to match translocation data under a single experimental condition, but none has been tested against an independent constraint capable of distinguishing genuine nanoscale surface physics from curve-fitting. Here, we resolve this ambiguity using pressure-driven counterflow as such an independent test by measuring how the event-charge-deficit distribution of DNA translocations responds to a controlled counterpressure. Our model combining a sub-nanometer stagnant fluid layer (δ ≈ 0.18 nm, consistent across all lengths tested) with coil drag scaled by the radius of gyration reproduces the measured pressure dependence; models omitting either effect require unphysical parameter values and break down as pressure increases. These results establish the stagnant layer as a physically real departure from continuum hydrodynamics rather than a fitting artifact, and yield an analytical expression for the pressure-dependent translocation velocity that offers a route toward more accurate velocity models for nanopore sequencing and sensing.