Direction-Aware Signal Analysis for Protein Translocation in Multilayer Solid-State Nanopores

Abstract

Solid-state nanopores are widely used for single-molecule sensing, yet transport direction is typically treated as a nuisance variable rather than an informative signal dimension. In this work, we investigate directional protein translocation in multilayer solid-state nanopores composed of$\operatorname{SiN}_{x}$, graphene, and a terminal two-dimensional material using a signal-centric analysis framework. Ionic current recordings obtained under forward and reverse bias conditions reveal polarity-dependent restructuring of the translocation signal space that cannot be captured by mean transport metrics alone. Unsupervised Gaussian mixture analysis identifies latent event subpopulations whose occupancies depend on transport direction, while feature-level evaluation using effect-size analysis shows that directionality is strongly voltage and feature-selective. In contrast, symmetric$\text{SiN}_{\mathrm{x}}$nanopores exhibit direction-independent behavior across all features. These results demonstrate that transport polarity can be treated as an additional sensing dimension in solid-state nanopore systems, enabling improved interpretation and operating-point selection without active gating or chemical functionalization.