Tri-Layer Solid-State Nanopore Arrays with Crosstalk Suppression for High-Throughput, Femtomolar-Level Biosensing

Solid-state nanopore arrays are emerging as powerful tools for label-free, ultrasensitive biosensing, yet their implementation has been constrained by inter-pore crosstalk and limited fabrication uniformity. A multilayer Al₂O₃/Au/Si₃N₄ nanopore architecture, produced via helium ion beam lithography, is introduced to address these limitations through structural and materials-level innovation. Finite-element analysis identifies a critical inter-pore spacing approximately 20 times the pore radius as necessary to minimize electric field coupling, enabling rational array design. The membrane structure incorporates a dielectric Al₂O₃ layer for electrical isolation and an intermediate gold layer for site-specific aptamer immobilization, confining molecular recognition to the nanopore interior. Arrays with ∼30 nm pores and <5% size variation achieve 300 nm spacing and support statistically independent, parallel signal acquisition. Diverse nanopore arrays with 75 nm pores and 800 nm spacing are utilized for the specific detection of alpha-fetoprotein. Detection of alpha-fetoprotein demonstrates label-free sensing at concentrations down to ∼3 fM across six orders of magnitude in dynamic range. This platform defines a closed-loop pathway from theoretical modeling to scalable fabrication, establishing a foundation for rational design and high-throughput deployment of solid-state nanopore biosensors.