Robust Fabrication Methods for Solid-state Nanopores

Abstract

Solid-state nanopores represent a versatile and robust technology for high-precision single-molecule detection, offering distinct advantages over biological nanopores in terms of durability, size control, and integration with microfabrication techniques. This research investigates the reliable fabrication of solid-state nanopores for single-molecule detection, particularly in the fields of genomics, proteomics, and molecular diagnostics. These nanometer-scale channels in thin membranes detect biomolecules by monitoring disruptions in ionic current as they pass through. Although biological nanopores have proven effective for DNA sequencing, the limited structural stability of their membranes, limited pore sizes, and sensitivity to environmental conditions restrict broader applications. In contrast, solid-state nanopores offer enhanced customization, durability, and a longer operational lifespan. However, challenges persist in maintaining stable membranes, addressing higher electrical noise levels during measurements, reducing fabrication costs, and meeting the operating bandwidth requirements of existing measurement systems. This study addresses these challenges by exploring and developing robust protocols for fabricating nanopores optimized for improved performance and analysis. The study begins with a fabrication process that employs Transmission Electron Microscopy (TEM) to create nanopores in silicon nitride membranes, offering both drilling precision and real-time monitoring. This approach tackles significant fabrication challenges, including membrane suspension, prevention of pore expansion, and optimization of parameters for reproducible and stable pore formation. A comprehensive analysis of failure modes guides necessary protocol adjustments, thereby improving the reliability and yield of the process. Furthermore, the research explores Controlled Dielectric Breakdown (CBD) as a cost-effective alternative for nanopore fabrication using TEM. We evaluate CBD's effectiveness in terms of pore formation times and structural stability, along with a detailed analysis of software control parameters that influence the reproducibility and accuracy of nanopores. Additionally, we present statistical data on nanopore device performance, providing insights into CBD's potential to streamline nanopore manufacturing. Despite its advantages, CBD has several drawbacks, including issues with membrane structural stability and stochastic variability in the location of the nanopore formed due to factors involved in the breakdown process. These issues can impact the reproducibility and reliability of nanopores in biomolecule sensing applications. To address these limitations, we introduce a novel AFM-assisted nanoindentation approach that enables precise, localized membrane thinning prior to dielectric breakdown, allowing for controlled nanopore formation. By precisely regulating the indentation force, we achieve targeted pore formation, allowing for fine-tuning of pore location, geometry and stability—a crucial factor in reducing variability in nanopore device performance. Results from AFM nanoindentation indicate significant improvements in pore robustness, leading to more stable ionic current readings and an extended lifespan for the nanopores. Additionally, the study investigates feedback-controlled silicon etching as a novel strategy to explore in-situ fabrication capabilities, thereby eliminating the need for handling or transferring the devices, reducing the risk of membrane breakage, and enhancing device robustness. This method employs real-time feedback mechanisms to dynamically adjust etching parameters, ensuring precise control over nanopore location and dimension. By continuously monitoring the ionic current feedback measurements during the etching process in the fluidic cell for nanopore fabrication, we can respond to variations in the etching environment that often influence the final characteristics of the nanopores. This technique provides a robust framework for robust nanopore fabrication, paving the way for future advancements in nanopore-based biosensors. By refining the methods for nanopore fabrication, this work advances the reliability, affordability, and scalability of solid-state nanopore devices. These advancements enhance the potential of nanopore-based technologies in biomolecule analysis, opening avenues for broader applications in biotechnology, clinical diagnostics, and personalized medicine.