Fabrication and Characterization of Glass Nanopores.

Nanopore technology has emerged as a transformative tool for single-molecule detection, enabling label-free, real-time analysis of nucleic acids, proteins, and other biomolecules. Among various nanopore platforms, glass nanopores fabricated by laser-assisted pulling offer distinct advantages, including exceptionally low electrical noise, excellent chemical stability, and rapid, cost-effective production. However, achieving reproducible nanopore fabrication with precise diameter control remains challenging, often limiting their widespread adoption. This chapter presents a detailed protocol for fabricating high-quality glass nanopores with diameters ranging from approximately 4 nm to 14 nm, suitable for diverse single-molecule detection applications. By following this standardized workflow, researchers can reproducibly generate size-controlled glass nanopores and achieve enhanced success rates in biomolecule detection experiments. This protocol is designed to be accessible to laboratories without specialized nanofabrication facilities, democratizing access to nanopore technology for the broader research community.