Zhaowei Guan, Dezhi Feng, Jinbo Zhu
Detection of nucleic acids at the single-molecule level is a cornerstone of next-generation diagnostics. Unmodified glass nanopore-based sensing stands out among single-molecule detection techniques for its simplicity and versatility. This review presents the key advantages of unmodified glass nanopore, including its chemically homogeneous interface, tunable conical geometry, and inherent ion current rectification, which together enable high-fidelity resistive pulse sensing of nucleic acids. We propose a classification framework that differentiates two categories of methods. Amplification-free strategies retain molecular fidelity and conformational dynamics, while amplification-coupled approaches overcome limitations of low target concentrations to achieve ultrasensitive detection. Furthermore, we highlight recent breakthroughs, including standardized encapsulation for long-term stability, automated declogging systems, and the combination of DNA nanotechnology approaches to enhance specificity. By integrating these advancements, this review provides a comprehensive framework for understanding unmodified glass nanopore technology and outlines a strategic roadmap for developing integrated, intelligent, and portable single-molecule analytical tools for precision medicine.