Nanopore‐Based Label‐Free and Single‐Molecule Sequencing toward Precision Diagnosis

Abstract Nanopore‐based single‐molecule sequencing (SMS) is a powerful tool for acquiring detailed heterogeneous information on critical building blocks of life, such as nucleic acids, proteins, and a wide array of biomolecules, at the single‐molecule level. Real‐time current‐intensity fluctuations corresponding to the passage of target molecules through nanopores enable long‐read length, high‐throughput, and high‐accuracy detection, thus meeting the stringent demands of precision diagnosis. Herein, a concise overview of various principles and fabrication methods is provided for nanopores, with particular emphasis on recent advancements in nanopore‐based DNA and protein sequencing, revealing innovative approaches for effectively capturing and translocating target molecules, and rapid and accurate identification. Further typical cases of nanopore‐based SMS applications in precision diagnosis are analyzed, focusing on genetic disorders, infectious diseases, cancers, and abnormal post‐translational modifications to highlight the clinical potential of nanopores. Additionally, the inherent limitations and challenges of nanopores in terms of sensitivity, detection range, and selectivity are discussed and present the latest strategies for enhancing nanopore performance. Finally, perspectives are provided on the future of nanopore‐based SMS, particularly at the intersection of microfluidic devices, surface functionalization, and machine learning, to facilitate more diverse and advanced developments in this field.