Advanced nucleic acid detection with solid-state nanopores: from fundamental understanding to cutting-edge platforms

Solid-state glass nanopore sensing has emerged as a powerful technique for single molecule detection, due to its operational simplicity, high sensitivity, and real-time analytical capability. Despite these advantages, significant challenges remain in achieving multiplexed detection of diverse targets and in enabling precise molecular characterization at the single nucleotide level. This thesis presents advancements in glass nanopore sensing for both DNA and RNA analysis by leveraging nucleic acid self-assembly, all without requiring enzymatic amplification or fluorescence labelling. To establish a fundamental understanding of signal generation mechanisms, I first conducted a systematic investigation into the relationship between nanopore current signals and molecular characteristics using M13mp18 DNA carriers. By precisely controlling parameters such as analyte DNA length, strand duplications, sequence composition, unpaired nucleotides, and structural rigidity, I uncovered key correlations between signal features and molecular properties, including molecular volume, topology, and conformational flexibility. This study further demonstrated the ability to discriminate between circular and linear DNA molecules of identical lengths, underscoring the potential of carrier-based nanopore sensing for advanced molecular discrimination. These insights provide essential guidance for the rational design of high resolution, structure-sensitive nanopore detection methods. Building upon this foundation, I developed two innovative nucleic acid sensing platforms that address critical needs in pathogen diagnostics and disease-related genetic analysis. The first platform introduces a poly(dT) enhancement strategy that enables simultaneous detection of up to 81 different viruses and bacteria on 27 barcoded M13mp18 carriers in a single assay. This multiplex approach significantly improves detection efficiency compared to traditional single target methods while maintaining high sensitivity and specificity. Moreover, the system accommodates a wide range of RNA lengths from short regulatory RNAs to fragmented long genomic sequences, overcoming a long-standing limitation of existing detection technologies. The second platform, RNA Single-nucleotide Characterization and Analysis Nanolatch (RNA-SCAN), enables high-resolution detection of base mutations and chemical modifications in long RNA molecules. Using viral and bacterial templates, including MS2 RNA and 16S rRNA from E. coli, Salmonella, and A. baumannii, I demonstrated the platform’s capability to identify single-nucleotide variations and modification patterns within complex biological matrices. These results highlight RNA-SCAN as a powerful tool for advancing RNA biology research and RNA-related disease diagnostic applications. These technological advances establish carrier-based glass nanopore sensing as a versatile platform combining multiplexed detection capability with single-nucleotide resolution for nucleic acid analysis. The developed methods not only contribute to our understanding of nanopore-based single-molecule detection, but also show great potential for applications in clinical diagnostics, pathogen surveillance, and fundamental research in molecular biology, offering practical solutions to critical challenges in biological and medical sciences. Looking ahead, future development of these technologies may enable rapid, cost-effective, and comprehensive analysis of complex biological samples in both research and clinical settings.

Name dc:type.qualificationname

Doctor of Philosophy (PhD)

Level dc:type.qualificationlevel

Doctoral

Grantor dc:publisher.institution

University of Cambridge

Year dc:date.issued

2025

Author dc:creator

• Li, Yunxuan

Advisor dc:contributor.advisor

• Keyser, Ulrich

• DNA Nanotechnology
• Nanopore Sensing
• RNA Nanotechnology

Licence

• https://www.repository.cam.ac.uk/bitstreams/0c4e3a79-8410-4937-8389-6b189b889ce6/download
• http://purl.org/NET/rdflicense/allrightsreserved

Language dc:language

eng

Author Identifier

0009-0005-8362-5173

OAI identifier oai:identifier

oai:www.repository.cam.ac.uk:1810/393093