Applications of nanopore sensing and DNA nanotechnology for the analysis of nucleic acids

Nanopore sensing is a versatile technology for biomolecule analysis based on resistive pulse sensing. It includes biological nanopores, primarily used for long-read direct sequencing, and solid-state nanopores, which have been applied to biosensing molecules such as proteins, RNA, and DNA. While solid-state nanopores hold immense promise for sensing applications beyond sequencing, their lack of inherent specificity poses a major challenge. Combining nanopore sensing with DNA nanotechnology enhances the specificity, enabling the detection of biomolecules like proteins and nucleic acids. This thesis explores various applications of nanopore sensing for direct DNA and RNA biosensing, both independently and in combination with DNA nanotechnology. Chapters 1 and 2 introduce the topic and methods. In Chapter 3, I optimised nanopore sensing chips, establishing criteria for biosensing-capable nanopores. Enhancements to chip fabrication and storage significantly reduced the need for frequent chip replacement, allowing for prolonged usability of nanopore chips. Chapter 4 focuses on using nanopore sensing to detect and quantify short DNA oligonucleotides. Leveraging a designed DNA nanostructure, I successfully detected single- and double-stranded DNA oligonucleotides with lengths of up to 65 nucleotides. In Chapter 5, I discuss pathways towards RNA detection for microbial identification and optimising long RNA labelling methods to reduce damage during sample preparation. Using DNA nanotechnology, I present proof-of-concept experiments on ribosomal RNA from bacteria and fungi, applying these methods to blood-spiked samples for bacterial detection. In Chapter 6, I investigate the direct DNA integrity and purity analysis using nanopore sensing. I studied the behaviour of nanopores with a DNA carrier and DNA ladders of varying sizes and developed a protocol for accurate DNA integrity and size estimation in complex samples. The reliability was validated by comparing results with existing integrity analysis tools. This thesis ends with a conclusion and outlook focusing on the potential of nanopore sensing and DNA nanotechnology for diverse applications, including miRNA quantification, oligonucleotide drug analysis, DNA sequencing library preparation, and RNA diagnostics. Finally, I discuss exciting possibilities for biosensing applications.

Name dc:type.qualificationname

Doctor of Philosophy (PhD)

Level dc:type.qualificationlevel

Doctoral

Grantor dc:publisher.institution

University of Cambridge

Year dc:date.issued

2024

Author dc:creator

• Alawami, Mohammed

Advisor dc:contributor.advisor

• Ulrich, Keyser

• Nanopore
• Sensing
• DNA Nanotechnology

Licence

• https://www.repository.cam.ac.uk/bitstreams/44ad11b8-e18a-4265-b7ed-ce2369f0d9d1/download
• http://purl.org/NET/rdflicense/allrightsreserved

Language dc:language

eng

Author Identifier

0000-0003-2741-5436

OAI identifier oai:identifier

oai:www.repository.cam.ac.uk:1810/396936