Nanopores have displayed immense potential as an analytical platform for label-free single-molecule biosensing, particularly evident in their successful application for direct long-read DNA sequencing. However, the analysis of proteins using nanopores presents unique challenges due to inherent limitations. This thesis aims to address these challenges and advance the performance of protein nanopore sensing.
The thesis commences by exploring the potential of nanopipettes in studying proteins and their interactions, with a specific focus on actin, a crucial protein in eukaryotic cells. Nanopipettes offer insights into the ATP-activated filamentation, urea-induced unfolding, and drug-induced structural alteration. However, direct analysis using nanopipettes still faces limitations in terms of spatiotemporal resolution and selectivity.
In the subsequent part of the thesis, molecular carriers including DNA and peptides are explored as powerful tools to enhance protein analysis through nanopores. To overcome the issue of carrier folding, a novel strategy of protein-binding-induced carrier dimerisation is developed, enabling precise detection of target proteins.
The use of cationic supercharged unstructured polypeptide (SUP) carriers proves highly beneficial in enhancing protein capture and detection, leading to significant improvements in nanopore measurements. The electrostatic attraction between the SUP carrier and the nanopore not only increases protein capture but also decelerates protein translocation through the pore. This approach successfully realises the size identification of small proteins and precise recognition of protein-protein interactions at the single-molecule level.
In summary, the integration of nanopipettes and molecular carriers show great promise for advancing single-molecule protein analysis. By overcoming the inherent limitations of protein nanopore sensing, these advancements have the potential to significantly impact the field of proteomics and contribute to precise early-stage diagnostics and therapeutics.