Following the tide – the electroosmotic transport of proteins across nanopores: engineering, biophysics and potential applications

Proteins are essential workhorses within the cell, performing a wide range of functions, ranging from cell division to metabolism. They are composed of chains of building blocks called amino acids, which must fold into specific shapes for the protein to function properly. An imbalance in protein concentration, or a change in the amino acid sequence, can disrupt this folding and function, potentially leading to disease. Because these changes are often subtle, highly sensitive methods are needed to detect and characterise them. Nanopores - nanometre-sized holes in a membrane - hold promise in this area because they can detect changes in single molecules, as they have successfully been employed in DNA sequencing. This thesis addresses a key challenge in protein sequencing: how to transport the unfolded protein across the nanopore. By engineering nanopores to generate a strong driving force, named the electroosmotic flow, the unidirectional transport of proteins was achieved. Next, this mechanism of transport was investigated in terms of 1) the nanopore characteristics required for single-file protein transport and 2) controlling the transport speed. Finally, a proof of concept demonstrated that this approach could detect proteins of interest in complex mixtures, such as bacterial cell contents.