Engineering nanopore sensors for proteomic and metabolomic analysis
|PhD ceremony:||Mr F.L.R. (Florian) Lucas|
|When:||January 18, 2022|
|Supervisor:||G. (Giovanni) Maglia, Prof Dr PhD|
|Co-supervisor:||dr. C. Wloka|
|Where:||Academy building RUG|
|Faculty:||Science and Engineering|
This thesis describes experimental and computational analysis for the creation of biological nanopore-based single-molecule sensors. In essence, nanopore sensors consist of a non-conductive sheet with a nanometre-sized aperture connecting two electrolyte filled compartments. The conductance across this aperture is reduced due to ion exclusion whenever a molecule translocates through it, which can be observed as a reduction in current when a potential is applied.
Here, we engineered membrane-spanning proteins for the creation of these nanometre-sized apertures in order to observe and characterize peptides. By engineering the constriction inside the nanopore to interact with translocating peptides, we were able to increase the residence time of the analyte inside the nanopore. By characterizing the current fluctuations over time, we were able to fingerprint trypsin digested proteins based on their translocating peptides.
In addition, we were able to trap substrate-binding proteins inside a nanopore, which undergo a change in their ion exclusion when bound to an analyte. We sought to rapidly detect vitamin B1 in human urine, using a single nanopore in combination with a binding protein. We combined a physical understanding of the system with an empirical Bayesian approach, allowing the detection of thiamine in approximately 20 seconds.