NOTE · Single molecule characterisation of biological nanopores in action
Horizon 2020 — Marie Skłodowska-Curie Actions
- Duration
- 2021-10-01 → 2023-09-30
- EU contribution
- €175,572
- Participants
- 1
- Scheme
- MSCA-IF
Lines connect the coordinator with its partners.
Results in brief
Single molecule characterisation of biological nanopores in action
Biological nanopores are simply nanometre-scale diameter holes, created by a transmembrane protein puncturing a cellular membrane. There are numerous examples of pore-forming proteins in nature, many originating from pore-forming toxins or pore-forming proteins found on the outer membrane of Gram-negative bacteria, which enable passive transport across this membrane. These nanopores can be used to perform electrical measurements of ion flows, i.e., for characterising the translocation of molecules. In such measurements, two key parameters are typically characterised: the ionic current and how it changes over time. For example, a larger molecule entering the nanopore lowers the ionic current for a long time, whereas a smaller molecule passing through the pore creates only a short dip in the ionic current. Further, the level of ionic current passing through a nanopore also differs between the type and conformational state of molecule inside it. This is why nanopores are extensively used for DNA sequencing and engineered for sensing analytes from bodily fluids. While the promise of biological nanopores as innovative and fast analytical tools in biotechnology and biophysics is clear, many open questions relating to their exact physico-chemical mechanisms still remain. Specifically, while we know that it is the electro-osmotic flow, not directly the applied electric field, that forces substrates to be pulled through biological nanopores, the magnitudes of the forces acting inside nanopores and how these differ between peptides and proteins with different amino acid compositions and particularly surface charges are not known. In addition to unravelling the fundamental principles, advanced physico-chemical insights into the translocation are of utmost importance for the development of biological nanopore-based biosensors. Therefore, we need to be able to understand at the molecular level which forces apply when a biological sample such as sweat or blood is injected into the array of nanopores; however, this is not possible using existing experimental measurement techniques. The overall objectives of the research are twofold, i.e., to create devices that enable (i) the stable insertion of biological nanopores into a membrane for extended periods of time and (ii) the measurement of forces exerted by biological nanopores on different substrates. The work will greatly contribute to the use of biological nanopores in miniaturised and wearable biosensors, for instance for i) direct detection of analytes from patient’s sweat, urine or blood samples, ii) the development of portable personalised health monitors, e.g. to detect glucose levels.
Data: CORDIS, © European Union
Project objective
Biological nanopores are nanometre-scale holes in membranes created by transmembrane proteins. In nature, nanopores come from pore-forming toxins or exist as transporters in bacteria. To date, they have been successfully used for an impressive variety of applications ranging from DNA sequencing through to the measurement of function-related motions of enzymes. Planned developments using nanopores include their integration into biosensors for the detection of analytes from blood. However, to reach their potential, miniaturisation and parallelisation of electrophysiology setups and an in-depth understanding of how nanopores exert forces on their substrates during trapping and translocation are required. There are currently no experimental techniques which provide access to this information. Single molecule studies using the optical tweezers (OT) have, over the past decade, become the gold standard in high resolution measurements of forces in biological systems, including observations of sub-nanometre enzyme kinetics, protein folding and protein degradation machinery in action. I propose the development of low-cost, microfluidic-based tools for the study of biological nanopores using OT. The technologies arising from this work will provide an unprecedented insight into the function of biological nanopores and will enable studies of any other transmembrane protein system, many of which are also of great medical importance. The findings will also lay the groundwork for peptide-sequencing and biosensor technologies for personalised medicine. The combination of my skills in single molecule biophysics and Prof. Maglia’s expertise at the forefront of biological nanopore technologies is ideal for the successful completion of the research objectives. Through the planned interdisciplinary work and high-quality training, I will increase both my research- and transferable skill sets enabling me to reach my goal of establishing a leading independent research group within the EU.
Original text from CORDIS.
Participants
- RIJKSUNIVERSITEIT GRONINGEN · GroningenCoordinatorNetherlands
Links
Data: CORDIS, © European Union