Periodic Reporting for period 1 - NOTE (Single molecule characterisation of biological nanopores in action)
Reporting period: 2021-10-01 to 2023-09-30
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.
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.
The first stage of the work involved the prototyping of different designs of microfluidic chips for the formation of stable free-standing lipid bilayers. While the designs were found to work well (main result 1), with single nanopore insertion events observed via electrophysiological measurements, the stability of the bilayers was not sufficient for the length of the planned optical tweezers measurements.
The second stage of the work involved the development of more stable membranes for nanopore insertion and measurement. This took us in the direction of testing hybrid polymer-lipid membranes and performing a systematic characterisation of how these supported insertion of different biological nanopores and were stable against biological fluids, making them suitable for biosensor applications (main result 2, manuscript currently being finalised).
The final stage of the work involves the combination of the microfluidic chips with the stable membranes to enable characterisation using optical tweezers – this is still being finalised and will constitute main result 3 of the project.
The second stage of the work involved the development of more stable membranes for nanopore insertion and measurement. This took us in the direction of testing hybrid polymer-lipid membranes and performing a systematic characterisation of how these supported insertion of different biological nanopores and were stable against biological fluids, making them suitable for biosensor applications (main result 2, manuscript currently being finalised).
The final stage of the work involves the combination of the microfluidic chips with the stable membranes to enable characterisation using optical tweezers – this is still being finalised and will constitute main result 3 of the project.
Significant progress has been made during this project towards enabling measurements of mixed biological samples using biological nanopores and creating devices for miniaturisation of electrophysiology measurements using biological nanopores.
These results will push forward the development of biological nanopore-based biosensors for health applications, for example wearable devices that constantly monitor a range of analytes from the blood.
These results will push forward the development of biological nanopore-based biosensors for health applications, for example wearable devices that constantly monitor a range of analytes from the blood.