Slowing down to improve nanopore measurements
Synthetic nanopores are a promising method for characterizing individual molecules such as proteins, but interactions between the pore and the molecules being studied often interfere with the outcome. Researchers from Professor Michael Mayer’s Biophysics group have sought inspiration from nature to make the process more efficient and precise, ultimately yielding more accurate measurements.
Nanopores, surface passages just 10 to 30 nanometers wide, are useful tools for characterizing single molecules such as DNA strands, nanoparticles, or proteins. There are two types: biological nanopores found in nature, and synthetic nanopores, which are made in the lab out of materials such as silicon nitride. When they are present in an electrically insulating membrane, synthetic nanopores allow for the characterization of a wide variety of molecules because their size can be adjusted during fabrication. Detection of molecules requires monitoring the ionic current passing through the nanopore as a voltage is applied across the membrane. A molecule passing through the nanopore causes fluctuations in the current that can be interpreted, giving details of its shape, volume, electrical charge, rotation speed, and propensity to bind with other molecules. This information, in turn, helps allows researchers to identify the molecule.
Synthetic nanopores suffer from one major downside, though: their material tends to interact with the proteins being studied, leading to adsorption to the pore wall and clogging. To overcome this, the AMI researchers investigated lipid coatings to ease the translocation of proteins through the pore, allowing for more advanced characterization than previously possible.
In the context of sensing and characterizing single proteins with synthetic nanopores, these coatings provide at least four benefits: first, they minimize unwanted protein adhesion to the pore walls. Second, they can slow down protein translocation and rotation through their ability to tether proteins with a lipid anchor to the fluid bilayer coating. Third, they provide the possibility of imparting analyte specificity by including lipid anchors with a specific receptor or ligand in the coating. Finally, they offer a method for tuning nanopore diameters. The AMI study, which considered a wide variety of lipid membrane compositions, focused on changes in the nanopore’s signal noise properties with the application of a lipid coating, the stability of the lipid coating, its viscosity and translocation time, and the ability to successfully apply the lipid coating to a nanopore.
The most significant results from this study relate to lipids inspired by nature, in this case those produced by archaea, single-celled microorganisms with a structure similar to bacteria. Archaea are found in environments once thought to be entirely uninhabitable, such as volcanic vents, where most bacteria cannot survive. “These membrane-spanning lipids were the most viscous of those tested and led to the longest translocation times – a critical improvement with regard to advanced characterization of proteins,” explains BioPhysics alumna Dr. Olivia Eggenberger. “Additionally, we expanded our previous analysis to account for the lower event frequency that resulted from this high viscosity, allowing for the analysis of multiple populations within one dataset.”
According to Eggenberger, these findings are of particular use in the synthetic nanopore field, but the principles are universal and apply to lipid coatings in any function. In the long term, synthetic nanopores could be used to detect proteins such as amyloids that are markers of Alzheimer’s disease.
Reference: Eggenberger, O. M.; Leriche, G.; Koyanagi, T.; Ying, C.; Houghtaling, J.; Schroeder, T. B. H.; Yang, J.; Li, J.; Hall, A.; Mayer, M. Fluid Surface Coatings for Solid-State Nanopores: Comparison of Phospholipid Bilayers and Archaea-Inspired Lipid Monolayers. Nanotechnology 2019, 30 (32), 325504.