Exploring the water osmosis within physical and chemical controlled confinement for protein sensing.

Abstract

Proteins play a prominent role in life activities and the occurrence of disease, which is highly determined by their amino acid sequences and conformations. Therefore, protein sensing, including detection, sequencing, and confirmation characterization, has been attracting increasing attention in almost all areas. While nanopore sensing is regarded as one of the most promising approaches for single-molecule protein sensing, the heterogeneity of the protein charges raises a great challenge for capturing individual proteins into nanopores. Conventionally, the main forces to drive a molecule into the nanopore are the electrophoresis force determined by the charge carried by the molecule with an applied voltage, and the electroosmotic flow determined by the asymmetric anion/cation flow inside the nanopore also with an applied voltage. Here, we proposed water osmosis that is independent of the applied voltage to drive the protein/peptide with all kinds of charges. With the highly physical and chemical controlled confinement of aerolysin nanopore, by constructing an asymmetric salt concentration between two sides of the detection system, water osmosis occupied the dominance of all the fluidics inside aerolysin, further promoting the capturing of individual peptides without applied voltage. By enhancing the salt concentration gradient, the capture efficiency of a neutral model peptide, Ang II, was observed to be improved by nearly 13-fold. Furthermore, we explore the factors beyond salt concentration gradient, including charge distribution of the nanopore, solution composition, and lipid composition to precisely regulate the water osmosis and promote the peptide characterization with only one amino acid difference. Overall, the application of water osmosis in aerolysin nanopore can not only increase peptide capture frequency but also promote the development of proteomics research.