Electro-Osmotic Vortices Promote the Capture of Folded Proteins by PlyAB Nanopores

Abstract

Biological nanopores are emerging as powerful tools for single-molecule analysis and sequencing. Here, we engineered the two-component pleurotolysin (PlyAB) toxin to assemble into 7.2 × 10.5 nm cylindrical nanopores with a low level of electrical noise in lipid bilayers, and we addressed the nanofluidic properties of the nanopore by continuum simulations. Surprisingly, proteins such as human albumin (66.5 kDa) and human transferrin (76–81 kDa) did not enter the nanopore. We found that the precise engineering of the inner surface charge of the PlyAB induced electro-osmotic vortices that allowed the electrophoretic capture of the proteins. Once inside the nanopore, two human plasma proteins could be distinguished by the characteristics of their current blockades. This fundamental understanding of the nanofluidic properties of nanopores provides a practical method to promote the capture and analysis of folded proteins by nanopores.

Analytical Model for Particle Capture in Nanopores Elucidates Competition among Electrophoresis, Electroosmosis, and Dielectrophoresis

“…Electroosmosis is hence deeply entangled with the ionic flow, as both depend on pore shapes and their charge distribution, leading to complex electrohydrodynamic patterns. 25 − 27 Electroosmosis can either compete or cooperate with electrophoresis in particle capture. 28 − 31 An example of competition is discussed in ref ( 30 ), where the electroosmosis was shown to induce capture of peptides against electrophoresis.…”

Biological Nanopores: Engineering on Demand

“…Analogous mutations in PlyAB also lead to a change in ion selectivity, impacting the EOF direction. Furthermore, in PlyAB they also cause the appearance of electroosmotic vortices, which are important for protein capture and are already appreciated in solid-state nanopores [111,150].…”

“…Another viral protein, the thermophage G20c portal protein, can be inserted into lipid bilayers when conjugated with porphyrin [110]. Other potentially useful pores include pleurotolysin (PlyAB, [111]) and the outer membrane protein F (OmpF; [112]), which have been explored for the detection of proteins and ampicillin, respectively ( Figure 2).…”

“…Analogous mutations in PlyAB also lead to a change in ion selectivity, impacting the EOF direction. Furthermore, in PlyAB they also cause the appearance of electroosmotic vortices, which are important for protein capture and are already appreciated in solid-state nanopores [111,150]. While it is relatively straightforward to predict which pore residues might prevent nucleic acid passage through charge repulsion, charge alterations may have unpredictable effects on peptide translocation (see above).…”

“…The α-HL pores also remain stable in the presence of moderate concentrations of While it is relatively straightforward to predict which pore residues might prevent nucleic acid passage through charge repulsion, charge alterations may have unpredictable effects on peptide translocation (see above). Because of the intricate interplay between the electroosmotic and electrophoretic transport of proteins and peptides, current efforts appear to be more focused on pH optimization that allows appropriate sampling of protein analytes [103,111,148]. However, the transport of analytes larger than the pore channel may be dictated by electrophoresis, rather than EOF, as demonstrated by the analysis of 540 amino acid-large pertactin passenger domain using the AeL nanopore [151].…”