Is the Volume Exclusion Model Practicable for Nanopore Protein Sequencing?

Abstract

The nanopore approach holds the possibility for achieving single-molecule protein sequencing. However, ongoing challenges still remain in the biological nanopore technology, which aims to identify 20 natural amino acids by reading the ionic current difference with the traditional current-sensing model. In this paper, taking aerolysin nanopores as an example, we calculate and compare the current blockage of each of 20 natural amino acids, which are all far from producing a detectable current blockage difference. Then, we propose a modified solution conductivity of σ′ in the traditional volume exclusion model for nanopore sensing of a peptide. The σ′ value describes the comprehensive result of ion mobility inside a nanopore, which is related to but not limited to nanopore–peptide interactions, and the positions, orientations, and conformations of peptides inside the nanopore. The nanopore experiments of a short peptide (VQIVYK) in wild type and mutant nanopores further demonstrate that the traditional volume exclusion model is not enough to fully explain the current blockage contribution and that many other factors such as enhanced nanopore–peptide interactions could contribute to a dominant part of the current change. This modified sensing model provides insights into the further development of nanopore protein sequencing methods.

β-Barrel Nanopores with an Acidic–Aromatic Sensing Region Identify Proteinogenic Peptides at Low pH

“…These discrepancies could be explained by considering that I ex % reflects the excluded volume rather than the mass of the peptide. 42 , 43 …”

Protein identification by nanopore peptide profiling

“…In nanopore analysis, analytes are measured as they disrupt an ionic current passing through individual nanopores under an applied potential. Importantly, the magnitude of the current blockade (I B ) is mainly, altought not exclusively 11 , proportional to the volume the analyte excludes, allowing size discrimination of chemically similar (bio)polymers such as PEG chains, DNA, proteins, and peptides [12][13][14][15][16][17][18][19][20][21][22][23][24] . Furthermore, nanopores are capable of accurately detecting a variety of molecules, including proteins and DNA, with high precision at the single-molecule level 17,21,[24][25][26][27][28] .…”

Biological nanopores for sensing applications

“…When analytes translocate through the constriction region of nanopore, factors such as their hydrophobic volume, surface charge, and analyteprotein interaction together contribute to the current change. 22,23 Commonly, the current blockade (ΔI/I 0 ) and dwell time (Δt) are analyzed to characterize the signals of a certain analyte (Figure 1C). For discrimination of complicated signals, machine learning algorithm that extracts the features of signals is preferred.…”

“…While for neutral or weakly charged analytes, electroosmotic flow dominates. When analytes translocate through the constriction region of nanopore, factors such as their hydrophobic volume, surface charge, and analyte‐protein interaction together contribute to the current change 22,23 . Commonly, the current blockade (∆ I / I 0 ) and dwell time (∆ t ) are analyzed to characterize the signals of a certain analyte (Figure 1C).…”