Investigating protein translocation in the presence of an electrolyte concentration gradient across a solid‐state nanopore

Abstract

Electrolyte chemistry plays an important role in the transport properties of analytes through nanopores. Here, we report the translocation properties of the protein human serum transferrin (hSTf) in asymmetric LiCl salt concentrations with either positive (Ctrans/Ccis < 1) or negative chemical gradients (Ctrans/Ccis > 1). The cis side concentration was fixed at 4 M for positive chemical gradients and at 0.5 M LiCl for negative chemical gradients, while the trans side concentration varied between 0.5 to 4 M which resulted in six different configurations, respectively, for both positive and negative gradient types. For positive chemical gradient conditions, translocations were observed in all six configurations for at least one voltage polarity whereas with negative gradient conditions, dead concentrations where no events at either polarity were observed. The flux of Li+ and Cl− ions and their resultant cation or anion enrichment zones, as well as the interplay of electrophoretic and electroosmotic transport directions, would determine whether hSTf can traverse across the pore.

Enhanced Signal to Noise Ratio Enables High Bandwidth Nanopore Recordings and Molecular Weight Profiling of Proteins

“…While EP is sensitive to the charge of the protein, EO is sensitive to the charge of the nanopore surfacethe intricate interplay of the two forces would dictate the translocation characteristics . While KCl has been the choice of electrolyte in a myriad of nanopore-based translocation experiments, LiCl has been shown to promote events in the EOF (EO flow) direction at both relatively higher concentrations and asymmetric conditions (i.e., gradients). , LiCl has also been shown to reduce the translocation speed of DNA. , Unlike EPF (electrophoretic force) dominant transport, EOF can also transport net-neutral protein at their isoelectric point . Thus, at a high enough pH where the nanopore surface is charged, EOF could permit the translocation of a myriad of proteins irrespective of their net charge .…”

“…24 While KCl has been the choice of electrolyte in a myriad of nanopore-based translocation experiments, LiCl has been shown to promote events in the EOF (EO flow) direction at both relatively higher concentrations and asymmetric conditions (i.e., gradients). 21,25 LiCl has also been shown to reduce the translocation speed of DNA. 26,27 Unlike EPF (electrophoretic force) dominant transport, EOF can also transport net-neutral protein at their isoelectric point.…”

“…Moreover, the three larger proteins (i.e., catalase, ferreting, and alpha-macroglobulin) preferred a conventional gradient (i.e., higher trans electrolyte concentration) over symmetric electrolyte conditions in which the former easily produced ≳50 000 events/pipette, while the latter led to irreversible clogging, often yielding ∼3 times fewer events/pipette. Recently, Saharia et al showed, with an electrolyte gradient in which the cis electrolyte concentration is higher than the trans chamber (i.e., reverse gradient), both the signal amplitude (and by extension SNR) and C R of proteins could be increased in the electroosmotic transport direction (electrolyte chemistry-centric approach) . Thus, with such an approach, high LPF sensing could be done without resorting to pore sizes that lead to irreversible pore clogging by the analyte.…”

“…Recently, Saharia et al showed, with an electrolyte gradient in which the cis electrolyte concentration is higher than the trans chamber (i.e., reverse gradient), both the signal amplitude (and by extension SNR) and C R of proteins could be increased in the electroosmotic transport direction (electrolyte chemistrycentric approach). 21 Thus, with such an approach, high LPF sensing could be done without resorting to pore sizes that lead to irreversible pore clogging by the analyte. For example, the smallest protein (i.e., cytochrome c) failed to produce high enough SNR events under symmetric conditions which, with the use of reverse gradient (i.e., higher cis electrolyte concentration), easily produced >50 000 events/pipette.…”

Protein and DNA Yield Current Enhancements, Slow Translocations, and an Enhanced Signal-to-Noise Ratio under a Salt Imbalance

“…15 Notably, the cis chamber was always at a low salt concentration (e.g., ∼0.2 M and below mostly) except in a few select cases. 16 Typically, the trans side (i.e., voltage application chamber) is kept at a higher concentration compared to the cis chamber (grounded side) and a positive bias (in the case of DNA) is applied to the trans side, which causes the positive ions to flow in favor of chemical and electrical gradients (trans to cis) and polarizes the cis side pore opening, leading to a higher capture rate and slower translocation time. However, to the best of our knowledge, no prior work exists with the use of asymmetric salt conditions with nanopipettes.…”