An Ion-Specific Effect on Polymer-Protein Interaction Enhances Resolution of Nanopore-Based Detection

Abstract

Poly(ethyleneglycol) (PEG) oligomers partition into acqueous transmembrane channels formed by alpha-Hemolysin (aHL) and Aerolysin (AeL) proteins and transiently bind the pore to induce reductions in conductance, i.e. resistive pulses, the amplitude of which is strongly related to polymer chain length. Histograms of such data for PEG yield mass spectrograms with single monomer resolution between approximately 20 and 60 repeat units (r.u.). For chain lengths <20 the duration of resistive pulses becomes too short (e.g.<50 µs) to be fully resolved using state-of-the art electrophysiological recording technology. Recently, computational reconstruction of unresolved events has revealed the presence in a sample of PEG species down to 9 r.u. from data acquired with a bandwidth of 0-100 kHz(1), showing that small PEGs also interact with the pore. We were interested in prolonging the interaction of pore and polymer in order to enable a quantitative detection of PEG species <20 r.u. Krasilnikov and coworkers reported in 2011 (2) that the anion species in the electrolyte strongly affected the dwell times of the aHL pore by PEG, with strongly hydrated F- promoting long-lasting events. Here, using 1:1 mixtures of KF and KCl (4 M) solutions, we report direct resolution of PEG oligomers down to 8 r.u. from data obtained at filter settings of 20 kHz (see figure), due to a significant increase in the characteristic dwell times (tau) of events: for PEG28 tau increased from approx. 1.5 ms in 4M KCl to 7 ms in 4M 1:1 KCl/KF while tau for PEG12 was not directly measurable in KCl (estimated at 18 µs in Ref. 1) but found at ca. 90 µs in KCl/KF.(1) Balijepalli et al. ACS Nano 2014, 8, 1547-1553.(2) Rodrigues et al. Biophys. J. 2011, 100, 2929-2935.