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Nanopore probing of molecular level transport of proteins is strongly influenced by electrolyte type, concentration, and solution pH. As a result, electrolyte chemistry and applied voltage are critical for protein transport and impact, for example, capture rate ( C R ), transport mechanism ( i.e. , electrophoresis, electroosmosis or diffusion), and 3D conformation ( e.g. , chaotropic vs. kosmotropic effects). In this study, we explored these using 0.5–4 M LiCl and KCl electrolytes with holo-human serum transferrin (hSTf) protein as the model protein in both low (±50 mV) and high (±400 mV) electric field regimes. Unlike in KCl, where events were purely electrophoretic, the transport in LiCl transitioned from electrophoretic to electroosmotic with decreasing salt concentration while intermediate concentrations ( i.e. , 2 M and 2.5 M) were influenced by diffusion. Segregating diffusion-limited capture rate ( R diff ) into electrophoretic ( R diff,EP ) and electroosmotic ( R diff,EO ) components provided an approach to calculate the zeta-potential of hSTf ( ζ hSTf ) with the aid of C R and zeta potential of the nanopore surface ( ζ pore ) with ( ζ pore – ζ hSTf ) governing the transport mechanism. Scrutinization of the conventional excluded volume model revealed its shortcomings in capturing surface contributions and a new model was then developed to fit the translocation characteristics of proteins.
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