Influence of ionic constituents and electrical conductivity on the propagation of charged nanoscale objects in passivated gel electrophoresis

Abstract

When determining the electric field E acting on charged objects in gel electrophoresis, the electrical conductivity of the buffer solution is often overlooked; E is typically calculated by dividing the applied voltage by a separation distance between electrodes. However, as a consequence of electrolytic reactions, which occur at the electrodes, gradients in the ionic content of the buffer solution and its conductivity can potentially develop over time, thereby impacting E and affecting propagation velocities of charged objects, v, directly. Here, we explore how the types and concentrations of ionic constituents of the buffer solution, which largely control its conductivity, when used in passivated gel electrophoresis (P-gelEP), can influence E, thereby altering v of charged nanospheres propagating through large-pore gels. We measure the conductivity of the buffer solution in the center of the gel region near propagating bands of nanospheres, and we show that predictions of E based on conductivity closely correlate with v. We also explore P-gelEP involving two different types of passivation agents: nonionic polyethylene glycol (PEG) and anionic sodium dodecyl sulfate (SDS). Our observations indicate that using a conductivity model to determine E from the local current density and the conductivity where spheres are propagating can lead to a better estimate than the standard approach of a voltage divided by a separation. Moreover, this conductivity model also provides a starting point for interpreting the complex behavior created by amphiphilic ionic passivation agents, such as SDS, on propagating nanospheres used in some P-gelEP experiments.

Band-collision gel electrophoresis

“…7i; Supplementary Movie 11). We have also used sulfate-stabilized polystyrene (SSPS) nanospheres as reagents in a passivated gel form of BCGE; the nonionic passivation agent PEG-1000 has been added at 3.25 mM to allow SSPS nanospheres to propagate in a large-pore agarose gel 45,61 (see Methods). Sulfate groups on a sphere becomes irreversibly bound to sulfate groups on other spheres, mediated by Sr 2+ cations 62 , leaving behind a narrow stationary band of nanosphere aggregates larger than the gel’s pore size (Fig.…”

“…All gels are prepared using Sigma-Aldrich Type I-A, low EEO agarose at 3.0% w/w in distilled water (conductivity measured to be <0.5 μS cm −1 ) 45 . Using this agarose concentration corresponds to characteristic pore sizes of the gel of ≈50 nm, since nanospheres having diameters smaller than ≈50 nm will propagate through the gel 61 .…”

“…The chamber is filled with buffer solution (typically 5.0 mM sodium borate buffer at pH = 9.0) to a height of 2.5 cm above the gel surface. While we report electric field strengths to enable comparison with other GE experiments, we operate our power supply in constant current mode throughout all experiments to keep band velocities constant over longer durations 45 . All infusions of samples into wells are 4 μL in volume unless otherwise stated.…”

“…1 and the Methods section), thereby revealing both attractive interactions and also, more generally with other reagent types, irreversible chemical reactions. We extend this approach, which we call band-collision gel electrophoresis (BCGE), to include invisible (i.e., optically non-absorbing or only very weakly absorbing) molecules, as well as colloidal species that scatter visible light such as polymer nanospheres 45 . We show that a wide range of complex spatiotemporal patterns form and evolve when bands of different species collide.…”

Ionic conductivity and hydrodynamic permeability of inhomogeneous (cavity doped) polyelectrolyte hydrogels

“…These include electro-osmotic advection and ion diffusion, which are absent in ideal homogeneous media, but may arise from inhomogeneities of charge and/or permeability. Ion transport in porous media with hierarchical architectures is important in a diversity of fields, including deep brain stimulation (Pomfret, Sillay & Miranpuri 2013) and gel electrophoresis (Bikos & Mason 2018). Electro-osmotic flow that accompanies ion transport has been harnessed to great benefit for capillary electrochromatography, significantly enhancing molecular separations by electro-osmotic flow suppressing hydrodynamic dispersion (Tallarek et al 2001).…”