Orders-of-Magnitude Larger Force Demonstrated for Dielectrophoresis of Proteins Enabling High-Resolution Separations Based on New Mechanisms

Abstract

Proteins are perhaps the most important yet frustratingly complicated and difficult class of compounds to analyze, manipulate, and use. One very attractive option to characterize and differentially concentrate proteins is dielectrophoresis, but according to accepted theory, the force on smaller particles the size of proteins is too low to overcome diffusive action. Here, three model proteins, immunoglobulin G, α-chymotrypsinogen A, and lysozyme, are shown to generate forces much larger than predicted by established theory are more consistent with new theoretical constructs, which include the dipole moment and interfacial polarizability. The forces exerted on the proteins are quantitatively measured against well-established electrophoretic and diffusive processes and differ for each. These forces are orders of magnitude larger than previously predicted and enable the selective isolation and concentration of proteins consistent with an extremely high-resolution separation and concentration system based on the higher-order electric properties. The separations occur over a small footprint, happen quickly, and can be made in series or parallel (and in any order) on simple devices.

Protein Dielectrophoresis: A Tale of Two Clausius-Mossottis—Or Something Else?

“…An important example is provided by Quevedo et al [109], who show that synthetic lysozyme and BSA sub-micron particles, as well as their blends, are separable based on differences in their isoelectric points and as manifested in large differences of the voltage required to selectively trap them. These results mirror to some extent the unique electrokinetic signatures found by Liu and Hayes for α-chymotrypsinogen, immunoglobulin G and lysozyme in their innovative gradient insulator-based (g-iDEP) device operated under DC conditions [23]. Although these proteins exhibited behaviours consistent with negative DEP, non-linear electrokinetic effects may have complicated this interpretation.…”

“…For example, BSA can be driven to a metastable state at a high mass concentration and high values of buffer pH and conductivity. In this respect, it is also of interest to note that in their iDEP experiments with high protein concentrations, Liu and Hayes [23] reported negative DEP responses for α-chymotrypsinogen (4 mM), immunoglobulin G (0.7 mM) and lysozyme (7 mM). Proteins in an iDEP experiment can also be subjected to shear stresses and high electric field strengths, factors known to influence initial protein crystal growth [102,103].…”

“…In such cases, negative rather than positive protein DEP has been reported (e.g. [21][22][23]). As indicated in Figure 1, and discussed in Section 2, the theory underpinning [CM] macro employs the Maxwell cavity field, with the limitation of a minimum cavity size below which the rules of classical electrostatics theory may not hold.…”

iDEP-assisted isolation of insulin secretory vesicles

“…Accordingly, in an overlapping pattern of subpopulations, those with higher EKMr values overcome the weaker opposing forces at a given gate once the voltage is lowered. 16,59 Hence, the signal which was previously averaged with signals from the other overlapping subpopulations is now discernible at a higher EKMr value. Considering the high resolution of the technique, homogeneous subpopulations of vesicles undoubtably consist of a very narrow range of EKMr values.…”

“…Thus, an alternative approach to organelle isolation will be beneficial to define organelle subpopulations more accurately. Direct current insulator-based dielectrophoresis (DC-iDEP) provides a valuable tool for separating subpopulations of bioparticles with high resolution including viruses, bacteria, organelles, and proteins [13][14][15][16] . This approach offers a wide dynamic range, as it has been used for separations ranging from neural progenitors and stem cells 17 to resistant versus susceptible strains of cellular pathogens 15,18 .…”