Asuka Nakano

Protein dielectrophoresis: Advances, challenges, and applications

Protein dielectrophoresis (DEP) has the potential to play an important role as a manipulation, fractionation, preconcentration, and separation method in bioanalysis and as manipulation tool for nanotechnological applications. The first demonstrations of protein DEP have been reported almost 20 years ago. Since then various experimental realizations to manipulate proteins by DEP as well as more targeted applications employing protein DEP have been demonstrated. This review summarizes the experimental studies in the field of protein DEP trapping and focusing as well as specific applications in separation, molecular patterning, on bioprobes and biosensors. While a comprehensive theoretical model describing protein DEP is still lacking we also attempt to provide an overview of the factors influencing protein DEP and relate to currently available theoretical models. We further point out the variations in experimental conditions used in the past to study the somewhat 20 proteins as well as the implications of protein molecular structure to the DEP response.

Immunoglobulin G and bovine serum albumin streaming dielectrophoresis in a microfluidic device

Dielectrophoresis (DEP) has demonstrated to be a versatile tool to manipulate micro‐ and nanoparticles with applications for positioning, separation and fractionation. Recent developments of DEP have also shown that DEP can be used for the manipulation of biomolecules, such as DNA. Here, we focus on the manipulation of proteins using insulator‐based dielectrophoresis (iDEP). We designed suitable post arrays in a microfluidic channel and use numerical simulations to calculate the electric field distribution as well as concentration of proteins according to a convection–diffusion model for both negative and positive DEP. Experimentally, we find DEP trapping of mainly protein aggregates in phosphate buffer. However, when adding a charged zwitterionic detergent, we observed DEP streamlining of immunoglobulin G (IgG) and bovine serum albumin (BSA). Our experimental observations are in excellent agreement with numerical simulations and indicate positive DEP behavior of IgG and BSA under the employed experimental conditions. Our results demonstrate DEP streaming of proteins in an iDEP device for the first time and indicate the potential of protein DEP for separation and fractionation.

Tuning direct current streaming dielectrophoresis of proteins

Dielectrophoresis (DEP) of biomolecules has large potential to serve as a novel selectivity parameter for bioanalytical methods such as (pre)concentration, fractionation, and separation. However, in contrast to well-characterized biological cells and (nano)particles, the mechanism of protein DEP is poorly understood, limiting bioanalytical applications for proteins. Here, we demonstrate a detailed investigation of factors influencing DEP of diagnostically relevant immunoglobulin G (IgG) molecules using insulator-based DEP (iDEP) under DC conditions. We found that the pH range in which concentration of IgG due to streaming iDEP occurs without aggregate formation matches the pH range suitable for immunoreactions. Numerical simulations of the electrokinetic factors pertaining to DEP streaming in this range further suggested that the protein charge and electroosmotic flow significantly influence iDEP streaming. These predictions are in accordance with the experimentally observed pH-dependent iDEP streaming profiles as well as the determined IgG molecular properties. Moreover, we observed a transition in the streaming behavior caused by a change from positive to negative DEP induced through micelle formation for the first time experimentally, which is in excellent qualitative agreement with numerical simulations. Our study thus relates molecular immunoglobulin properties to observed iDEP, which will be useful for the future development of protein (pre)concentration or separation methods based on DEP.

Quantification of pH gradients and implications in insulator-based dielectrophoresis of biomolecules.

Direct current (DC) insulator‐based dielectrophoretic (iDEP) microdevices have the potential to replace traditional alternating current dielectrophoretic devices for many cellular and biomolecular separation applications. The use of large DC fields suggest that electrode reactions and ion transport mechanisms can become important and impact ion distributions in the nanoliters of fluid in iDEP microchannels. This work tracked natural pH gradient formation in a 100 μm wide, 1 cm‐long microchannel under applicable iDEP protein manipulation conditions. Using fluorescence microscopy with the pH‐sensitive dye FITC Isomer I and the pH‐insensitive dye TRITC as a reference, pH was observed to drop drastically in the microchannels within 1 min in a 3000 V/cm electric field; pH drops were observed in the range of 6–10 min within a 100 V/cm electric field and varied based on the buffer conductivity. To address concerns of dye transport impacting intensity data, electrokinetic mobilities of FITC were carefully examined and found to be (i) toward the anode and (ii) 1 to 2 orders of magnitude smaller than H+ transport which is responsible for pH drops from the anode toward the cathode. COMSOL simulations of ion transport showed qualitative agreement with experimental results. The results indicate that pH changes are severe enough and rapid enough to influence the net charge of a protein or cause aggregation during iDEP experiments. The results also elucidate reasonable time periods over which the phosphate buffering capacity can counter increases in H+ and OH− for unperturbed iDEP manipulations.

Temporal and Spatial Temperature Measurement in Insulator-Based Dielectrophoretic Devices

Insulator-based dielectrophoresis is a relatively new analytical technique with a large potential for a number of applications, such as sorting, separation, purification, fractionation, and preconcentration. The application of insulator-based dielectrophoresis (iDEP) for biological samples, however, requires the precise control of the microenvironment with temporal and spatial resolution. Temperature variations during an iDEP experiment are a critical aspect in iDEP since Joule heating could lead to various detrimental effects hampering reproducibility. Additionally, Joule heating can potentially induce thermal flow and more importantly can degrade biomolecules and other biological species. Here, we investigate temperature variations in iDEP devices experimentally employing the thermosensitive dye Rhodamin B (RhB) and compare the measured results with numerical simulations. We performed the temperature measurement experiments at a relevant buffer conductivity range commonly used for iDEP applications under applied electric potentials. To this aim, we employed an in-channel measurement method and an alternative method employing a thin film located slightly below the iDEP channel. We found that the temperature does not deviate significantly from room temperature at 100 μS/cm up to 3000 V applied such as in protein iDEP experiments. At a conductivity of 300 μS/cm, such as previously used for mitochondria iDEP experiments at 3000 V, the temperature never exceeds 34 °C. This observation suggests that temperature effects for iDEP of proteins and mitochondria under these conditions are marginal. However, at larger conductivities (1 mS/cm) and only at 3000 V applied, temperature increases were significant, reaching a regime in which degradation is likely to occur. Moreover, the thin layer method resulted in lower temperature enhancement which was also confirmed with numerical simulations. We thus conclude that the thin film method is preferable providing closer agreement with numerical simulations and further since it does not depend on the iDEP channel material. Overall, our study provides a thorough comparison of two experimental techniques for direct temperature measurement, which can be adapted to a variety of iDEP applications in the future. The good agreement between simulation and experiment will also allow one to assess temperature variations for iDEP devices prior to experiments.